Hemostasis, the process of blood clot formation, is a coordinated series of responses to vessel injury. It requires complex interactions between platelets, the clotting cascade, blood flow and shear, endothelial cells, and fibrinolysis.
Platelet Plug Formation
Platelets are activated at the site of vascular injury to form a plug to stop bleeding. Physiologic platelet stimuli include adenosine diphosphate (ADP), epinephrine, thrombin, and collagen. ADP and epinephrine are relatively weak platelet stimulators; thrombin and collagen are strong agonists. Thrombin activation is mediated by G protein-coupled protease-activated receptors (PAR),1 specifically PAR-1 and PAR-4. Thrombin cleaves the external domain of the PAR to initiate transmembrane signaling [see Figure 1].2 Platelet responses to ADP require the coordinated activation of two G-protein-coupled receptors, P2Y1 and P2Y12, which lead to activation of phospholipase C and suppression of cyclic adenosine monophosphate (cAMP) formation, respectively. Antiplatelet drugs such as ticlopidine and clopidogrel block activation of P2Y12.3 There are also specific receptors for epinephrine, thromboxane A2, and collagen.
Platelet activation involves four distinct processes: adhesion (deposition of platelets on subendothelial matrix); aggregation (cohesion of platelets); secretion (release of platelet granule proteins); and procoagulant activity (enhancement of thrombin generation) [see Figure 2].
Adhesion
Platelet adhesion is primarily mediated by the binding of platelet surface receptor glycoprotein (GP) Ib-IX-V complex to the adhesive protein von Willebrand factor (vWF) in the subendothelial matrix.4 Deficiency of GPIb-IX-V complex or vWF leads to two congenital bleeding disorders, Bernard-Soulier disease and von Willebrand disease, respectively [see 5:XIII Hemorrhagic Disorders]. Other adhesive interactions (e.g., binding of platelet collagen receptor GPIa-IIa to collagen fibrils in the matrix) also contribute to platelet adhesion.5
Aggregation
Platelet aggregation involves binding of fibrinogen to the platelet fibrinogen receptor (i.e., the GPIIb-IIIa complex). GPIIb-IIIa (also termed aIIbb3) is a member of a superfamily of adhesive protein receptors, called integrins, which are found in many different cell types. It is the most abundant receptor on the platelet surface. GPIIb-IIIa does not bind fibrinogen on nonstim-ulated platelets. After platelet stimulation, GPIIb-IIIa undergoes a conformational change and is converted from a low-affinity fibrinogen receptor to a high-affinity receptor in a process termed inside-out signaling. Fibrinogen, a divalent molecule, serves to bridge the activated platelets [see Figure 3]. The cyto-solic portion of the activated GPIIb-IIIa complex binds to the platelet cytoskeleton and can mediate platelet spreading and clot retraction (in a process termed outside-in signaling).6 Congenital deficiency of GPIIb-IIIa or fibrinogen leads to Glanzmann thrombasthenia and afibrinogenemia. The GPIIb-IIIa-fibrinogen pathway is the final common course for platelet aggregation. Blockade of this pathway is the basis of an important class of an-tiplatelet drugs.
Protein secretion
After stimulation, platelet granules release ADP and serotonin, which stimulate and recruit additional platelets; adhesive proteins such as fibronectin and thrombospondin, which reinforce and stabilize platelet aggregates; factor V, a component of the clotting cascade; thromboxane, which stimulates vasoconstriction; and growth factors such as platelet-derived growth factor (PDGF), which stimulate proliferation of smooth muscle cells and mediate tissue repair. PDGF may also contribute to the development of atherosclerosis and reocclusion after coronary angioplasty.
Figure 1 Thrombin activation is mediated by G protein-coupled protease-activated receptor (PAR). Thrombin cleaves the NH2-terminal exodomain of the PAR, exposing a new NH2 terminus, which then serves as a tethered ligand to bind intramolecularly to the body of the receptor to initiate transmembrane signaling.
Figure 2 After platelets are activated, they undergo significant morphologic changes, producing elongated pseudopods. They also become extremely adhesive. The functional response of activated platelets involves four distinct processes: adhesion (deposition of platelets on subendothelial matrix); aggregation (cohesion of platelets); secretion (release of platelet granule proteins); and procoagulant activity (enhancement of thrombin generation).
Procoagulation
Platelet procoagulation involves the assembly of the enzyme complexes of the clotting cascade on the platelet surface. It is an important example of the close interrelationship between platelet activation and the activation of the clotting cascade.
Clotting Cascade
The central feature of the clotting cascade is the sequential activation of a series of proenzymes (zymogens) to enzymes, ultimately generating fibrin and reinforcing the platelet plug. Another key feature, amplification, ensures rapid response for effective hemostasis but demands tight regulation to prevent untoward thrombosis.
The clotting cascade is usually depicted as comprising intrinsic and extrinsic pathways [see Figure 4]. The intrinsic pathway is initiated by the exposure of blood to a negatively charged surface (e.g., glass), whereas the extrinsic pathway is activated by tissue factor or thromboplastin. Both pathways converge on the activation of factor X, which then activates prothrombin (factor II) to thrombin, the final enzyme of the clotting cascade.
Although this classic view of the clotting cascade has been useful in the interpretation of clotting times, it is not completely accurate. Patients who are severely deficient in factor XII—as well as many patients deficient in factor XII—do not bleed clinically, which indicates that the initiation part of the intrinsic path way (the contact phase) is not important in vivo. It is now established that generation or exposure of tissue factor at the wound site is the primary physiologic event that initiates clotting [see Figure 4].7 Tissue factor functions as a cofactor that is absolutely required by factor VII/factor VIIa to initiate clotting. Factor VIIa activates factor X directly and indirectly via the activation of factor IX. This dual pathway of factor X activation is necessary apparently because of the limited amount of tissue factor generated in vivo and the presence of the tissue factor pathway inhibitor (see below), which, when complexed with factor Xa, inhibits the tissue factor/factor VIIa complex.
All of the procoagulants are synthesized in the liver except vWF, which is synthesized in megakaryocytes and endothelial cells. The vitamin K-dependent procoagulants are prothrombin, factor VII, factor IX, and factor X; the vitamin K-dependent anticoagulants are protein C and protein S. For these factors, the formation of a-carboxyglutamic acid residues by vitamin K-dependent carboxylation of glutamic acid residues endows them with calcium-binding properties and the ability to interact with phospholipid membrane surfaces, which are required for biologic activity.
Interaction between activated platelets and the clotting cascade
There is an extremely close interaction between the clotting cascade and activated platelet surface in vivo. When platelets are activated, anionic lipids become exposed on the platelet surface, and factor V (stored in platelet granules) is released and bound on the anionic lipids. The factor V on the platelet surface is activated to factor Va and acts as an assembly site for the binding of factor Xa (enzyme) and prothrombin (substrate) known as the prothrombinase complex. At the assembly site, thrombin generation by the prothrombinase complex is approximately 300,000 times more efficient than thrombin generation by fluid-phase factor Xa and prothrombin alone, and the platelet plug keeps the thrombin localized. Factor Xa bound on factor Va is also relatively protected from inhibition by circulating inhibitors such as an-tithrombin III (AT-III) (see below). Similar enzyme complex assembly applies to the activation of factor X by factor VIIIa (cofac-tor) and factor IXa (the intrinsic tenase). The result of these processes is efficient amplification and localization of clotting.
Control Mechanisms
Coagulation is modulated by a number of mechanisms: dilution of procoagulants in flowing blood; removal of activated factors through the reticuloendothelial system, especially in the liver; and control by natural antithrombotic pathways. At least seven separate and distinct control systems modulate each phase of hemostasis and protect against thrombosis, vascular inflammation, and tissue damage [see Table 1]. Antithrombin III, protein C, protein S, and tissue factor pathway inhibitor (TFPI) collectively regulate the clotting cascade; prostacyclin and nitric oxide modulate vascular and platelet reactivity; ecto-ADPase inhibits platelet recruitment; and fibrinolysis removes the fibrin clot.
Antithrombin iii—heparan sulfate system
Antithrombin III is a circulating plasma protease inhibitor. It inhibits thrombin and factor Xa, the two key enzymes in the clotting cascade. AT-III also inhibits activated factor XII and factor XI. In the absence of the glycosaminoglycan heparin, AT-III inhibits thrombin and factor Xa relatively slowly (complete inhibition requires a few minutes). When present, heparin binds to a discrete binding site on AT-III that causes a conformational change in AT-III, which then inhibits thrombin instantaneously and irreversibly. This augmentation of the inhibition of throm-bin and factor Xa is the basis for the therapeutic use of heparin as an anticoagulant. Heparan sulfate proteoglycans on the luminal surface of endothelial cells appear to activate AT-III in a manner similar to that of heparin [see Figure 5].9
Thus, the endothelial surface is normally coated with a layer of AT-III that is already activated by the endogenous heparan sul-fate. Because 1 ml of blood can be exposed to as much as 5,000 cm2 of endothelial surface, the AT-III-heparan sulfate system is poised to rapidly inactivate any thrombin in the general circulation.
Protein c and protein s-thrombomodulin system
Thrombomodulin is an integral membrane protein found on the luminal surface of the vascular endothelium in the microcir-culation. The binding of thrombin to thrombomodulin results in a remarkable switch in thrombin’s substrate specificities: it no longer clots fibrinogen or activates platelets [see Figure 6]. On the other hand, it acquires the ability to activate protein C in plasma.10 A distinct endothelial receptor for protein C has been found that enhances the activation of protein C by the thrombin-thrombomodulin complex.11 Activated protein C degrades factor Va and factor Villa, the two cofactors responsible for the assembly of the prothrombinase and intrinsic tenase complex in the clotting cascade. Protein S serves as a cofactor for activated protein C. Deficiencies of AT-III, protein C, and protein S are important causes of a hypercoagulable state.
Figure 3 Platelet aggregation involves binding of the divalent molecule fibrinogen to the platelet fibrinogen receptor (the GPIIb-IIIa complex). After platelet stimulation, GPIIb-IIIa is converted from a low-affinity fibrinogen receptor to a high-affinity receptor (inside-out signaling). The cytosolic portion of the activated GPIIb-IIIa complex can mediate platelet spreading and clot retraction (outside-in signaling).
Figure 4 In the classic view of the clotting cascade (left), the intrinsic pathway is initiated by the exposure of blood to a negatively charged surface (e.g., glass) and the extrinsic pathway is activated by tissue factor or thromboplastin. In the modified view (right), generation or exposure of tissue factor at the wound site is the primary physiologic event that initiates clotting.
Protein C and protein S both show some structural similarity to the vitamin K-dependent clotting factors (prothrombin, factor VII, factor IX, and factor X). Protein S circulates in two forms: a free form, in which it is active as an anticoagulant, and a bound, inactive form, in which it is complexed to C4b-binding protein of the complement system. C4b-binding protein acts as an acute-phase reactant. The resultant increase in inflammatory states reduces the activity of free protein S, enhancing the likelihood of thrombosis.
Tissue factor pathway inhibitor
Tissue factor pathway inhibitor is a circulating plasma protease inhibitor that is synthesized by the microvascular en-dothelium. Unlike AT-III, TFPI has a very low plasma concentration. TFPI inhibits factor Xa. The TFPI/factor Xa complex becomes an effective inhibitor of tissue factor/factor VIIa, thus mediating feedback inhibition of tissue factor/factor VIIa [see Figure 7]. Animal studies have shown that depletion of the endogenous TFPI sensitizes the animals to the development of disseminated intravascular coagulation induced by tissue factor or endotoxin.12
TFPI is primarily synthesized by the microvascular endotheli-um. Approximately 20% of TFPI circulates in plasma associated with lipoproteins; the majority remains associated with the en-dothelial surface, apparently bound to the cell-surface glycosaminoglycans. The plasma level of TFPI is greatly increased after intravenous administration of heparin. This release of en-dothelial TFPI may contribute to the antithrombotic efficacy of heparin and low-molecular-weight heparin. Recombinant TFPI is now in early clinical trials.
Prostacyclin
Upon cell perturbation, the fatty acid arachidonic acid is released from cell membrane phospholipids by phospholipase A2. The enzyme prostaglandin endoperoxide H synthase-1 (PGHS-1) converts arachidonic acid into prostaglandin endoperoxides and finally to thromboxane A2 (TXA2) in platelets and prostacy-clin (PGI2) in endothelial cells.
Table 1 Natural Antithrombotic Mechanisms of Endothelial Cells
|
Regulation of clotting cascade |
Tissue factor pathway inhibitor Antithrombin III |
|
Protein C/Protein S |
|
|
Modulation of vessel and platelet reactivity |
Prostacyclin Nitric oxide |
|
Inhibition of platelet recruitment |
Ecto-ADPase (CD39) |
|
Removal of fibrin clot |
Fibrinolysis |
Figure 5 In the absence of heparan sulfate (HS), antithrombin III (AT-III) inhibits thrombin slowly. When HS is present, it binds to a specific site on AT-III that causes a conformational change in AT-III, allowing it to reach the active site of thrombin and inhibit the enzyme instantaneously. HS also binds to a specific site on thrombin, positioning it for optimal inhibition by AT-III.
TXA2 and PGI2 have opposite functions. TXA2 is a potent stimulator of platelet aggregation and causes vasoconstriction, whereas PGI2 inhibits platelet aggregation and induces vasodilatation. PGI2 functions by activating adenylate cyclase, which leads to an increase in intracellular cAMP [see Figure 8].
Figure 6 The protein C/protein S pathway is complementary to the AT-III pathway. When thrombin binds to thrombomodulin, thrombin undergoes a conformational change and no longer clots fibrinogen or activates platelets. However, it acquires the ability to activate protein C in plasma. Protein S serves as a cofactor for activated protein C. Activated protein C degrades activated factors V and VIII, the two cofactors in the clotting cascade.
Figure 7 Tissue factor pathway inhibitor (TFPI) binds to and inhibits factor Xa. After binding to factor Xa, TFPI undergoes a conformational change. The TFPI/factor Xa complex then mediates feedback inhibition of tissue factor/factor VIIa.
