Hemostasis is the cessation of bleeding from a cut or severed vessel, whereas thrombosis occurs when the endothelium lining blood vessels is damaged or removed (eg, upon rupture of an atherosclerotic plaque). These processes involve blood vessels, platelet aggregation, and plasma proteins that cause formation or dissolution of platelet aggregates and fibrin.
In hemostasis, there is initial vasoconstriction of the injured vessel, causing diminished blood flow distal to the injury. Then, hemostasis and thrombosis share three phases:
Formation of a loose and temporary platelet aggregate at the site of injury. Platelets bind to collagen at the site of vessel wall injury, form thromboxane A2, and release ADP, which activate other platelets flowing by the vicinity of the injury. (The mechanism of platelet activation is described below.) Thrombin, formed during coagulation at the same site, causes further platelet activation. Upon activation, platelets change shape and, in the presence of fibrinogen and/or von Willebrand factor, aggregate to form the hemostatic plug (in hemostasis) or thrombus (in thrombosis).
Formation of a fibrin mesh that binds to the platelet aggregate, forming a more stable hemostatic plug or thrombus.
Partial or complete dissolution of the hemostatic plug or thrombus by plasmin.
There Are Three Types of Thrombi
Three types of thrombi or clots are distinguished. All three contain fibrin in various proportions.
The white thrombus is composed of platelets and fibrin and is relatively poor in erythrocytes. It forms at the site of an injury or abnormal vessel wall, particularly in areas where blood flow is rapid (arteries).
The red thrombus consists primarily of red cells and fibrin. It morphologically resembles the clot formed in a test tube and may form in vivo in areas of retarded blood flow or stasis (eg, veins) with or without vascular injury, or it may form at a site of injury or in an abnormal vessel in conjunction with an initiating platelet plug.
A third type is fibrin deposits in very small blood vessels or capillaries.
We shall first describe the coagulation pathway leading to the formation of fibrin. Then, we shall briefly describe some aspects of the involvement of platelets and blood vessel walls in the overall process. This separation of clotting factors and platelets is artificial since both play intimate and often mutually interdependent roles in hemostasis and thrombosis; this strategy facilitates description of the overall processes involved.
Both Extrinsic & Intrinsic Pathways Result in the Formation of Fibrin
Two pathways lead to fibrin clot formation: the extrinsic and the intrinsic pathways. These pathways are not independent, as previously thought. However, this artificial distinction is retained in the following text to facilitate their description.
Initiation of fibrin clot formation in response to tissue injury is carried out by the extrinsic pathway. The intrinsic pathway can be activated by negatively charged surfaces in vitro, for example glass. Both pathways lead to the proteolytic conversion of prothrombin to thrombin. Thrombin catalyzes cleavage of fibrinogen to initiate fibrin clot formation. The extrinsic and intrinsic pathways are complex and involve many different proteins (Figures 55–1 and 55–2; Table 55–1). The coagulation factors are another example of multidomain proteins sharing conserved domains (see, Figure 5–9). In general, as shown in Table 55–2, these proteins can be classified into five types: (1) zymogens of serine-dependent proteases that are activated during the process of coagulation; (2) cofactors; (3) fibrinogen; (4) a transglutaminase that covalently crosslinks fibrin and stabilizes the fibrin clot; and (5) regulatory and other proteins.
The pathways of blood coagulation, with the extrinsic pathway indicated at the top left and the intrinsic pathway at the top right. The pathways converge in the formation of factor Xa and culminate in the formation of cross-linked fibrin. Complexes of tissue factor and factor VIIa activate not only factor X (extrinsic Xase [tenase]) but also factor IX in the intrinsic pathway (dotted arrow). In addition, thrombin feedback activates at the sites indicated (dashed arrows) and also activates factor VII to factor VIIa (not shown). The three predominant complexes, extrinsic Xase, intrinsic Xase, and prothrombinase, are indicated in the arrows; these reactions require anionic procoagulant phospholipid membrane and calcium. Activated proteases are in solid-outlined boxes; active cofactors are in dash-outlined boxes and inactive factors are not in boxes. (HK, high-molecular-weight kininogen; PK, prekallikrein.)
The structural domains of selected proteins involved in coagulation and fibrinolysis. Shared domains are a result of gene duplication and exon shuffling that contributed to the molecular evolution of the coagulation system. The domains are as identified at the bottom of the figure and include signal peptide, propeptide, Gla (γ-carboxyglutamate) domain, epidermal growth factor (EGF) domain, apple domain, kringle domain, fibronectin (types I and II) domain, the zymogen activation region, aromatic amino acid stack, and the catalytic domain. Interdomain disulfide bonds are indicated, but numerous intradomain disulfide bonds are not. Sites of proteolytic cleavage in synthesis or activation are indicated by arrows (dashed and solid, respectively). FVII, factor VII; FIX, factor IX; FX, factor X, FXI; factor XI; FXII, factor XII; tPA, tissue plasminogen activator. (Adapted, with permission, from Furie B, Furie BC: The molecular basis of blood coagulation. Cell 1988;53:505.)
TABLE 55–1Numerical System for Nomenclature of Blood Clotting Factors ||Download (.pdf) TABLE 55–1 Numerical System for Nomenclature of Blood Clotting Factors
|Factor ||Common Name |
| ||These factors are usually referred to by their common names |
|IV ||Ca2+ ||Ca2+ is usually not referred to as a coagulation factor |
|V ||Proaccelerin, labile factor, accelerator (Ac-) globulin |
|VIIa ||Proconvertin, serum prothrombin conversion accelerator (SPCA), cothromboplastin |
|VIII ||Antihemophilic factor A, antihemophilic globulin (AHG) |
|IX ||Antihemophilic factor B, Christmas factor, plasma thromboplastin component (PTC) |
|X ||Stuart-Prower factor |
|XI ||Plasma thromboplastin antecedent (PTA) |
|XII ||Hageman factor |
|XIII ||Fibrin stabilizing factor (FSF), fibrinoligase |
TABLE 55–2The Functions of the Proteins Involved in Blood Coagulation ||Download (.pdf) TABLE 55–2 The Functions of the Proteins Involved in Blood Coagulation
|Zymogens of Serine Proteases |
|Factor XII ||Binds to negatively charged surface, eg, kaolin, glass; activated by high-molecular-weight kininogen and kallikrein |
|Factor XI ||Activated by factor XIIa |
|Factor IX ||Activated by factor XIa and factor VIIa |
|Factor VII ||Activated by factor VIIa, factor Xa, and thrombin |
|Factor X ||Activated on the surface of activated platelets by tenase complex (Ca2+, ffactors VIIIa and IXa) and by the extrinsic tenase complex Ca2+, tissue factor and factor VIIa) |
|Prothrombin Factor II ||Activated on the surface of activated platelets by prothrombinase complex (Ca2+, factors Va and Xa) to form thrombin [Factors II, VII, IX, and X are Gla-containing zymogens] (Gla = γ-carboxyglutamate) |
|Factor VIII ||Activated by thrombin; factor VIIIa is a cofactor in the activation of factor X by factor IXa |
|Factor V ||Activated by thrombin; factor Va is a cofactor in the activation of prothrombin by factor Xa |
|Tissue factor (factor III) ||A glycoprotein located in the subendothelium and expressed on activated monocytes to act as a cofactor for factor VIIa |
|Factor I ||Cleaved by thrombin to form fibrin clot |
|Thiol-Dependent Transglutaminase |
|Factor XIII ||Activated by thrombin; stabilizes fibrin clot by covalent cross-linking |
|Regulatory and Other Proteins |
|Protein C ||Activated to activated protein C (APC) by thrombin bound to thrombomodulin; then degrades factors VIIIa and Va |
|Protein S ||Acts as a cofactor of protein C; both proteins contain Gla (γ-carboxyglutamate) residues |
|Thrombomodulin ||Protein on the surface of endothelial cells; binds thrombin, which then activates protein C |
The Extrinsic Pathway Leads to Activation of Factor X
The extrinsic pathway involves tissue factor, factors VII and X, and Ca2+ and results in the production of factor Xa (by convention, activated clotting factors are referred to by use of the suffix a). The extrinsic pathway is initiated at the site of tissue injury with the exposure of tissue factor (TF; Figure 55–1), located in the subendothelium and on activated monocytes. TF interacts with and activates factor VII (53 kDa, a zymogen containing vitamin K–dependent γ-carboxyglutamate [Gla] residues; see Chapter 44), synthesized in the liver. It should be noted that in the Gla-containing zymogens (factors II, VII, IX, and X), the Gla residues in the amino terminal regions of the molecules serve as high-affinity binding sites for Ca2+. TF acts as a cofactor for factor VIIa, enhancing its enzymatic activity to activate factor X (56 kDa). The reaction by which factor X is activated requires the assembly of the extrinsic tenase complex (Ca2+-TF-factor VIIa) formed on a cell membrane surface exposing the procoagulant anionic aminophospholipid phosphatidylserine. Factor VIIa cleaves an Arg-Ile bond in factor X to produce the two-chain serine protease, factor Xa. TF and factor VIIa also activate factor IX in the intrinsic pathway. Indeed, the formation of complexes between membrane-bound TF and factor VIIa is now considered to be the key process involved in initiation of blood coagulation in vivo.
Tissue factor pathway inhibitor (TFPI) is a major physiologic inhibitor of coagulation. TFPI is a protein that circulates in the blood where it directly inhibits factor Xa by binding to the enzyme near its active site. This factor Xa-TFPI complex then inhibits the factor VIIa-TF complex.
The Intrinsic Pathway Also Leads to Activation of Factor X
The formation of factor Xa is the major site where the intrinsic and extrinsic pathways converge (Figure 55–1). The intrinsic pathway (Figure 55–1) involves factors XII, XI, IX, VIII, and X, as well as prekallikrein, high-molecular-weight (HMW) kininogen, Ca2+, and cell-surface exposed phosphatidylserine. This pathway results in the production of factor Xa by the intrinsic tenase complex (see below for composition), in which factor IXa serves as the serine protease and factor VIIIa as the cofactor. As noted above, the activation of factor X provides an important link between the intrinsic and extrinsic pathways.
The intrinsic pathway can be initiated by “contact” in which prekallikrein, HMW kininogen, factor XII, and factor XI are exposed to a negatively charged activating surface. In vivo, polymers of phosphates, such as extracellular DNA, RNA, and polyphosphate (macromolecules available only following cell damage) may serve as this negatively charged activating surface. Kaolin, a highly negatively charged hydrated aluminum silicate, can be used for in vitro tests as an initiator of the intrinsic pathway. When the components of the contact phase assemble on the activating surface, factor XII is activated to factor XIIa upon proteolysis by kallikrein. This factor XIIa, generated by kallikrein, attacks prekallikrein to generate more kallikrein, setting up a positive feedback activation loop. Factor XIIa, once formed, activates factor XI to XIa and also releases bradykinin (a peptide with potent vasodilator action) from HMW kininogen.
In the presence of Ca2+, factor XIa activates factor IX (55 kDa, a Gla-containing zymogen), to the serine protease, factor IXa. This, in turn, also cleaves an Arg-Ile bond in factor X to produce factor Xa. This latter reaction requires the assembly of components, called the intrinsic tenase complex, composed of Ca2+-factor VIIIa-factor X, which forms on the membrane surface of platelets activated to expose procoagulant phosphatidylserine. (Note that this phospholipid is normally on the internal side of the plasma membrane of resting, nonactivated platelets.)
Factor VIII (330 kDa), a circulating glycoprotein, is not a protease precursor but a cofactor that serves as a receptor on the platelet surface for factors IXa and X. Factor VIII is activated by minute quantities of thrombin to form factor VIIIa, which is in turn inactivated upon further cleavage by thrombin.
The role of the initial steps of the intrinsic pathway in initiating coagulation has been called into question because patients with a hereditary deficiency of factor XII, prekallikrein or HMW kininogen do not exhibit bleeding problems. Similarly, patients with a deficiency of factor XI may not have bleeding problems. In experimental thrombosis models, deficiencies in the intrinsic pathway are protective against thrombosis. The intrinsic pathway largely serves to amplify factor Xa and ultimately thrombin formation, through feedback mechanisms (see below). The intrinsic pathway may also be important in fibrinolysis (see below) since kallikrein, factor XIIa, and factor XIa can cleave plasminogen and kallikrein can activate single-chain urokinase.
Factor Xa Leads to Activation of Prothrombin to Thrombin
Factor Xa, produced by either the extrinsic or the intrinsic pathway, activates prothrombin (factor II) to thrombin (factor IIa) (Figure 55–1).
The activation of prothrombin, like that of factor X, occurs on a membrane surface and requires the assembly of a prothrombinase complex, consisting of Ca2+, factor Va, and factor Xa. The assembly of the prothrombinase complex, like that of the tenase complex, takes place on the phosphatidylserine-exposing membrane surface of activated platelets.
Factor V (330 kDa) is synthesized in the liver, spleen, and kidney and is found in platelets as well as in plasma. Factor V functions as a cofactor in a manner similar to that of factor VIII in the tenase complex. When activated to factor Va by traces of thrombin, it binds specifically to the platelet membrane (Figure 55–3) and forms a complex with factor Xa and prothrombin. It is subsequently inactivated by activated protein C (see below), thereby providing a means of limiting the activation of prothrombin to thrombin. Prothrombin (72 kDa; Figure 55–3) is a single-chain glycoprotein synthesized in the liver. The amino terminal region of prothrombin (Figure 55–2) contains 10 Gla residues, and the serine-dependent active protease site is in the catalytic domain close to the carboxyl terminal region of the molecule. Upon binding to the complex of factors Va and Xa on the platelet membrane (Figure 55–3), prothrombin is cleaved by factor Xa at two sites to generate the active, two-chain thrombin molecule, which is then released from the platelet surface.
Diagrammatic representation (not to scale) of the binding of factors Va, Xa, and prothrombin (PT) to the plasma membrane of the activated platelet in the prothrombinase complex. A central theme in blood coagulation is the assembly of protein complexes, ie, the tenase complexes and the prothrombinase complex, on membrane surfaces on which phosphatidylserine is exposed in a Ca2+-dependent fashion; the catalytic efficiency of zymogen activation is increased by many orders of magnitude by the membrane-bound complexes. Gamma-carboxyglutamate residues (indicated by Y) on vitamin K–dependent proteins bind calcium and contribute to the exposure of membrane binding sites on these proteins. (Adapted, with permission, from Furie B, Furie BC: The molecular basis of blood coagulation. Cell 1988;53:505.)
Conversion of Fibrinogen to Fibrin Is Catalyzed by Thrombin
Thrombin, produced by the prothrombinase complex, in addition to having a potent stimulatory effect on platelets (see below), converts fibrinogen to fibrin (Figure 55–1). Fibrinogen (aka factor I, 340 kDa; see Figures 55–1 and 55–4; Tables 55–1 and 55–2) is an abundant (3 mg/mL) soluble plasma glycoprotein that consists of a dimer of three polypeptide chains, (Aα, Bβ, γ)2, that is covalently linked by 29 disulfide bonds. The Bβ and γ chains contain asparagine-linked complex oligosaccharides (see Chapter 46). All three chains are synthesized in the liver; the three genes encoding these proteins are on the same chromosome where their expression is coordinately regulated in humans. The amino terminal regions of the six chains are held in close proximity by a number of disulfide bonds (a subset is shown in Figure 55–4), while the carboxyl terminal regions are spread apart. Thus, the fibrinogen molecule has a trinodular, elongated structure with a central E domain that is linked to lateral D domains via coiled coil regions (Figures 55–4 and 55–5A). The N-terminal A and B portions of the Aα and Bβ chains are termed fibrinopeptide A (FPA) and fibrinopeptide B (FPB), respectively; these domains are highly negatively charged as a result of an abundance of aspartate and glutamate residues (see below). The negative charges contribute to the solubility of fibrinogen in plasma and importantly also serve to prevent aggregation by causing electrostatic repulsion between fibrinogen molecules.
Diagrammatic representation of fibrinogen. (A) Fibrinogen is a dimeric molecule, with each half composed of three polypeptide chains: Aα, Bβ, and γ. Disulfide bonds join together the chains and the two halves of the molecule. (B) Fibrinogen forms a trinodular structure with a central E domain linked via coiled coil regions to two lateral D domains each of which contains a flexible Aa chain αC domain. The thrombin-cleaved regulatory peptides fibrinopeptide A (FPA) and fibrinopeptide B (FPB) reside within the E nodule as shown. (Figure modified from Weitz JI: Overview of hemostasis and thrombosis. In: Hoffman R, Benz EJ Jr, Silberstein LR, et al [editors]: Hematology: Basic Principals and Practice, 6th ed. Elsevier Saunders, 2013:1779.)
Fibrin polymerization and degradation. (A) The formation of fibrin monomer via cleavage of fibrinopeptide A (FPA) and fibrinopeptide B (FPB) from fibrinogen by thrombin; the spontaneous polymerization of fibrin monomers to dimers and higher oligomers: followed by the stabilization of fibrin oligomers by factor XIIIa-mediated covalent crosslinking of adjacent fibrin monomers. Finally (bottom), is illustrated the degradation of fibrin polymers into soluble degradation products by plasmin digestion, which leads to blot dissolution. Figure modified from Weitz JI: Overview of hemostasis and thrombosis. In Hoffman R, Benz Jr EJ, Silberstein LR, et al (editors): Hematology: Basic Principals and Practice, 6th ed. Elsevier Saunders, 2013, pp. 1779.) (B) Thrombin cleavage site of the Aα and Bβ chains of fibrinogen to yield FPA/FPB (left; green) and the α and β chains of fibrin monomer (right; black). (C) Schematic of factor XIIIa (transglutaminase)-mediated crosslinking of fibrin molecules. (Figure modified from Weitz JI: Overview of hemostasis and thrombosis. In: Hoffman R, Benz EJ Jr, Silberstein LR, et al (editors): Hematology: Basic Principles and Practice, 6th ed. Elsevier Saunders, 2013:1779.)
Thrombin (34 kDa), the serine protease formed by the prothrombinase complex, hydrolyzes the four Arg-Gly bonds between the N-terminal fibrinopeptides and the α and β portions of the Aα and Bβ chains of fibrinogen (Figure 55–5A, B). The release of FPA and FPB by thrombin generates fibrin monomer, which has the subunit structure (α, β, γ)2. Since FPA and FPB contain only 16 and 14 residues, respectively, the fibrin molecule retains 98% of the residues present in fibrinogen. The removal of the fibrinopeptides exposes binding sites within the E-domain of fibrin monomers that specifically interact with complementary domains within the D-domains of other fibrin monomers. In this way, fibrin monomers spontaneously polymerize in a half-staggered pattern to form long strands (protofibrils) (Figure 55–5A). Although insoluble, this initial fibrin clot is unstable, held together only by the noncovalent association of fibrin monomers.
In addition to converting fibrinogen to fibrin, thrombin also activates factor XIII to factor XIIIa. Factor XIIIa is a highly specific transglutaminase that covalently cross-links γ–chains and, more slowly, α-chains of fibrin molecules by forming peptide bonds between the amide groups of glutamine and the ε-amino groups of lysine residues (see Figure 51–5C). Such crosslinking yields a more stable fibrin clot with increased resistance to proteolysis. This fibrin mesh serves to stabilize the hemostatic plug or thrombus.
Levels of Circulating Thrombin Are Carefully Controlled
Once active thrombin is formed in the course of hemostasis or thrombosis, its concentration must be carefully controlled to prevent further fibrin formation or platelet activation. This is achieved in two ways. Thrombin circulates as its inactive precursor, prothrombin, which is activated as a result of a cascade of enzymatic reactions, each converting an inactive zymogen to an active enzyme and leading finally to the conversion of prothrombin to thrombin (Figure 55–1). At each point in the cascade, feedback mechanisms produce a delicate balance of activation and inhibition. The concentration of factor XII in plasma is approximately 30 μg/mL, while that of fibrinogen is 3 mg/mL, with intermediate clotting factors increasing in concentration as one proceeds down the cascade; these facts illustrate that the clotting cascade provides amplification. The second means of controlling thrombin activity is the inactivation of any thrombin formed by circulating inhibitors, the most important of which is antithrombin (see below).
Four naturally occurring thrombin inhibitors exist in normal plasma. The most important is antithrombin, which contributes approximately 75% of the antithrombin activity. Antithrombin can also inhibit the activities of factors IXa, Xa, XIa, XIIa, and VIIa complexed with tissue factor. α2-Macroglobulin contributes most of the remainder of the antithrombin activity, with heparin cofactor II and α1-antitrypsin acting as minor inhibitors under physiologic conditions.
The endogenous activity of antithrombin is greatly potentiated by the presence of sulfated glycosaminoglycans (heparans) (see Chapter 48). Heparans bind to a specific cationic site of antithrombin, which induces a conformational change that promotes binding of antithrombin to thrombin, as well as to its other substrates. This mechanism is the basis for the use of heparin, a derivatized heparan, in clinical medicine to inhibit coagulation. The anticoagulant effects of heparin can be antagonized by strongly cationic polypeptides such as protamine, which bind strongly to heparin, thus inhibiting heparin binding to antithrombin.
Low-molecular-weight heparins (LMWHs), derived from enzymatic or chemical cleavage of unfractionated heparin, are finding increasing clinical use. They can be administered subcutaneously at home, have greater bioavailability than unfractionated heparin, and do not need frequent laboratory monitoring.
Individuals with inherited deficiencies of antithrombin are prone to develop venous thrombosis, providing evidence that antithrombin has a physiologic function and that the coagulation system in humans is normally in a dynamic state.
Thrombin is involved in an additional regulatory mechanism that operates in coagulation. It combines with thrombomodulin, a glycoprotein present on the surfaces of endothelial cells. The complex activates protein C on the endothelial protein C receptor. In combination with protein S, activated protein C (APC) degrades factors Va and VIIIa, thereby limiting their actions in coagulation. A genetic deficiency of either protein C or protein S can cause venous thrombosis. Furthermore, patients with factor V Leiden (which has a glutamine residue in place of an arginine at position 506) have an increased risk of venous thrombotic disease because factor V Leiden is resistant to inactivation by APC; this condition is.
Coumarin Anticoagulants Inhibit the Vitamin K–Dependent Carboxylation of Factors II, VII, IX, & X
The coumarin drugs (eg, warfarin), which are used as anticoagulants, inhibit the vitamin K–dependent carboxylation of Glu to Gla residues (see Chapter 44) in the amino terminal regions of factors II, VII, IX, and X and also proteins C and S. These proteins, all of which are synthesized in the liver, are dependent on the Ca2+-binding properties of the Gla residues for their normal function in the coagulation pathways. Coumarins inhibit the reduction of the quinone derivatives of vitamin K to the active hydroquinone forms (see Chapter 44). Thus, the administration of vitamin K will bypass the coumarin-induced inhibition and allow the post-translational modification of carboxylation to occur. Reversal of coumarin inhibition by vitamin K requires 12 to 24 hours, whereas reversal of the anticoagulant effects of heparin by protamine is almost instantaneous.
Heparin and warfarin are used in the treatment of thrombotic and thromboembolic conditions, such as deep vein thrombosis and pulmonary embolism. Heparin is administered first, because of its prompt onset of action, whereas warfarin takes several days to reach full effect. Their effect is not well predictable by dosage, and thus because of the risk of producing hemorrhage, these drugs are closely monitored by use of appropriate tests of coagulation (see below).
New oral inhibitors of thrombin (dabigatran) or of factor Xa (rivaroxaban, apixaban and others) are also used in the prevention and treatment of thrombotic conditions. These drugs are advantageous because their effect is predictable based on the dose, and some do not require routine monitoring by laboratory tests.
There Are Several Hereditary Bleeding Disorders, Including Hemophilia A
Inherited deficiencies of the clotting system that result in bleeding are found in humans. The most common is deficiency of factor VIII, causing hemophilia A, an X chromosome-linked disease. Hemophilia B, also X chromosome-linked, is due to a deficiency of factor IX and has recently been identified as the form of hemophilia that played a major role in the history of the royal families of Europe. The clinical features of hemophilia A and B are almost identical, but these two diseases can be readily distinguished on the basis of specific assays for the two factors.
The gene for human factor VIII measures 186 kb in length, and contains 26 exons that encode a protein of 2351 amino acids. A variety of mutations in the factor VIII and IX genes have been detected leading to diminished activities of the factor VIII and IX proteins; these include partial gene deletions and point and missense mutations. Prenatal diagnosis by DNA analysis after chorionic villus sampling is now possible (see Figure 39–9).
In the past, treatment for patients with hemophilia A and B consisted of administration of cryoprecipitates (enriched in factor VIII) prepared from individual donors or lyophilized factor VIII or IX concentrates prepared from very large plasma pools. It is now possible to prepare factors VIII and IX by recombinant DNA technology (see Chapter 39). Such preparations are free of contaminating viruses (eg, hepatitis A, B, C, or HIV-1) found in human plasma, but are expensive; their use will increase as the cost of production decreases.
The most common hereditary bleeding disorder is von Willebrand disease, with a prevalence of up to 1% of the population. It results from a deficiency or defect in von Willebrand factor, a large multimeric glycoprotein that is secreted by endothelial cells and platelets into the plasma, where it stabilizes factor VIII. von Willebrand factor also promotes platelet adhesion at sites of vessel wall injury (see below).
Fibrin Clots Are Dissolved by Plasmin
As stated above, the coagulation system is normally in a state of dynamic equilibrium in which fibrin clots are constantly being laid down and dissolved. This latter process is termed fibrinolysis. Plasmin, the serine protease mainly responsible for degrading fibrin and fibrinogen, circulates in the form of its inactive zymogen, plasminogen (90 kDa), and any small amounts of plasmin that are formed in the fluid phase under physiologic conditions are rapidly inactivated by the fast-acting plasmin inhibitor, α2-antiplasmin. Plasminogen binds to fibrin and thus becomes incorporated in clots as they are produced; since plasmin that is formed when bound to fibrin is protected from α2-antiplasmin, it remains active. Activators of plasminogen of various types are found in most body tissues, and all cleave the same Arg-Val bond in plasminogen to produce the disulfide bridge-linked two-chain serine protease, plasmin (Figure 55–6). The specificity of plasmin for fibrin is another mechanism to regulate fibrinolysis. Via one of its kringle domains, plasmin(ogen) specifically binds lysine residues on fibrin and so is increasingly incorporated into the fibrin mesh as it cleaves it. (Kringle domains [Figure 55–2] are common protein motifs of about 100-amino-acid residues in length; they have a characteristic covalent structure defined by a pattern of three disulfide bonds.) Thus, the carboxypeptidase TAFIa (activated thrombin activatable fibrinolysis inhibitor) (Figure 55–6), which removes terminal lysines from fibrin, is able to inhibit fibrinolysis. Thrombin activates TAFI to TAFIa, thereby inhibiting fibrinolysis during clot formation.
Initiation of fibrinolysis by the activation of plasminogen to plasmin. Scheme of sites and modes of action of tissue plasminogen activator (t-PA), urokinase, plasminogen activator inhibitor, α2-antiplasmin, and thrombin-activatable fibrinolysis inhibitor (TAFIa).
Tissue plasminogen activator (t-PA) (Figures 55–2 and 55–6) is a serine protease that is released into the circulation from vascular endothelium under conditions of injury or stress and is catalytically inactive unless bound to fibrin. Upon binding to fibrin, t-PA cleaves plasminogen within the clot to generate plasmin, which in turn digests the fibrin to form soluble degradation products and thus dissolves the clot. Neither plasmin nor the plasminogen activator can remain bound to these degradation products, and so they are released into the fluid phase where they are inactivated by their natural inhibitors. Prourokinase is the precursor of a second activator of plasminogen, urokinase. Originally isolated from urine, it is now known that urokinase is synthesized by various cell types including monocytes and macrophages, fibroblasts, and epithelial cells. The main action of urokinase appears to be the degradation of extracellular matrix. Figure 55–6 indicates the sites of action of five proteins that influence the formation and action of plasmin.
Recombinant t-PA & Streptokinase Are Used as Clot Busters
t-PA, marketed as Alteplase, is produced by recombinant DNA methods. It is used therapeutically as a fibrinolytic agent, as is streptokinase, an enzyme secreted by a number of streptococcal bacterial strains. However, the latter is less selective than t-PA, activating plasminogen in the fluid phase (where it can degrade circulating fibrinogen) as well as plasminogen that is bound to a fibrin clot. The amount of plasmin produced by therapeutic doses of streptokinase may exceed the capacity of the circulating α2-antiplasmin, causing fibrinogen as well as fibrin to be degraded and resulting in the bleeding often encountered during fibrinolytic therapy. Because of its relative selectivity for degrading fibrin, recombinant t-PA has been widely used to restore the patency of coronary arteries following thrombosis. If administered early enough, before irreversible damage of heart muscle occurs (about 6 hours after onset of thrombosis), t-PA can significantly reduce the mortality rate from myocardial damage following coronary thrombosis. Streptokinase has also been widely used in the treatment of coronary thrombosis, but has the disadvantage of being antigenic. t-PA has also been used in the treatment of ischemic stroke, peripheral arterial occlusion, deep vein thrombosis and pulmonary embolism.
There are a number of disorders, including cancer and sepsis, in which the concentrations of plasminogen activators increase. In addition, the antiplasmin activities contributed by α1-antitrypsin and α2-antiplasmin may be impaired in diseases such as cirrhosis. Since certain bacterial proteins, such as streptokinase, are capable of activating plasminogen, they may be responsible for the diffuse hemorrhage sometimes observed in patients with disseminated bacterial infections.
Platelet Aggregation Requires Outside-In and Inside-Out Transmembrane Signaling
Platelets normally circulate in an unstimulated disk-shaped form. During hemostasis or thrombosis, platelets become activated and help form hemostatic plugs or thrombi (Figure 55–7). Three major steps are involved: (1) adhesion to exposed collagen in blood vessels, (2) release (exocytosis) of the contents of their storage granules, and (3) aggregation.
(A) Diagrammatic representation of platelet activation by collagen, thrombin, thromboxane A2 and ADP, and inhibition by prostacyclin. The external environment, the plasma membrane, and the inside of a platelet are depicted from top to bottom. Platelet responses include, depending on the agonist, change of platelet shape, release of the contents of the storage granules, and aggregation. (AC, adenylyl cyclase; cAMP, cyclic AMP; COX-1, cyclooxgenase-1; cPLA2, cytosolic phospholipase A2; DAG, 1,2-diacylglycerol; GP, glycoprotein; IP, prostacyclin receptor; IP3, inositol 1,4,5-trisphosphate; P2Y1, P2Y12, purinoceptors; PAR, protease activated receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PL, phospholipid; PLCβ, phospholipase Cβ; PLCγ, phospholipase Cγ; TP, thromboxane A2 receptor; TxA2, thromboxane A2; VWF, von Willebrand factor.) The G proteins that are involved are not shown. (B) Diagrammatic representation of platelet aggregation mediated by fibrinogen binding to activated GPIIb-IIIa molecules on adjacent platelets. Signaling events initiated by all aggregating agents transform GPIIb-IIIa from its resting state to an activated form that can bind divalent fibrinogen or, at the high shear that occurs in small vessels, multivalent von Willebrand factor.
Platelets adhere to collagen via specific receptors on the platelet surface, including the glycoprotein complexes GPIa-IIa (α2β1 integrin; Chapter 52) and GPIb-IX-V, and GPVI. The binding of GPIb-IX-V to collagen is mediated via von Willebrand factor; this interaction is especially important in platelet adherence to the subendothelium under conditions of high shear stress that occur in small vessels and partially stenosed arteries.
Platelets that are bound to collagen change shape and spread out on the subendothelium. These adherent platelets release the contents of their storage granules (the dense granules and the alpha granules); some of the molecules released amplify the responses to vessel wall injury. Granule release is also stimulated by thrombin.
Thrombin, formed from the coagulation cascade, is the most potent activator of platelets and initiates activation by interacting with its receptors PAR (protease-activated receptor)-1, PAR-4, and GPIb-IX-V on the platelet plasma membrane (Figure 55–7A). The further events leading to platelet activation upon binding to PAR-1 and PAR-4 are examples of outside-in transmembrane signaling, in which a chemical messenger outside the cell generates effector molecules inside the cell. In this instance, thrombin acts as the external chemical messenger (stimulus or agonist). The interaction of thrombin with its G protein–coupled receptors PAR-1 and PAR-4 stimulates the activity of an intracellular phospholipase Cβ (PLCβ). This enzyme hydrolyzes the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2, a polyphosphoinositide) to form the two internal effector molecules (1,2-diacylglycerol (DAG) and 1,4,5-inositol trisphosphate (IP3); see Figure 42–7).
Hydrolysis of PIP2 is also involved in the action of many hormones and drugs. DAG stimulates protein kinase C, which phosphorylates the protein pleckstrin (47 kDa). This results in aggregation and release of the contents of the storage granules. ADP released from dense granules can also activate platelets via its specific G protein–coupled receptors (Figure 55–7A), resulting in aggregation of additional platelets. IP3 causes release of Ca2+ into the cytosol mainly from the dense tubular system (or residual smooth endoplasmic reticulum from the megakaryocyte), which then interacts with calmodulin and myosin light chain kinase, leading to phosphorylation of the light chains of myosin. These chains then interact with actin, causing changes of platelet shape.
Collagen-induced activation of a platelet cytosolic phospholipase A2 by increased levels of intracellular Ca2+ results in liberation of arachidonic acid from platelet membrane phospholipids, leading to the formation of thromboxane A2 (see Chapter 23). Thromboxane A2, in turn, by binding to its specific G protein–coupled receptor, can further activate PLCβ, promoting platelet aggregation (Figure 55–6A).
Activated platelets, besides forming a platelet aggregate, accelerate the activation of factor X and prothrombin by exposing the anionic phospholipid phosphatidylserine on their membrane surface (Figure 55–1). Polyphosphate, released from the dense granules, accelerates factor V activation and also accelerates factor XI activation by thrombin.
All of the aggregating agents, including thrombin, collagen, ADP, and others such as platelet-activating factor, via an inside-out signaling pathway, modify the platelet surface glycoprotein complex GPIIb-IIIa (αIIbβ3; see Chapter 52) so that the receptor has a higher affinity for fibrinogen or von Willebrand factor (Figure 55–7B). Molecules of divalent fibrinogen or multivalent von Willebrand factor then link adjacent activated platelets to each other, forming a platelet aggregate. von Willebrand factor-mediated platelet aggregation occurs under conditions of high shear stress. Some agents, including epinephrine, serotonin, and vasopressin, exert synergistic effects with other aggregating agents.
Endothelial Cells Synthesize Prostacyclin & Other Compounds That Affect Clotting & Thrombosis
The endothelial cells in the walls of blood vessels make important contributions to the overall regulation of hemostasis and thrombosis. As described in Chapter 23, these cells synthesize the prostanoid prostacyclin (PGI2), a potent inhibitor of platelet aggregation. Prostacyclin acts by stimulating the activity of adenylyl cyclase in the surface membranes of platelets via its G protein-coupled receptor (Figure 55–7A). The resulting increase of intraplatelet cAMP opposes the increase in the level of intracellular Ca2+ produced by IP3 and thus inhibits platelet activation. This is in contrast with the effect of the prostanoid thromboxane A2 that is formed by activated platelets, which is that of promoting aggregation. Endothelial cells play other roles in the regulation of thrombosis. For instance, these cells possess an ADPase, which hydrolyzes ADP, and thus opposes its aggregating effect on platelets. In addition, these cells appear to synthesize heparan sulfate, an anticoagulant, and they also synthesize plasminogen activators, which may help dissolve thrombi. Table 55–3 lists some molecules produced by endothelial cells that affect thrombosis and fibrinolysis. Nitric oxide (endothelium-derived relaxing factor) is discussed in Chapter 49.
TABLE 55–3Molecules Synthesized by Endothelial Cells That Play a Role in the Regulation of Thrombosis and Fibrinolysis ||Download (.pdf) TABLE 55–3 Molecules Synthesized by Endothelial Cells That Play a Role in the Regulation of Thrombosis and Fibrinolysis
|Molecule ||Action |
|ADPase (CD39, an ectoenzyme) ||Degrades ADP (an aggregating agent of platelets) to AMP + Pi |
|Nitric oxide (NO) ||Inhibits platelet adhesion and aggregation by elevating levels of cGMP |
|Prostacyclin (PGI2, a prostaglandin) ||Inhibits platelet aggregation by increasing levels of cAMP |
|Thrombomodulin (a glycoprotein) ||Binds thrombin, which then cleaves protein C to yield activated protein C; this in combination with protein S degrades factors Va and VIIIa, limiting their actions |
|Endothelial protein C receptor (EPCR, a glycoprotein) ||Facilitates protein C activation by the thrombin-thrombomodulin complex |
|Tissue plasminogen activator (t-PA, a protease) ||Activates plasminogen to plasmin, which digests fibrin; the action of t-PA is opposed by plasminogen activator inhibitor-1 (PAI-1) |
Analysis of the mechanisms of uptake of atherogenic lipoproteins, such as LDL, by endothelial, smooth muscle, and monocytic cells of arteries, along with detailed studies of how these lipoproteins damage such cells is a key area of study in elucidating the mechanisms of atherosclerosis (see Chapter 26).
Aspirin Is One of Several Effective Antiplatelet Drugs
Antiplatelet drugs inhibit platelet responses. The most commonly used antiplatelet drug is aspirin (acetylsalicylic acid), which irreversibly acetylates and thus inhibits the platelet cyclooxygenase (COX-1) involved in formation of thromboxane A2 (see Chapter 15), a potent aggregator of platelets and also a vasoconstrictor. Platelets are very sensitive to aspirin; as little as 30 mg/d (one regular aspirin tablet contains 325 mg) effectively eliminates the synthesis of thromboxane A2. Aspirin also inhibits production of prostacyclin (PGI2, which opposes platelet aggregation and is a vasodilator) by endothelial cells, but unlike platelets, these cells regenerate cyclooxygenase within a few hours. Thus, the overall balance between thromboxane A2 and prostacyclin can be shifted in favor of the latter, opposing platelet aggregation. Indications for treatment with aspirin include management of acute coronary syndromes (angina, myocardial infarction), acute stroke syndromes (transient ischemic attacks, acute stroke), severe carotid artery stenosis, and primary prevention of these and other atherothrombotic diseases.
Other antiplatelet drugs include clopidogrel, prasugrel, and ticagrelor, specific inhibitors of the P2Y12 receptor for ADP, and antagonists of ligand binding to GPIIb-IIIa (eg, abciximab) that interfere with fibrinogen and von Willebrand factor binding and thus platelet aggregation.
Laboratory Tests Measure Coagulation, Thrombolysis, & Platelet Aggregation
A number of laboratory tests are available to measure the phases of hemostasis described above. The tests include platelet count, bleeding time/closure time, platelet aggregation, activated partial thromboplastin time (aPTT or PTT), prothrombin time (PT), thrombin time (TT), concentration of fibrinogen, fibrin clot stability, and measurement of fibrin degradation products. The platelet count quantitates the number of platelets. The skin bleeding time is an overall test of platelet and vessel wall function, while the closure time measured using the platelet function analyzer PFA-100 is an in vitro test of platelet-related hemostasis. Platelet aggregation measures responses to specific aggregating agents. aPTT is a measure of the intrinsic pathway and PT of the extrinsic pathway, with aPTT being used to monitor heparin therapy and PT, to measure the effectiveness of warfarin. The reader is referred to a textbook of hematology for a discussion of these tests.