Adhesion

Hemostasis Mechanism – Platelet Structure and Function

August 24, 2016 Cardiology, Hematology No comments , , , , , , , , , , , , , , , , , , , , , ,

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
  • 𝛼IIb𝛽3 [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, 𝛼IIb𝛽3, ð›¼2𝛽1, ð›¼5𝛽1, ð›¼6𝛽1
  • Interaction of GPVI with collagen activates platelet intergrins
  • At low shear conditions, fibrinogen is the primary ligand (interacting with 𝛼IIb𝛽3), 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 𝛼IIb𝛽3, 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 𝛼IIb𝛽3, 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 𝛼IIb𝛽3, 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 𝛼IIb𝛽3, 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

Screen Shot 2016-08-18 at 4.01.30 PMThe interaction of the platelet GPIb "complex" (the polypeptides GPIb𝛼, GPIb𝛽, 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 GPIb 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 𝛼IIb𝛽3. 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 𝛼IIb𝛽3 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 𝛼IIb𝛽3.

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, ð›¼2b𝛽1
  • 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

Screen Shot 2016-08-21 at 12.46.55 PMPARs 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 Gq and Gi, 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 Gq 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 𝛼IIb𝛽3; 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 Gs 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: 𝛼IIb𝛽3 (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 ð›¼IIb𝛽3 for its ligands. Fibrinogen and vWF play dominant roles in platelet aggregation through binding to ð›¼IIb𝛽3, and also by the ability of the former to generate polymerized fibrin as support for the platelets in a thrombus.

The signaling pathways of 𝛼IIb𝛽3 are 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 𝛼IIb𝛽3 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 𝛼IIb𝛽3), 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 , , , , , , , , , , , ,

1163px-Flag_of_the_Commandant_of_the_United_States_Marine_Corps.svgInflammation 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.


Screen Shot 2015-11-11 at 7.15.10 PMTissue 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.

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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

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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.

Infectious Diseases – Sites of Infections and the Virulence

September 14, 2014 Infectious Diseases, Physiology and Pathophysiology No comments , , , ,

CaduceusThe topics of sites of infections and virulence are two fundamental content in the discipline of infectious diseases. As a pharmacist and clinician we have to read and understand them, which can direct our clinical practice at the right direction.

Site of Infection

The type of pathogen (the number of infectious agents absorbed by the host and the virulence of the pathogen), the portal of entry, and the competence of the host’s immunologic defense system ultimately determine the site of an infectious disease. Some pathogens cause local infectious diseases, some tend to disseminate (though the circulatory system) from the primary site of infection to involve other locations and organ systems, which we call systemic infections. In particular situation, the abscess or pus, is a purulent exudate or a localized pocket rich in leukocytes (mostly neutrophils), devitalized tissue and debris of dead cells, and in many cases, microbes.

In this case, the dissemination of the pathogen has been contained by the host, but white cell function within the toxic environment of the abscess is hampered, and the elimination of microorganisms is slowed if not actually stopped. Similarly, infections of biomedical implants such as catheters, artificial heart valves, and prosthetic bone implants are seldom cured by the host’s immune response and antimicrobial therapy (due to the biofilm formed on these devices), which necessitates the removal of the device.

Virulence

Virulence factors are substances or products generated by infectious agents that enhance their ability to cause disease. Generally, virulence factors can be summarized into four primary categories including toxins, adhesion factors, evasive factors, and invasive factors.

Toxins

Toxins are substances that alter or destroy the normal function of the host or host’s cells. Toxin production is a trait chiefly monopolized by bacterial pathogens, although certain fungal and protozoan pathogens also elaborate substances toxic to humans. Bacterial toxins have diverse spectrum of activity and exert their effects on a wide variety of host target cells. For classification purposes, however, the bacterial toxins can be divided into two main types: exotoxins, and endotoxins.

Exotoxins are proteins released from the bacterial cell during growth. Bacterial exotoxins enzymatically inactivate or modify key cellular constituents, leading to cell death or dysfunction. Diphtheria toxin, for example, inhibits cellular protein synthesis; botulism toxin decreases the release of neurotransmitter from cholinergic neurons, causing flaccid paralysis; tetanus toxin decreases the release of neurotransmitter from inhibitory neurons, producing spastic paralysis; and cholera toxin induces fluid secretion into the lumen of the intestine, causing diarrhea. Other examples of exotoxin-induced diseases include pertussis (whooping cough), anthrax, traveler’s diarrhea, toxic shock syndrome, and a host of food-borne illnesses (i.e., food poisoning).

In contrast to exotoxins, endotoxins do not contain protein, are not actively released from the bacterium during growth, and have no enzymatic activity. Rather, endotoxins are complex molecules composed of lipid and polysaccharides found in the cell wall of gram-negative bacteria. Studies of different endotoxins have indicated that the lipid portion of the endotoxin confers the toxic properties to the molecule. Endotoxins are potent activators of a number of regulatory systems in humans. A small amount of endotoxin in the circulatory system (endotoxemia) can induce clotting bleeding, inflammation, hypotension, and fever.

Adhesion Factors

No interaction between microorganisms and humans can progress to infection or disease if the pathogen is unable to attach and colonize the host. The process of microbial attachment may be site specific (e.g., mucous membranes, skin surfaces), cell specific (e.g., T lymphocytes, respiratory, epithelium, intestinal epithelium), or nonspecific (e.g., moist areas, charged surfaces). In any of these cases, adhesion requires a positive interaction between the surfaces of host cells and the infectious agent.

The site to which microorganisms adhere is called a receptor, and the reciprocal molecule or substance that binds to the receptor is called a ligand or adhesin. Receptors may be proteins, carbohydrates, lipids, or complex molecules composed of all three. Similarly, ligands may be simple or complex molecules and, in some cases, highly specific structures. After initial attachment, a number of bacterial agents become embedded in a gelatinous matrix of polysaccharides called a slime or mucous layer. The slime layer serves two purposes: It anchors the agent firmly to host tissue surfaces, and it protects the agent from the immunologic defenses of the host. Many viral agents produce filamentous appendages or spikes that recognize carbohydrate receptors on the surfaces of specific cells in human body.

Evasive Factors

A number of factors produced by pathogens enhance virulence by evading various components of the host’s immune system. The are briefly summarized below.

1.Extracellular polysaccharides, including capsules, slime, and mucous layer, discourage engulfment and killing of pathogens by the host’s phagocytic white blood cells.

2.Some pathogens can avoid phagocytosis by excreting leukocidin C toxins, which cause specific and lethal damage to the cell membrane of host neutrophils and macrophages, etc.

3.Some pahogens are adapted to survive and reproduce within phagocytic white blood cells after ingestion, avoiding or neutralizing the usually lethal products contained within the lysosomes of the cell. An extreme example is the Helicobacter pylori, which produces a urease enzyme on its outer cell wall. The urease converts gastric urea into ammonia, thus neutralizing the acidic environment of the stomach and allowing the organism to survive in this hostile environment.

4.Some pathogens evading immunologic surveillance have evolved ways to avoid recognition by host antibodies. Strains of S. aureus produce a surface protein (protein A) that immobilizes immunoglobulin G (IgG), holding the antigen-binding region harmlessly away from the organisms. Also, this pathogen secretes a unique enzyme called coagulase which converts soluble human coagulation factors into solid clot, which envelops and protects the organism from phagocytic host cells and antibodies.

5.Some agents secrete enzymes that cleave and inactivate secretory IgA, neutralizing the primary defense of the respiratory and genital tracts at the site of infection.

6.Some agents can alter surface antigens during the disease course so that the immunological detection has been avoided.

7.Some viruses, such as HIV, impair the function of immunoregulatory cells. Although this property increases the virulence of these agents, it is not considered a virulence factor in the true sense of the definition.

Invasive Factors

Invasive factors are products produced by infectious agents that facilitate the penetration of anatomic barriers and host tissue. Most invasive factors are enzymes capable of destroying cellular membranes (e.g., phospholipases), connective tissue (e.g., elastases, collagenases), intercellular matrices (e.g., hyaluronidase), and structural protein complexes (e.g., proteases).

At the end, I want to emphasize that it is the combined effects of these factors above, the amount of pathogen the host absorbing, and the antimicrobial and inflammatory substances released by host cells mediate the pathophysiology of the infectious diseases.