Month: July 2016

Renal Potassium Reabsorption and Secretion

July 24, 2016 Cardiology, Critical Care, Nephrology, Physiology and Pathophysiology No comments , , , , , , , , , , ,

Percentage of fitered load transported at different locations depending on diet

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The Importance of Potassium Balance

The vast majority of body potassium is freely dissolved in the cytosol of tissue cells and constitutes the major osmotic component of the intracellular fluid (ICF). Only about 2% of total-body potassium is in the extracellular fluid (ECF). This small fraction, however, is absolutely crucial for body function, and the concentration of potassium in the ECF is a closely regulated quantitiy. Major increases and decreases (called hyperkalemia and hypokalemia) in plasma values are cause for medical intervention. The importance of maintaining this concentration stems primarily from the role of potassium in the excitability of nerve and muscle, especially the heart.

The ratio of the intracellular to extracellular concentration of potassium is the major determinant of the resting membrane potential in these cells. A significant rise in the extracellular potassium concentration causes a sustained depolarization. Low extracellular potassium may hyperpolarize or depolarize depending on how changes in extracellular potassium affect membrane permeability. Both conditions lead to muscle and cardiac disturbances.

Potassium Movement Between the ICF and ECF

Given that the vast majority of body potassium is contained within cells, the extracellular potassium concentration is cruically dependent on 1).the total amount of potassium in the body and 2).the distribution of this potassium between the extracellular and intracellular fluid compartments. Total-body potassium is determined by the balance between potassium intake and excretion. Healthy individuals remain in potassium balance, as they do in sodium balance, by excreting potassium in response to dietary loads and withholdin g excretion when body potassium is depleted. The urine is the major route of potassium excretion, although some is lost in the feces and sweat. Normally the losses via sweat and the gastrointestinal tract are small, but large quantities can be the lost from digestive tract during vomiting or diarrhea. The control of renal potassium transport is the major mechanism by which total-body potassium is maintained in balance.

The high level of potassium within cells is maintained by the collective operation of the Na-K-ATPase plasma membrane pumps, which actively transport potassium into cells. Because the total amount of potassium in the extracellular compartment is so small (40-60 mEq total), even very slight shifts of potassium into or out of cells produce large changes in extracellular potassium concentration. Similarly, a meal rich in potassium (e.g., steak, potato, and spinach) could easily double the extracellular concentration of potassium if most of that potassium were not transferred from the blood to the intracellular compartment. It is crucial, therefore, that dietary loads be taken up into the intracellular compartment rapidly to prevent major changes in plasma potassium concentration.

The tissue contributing most to the sequestration of potassium is skeletal muscle, simply because muscle cells collectively contain the largest intracellular volume. Muscle effectively buffers extracellular potassium by taking up or releasing it to keep the plasma potassium concentration close to normal. On a moment-to-moment basis, this is what protects the ECF from large swings in potassium concentration. Major factors involved in these homeostatic processes include insulin and epinephrine, both of which cause increased potassium uptake by muscle and certain other cells through stimulation of plasma membrane Na-K-ATPases. Another influence is the gastrointestinal tract, which contains an elaborate neural network (the "gut brain") that sends signals to the central nervous system. It also contains a complement of enteroendocrine cells that release an array of peptide hormones. Together these neural and hormonal signals affect many target organs, including the kidneys in response to dietary input.

The increase in plasma insulin concentration after a meal is a crucial factor in moving potassium absorbed from the GI tract into cells rather than allowing to accumulate in the ECF. This newly ingested potassium then slowly comes out of cells between meals to be excreted in the urine. Moreover, a large increase in plasma potassium concentration facilitates insulin secretion at any time, and the additional insulin induces greater potassium uptake by the cells.

The effect of epinephrine on cellular potassium uptake is probably of greatest physiological importance during exercise when potassium moves out of muscle cells that are rapidly firing action potentials. In fact, very intense intermittent exercises such as wind sprints can actually double plasma potassium for a brief period. However, at the same time, exercise increases adrenal secretion of epinephrine, which stimulates potassium uptake bu the Na-K-ATPase in muscle and other cells and transiently high potassium levels are restored to normal with a few minutes of rest. Similarly, trauma causes of loss of potassium from damaged cells and epinephrine released due to stress stimulates other cells to take up plasma potassium.

Another influence on the distribution of potassium between the ICF and ECF is the ECF hydrogen ion concentration: An increase in ECF hydrogen ion concentration is often associated with net potassium movement out of cells, whereas a decrease in ECF hydrogen ion concentration causes net potassium movement into them. It is as though potassium and hydrogen ions were exchanging across plasma membranes.

Renal Potassium Handling

Althgouh skeletal msucle and other tissues play an important role in the moment-to-moment control of plasma potassium concentration, in the final analysis, the kidney determines total-body potassium content. It is helpful to keep in mind several major differences between teh renal handling of sodium and potassium.

  • The filtered load of sodium is 30 to 40 times greater than the filtered load of potassium and the tubules always have to recover the majority of filtered sodium. This is not the case for potassium.
  • Sodium is only reabsorbed, never sereted. In contrast, potassium is both reabsorbed and secreted, ant its regulation is primarily focused on secretion.
  • The renal handling of sodium has a much greater effect on potassium than vice versa, which is a major feature of control.

Potassium is freely filtered into Bowman's space. Under all conditions, almost all the filtered load (~90%) is reabsorbed by the proximal tubule and thick ascending limb of the loop of Henle. Then, if the body is conserving potassium, most of the rest is reabsorbed in the distal nephron and medullary collecting ducts, leaving almost none in the urine. In contrast, if the body is ridding itself of potassium, a large amount is secreted in the distal nephron, resulting in a substantial excretion, of which the amount excreted may exceed the filtered load when secretion occurs at high rates.

PS: Proximal tubule reabsorption percentage: 65%; thick ascending limb of the loop of Henle reabsorption percentage: 25%

Proximal tubule

In the proximal tubule about 65% of the filtered load is reabsorbed, mostly via the paracellular route. The flux is driven by the concentration gradient set up when water is reabsorbed, which concentrates potassium and other solutes remaining in the tubular lumen. This flux is essentially unregulated and varies mostly with how much sodium, and therefore water, is reabsorbed.

The active transport of potassium is always coupled to the active transport of another solute, either sodium or hydrogen. In the proximal tubule the efflux of sodium by the Na-K-ATPase is very vigorous, requiring a high rate of potassium uptake from the interstitium. Since we know that there is net potassium transport into the interstitium, this pumped potassium must therefore recycle right back by passive flux through channels in the basolateral membrane. In some regions influx of potassium across apical membranes occurs via H-K antiporters that are simultaneously secreting protons.

The loop of Henle

The loop of Henle continues the reabsorption of potassium. The major events take place in the thick ascending limb, where the Na-K-2Cl multiporter in the apical membrane of the tubular cells takes up potassium. The interaction with sodium in these cells is even more complicated than in the proximal tubule because potassium is transported into the bubular cells both from the lumen with sodium via Na-K-2Cl symporters and from the interstitium via the Na-K-ATPase. The tubule contains far less potassium than sodium, but the Na-K-2Cl transporter moves equal amounts of each one. Therefore to supply enough potassium to accompany the large amount of sodium being reabsorbed by the symporter, potassium must recycle back to the lumen by passive channel flux. If this did not happen then sodium reabsorption would be limited only to the amount of potassium present in the tubular fluid.

Some potassium entering from the lumen does move through the cells and exit across the basolateral membrane along with the potassium entering via the Na-K-ATPase. It exits by a combination of passive flux through channels and through K-Cl symporters with chloride, thus yielding net transcellular reabsorption. Some potassium is also reabsorbed by the paracellular route in this segment, driven by a lumen-positive voltage.

The distal nephron

The distal convoluted tubule and connecting tubule stand out as being particularly imporant in potassium handling because of their rich complement of transport elements and their location prior to segments where most of water is absorbed. These regions play a major role in potassium secretion when total body potassium is high (high potassium diet). The distal nephron expresses both reabsorptive and secretory mechanisms, and it is the quantitative amount of each that determines net potassium excretion. There are several cell types in the epithelium of the connecting tubule and cortical collecting duct. Principal cells (approximately 70% of the cells) and intercalated cells. The intercalcated cells are further subdivided into type A (more numerous), type B (sparse) and a third type called non-A non-B cells. Potassium secretion occurs in principal cells, whereas the type A intercalated cells reabsorb potassium. The mechanisms of both secretion and reabsorption are straightforward. Secretion of potassium by principal cells involves the uptake of potassium from the interstitium via the Na-K-ATPase and secretion into the tubular lumen through channels. Type A intercalated cells reabsorb potassium via the H-K-ATPase in the apical membrane, which actively takes up potassium from the lumen. They then allow potassium to enter the interstitium across the basolateral membrane via potassium channels.

Finally, the medullary collecting ducts reabsorb small amounts of potassium under all conditions. When the sum of upstream processes has already reabsorbed almost all the potassium, the medullary collecting ducts bring the final urine excretion down to a few percent of the filtered load, for an excretion of about 10 to 15 mEq/day. On the other hand, if upstream segments are secreting avidly, the modest reabsorption in the medullary collecting ducts does little to prevent an excretion that can reach 1000 mEq/day.

Regulation of Potassium Excretion

The mechanisms regulating potassium excretion are as complex, and perhaps more so, than those regulating sodium excretion. And as pointed out earlier, active potassium trasnport is intertwined with sodium and hydrogen transport. But within the complexity one thing is abundantly clear – the healthy kidneys do a remarkable job of integrating signals to increase potassium excretion in response to high dietary loads and reduce excretion in the face of restricted diets.

The key regulated variable is potassium secretion by principal cells in the distal nephron. There are 3 transport processes in these cells that determine the amount of secretion: potassium influx by the Na-K-ATPase, potassium efflux into the lumen, and potassium efflux back into the interstitium (recycling). Much of the control is exerted on the activity of potassium channels. The kidneys and other body organs express numerous potassium channel species; for simplicity we do not usually differentiate between types. However in the apical membrane of principal cells in the distal nephrone, 2 types of channels stand out as being those that secrete potassium in a regulated manner: ROMK and BK. Although ROMK and BK channels are both permeable to potassium, they play different mechanisms. At very low dietary loads of potassium, there is virtually no secretion by either kind of channel. ROMK channels are sequestered in intracellular vesicles and BK channels are closed. At normal potassium loads, ROMK channels are moved to the apical membrane and secrete potassium at a modest rate. BK channels are still closed, held in reserve and ready to respond to appropriate signals when needed. At high excretion rates, both types of channel are present in the luminal membrane and avidly secreting potassium being pumped in by the Na-K-ATPase.

Plasma potassium

First, the filtered load is directly proportional to plasma concentration. Second, the environment of the principal cells that secrete potassium, that is, the cortical interstitium, has a potassium concentration that is nearly the same as in plasma. The Na-K-ATPase that takes up potassium is highly sensitive to the potassium concentration in this space, and varies its pump rate up and down when potassium levels in the plasma vary up and down. Thus plasma potassium concentration exerts an influence on potassium excretion, but is not the dominant factor under normal conditions.

Dietary potassium

Dietary potassium must be matched by renal excretion. The healthy kidneys do this very well by increasing and decreasing potassium excretion in parallel with dietary load. Just how the kidneys "know" about dietary input is still somewhat mysterious. Although very large potassium loads can increase plasma potassium somewhat, the changes in excretion assocaited wtih ordinary fluctuations in dietary input do not seem to be accounted for on the basis of either changes in plasma potassium or the other identified factors. One factor known to exert an influence, but not the major one, is the previously mentioned gastrointestinal peptide hormones released in response to ingested potassium. They influence not only the cellular uptake of potassium absorbed from the GI tract, but also the renal handling ot potassium, and seem to be one of the links between dietary load and excretion.

A manifestation of changing dietary loads over time is to regulate the distribution of ROMK channels between the apical membrane and intracellular storage, that is, high-potassium diets lead to insertion of apical channels and therefore highest potassium secretion. In contrast, during periods of prolonged low potassium ingestion, there are few ROMK channels in the apical membrane. Yet another adaptation to prolonged periods of low potassium ingestion is an increase in H-K-ATPase activity in intercalated cells, resulting in even more efficient reabsortpion of filtered potassium.


A stimulator of aldosterone secretion by the adrenal cortex, in addition to AII, is an increase in plasma potassium concentration. This is a direct action of potassium and does not involve the renin-angiotensin system. If anything, high levels of potassium decrease the formation of AII. Aldosterone, as well as increasing expression of the Na-K-ATPase and ENaC sodium channels, also stimulates the activity of ROMK channels in principal cells of the distal nephron. Both actions have the effect of increasing potassium secretion. Greater pumping by the Na-K-ATPase supplies more potassium from the interstitium to the cytosol of the principal cells, and more functioning ROMK channels provide more pathways for secretion. Conversely, low levels of aldosterone deter potassium secretion.

Angiotensin II

AII is an inhibitor of potassium secretion. Its mechanism of action is to decrease the activity of ROMK channels in principal cells and distal convoluted cells, thereby limiting the potassium flux from cell to lumen. Thus AII and aldosterone exert influences on potassium excretion in opposite directions.

Delivery of sodium to principal cells

Sodium delivery to principal cells in the connecting tubule and cortical collecting duct is a major regulator. High sodium delivery stimulates potassium secretion. It does so in 2 ways. First, sodium entry via sodium channels (ENaC?) in principal cells depolarizes the apical membrane and thereby increases the electrochemical gradient driving the outward flow of potassium through channels. Second, more sodium delivered means more sodium taken up, and therefore more sodium pumped out by the Na-K-ATPase, in turn causing more potassium to be pumped in. Sodium delivery to principal cells, and hence potassium secretion, is strongly affected by the amount of sodium reabsorption in prior segments.

Regulation of Water Excretion

July 23, 2016 Cardiology, Critical Care, Nephrology, Physiology and Pathophysiology No comments , , , , ,

Water excretion, as with sodium excretion, is regulated in partnership with the CV system. Central goals in regulating both salt and water excretion are to: 1).preserve vascular volume and 2).maintain plasma osmolality at a level that is healthy for tissue cells. The main regulators of water excretion, not surprisingly, relate to osmolality and volume.

Quantitatively, renal water excretion is determined by 2 values: 1).the amount of solute in the urine and 2).the osmolality of the urine.

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Urine water excretion = urine solute excretion/urine osmolality

Excreted solute consists mostly of organic waste and excess electrolytes. In a given metabolic state, the rate of organic waste excretion is more or less constant, and is not altered for purposes of controlling water excretion. Electrolyte excretion is highly regulated, but more to achieve balance of individual substances like sodium and potassium than to control water excretion per se. Given that solute excretion is so variable, the main way the body controls water excretion in normal circumstances is to control urine osmolality. In other words, given a certain amount of solute that is excreted, the body controls the amount of water accompanying it by controlling urine osmolality.

When the body excretes urine that is more dilute than plasma (osmolality below 285 mOsm/kg H2O), the body is excreting "free water" (like adding pure water to otherwise isosmotic urine). Conversely, when the excreted urine is more concentrated than plasma, there is "negative free water" excretion. It is as if the body has reclaimed pure water from otherwise isosmotic urine.

The kidneys first generate hypo-osmotic tubular fluid in the loop of Henle. Then, as the fluid subsequently flows through the collecting duct system, variable amounts of water are reabsorbed by allowing the tubular fluid to equilibrate to varying degrees with the surrounding interstitum. The final urine osmolality, and hence final volume, depends on the peak medullary osmolality and how closely the tubular osmolality approaches that value. We also know that equilibration with the interstitium is a function of water permeability in the collecting ducts under the control of the hormone antidiuretic hormone (ADH). Therefore the regulation of water excretion, that is independent of solute excretion, focuses on control over ADH secretion.

Osmoreceptor Control of ADH Secretion

Plasma osmolality is one of the most tightly regulated variables in the body. Plasma osmolality is set mainly by the ratio of ECF sodium (plus its assocaited anions) to water. Other solutes (e.g., glucose and potassium) make some contribution, but those other solutes are regulated for reasons other than plasma osmolality. Thus, except under unusual circumstances such as severe hyperglycemia, variations in plasma osmolality mostly reflect variations in sodium concentration. If the body keeps the inputs and outputs of sodium and water matched in lock step, osmolality remains constant. But inputs are often not matched. The major effect of gaining or losing water or salt without corresponding changes in the other is a change in the osmolality of the body fluids. When osmolality deviates from normal, strong reflexes come into play to change ADH secretion, and thus change the excretion of water.

Baroreceptor Control of ADH Secretion

There is another major influence on ADH secretion. This originates in systemic baroreceptors. A decreased extracellular volume or major decrease in arterial pressure reflexively activates increased ADH secretion. The response is mediated by neural pathways originating in cardiopulmonary baroreceptors, and if arterial pressure decreases, from arterial baroreceptors.

Decreased CV pressures cause less firing by the baroreceptors, which relieves inhibition of stimulatory pathways and results in more ADH secretion. In effect, the low CV pressures are interpreted as low volume, and the response of increased ADH appropriately serves to minimize loss of water. Conversely, baroreceptors are stimualted by increased CV pressures, interpreted as excess volume, and this causes inhibition of ADH secretion. The decrease in ADH results in decreased reabsorption of water in the collecting ducts, and more excretion. The adaptive value of these baroreceptor reflexes is to help stabilize ECF volume and, hence, blood pressure.

There is a second adaptive value to this reflex: Large decreases in plasma volume, as might occur after a major hemorrhage, elicit such high concentrations of ADH – much higher than those needed to produce maximal antidiuresis – that the hormone is able to exert direct vasoconstrictor effects on arteriolar smooth muscle. The result is increased total peripheral resistance, which helps restore arterial blood pressure independently of the slower restoration of body fluid volumes. Renal arterioles and mesangial cells also participate in this constrictor response, and so a high plasma concentration of ADH, quite apart form its effect on water permeability and sodium reabsorption in the distal nephron, promotes retention of both sodium and water by lowering GFR.


The cells that synthesize ADH in the hypothalamus also receive synaptic input from many other brain areas. Thus, ADH secretion and, hence, urine flow can be altered by pain, fear, and a variety of other factors, including drugs such as alcohol, which inhibits ADH release. However, this complexity should not obscure the generalization that ADH secretion is determined over the long term primarily by the states of body fluid osmolality and plasma volume.

We have described 2 different major afferent pathways controlling the ADH-secreting hypothalamic cells: 1 from baroreceptors and 1 from osmoreceptors. These hypothalamic cells are, therefore, true integrators, whose activity is determined by the total synaptic input to them. Thus, a simultaneous increase in plasma volume and decrease in body fluid osmolality causes strong inhibition of ADH secretion. Conversely, a simultaneous decrease in plasma volume and increase in osmolality produces very marked stimulation of ADH secretion. However, what happens when baroreceptor and osmoreceptor inputs oppose each other? In general, because of the high sensitivity of the osmoreceptors, the osmoreceptor influence predominates over that of the baroreceptors when changes in osmolality and plasma volume are small to moderate. However, a dangerous reduction in plasma volume will take precedence over decreased body fluid osmolality in influencing ADH secretion; under such conditions, water is retained in excess of solute even though the body fluids become hypo-osmotic (for the same reason, plasma sodium concentration decreases). In essence, when blood volume reaches a life-threatening low level, it is more important for the body to preserve vascular volume and thus ensure an adequate cardiac output than it is to preserve normal osmolality.

Thirst and Salt Appetite

Deficits of salt and water cannot be corrected by renal conservation, and ingestion is the ultimate compensatory mechanism. The subjective feeling of thirst, which drives one to obtain and ingest water, is stimulated both by reduced plasma volume and by increased body fluid osmolality. Note that these are precisely the same changes that stimulate ADH production, and the receptors – osmoreceptors and the nerve cells that respond to the CV baroreceptors – that initiate the ADH-controlling reflexes are near those that initiate thirst. The thirst response, however, is significantly less sensitive than the ADH response.

There are also other pathways controlling thirst. For example, dryness of the mouth and throat causes profound thirst, which is relieved by merely moistening them. Also, when animals such as the camel (and humans, to a lesser extent) become markedly dehydrated, they will rapidly drink just enough water to replace their previous losses and then stop. What is amazing is that when the stop, the water has not yet had time to be absorbed from the gastrointestinal tract into the blood.

Specific Immunosuppressive Therapy

July 20, 2016 Hematology, Immunology, Infectious Diseases, Oncology, Pharmacology, Transplantation No comments , , , , , , , , , , , , , , , ,

The ideal immunosuppressant would be antigen-specific, inhibiting the immune response to the alloantigens present in the graft (or vice versa alloantigens present in recipient in GVHD) while preserving the recipient's ability to respond to other foreign antigens. Although this goal has not yet been achieved, several more targeted immunosuppressive agents have been developed. Most involve the use of monoclonal antibodies (mAbs) or soluble ligands that bind specific cell-surface molecules. On limitation of most first-generation of mAbs came from their origin in animals. Recipients of these frequently developed an immune response to the nonhuman epitopes, rapidly clearing the mAbs from the body. This limitation has been overcome by the construction of humanized mAbs and mouse-human chimeric antibodies.

Many different mAbs have been tested in transplantation settings, and the majority work by either depleting the recipient of a particular cell population or by blocking a key step in immune signaling. Antithymocyte globulin (ATG), prepared from animals exposed to human lymphocytes, can be used to deplete lymphocytes in patients prior to transplantation, but has significant side effects. A more subset-specific strategy uses a mAb to the CD3 molecule of the TCR, called OKT3, and rapidly depletes mature T cells from the circulation. This depletion appears to be caused by binding of antibody-coated T cells to Fc receptors on phagocytic cells, which then phagocytose and clear the T cells from the circulation. In a further refinement of this strategy, a cytotoxic agent such as diphtheria toxin is coupled with the mAb. Antibody-bound cells then internalize the toxin and die. Another technique uses mAbs specific for the high-affinity IL-2 receptor CD25. Since this receptor is expressed only on activated T cells, this treatment specifically blocks proliferation of T cells activated in response to the alloantigens of the graft. However, since TREG cells also express CD25 and may aid in alloantigen tolerance, this strategy may have drawbacks. More recently, a mAb against CD20 has been used to deplete mature B cells and is aimed at suppressing AMR (antibody-mediated rejection) responses. Finally, in cases of bone marrow transplantation, mAbs against T-cell-specific markers have been used to pretreat the donor's bone marrow to destory immunocompetent T cells that may react with the recipient tissues, causing GVHD.

Because cytokines appear to play an important role in allograft rejection, these compounds can also be specifically targeted. Animal studies have explored the use of mAbs specific for the cytokines implicated in transplant rejection, particularly TNF-alpha, IFN-gamma, and IL-2. In mice, anti-TNF-alpha mAbs prolong bone marrow transplants and reduce the incidence of GVHD. Antibodies to IFN-gamma and to IL-2 have each been reported in some cases to prolong cardiac transplants in rats.

TH-cell activation requires a costimulatory signal in addition to the signal mediated by the TCR. The interaction between CD80/86 on the membrane of APCs and the CD28 or CTLA-4 molecule on T cells provides one such signal. Without this costimulatory signal, antigen-activated T cells become anergic. CD28 is expressed on both resting and activated T cells, while CTLA-4 is expressed only on activated T cells and binds CD80/86 with a 20-fold-higher affinity. In mice, D. J. Lenschow, J. A. Bluestone, and colleagues demonstrated prolonged graft survival by blocking CD80/86 signaling with a soluble fusion protein consisting of the extracellular domain of CTLA-4 fused to human IgG1 heavy chain. This new drug, belatacept, was shown to induce anergy in T cells directed against the graft tissue and has been approved by the FDA for prevention of organ rejection in adult kidney transplant pateints.

[Physiology][Hematology] Coagulation Factors, Anticoagulation Factors, and Pathways of Hemostasis and Thrombosis

July 14, 2016 Hematology, Molecular Biology No comments , , , , , , , , , , , , , , , , , ,

The role of surfaces in coagulation and coagulation inhibition and fibrinolysis

Functionally, the relationship between clotting and surface is striking. For a process that is typically termed humoral, most of the coagulation reactions take place on biologic surfaces.

  • The presence of a phospholipid surface increases the rate of activation of prothrombin by several orders of magnitude.
  • The presence of a phospholipid surface also localizes the reaction to the site of injury and may protect the reaction from inhibitors.
  • The activation of protein C by thrombin is a reaction that occurs on a cellular surface.
  • Coagulation inhibitors such as antithrombin and heparin cofactor II are also more efficient when the reactions occur on surfaces. The typical surface in these reactions is glycosaminoglycans like heparin, heparin sulfate, and derma tan sulfate.

The Vitamin K-dependent Zymogens


Tenase/Intrinsic tenase complex: factor VIIIa-factor IXa complex

Extrinsic tenase complex: tissue factor-factor VIIa complex

Prothrombinase compelx: factor Va-factor Xa complex

These zymogens all a similar domain structure of a C-terminal serine protease domain and an N-terminal γ-carboxy glutamic acid (Gla) domain, which are connected by two epidermal growth factor (EGF)-like domains or kringle domains. The Gla domain mediates the binding of zymogens to a negatively charged lipid surface (in a calcium-dependent manner), a domain that is characteristic to the vitamin K–dependent proteins.

Screen Shot 2016-08-11 at 7.36.50 PMThe Gla domain refers to the 42-residue region located in the N-terminus of the mature protein that comprises 9 to 12 glutamic acid residues that are posttranslationally γ-carboxylated into Gla residues by a specific γ-glutamyl carboxylase in the endoplasmatic reticulum of hepatocytes. This γ-carboxylase requires oxygen, carbon dioxide, and the reduced form of vitamin K for its action, hence the name vitamin K–dependent proteins. For each Glu residue that is carboxylated, one molecule of reduced vitamin K is converted to the epoxide form. Warfarin inhibits the activity of vitamin K epoxide reductase, thereby preventing vitamin K recycling and hinibiting γ-carboxylation, which results in a heterogeneous population of circulaing undercarboxylated forms of the vitamin K-dependent proteins with reduced activity. Recognition by and interaction with γ-carboxylase is facilitated by the propeptide sequence that is located C-terminal to the signal peptide.

The serine protease domains of the vitamin K-dependent proteins are highly homologous, as they bear a chymotrypsin-like fold and display trypsin-like activity.

Function of cofactors

Interaction of the vitamin K-dependent proteases with specific cofactors on a anionic membrane surface enhance substrate recognition, as the cofactors interact with both the protease and the substrate, bridging the two together, which results in a dramatic enhancement of the catalytic activity. Also the increase in catalytic rate has been attributed to a cofactor-induced conformational change in the protease. Cofactors are not always enhance coagulation, as in the example of thrombin, cofactor of throbbomodulin help thrombin to activate protein C.


Activates TAFI, platelet, fibrinogen, FV, FVIII, FXI, FXIII, and protein C

Activated by prothrombinase complex

Inhibited by serpins (enhanced by glycosaminoglycans like heparin)

Screen Shot 2016-05-20 at 2.19.12 PMProthrombin is composed of fragment 1 (F1: Gla and kringle 1), fragment 2 (F2: kringle 2), and the serine protease domain. The primary function of kringle 1 and kringle 2 domain is to be bound by prothrombinase complex. PS: Gla and kringle, kringle 2, and serine protease.

Prothrombin is proteolytically activated by the prothrombinase complex that cleaves at Arg271 and Arg320, both of which are necessary to generate procoagulant α-thrombin (IIa). Thrombin's main function is to induce the formation of a fibrin clot by removing fibrinopeptides A and B from fibrinogen to form fibrin monomers, which then spontaneously polymerize. The dynamic structural conformation of thrombin allows for binding to diverse ligands, and the subsequent ligand-indued conformational stabilization, known as thrombin allostery, regulates and controls thrombin activity. Thrombin also is able to cleave a wide variety of substrates with high specificity (TAFI, platelet, FV, FVIII, FXI, FXIII, protein C).

The physiologic inhibitors of thrombin are the serine protease inhibitors (serpins) antithrombin, heparin cofactor II, protein C inhibitor, and protease nexin 1, with antithrombin being the primary plasma inhibitor. For all four serpins, the rate of thrombin inhibition can be accelerated by glycosaminoglycans, such as heparin, through mutual binding to the serpin and thrombin, which ensures rapid inhibition of thrombin at the intact endothelial cell surface where heparin-like glycosaminoglycans are found.


Activates FIX, FX (in the form of extrinsic tenase)

Activated by Xa, thrombin, IXa, and XIIa

Inhibited by TFPI; antithrombin (only in the presence of heparin)

Screen Shot 2016-05-20 at 2.38.51 PMFactor VII consists of a Gla domain with 10 Gla residues, two EGF-like domains, a connecting region, and the serine protease domain.

Factor VII is proteolytically activated once it has formed a high-affinity complex with its cofactor tissue factor (there are small amount of VIIa in the circulation by unknown mechanism). A number of coagulation proteases including factor Xa, thrombin, IXa, and XIIa are capable of cleaving factor VII at Arg152 to generate factor VIIa, with factor Xa being considered the most potent and physiologically relevant activator of factor VII. Autoactivation can also occur, which is initiated by minute amounts of preexisting factor VIIa.

The extrinsic tenase complex activates both FIX and X.

The extrinsic tenase complex is inhibited by the tissue factor pathway inhibitor (TFPI). Antihrombin (only in the presence of heparin) also can inhibits the extrinsic tenase complex.


Aactivates FX in the form of intrinsic tenase

Activated by extrinsic tenase, factor XIa

Inhibited by antithrombin (enhanced by heparin)

Screen Shot 2016-05-20 at 3.08.29 PMFactor IX consists of a Gla domain, two EGF-like domains, a 35-residue activation peptide, and the serine protease domain. The Gla domain contains 12 Gla residues, of which the 11th and 12th Gla (Glu36 and Glu40) are not evolutionary conserved in other vitamin K-dependent proteins and are not essential for normal factor IX function.

Limited proteolysis of factor IX at both Arg145 and Arg180 by either the extrinsic tenase or factor XIa results in the release of the activation peptide and generation of factor IXa.

Factor IXa has a low catalytic efficiency as a result of impaired access of substrates to the active site that results from steric and repulsion. Reversible interaction with the cofactor VIIIa on anionic membranes and subsequent factor  X binding leads to rearrangement of the regions surrounding the active site and proteolytic factor X activation.

The primary plasma inhibitor of factor IXa is the serpin antihrombin, and this inhibition is enhanced by heparin, which induces a conformational change in antithrombin that is required for simultaneous active site and exosite interactions with factor IXa.


Activates prothrombin (prothrombinase complex); FV, VII, and VIII

Activated by extrinsic tenase; intrinsic tenase

Inhibited by antithrombin (enhanced by heparin); TFPI

Factor X is a two-chain zymogen consisting of a light chain which comprises the Gla domain with 11 Gla residues and the EGF domain, and a heavy chain that consists of a 52-residue activation peptide and the serine protease domain. The two chains are linked via a disulfide bond.

Factor X is activated by intrinsic tenase or extrinsic tenase, following cleavage at Arg194 in the heavy chain. After activated, Xa reversibly associates with its cofactor Va on an anionic membrane surface in the presence of calcium ions to form prothrombinase, the physiologic activator of prothrombin. Factor Xa is also involved in the proteolytic activation of FV, FVII, and VIII.

Further autocatalytic cleavage at Arg429 near the C-terminus of the factor Xa heavy chain leads to release of a 19-residue peptide, yielding the enzymatically active factor Xaβ. Plasmin-mediated cleavage of factor Xa at adjacent C-terminal Arg or Lys residues also results in the generation of factor Xaβ and factor Xaβ derivatives. While the coagulation activity is eliminated in the factor Xaβ derivatives, they are capable of interacting with the zymogen plasminogen and enhance its tissue plasminogen activator-mediated conversion to plasmin, thereby promoting fibrinolysis.

A primary plasma inhibitor of factor Xa is the serpin antithrombin, and this inhibition is enhanced by heparin. Another potent factor Xa inhibitor is TFPI, which inhibits both the extrinsic tenase-factor Xa complex as well as free factor Xa, for which protein S function as a cofactor.

Protein C

Inactivates FVa, FVIIIa (cofactor protein S, with calcium and surface) 

Activated by thrombin-thrombomodulin complex (enhanced by EPCR and PF4)

Inhibited by heparin-dependent serpin protein C inhibitor and by PAI-1

Protein C is synthesized as a single-chain precursor and during intracellular processing amino acids Lys146-Arg147 are excised. The zymogen consists of a light chain comprising the Gla domain and the EGF domains, which is linked via a disulfide bond to the heavy chain that consists of the activation peptide and the serine protease domain.

Screen Shot 2016-05-24 at 8.45.18 PMProtein C is proteolytically activated by alpha-thrombin in complex with the endothelial cell surface protein thrombomodulin following cleavage at Arg169. The activation peptide is released and the mature serine protease activated protein C (APC) is formed. Activation of protein C is enhanced by its localization on the endothelial surface through association with the endothelial cell protein C receptor (EPCR). Also, protein C activation is accelerated by platelet factor 4 (PF4), which is secreted by activated platelets. Upon interaction with the Gla domain of protein C, PF4 modifies the conformation of protein C, thereby enhancing its affinity for the thrombomodulin-thrombin complex.

APC consists of the disulfide-linked light chain comprising the Gla and EGF domains and the catalytic heavy chain. In complex with its cofactor protein S, APC proteolytically inactivates factors Va and VIIIa in a calcium- and membrane-dependent manner. Intact factor V has been reported to function as a cofactor for the inactivation of factor VIIIa in the presence of protein S.

APC is primarily inhibited by the the heparin-dependent serpin protein C inhibitor and by plasminogen activator inhibitor-1 (PAI-1).

The Procoagulant Cofactors V and VIII

Factors V and VIII both function as cofactors in coagulation and dramatically enhance the catalytic rate of their macromolecular enzyme complexes, resulting in the generation of thrombin (via prothrombinase) and factor Xa (via intrinsic tenase), respectively. Apart from their functional equivalence, they also share similar gene structure, amino aacid sequences, and protein domain structures.

Function of cofactors

Interaction of the vitamin K-dependent proteases with specific cofactors on a anionic membrane surface enhance substrate recognition, as the cofactors interact with both the protease and the substrate, bridging the two together, which results in a dramatic enhancement of the catalytic activity. Also the increase in catalytic rate has been attributed to a cofactor-induced conformational change in the protease. Cofactors are not always enhance coagulation, as in the example of thrombin, cofactor of throbbomodulin help thrombin to activate protein C.


Accelerates the ability of FXa to rapidly convert prothrombin to thrombin

Activated by thrombin (principal activator), FXa (primarily in initiation phase)

Inhibited by APC

FV and factor V-short interact with full-length TFPI

Approximately 20% percent of the total factor V in blood is stored in the alpha-granules of platelets. Although it was originally thought that megakaryocytes synthesize factor V, studies in humans indicate that platelet factor V originates from plasma through endocytic uptake. Platelet factor V is modified intracellularly such that it is functionally unique compared to its plasma-derived counterpart. It is partially activated, more resistant to inactivation by APC, and has several different posttranslational modifications.

Screen Shot 2016-05-29 at 8.22.28 PMFactor V has an A1-A2-B-A3-C1-C2 domain structure. The two C-type domains belong to the family of discoidin domains, which are generally involved in cell adhesion, and share approximately 55 percent sequence identity with the factor VIII C domains. The C domain mediate binding to the anionic phospholipid surface, thereby localizing factor V to the site of injury and facilitating interaction with factor Xa and prothrombin. In contrast, large central B domain of factor V shows weak homology to the factor VIII B domain or to any other known protein domain.

Factor V undergoes extensive postranslational modifications, including sulfation, phosphorylation, and N-linked glycoslation. Sulfation at sites in teh A2 and B domain are involved in the thrombin mediated proteolytic activation of factor V. Phosphorylation at Ser692 in the A2 domain enhances the APC-dependent inactivation of the cofactor Va.

Sequential proteolytic cleavage of the procofactor factor V at Arg709, Arg1018, and Arg1545 in the B domain results in release of the inhibitory constraints exerted by the B domain and in the generation of the heterodimeric cofactor Va, where maximal cofactor activity correlates with cleavage at Arg1545. Thrombin has generally been recognized to be the principal activator of factor V. However, recent findings suggest that in the initiation phase of coagulation factor V is primarily activated by factor Xa. Factor Xa initially cleaves factor V at Arg1018, followed by proteolysis at Arg709 and Arg1545.

Factor Va is composed of a heavy chain comprising the A1-A2 domains and the A3-C1-C2 light chain, which are noncovalently associated via calcium ions. Factor Va is a nonenzymatic cofactor within the prothrombinase complex that greatly accelerates the ability of factor Xa to rapidly convert prothrombin to thrombin.

APC catalyzes the inactivation of factor Va by cleavage at the main sites Arg306 and Arg506, upon which the cleaved A2 fragment dissociates and factor Va can no longer associated with factor Xa.

Both factor V and an alternativel spliced isoform of factor V (factor V-short), which lacks the major part of the B domain (residues 756 to 1458) and normally circulates in low abundance, interact with full-length TFPI (TFPIalpha), most likely through the acidic B domain region. The linkage of factor V and TFPIalpha is considered to attenuate the bleeding phenotype in factor V-deficient patients, as the low TFPIalpha levels in these patients allow the residual platelet factor to be sufficient for coagulation. Conversely, increased factor V-short expression caused by an A2440G mutation in the factor V gene leads to a dramatic increase in plasma TFPIalpha, resulting in a bleeding disorder.


Accelerates the ability of FIXa to rapidly convert FX to FXa

Activated by thrombin (principal) and FXa (also principal)

Activity downregulated spontaneously, by APC, FXa or FIXa

Factor VIII (antihemophilic factor) was first discovered in 1937, but it was not until 1979 that its purification by Tuddenham and coworkers led to the molecular identification of the protein. The mature factor VIII procofactor comprises 2332 amino acids and circulates in a high-affinity complex with its carrier protein VWF at a concentration of approximately 0.7 nM and a circulatory half-life of 8 to 12 hours. Complex formation with VWF protects factor VIII from proteolytic degradation, premature ligand binding, and rapid clearance from the circulation.

The primary source of factor VIII is the liver, but extrahepatic synthesis of factor VIII also occurs. While contradictory evidence exists on the cellular origin of both hepatic and extrahepatic factor VIII synthesis, recent studies in mice support that endothelial cells from many tissues and vascular beds synthesize factor VIII, with a large contribution from hepatic sinusoidal endothelial cells. This is consistent with observations on factor VIII expression in human endothelial cells from the liver and lung.

Screen Shot 2016-05-29 at 8.22.42 PMThe A1-A2-B-A3-C1-C2 domain structure of factor VIII shares significant homology with factor V except in the B domain region. In contrast to factor V, factor VIII B domain is dispensable for procoagulant activity. The C-terminal regions of the A1 and A2 domians and the N-terminal portion of the A3 domain contain short segments of 30 to 40 negatively charged residues known as the a1, a2, and a3 regions. Interaction with VWF is faciliated by the a3 region and C1 domain. The C domains mediate binding to the anionic phospholipid surface, thereby localizing factor VIII to the site of injury and facilitating interaction with factor IXa and factor X.

Factor VIII is heavily glycosylated and the majority of the N-linked glycosylation sites are found in the B domain, which mediate interaction with the chaperons calnexin and calreticulin and, in part, with the LMAN1-MCDF2 receptor complex. Sulfation of tyrosin residues is required for optimal activation by thrombin, maximal activity in complex with factor IXa, and maximal affinity of factor VIIIa for VWF.

Thrombin and factor Xa are the principal activators of the procofactor VIII and generate the cofactor VIIIa through sequential proteolysis at Arg740, Arg372, and Arg1689. The heterotrimeric factor VIIIa is composed of the A1, A2, and the A3-C1-C2 light chain subunits. The A1 and A3-C1-C2 subunites are noncovalently linked through calcium ions, whereas A2 is associated with weak affinity primarily by electrostatic interactions. Once activated, factor VIIIa functions as a cofactor for factor IXa in the phospholipid-dependent conversion of factor X to factor Xa. The rapid and spontaneous loss of factor VIIIa activity is attributed to A2 domain dissociation from the heterotrimer. Additional proteolysis by APC, factor Xa, or factor IXa also results in the downregulation of factor VIIIa cofactor activity.

The Soluble Cofactors Protein S and Von Willebrand Factor

Protein S

Protein S is a vitamin K-dependent single-chain GP of 635 amino acids that circulates with a plasma half-life of 42 hours. Part of the total protein S pool circulates in a free form at a concentration of 150 nM, whereas the majority (~60%; 200 nM) circulates bound to the complement regulatory protein C4b-binding protein (C4BP). Protein S is primarily synthesized in the liver by hepatocytes, in addition to endothelial cells, megakaryocytes, testicular Leydig cells, and osteoblasts.

The protein structure of protein S differs from the other vitamin K-dependent proteins as it lacks a serine protease domain and, consequently, is not capable of catalytic activity. Protein S is composed of a Gla domain comprising 11 Gla residues, a thrombin-sensitive region (TSR), four EGF domains, and a C-terminal sex hormone-binding globulin (SHBG)-like region that consists of two laminin G-type domains. The SHBG-like domain is involved in the interaction with beta-subunit of C4BP.

Apart from gamma-carboxylation of Glu residues, protein S is posttranslationally modified via N-glycosylation in the second laminin G-type domain of the SHBG-like region. beta-Hydroxylation of Asp95 or Asn residues in each EGF domain allows for calcium binding that orients the four EGF domains relative to each other.

Free protein S serves as a cofactor for APC in the proteolytic inactivation of FVa and FVIIIa. Interaction of protein S with APC on a negatively charged membrane surface alters the location of the APC active site relative to FVa, which accounts for the selective protein S-dependent rate enhancement of APC cleavage at Arg306 in FVa. C4BP-bound protein S also exerts a similar stimulatory effect on Arg306 cleavage, albeit to lower extent, whereas it inhibits the initial APC-mediated FVa cleavage at Arg506, resulting in an overall inhibition of FVa inactivation. Cleavage of the TSR by thrombin and/or FXa results in a loss of APC-cofactor activity. Protein S also functions as a cofactor for TFPIalpha in the inhibition of factor Xa, which is mediated by the SHBG-like region in protein S.


VWF is a large multimeric GP that is required for normal platelet adhesion to components of the vessel wall and that serves as a carrier for factor VIII. It is exclusively synthesized in megakaryocytes and endothelial cells and stored in specialized organelles in platelets and endothelial cells. VWF multimers circulate at a concentration of 10 nM with a half-life of 8 to 12 hours. Clearance of VWF multimers is mainly mediated by macrophages from the liver and spleen.

Large VWF multimers are cleaved by the plasma protease ADAMTS-13. This cleavage produces the smaller size VWF multimers that circulate in plasma. Reduced ADAMTS-13 activity is linked to various microangiopathies with increased platelet activity.

The precursor protein of VWF is composed of a 22-residue signal peptide and of a proVWF protein comprising 2791 amino acids that has 14 distinct domains. Upon translocation to the ER, the signal peptide is cleaved off, and the proVWF dimerizes in a tail-to-tail fashion through cysteines in its cysteine knot (CK) domain. During transit through the Golgi apparatus, proVWF dimers multimerize in a head-to-head fashion through the formation of disulfide bonds between cysteine residues in the D3 domain. At the same time, D1 and D2 domains are cleaved off as a single fragment to form the VWF propeptide (741 amino acids), while the remaining domains comprising 2050 amino acid residues and up to 22 carbohydrate chains form mature VWF. In the trans-Golgi network, the VWF propeptide promotes mature VWF to assemble into high-molecular-weight multimers. These multimers subsequently aggregate into tubular structures that are packaged into alpha-granules in megakaryocytes and into Weibel-Palade bodies in endothelial cells.

Upon exocytosis from Weibel-Palade bodies and at high shear rates, multimeric VWF unrolls from a globular to a filamental conformation, up to many microns long, which becomes a high-affinity surface for the platelet GPIb-V-IX complex. Large VWF multimers are more active than smaller multimers, which is explained by the fact that the former contain multiple domains that support the interactions between platelets, endothelial cells, and subendothelial collagen. VWF binds to matrix collagens via its A1 and A3 domains. The A1 domain also mediates binding to platelet GPIb, which is required for the fast capture of platelets. Platelet adhesion to VWF is further supported by VWF immobilization on surface and by high shear stress.

VWF complexes with factor VIII through the first 272 residues in the N-terminal region of the mature VWF protein subunit, thereby protecting factor VIII from proteolytic degradation, premature ligand binding, and rapid dlearance from the circulation.

Factor XI and The Contact System


Activates FIX with cofactor HK (calicum-dependent, phospholipid-independent)

Activated by FXIIa, thrombin, and FXIa

Inhibited by nexin 1, nexin 2, antithrombin, C1-inhibitor, alpha1-protease inhibitor, protein Z-dependent protease inhibitor, and alpha2-antiplasmin

Screen Shot 2016-06-11 at 2.57.05 PMFactor XI is synthesized in the liver and secreted as a single-chain zymogen of 607 amino acids. In the circulation, FXI is found as a homodimer at a concentration of 30 nM with a plasma half-life of 60 to 80 hours. All FXI homodimers circulate in complex with high-molecular-weight kininogen (HK). HK is thought to mediate binding of factor XI to negatively charged surfaces, thereby facilitating factor XI activation.

Each FXI subunit comprises four apple domians and a serine protease domain. The apple domains are structured by three disulfide bonds and form a disk-like platform on which the serine protease domain rests. The dimerization of two FXI subunits is mediated by interactions between the two apple 4 domains that involve a disulfide bond between the Cys321 residues, hydrophobic interactions, and a salt bridge, of which only the latter two are required for dimerization. The domain structure of FXI is highly similar to that of the monomer prekallikrein (PK), the zymogen of the protease kallikrein, which also circulates in complex with HK.

FXI does not bear a Gla domain and thus does not require gamma-carboxylation to exert its procoagulant activity.

Activation of a FXI subunit to FXIa proceeds through proteolysis at Arg369 in the N-terminal region of the serine protease domain and yields two-chain activated factor XIa. There are several catalysts capable of FXI activation, which include the contact factor XIIa, thrombin, or factor XIa itself in the presence of negatively charged surfaces. FXI must be a dimer to be activated by FXIIa, whereas thrombin and factor XIa lack this requirement.

Following activation of FXI, binding sites for the substrate FIX become available in the apple 3 domain and serine protease domian of factor XIa. FXIa proteolytically activates FIX to factor FIXa in a calcium-dependent but phospholipid-independent manner. Both forms of the FXIa dimer as well as monomeric FXIa activate FIX in a similar manner.

Accumulating evidence supports the notion that FXIIa-dependent activation of FXI is not essential to normal hemostasis, but is important in pathologic thrombus formation. Thrombin-mediated activation of FXI, on the other hand, seems most significant under conditions of low tissue factor and is assumed to enhance clot stability thorugh thrombin-activation of TAFI.

FXIa function is regulated by the serpins protease nexin 1, antithrombin, C1-inhibitor, alpha1-protease inhibitor, protein Z-dependent protease inhibitor, and alpha2-antiplasmin. Platelets also contain a FXIa inhibitor, the Kunitz-type inhibitor protease nexin 2.

The Contact System: FXII, Prekallikrein, and High-Molecular Weight Kininogen

FXII, HK, and PK are part of the contact system in blood coagulation, which is triggered following contact activation of FXII mediated via negatively charged surface.


Activates FXII

Activated by FXIIa

PK is synthesized in the liver, circulates as a zymogen, and is highly homologous to FXI. Conversion into the serine protease proceeds through limited proteolysis by FXIIa, and the generated kallikrein reciprocally activates more FXII.


HK, which is also synthesized in the liver, is a nonenzumatic cofactor that circulates in complex with FXI, which then activates FIX.

Contact system

The contact system is at the basis of the activated partial thromboplastin time (APTT) assay that is widely used in clinical practice. In this clinical laboratory test, the negatively charged surface is provided by reagents. FXIIa activates FXI, which then activates FIX. Despite HK and PK being required for a normal APTT, they appear to be dispensable for coagulation in vivo. Individuals who are deficient in any of these factors do not have a bleeding tendency, even after significant trauma or surgery.


Activates FXI, PK

Activated by negatively charged surface (platelet polyphosphate, microparticles derived from platelets and erythrocytes, RNA, and colalgen), kallikrein

Inhibited by serpin C1 inhibitor, antithrombin, and PAI-1

FXII, which is homologous to plasminogen activators, consists of an N-terminal fibronectin type I domain, an EGF-like domain, a fibronectin type II domain, a second EGF-like domain, a kringle domain, a proline-rich region, and a C-terminal serine protease domain. The proline-rich region is unique to FXII, as it is not found in any of the other serine proteases.

Limited proteolysis by kallikrein at Arg353 in FXII yields the activated two-chain alpha-FXIIa. Once activated, alpha-FXIIa activates FXI to FXIa. Furthermore, alpha-FXIIa activates PK, thereby contributing to its own feedback activation. Also FXI is known to acquire alpha-FXIIa activity upon contact with a negatively charged surface, the latter inducing a conformation change in FXII. This conformational change induces a limited amount of proteolytic activity in FXII, known as auto-activation. Furthermore, the surface-induced active conformation of FXII is suggested to enhance the proteolytic conversion to alpha-FXIIa. The fibronectin type I and II domains, EGF-2, the kringle domain, and the proline-rich region are reported to conribute to interaction with a negatively charged surface.

Further cleavage of alpha-FXIIa by kallikrein at Arg334 and Arg343 in the light chain results in the generation of beta-FXIIa, which comprises a nine-residue heavy-chain fragment that is disulfide-linked to the light chain. Given the absence of the heavy chain, beta-FXIIa does not interact with anionic surfaces. Even though beta-FXIIa is still capable of activating PK, it is no longer activates FXI.

The serpin C1 inhibitor is the main plasm inhibitor of alpha-FXIIa and beta-FXIIa. In addition, antithrombin (AT) and PAI-1 also inhibit FXIIa activity.

Revised Model of Coagulation

Screen Shot 2016-07-14 at 6.11.20 PM


Hemostasis is the process through which bleeding is controlled at a site of damaged vascular endothelium and represents a dynamic interplay between the subendothelium, endotheliumcirculating cells, and plasma proteins. The hemostatic process often is divided into three phases: the vascular, platelet, and plasma phases. Although it is helpful to divide coagulation into these phases for didactic purposes, in vivo, they are intimately linked and occur in a continuum.

The vascular phase is mediated by the release of locally active vasoactive agents that result in vasoconstriction at the site of injury and reduced blood flow. Vascular injury exposes the underlying subendothelium and procoagulant proteins, including von Willebrand factor (vWF), collagenn and tissue factor (TF) that then come into contact with blood.

During the platelet phase, platelets bind to vWF incorporated into the subendothelial matrix thorugh their expression of glycoprotein Iba (GPIbalpha). Platelets bound to vWF form a layer across the exposed subendothelium, a process termed platelet adhesion, and subsequently are activated via receptors, such as collagen receptors integrin alpha2beta1 and glycoprotein (GPVI), resulting in calcium mobilization, granule release, activation of the fibrinogen receptor, integrin alpha(IIb)b3, and subsequent platelet aggregation.

The plasma phase of coagulation can be further subdivided into initiation, priming, and propagation. Initiation begins when vascular injury also leads to exposure of TF in the subendothelium and on damaged endothelial cells. TF binds to the small amounts of circulating activated factor VII (FVIIa), resulting in formation of teh TF:FVIIa complex (extrinsic tenase complex); this complex binds to and activates factor X (FX) to activated FX (FXa). The TF:FVIIa:FXa complex converts a small amount of prothrombin (factor II/FII) to thrombin (activated factor II/FIIa). This small amount of thrombin is able to initiate coagulation and generate an amplification loop by cleaving factor VIII (FVIII) from vWF, activating clotting factors FVIII, XI (FXI), and platelets, which result in exposure of membrane phospholipids and release of partially activated factor V (FVa). At the end of the initiation and priming phases, the platelet is primed with an exposed phospholipid surface with bound activated cofactors (FVa and FVIIIa).

During the propagation phase, FIXa, generated either by the action of FXIa on the platelet surface or TF-VIIa complex on the TF bearing cell, bind to its cofactor, FVIIIa, to form the potent intrinsic tenase complex. FX is then bound and cleaved by the intrinsic tenase complex (FIXa:FVIIIa) leading to large amounts of FXa, which in association with its cofactor, FVa, forms the prothrombinase complex on the activated platelet surface. The prothrombinase complex (FXa:FVa) then binds and cleaves prothrombin leading to an ultimate burst of thrombin sufficient to convert fibrinogen to fibrin and to result in subsequent clot formation. The formed clot is stabilized by the thrombin-mediated activation of factor XIII (FXIII), which acts to cross-link fibrin, and thrombin-activatable fibrinolysis inhibitor (TAFI), which acts to remove lysine residues from the fibrin clot, thereby limiting plasmin binding. Utimately, the clot undergoes fibrinolysis, resulting in the restoration of normal blood vessel architecture. The fibrinolytic process is initiated by the release of tissue plasminogen activator (tPA) near the site of injury. tPA converts plasminogen to plasmin, which (via interactions with lysine and arginine residues on fibrin) cleaves the fibrin into dissolvable fragments.


Hemostasis – Plasma Phase – Initiation

  • FX > FXa via TF:FVIIa complex
  • FII > FIIa (small amount) via TF:FVIIa:FXa complex

Hemostasis – Plasma Phase – Priming

  • FVIII:vWF complex > FVIII + vWF via FIIa (small amount)
  • FVIII > FVIIIa via FIIa (small amount)
  • FXI > FXIa via FIIa (small amount)
  • Platelet > activated platelet via FIIa (small amount)
  • Activated platelet > partially activated FV via release

Hemostasis – Plasma Phase – Propagation

  • FIX > FIXa via FXIa or TF:VII complex
  • FX > FXa (large amount) via FIXa:FVIIIa complex
  • FII > FIIa (large amount) via FXa:FVa complex
  • FXIII > FXIIIa via FIIa (large amount)

Regulation of Hemostasis

Both the hemostatic and fibrinoglytic processes are regulated by inhibitors that limit coagulation at the site of injury and quench the reactions, thereby preventing systemic activation and pathologic propagation of coagulation. The hemostatic system has three main inhibitory pathways: antihrombin (AT), the protein C:protein S complex, and tissue factor pathway inhibitor (TFPI).

AT/Antithrombin target at thrombin and FXa

AT (antithrombin) released at the margins of endothelial injury binds in a 1:1 complex with thrombin, inactivating thrombin not bound by the developing clot. Antithrombin also rapidly inactivates FXa; thus, any excess FXa generated by the TF:VIIa complex during initiation is inactivated and unable to migrate to the activated platelet surface.

Thrombomodulin, protein C and S, target at FVa and FVIIIa

Excess free thrombin at the clot margins binds to thrombomodulin, a receptor expressed on the surface of intact endothelial cells that when complexed with thrombin activates protein C; activated protein C complexes with its cofactor protein S and inactivates FVa and FVIIIa.

Tissue factor pathway inhibitor/TFPI, target at TF:FVIIa and FXa

TFPI is a protein produced by endothelial cells that inhibits the TF:FVIIa complex and FXa. Binding to FXa is required for the inhibitory effect on TF:FVIIa. This negative feedback results in reduced subsequent thrombin generation and quenching of fibrin generation. The action of both AT and TFPI inhibits FXa during the initiation phase leading to dependence of platelet-surface FXa generation during the propagation phase for adequate hemostasis.


The fibrinolytic system also includes two inhibitors, principally plasminogen activator inhibitor-1 (PAI-1), and alpha2-antiplasmin (alpha2AP), which inhibit tPA and plasmin, respectively.

ASH Guideline for RBC Transfusion

July 13, 2016 Critical Care, Hematology, Transfusion No comments , , , , , ,

The Guideline

The development of clinical practice guidelines for RBC transfusion has been challenged by a limited availability of high-quanlity evidence to support practice recommendations. There is general agreement that RBC transfusion is typically not indicated for hemoglobin (Hgb) levels of >10 g/dL and that transfusion of RBCs should be considered when Hb is <7 to 8 g/dL depending on patient characteristics. The decision to transfuse RBCs should be based on a clinical assessment of the patient that weighs the risks associated with transfusion aganist the anticipated benefit. As more studies addressing RBC transfusion become available, it becomes increasely clear that liberal transfusion strategies are not necessarily associated with superior outcomes and may expose patients to unnecessary risks.

The most recently published guidelines from the AABB (formerly the American Association of Blood Bank) are based on a systematic review of randomized, controlled trials evaluating transfusion thresholds. These guidelines recommend adhering to a restrictive transfusion stratety and consider transfusion when Hb is 7 to 8 g/dL in hospitalized, stable patients. This strong recommendation is based on high-quality evidence from clinical trials comparing outcomes in liberal versus restrictive transfusion strategies in this patient population. A restrictive transfusion strategy is also recommended for patients with preexisting cardiovascular disease. In this population, transfusion should be considered when Hb levels are <8 g/dL or for symptoms such as chest pain, orthostatic hypotension, tachycardia unresponsive to fluid resuscitation, or congestive heart failure. This weak recommendation is based on moderate-quality evidence due to limited clinical trial data directly addressing this population of patients. Additional clinical practice guidelines exist that specify Hb targets for critical care patients with conditions including sepsis, ischemic stroke, and acute coronary syndrome.

RBC transfusion is indicated in patients who are actively bleeding and should be based on clinical assessment of the patient in addition to laboratory testing. Much remains to be learned about the optimal resuscitation of the bleeding patient. However, a recent study examining transfusion in patients with active upper gastrointestinal bleeding showed superior outcomes in patients treated with a restrictive transfusion strategy (<7 g/dL).

The Physiologic Response to Anemia

The initial response to anemia is a shift in the oxygen dissociation curve to the right as modulated by an increase in production of 2,3-DPG in RBCs. This shift allows for the unloading of oxygen to the tissues at higher partial pressures of oxygen, ensuring adequate oxygen delivery despite the reduction in RBC mass.

As anemia progresses, the cardiac output will increase by an increase in the heart rate to preserve the delivery of oxygen in the setting of decreased oxygen content. As RBC mass is reduced in anemia, the viscosity of the blood decreases. This reduction in viscosity leads to an increase in regional blood flow at the tissue and organ level, driving up local perfusion area and pressures leading to increased oxygen extraction. While a change in viscosity may be the trigger for increased regional blood flow, there has been suggestion that local blood vessel dilatation may be mediated by the release of nitric oxide (NO) from the RBCs. In order for these mechanisms to work properly, the patient must be at or near a euvolemic state. In considering these regulatory mechanisms, it is important to understand that the transfusion of RBCs will incease viscosity by adding stored RBCs that may not have the same vasoactive capabilities of native RBCs. As such, a transfusion of RBCs may inhibit compensatory mechanisms for low oxygen states, without significiantly increasing oxygen delivery.

There is evidence that low levels of Hb can be tolerated in healthy subjects. Hematocrits of 10% to 20% have been achieved in experimental studies using normovolemic hemodilution without untoward effects. Weiskopf and colleagues studied patients who underwent isovolemic reduction of Hb to 7, 6, and 5 g/dL. No evidence of reduced oxygen delivery was detected at any of the tested values of Hb; however, there was a subtle reversible reduction in reaction time and impaired immediate and delayed memory observed at Hb below 6 g/dL. An important source of data regarding the impact of anemia on surgical outcome comes from studies of Jehovah's Witness patients. Carson has demonstrated that the risk of death in these pateints at Hb between 7 and 8 g/dL is low. However, the odds of death increase by 2.5 for each gram decrease in Hb below 8 g/dL. The mortality is very high at Hb levels below 5 g/dL. It should be noted that these data are from patients who refuse all RBC transfusions. There is time to intervene between a low Hb and resulting morbidity or mortality in most patients.