Tom "Prophet" Hsiung » Recruitment

Diffusion of Gases

September 28, 2016 Uncategorized No comments bulk flow, Diffusion Coefficient, diffusion limitation, diffusivity, equilibrium, Fick's law, match of ventilation and perfusion, partial pressure difference, perfusion limitation, Recruitment, Surface Area, temperature, thickness of barrier, total cross-sectional area

Diffusion of a gas occurs when there is a net movement of molecules from an area in which that particular gas exerts a high partial pressure to an area in which it exerts a lower partial pressure. Movement of a gas by diffusion is therefore different from the movement of gases through the conducting airways, which occurs by "bulk flow" (mass movement or convection). During bulk flow, gas movement results from differences in total pressure, and molecules of different gases move together along the total pressure gradient. During diffusion, different gases move according to their own individual partial pressure gradients. Gas transfer during diffusion occurs by random molecular movement. It is therefore dependent on temperature because molecular movement increases at higher temperatures. Gases move in both directions during diffusion, but the area of higher partial pressure, because of its greater number of molecules per unit volume, has proportionately more random "departures." Thus, the net movement of gas is dependent on the partial pressure difference between the 2 areas. In a static situation, diffusion continues until no partial pressure differences exist for any gases in the 2 areas; in the lungs, oxygen and carbon dioxide continuously enter and leave the alveoli, and so such an equilibrium does not take place.

Fick's Law for Diffusion

Oxygen is brought into the alveoli by bulk flow through the conducting airways. When air flows through the conducting airway during inspiration, the linear veocity of the bulk flow decreases as the air approaches the alveoli. This is because the total cross-sectional area increases dramatically in the distal protions of the tracheobronchial tree.

By the time the air reaches the alveoli, bulk flow probably ceases, and further gas movement occurs by diffusion. Oxygen then moves through the gas phase in the alveoli according to its own partial pressure gradient. The distance from the alveolar duct to the alveolar-capillary interface is usually less than 1 mm. Diffusion in the alveolar gas phase is believed to be greatly assisted by the pulsations of the heart and blood flow, which are transmitted to the alveoli and increase molecular motion.

Oxygen the diffuses through the alveolar-capillary interface. It must first, therefore, move from the gas phase to the liquid phase, according to Henry's law. Oxygen must dissolve in and diffuse through the thin layer of pulmonary surfactant, the alveolar epithelium, the interstitium, and the capillary endothelium. It must then diffuse through the plasma, where some remains dissolved and the majority enters the erythrocyte and combines with hemoglobin. The blood then carries the oxygen out of the lung by bulk flow and distributes it to the other tissues of the body. At tissues, oxygen diffuses from the erythrocyte through the plasma, capillary endothelium, interstitium, tissue cell membrane, and cell interior and into the mitochondrial membrane. The process is almost entirely reversed for carbon dioxide.

The factors that determine the rate of diffusion of gas through the alveolar-capillary barrier are described by Fick's law for diffusion, shown here in a simpified form:

V_gas = [A X D X (P₁ – P₂)] / T [Equation 1]

where V_gas = volume of gas diffusing through the tissue barrier per time, mL/min

A = surface area of the barrier available for diffusion

D = diffusion coefficient, or diffusivity, of the particular gas in the barrier

T = thickness of barrier of the diffusion distance

P₁– P₂ = partial pressure difference of the gas across the barrier

That is, the volume of gas per unit of time moving across the alveolar-capillary barrier is directly proportional to the surface area of the barrier, the diffusivity, and the difference in concentration between the 2 sides, but is inversely proportional to the barrier thickness.

Surface area of barrier

The surface area of the blood-gas barrier is believed to be at least 70 m² in a healthy average sized adult at rest. That is, about 70 m² of the potential surface area is both ventilated and perfused at rest. If more capillaries are recruited, as in exercise, the surface area available for diffusion increase; if venous return falls, for example, because of hemorrhage, or if alveolar pressure is raised by positive-pressure ventilation, then capillaries may be derecruited and the surface available for diffusion may decrease.

Thickness of barrier

The thickness of the alveolar-capillary diffusion barrier is only about 0.2 to 0.5 um. This barrier thickness can increase in interstitial fibrosis or interstitial edema, thus interfering with diffusion. Diffusion probably increase at higher lung volumes as alveoli are stretched, the diffusion distance decreases slightly (and also because small airways subject to closure may be open at higher lung volumes).

Diffusion coefficient/Diffusivity

The diffusivity, or diffusion constant, for a gas is directly proportional to the solubility of the gas in the diffusion barrier and is inversely proportional to the square root of the molecular weight (MW) of the gas:

The relationship between solubility and diffusion through the barrier has already been discussed. The diffusivity is inversely propprtional to the square root of the MW of the gas because different gases with equal numbers of molecules in equal volumes have the same molecular energy if they are at the same temperature. Therefore, light molecules travel faster, have more frequent collisions, and diffuse more rapidly. Thus, Graham's law states that the relative rates of diffusion of 2 gases are inversely proportional to the square roots of their MWs, if all else is equal.

Because the difference in MWs of oxygen and carbon dioxide, it should diffuse 1.2 times as fast as carbon dioxide. In hte alveolar-capillary barrier, however, the relative solubilities of oxygen and carbon dioxide must also be considered. The solubility of carbon dioxide in the liquid phase is about 24 times that of oxygen, and so carbon dioxide diffuse about 20 times more rapidly through the alveolar-capillary barrier than does oxygen. For this reason, patients develop problems in oxygen diffusion through the alveolar-capillary barrier before carbon dioxide retention due to diffusion impairment occurs.

Limitations of Gas Transfer

The factors that limit the movement of a gas through the alveolar-capillary barrier, as described by Fick's law for diffusion, can be divided into 3 components: the diffusion coefficient, the surface area and thickness of the alveolar-capillary membrane, and the partial pressure difference across the barrier for each particular gas.

Diffusion Limitation

An erythrocyte and its attendant plasma spend an average of about 0.75 to 1.2 seconds inside the pulmonary capillaries at resting cardiac outputs. This time can be estimated by dividing the pulmonary capillary blood volume by the pulmonary blood flow. Some erythrocytes may take less time to traverse the pulmonary capillaries; others may take longer. Figure 6-1 shows schematically the calculated change with time in the partial pressures in the blood of 3 gases: oxygen, carbon monoxide, and nitrous oxide. These are shown in comparision to the alveolar partial pressures for each gas, as indicated by the dotted line. This alveolar partial pressure is different for each of the 3 gases, and it depends on its concentration in the inspired gas mixture and on how rapidly it is removed by the pulmonary capillary blood. The schematic is drawn as though all 3 gases were administered simultaneously, but this is not necessarily the case. Consider each gas as though it were acting independently of the others.

The partial pressure of carbon monoxide in the pulmonary capillary blood rises very slowly compared with that of the other 2 gases in the figure. (Obviously, a low inspired concentration of carbon monoxide must be used for a very short time in such an expirement.) Nevertheless, if the content of carbon monoxide were measured simultaneously, it would be rising very rapidly. The reason for this rapid rise is that carbon monoxide combines chemically with the hemoglobin in the erythrocytes. Indeed, the affinity of carbon monoxide for hemoglobin is about 210 times that of oxygen for hemoglobin. The carbon monoxide that is cheically combined with hemoglobin does not contribute to the partial pressure of carbon monoxide in the blood because it is no longer physically dissolved in it.

Therefore, the partial pressure of carbon monoxide in the pulmonary capillary blood does not come close to the partial pressure of carbon monoxide in the alveoli during the time that the blood is exposed to the alveolar carbon monoxide. (If the alveolar partial pressure of carbon monoxide were great enough to saturate the hemoglobin, the pulmonary capillary partial pressure would rise rapidly.) The partial pressure difference across the alveolar-capillary barrier for carbon monoxide is thus well maintained for the entire time the blood spends in the pulmonary capillary, and the diffusion of carbon monoxide is limited only by its diffusivity in the barrier and by the surface area and thickness of the barrier – that is, the diffusion characteristics of the barrier itself. Carbon monoxide transfer from the alveolus to the pulmonary capillary blood is referred to as diffusion-limited rather than perfusion-limited.

Perfusion Limitation

The partial pressure of nitrous oxide in the pulmonary capillary blood equilibrates very rapidly with the partial pressure of nitrous oxide in the alveolus because nitrous oxide moves through the alveolar-capillary barrier very easily and because it does not combine chemically with the hemoglobin in the erythrocytes. After only about 0.1 of a second of exposure of the pulmonary capillary blood to the alveolar nitrous oxide, the partial pressure difference across the alveolar-capillary barrier has been abolished. From this point on, no further nitrous oxide transfer occurs from the alveolus to that portion of the blood in the capillary that has already equilibrated with the alveolar nitrous oxide partial pressure; duirng the last 0.6 to 0.7 of a second, no net diffusion occurs between the alveolus and the blood as it travels through the pulmonary capillary. Of course, blood just entering the capillary at the arterial end will not be equilibrated with the alveolar partial pressure of nitrous oxide, and so nitrous oxide can diffuse into the blood at the arterial end. The transfer of nitrous oxide is therefore perfusion-limited. Nitrous oxide transfer from a particular alveolus to one of its pulmonary capillaries can be increased by increasing the cardiac output and thus reducing the amount of time the blood stays in the pulmonary capillary after equilibration with the alveolar partial pressure of nitrous oxide has occurred. (Because increasing the cardiac output may recruit previously unperfused capillaries, the total diffusion of both carbon monoxide and nitrous oxide may increase as the surface area for diffusion increases.)

Diffusion of Oxygen

As can be seen in Figure 6-1, the time course for oxygen transfer falls between those for carbon monoxide and nitrous oxide. The partial pressure of oxgen rises fairly rapidly (note that it starts at the PO₂ of 40 mm Hg, rather than at zero), and equilibration with the alveolar PO₂ of about 100 mm Hg occurs within about 0.25 of a second, or about one third of the time the blood is in the pulmonary capillary at normal resting cardiac outputs. Oxygen moves easily through the alveolar-capillary barrier and into the erythrocytes, where it combines chemically with hemoglobin. The partial pressure of oxygen rises more rapidly than the partial pressure of carbon monoxide (at very low partial pressure carbon monoxide that would be used). Nonetheless, the oxygen chemically bound to hemoglobin (and therefore no longer physically dissolved) exerts no partial pressure, and so the partial pressure difference across the alveolar-capillary membrane is initially well maintained and oxygen transfer occurs. The chemical combination of oxygen and hemoglobin, however, occurs rapidly (within hundredths of a second), and at the normal alveolar partial pressure of oxygen, the hemoglobin becomes nearly saturated with oxygen very quickly. As this happens, the partial pressure of oxygen in the blood rises rapidly to that in alveolus, and from that point, no further oxygen transfer from the alveolus to the quilibrated blood cna occur. Therefore, under the conditions of normal alveolar PO₂ and a normal resting cardiac output, oxygen transfer from alveolus to pulmonary capillary is perfusion-limited.

Figure 2-6A shows similar graphs of calculated changes in the partial pressure of oxygen in the blood as it moves through a pulmonary capillary. The alveolar PO₂ is normal. During exercise, blood moves through the pulmonary capillary much more rapidly than it does at resting cardiac outputs. In fact, the blood may stay in the "functional" pulmonary capillary on an average of only about 0.25 of a second during strenuous exercise, as indicated on the graph. Oxygen transfer into the blood per time will be greatly increased because there is little or no perfusion limitation of oxygen transfer. (Indeed, that part of the blood that stays in the capillary less than the average may be subjected to diffusion limitation of oxygen transfer.) Of course, total oxygen transfer is also increased during exercise because of recruitment of previously unperfused capillaries, which increases the surface area for diffusion, and because of better matching of ventilation and perfusion. A person with an abnormal alveolar-capillary barrier due to a fibrotic thickening or interstitial edema may approach diffusion limitation of oxygen transfer at rest and may have a serious diffusion limitation of oxygen transfer during strenuous exercise, as can be seen in the middle curve in Figure 2-6A. A person with an extremely abnormal alveolar-capillary barrier might have diffusion limitation of oxygen transfer even at rest, as seen on the right in the figure.

The effect of a low alveolar partial pressure of oxygen on oxygen transfer from the alveolus to the capillary is seen in Figure 6-2B. The low alveolar PO₂ sets the upper limit for the end-capillary blood PO₂. Because the oxygen content of the arterial blood is decreased, the mixed venous PO₂ is also decreased. The even greater decrease in the alveolar partial pressure of oxygen, however, causes a decreased alveolar-capillary partial pressure gradient, and the blood PO₂ takes longer to equilibrate with the alveolar PO₂. For this reason, a normal person exerting himself or herself at high altitude might be subject to diffusion limitation of oxygen transfer.

Diffusion of Carbon Dioxide

The time course of carbon dioxide transfer from the pulmonary capillary blood to the alveolus is shown in Figure 6-3. In a normal person with a mixed venous partial pressure of carbon dioxide of 45 mm Hg an dan alveolar partial partial pressure of carbon dioxide of 40 mm Hg, an equilibrium is reached in a little more than 0.25 of a second, or about the same time as that for oxygen. This may seem surprising, considering that the diffusivity of carbon dioxide is about 20 times that of oxygen, but the partial pressure gradient is normally only about 5 mm Hg for carbon dioxide, whereas it is about 60 mm Hg for oxygen. Carbon dioxide transfer is therefore normally perfusion-limited, although it may be diffusion-limited in a person with an abnormal alveolar-capillary barrier, as shown in the figure.

Measurement of Diffusing Capacity

If is often useful to determine the diffusion characteristics of a patient's lungs during their assessment in the pulmonary function laboratory. It may be particularly important to determine whether an apparent impairment in diffusion is a result of perfusion limitation or diffusion limitation.

The diffusion capacity (or transfer factor) is the rate at which oxygen or carbon monoxide is absorbed from the alveolar gas into the pulmonary capillaries (in milliliters per minute) per unit of partial pressure difference (in millimeters of mercury). The diffusing capacity of the lung (for gas x), D_Lx, is therefore equal to the uptake of gas x, V_x, divided by the difference between the alveolar partial pressure of gas x, P_Ax, and the mean capillary partial pressure of gas x, Pc_x:

The mean partial pressure of oxygen or carbon monoxide is, as already discussed, affected by their chemical reactions with hemoglobin, as well as by their transfer through the alveolar-capillary barrier. For this reason, the diffusing capacity of the lung is determined by both the diffusing capacity of the membrane (both the alveolar-capillary membrane and the plasma membrane of the erythrocyte), D_M, and the reaction with hemoglobin, expressed as 𝜃 X Vc, where 𝜃 is the volume of gas in mililiters per minute taken up by the erythrocytes in 1 mL of blood per millimeter of mercury partial pressure gradient between the plasma and the erythrocyte and Vc is the capillary blood volume in milliliters. (The units of 𝜃 X Vc are therefore mL/min/mm Hg.) The diffusing capacity of the lung, D_L, can be shown to be related to D_M and 𝜃 X Vc as follows:

D_A, or diffusion through the alveolus, is normally very rapid and usually can be disregarded; however, in conditions such as alveolar pulmonary edema or pneumonia it may be a major problem.

Carbon monoxide is most frequently used in determinations of the diffusing capacity because the mean pulmonary capillary partial pressure of carbon monoxide is virtually zero when nonlethal alveolar partial pressures of carbon monoxide are used:

Several different methods are used clinically to measure the carbon monoxide diffusing capacity and involve both single-breath and steady-state techniques, sometimes during exercise. The D_Lco is decreased in diseases associated with interstitial or alveolar fibrosis, such as idiopathic pulmonary fibrosis, sarcoidosis, scleroderma, and asbestosis, or with conditions causing interstitial or alveolar pulmonary edema. It is also decreased in conditions causing a decrease in the surface area available for diffusion, such as emphysema, tumors, a low cadiac output, or a low pulmonary capillary blood volume, as well as in conditions leading to ventilation-perfusion mismatch, which effectively decreases the surface area available for diffusion.

[Respiration][Circulation] Blood Flow to the Lung – General and Pulmonary Vascular Resistance

September 11, 2016 Cardiology, Critical Care, Hemodynamics, Mechanical Ventilation, Physiology and Pathophysiology, Pulmonary Medicine, Respirology No comments bronchial circulation, distention, extravascular effects, functional pulmonary capillary, gas exchange, lung volume, PEEP, pulmonary circulation, PVR, Recruitment, SVR, Transmural Pressure, U-shape curve

The lung receives blood flow via both the bronchial circulation and the pulmonary circulation. Bronchial blood flow constitutes a very small portion of the output of the left ventricle and supplies part of the tracheobronchial tree with systemic arterial blood. Pulmonary blood flow (PBF) constitutes the entire output of the right venticle and supplies the lung with the mixed venous blood draining all the tissues of the body.

There is about 250 to 300 mL of blood per square meter of body surface area in the pulmonary circulation. About 60 to 70 mL/m² of this blood is located in the pulmonary capillaries.

Gas exchange starts to take place in smaller pulmonary arterial vessels, which are not truly capillaries by histologic standards. These arterial segments and successive capillaries may be thought of as functional pulmonary capillaries.

About 280 billion pulmonary capillaries supply approximately 300 million alveoli, resulting in a potential surface area for gas exchange estimated to be 50 to 100 m².

Bronchial Circulation

The bronchial arteries arise variably, either directly from the aorta or from the intercostal arteries. They supply arterial blood to the tracheobronchial tree and to other structures of the lung down to the level of the terminal bronchioles. They also provide blood flow to the hilar lymph nodes, visceral pleura, pulmoonary arteries and veins, vagus, and esophagus. The bronchial circulation may be important in the "air-conditioning" of inspired air. The blood flow in the bronchial circulation constitutes about 2% of left ventricle output of the left ventricle. Blood pressure in the bronchial arteries is the same as that in other systemic arteries.

The venous drainage of the bronchial circulation is unusual. Although some of the bronchial venous blood enters the azygos and hemiazygos veins, a substantial portion of bronchial venous blood enters the pulmonary veins. Therefore, the bronchial venous blood entering the pulmonary venous blood is part of the normal anatomic right-to-left shunt. Histologists have also identified anastomoses, or connections, between some bronchial capillaries and pulmonary capillaries and between bronchial arteries and branches of the pulmonary artery. Thse connections probably play little role in a healthy person but may open in pathologic states, such as when either bronchial or PBF to a protion of lung is occluded. For example, if PBF to an area of the lung is blocked by a pulmonary embolus, bronchial blood flow to that area increases.

Pulmonary Circuation

The pulmonary vessels offer much less resistance to blood flow than do the systemic arterial tree. They are also much more distensible than systemic arterial vessels. These factors lead to much lower intravascular pressures than those found in the systemic arteries, which makes them more compressible. The pulmonary vessels are located in the thorax and are subject to alveolar and intrapleural pressures that can change greatly. Therefore, factors other than the tone of the pulmonary vascular smooth muscle may have profound effects on pulmonary vascular resistance (PVR). The transmural pressure difference across vessel walls is therefore a major determinant of PVR.

Because the right and left circulations are in series, the outputs of the right and left venticles must be approximately equal to each other over the long run. If the 2 outputs are the same and the measured pressure drops across the systemic circulation and the pulmonary circulation are bout 98 and 10 mm Hg, respectively, then the PVR must be about one tenth that of the systemic vascular resistance (SVR). Again, the low resistance to blood flow offered by the pulmonary circulation is due to the structural aspects of the pulmonary circulation.

The resistance is fairly evenly distributed among the pulmonary arteries, the pulmonary capillaries, and the pulmonary veins (from 15 mm Hg to 12 mm Hg, from 12 mm Hg to 8 mm Hg, from 8 mm Hg to 5 mm Hg, respectively). At rest, about one third of the resistance (PVR) is located in the pulmonary arteries, about one third is located in the pulmonary capillaries, and about one third is located in the pulmonary veins.

PVR

The relative small amounts of vascular smooth muscle, low intravascular pressures, and high distensibility of the pulmonary circulation lead to a much greater importance of extravascular effects ("passive factors") on PVR. Gravity, body position, lung volume, alveolar and intrapleural pressures, intravascular pressures, and right ventricular output all can have profound effects on PVR without any alteration in the tone of the pulmonary vascular smooth muscle.

Transmural Pressure On PVR

For distensible-compressible vessels, the transmural pressure difference is an important determinant of the vessel diameter. As the transmural pressure difference (which is equal to pressure inside minus pressure outside) increases, the vessel diameter increases and resistance falls; as the transmural pressure difference decreases, the vessel diameter decreases and the resistance increases. Negative transmural pressure differences lead to compression or even collapse of the vessel.

Lung Volume on PVR

Two different groups of pulmonary vessels must be considered when the effects of changes in lung volume on PVR are analyzed: those vessels that are exposed to the mechanical influences of the alveoli and the larger vessels that are not – the alveolar and extraalevolar vessels.

As lung volume increases during a normal negative-pressure inspiration, the alveoli increase in volume. While he alveoli expand, the vessels found between them, mainly pulmonary capillaries, are elongated. As these vessels are stretched, their diameters decrease, just as stretching a rubber tube causes its diameter to narrow. Resistance to blood flow through the alveolar vessels increases as the alveoli expand because the alveolar vessels are longer (resistance is directly proportional to length) and because their radii are smaller (resistance is inversely proportional to radius to the fourth power). At high lung volumes, then, the resistance to blood flow offered by the alveolar vessels increases greatly; at low lung volumes, the resistance to blood flow offered by the alveolar vessels decreases.

One group of the extraalveolar vessels, the larger arteries and veins, is exposed to the intrapleural pressure. As lung volume is increased by making the intrapleural pressure more negative, the transmural pressure difference of the larger arteries and veins increase and they distend. Another factor tending to decrease the resistance to blood flow offered by the extraalveolar vessels at higher lung volumes is radial traction by the connective tissue and alveolar septa holding the larger vessels in place in the lung. Thus, at high lung volumes, the resistance to blood flow offered by the extraalveolar vessels decreases. During a forced expiration to low lung volumes, however, intrapleural pressure becomes very positive. Extraalveolar vessels are compressed, and as the alveoli decrease in size, they exert less radial traction on the extraalveolar vessels. The resistance to blood flow offered by the extraalveolar vessels increase greatly.

Because the alveolar and extraalveolar vessels may be thought of a 2 groups of resistances in series with each other, the resistances of the alveolar and extraalveolar vessels are additive at any lung volume. Thus, the effect of changes in lung volume on the total PVR gives the U-shape cruve. PVR is lowest near the functional residual capacity and increases at both high and low lung volumes.

There is another type of extraalveolar vessel called corner vessel, or extraalveolar capillary. Although these vessels are found between alveoli, their locations at junctions of alveolar septa give them different mechanical properties. Expansion of the alveoli during inspiration increases the wall tension of the alveolar septa, and the corner vessels are distended by increased radial traction, whereas the alveolar capillaries are compressed.

Also note that during mechanical positive-pressure ventilation, alveolar pressure (PA) and intrapleural pressure are positive during inspiration. In this case, and the resistance to blood flow offered by both alveolar and extraalveolar vessels increases during lung inflation. This is especially a problem during mechanical positive-pressure ventilation with positve end-expiratory pressure (PEEP). During PEEP, airway pressure (and thus alveolar pressure) is kept positive at end expiratory to help prevent atelectasis. In this situation, alveolar pressure and intrapleural pressure are positive during both inspiration and expriation. PVR is elevated in both alveolar and extraalveolar vessels throughout the respiratory cycle. In addition, because intrapleural pressure is always positive, the other intrathoracic blood vessels are subjected to decreased transmural pressure differences; the venae cavae, which have low intravascular pressure, are also compressed. If cardiovascular reflexes are unable to adjust to this situation, cardiac output may fall precipitously because of decreased venous return (for the reason see thread "Effects of Pressure Outside the Heart on Cardiac Output" at http://www.tomhsiung.com/wordpress/2016/09/effects-of-pressure-outside-the-heart-on-cardiac-output/) and high PVR.

Recruitment and Distention

During exercise, cardiac output can increase several-fold without a correspondingly great increase in MPAP. Although the MPAP does increase, the increase is only a few millimeters of mercury, even if cardiac output has doubled or tripled. Since the pressure drop across the pulmonary circulation is proportional to the cardiac output times the PVR, this must indicate a decrease in PVR.

Like the effects of lung volume on PVR, this decrease appears to be passive – that is, it is not a result of changes in the tone of pulmonary vascular smooth muscle caused by neural mechanisms or humoral agents. In fact, a fall in PVR in response to increased blood flow or even an increase perfusion pressure can be demonstrated in a vascularly isolated perfused lung. There are two different mechanisms that can explain this decrease in PVR in response to elevated blood flow and perfusion pressure: recruitment and distention.

Recruitment

At resting cardiac outputs, not all the pulmonary capillaries are perfused. A substantial proportion of capillaries, perhaps as large as one half to two thirds, is probably not perfused because of hydrostatic effects. Others may be unperfused because they have a relatively high critical opening pressure. That is, these vessels, because of their high vascular smooth muscle tone or other factors such as positive alveolar pressure, require a higher perfusion pressure than that solely necessary to overcome hydrostatic forces. Under normal circumstances, it is not likely that the critical opening pressures for pulmonary blood vessels are very great because they have so little smooth muscle. Increased blood flow increases the MPAP, which opposes hydrostatic forces and exceeds the critical opening pressure in previously unopened vesels. This series of events opens new parallel pathways for blood flow, which lowers the PVR. This opening of new pathways is called recruitment. Note that decreasing the cardiac output or pulmonary artery pressure can result in a derecruitment of pulmonary capillaries.

Distention

As perfusion pressure increases, the transmural pressure gradient of the pulmonary blood vessels increases, causing distention of the vessels. This increases their radii and decreases their resistance to blood flow.

Control of Pulmonary Vascular Smooth Muscle

Pulmonary vascular smooth muscle is responsive to both neural and humoral influences. These produce "active" alterations in PVR, as opposed to those "passive" factors discussed in the previous section.

The pulmonary vasculature is innervated by both sympathetic and parasympathetic fibers of the autonomic nervous system. The innervation of pulmonary vessels is relatively sparse in comparsion with that of systemic vessels. There is relatively more innervation of the larger vessels and less of the smaller, more muscular vessels. There appears to be no innervation of vessels smaller than 30 um in diameter. There does not appear to be much innervation of intrapulmonary veins and venules.

The effects of stimulation of the sympathetic innervation of the pulmonary vasulature are somewhat controversial. Some investigators have demonstrated an increase in PVR with sympathetic stimulation of the innervation of the pulmonary vasculature, whereas others have shown only a decreased distensibility with no change in calculated PVR. Stimulation of the parasympathetic innervation of the pulmonary vessels generally causes vasodilation, although its physiologic function is not known.

The catecholamines epinephrine and norepinephrine both increase PVR when injected into the pulmonary circulation. Histamine, found in the lung in mast cells, is a pulmonary vasoconstrictor. Certain prostaglandins and related substances, such as PGF_2alpha, PGE₂, and thromboxane, are also pulmonary vasoconstrictors, as is endothelin, a 21-amino acid peptide synthesized by the vascular endothelium. Alveolar hypoxia and hypercapnia also cause pulmonary vasoconstriction. Acetylcholine, the beta-adrenergic agonist isoproterenol, nitric oxide (NO), and certain prostaglandins, such as PGE₁, and PGI₂ (prostacyclin), are pulmonary vasodilators.

Gravity's Impact on PVR

Determinations of the regional distribution of PBF (see discussion below) have shown that gravity is another important "passive" factor affecting local PVR and the relative perfusion of different regions of the lung (see discussion below). The interaction of the effects of gravity and extravascular pressures may have a profound influence on the relative perfusion of different areas of the lung.

The Regional Distribution of Pulmonary Blood Flow

Interaction of Gravity and Extravascular Pressure

Experiments done on excised, perfused, upright animal lungs have demonstrated the same gradient of increased perfusion per unit volume from the top of the lung to the bottom. When the experiments were done at low pump outputs so that the pulmonary artery pressure was low, the uppermost regions of the lung received no blood flow. Perfusion of the lung ceased at the point at which alveolar pressure (PA) was just equal to pulmonary arterial pressure (Pa). Above this point, there was no perfusion because alveolar pressure exceeded pulmonary artery pressure, and so the transmural pressure across capillary walls was negative. Below this point, perfusion per unit volume increased steadily with increased distance down the lung.

Thus, under circumstances in which alveolar pressure is greater than pulmonary artery pressure in the upper parts of the lung, no blood flow occurs in that region, and the region is referred to as being in zone 1, as shown in Fingure 4-9. Any zone 1, then, is ventilated but not perfused. It is alveolar dead space. Fortunately, during normal, quiet breathing in a person with a normal cardiac output, pulmonary artery pressure, even in the uppermost regions of the lung, is greater than alveolar pressure, and so there is no zone 1. Some experiments have also demonstrated perfusion of the corner vessels under zone 1 conditions.

The lower portion of the lung in Figure 4-9 is said to be in zone 3. In this region, the pulmonary artery pressure and the pulmonary vein pressure (Pv) are both greater than alveolar pressure. The driving pressure for blood flow through the lung in this region is simply pulmonary artery pressure minus pulmonary vein pressure. Note that this driving pressure stays constant as one moves further down the lung in zone 3 because the hydrostatic pressure effects are the same for both the arteries and the veins.

The middle portion of the lung in Figure 4-9 is in zone 2. In zone 2, pulmonary artery pressure is greater than alveolar pressure, and so blood flow does occur. However, because alveolar pressure is greater than pulmonary vein pressure, the effective driving pressure for blood flow is pulmonary artery pressure minus alveolar pressure in zone 2. Notice that in zone 2 the increase in blood flow per distance down the lung is greater than it is in zone 3. This because the upstream driving pressure, the pulmonary artery pressure, increases according to the hydrostatic pressure increase, but the effective downstream pressure, alveolar pressure, is constant throughout the lung at any instant.

It is important to realize that the boundaries between the zones are dependent on physiologic conditions – they are not fixed anatomic landmarks. Alveolar pressure changes during the course of each breath. During eupneic breathing these changes are only a few centimeters of water, but they may be much greater during speech, exercise, and other conditions. A patient on a positive-pressure ventilator with PEEP may have substantial amounts of zone 1 because alveolar pressure is always high. Similarly, after a hemorrhage or during general anesthesia, PBF and pulmonary artery pressure are low and zone 1 conditions are also likely. During exercise, cardiac output and pulmonary artery pressure increase and any existing zone 1 should be recruited to zone 2. The boundary between zones 2 and 3 will move upward as well. Pulmonary artery pressure is highly pulsatile, and so the borders between the zones probably even move up a bit with each contraction of the right ventricle.

Changes in lung volume also affect the regional distribution of PBF and will therefore affect the boundaries between zones. Finally, changes in body position alter the orientation of the zones with respect to the anatomic locations in the lung, but the same relationships exist with respect to gravity and alveolar pressure.

Hypoxic Pulmonary Vasoconstriction

Alveolar hypoxia or atelectasis causes an active vasoconstriction in the pulmonary circulation. The site of vascular smooth muscle constriction appears to be in the arterial (precapillary) vessels very close to the alveoli.

The mechanism of hypoxic pulmonary vasoconstriction is not completely understood. The response occurs locally, that is, only in the area of the alveolar hypoxia. Connections to the central nervous system are not necessary: An isolated, excised lung, perfused with blood by a mechanical pump with a constant output, exhibits an increased perfusion pressure when ventilated with hypoxic gas mixtures. This indicates that the increase in PVR can occur without the influence of extrinsic nerves. Thus, it is not surprising that hypoxic pulmonary vasoconstriction persists in human patients who had received heart-lung transplants. Hypoxia may cause the release of a vasoactive substance from the pulmonary parenchyma or mast cells in the area. Histamine, serotonin, catecholamines, and prostaglandins have all been suggested as the mediator substance, but none appears to completely mimic the response. Decreased release of a vasodilator such as nitric oxide may also be involved in hypoxic pulmonary vasoconstriction. Possibly several mediators act together. More recent studies have strongly indicated that hypoxia acts directly on pulmonary vascular smooth muscle to produce hypoxic pulmonary vasoconstriction.

Physiologic Function of Hypoxic Pulmonary Vasoconstriction

The function of hypoxic pulmonary vasoconstriction in localized hypoxia is fairly obvious. If an area of the lung becomes hypoxic because of airway obstruction or if localized atelectasis occurs, any mixed venous blood flowing to that area will undergo little or no gas exchange and will mix with blood draining well-ventilated areas of the lung as it enters the left atrium. This mixing will lower the overall arterial PO₂ (PaO₂) and may even increase the arterial PCO₂ (PaCO₂). The hypoxic pulmomary vasoconstriction diverts mixed venous blood flow away from poorly ventilated areas of the lung by locally increasing vascular resistance. Therefore, mixed venous blood is sent to better-ventilated areas of the lung. The problem with hypoxic pulmonary vasoconstriction is that it is not a very strong response because there is so little smooth muscle in the pulmonary vasculature. Very high pulmonary artery pressures can interfere with hypoxic pulmonary vasoconstriction, as can other physiologic disturbances, such as alkalosis.

In hypoxia of the whole lung, such as might be encountered at high altitude or in hypoventilation, hypoxic pulmonary vasoconstriction occurs throughout the lung. Even this may be useful in increasing gas exchange because greatly increasing the pulmonary artery pressure recruits many previously unperfused pulmonary capillaries. This increases the surface area available for gas difusion and improves the matching of ventilation and perfusion. On the other hand, such a whole-lung hypoxic pulmonary vasoconstriction greatly increases the workload on the right venticle, and the high pulmonary artery pressure may overwhelm hypoxic pulmonary vasoconstriction in some parts of the lung, increase the capillary hydrostatic pressure in those vessels, and lead to pulmonary edema.

Hemostasis Mechanism – Platelet Structure and Function

August 24, 2016 Cardiology, Hematology No comments activation, Adhesion, ADP, aggregation, beta-thromboglobulin, cAMP, dense bodies, epinephrine, fibrinogen, GPVI, granules, high shear conditions, low shear conditions, organelles, P2Y12, PAR, PECAM-1, platelet kinetics, Recruitment, secretion, thrombospondin, VWF, 𝛼IIb𝛽3

Platelet Granules and Organelles

Platelets possess secretory granules and mechanisms for cargo release to amplify responses to stimuli and influence the surrounding environment. Platelet granule structures include 𝛼- and dense granules, lysosomes, and peroxisomes. 𝛼-Granules and the dense bodies are the main secretory granules that release cargo (e.g., fibrinogen and adenosine diphosphate [ADP]) upon platelet activation.

Platelet granule secretion begins with a dramatic increase in platelet metabolic activity, set off by a wave of calcium release and marked by increased adenosine triphosphate (ATP) production. After platelet stimulation by agonists, a "contractile ring" develops around centralized granules, the granules fuse with the surface membranes, and then they extrude their contents. Granule secretion in platelets is a graded process that depends on the number, concentration, and nature of the original stimulus/stimuli, either strong (e.g., thrombin and collagen) or weak (e.g., ADP and epinephrine).

𝛼-Granules

What the 𝛼-granules have include:

𝛽-thromboglobulin
PF4
thrombospondin
vWF
Fibrinogen
Other plasma proteins (small amount)
Growth factors
𝛼_IIb𝛽₃ [platelet receptor]

𝛼-Granules, with a cross-sectional diameter of approximately 300 nm and numbering approximately 50 per platelet, are the predominant platelet granules. They are approximately spherical in shape, with an outer membrane enclosing two distinct intragranular zones that vary in electron density. The larger, electron-dense region is often eccentrically placed and consists of a nucleoid material that is rich in platelet-specific proteins such as 𝛽-thromboglobulin. The second zone, of lower electron density, lies in the periphery adjacent to the granule membrane and contains tubular structures with adhesive GPs such as von Willebrand factor (vWF) and multimerin, along with factor V. Platelets take up plasma proteins and store them in their 𝛼-granules.

Three proteins, 𝛽-thromboglobulin, PF4, and thrombospondin, are synthesized in megakaryocytes and highly concentrated in 𝛼-granules. The first two, 𝛽-thromboglobulin and PF4, show homology in amino acid sequence and share the additional features of localization in the dense nucleoid of 𝛼-granules, heparin-binding properties, and membership in the CXC family of chemokines. Together, they constitute approximately 5% of total platelet protein, and they can serve as useful markers for platelet activation in serum or plasma. Thrombospondin may comprise up to 20% of the total platelet protein released in response to thrombin, and likely participates in multiple biologic prcesses.

vWF is also synthesized by megakaryocytes and is present in the tubular structures of the 𝛼-granule peripheral zone, similar to its localization within Weibel-Palade bodies of vascular endothelial cells. Factor V and multimerin, a factor V/Va-binding protein, co-localize with vWF in platelets but not in endothelial cells. Fibrinogen is also found in 𝛼-granules, but is incorporated actively from plasma and not synthesized by megakaryocytes. In fact, small amounts of virtually all plasma proteins, such as albumin, immunoglobulin G (IgG), fibronectin, and 𝛽-amyloid protein precursor, may be taken up into the platelet 𝛼-granules. 𝛼-Granules also contain many growth factors, including platelet-derived growth factor, transforming growth factor-𝛽1 (TGF-𝛽₁), and vascular endothelial growth factor. These signaling molecules may contribute to the mitogenic activity of platelets.

Platelet 𝛼-granules serve as an important reservoir for 𝛼(IIb)𝛽₃ that contributes significantly to the surface fibrinogen receptors present on activated platelets. The 𝛼-granule membrane protein, P-selectin (granule membrane protein-140) is translocated to the plasma membrane after platelet activation. Finally, a number of additional proteins have been located to the surface of 𝛼-granules alone, including CD9, platelet endothelial cell adhesion molecule-1 (PECAM-1), Rap 1b, GPIb-IX-V, and osteonectin.

The platelets and megakaryocytes of patients with gray platelet syndrome have decreased numbers of 𝛼-granules and reduced levels of some proteins. It is proposed that there is incorrect targeting of 𝛼-granule proteins to the 𝛼-granule in the megakaryocyte in this disease.

Dense Bodies

Dense bodies contain a large reservoir of ADP, a crtitical agnoist for platelet activation that amplifies the effect of other stimuli. In addition to this nonmetabolic pool of ADP, the dense bodies are rich in ATP, pyrophosphate, calcium, and serotonin (5-hydroxytryptamine), with lesser amounts of guanosine triphosphate (GTP), guanosine diphosphate (GDP), and magnesium. The adenine nucleotides are synthesized and segregated by megakaryocytes, whereas serotonin is incorporated into dense granules from the plasma by circulating platelets. There is more ADP than ATP in dense bodies, and both can lead to adenosine monophosphate (AMP). In turn, AMP can be dephosphorylated to adenosine or cyclized to produce cyclic AMP, an inhibitor of the platelet-stimulatory response. The dense granule membrane contains P-selectin and granulophysin.

Compared ADP/ATP within the metabolic/cytoplasmic pool (at least two different pools: metabolic pool and cytoplasmic pool), ADP/ATP in storage pool (dense bodies) contains approximately two-thirds of the total platelet nucleotides, mainly in the form of ADP and ATP, and is metabolically inactive, does not rapidly incorporate exogenous adenine or phosphate, and equilibrates slowly with the metabolic pool. Nucleotides in this pool (storage pool) are extruded fromt the platelet during the release reaction and cannot be replenished after release.

The ATP (metabolic pool) that is broken down to provide energy for the release reaction is not rephosphorylated; rather, it is irreversibly degraded to hypoxanthine, which diffuses out of the cell.

Lysosomes

Lysosomes are small, acidified vesicles, approximately 200 nm in diameter, that contain acid hydrolases with pH optima of 3.5 to 5.5, including 𝛽-glucuronidase, cathepsins, aryl sulfatase, 𝛽-hexosaminidase, 𝛽-galactosidase, heparitinase, and 𝛽-glycerophosphatase. Additional protein found in lysosomes include cathepsin D and lysosome-associated membrane proteins (LAMP-1/LAMP-2, which are expressed on the plasma membrane after activation). Lysosomal constituents are released more slowly and incompletely (maximally, 60% of the granules) than 𝛼-granules or dense-body components after platelet stimulation, and their release also requires stronger agonists such as thrombin or collagen.

Organelles

Peroxisomes are rare, small granules, demonstrable with alkaline diaminobenzidine as a result of their catalase activity. The structure may participate in the synthesis of platelet-activating factor.

Mitochondria in platelets are similar, with the exception of their smaller size, to those in other cell types. There are approximately seven per human platelet, and they serve as the site for the actions of the respiratory chain and the citric acid cycle. Glycogen is found in small particles or in masses of closely associated particles, playing an essential role in platelet metabolism.

Platelet Kinetics

Approximately one-third of the total platelet mass appears to pool in the spleen. The splenic pool exchanges freely with the platelets in the peripheral circulation. Administration of epinephrine, which evacuates platelets from the spleen, increases the peripheral platelet count 30% to 50%, and platelet counts in asplenic patients are not affected by epinephrine. Some studies suggest that the splenic pool consists of the youngest, largest platelets. Pathophysiologi states can result in 80% to 90% of platelets being sequestered in the spleen, resulting in thrombocytopenia.

Other organs that have pool of platelets (accounting for about 16% of total platelets) include the lungs and the liver and so on.

The life span of platelet has been estimated to be 8 to 12 days in humans. In steady state, when platelet production equals destruction, platelet turnover has been estmated at 1.2 to 1.5 x 10¹¹ cells per day.

PS: Details of various platelet products can be found in thread "Platelet Transfusion for Patients w/ Cancer" at http://www.tomhsiung.com/wordpress/2013/04/platelet-transfusion-for-patients-with-cancer-part-one/

Platelet Adhesion and Activation

Part I – Adhesion

Adhesive ligands: vWF, collagen, fibronectin, thrombospondin, laminin (perphaps)
Platelet surface receptors: GPIb/V/IX complex, GPVI, 𝛼_IIb𝛽₃, 𝛼₂𝛽₁, 𝛼₅𝛽₁, 𝛼₆𝛽₁
Interaction of GPVI with collagen activates platelet intergrins
At low shear conditions, fibrinogen is the primary ligand (interacting with 𝛼_IIb𝛽₃), but other ligands may also be involved

Platelet adhesion to exposed subendothelium is a complex multistep process that involves a diverse array of adhesive ligands (vWF, collagen, fibronectin, thrombospondin, and perphaps laminin) and surface receptors (GPIb/V/IX, GPVI, integrins 𝛼_IIb𝛽₃, 𝛼₂𝛽₁, 𝛼₅𝛽₁, and 𝛼₆𝛽₁). The specific ligand/receptor palyers in primary platelet adhesion are largely dependent on the arterial flow conditions present. In high shear conditions, platelet tethering is dependent on the unique shear-dependent interaction between GPIb/V/IX and subendothelial vWF, derived either from plasma or released by endothelial cells and/or platelets. A tether forms between GPIb and vWF that either halts platelet movement or reduces it such that other interactions can proceed. Subsequent interactions are mediated by GPVI binding to glycineproline-hydroxyproline sites on collagen and perhaps to exposed laminin. The interaction of GPVI with collagen strongly activates platelets such that 𝛼_IIb𝛽₃, can engage in high-affinity interactions with ligands. At low shear conditions, fibrinogen is thought to be the primary ligand supporting platelet plug formation through its interaction with 𝛼_IIb𝛽₃, although thrombus formation can take place in the absence of vWF and fibrinogen, so other ligands may also be involved.

Following initial platelet adhesion, subsequent platelet-platelet interactions are intially mediated by two receptors, GPIb/V/IX and 𝛼_IIb𝛽₃, and their respective contributions are dependent on the flow conditions present. In high shear stress conditions, GPIb/V/IX receptor and vWF ligand action are predominant, with fibrinogen playing a stabilizing role.

Platelet Glycoprotein Ib Complex-von Willebrand Factor Interaction and Signaling

The interaction of the platelet GP_Ib "complex" (the polypeptides GP_Ib𝛼, GP_Ib𝛽, GPIX, and GPV) with its primary ligand. vWF, is the receptor-ligand pairing that initiates platelet adhesion followed by a cascade of events leading to pathologic thrombosis or physiologic hemostasis. A unique aspect of this receptor-ligand interaction is that it requires the presence of high arterial shear rates to take place, thus explaining the predisposition of platelet-rich "white clots" in the arterial circulation over clots found in the venous circulation, with its relatively lower shear forces, in which clot formation takes place independent of the GP_Ib complex.

The binding site for vWF is present in the N-terminal 282 residues of GPIb. Important to the interactions are a cluster between residues Asp 252 and Arg 293 containing sulfated tyrosine residues and important anionic residues, a disulfide loop between Cys 209 and Cys 248, and an N-terminal flanking sequence of the leucine-rich repeats (LRG). Mutations involving single amino acid residues within these LRGs account for some cases of the congential bleeding disorder Bernard-Soulier syndrome, in which the GPIb complex binds poorly, or not at all, to vWF.

Unlike other receptors, GPIb does not require platelet activation for its interactions with vWF. In vitro, the interactions of vWF and binding with the GPIb complex occur with generally very low affinity in the absence of shear. The presence of the vancomycin-like antibiotic ristocetin or viper venom proteins, such as botrocetin, promotes the interactions. Mobilization may uncoil vWF to promote interactions with GPIb. The addition of shear, in a parallel-plate flow system, results in platelet interaction with subendothelial vWF that occurs in a biphasic fashion. Likewise, the rate of translocation of platelets from blood to the endothelial cell surface, which is dependent, increases linearly up to wall shear rates of 1,500 s^-1, whereas the translocation rate remains relatively constant with the wall shear rate between 1,500 and 6,000 s^-1. Thus, the presence of shear is important for promoting the interactions between the GPIb complex and vWF. Studies of real-time thrombus formation in the absence of platelet GPIb complex and in blood from individuals with severe (type 3) von Willebrand disease indicate that GPIb and vWF interaction are required for platelet surface interaction at high shear rates (>1,210 s^-1), whereas GPIb deficiency results in poor platelet adhesion at lower shear. Shear accelerates thrombus formation likely by promoting this receptor-ligand interaction.

When the GPIb complex interacts with its vWF ligand under conditions of elevated shear stress, signals are initiated that activate integrin 𝛼_IIb𝛽₃. The pathways involved lead to a) elevation of intracellular calcium; b) activation of a tyrosine kinase signaling pathway that incorporates nonreceptor tyrosine kinases such as Src, Fyn, Lyn, and Syk, phospholipase C(gamma)2, and adaptor protein such as SHC, LAT, and SLP-76; c) inside-out signaling through the 𝛼_IIb𝛽₃ integrin followed by platelet aggregation; and d) activation of protein kinase C (PKC), protein kinase G (PKG), and phosphoinositide 3-kinase (PI3K), …… and so on.

Once vWF binds to GPIb-V-IX, signaling complexes form in the vicinity of the GPIb𝛼 cytoplasmic tail consisting of cytoskeletal proteins such as 14-3-3ζ as well as signaling protein like Src and PI 3-kinase. This process leads to Syk activation, protein tyrosine phosphorylation, and recruitment of other cytoplasmic proteins with pleckstrin homology domains that can support interactions with 3-phosphorylated phosphoinositides and ultimately activation of integrin 𝛼_IIb𝛽₃.

Glycoprotein Ib Complex Interaction with Thrombin and Other Molecules

The GPIb complex serves as an 𝛼-thrombin binding site on platelets, although the physiologic relevance of the interactions is not clear. The density of GPIb complexes (~20,000/platelet) far exceeds the number of thrombin binding sites reported on platelets (~6,000/platelet). Studies have identified interaction of the GPIb complex with ligands other than vWF. These include a study a reversible association of GPIb with P-selectin, which is examined in more detail in the section "Platelets and Endothelium." The interaction of platelet GPIb with the neutrophil adhesion receptor 𝛼(M)𝛽2 (Mac-1) is discussed in the section "Platelets and White Blood Cells." Additionally, GPIb reportedly interacts with high-moecular weight kininogen, factor XII, and factor XI.

Platelet-Collagen Interaction and Signaling

Receptors: GPVI, 𝛼_2b𝛽₁
Ligands: collagens

Collagens, one of the most thrombogenic substance in vessels, are very important activators of platelets in the vascular subendothelium and vessel wall, and thus are prime targets for therapeutic intervention in patients experience a pathologic arterial thrombotic event such as MI or stroke. Platelets have two major surface receptors for collagen, the immunoglobulin superfamily member GPVI and the integrin 𝛼₂𝛽₁. The former is considered to be the primary palyer in platelet adhesion. In additon to these two surface receptors, the GPIb complex can also be considered an indirect collagen receptor because its subendothelial vWF ligand essentially acts as bridging molecule between platelets and collagen by fixing itself to the latter, which, in turn, acts as scaffolding for the multimers. Collagen adhesion also results in indirect activation of the protease-activated receptor 1 via MMP-1. Other molecules, such as CD 36, may also sustain collagen interaction.

Glycoprotein VI receptor

GPVI is the main receptor involved in collagen-mediated platelet activation. Studies of mice lacking platelet GPVI show that they lose collagen-induced platelet activation due to a defect in platelet adhesion. Thus, GPVI appears to serve as teh initial receptor involved in platelet adhesion, and it activates integrin binding. GPVI alone supports adhesion to insoluble collagens, and works with 𝛼₂𝛽₁ to promote platelet adhesion to soluble collagen microfibrils. GPVI can also be engaged by collagen-related peptides (arranged in triple helical structures with sequences similar to collagen) and teh snake venom convulxin, which elicit signals through GPVI.

Synergism between GPVI pathways and those related to other adhesion receptors such as GPIb-V-IX and soluble agonists released by activated platelets are likely necessary for the full repertoire of platelet-collagen signaling. Exposure of platelets to collagen surfaces likely results in GPVI clustering that in turn triggers the tyrosine phosphorylation of the FcRγ chain. The GPVI/FcRγ-chain complex leads to platelet activation through a pathway that has many aspects in common with signaling by immune receptors, such as the Fc receptor family and the B- and T-cell antigen receptors.

α₂β₁ receptor

The first platelet collagen receptor identified was the integrin 𝛼₂𝛽₁ receptor, also known as platelet GPIa/IIa and lymphocyte VLA-2.

When compared to vWF, collagen is a more efficient substrate when it comes to supporting stable platelet adhesion and thrombus formation. The fact that initial platelet tethering to collagen under high shear flow first requires interaction between vWF and platelet GPIb serves to underscore the importance of the two major collagen receptors, GPVI and 𝛼₂𝛽₁, in promoting platelet adhesion and activation under shear conditions.

In addition to GPVI, the α₂β₁ receptor also propagates signaling. The use of α₂β₁ selective ligands has demonstrated calcium-dependent spreading and tyrosine phosphorylation of several proteins when interaction with platelets takes place.

Physiologic Inhibition of Platelet Adhesion

Negative regulation of platelets is essential to set the stimulus threshold for thrombus formation, determine final clot size and stability, and prevent uncontrolled thrombosis. The mechanisms behind the negative regulation of platelet activation are described later, and in this respect, roles of players such as nitric oxide and prostacyclin have been well characterized. Platelet activation can also be inhibited by signaling through the adhesion moleculde PECAM-1 (CD31). Expressed on a number of blood cells and endothelial cells, PECAM has a wide array of regulatory functions in processes such as apoptosis and cell activation. Following homophilic interactions and/or clustering, PECAM-1 is tyrosine phosphorylated in its cytoplasmic tail ITIM domain. Phosphorylation of PECAM-1 recruits and activates the SH2 domain-containing protein-tyrosine phosphatase, SHP-2. Studies suggest that the PECAM-1/SH-2 complex functions to counteract platelet activating, most particularly for collagen by inhibiting GPVI/FcRγ chain signaling.

Part II – Activation

PAR Thrombin Interactions

See Figure 16.9

Platelet thrombin receptors/platelet protease-activated receptors/PARs and signaling

PARs are G-protein-coupled receptors that use a unique mechanism to convert an extracellular protein cleavage event into an intracellular activation signal. In this case, the ligand is already part of the receptor per se, by virtue of the fact that it is represented by the amino acid sequence SFLLRN (residues 42 through 47) and is unmashed as a new amino terminus after thrombin cleaves the peptide bond between Arg 41 and Ser 42 (Figure 16.9). This "tethered ligand" then proceeds to irreversibly dock with the body of its down receptor to effect transmembrane signaling, as shown in Figure 16.9.

Thrombin signaling in platelet is mediated, at least in part, by four members of a family of G-protein-coupled PARs (PAR-1, -2, -3, and -4). Human platelets express PAR-1 and PAR-4, and activation of either is sufficient to trigger platelet aggregation. PAR-1, -3, and -4 can be activated by thrombin, whereas PAR-2 can be activated by trypsin, tryptase, and coagulation factors VIIa and Xa. Presumably, other proteases are capable of recognizing the active sites of these receptors and can thus also trigger PAR signaling.

Once activated, PAR-1 is rapidly uncoupled from signaling and internalized into the cell. It is then transported to lysosomes and degraded. Platelet presumably have no need for a thrombin receptor recycling mechanism, becuase once activated, they are irreversibly incorporated into blood clots. Conversely, in cell lines with characteristics similar to megakaryocytes, new protein synthesis is needed for recovery of PAR-1 signaling, and in endothelial cells, sensitivity to thrombin is maintained by delivery of naive PAR-1 to the cell surface from a preformed intracellular pool.

Platelet ADP (Purinergic) Receptors and Signaling

P2Y1
P2Y12
P2X

Evidence that ADP plays an important role in both the formation of the platelet plug and the pathogenesis of arterial thrombosis has been accumulating since its initial characterization in 1960 as a factor derived from red blood cells that influences platelet adhesion. ADP is present in high concentratons (molar) in platelet-dense granules and is released when platelet stimulation takes place with other agonists, such as collagen; thus, ADP serves to further amplify the biochemical and physiologic changes associated with platelet activation and aggregation. Inhibitors of this ADP-associated aggregation include commonly used clinical agents, including ticlopidine, clopidogrel, prasugrel, and ticagrelor, proven to be very effective antithrombotic drugs.

Adenine nucleotides interact with P2 receptors that are ubiquitous among different cell types and have been found to regulate a wide range physiologic processes. They are divided into two groups, the G-protein-coupled superfamily named P2Y and the ligand-gated ion channel superfamily termed P2X. Two G-protein-coupled (P2Y) receptors contribute to platelet aggregation. The P2Y₁ receptor initiates aggregation through mobilization of calcium stores, and the P2Y₁₂ receptor is coupled to inhibition of adenylate cyclase and is essential for a full aggregation response to ADP with stabilization of the platelet plug.

PS: ADP >>> P2Y₁₂ >>> inhibition of adenylate cyclase >>> decreased cAMP production >>> decreased intensity of aggregation

Inhibition of either P2Y₁ or P2Y₁₂ receptors is sufficient to block ADP-mediated platelet aggregation, and coactivation of both receptors is therefore necessary, through the G proteins G_q and G_i, respectively, for ADP to activate and aggregate the platelet.

Although considered a weak agonist in comparison to collagen or thrombin, ADP clearly palys an important role in thrombus stabilization, likely by contributing to the recruittment of additional platelets to growing thrombi. Aggregation is often reversible when platelets are stimulated by ADP alone. In addition, low concentrations of ADP serve to amplify the effects of both strong and weaker agonists, the latter inlcuding serotonin and adrenaline, among others.

Platelet Activation by Soluble Agonists

Epinephrine

Epinephrine is unique among platelet agonists because it is considered to be capable of stimulating secretion and aggregation, but not cytoskeletal reorganization responsible for shape change. Platelet responses to epinephrine are mediated through 𝛼₂-adrenergic receptors, and these responses have been found to vary among individuals, with some donors with otherwise normal platelets manifesting delayed or absent responses.

Arachidonic acid, thromboxane A₂, and thromboxane receptors

After platelet stimulation by a number of agonists, arachidonic acid is generated directly by phospholipase A from its membrane phospholipid precursors (PC, PS, and PI) and indirectly by PLC generation of DAG followed by DAG lipase action. Most platelet agonists are believed to activate this pathway. Three known eicosanoid subsetsl of biochemical compounds are known to be derived from the formation of arachidonic acid – the prostanoids, leukotrienes, and epoxides. Although all three of these pathways are present in platelets, most arachidonic acid ends up being metabolized to thromboxane A₂ (TxA₂).

TxA₂ is produced in platelets from arachidonic acid through the generation of PGH₂ by the enzyme cyclo-oxygenase, which is irreversibly inhibited by aspirin through acetylation of a serine residue near its C terminus. PGE₂ and PGI₂ act to inhibit platelet activation by generating intracellular cAMP, whereas TxA₂ activates platelets. Platelets primarily synthesize thromboxane, and endothelial cells mainly synthesize prostaglandins such as PGI₂.

Like ADP and epinephrine, TxA₂ is also capable of activating nearby platelets after its release into plasma. It has a very short half-life of 30 seconds before its conversion to the inactive metabolite thromboxane B₂ prevents widespread platelet activation beyond the vicinity of thrombus formation. Both arachidonic acid and analogs of TxA₂ have been found to activate and aggregate platelets by mediating shape change and phosphorylation of signaling enzymes. The thromboxane receptor (TP) is a member of the seven-transmembrane G-protein-coupled receptor family and has been localized to the plasma membrane. Two isoforms of the receptor have been identified in platelets TP𝛼 and TP𝛽 – and they activate platelets through ghe G_q pathway.

Physiologic Inhibition of Platelet Activation

One of the many remarkable features of platelets is their ability to remain in a physiologic resting state and resist becoming activated while navigating the heart, arterial, and venous circulations. Indeed, the pathologic consequences associated with widespread inappropriate platelet activation are life- and limb-threatening in the settings of well-characterized clinical disorders, such as thrombotic thrombocytopenic purpura and heparin-induced thrombocytopenia. The mechanisms responsible for maintaining the fine balance of keeping platelets in a resting state until they encounter a genuine need to undergo adhesion, activation, and aggregation at the site of vascular injury are nearly as diverse as those responsible for mediating these physiologic phenomena.

Some general mechanisms involved in physiologic inhibition of platelet activation include phenomena such as a) generation of negative-regulating molecules by the platelet (e.g., cAMP), endothelium (e.g., PGI₂, nitric oxide, heparan sulfate), and at distant sites (e.g., antithrombin); b) barrier of endothelial cells that prevents direct contact of circulating platelets with collagen; c) ecto-ADPase (CD39) expression by endothelial cells that metabolizes ADP secreted from platelets; d) tendency for blood flow to wash away unbound thrombin and other soluble mediators from the site of platelet plug formation; e) brief half-life of certain key platelet activators such as TxA₂; f) tight regulation of the affinity state of receptors such as 𝛼_IIb𝛽₃; g) downregulation of signaling receptors to limit their actions; and h) inhibitory pathways mediated by ITIM-containing and/or contact-dependent adhesion receptors, such as PECAM, CECAM-1, JAM-A, and potentially others.

Receptor downregulation and desensitization

Signaling through G-protein-coupled receptors present on the surface of platelets is limited by their phosphorylation, which triggers desensitization, that is, uncoupling from G proteins, and internalization via Claritin-mediated endocytosis (for detail about G-protein coupled receptors please refers to thread "G Protein-Coupled Receptors and Second Messengers" at http://www.tomhsiung.com/wordpress/2014/09/g-protein-coupled-receptors-and-second-messengers/). G-protein kinases and 𝛽-arrestin are central to these processes. In addition, G-protein-coupled receptors interact with a myriad of other molecules that finely tune their signaling, including regulators of G-protein signaling (RGS) and GPCR-associated sorting proteins.

Inhibitory prostaglandins

Generation of the prostaglandins from arachidonic acid metabolism, such as PGI₂ and PGE₂ (at high concentrations), results in inhibition of platelet activation and aggregation, and counterbalances the actions of thromboxanes derived from the same pathway. While PGI₂ and PGD₂ inhibit platelet function at low doses, PGE2 displays a biphasic reponse, and inhibits platelet function only at higher concentrations, likely via the EP4 receptor. The inhibitory effects are mediated via G-protein-coupled receptors (IP and EP receptors, respectively) that couple to the 𝛼 subunits of G_s to regulate adenylate cyclase-mediated generation of cAMP. cAMP levels in platelets are also governed by the activity of phosphodiesterase, the enzyme responsible for cAMP metabolism. This enzyme activity is inhibited drugs such as the weak antiplatelet agent dipyridamole, the bronchodilator theophylline, and sildenafil, used to treat erectile dysfunction in men.

Nitric oxide

NO is generated by endothelial cells and platelets from L-arginine in response to shear stress forces and other platelet agonists, such as thrombin and ADP. The bulk of the evidence suggests that at high concentrations NO functions to inhibit platelet activation through the cyclic guanosine monophosphate (cGMP) second messenger generated by guanylate cyclase activation. Elevations in cGMP, by modulating phosphodiesterase activity, can raise intraplatelet cAMP. Paradoxically, low levels of NO may elicit platelet activation pathways. Endothelial NO synthase activity is enhanced during platelet activation, presumably as an additional means for limiting platelet aggregation.

Platelet Aggregation: 𝛼_IIb𝛽₃ (GPIIb/IIIa) Receptor and Its Signaling Mechanisms

Platelet aggregation is a complex phenomenon that is the end result of a series of adhesion- and activation-related processes. Essential components of this process include an agonist, calcium, and the adhesive proteins fibrinogen and vWF. Divalent cations, such as calcium and magnesium, are required for platelet aggregation in trace amounts, and these alter the specificity of the integrin 𝛼_IIb𝛽₃ for its ligands. Fibrinogen and vWF play dominant roles in platelet aggregation through binding to 𝛼_IIb𝛽₃, and also by the ability of the former to generate polymerized fibrin as support for the platelets in a thrombus.

The signaling pathways of 𝛼_IIb𝛽₃are complex. Central concepts of the signaling pathway include inside-out signaling, which involves the processes termed affinity and avidity modulation, and outside-in signaling, in which messages are transmitted to the inside of the platelet via the events occurring outside the membrane through 𝛼_IIb𝛽₃ activation. Primary platelet agonists such as ADP, thrombin, and matrix proteins collagen and vWF affect platelet aggregation through a process known as inside-out signaling. In the inside-out signaling, agonist-dependent intracellular signals stimulate the interaction of key regulatory ligands (such as talin) with integrin cytoplasmic tails. This leads to conformational changes in the extracellular domain that result in increased affinity for adhesive ligands such as fibrinogen, vWF, and fibronectin. In the outside-in signaling, extracellular ligand binding, initially reversible, becomes progressively irreversible and promotes integrin clustering and further conformational changes that are transmitted to the cytoplasmic tail. This results in the recruitment and/or activation of enzymes, adaptors, and effectors to form integrin-based signaling complexes.

Brief Review of Physiology of Platelet

Following injury to the blood vessel, platelets interact with collagen fibrils in the exposed subendothelium by a process (adhesion) that involves, among other events, the interaction of a plasma protein ,vWF, and a specific glycoprotein (GP) complex on the platelet surface, GP Ib-IX-V (GPIb-IX). This interaction is particularly important for platelet adhesion under conditions of high shear stress. After adherence to the vessel wall via vWF and the long GP Ib-IX-V receptor, other platelet receptors interact with proteins of the subendothelial matrix. Hereby collagen provides not only a surface for adhesion but also serves as a strong stimulus for platelet activation.

Activated platelets release the contents of their granules (secretion), including ADP and serotonin from the dense granules, which causes the recruitment of additional platelets. These additional platelets form clumps at the site of vessel injury, a process called aggregation (cohesion). Aggregation involves binding of fibrinogen to specific platelet surface receptors, a complex composed of GPIIb-IIIa (integrin 𝛼_IIb𝛽₃), an integrin that normally exists in a resting (low-affinity) state but that transforms into an activated (high-affinity) state when stimulated by the appropriate signal transduction cascade. GPIIb-IIIa is platelet specific and has the ability to bind vWF as well. Although resting platelets do not bind fibrinogen, platelet activation induces a conformational change in the GPIIb-IIIa complex that leads to fibrinogen binding.

Moreover, platelets play a major role in coagulation mechanisms; several key enzymatic reactions occur on the platelet membrane lipoprotein surface. During platelet activation, the negatively charged phospholipids, especially PS, become exposed on the platelet surface, and essential step for accelerating specific coagulation reactions by promoting the binding of coagulation factors involved in thrombin generation.

A number of physiologic agonists interact with specific receptors on the platelet surface to induce responses, including a change in platelet shape from discoid to spherical, aggregation, secretion, and thromboxane A₂ production. Other agonists, such as prostacyclin, inhibit these responses. Binding of agonists to platelet receptors initiates the production or release of several intracellular messenger molecules, including products of hydrolysis of phosphoinositide (PI) by phospholipase C, TxA₂, and cyclic nucleotides. These induce or modulate the various platelet responses of Ca²⁺ mobilization, protein phosphorylation, aggregation, secretion, and thromboxane production.

Inflammation – The Beginning and Ongoing

March 1, 2015 Infectious Diseases, Physiology and Pathophysiology No comments Adhesion, Chemotaxis, Foreign Bodies, Immune Reactions, Infections, Inflammation, Migration, Recognition, Recruitment, Tissue Necrosis, Vascular Caliber, Vascular Flow, Vascular Permeability

Inflammation is a response of vascularized tissues to infections, foreign invaders, and damaged tissues that brings cells and molecules of host defines from the circulation to the sites where they are needed, in order to eliminate the offending agents. Inflammation generally is a defensive response that is essential for survival, where mediators of this response include phagocytic leukocytes and cytokines/substances produced by them, antibodies, and complement proteins. Most of these mediators normally circulate in the blood, from which they can be rapidly recruited to any site in the body;some of the cells also reside in tissues. The process of inflammation delivers these cells and proteins to damaged or necrotic tissues and foreign invaders, and activates the recruited cells and molecules, which then function to get rid of the harmful or unwanted substances. Without inflammation, infections would go unchecked, wounds would never heal, and injured tissues might remain permanent festering sores.

On the other hand, the primary function of the inflammatory response is to eliminate a pathogenic insult and remove injured tissue components, thus allowing tissue repair to take place. In teleologic terms, the body attempts to contain or eliminate offending agents to protect tissues, organs and, ultimately, the whole body from damage. Specific cells are imported to attack and destory injurious agents, enzymatically digest and remove them, or wall them off. In the process, damaged cells and tissues are digested and removed to allow repaire to occur.

Causes of Inflammation

Inflammatory reactions may be triggered by a variety of stimuli, including:

1.Infections (bacterial, viral, fungal, parasitic, rickettsiaceae and so on) and microbial toxins are among the most common and medically important causes of inflammation. Different infectious pathogens elicit varied inflammatory responses, from mild acute inflammation that causes little or no lasting damage and successfully eradicates the infection, to severe systemic reactions that can be fatal, to prolonged chronic reactions that cause extensive tissue injury. The outcomes are determined largely by the type of pathogen and, to some extent, by characteristics of the host that remain poorly defined (relative post: http://forum.tomhsiung.com/pharmacy-practice/pharmacotherapy/416-severe-sepsis-septic-shock.html).

PS: In the article of NEJMra1208623, the specific response in any patient depends on the causative pathogen (load and virulence) and the host (genetic characteristics and coexisting illnesses), with differential responses at local, regional, and systemic levels.

2.Tissue necrosis elicits inflammation regardless of the cause of cell death, which may include ischemia (reduced blood flow, the cause of myocardial infarction, etc.) trauma, and physical and chemical injury (e.g., thermal injury, as in burns or frostbite; irradiation; exposure to some environmental chemicals). Several molecules released from necrotic cells are known to trigger inflammation。

3.Foreign bodies (splinters, dirt, sutures) may elicit inflammation by themselves or because they cause traumatic tissue injury or carry microbes. Even some endogenous substances can be considered potentially harmful if large amounts are deposited in tissues; such as substances include urate crystals (in the disease gout), cholesterol crystals (in atherosclerosis), and lipids (in obesity-associated metabolic syndrome).

4.Immune reactions (also called hypersensitivity) are reactions in which the normally protective immune system damages the individual's own tissues. The injurious immune responses may be directed against self antigens, causing autoimmune diseases, or may be inappropriate reactions against microbes. Also, the textbook of Drug-Induced Diseases by James E. Tisdale, PharmD, describes the hypersensitivity as reactions mediated by the immune system. In inflammations caused by immune reactions or hypersensitivity, self and environmental antigens act as the triggers of inflammations and since that these stimuli cannot be eliminated, autoimmune and allergic reactions tend to be persistent and difficult to cure, and they are often associated with chronic inflammations.

Tissue Injury

On the other hand, inflammation could be described as the result of tissue injury.

Cell must be able to adapt to fluctuating environmental conditions (e.g., temperature, solute concentrations, oxygen supply, noxious agents, etc.). The evolution of multicellular organisms eased the precarious lot of individual cells by establishing a controlled extracellular environment where the "inner" environmental conditions remain relatively constant. If a change in the environment is too huge, a cell can be injuried; and if the injury exceeds the cell's adaptive capacity, the cell dies. A cell exposed to persistent sublethal injury has limited available responses, expression of which we interpret as cell injury.

All cells have efficient mechanisms to deal with shifts in environmental conditions. When environmental changes exceed the cell's capacity to maintain normal homeostasis, we recognize acute cell injury. If these stress is removed in time or if the cell can withstand the assault, the damage is reversible, and complete structural and functional integrity is restored. For example, when circulation to the heart is interrupted for less than 30 minutes, all structural and functional alterations prove to be reversible. The cell can also be exposed to persistent sublethal stress, as in mechanical irritation of the skin or exposure of the bronchial mucosa to tobacco smoke. Cell have time to adapt to reversible injury in a number of ways, each of which has a morphologic counterpart. On the other hand, if the stress is sufficiently severe, irreversible injury leads to cell death. The moment when reversible injury becomes irreversible injury, the "point of no return," is not known at present.

Inflammation Mediators

The mediators of inflammation are the substances that initiate and regulate inflammatory reactions. The most important inflammation mediators include vasoactive amines, lipid products (prostaglandins and leukotrienes), cytokines (including chemokines), and products of complement activation. These mediators induce various components of the inflammatory response typically by distinct mechanisms, which is why inhibiting each has been therapeutically beneficial. However, there is also some overlap (redundancy) in the actions of the mediators.

The inflammation mediators have some common characteristics, like

Mediators are either secreted by cells or generated from plasma proteins. Cell-derived mediators are normally sequestered in intracellular granules and can be rapidly secreted by granule exocytosis (e.g., histamine in mast cell granules) or are synthesised de novo (e.g., prostaglandins and leukotrienes, cytokines) in response to a stimulus. The major cell types that produce mediators of acute inflammation are the sentinels that detect invaders and damage in tissues, that is, macrophages, dendritic cells, and mast cells, but platelets, neutrophils, endothelial cells, and most epithelia can also be induced to elaborate some of the mediators. Plasma derived mediators (e.g., complement proteins) are produced mainly in the liver and are present in the circulation as inactive precursors that must be activated. When activated a series of proteolytic and protein-protein interactions are initiated that ultimately to acquire their biologic properties.
Ative mediators are produced only in response to various stimuli. These stimuli include microbial products and substances released from necrotic cells. Some of the stimuli trigger well-defined receptors and signalling pathways.
Most of the mediators are short-lived. They quickly decay, or are inactivated by enzymes, or they are otherwise scavenged or inhibited. There is thus a system of checks and balances that regulates mediator actions.
One mediator can stimulate the release of other mediators. The secondary mediators may have the same actions as the initial mediators but may also have different and even opposing activities. Such cascades provide mechanisms for amplifying or, in certain instances, counteracting the initial action off a mediator.

A detail about inflammation mediators can be found here, .

The Process of Inflammation

The steps of the inflammatory response can be divided as the five sections: 1.recognition of the injurious agent;2.recruitment of leukocytes;3.removal of the agent;4.regulation (control) of the response;and 5.resolution (repair).

When an individual encounters an injurious agent, as described above, phagocytes that reside in all tissues try to eliminate these agents. At the same time, phagocytes and other sentinel cells in the tissues recognise the presence of the inflammation triggers and react by liberating cytokines, lipid messengers, and other mediators of inflammation. Some of these mediators act on small blood vessels in the vicinity and promote the efflux of plasma and the recruitment of leukocytes (as demonstrated as dilation of small vessels leading to an increase in blood flow [vasodilation/resulting in more blood cells and plasma proteins], increased permeability of the endothelia, and emigration and accumulation of the leukocytes [stasis]) to the site where the offending agent is located.

PS: the following figure shows the inherent differences between exudate and transudate. Edema denotes an excess of fluid in the interstitial tissue or serous cavities; it can be either an exudate or a transudate. Purulent is a inflammatory exudate rich in leukocytes, the debris of dead cells and, in many cases, microbes.

Recognition of Inflammation-Causative Substances

Cellular Receptors for Microbes

Several cellular receptors and circulating proteins are capable of recognising microbes and products of cell damage and triggering inflammation. Cells express receptors in the plasma membrane, the endosomes (note, it's not the lysosomes), and the cytosol that enable the cells to sense the presence of foreign invaders in any cellular compartment. The most-documentted such receptors are the family of Toll-like receptors (TLRs). These receptors are expressed on many cell types, including epithelial cells, dendritic cells, macrophages, and other leukocytes. Engagement of these receptors triggers production of molecules involved in inflammation, including adhesion molecules on endothelial cells, cytokines, and other mediators.

Sensors of Cell Damage

All cells have cytosolic receptors that recognise a diverse set of molecules that liberated or altered as a consequence of cell damage. These molecules include uric acid, ATP, reduced intracellular K⁺ concentrations, even DNA when it is released into the cytoplasm and not sequestered in nuclei, as it should be normally, and many others. These receptors activate a multi protein cytosolic complex called inflammasome which induces the produce of the cytokine interleukin-1 (IL-1).

Indirect Recognizing

In addition to directly recognising microbes, many leukocytes express receptors for the Fc tails of antibodies and for complement proteins. It is likely that the binding of antibodies and complement proteins by microbes will change the conformation of the Fc tails and activated complement proteins, respectively, and this change of conformation provides the chance of them to bind these leukocyte receptors. These receptors recognise microbes coated with antibodies and complement and promote ingestion and destruction of the microbes as well as inflammation. Some circulating proteins like complements reacts against microbes and produces mediators of inflammation. These proteins act indirectly to the recognition of inflammation-causative substances, with the help of which the cells recognise these substances more effectively.

Reactions of Blood Vessels in Acute Inflammation

The vascular reactions of acute inflammation consist of changes in the flow of blood and the permeability of vessels, both designed to maximise the movement of plasma proteins and leukocytes out of the circulation and into the site of infection or injury. Vasoactive mediators originating from plasma and cells are generated at sites of tissue injury. These vasoactive molecules bind specific receptors on vascular endothelial and smooth muscle cells, causing vasoconstriction or vasodilation. Vasodilation of arterioles increases blood flow and exacerbates fluid leakage into the tissue. Vasoconstriction of postcapillary venules increases capillary bed hydrostatic pressure, further stimulating edema formation. Also vasoactive mediators bind specific receptors on endothelial cells, causing reversible endothelial cell contraction and gap formation.

Changes in Vascular Flow and Caliber

Changes in vascular flow and caliber begin early after injury and consist of the following. Vasodilation is induced by the action of several mediators, notably histamine, on vascular smooth muscle. It is one of the earliest manifestations of acute inflammation. Vasodilation first involves the arterioles and then leads to opening of new capillary beds in the area. The result is increased blood flow, which is the cause of heat and redness.

PS: Critical Closing Pressure & Active/Inactive Capillaries
In rigid tubes, the relationship between pressure and flow of homogeneous fluids is liner, but in thin-walled blood vessels in vivio it is not. When the pressure in a small blood vessel is reduced, a point is reached at which no blood flows, even though the pressure is not zero. This is because the vessels are surrounded by tissues that exert a small but definite pressure on them, and when the intraluminal pressure falls below the tissue bpressure, they collapse. The threshold press is called critical closing pressure.
So in resting tissues, most of the capillaries are collapsed, these capillaries are inactive cappliaries. In active tissues, the metarterioles and the precapillary sphinctersdilate. The result is that the intracapillary pressure rises, overcoming the critical closing pressure of the vessels, and blood flows through all of the capillaries. Relaxation of the smooth muscle of the metarterioles and precapillary sphincters is due to the action of vasodilator metabolites formed in active tissue.

Vasodilation is quickly followed by increased permeability of the microvasculature, with the outpouring of protein-rich fluid into the extravascular tissues. The loss of fluid caused by increased permeability and the increased vessel diameter lead to slower blood flow, concentration of red cells in small vessels, and increased viscosity of the blood. These changes result in engorgement of small vessels with slowly moving red cells, a condition termed stasis, which is seen as vascular congestion and localised redness of the involved tissue.

As stasis develops, blood leukocytes, principally neutrophils, accumulate along the vascular endothelium. At the same time endothelia cells are activated by mediators produced at sites of infection and tissue damage, and express increased levels of adhesion molecules. Leukocytes then adhere to the endothelium, and soon afterward they migrate through the vascular wall into the interstitial tissue.

Increased Vascular Permeability (Vascular Leakage)

Several mechanisms are responsible for the increased permeability of post capillary venules, a hallmark of acute inflammation. These mechanisms of increased vascular permeability are described separately, all probably contribute in varying degrees in responses to most stimuli. For example, at different stages of a thermal burn, leakage results from chemically mediated endothelial contraction and direct and leukocyte-dependent endothelia injury. The vascular leakage induced by these mechanisms can cause life-threatening loss of fluid in severely burned patients.

Contraction of endothelial cells resulting in increased inter endothelial spaces is the most common mechanisms of vascular leakage. It is elicited by histamine, bradykinin, leukotrienes, and other chemical mediators. It is called the immediate transient response because it occurs rapidly after exposure to the mediator and is usually short-lived (15 to 30 minutes). In some forms of mild injury (e.g., after burns, irradiation or ultraviolet radiation, and exposure to certain bacterial toxins), vascular leakage begins after a delay of 2 to 12 hours and lasts for several hours or even days;this delayed prolonged leakage may be caused by contraction of endothelial cells or mild endothelial damage. Late-appearing sunburn is a good example of this type of leakage.

Endothelial injury, resulting in endothelia cell necrosis and detachment. Direct damage to the endothelium is encountered in severe injuries, for example, in burns, or is induced by the actions of microbes and microbial toxins that target endothelial cells. Neutrophils that adhere to the endothelium during inflammation may also injure the endothelial cells and thus amplify the reaction. In most instances leakage starts immediately after injury and is sustained for several hours until the damage vessels are thromboses or repaired.

Increased transport of fluids and proteins, called transcytosis, through the endothelial cell. This process may involve intracellular channels that may be stimulated by certain factors, such as vascular endothelial growth factor (VEGF), that promote vascular leakage. However, the contribution of this process to the vascular permeability of acute inflammation is uncertain.

PS: In addition to blood vessels, lymphatic vessels also participate in acute inflammation. The system of lymphatics and lymph nodes filters and polices the extravascular fluids. Lymphatics normally drain the small amount of extravascular fluid that has seeped out of capillaries. In inflammation, lymph flow is increased and helps drain deem fluid that accumulates because of increased vascular permeability. In addition to fluid, leukocytes and cell debris, as well as microbes, may find their way into lymph. Therefore the lymphatics may become secondarily inflamed (lymphangitis), as may the draining lymph nodes (lymphadenitis). Inflamed lymph nodes are often enlarged because of hyperplasia of the lymphoid follicles and increased numbers of lymphocytes and macrophages. This constellation of pathologic changes is termed reactive, or inflammatory, lymphadenitis. For clinicians the presence of red streaks near a skin would is telltale sign of an infection in the wound. This streaking follows the course of the lymphatic channels and is diagnostic of lymphangitis;it may be accompanied by painful enlargement of the draining lymph nodes, indicating lymphadenitis.

Leukocyte Recruitment to Sites of Inflammation

The changes in blood flow and vascular permeability are quickly followed by an influx of leukocytes into the tissue. The most important leukocytes in typical inflammatory reactions include neutrophils and macrophages, both termed phagocytosis. These leukocytes (but no limited to) ingest and destroy bacteria and other microbes, as well as necrotic tissue and foreign substances. Leukocytes also produce growth factors that aid in repair.

But a price that is paid for the defensive potency of leukocytes is that, when strongly activated, they may induce tissue damage and prolong inflammation, because the leukocyte products that destroy microbes and help "clean up" necrotic tissues can also injure normal bystander host tissues.

The journey of leukocytes from the vessel lumen to the tissue is a multistep process that is mediated and controlled by adhesion molecules and cytokines called chemokines. Briefly, the recruitment of leukocyte to sites of inflammation is a multistep process, which can be divided into adhesion to endothelium, migration through endothelium, and chemotaxis.

Leukocyte Adhesion to Endothelium

The whole process includes the margination, rolling, and adhesion of leukocytes to endothelium. In normal and unactivated status, vascular endothelium does not bind circulating cells or impede their passage. In inflammation, the endothelium is activated and can bind leukocytes as a prelude to their exit from the blood vessels, which follows those mechanisms below.

In normally flowing blood in venues, red cells are confined to a central axial column, displacing the leukocytes toward the wall of the vessel. Because blood flow slows early in inflammation (stasis), hemodynamic conditions change (wall shear stress decreaes), and more white cells assume a peripheral position along the endothelial surface. This process of leukocyte redistribution is called margination. Subsequently, leukocytes adhere transiently to the endothelium, detach and bind again, thus rolling on the vessel wall. Finally the cells come to rest at some point where they adhere firmly (adhesion).

The attachment of leukocytes to endothelial cell is mediated by complementary adhesion molecules on the two cell types (leukocytes and endothelium) whose expression is enhanced by cytokines. The two major families of molecules involved in leukocyte adhesion and migration are the selections and interns, and their ligands.

Table 1 Endothelia and Leukocyte Adhesion Molecules

The initial rolling interactions are mediated by a family of proteins called selectins. There are three types of selectins: L-selectin expressed on leukocytes, E-selectin expressed on endothelium, and P-selectin expressed in platelets and on endothelium. There are ligands for each selectin, which are expressed on leukocytes and endothelium, respectively. The expression of selecting and their ligands is regulated by cytokines produced in response to infection and injury. Within 1 to 2 hours the endothelial cells begin to express E-selectin and the ligands for L-selectin. Other mediators such as histamine and thrombin stimulate the redistribution of P-selectin from its normal intracellular stores in endothelial cell granules (called Weibel-Palade bodies) to the cell surface.

The interactions between selections and their ligands are low-affinity with a fast off-rate, and they are easily disrupted by the flowing blood. As a result, the bound leukocytes bind, detach, and bind again, and thus begin to roll along the endothelial surface.

These weak rolling interactions slow down the leukocytes and give them the opportunity to bind more firmly to the endothelium. Firm adhesion is mediated by a family of heterodimeric leukocyte surface proteins called integrins such as VLA-4, LFA-1 etc, where they are expressed on leukocytes with ligands such as VCAM-1, ICAM-1, etc. Leukocytes normally express interns in a low affinity state. Chemokines that were produced a the site of injury bind to endothelial cell proteoglycans, and are displayed at high concentrations on the endothelial surface. Meanwhile, these chemokines bind to and activate the rolling leukocytes which induces the conversion of integrins from low-affinity state to hight-affinity state. Finally, high level of ligand on endothelial surface and high -affinity of integrin on leukocytes results in firm integrin-mediated binding of these two cell types at the site of inflammation. The leukocytes stop rolling, their cytoskeleton is reorganised, and the spread out on the endothelial surface.

Leukocyte Migration Through Endothelium

Transmigration of leukocytes occurs mainly in postcapillary venules. Chemokines act on the adherent leukocytes and stimulate the cells to migrate through interendothelial spaces toward the chemical concentration gradient, that is, toward the site of injury or infection where the cheekiness are being produced. Several adhesion molecules present in the intercellular junctions between endothelia cells are involved in the migration of leukocytes, including a member of the immunoglobulin superfamily called CD31 or PECAM-1 (platelet endothelial cell adhesion molecule). After traversing the endothelium, leukocytes pierce the basement membrane, probably by secreting collagenases, and enter the extravascular tissue. The cells then migrate toward the chemotactic gradient created by cheekiness and other chemoattractants and accumulate in the extravascular site.

Chemotaxis of Leukocytes

After exiting the circulation, leukocytes move in the tissue word the site of injury by a process called chemotaxis, which is defined as locomotion along a chemical gradient. Both exogenous and endogenous substances can act as chemoattractants. The most common exogenous agents are bacterial products, including peptides that possess an N-formylmethionine terminal amino acid and some lipids. Endogenous chemoattractants include several chemical mediators like: 1.cytokines, particularly those of the chemokine family (e.g., IL-8);2.components of the complement system, particularly C5a;and 3.arachidonic acid (AA) metabolites, mainly leukotriene B4 (LTB4). All these chemotactic agents bind to specific seven-transmembrane G protein-coupled receptors on the surface of leukocytes. Signals initiated from these receptors result in activation of second messengers (check thread "G Protein-Coupled Receptors" at http://www.tomhsiung.com/wordpress/2014/09/g-protein-coupled-receptors/) that increase cytosolic calcium and activate small guanosine triphosphatases of the Rac/Rho/cdc42 family as well as numerous kinases. These signals induce polymerization of actin, resulting in increased amounts of polymerized actin at the leading edge of the cell and localization of myosin filaments at the back. The leukocyte moves by extending filopodia that pull the back of the cell in the direction of extension, much as an automobile with front-wheel drive is pulled by the wheels in front. The net result is that leukocytes migrate toward the inflammatory stimulus in the direction of the locally produced chemoattractants.

The Nature of the Leukocyte Infiltrate

The nature of the leukocyte infiltrate varies with the age of the inflammatory response and the type of stimulus. In most forms of acute inflammation neutrophils predominate in the inflammatory infiltrate during the first 6 to 24 hours and are replaced by monocytes in 24 to 48 hours. There are several reasons for the early preponderance of neutrophils: they are more numerous in the blood than other leukocytes, they respond more rapidly to chemokines, and they may attach more firmly to the adhesion molecules that are rapidly induced on endothelial cells, such as P- and E-selectins. After entering tissues, neutrophils are short-lived;they undergo apoptosis and disappear within 24 to 48 hours. Monocytes not only survive longer but may also proliferate in the tissues, and thus they become the dominant population in prolonged inflammatory reactions.

There are, however, exceptions to this stereotypic pattern of cellular infiltration. In certain infections like those produced by Pseudomonas bacteria, the cellular infiltrate is dominated by continuously recruited neutrophils for several days;in viral infections, lymphocytes may be the first cells to arrive; some hypersensitivity reactions are dominated by activated lymphocytes, macrophages, and plasma cells;and in allergic reaction, eosinophils may be the main cell type.

Complement System

The complement system is a collection of soluble proteins and membrane receptors that function mainly in host defines against microbes and in pathologic inflammatory reactions. This system of complement functions in both innate and adaptive immunity for defines against microbial pathogens. In the process of complement activation, several cleavage products of complement proteins are elaborated that cause increased vascular permeability, chemotaxis, and opsonization.

Complement system acts as the bridge between innate and adaptive immune system. This concept is due to the fact that complement proteins can be activated directly by antigen-antibody complexes.

Primary Functions

There are three main effects of complement: 1.lysis of cells such as bacteria, allografts, and tumor cells; 2.generation of mediators that participate in inflammation and attract neutrophils; and 3.opsonization – enhancement of phagocytosis.

C3b is the central molecule of the complement cascade. It has two core functions: 1.it combines with other complement components to generate C5 convertase, the enzyme that leads to the production of the membrane attack complex (first it adhere to the surface of the targets); and 2.it opsonises bacteria because phagocytes have receptors for C3b on their surface.

How to activate?

In the classic pathway, antigen-antibody complexes activate C1² to form a protease and thereafter the complement cascade starts. In the lectin pathway, MBL (mannas-binding lectin/mannose-binding protein) binds to the surface of microbes bearing mannan. This activates proteases associated with MBL that activates complement cascade. In the alternative pathway, many unrelated cll surface substances can initiate the process by binding C3 and factor B. This complex is cleaved by a protease and finally the complement cascade initiates.

Negative Feedback Mechanisms

Innate and inflammatory responses are regulated by either enhancing or inhibiting mechanisms. The inhibiting mechanisms controls the degree of inflammation and terminate it when appropriate so that the causative substances of inflammation are eliminated while harmful effects to body could be limited to minimize the tissue damage.

In part, inflammation declines after the offending agents are removed simply because the mediators of inflammation are produced in rapid bursts, only as long as the stimulus persists, have short half-lives, and are degraded after their release. On the other hand, as inflammation develops, the process itself triggers a variety of stop signals that actively control and inhibit the inflammatory reaction. Some substances like lipoxins derived from arachidonic acid (AA), transforming growth factor-β (TGF-β), and IL-10 act as anti-inflammatory mediators to obtain the purpose of controlling and inhibiting the inflammation.

A Price Paid to Inflammation

General Symptoms and Signs of Inflammation

Although inflammation serves to protect and control infections and other harmful insults, it can also cause further tissue damage, which is manifested as the disease symptoms of redness, swelling, heat, and pain. The increased blood flow due to vasodilation results in redness and increased temperature in the area. The increased vascular permeability causes blood fluids to leak out of the vessels as the phagocytes transmigrate and thereby also cause edema (swelling) of the surrounding tissue. The source of the pain is still not clearly understood, but it is probably due to the combined effects of cytokines (e.g.,, prostaglandins) and coagulation cascade components on nerve endings in the inflamed region. Bradykinin also appears to increase sensitivity to pain. Pus, a common sign of infection, is composed mainly of dead PMNs and tissue cells.

Although phagocytic cells are effective killers of bacteria and are essential for clearing the invading bacteria from an infected area, the body can pay a high price for this service. During active killing of a bacterium, lysosomal enzymes are released into the surrounding area, as well as into the phagolysosome. Released lysosomal enzymes damage adjacent tissues and can be the main cause of tissue damage that results from a bacterial infection. Also, PMNs kill themselves as a result of their killing activities, and lysosomal granules released by dying PMNs contribute further to tissue destruction.

Recruitment

Diffusion of Gases

[Respiration][Circulation] Blood Flow to the Lung – General and Pulmonary Vascular Resistance

Hemostasis Mechanism – Platelet Structure and Function

Inflammation – The Beginning and Ongoing

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