P2Y12

Overview of the Hemostasis System

March 15, 2017 Hematology, Physiology and Pathophysiology No comments , , , , , , , , , , , , , , , , , , , , ,

Procoagulant Pathways

Two procoagulant pathway have been identified which converge at the “intrinsic” (accessory) fXase (fIXa*fVIIIa) complex. The contact or “intrinsic” pathway is activated by the interaction of blood with a foreign surface. This pathway is activated by the factor XIIa-high-molecular-weight kininogen (HMWK)-prekallikrein complex in association with foreign surfaces including glass, dextran sulfate, or kaolin. The complex catalyst activates factor XI leading to the factor XIa-HMWK complex which activates fIX to fIXa. The “intrinsic” fXase rapidly cleaves fX to fXa.

Factor Xa is directly but less effectively produced by the “extrinsic” fXase, which is composed of plasma-derived fVIIa and tissue factor and expressed when the latter is exposed to blood. The “extrinsic” fXase also activates fIX to fIXa as the reaction progresses, with suppression of the quaternary complex occurring by TFPI. The “intrinsic” fXase complex is kinetically superior and ultimately produces the majority of fXa.

Since bleeding pathology is not ordinarily associated with defects of the initiation protein complexes of the contact pathway, most investigations conclude that the “intrinsic” pathway is not the primary provider for hemostasis. The “intrinsic” pathway, however, has been implicated in some forms of thrombosis. The primary (“extrinsic”) and accessory (“intrinsic”) pathways, initiated by independent routes, both lead to the activation of factor IX and converge at the “intrinsic” fXase complex. Regardless of the path, the outcome is the formation of the prothrombinase complex and thrombin generation. Each reaction of the primary pathway of coagulation involves the vitamin K-dependent zymogens and serine proteases, cofactor proteins, and Ca2+ ions assembled on membranes. The complexes display reaction rates 10^5 to 10^9 times greater than the respective serine proteases alone.

Clinical laboratory tests differentiate between the pathways. The activated partial thromboplastin time (aPTT) initiates coagulation through the accessory pathway, whereas the prothrombin time (PT) assay initiates coagulation through the primary pathway. The designations of primary and accessory pathways are based on clinical evidence of bleeding diseases. Deficiencies of proteins associated with the “intrinsic” or accessory pathway (factor XII, prekallikrein, and HMWK) exist but are not associated with abnormal bleeding events, even after surgical challenge. However, deficiencies of the protein components of the “extrinsic” or primary pathway (prothrombin and factors V, VII, VIII, IX, and X) can lead to severe bleeding diatheses. Factor XI deficiency may also result in bleeding episodes subsequent to trauma or surgery. The role of the accessory pathway is therefore not clearly understood.

Factor XII, prekallikrein, and HMWK are required for activity of the contact or accessory pathway and deficiencies are reported by the aPTT. Factor XII and prekallirein are zymogens that are activated to sarin proteases, while HMWK is a cofactor. The accessory pathway factors are hypothesized to play a role in disseminated intravascular coagulation (DIC) associated with the systemic inflammatory response syndrome, and may also be involved in the promotion of thrombus stability. The accessory pathway may also be important in cardiopulmonary bypass because of contact between blood components and synthetic surfaces.

The importance of the membrane component in coagulation was initially identified by kinetic studies of the prothrombinase complex. In the absence of the membrane surface, the cofactor (factor Va)-enzyme (factor Xa) interaction is relatively weak, with dissociation constant (Kd) of 800 nmol/L. The factor Va-lipid interaction (Kd = 3 nmol/L) and factor Xa-lipid interaction (Kd = 110 nmol/L) show higher affinity. However, all of the components must be present to generate the high-affinity factor Va-factor Xa-Ca2+ membrane complex, with a Kd of 1 nmol/L. The fully assembled complex is stabilized through factor Va-factor Xa, factor Va-lipid, and factor Xa-lipid interactions. Similar properties have been observed for the fIXa/fVIIIa, TF/fVIIa, and Tm/fIIa complexes.

The primary pathway of coagulation is initiated or triggered by the interaction of circulating factor VIIa with its cofactor tissue factor (TF). In general, the serine proteases associated with hemostasis circulate in their zymogen or inactive forms; however, low levels of circulate in their zymogen or inactive forms; however, low levels of circulating factor VIIa are present in blood. This factor VIIa binds to tissue factor expressed by pathology and initiates the procoagulant response. Free factor VIIa is a poor enzyme with virtually no proteolytic activity, but as a consequence is protected from interacting with the circulating inhibitors in the absence of tissue factor. Tissue factor, an integral membrane protein not normally expressed on vascular cell surfaces, is constitutively expressed on extravascular cellular surfaces and thus becomes exposed upon damage to the endothelial cell layer. Tissue factor is also expressed on peripheral blood cells and endothelial cells stimulated by inflammatory cytokines.

Upon interaction of plasma factor VIIa and the injury/pathology present, tissue factor, the “extrinsic” fXase complex (factor VIIa-tissue factor) is formed and initiates coagulation by activating factor IX and X. Factor IXa forms a complex with its cofactor, factor VIIIa, to generate the “intrinsic” fXase complex, and factor Xa combines with factor Va to form the prothrombinase complex. The factor VIIIa-factor IXa complex more efficiently activates factor X to factor Xa, providing a robust source of the enzyme component (factor Xa) of the prothrombinase complex.

Deficiencies of the “intrinsic” fXase components factor VIIIa (hemophilia A) and factor IXa (hemophilia B) illustrate the significance of the factor IX activation by the fVIIa/TF complex and the enhanced rate of factor X activation by the intrinsic fXase and the inhibition of the extrinsic fXase by TFPI. Hemophilia A and B are detected using the aPTT. It would appear activation of factor X by the extrinsic fXase should compensate for the lack of factor activation by the intrinsic fXase in hemophilias A and B. However, this compensatory mechanism only occurs during clinical administration of supraphysiologic concentrations of recombinant factor VIIa during replacement therapy for hemophilia with inhibitors. The natural physiologic levels of factor VIIa are not able to provide sufficient levels of factor Xa to support normal coagulation. Factor Xa generation is suppressed to approximately one-half the level observed when factor X is the only substrate presented.

Factor IX, not factor X, appears to be the preferred substrate of the extrinsic fXase. In addition, factor IXalpha, the intermediate species in factor IX activation, is generated more rapidly in the presence of factor X. Factor IXalpha activation to the final product factor IXa occurs at a higher rate than factor IX activation, thereby providing a burst of factor IXa to form the intrinsic fXase complex. The low level of factor Xa generated by the tissue factor-factor VIIa complex most likely functions in the activation of factor IX. A model of extrinsic fXase behavior suggests that factor IX is converted to factor IXalpha by the extrinsic fXase or factor Xa-phospholipid complex. Factor IXalpha is then rapidly converted to factor IXa by the extrinsic fXase. The factor VIIIa-factor IXa complex subsequently activates the major fraction of factor X to factor Xa and provides the enzyme component for the prothrombinase complex. Measurements of second-order rate constants for factor Xa generation by the intrinsic and extrinsic fXase complexes also support this model. The rate of factor Xa generation by the tissue factor-factor VIIa complex is 1/50th the rate of factor Xa generation by the factor VIIIa-factor IXa complex. Both complexes thus have distinct roles in the procoagulant response.

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Accessory Pathway

The procoagulant proteins that make up the intrinsic or accessory pathway consist of factor XII, plasma prekallikrein, HMWK, and factor XI. These proteins are responsible for the contact activation of blood coagulation. The physiologic role of the intrinsic pathway is not clearly understood, but does not appear to be essential for hemostasis because individuals deficient in factor XII, plasma prekallirein, or HMWK do not manifest abnormal bleeding. Factor XI appears to play a more prominent role, unrelated to its activities in the intrinsic pathway, after activation by thrombin.

Update on March 15 2017

Hemostatic System

The major components of the hemostatic system are the vascular endothelium, platelets, and the coagulation and fibrinolytic systems.

Vascular Endothelium

A monolayer of endothelial cells lines the intimal surface of the circulatory tree and separates the blood from the prothrombotic sub endothelial components of the vessel wall. As such, the vascular endothelium encompasses about 10^13 cells and covers a vast surface area. Rather than serving as a static barrier, the healthy vascular endothelium is a dynamic organ that actively regulates hemostasis by inhibiting platelets, suppressing coagulation, promoting fibrinolysis, and modulating vascular tone and permeability.

Platelet Inhibition

Endothelial cells synthesize prostacyclin and nitric oxide and release them into the blood.These agents not only serve as potent vasodilators but also inhibit platelet activation and subsequent aggregation by stimulating adenylate cyclase and increasing intracellular levels of cyclic adenosine monophosphate (cAMP). In addition, endothelial cells express (CD39) on their surface, a membrane-associated ecto-adenosine diphosphatase (ADPase). By degrading ADP, which is a platelet agonist, CD39 attenuates platelet activation.

  • Prostacyclin
  • Nitric oxide
  • CD39

Anticoagulant Activity

Intact endothelial cells play an essential part in the regulation of thrombin generation through a variety of mechanisms. Endothelial cells produce heparan sulfate proteoglycans, which bind circulating antithrombin and accelerate the rate at which it inhibits thrombin and other coagulation enzymes. Tissue factor pathway inhibitor (TFPI), a naturally occurring inhibitor of coagulation, binds heparan sulfate on the endothelia cell surface. Administration of heparin or low-molecular-weight heparin (LMWH) displaces glycosaminoglycan-bound TFPI from the vascular endothelium, and released TFPI may contribute to the antithrombotic activity of these drugs.

Endothelial cells regulate thrombin generation by expressing thrombomodulin and endothelial cell protein C receptor (EPCR) on their surfaces. Thrombomodulin binds thrombin and alters this enzyme’s substrate specificity such that it no longer acts as a procoagulant but becomes a potent activator of protein C. Activated protein C serves as an anticoagulant by degrading and inactivating activated factor V (FVa) and factor VIII (FVIIIa), key cofactors involved in thrombin generation. Protein S acts as a cofactor in this reaction, and EPCR enhances this pathway by binding protein C and presenting it to the thrombin-thrombomodulin complex for activation. In addition to its role as an anticoagulant, activated protein C also regulates inflammation and preserves the barrier function of the endothelium.

  • Heparan
  • TFPI
  • Thrombomodulin
  • EPCR

Fibrinolytic Activity

The vascular endothelium promotes fibrinolysis by synthesizing and releasing tissue-type and urokinase-type plasminogen activator (t-PA and u-PA, respectively), which initiate fibrinolysis by converting plasminogen to plasmin. Endothelial cells in most vascular beds synthesize t-PA constitutively. In contrast, perturbed endothelial cells produce u-PA in the settings of inflammation and would repair.

Endothelial cell also produce type 1 plasminogen activator inhibitor 1 (PAI-1), the major regulator of both t-PA and u-PA. Therefore, net fibrinolytic activity depends on the dynamic balance between the release of plasminogen activators and PAI-1.  Fibrinolysis localizes to the endothelial cell surface because these cells express annexin II, a coreceptor for plasminogen and t-PA that promotes their interaction. Therefore, healthy vessels actively resist thrombosis and help maintain platelets in quiescent state.

  • t-PA
  • u-PA
  • PAI-1
  • Annexin II

Vascular Tone and Permeability

In addition to synthesizing potent vasodilators, such as prostacyclin and nitric oxide, endothelial cells also produce a group of counter-regulatory peptides known as endothelins that induce vasoconstriction. Endothelial cell permeability is influenced by the connections that join endothelial cells to their neighbors. Macromolecules traverse the endothelium via patent intercellular junctions, by endocytosis, or through transendothelial pores. Vasodilatation, servers thrombocytopenia, and high doses of heparin can increase endothelial permeability, which may contribute to bleeding. Activated protein C may also contribute to the barrier function of the endothelium.

Platelets

Platelets are anucleate particles released into the circulation after fragmentation of bone marrow megakaryocytes. Because they are anucleate, platelets have limited capacity to synthesize proteins. Thrombopoietin, a glycoprotein synthesized in the liver and kidneys, regulates megakaryocytic proliferation and maturation as well as platelet production. After they enter the circulation, platelet have a life span of 7 to 10 days.

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Damage to the intimal lining of the vessel exposes the underlying subendothelial matrix. Platelets home to sites of vascular disruption and adhere to the exposed matrix proteins. Adherent platelets undergo activation and not only release substances that recruit additional platelets to the site of injury but also promote thrombin generation and subsequent fibrin formation. A potent platelet agonist, thrombin amplifies platelet recruitment and activation. Activated platelets then aggregate to form a plug that seals the leak in the vasculature. An understanding of the steps in these highly integrated processes helps pinpoint the sites of action of the antiplatelet drugs and rationalizes the utility of anticoagulants for the treatment of arterial thrombosis and venous thrombosis.

Adhesion

Platelets adhere to exposed collagen and von Willebrand factor (vWF) and form a monolayer that supports and promotes thrombin generation and subsequent fibrin formation. These events depend on constitutively expressed receptors on the platelet surface, alpha2beta1 and glycoprotein (GP) VI, which bind collagen, and GPIbalpha and GPIIb/IIIa (alphaIIbbeta3), which bind vWF. The platelet surface is crowded with receptors, but those involved in adhesion are the most abundant: every platelet has about 40,000 to 80,000 copies of GPIIb/IIIa and 25,000 copies of GPIbalpha. Receptors cluster in cholesterol-enriched subdomains, which render them more mobile, thereby increasing the efficiency of platelet adhesion and subsequent activation.

  • Platelet receptors: alpha2beta1, GP VI, GPIbalpha, GPIIb/IIIa
  • Platelet receptors’ ligands: collagen (alpha2beta1, GP VI), vWF (GPIbalpha, GPIIb/IIIa)

Under low shear conditions, collagen can capture and activate platelets on its own. Captured platelets undergo cytoskeletal reorganization that causes them to flatten and to adhere more closely to the damaged vessel wall. Under high shear conditions, however, collagen and vWF must act in concert to support optimal platelet adhesion and activation. vWF synthesized by endothelial cells and megakaryocytes assembles into multimers that range from 550 to more than 10,000 kDa. When released from storage in the Weibel-Palade bodies of endothelial cells or the alpha-granules of platelets, most of the vWF enters the circulation, but the vWF released from the abluminal surface of endothelial cells accumulates in the subendothelial matrix, where it binds collagen via its A3 domain. This surface-immobilized vWF can simultaneously bind platelets via its A1 domain. In contrast, circulating vWF does not react with unstimulated platelets. This difference in reactivity likely reflects vWF conformation; whereas circulating vWF is in a coiled conformation that prevents access of its platelet-binding domain to vWF receptors on the platelet surface, immobilized vWF assumes an elongated shape that exposes its A1 domain. In this extended conformation, large vWF multimers serve as the molecular glue that tethers platelets to the damaged vessel wall with sufficient strength to withstand higher shear forces. Large vWF multimers provide additional binding sites for collagen and heighten platelet adhesion because platelets have more vWF results in platelet activation, the next step in platelet plug formation.

  • Low shear conditions
  • High shear conditions
  • Free vWF
  • Surface-immobilized vWF
  • Interaction: vWF and collagen

Activation and Secretion

Adhesion to collagen and vWF initiates signaling pathways that result in platelet activation. These pathway induce cyclooxyrgenase-1 (COX-1)-dependent synthesis and release of thromboxane A2, and trigger the release of adenosine diphosphate (ADP) from storage granules. Thromboxane A2 is a potent vasoconstrictor, and similar to ADP, locally activates ambient platelets and recruits them to the site of injury. This process results in expansion of the platelet plug. To activate platelets, thromboxane A2 and ADP must bind to their respective receptors on the platelet membrane. The thromboxane receptor (TP) is a G-protein coupled receptor that is found on platelets and on the endothelium, which explains why thromboxane A2 induces vasoconstriction as well as platelet activation. ADP interacts with a family of G protein-coupled receptors on the platelet membrane. Most important of these is P2Y12, which is the target of the theienopyridines, but P2Y1 also contributes to ADP-induced platelet activation, and maximal ADP-induced platelet activation requires activation of both receptors. A third ADP receptor, P2X1, is an adenosine triphosphate (ATP)-gated calcium channel. Platelet storage granules contain ATP as well as ADP; ATP released during the platelet activation process may contribute to the platelet recruitment process in a P2X1-dependent fashion.

  • TXA2
  • Thromboxane receptor
  • ADP
  • ADP receptors (P2Y12, P2Y1, P2X1)
  • Pathway: collagen and/or vWF > thromboxane A2 release, and ADP release

Although TP and the various ADP receptors signal through different pathways, they all trigger an increase in the intracellular calcium concentration in platelets. This in turn induces shape change via cytoskeletal rearrangement, granule mobilization and release, and subsequent platelet aggregation. Activated platelets promote coagulation by expressing phosphatidylserine on their surfaces, and anionic phospholipid that supports assembly of coagulation factor complexes. After being assembled, these clotting factor complexes trigger a burst of thrombin generation and subsequent fibrin formation. In addition to converting fibrinogen to fibrin, thrombin amplifies platelet recruitment and activation and promotes expansion of the platelet plug. Thrombin binds to protease-activated receptors types 1 and 4 (PAR1 and PAR4, respectively) on the platelet surface and cleaves their extended amino-termini, thereby generating new amino-termini that serve as tethered ligands that bind and activate the receptors. Whereas low concentrations of thrombin cleave PAR1, PAR4 cleavage requires higher thrombin concentrations. Cleavage of either receptor triggers platelet activation.

  • Pathway: thrombin (ligand), and PAR1 and PAR4 (receptors)

In addition to providing a surface on which clotting factors assemble, activated platelets also promote fibrin formation and subsequent stabilization by releasing factor V, factor XI, fibrinogen, and factor XIII. Thus, there is coordinated activation of platelets and coagulation, and the fibrin network that results from thrombin action helps anchor the platelet aggregates at the site of injury. Activated platelets also release adhesive proteins, such as vWF, thrombospondin, and fibronectin, which may augment platelet adhesion at sites of injury, as well as growth factors, such as platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGFbeta), which promote wound healing. Platelet aggregation is the final step in the formation of the platelet plug.

  • Secretion: platelet derived factor V, factor XI, fibrinogen, and factor XIII, vWF

Aggregation

Platelet aggregation links platelets to each other to form clumps. GPIIb/IIIa mediates these platelet-to-platelet linkages. On inactivated platelets, GPIIb/IIIa exhibits minimal affinity for its ligands. Upon platelet activation, GPIIb/IIIa undergoes a conformational transformation, which reflects transmission of inside-out signals from its cytoplasmic domain to its extracellular domain. This transformation enhances the affinity of GPIIb/IIIa for its ligands; fibrinogen; and, under high shear conditions, vWF. Arginine-glycine-aspartic acid (RGD) sequences located on fibrinogen and vWF, as well as platelet-binding lycine-glycine-aspartic acid (KGD) sequence on fibrinogen, mediate their interaction with GPIIb/IIIa. When subjected to high shear, circulating vWF elongates and exposes its platelet-binding domain, which enables its interaction with the conformationally activated GPIIb/IIIa. Divalent fibrinogen and multivalent vWF molecules serve as bridges and bind adjacent platelets together. After being bound to GPIIb/IIIa, fibrinogen and vWF induce outside-inside signals that augment platelet activation and result in the activation of additional GPIIb/IIIa receptors, creating a positive feedback loop. Because GPIIb/IIIa acts as the final effector in platelet aggregation, it is a logical target for potent antiplatelet drugs. Fibrin, the ultimate product of the coagulation system, tethers the platelet aggregates together and anchors them to the site of injury.

  • Platelet (activated) receptors: GPIIb/IIIb (fibrinogen, vWF)

Coagulation

Coagulation results in the generation of thrombin, which converts soluble fibrinogen to fibrin. Coagulation occurs through the action of discrete enzyme complexes, which are composed of a vitamin K-dependent enzyme and a non-enzyme cofactor, and assemble on anionic phospholipid membranes in a calcium-dependent fashion. Each enzyme complex activates a vitamin K-dependent substrate that becomes the enzyme component of the subsequent complex. Together, these complexes generate a small amount of thrombin, which amplifies its own generation by activating the non-enzyme cofactors and platelets, which then provide an anionic surface on which the complexes assemble. The three enzyme complexes involved in thrombin generation are extrinsic tenase, intrinsic tenase, and prothrombinase. Although extrinsic tenase initiates the system under most circumstances, the contact system also plays a role in some situations.

Extrinsic Tenase (FVIIa-TF complex)

This complex forms upon exposure of tissue factor-expressing cells to the blood. Tissue factor exposure occurs after atherosclerotic plaque rupture because the core of the plaque is rich in cells that express tissue factor. Denuding injury to the vessel wall also exposes tissue factor constitutively expressed by subendothelial fibroblasts and smooth muscle cells. In addition to cells in the vessel wall, circulating monocytes and monocyte-derived microparticles (small membrane fragments) also provide a source of tissue factor. When tissue factor-bearing monocytes or microparticles bind to platelets or other leukocytes and their plasma membranes fuse, tissue factor transfer occurs. By binding to adhesion molecules expressed on activated endothelial cells or to P-selectin on activated platelets, these tissue factor-bearing cells or microparticles can initiate or augment coagulation. This phenomenon likely explains how venous thrombi develop in the absence of obvious vessel wall injury.

Tissue factor is an integral membrane protein that serves as a receptor for factor VIIa. The blood contains trace amounts of factor VIIa, which has negligible activity in the absence of tissue factor. With tissue factor exposure on anionic cell surfaces, factor VIIa binds in a calcium-dependent fashion to form the extrinsic tenase complex, which is a potent activator of factor IX and X. After being activated, factor IXa and factor Xa serve as the enzyme components of intrinsic tenase and prothrombinase, respectively.

Intrinsic Tenase (FVIIIa-FIXa complex)

Factor IXa binds to factor VIIIa on anionic cell surfaces to form the intrinsic tenase complex. Factor VIII circulates in blood in complex with vWF. Thrombin cleaves factor VIII and releases it from vWF, converting it to its activated form. Activated platelets express binding sites for factor VIIIa. After being bound, factor VIIIa binds factor IXa in a calcium-dependent fashion to form the intrinsic tenase complex, which then activates factor X. The change in catalytic efficiency of factor IXa-mediated activation of factor X that occurs with deletion of individual components of the intrinsic tenase complex highlights their importance. Absence of the membrane or factor VIIIa almost completely abolishes enzymatic activity, and the catalytic efficiency of the complete complex is 10^9-fold greater than that of factor IX alone. Because intrinsic tenase activates factor X at a rate 50- to 100-fold faster than extrinsic tenase, it plays a critical role in the amplification of factor Xa and subsequent thrombin generation.

Prothrombinase (FXa-FVa complex)

Factor Xa binds to factor Va, its activated cofactor, on anionic phospholipid membrane surfaces to form the prothrombinase complex. Activated platelets release factor V from their alpha-granules, and this platelet-derived factor V may play a more important role in hemostasis than its plasma counterpart. Whereas plasma factor V requires thrombin activation to exert its cofactor activity, the partially activated factor V released from platelets already exhibits substantial cofactor activity. Activated platelets express specific factor Va binding sites on their surface, and bound factor Va serves as a receptor for factor Xa. The catalytic efficiency of factor Xa activation of prothrombin increase by 10^9-fold when factor Xa incorporates into the prothrombinase complex. Prothrombin binds to the prothrombinase complex, where it undergoes conversion to thrombin in a reaction that release prothrombin fragment 1.2. Plasma levels of F1.2, therefore, provide a marker of prothrombin activation.

Fibrin Formation

The final effector in coagulation is thrombin. Thrombin converts soluble fibrinogen into insoluble fibrin. Fibrinogen is a dimeric molecule, each half of which is composed of three polypeptide chains – the Aalpha, Bbeta, and gamma chains. Numerous disulfide bonds covalently link the chains together and join the two halves of the fibrinogen molecule. Electron micrographic studies of fibrinogen reveal a trinodular structure with a central E domain flanked by two D domains. Crystal structures show symmetry of design with the central E domain, which contains the amino termini of the fibrinogen chains, joined to the lateral D domains by coiled-coil regions.

Fibrinogen circulates in an inactive form. Thrombin binds to the amino termini of the Aalpha and Bbeta chains of fibrinogen, where it cleaves specific peptide bonds to release fibrinopeptide A and fibrinopeptide B and generates fibrin monomer. Because they are products of thrombin action on fibrinogen, plasma levels of these fibrinopeptides provide an index of thrombin activity. Fibrinopeptide release creates new amino termini that extend as knobs from the E domain of one fibrin monomer and insert into preformed holes in the D domains of other fibrin monomers. This creates long strands known as protofibrils, consisting of fibrin monomers noncovalently linked together in a half-staggered overlapping fashion.

Noncovalently linked fibrin protofibrils are unstable. The stability of the fibrin network is enhanced by platelets and procoagulant cells. Platelets not only bind fibrin via GPIIb/IIIa and promote the formation of a dense fibrin network, but the also release factor XIII. By covalently cross-linking the alpha and gamma chains of adjacent fibrin monomers, factor XIIIa stabilizes the fibrin in a calcium-dependent fashion and renders it relatively resistant to degradation. Factor XIII circulates in blood as a heterodimer consisting of pairs of A and B subunits. The active and calcium binding sites on factor XIII are localized to the A subunit. Platelets contain large amounts of factor XIII in their cytoplasms, but platelet-derived factor XIII consists only of the A subunits. Both plasma and platelet factor XIII are activated by thrombin.

Contact Pathway

Current thinking is that tissue factor exposure represents the sole pathway for activation of coagulation and that the contact system – which includes factor XII, prekallikrein, and high-molecular-weight kininogen – is unimportant for hemostasis because patients deficient in these factors do not have bleeding problems. The physiologic role of factor XI is more difficult to assess because the plasma level of factor XI does not predict the propensity for bleeding. Although the capacity of thrombin to feed back and activate platelet-bound factor XI may explain this phenomenon, platelet-derived factor XI may be more important for hemostasis than circulating factor XI.

We cannot ignore the contact pathway, however, because coronary catheters and other blood-contacting medical devices, such as stents or mechanical valves, likely trigger clotting through this mechanism. Factor XII bound to the surface of catheters or devices undergoes a conformational change that results in it activation. Factor XIIa converts prekallikrein to kallikrein in a reaction accelerated by high-molecular-weight kininogen, and factor XIIa and kallikrein then feedback to activate additional factor XII. Factor XIIa propagates coagulation by activating factor XI.

In addition to its role in device-related thrombosis, the contact pathway may also contribute to the stability of arterial and venous thrombi. DNA and RNA released from damaged cells in atherosclerotic plaques activates factor XII, and mice DNA- or RNA-degrading enzymes exhibit attenuated thrombosis at site of arterial injury. Polyphosphates released from activated platelets also activate factor XII and may provide another stimulus for contact pathway activation. Mice deficient in factor XII or factor XI form small unstable thrombi at sites of arterial or venous damage, suggesting that factor XII and factor XI contribute to thrombogenesis. It is unknown whether the same is true in humans. Patients with unstable angina have increased plasma levels of factor XIa, but it is unclear whether this reflects activation by factor XIIa or by thrombin. Although the contribution of the contact pathway to thrombin generation remains uncertain, the final product of coagulation is fibrin. Hemostasis depends on the dynamic balance between the formation of fibrin and its degradation.

Interactions Between Platelet and Coagulation System (Summary)

Platelet and coagulation system act in synergism to maintain hemostasis.

Platelet for coagulation system

  • Platelet provides membrane where clotting factors reactions could take place quickly
  • Platelet secrets FV (partially activated), FXI, fibrinogen, FXIII, and vWF
  • Polyphosphates released from activated platelets activate FXII

Coagulation system for platelet

  • Thrombin activates platelet via PAR-1 and PAR-4
  • Fibrinogen helps platelet binds adjacent platelet together

Update on Mar 25 2017

Fibrinolytic System

Fibrinolysis initiates when plasminogen activators convert plasminogen to plasmin, which then degrades fibrin into soluble fragments. Blood contains two immunologically and functionally distinct plasminogen activators, t-PA and u-PA. Whereas t-PA mediates intravascular fibrin degradation, u-PA binds to a specific u-PA receptor (u-PAR) on the surface of cells, where it activates cell-bound plasminogen. Consequently, pericellular proteolysis during cell migration and tissue remodeling and repair are the major functions of u-PA.

Regulation of fibrinolysis occurs at two levels. PAI-1, and to a lesser extent, PAI-2, inhibit the plasminogen activators, and alpha2-antiplasmin inhibits plasmin. Endothelial cells synthesize PAI-1, which inhibits both t-PA and u-PA, whereas monocytes and the placenta synthesize PAI-2, which specifically inhibits u-PA. Thrombin-activated fibrinolysis inhibitor (TAFI) also modulates fibrinolysis and provides a link between fibrinolysis and coagulation. Whereas thrombosis can occur if there is impaired activation of the fibrinolytic system, excessive activation leads to bleeding.

  • Plasminogen
  • t-PA
  • u-PA
  • PAI-1
  • PAI-2
  • TAFI

Mechanism of Action of Tissue Plasminogen Activator

Tissue plasminogen activator, a serine protease, contains five discrete domains: a fibronectin-like finger domain, and epidermal growth factor (EGF) domain, two kringle domains, and a protease domain. Synthesized as a single-chain polypeptide, plasmin readily converts t-PA into a two-chain form. Single- and two-chain forms of t-PA convert plasminogen to plasmin. Native Glu-plasminogen is a single-chain polypeptide with a Glue residue at its amino-terminus. Plasmin cleavage at the amino-terminus generates lysine-plasminogen, a truncated form with a Lys residue at its new amino terminus. t-PA cleaves a single peptide bond to convert single-chian glutamine- or lysine-plasminogen into two-chain plasmin composed of a heavy chain containing five kringle domains and a light chain containing the catalytic domain. Because its open conformation exposes the t-PA cleavage site, lysine-plasminogen is a better substrate than glutamic acid-plasminogen, which assumes a circular conformation that renders this bond less accessible.

Tissue plasminogen activator has little enzymatic activity in the absence of fibrin, but its activity increases by at least three orders of magnitude when fibrin is present. This increase in activity reflects the capacity of fibrin to serve as a template that binds t-PA and plasminogen and promotes their interaction. Whereas t-PA binds fibrin via its finger and second kringle domains, plasminogen binds fibrin via its kringle domains. Kringle domains are loop-like structures that bind Lys residues on fibrin. As fibrin undergoes degradation, more Lys residues are exposed, which provide additional binding sites for t-PA and plasminogen. Consequently, degraded fibrin stimulates t-PA activation of plasminogen more than intact fibrin.

alpha2-antiplasmin rapidly inhibits circulating plasmin by docking to its first kringle domain and then inhibiting the active site. Because plasmin binds to fibrin via its kringle domains, plasmin generated on the fibrin surface resists inhibition by alpha2-antiplasmin. This phenomenon endows fibrin-bound plasmin with the capacity to degrade fibrin. Factor XIIIa cross-links small amounts of alpha2-antiplasmin onto fibrin, which prevents premature fibrinolysis.

Similar to fibrin, annexin II on endothelial cells binds t-PA and plasminogen and promotes their interaction. Cell-surface gangliosides and alpha-enolase also may bind plasminogen and promote its activation by altering its conformation into the more readily activated open form. Plasminogen binds to endothelial cells via its kringle domains. Lipoprotein a, which also possesses kringle domains, impairs cell-based fibrinolysis by competing with plasminogen for cell-surface binding. This phenomenon may explain the association between elevated lipoprotein a levels and atherosclerosis.

Mechanism of Action of Urokinase-Type Plasminogen Activator

Synthesized as a single-chain polypeptide, single-chain u-PA (scu-PA) has minimal enzymatic activity. Plasmin converts scu-PA into a two-chain form that is enzymatically active and capable of binding u-PAR, the u-PA receptor on cell surfaces. Further cleavage at the amino terminus of two-chain u-PA yields a truncated, lower-molecular-weight form that lacks the u-PAR binding domain.

Two-chain forms of u-PA readily convert plasminogen to plasmin in the absence or presence of fibrin. In contrast, scu-PA does not activate plasminogen in the absence of fibrin, but it can activate fibrin-bound plasminogen because plasminogen adopts a more open and readily activatable conformation when immobilized on fibrin. Similar to the higher-molecular-weight form of two-chain u-PA, scu-PA binds to cell surface u-PAR, where plasmin can activate it. Many tumor cells elaborate u-PA and express u-PAR on their surfaces. Plasmin generated on these cells endows them with the capacity for metastasis.

Mechanism of Action of TAFI

Thrombin-activated fibrinolysis inhibitor, a procarboxpeptidase B-like molecule synthesized in the liver, circulates in blood in a latent form while thrombin bound to thrombomodulin can activate it. Unless bound to thrombomodulin, thrombin activates TAFI inefficiently. TAFIa attenuates fibrinolysis by cleaving lysine residues form the carboxy termini of chains of degrading fibrin, thereby removing binding sites for plasminogen, plasmin, and t-PA. TAFI links fibrinolysis to coagulation because the thrombin-thrombomodulin complex not only activates TAFI, which attenuates fibrinolysis, but also activates protein C, which mutes thrombi in generation.

Activated TAFI (TAFIa) has a short half-life in plasma because the enzyme is unstable. Genetic polymorphisms can result in synthesis of more stable forms of TAFIa. Persistent attenuation of fibrinolysis by these variant forms of TAFIa may render patients susceptible to thrombosis.

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, 𝛼2𝛽1, 𝛼5𝛽1, 𝛼6𝛽1
  • 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, 𝛼2b𝛽1
  • 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 𝛼IIb𝛽3 for its ligands. Fibrinogen and vWF play dominant roles in platelet aggregation through binding to 𝛼IIb𝛽3, 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.