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Hemostasis is a precisely orchestrated process involving platelets, clotting factors, and endothelium that occurs at the site of vascular injury and culminates in the forma- tion of a blood clot, which serves to prevent or limit the extent of bleeding. The general sequence of events leading to hemostasis at a site of vascular injury is shown in Fig. 4.5. #102 | | |
Arteriolar vasoconstriction occurs immediately and mark- edly reduces blood flow to the injured area (Fig. 4.5A). It is mediated by reflex neurogenic mechanisms and aug- mented by the local secretion of factors such as endothe- lin, a potent endothelium-derived vasoconstrictor. This effect is transient, however, and bleeding would resume if not for activation of platelets and coagulation factors. #103 | | |
Primary hemostasis: the formation of the platelet plug. Dis- ruption of the endothelium exposes subendothelial von Willebrand factor (vWF) and collagen, which promote platelet adherence and activation. Activation of platelets results in a dramatic shape change (from small rounded discs to flat plates with spiky protrusions that markedly increased surface area), as well as the release of secre- tory granules. Within minutes the secreted products recruit additional platelets, which undergo aggregation to form a primary hemostatic plug (Fig. 4.5B). #104 | | |
Secondary hemostasis: deposition of fibrin. Vascular injury exposes tissue factor at the site of injury. Tissue factor is a membrane-bound procoagulant glycoprotein that is normally expressed by subendothelial cells in the vessel wall, such as smooth muscle cells and fibroblasts. Tissue factor binds and activates factor VII (see later), setting in motion a cascade of reactions that culiminates in throm- bin generation. Thrombin cleaves circulating fibrinogen into insoluble fibrin, creating a fibrin meshwork, and also is a potent activator of platelets, leading to addi- tional platelet aggregation at the site of injury. This sequence, referred to as secondary hemostasis, consoli- dates the initial platelet plug (Fig. 4.5C). #105 | | |
Clot stabilization and resorption. Polymerized fibrin and platelet aggregates undergo contraction to form a solid, permanent plug that prevents further hemorrhage. At this stage, counterregulatory mechanisms (e.g., tissue plas- minogen activator, t-PA made by endothelial cells) are set into motion that limit clotting to the site of injury (Fig. 4.5D) and eventually lead to clot resorption and tissue repair. #106 | | |
Fig. 4.5 Normal hemostasis. (A) After vascular injury, local neurohumoral #107 | | |
factors induce a transient vasoconstriction. (B) Platelets bind via glycoprotein Ib (GpIb) receptors to von Willebrand factor (VWF) on exposed ECM and are activated, undergoing a shape change and granule release. Released ADP and thromboxane A2 (TXA2) induce additional platelet aggregation through platelet GpIIb-IIIa receptor binding to fibrinogen, and form the primary hemostatic plug. (C) Local activation of the coagulation cascade (involving tissue factor and platelet phospholipids) results in fibrin polymerization, “cementing” the platelets into a definitive secondary hemostatic plug. (D) Counterregulatory mechanisms, mediated by tissue plasminogen activator (t-PA, a fibrinolytic product) and thrombomodulin, confine the hemostatic process to the site of injury. #108 | | |
It should be emphasized that endothelial cells are central regulators of hemostasis; the balance between the anti-thrombic and prothrombotic activities of endo- thelium determines whether thrombus formation, propa- gation, or dissolution occurs. Normal endothelial cells express a variety of anticoagulant factors that inhibit platelet aggregation and coagulation and promote fibrinolysis; after injury or activation, however, this balance shifts, and endothelial cells acquire numerous procoagulant activities (activation of platelets and clotting factor, described above, see also Fig. 4.11). Besides trauma, endothelium can be activated by microbial pathogens, hemodynamic forces, and a number of pro-inflammatory mediators. We will return to the pro-coagulant and anti-coagulant roles of endothelium after a detailed discussion of the role of plate- lets and coagulation factors in hemostasis since endothe- lium modulates the functions of platelets and can trigger coagulation. #109 | | |
The following sections describe roles of platelets, coagu- lation factors and endothelium in hemostasis in greater detail, following the scheme illustrated in Fig. 4.5. #110 | | |
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Platelets play a critical role in hemostasis by forming the #112 | | |
primary plug that initially seals vascular defects and by providing a surface that binds and concentrates activated coagulation factors. Platelets are disc-shaped anucleate cell fragments that are shed from megakaryocytes in the bone marrow into the bloodstream. Their function depends on several glycoprotein receptors, a contractile cytoskele- ton, and two types of cytoplasmic granules. α-Granules have the adhesion molecule P-selectin on their membranes (Chapter 3) and contain proteins involved in coagulation, such as fibrinogen, coagulation factor V, and vWF, as well as protein factors that may be involved in wound healing, such as fibronectin, platelet factor 4 (a heparin-binding chemokine), platelet-derived growth factor (PDGF), and transforming growth factor-β. Dense (or δ) granules contain adenosine diphosphate (ADP) and adenosine triphosphate, ionized calcium, serotonin, and epinephrine. #113 | | |
After a traumatic vascular injury, platelets encounter constituents of the subendothelial connective tissue, such as vWF and collagen. On contact with these proteins, plate- lets undergo a sequence of reactions that culminate in the formation of a platelet plug (Fig. 4.5B). #114 | | |
Platelet adhesion is mediated largely via interactions with vWF, which acts as a bridge between the platelet surface receptor glycoprotein Ib (GpIb) and exposed collagen (Fig. 4.6). Notably, genetic deficiencies of vWF (von Willebrand disease, Chapter 14) or GpIb (Bernard- Soulier syndrome) result in bleeding disorders, attesting to the importance of these factors. #115 | | |
Platelets rapidly change shape following adhesion, being converted from smooth discs to spiky “sea urchins” with greatly increased surface area. This change is accompanied by alterations in glycoprotein IIb/IIIa that increase its affinity for fibrinogen (see later), and by the translocation of negatively charged phospholipids (particu- larly phosphatidylserine) to the platelet surface. These phospholipids bind calcium and serve as nucleation sites for the assembly of coagulation factor complexes. #116 | | |
Secretion (release reaction) of granule contents occurs along with changes in shape; these two events are often referred to together as platelet activation. Platelet activa- tion is triggered by a number of factors, including he coagulation factor thrombin and ADP. Thrombin acti- vates platelets through a special type of G-protein– coupled receptor referred to as a protease-activated receptor (PAR), which is switched on by a proteolytic cleavage carried out by thrombin. ADP is a component of dense-body granules; thus, platelet activation and ADP release begets additional rounds of platelet activa- tion, a phenomenon referred to as recruitment. Activated platelets also produce the prostaglandin thromboxane A2 (TXA2), a potent inducer of platelet aggregation. Aspirin inhibits platelet aggregation and produces a mild bleed- ing defect by inhibiting cyclooxygenase, a platelet enzyme that is required for TXA2 synthesis. Although the phenomenon is less well characterized, it is also suspected that growth factors released from platelets contribute to the repair of the vessel wall following injury. #117 | | |
Platelet aggregation follows their activation. The con- formational change in glycoprotein IIb/IIIa that occurs with platelet activation allows binding of fibrinogen, a large bivalent plasma polypeptide that forms bridges between adjacent platelets, leading to their aggregation. Predictably, inherited deficiency of GpIIb-IIIa results in a bleeding disorder called Glanzmann thrombasthenia. The initial wave of aggregation is reversible, but concur- rent activation of thrombin stabilizes the platelet plug by causing further platelet activation and aggregation, and by promoting irreversible platelet contraction. Plate- let contraction is dependent on the cytoskeleton and consolidates the aggregated platelets. In parallel, throm- bin also converts fibrinogen into insoluble fibrin, cement- ing the platelets in place and creating the definitive secondary hemostatic plug. Entrapped red cells and leuko- cytes are also found in hemostatic plugs, in part due to adherence of leukocytes to P-selectin expressed on acti- vated platelets. #118 | | |
Fig. 4.6 Platelet adhesion and aggregation. VWF functions as an adhesion #119 | | |
bridge between subendothelial collagen and the glycoprotein Ib (GpIb) plate- let receptor. Platelet aggregation is accomplished by fibrinogen binding to platelet GpIIb-IIIa receptors on different platelets. Congenital deficiencies in the various receptors or bridging molecules lead to the diseases indicated in the colored boxes. ADP, Adenosine diphosphate. #120 | | |
SUMMARY PLATELET ADHESION, ACTIVATION, #121 | | |
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Endothelial injury exposes the underlying basement membrane ECM; platelets adhere to the ECM primarily through the binding of platelet GpIb receptors to VWF. #124 | | |
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Adhesion leads to platelet activation, an event associated with secretion of platelet granule contents, including calcium (a cofactor for several coagulation proteins) and ADP (a mediator of further platelet activation); dramatic changes in shape and membrane composition; and activation of GpIIb/IIIa receptors. #126 | | |
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The GpIIb/IIIa receptors on activated platelets form bridging crosslinks with fibrinogen, leading to platelet aggregation. #128 | | |
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Concomitant activation of thrombin promotes fibrin deposi- tion, cementing the platelet plug in place. #130 | | |
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The coagulation cascade is a series of amplifying enzy- #132 | | |
matic reactions that lead to the deposition of an insoluble fibrin clot. As discussed later, the dependency of clot for- mation on various factors differs in the laboratory test tube and in blood vessels in vivo (Fig. 4.7). However, clotting in vitro and in vivo both follow the same general principles, as follows. #133 | | |
The cascade of reactions in the pathway can be likened to a “dance,” in which coagulation factors are passed from one partner to the next (Fig. 4.8). Each reaction step involves an enzyme (an activated coagulation factor), a substrate (an inactive proenzyme form of a coagulation factor), and a cofactor (a reaction accelerator). These components are assembled on a negatively charged phospholipid surface, which is provided by activated platelets. Assembly of reac- tion complexes also depends on calcium, which binds to γ-carboxylated glutamic acid residues that are present in factors II, VII, IX, and X. The enzymatic reactions that produce γ-carboxylated glutamic acid use vitamin K as a cofactor and are antagonized by drugs such as Coumadin, a widely used anti-coagulant. #134 | | |
Fig. 4.7 The coagulation cascade in the laboratory and in vivo. (A) Clotting is initiated in the laboratory by adding phospholipids, calcium, and either a negative-charged substance such as glass beads (intrinsic pathway) or a source of tissue factor (extrinsic pathway). (B) In vivo, tissue factor is the major initia- tor of coagulation, which is amplified by feedback loops involving thrombin (dotted lines). The red polypeptides are inactive factors, the dark green polypeptides are active factors, whereas the light green polypeptides correspond to cofactors. #135 | | |
Based on assays performed in clinical laboratories, the coagulation cascade has traditionally been divided into the extrinsic and intrinsic pathways (Fig. 4.7A). #136 | | |
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The prothrombin time (PT) assay assesses the function of the proteins in the extrinsic pathway (factors VII, X, V, II (prothrombin), and fibrinogen). In brief, tissue factor, phospholipids, and calcium are added to plasma and the time for a fibrin clot to form is recorded. #138 | | |
The partial thromboplastin time (PTT) assay screens the function of the proteins in the intrinsic pathway (factors XII, XI, IX, VIII, X, V, II, and fibrinogen). In this assay, clotting of plasma is initiated by the addition of negative- charged particles (e.g., ground glass) that activate factor XII (Hageman factor) together with phospholipids and calcium, and the time to fibrin clot formation is recorded. #139 | | |
Fig. 4.8 Sequential conversion of factor X to factor Xa by way of the extrinsic pathway, followed by conversion of factor II (prothrombin) to factor IIa (throm- bin). The initial reaction complex consists of a protease (factor VIIa), a substrate (factor X), and a reaction accelerator (tissue factor) assembled on a platelet phospholipid surface. Calcium ions hold the assembled components together and are essential for the reaction. Activated factor Xa then becomes the protease component of the next complex in the cascade, converting prothrombin to thrombin (factor IIa) in the presence of a different reaction accelerator, factor Va. #140 | | |
Although the PT and PTT assays are of great utility in evaluating coagulation factor function in patients, they do not recapitulate the events that lead to coagulation in vivo. This point is most clearly made by considering the clinical effects of deficiencies of various coagulation factors. Defi- ciencies of factors V, VII, VIII, IX, and X are associated with moderate to severe bleeding disorders, and prothrombin deficiency is likely incompatible with life. In contrast, factor XI deficiency is only associated with mild bleeding, and individuals with factor XII deficiency do not bleed and in fact may be susceptible to thrombosis. The paradoxical effect of factor XII deficiency may be explained by involve- ment of factor XII in the fibrinolysis pathway (discussed later); although there is also some evidence from experi- mental models suggesting that factor XII may promote thrombosis under certain circumstances, the relevance of these observations to human thrombotic disease remains to be determined. #141 | | |
Based on the effects of various factor deficiencies in humans, it is believed that, in vivo, factor VIIa/tissue factor complex is the most important activator of factor IX and that factor IXa/factor VIIIa complex is the most impor- tant activator of factor X (Fig. 4.7B). The mild bleeding tendency seen in patients with factor XI deficiency is likely explained by the ability of thrombin to activate factor XI (as well as factors V and VIII), a feedback mechanism that amplifies the coagulation cascade. #142 | | |
Among the coagulation factors, thrombin is the most important, because its various enzymatic activities control diverse aspects of hemostasis and link clotting to inflam- mation and repair. Among thrombin’s most important activities are the following: #143 | | |
Conversion of fibrinogen into crosslinked fibrin. Thrombin directly converts soluble fibrinogen into fibrin mono- mers that polymerize into an insoluble fibril, and also amplifies the coagulation process, not only by activating factor XI, but also by activating two critical cofactors: factors V and VIII. It also stabilizes the secondary hemo- static plug by activating factor XIII, which covalently crosslinks fibrin. #144 | | |
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Platelet activation. Thrombin is a potent inducer of platelet activation and aggregation through its ability to activate PARs, thereby linking platelet function to coagulation. #146 | | |
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Proinflammatory effects. PARs also are expressed on inflammatory cells, endothelium, and other cell types (Fig. 4.9), and activation of these receptors by thrombin is believed to mediate proinflammatory effects that con- tribute to tissue repair and angiogenesis. #148 | | |
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Anti-coagulant effects. Remarkably, through mechanisms described later, on encountering normal endothelium, thrombin changes from a procoagulant to an anti- coagulant; this reversal in function prevents clots from extending beyond the site of the vascular injury. #150 | | |
