The Molecular Basis of Blood Coagulation

The Molecular Basis of Blood Coagulation S Schulman and B Furie, Beth Israel Deaconess Medical Center, Boston, MA, USA Ó 2014 Elsevier Inc. All rights reserved.

Glossary aIIbb3 Integrin An integrin family protein on the surface of platelets that binds fibrinogen. Antithrombin III (AT-III) A serine protease inhibitor that irreversibly inhibits thrombin in a reaction accelerated by the anticoagulant heparin. Factor VIII A procofactor that, upon activation, interacts with factor IXa to form the ‘tenase’ complex; deficiency of factor VIII is the basis for hemophilia A. Fibrinogen A fibrous hexamer that polymerizes upon cleavage by thrombin to form fibrin clot. Protease-activated receptor-1 (PAR-1) A G proteincoupled receptor activated by thrombin on the surface of platelets. Protein C A vitamin K-dependent serine protease that, upon activation, downregulates coagulation by inactivating factors V and VIII.

Introduction

Molecular Basis of Coagulation

Human life depends upon a high-pressure circulatory system to maintain perfusion and thus requires a robust hemostatic response to prevent exsanguination following injury. The host defense to tissue damage is tripartite and well orchestrated, with critical contributions by cooperating platelets, endothelial cells, and the circulating blood coagulation proteins. Coagulation arose early in evolution as a primitive innate immune system and achieved its recognizable core in the jawless fish at least 450 million years ago (Davidson et al., 2003), since which time it has evolved multiple layers of regulation. Indeed, all vertebrates have a blood coagulation system, most of which are remarkably similar to the human system. The human blood coagulation cascade is an intricately complex web of positive and negative feedback loops finely tuned to ensure hemostasis without spontaneous hemorrhage or thrombosis. This delicate balance is underscored by the numerous inherited or acquired hematologic disorders predisposing to hemorrhage, as in hemophilia A caused by deficiency of factor VIII, or thrombosis, as with the common missense mutation R506Q better known as factor V Leiden. The medicinal leech Hirudo medicinalis perturbs this fine equilibrium by injecting its victim with the direct thrombin inhibitor hirudin prior to blood feeding, and a large panel of pharmacologic agents is now available to modulate human blood coagulation. This article will focus on the molecular basis of blood coagulation with particular attention to the biochemistry and regulation of this pathway as it relates to humans in health and disease.

Reference Module in Biomedical Research, 3rd edition

Protein S A vitamin K-dependent cofactor for protein C. Prothrombin The final zymogen of the blood coagulation cascade, which acts to convert fibrinogen to fibrin clot. P-selectin A cell surface molecule present on platelets and endothelial cells that facilitates an adhesive interaction with its receptor PSGL-1 on leukocytes. P-selectin glycoprotein ligand 1 (PSGL-1) A molecule on leukocytes that serves as the cell surface receptor for P-selectin. Thrombomodulin A cell surface cofactor that interacts with thrombin to activate protein C and halt blood coagulation. Tissue factor A cell surface molecule that initiates blood coagulation in vivo as well as an intracellular signaling cascade.

The Proteases The core of the evolutionarily conserved blood coagulation pathway comprises a cascade of proteases and a series of cofactors modulating these proteases to control the generation of fibrin at sites of injury (Table 1). Hemostasis is too rapid to tolerate transcriptional or translational regulation, so these proteins instead circulate in the plasma as inactive zymogens and procofactors that are activated by regulated limited proteolysis. The core vitamin K-dependent proteases involved in blood coagulation and the regulation of blood coagulation are prothrombin, factor VII, factor IX, factor X, and protein C. These zymogens are all serine proteases whose C-terminal catalytic domain has significant homology to trypsin and chymotrypsin (Furie et al., 1982). In comparison to these digestive proteases, which function ‘externally’, these plasma proteases require greater substrate specificity and spatial control of activation within the blood. These proteases achieve these constraints in part via their modular and diverse N-terminal regions, which include numerous self-folding protein–protein and protein–membrane interaction domains (Figure 1). A number of sequence features and structural domains are common to these proteases (Irwin et al., 1985; Degen and Davie, 1987; Leytus et al., 1986; Yoshitake et al., 1985; O’Hara et al., 1987; Foster et al., 1985). First, all are secretory proteins specified by an N-terminal signal sequence of 15–30 amino acids, ensuring their translocation into the endoplasmic reticulum en route to the plasma. While in the endoplasmic

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The Molecular Basis of Blood Coagulation

Table 1

Properties of the genes, mRNAs, and gene products of the components of the blood coagulation cascade

Prothrombin Factor X Factor IX Factor VII Factor VIII Factor V Factor XI Factor XII Fibrinogen Aa-chain Bb-chain g-chain Tissue factor Factor XIII

Molecular weight

Gene (kb)

Chromosome

mRNA

Exons (kb)

Plasma concentration (mg ml 1)

Function

72 000 56 000 56 000 50 000 330 000 330 000 160 000 80 000 340 000 64 000 56 000 47 000 45 000 320 000

21 22 34 13 185 7.0 23 12

11p11–q12 13q34 Xq26–27.3 13q34 Xq28 1q21–25 15 5

2.1 1.5 2.8 2.4 9.0 7.0

100 10 5 0.5 0.1 10

2.4

14 8 8 8 26 25 5 14

30 3000

Protease zymogen Protease zymogen Protease zymogen Protease zymogen Cofactor Cofactor Protease zymogen Protease zymogen Structural

12

4q26–q28 4q26–q28 4q26–q28 1pter–p12

5 8 9 2.1

6

0.1 60

Cofactor/initiator Fibrin stabilization

Adapted from Furie, B., Furie, B.C., 1988. The molecular basis of blood coagulation. Cell 53, 505–518 and reprinted with permission from Elsevier.

reticulum, a short propeptide (Jorgensen et al., 1987) of w18 residues directs these proteins to undergo an unusual vitamin K-dependent posttranslational modification in which 10–12 glutamate residues in the adjacent g-carboxyglutamic acid (Gla) domain are converted to Gla; this modification enables these proteins to chelate Ca2þ ions (Furie et al., 1979) and bind to negatively charged phospholipids that become exposed at sites of tissue injury (Nelsestuen, 1976; Prendergast and Mann, 1977; Borowski et al., 1986). With the exception of prothrombin, all of these core proteases also contain two epidermal growth factor (EGF)-like domains, a common structural motif of 43–50 amino acids that facilitates protein– protein interaction and complex formation (Stenflo, 1999). Prothrombin instead contains a pair of kringle domains of approximately 100 amino acids in length whose unique molecular surfaces provide specificity for assembly of the prothrombinase complex (Kotkow et al., 1995; Buddai et al., 2010). While not core proteases of the blood coagulation cascade, factors XI and XII are proteases involved in initiating the intrinsic pathway of coagulation in vitro (see below). Their role in blood coagulation in vivo is uncertain, but they may contribute more significantly to thrombosis than the normal hemostatic process (Zhang et al., 2010; Rosen et al., 2002; Renne et al., 2005; Wang et al., 2005). Both of these zymogens contain serine protease catalytic domains with homology to thrombin (Cool et al., 1985; Que and Davie, 1986; Fujikawa et al., 1986). Factor XII is a protein of 80 000 molecular weight consisting of a disulfide-linked light and heavy chain encoded by a single polypeptide chain. The light chain contains the serine protease catalytic domain, while the heavy chain includes a kringle domain flanked by two EGF-like domains and two fibronectin type domains. Factor XI is a protein composed of disulfide-linked homodimers of 160 000 molecular weight that is activated by factor XIIa. Each subunit of factor XIa contains a disulfide-bonded heavy and light chain following activation. A number of striking features of these proteins and the genes encoding them provide insight into the evolution of human blood coagulation. Both single nucleotide polymorphism analysis at highly conserved amino acids and dendrograms

indicate that prothrombin is the most ancient protease of coagulation. Additional proteases in the cascade likely arose through gene duplication events (Patthy, 1985), with those enzymes acting most remotely from prothrombin arising most recently (Davidson et al., 2003). The modular nature of the coagulation proteins also likely enabled their complexity; most of these small domains are encoded by single exons that could be shuffled to achieve diversity (Gilbert, 1978).

The Cofactors A number of cofactors without intrinsic enzymatic activity interact with the proteases of blood coagulation to alter either catalytic efficiency or substrate specificity. By this definition, protein cofactors regulating the coagulation cascade include tissue factor, factor V, factor VIII, protein S, and thrombomodulin. Factors V and VIII exist as procofactors of molecular weight 330 000 that require proteolytic cleavage at two sites in order to effectively stimulate factors Xa and IXa, respectively. These procofactors diverged after a gene duplication event and are thus closely related to one another but distinct from the other proteins involved in blood coagulation (Church et al., 1984). Cofactors V and VIII consist of three homologous A domains, a B domain, and two homologous C domains arranged linearly in primary sequence as A1-A2-B-A3-C1-C2. X-linked deficiency of factor VIII is the basis for hemophilia A. During downregulation of coagulation (see below), both factors Va and VIIIa are susceptible to inactivation by activated protein C via a proteolytic mechanism (van ’t Veer et al., 1997). The Arg506Gln polymorphism in factor V results in activated protein C resistance and is known as factor V Leiden, the most common inherited thrombophilia in people of European descent (Bertina et al., 1994). Tissue factor is an integral membrane protein that acts as a receptor for factors VII and VIIa to initiate blood coagulation. This 263 amino acid protein consists of an N-terminal ectodomain of 219 residues, a single transmembrane segment, and a 21-residue cytoplasmic tail lipidated through a thioester bond to cysteine (Morrissey et al., 1987; Bach et al., 1988). Tissue factor is constitutively expressed on the surface of most nonvascular cells and is inducible in endothelial cells and

The Molecular Basis of Blood Coagulation

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Figure 1 Modular domain organization of the proteases, cofactors, and regulatory proteins central to blood coagulation. The key highlights important sequence or structural elements common to many proteins involved in blood coagulation, including signal peptide, propeptide, Gla domain, aromatic amino acid stack domain, kringle region, epidermal growth factor (EGF) domain, fibronectin domains (types I and II), and the serine protease catalytic domain. Proteolytic cleavage sites related to protein maturation and zymogen activation are indicated with vertical or diagonal arrows, respectively. FIX, factor IX, etc.; PC, protein C; Pmg, plasminogen; PS, protein S; PT, prothrombin; PUK, prourokinase; tPA, tissue plasminogen activator; T, thrombin. Reprinted from Furie, B., Furie, B.C., 2009. Molecular basis of blood coagulation. In: Hoffman, R., Furie, B., Benz, E.J., Mcglave, P., Silberstein, L.E., Shattil, S.J. (Eds.), Hematology: Basic Principles and Practice, fifth ed. Churchill Livingstone Elsevier, Philadelphia, PA with permission from Elsevier.

circulating blood cells by injurious stimuli; tissue factor also circulates on the surface of microparticles and in trace amounts in a soluble form. In the presence of Ca2þ, tissue factor binds to factor VIIa, stimulating its catalytic activity remarkably, hence justifying its classification as a cofactor. Protein S is a vitamin K-dependent protein that serves as a cofactor to activated protein C, stimulating its ability to cleave and hence inactivate factors Va and VIIIa (Norstrom et al., 2003). Like protein C, protein S contains an N-terminal signal sequence, propeptide, and Gla domain. However, it lacks a catalytic protease domain, and the remainder of its structure is composed of a sequence of four EGF domains followed by

a pair of laminin G domains, an interaction motif common to proteins of the extracellular matrix (Dahlback et al., 1986). These EGF domains are curious in that they are the substrate for a posttranslational modification resulting in the unusual b-hydroxyaspartic acid and b-hydroxyasparagine amino acids (Stenflo et al., 1987). More than half of protein S circulates in blood bound to complement protein C4b (Dahlback, 1986), suggesting a provocative link between inflammation and thrombosis. Thrombomodulin is cell surface receptor that binds thrombin in a 1:1 stoichiometric complex to dramatically alter thrombin’s substrate specificity (Esmon et al., 1982). Whereas

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The Molecular Basis of Blood Coagulation

thrombin typically drives blood coagulation by cleaving fibrinogen, stimulating platelets via protease-activated receptors, and activating factors V, VIII, and XI, thrombomodulin inhibits the action of thrombin on each of these substrates and promotes the cleavage of protein C to downregulate the cascade. Thrombomodulin is a 75 000 molecular weight protein and is comprised of six EGF-like domains, a single transmembrane segment, and short cytosolic tail (Jackman et al., 1986; Wen et al., 1987). These EGF-like domains provide a binding site for both thrombin and protein C.

Generation of a Fibrin Clot The blood coagulation cascade culminates with the conversion of fibrinogen to fibrin, essentially transmitting the proteolytic injury signal into a fibrin clot capable of occluding the inciting tissue defect. Fibrinogen is the most abundant coagulation protein in plasma, consistent with its mechanical rather than signaling role. Fibrinogen is a 340 000 molecular weight hexamer comprising two Aa chains, two Bb chains, and two g chains (McKee et al., 1970). These chains are covalently stabilized by a series of disulfide bridges in a region known as the disulfide knot, which includes an N-terminal link between one set of Aa, Bb, and g chains as well as disulfides connecting neighboring Aa and g chains to one another. The fibrinogen molecule is nodular, comprising three globular domains connected by a fibrous linker (Fowler and Erickson, 1979). The N-terminal disulfide knot emerges from the E domain, where all six polypeptide chains converge. A single intertwined Aa, Bb, and g chain diverge to form each D domain, from which a short C-terminal tail protrudes from each polypeptide. This C-terminal tail of the g chain is the binding site for

glycoprotein IIb/IIIa on the surface of platelets and is thus a critical mediator of platelet aggregation. Thrombin proteolysis of the Aa and Bb chains releases short N-terminal fibrinopeptides A and B, respectively, to generate fibrin. After fibrinopeptide cleavage, the new conformation of the D domain enables association with both the D and E domains of neighboring fibrin molecules (Doolittle, 2003). These fibrin monomers then spontaneously polymerize via a series of DE and DD interactions to form long protofibrils that subsequently associate laterally to form thick and branched fibrils (Figure 2). These fibrin oligomers are initially only associated noncovalently, but can be stabilized covalently through the action of factor XIII (Ariens et al., 2002). Once activated by thrombin, factor XIIIa is a transglutaminase that cross-links lysine and glutamic acid residues on adjacent Aa and Bb chains to render fibrin polymerization irreversible and thus sturdy the nascent clot (Loewy, 1972). Factor XIII deficiency in humans results in moderate to severe bleeding owing to premature dissolution of unstable clot by fibrinolysis. Because fibrin itself is also able to interact with and compete for thrombin (Liu et al., 1979), fibrin helps to regulate its own accumulation. For this reason, some patients with inherited defects in fibrinogen are susceptible to thromboembolic disease in addition to bleeding. These disorders of fibrinogen are classified as quantitative (type I), which includes hypofibrinogenemia and afibrinogenemia, or as qualitative (type II), as in the dysfibrinogenemias.

The Vitamin K Cycle Sustains Blood Coagulation The vitamin K-dependent g-carboxylation of glutamic acid is fueled by a vitamin K redox cycle in the endoplasmic reticulum

Figure 2 Assembly of fibrin strands. (a) The fibrin hexamer (a2b2g2) has a trinodal domain structure consisting of an E domain connected through coiled-coil linkers to a pair of D domains. (b) Two fibrin molecules form a staggered dimer. (c) Polymerization of fibrin through DD and DE contacts to create long protofibrils that eventually associate laterally to create thick fibrils. (d) The transglutaminase activity of factor XIIIa covalently links the a and g chains of neighboring fibrin molecules to stabilize the fibrin assembly.

The Molecular Basis of Blood Coagulation membrane (Figure 3). The cycle begins with the g-carboxylation of select glutamic acid residues in secreted proteins specified by a unique propeptide containing a g-carboxylation recognition site (Kurachi and Davie, 1982; Jorgensen et al., 1987; Knobloch and Suttie, 1987). During the reaction, carbon dioxide is fixed to glutamic acid with the concomitant oxidation of vitamin K hydroquinone to vitamin K epoxide (Esmon et al., 1975). As mammals are unable to synthesize vitamin K de novo, this epoxide form of vitamin K must be recycled to regenerate the hydroquinone and thus complete the so-called vitamin K cycle. This reductive arm of the pathway is primarily contributed by the vitamin K epoxide reductase (VKOR) (Rost et al., 2004; Li et al., 2004). VKOR cooperates with a reduced thioredoxinlike protein (Silverman and Nandi, 1988; Wajih et al., 2007; Johan et al., 1987; Schulman et al., 2010; Li et al., 2010) to reduce vitamin K epoxide in two successive steps, first catalyzing the conversion to vitamin K quinone and then on to the hydroquinone (Jin et al., 2007). Both of these steps catalyzed by VKOR are inhibited by coumarin family anticoagulants including warfarin, the most commonly used oral anticoagulant and rodenticide. The g-carboxylation of glutamate is a rare posttranslational modification, with only 14 so-called vitaminK dependent proteins described in humans. Of these, exactly

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half are involved in blood coagulation, including prothrombin and factors VII, IX, and X, and regulatory proteins C, S, and Z (Furie et al., 1999). For these proteins, this peculiar posttranslational modification is required for Ca2þ-dependent activation where negatively charged phospholipids become exposed at sites of tissue injury (Soriano-Garcia et al., 1992; Zhang and Castellino, 1993). When warfarin is administered, the vitamin K-dependent proteins are undercarboxylated, decreasing the rate of thrombin generation and inhibiting blood coagulation. Deficiency of either the vitamin K-dependent g-carboxylase or VKOR results in the rare bleeding disorders vitamin K-dependent clotting factor deficiency types I and II, respectively (Weston and Monahan, 2008).

Activation of Blood Coagulation Activation In Vitro The biochemical cascade culminating in the thrombindependent deposition of fibrin clot has historically been divided into two independent pathways that converge to share the final steps in common (Davie and Ratnoff, 1964; Macfarlane, 1964) (Figure 4). These pathways are classified as

PT

PT

CO2 O2 GGCX

Warfarin KH 2

KO

VKOR

VKOR

SH SH

S–S

SH SH

K

S–S

SH SH

S S

Figure 3 The vitamin K cycle sustains blood coagulation. The vitamin K-dependent g-glutamyl carboxylase (GGCX) modifies propeptide-containing Gla proteins such as prothrombin (PT). Vitamin K epoxide (KO) is reduced by the vitamin K oxidoreductase (VKOR) in two successive steps, first to vitamin K quinone (K) and then to the hydroquinone (KH2) to complete the vitamin K cycle. The reducing equivalents for VKOR are provided by endoplasmic reticulum thioredoxin-like proteins, which become reduced by participation in oxidative protein folding of nascent secretory proteins (oxidative folding scheme only shown for K reduction for simplicity, but likely applies to KO reduction as well). Both of the reductive reactions catalyzed by VKOR are sensitive to inhibition by the anticoagulant warfarin.

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The Molecular Basis of Blood Coagulation

Intrinsic pathway

FXII

FXIIa

FXI

HMWK

Extrinsic pathway

FXIa

TF Ca2+ FIX T

FVIII

FVIIIa

FIXa

FVIIa

Ca2+

Ca2+

FIXa/VIIIa

FX

FVIIa/TF

Ca2+

FXa Ca2+

T

FV

FVII

FVa

FXa/Va

PT

Ca2+ T

FG

F

Figure 4 The blood coagulation cascade in vitro. The intrinsic pathway of blood coagulation is initiated by factor XII that, upon activation to XIIa, triggers cascade involving factors XI, IX, VIII, X, V, prothrombin, and fibrinogen. The extrinsic pathway is activated by tissue factor and drives cascade involving factors VII, X, V, prothrombin, and fibrinogen. Following generation of factor Xa, both the intrinsic and extrinsic pathways share a common pathway that ensures the conversion of fibrinogen to fibrin clot. Proenzymes are indicated by diamonds; procofactors are indicated by squares; enzymes and cofactors are indicated by circles; multiprotein complexes on membranes are indicated by rounded rectangles. Activated proteins and complexes are colored for emphasis. Ca2þ, calcium; F, fibrin; FG, fibrinogen; HMWK, high molecular weight kininogen; PT, prothrombin; T, thrombin; TF, tissue factor. Adapted from Furie, B., Furie, B.C., 1988. The molecular basis of blood coagulation. Cell 53, 505–518 with permission from Elsevier.

the intrinsic pathway, in which all essential components of the cascade are contained within whole blood itself, and the extrinsic pathway, in which a critical initiator of the cascade – tissue factor – must be expressed on an exogenous membrane such as a cell or microparticle surface. These two pathways are more useful in describing the coagulation cascade in vitro than in vivo, but merit further discussion because they are mechanistically insightful and critical to understanding clinical laboratory monitoring of blood coagulation. The intrinsic pathway is also known as the contact pathway because it is initiated when whole blood contacts a negatively charged surface such as glass or kaolin in vitro. The cascade begins when the serine protease kallikrein activates factor XII in a reaction requiring negatively charged surfaces and favored by high molecular weight kininogen (Meier et al., 1977). Factor XIIa then activates factor XI (Fujikawa et al., 1986). In a solution phase reaction strictly dependent on Ca2þ, factor XIa cleaves factor IX at two internal peptide bonds, generating the disulfide-bonded heavy and light chains comprising factor IXa and releasing a small activation peptide (Fujikawa et al., 1974).

Once generated, factor IXa interacts with factor VIIIa and Ca2þ on a membrane surface to generate the ‘tenase’ complex, converting factor X to factor Xa (van Dieijen et al., 1981). In what then becomes the common pathway shared between the intrinsic and extrinsic cascades, factor Xa joins with its cofactor Va on a membrane surface in a Ca2þ-dependent manner to form the ‘prothrombinase’ complex (Mann et al., 1988). The prothrombinase complex efficiently activates prothrombin to thrombin, which converts fibrinogen to fibrin clot. This intrinsic pathway is routinely monitored clinically by the addition of kaolin to plasma; the time required to form a clot is known as the activated partial thromboplastin time (aPTT). The extrinsic pathway is also known as the tissue factor pathway because it requires tissue factor present on cell or microparticle surfaces in addition to plasma to initiate the coagulation cascade. Although the mechanism of activation remains unclear, a trace amount of factor VIIa is always found in circulation. When tissue factor becomes exposed following injury, the extracellular domain forms a Ca2þ-dependent complex with factor VIIa (Guha et al., 1986; Broze et al., 1985).

The Molecular Basis of Blood Coagulation

This tissue factor/VIIa complex acts on two substrates to ramp up coagulation. First, it can activate additional factor VII to VIIa in a positive feedback loop (Pedersen et al., 1989), which is critical due to only trace amounts of factor VIIa present in resting plasma (Morrissey et al., 1993). The tissue factor–factor VIIa complex also activates the proenzyme factor X; factor Xa that is generated is also able to stimulate further activation of factor VII (Radcliffe and Nemerson, 1975). Once factor Xa is formed, the cascade is back in the familiar common coagulation pathway: factor Xa joins with factor Va to form the ‘prothrombinase’ complex, thrombin is generated, and fibrinogen cleaved to form fibrin clot. The extrinsic pathway of blood coagulation is monitored clinically by the addition of exogenous tissue factor to plasma; the time required to form a thrombus is known as the prothrombin time.

Activation In Vivo Although the intrinsic and extrinsic pathways neatly describe coagulation in vitro, a number of observations suggest that the physiologic mechanisms activating coagulation are distinct. Patients with inherited deficiencies of factor XII, prekallikrein, or high molecular weight kininogen have prolonged aPTTs, but these individuals have no bleeding diathesis (Ratnoff and Colopy, 1955; Colman et al., 1975; Wuepper, 1973); furthermore, individuals with factor XI deficiency may not exhibit spontaneous bleeding and may or may not be prone to postoperative bleeding. Together, these observations suggest some of these proximal components of the intrinsic pathway have a limited role in hemostasis in vivo. Next, it appears that the intrinsic and extrinsic pathways are more closely intertwined than our traditional portrait of the cascades would suggest: factor VIIa in complex with tissue factor can activate factor IX (Osterud and Rapaport, 1977), a key component of the intrinsic pathway, in addition to factors VII and X. This suggests a prominent role for the factor VIIIa–factor IXa complex in the tissue factor pathway, consistent with the robust bleeding encountered by individuals with hemophilia A or B (deficiency of factor VIII or factor IX, respectively). Finally, neither the intrinsic nor extrinsic pathways accounts for a subsequently identified P-selectin-dependent pathway of blood coagulation (Palabrica et al., 1992). In this pathway, microparticles decorated with tissue factor and P-selectin glycoprotein ligand 1 (PSGL-1) are targeted to sites of injury by platelet P-selectin (Falati et al., 2003; Giesen et al., 1999). This additional source of tissue factor can further stimulate factor VIIa. A physiologic pathway for blood coagulation in vivo is presented in Figure 5. Whether exposed at a site of tissue injury, on a microparticle, or by a stimulated monocyte, it is tissue factor that initiates the blood coagulation cascade by serving as a cofactor to factor VIIa. Some tissue factor in vivo may initially be in an inactive ‘encrypted’ conformation that requires rearrangement, with formation of a disulfide bond (Maynard et al., 1975; Chen et al., 2006), although there is no experimental proof of this hypothesis. The tissue factor–factor VIIa complex then activates factor IX, factor X, and additional factor VII. As both factor Xa and factor VIIa can activate further factor VII (Radcliffe and Nemerson, 1976), this procoagulant pathway can be rapidly upregulated in a positive feedback loop. Factor IXa will interact with factor VIIIa on a membrane surface to

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form the ‘tenase’ complex, further accelerating the generation of factor Xa. Once thrombin has been generated, it can also activate factor XI independent of the contact pathway (Naito and Fujikawa, 1991), with factor XIa further enhancing activation of factor IX and thus tenase activity. Irrespective of whether generated by the tissue factor–factor VIIa or factor VIIIa–factor IXa complex, factor Xa interacts with its cofactor Va to form the prothrombinase complex on cell membranes. Activation of prothrombin enables deposition of fibrin clot and feeds back to further activate factors Va, VIIIa, and XIa. This scheme still remains uncertain and is likely too simplistic. Recent studies have emphasized that initiation of the tissue factor pathway leads to factor Xa formation and the rapid inhibition of the tissue factor pathway due to the tissue factor pathway inhibitor (TFPI). Factor Xa binds to TFPI, and this complex inhibits the tissue factor–factor VIIa complex. At the same time, the so-called intrinsic pathway is primed to generate large amounts of thrombin with the generation of factor XIa, factor VIIIa, and factor Va (Orfeo et al., 2005). Another uncertainty revolves around the physiologic mechanism and significance of contact pathway activation in vivo. Activation of factor XII is supported by negatively charged surfaces including kaolin (as in measurement of the PTT), high molecular weight dextran sulfate (Citarella et al., 1997), extracellular RNA (Kannemeier et al., 2007), collagen (Wilner et al., 1968), and polyphosphates (Smith et al., 2006; Muller et al., 2009). Polyphosphates are long polymers of inorganic phosphate that are stored in platelet dense granules and secreted upon activation (Morrissey et al., 2012). The demonstration that mice lacking platelet polyphosphate secretion fail to efficiently activate the contact pathway suggests that polyphosphates may be a major physiologic activator of factor XII (Muller et al., 2009). Although the so-called contact pathway appears to have a minimal role in hemostasis in vivo, factors XIIa and XIa are critical for thrombus formation and deficiency of these factors can protect against pathologic thrombosis in multiple disease models (Kleinschnitz et al., 2006; Renne et al., 2005; Wang et al., 2005; Zhang et al., 2010; Rosen et al., 2002). Moreover, severe factor XI deficiency in humans protects against ischemic stroke (Salomon et al., 2008). This uncoupling of hemostasis and thrombosis makes the contact pathway an intriguing target for novel anticoagulant design (Renne, 2010). Another important but incompletely understood class of factors mediating thrombus formation in vivo are the thiol isomerase proteins such as protein disulfide isomerase (PDI). Although best understood for their role in disulfide exchange and oxidative protein folding in the endoplasmic reticulum (Hatahet and Ruddock, 2009), PDI and other related thioredoxin domain-containing proteins are released by activated platelets (Burgess et al., 2000; Cho et al., 2008) and injured endothelium (Hotchkiss et al., 1998; Jasuja et al., 2010). Inhibition of PDI with blocking antibodies (Jasuja et al., 2010; Bennett et al., 2000; Reinhardt et al., 2008; Cho et al., 2008) or small molecule inhibitors (Jasuja et al., 2012) robustly impedes thrombus formation. Some of this effect is related to inhibition of integrin aIIbb3 mediated platelet function (Burgess et al., 2000; Essex et al., 2001; Chen and Hogg, 2006), but fibrin generation due to tissue injury is also inhibited (Jasuja et al., 2010). It has been proposed that thiol isomerases modulate coagulation specifically via deencryption of an allosteric

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The Molecular Basis of Blood Coagulation

TF

FVIIa FVIIa/TF FVII

FVIIa

T

FXI

FVIIa/TF

FXIa

FIX

FIXa

T

FVIII

FVIIIa

FIXa/VIIIa

FX

FXa

FVa

FXa/Va

T

FV

PT T

FG

F

Figure 5 The blood coagulation cascade in vivo. Blood coagulation is initiated when tissue factor (TF) exposed on the cell surface comes into contact with activated factor VII. The TF/VIIa complex aids in the activation of further VIIa as well as IXa and Xa. Many steps of coagulation in vivo – including the generation of factors VIIa, XIa, VIIIa, and Va – are robustly accelerated by positive feedback after the activation of factors Xa, XIa, and thrombin. The final common pathway leading to fibrin clot formation in vivo is identical to that previously illustrated in vitro. Adapted from Furie, B., Furie, B.C., 1988. The molecular basis of blood coagulation. Cell 53, 505–518 with permission from Elsevier.

disulfide bond in tissue factor (Chen et al., 2006; Ahamed et al., 2006), but this mechanism remains controversial. Future work will be required to clarify the mediators by which PDI and other thiol isomerases participate in thrombus formation. The abundance of the blood coagulation factors in plasma provides further insight into the molecular logic that underlies the blood coagulation cascade (Furie and Furie, 1992, 2009; Dahlback, 2000; Davie, 1995). First, a general trend exists that the concentration of coagulation proteins increases as one moves down the cascade (Figure 6). Tissue factor, the initiator of coagulation in vivo, is found at or below concentrations of 100 ng ml1 in human plasma; prothrombin, the final zymogen in the sequence, circulates at 100 mg ml1. This increase in concentration parallels the aforementioned positive feedback loops that amplify the initial small signal that is translated into a major biological event. Next, at

a concentration of 3 mg ml1, fibrinogen is by far the most abundant coagulation protein in plasma. This is consistent with its structural rather than catalytic function in the creation of a fibrin clot. Finally, at a concentration of 100 ng ml1, cofactor VIII is by stoichiometry the limiting reagent in coagulation. This is consistent with its permissive regulatory role and the remarkable impact of its inactivation by activated protein C. An alternative regulatory mechanism in which a more abundant protein is destroyed to dampen coagulation would be much less efficient.

Inactivation and Regulation of Blood Coagulation It is crucial that blood coagulate rapidly at sites of tissue injury, but it is equally important that the process be rapidly halted

The Molecular Basis of Blood Coagulation

9

0.1 g ml–1

TF FVIII 0.5–5 g ml–1

FIX

FVII

FV FX

10–100 g ml–1

FXII FXIII

Prothrombin

3000 g ml–1

Fibrinogen

Figure 6 The exquisite regulation of blood coagulation is in large part due to the abundance of the participating proteins in plasma. Note the apical position of initiators of coagulation (i.e., tissue factor, TF) and key regulators of coagulation (i.e., factor VIII, FVIII). Fibrinogen’s role at the pyramid base is consistent with its structural rather than signaling role. The concentrations of these factors in human plasma are shown to the right of each tier of the pyramid.

once hemostasis is achieved. Moreover, it is critical that the thrombus form specifically at the site of tissue injury and nowhere else. Although some questions linger with respect to what signals the termination of coagulation, a number of mechanisms have been identified to ensure this exquisite spatial and temporal control of coagulation.

Spatial Checks on Coagulation Spatial mechanisms must limit thrombus formation to the site of tissue injury and prevent systemic coagulation. First, the majority of tissue factor, the critical initiating cofactor for the cascade, is embedded in the membranes of cells or microparticles (only a small fraction of tissue factor circulates in a soluble form). The interaction of tissue factor-associated microparticle PSGL-1 and P-selectin translocated to the plasma membrane of activated platelets directs these membranous vesicles to sites of injury (Larsen et al., 1989). Next, many of the clotting reactions proceed efficiently only on activated membrane surfaces, preventing extension beyond the margins of injury or metastasis to remote sites. For example, the tissue factor–factor VIIa complex (Guha et al., 1986), the ‘tenase complex’ (factor IXa–factor VIIIa) (Gilbert et al., 1990; Jones et al., 1985), and the ‘prothrombinase’ complex (factor Xa–factor Va) (Mann et al., 1992) only assemble on negatively charged membranes. Moreover, the vitamin K-dependent enzymes, which include prothrombin and factors VII, IX, and X, interact preferentially with membranes at sites of tissue injury; these membranes are enriched for negatively charged phospholipids such as phosphatidylserine that interact with these factors via their Gla domains in a Ca2þdependent manner (Furie et al., 1999). Many safeguards are in place to sequester activated serine proteases of the coagulation cascade that escape into systemic

circulation. These include a number of plasma protease inhibitors, of which antithrombin III is the most important (Figure 7). Antithrombin III is a glycoprotein comprised of 432 amino acids that is a component of the serpin (SErine Protease INhibitor) family. It forms a 1:1 stoichiometric complex with its target to inactivate it (Gettins, 2002). Serpins like antithrombin III contain a characteristic reactive site loop that is susceptible to proteolytic attack, with formation of a metastable covalent acyl-enzyme intermediate. The core of these inhibitors comprises a five stranded b-sheet, but following cleavage a dramatic conformational change occurs in which the free loop inserts into this structure to create an extremely stable sheet of six strands. When this rearrangement occurs prior to resolution of the covalent intermediate, the protease is inhibited irreversibly. Heparin is an endogenous sulfated mucopolysaccharide that is an essential cofactor for antithrombin III, increasing the rate of complex formation between antithrombin III and thrombin by up to 10 000-fold (Damus et al., 1973). While thrombin and factor Xa are physiologically the most important targets, antithrombin III can also inactivate factors VIIa, IXa, XIa, XIIa, and kallikrein. This mechanism is the basis for the widespread clinical use of unfractionated and low–molecular weight heparins for prophylactic and therapeutic anticoagulation. The importance of this regulatory mechanism in vivo is highlighted by patients with inherited autosomal dominant antithrombin III deficiency who are significantly predisposed to pathologic venous and arterial thrombosis (Lane et al., 1997). Other relevant plasma coagulation protease inhibitors include a-1-antitrypsin, heparin cofactor II, a2-macroglobulin, and C1-esterase inhibitor. In addition, scavenger receptors in the reticuloendothelial system of the liver efficiently take up and eliminate activated proteases returning via the portal circulation. Finally, while blood coagulation is promoted by stasis

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The Molecular Basis of Blood Coagulation

AT-III

FVIII

FVIIIa

FV

FVa

FG

F

PAR-1

PAR-1*

T

AT-III

AT-III

T

Heparin

T

tissue factor–factor VIIa complex to abrogate the tissue factordependent coagulation cascade.

Fibrinolysis Blood coagulation culminates in the generation of fibrin clot, but in vivo this insoluble meshwork is also susceptible to remodeling and dissolution as the demands for hemostasis and wound repair change. This enzymatic degradation of fibrin is termed fibrinolysis and, much like coagulation itself, is tightly regulated by a series of activators, inhibitors, and cofactors (Figure 9). Plasminogen is a zymogen that, upon activation to plasmin, is the primary catalyst of fibrin degradation. The proenzyme is a 92 000 molecular weight modular protein composed of an N-terminal PAN domain, five kringle domains, of which the first and fourth have the highest affinity for the lysine moieties of fibrin, and an S1 serine protease catalytic domain.

Plasminogen Activation AT-III

Figure 7 Heparin acts through antithrombin III to inhibit thrombin. Thrombin (T) favors blood coagulation by activating a number of clotting proteins including factor VIII, factor V, fibrinogen, and the proteaseactivated receptor-1 (PAR-1) on platelets. Thrombin is irreversibly inhibited by the serpin antithrombin III (AT-III) in a reaction that is accelerated more than 104-fold by heparin. Although heparin can be a polymer of variable molecular weight, a small pentasaccharide (shown in pink) provides the critical interaction with AT-III.

and/or shear stress common at sites of vessel injury, normally circulating blood in laminar flow does not encourage thrombus formation. A distinct but complementary set of mechanisms provides temporal control of coagulation. One of the best characterized systems halting coagulation is the thrombomodulin/protein C pathway (Figure 8). The major curiosity of this mechanism is that the protease activating the protein C zymogen is actually thrombin. Thrombin is ordinarily the definitive procoagulant protease of the cascade, cleaving fibrinogen and activating factors V, VIII, and XI as well as platelets. Upon stable interaction of thrombin with the cell surface receptor thrombomodulin, thrombin’s rate of protein C activation increases by three orders of magnitude and the cleavage of its procoagulant substrates is robustly inhibited (Esmon et al., 1982). Active protein C together with its cofactor protein S efficiently destroys rate-limiting cofactors Va and VIIIa to halt coagulation (Walker et al., 1979; Fulcher et al., 1984). Deficiency of protein C and protein S are associated with thromboembolic disease in humans (Griffin et al., 1981; Comp and Esmon, 1984), a testament to the importance of this anticoagulant pathway in vivo. TFPI exerts an important negative feedback mechanism to regulate coagulation driven by the extrinsic pathway (Broze, 1995). TFPI contains three tandem Kunitz domains, a serineprotease inhibitor motif present in the well characterized inhibitor aprotinin. The second of these Kunitz domains reversibly binds to factor Xa to inactivate it. This TFPI/Xa complex can then interact via the first Kunitz domain with the

Plasminogen is activated by cleavage of an Arg–Val bond at amino acid 560–561 that converts the enzyme from its single to its two chain form (Castellino, 1984). The two primary physiologic activators of plasminogen are tissue plasminogen activator (tPA) and urokinase, both of which are elaborated primarily by endothelial cells and together account for 85% of plasminogen activation. tPA is a 72 000 molecular weight glycoprotein that encompasses a fibronectin-like domain, an EGF-like domain, two kringle domains, and a serine protease catalytic domain (Pennica et al., 1983); tPA is interesting in that it actually requires fibrin itself as a cofactor for efficient catalysis. Urokinase is a 54 000 molecular weight protein sharing significant sequence and structural homology with tPA, albeit in miniature as it has no fibronectin-like domain and only a single kringle domain (Kasai et al., 1985). The kringle and fibronectin-like domains of these enzymes facilitate the interaction with fibrin and encourage their incorporation into the clot. Tissue plasminogen activator, urokinase and the related bacterial enzyme streptokinase have all been exploited in recombinant form to lyse occlusive clots in the coronary arteries or the neural vasculature of patients with acute myocardial infarction or stroke. A number of minor activators of plasminogen also contribute. In particular, the proximal enzymes of the contact pathway – factors XIIa, XIa, and kallikrein – activate plasminogen, highlighting the intimate relationship between coagulation and fibrinolysis (Colman, 1969; Mandle and Kaplan, 1979; Goldsmith et al., 1978). Conversely, factor XII is itself a substrate for plasmin, so fibrinolysis can also activate the contact pathway. Another link between coagulation and fibrinolysis is the profibrinolytic character of activated protein C, which by decreasing thrombin generation indirectly decreases the activity of a thrombin activatable fibrinolysis inhibitor (TAFI, see below).

Regulation of Fibrinolysis The generation and activity of plasmin are closely regulated by a series of inhibitors and receptors. First, active plasmin itself is

The Molecular Basis of Blood Coagulation

11

Figure 8 Thrombomodulin and protein C are central to an endogenous anticoagulant pathway. Cell surface thrombomodulin (TM) interacts with thrombin (T) to alter its substrate specificity, converting thrombin from a procoagulant to an anticoagulant protease. The thrombin–thrombomodulin complex converts protein C (PC) to activated protein C (APC). APC and its cofactor protein S (PS) efficiently inactivates factor Va (FVa) to FV-i and factor VIIIa (FVIIIa) to FVIII-i via proteolysis. The thrombin–thrombomodulin complex does not act on normal thrombin substrates such as factor V (FV), factor VIII (FVIII), fibrinogen (FG), or protease-activated receptor-1 (PAR-1). The EGF domains of protein C, protein S, and thrombomodulin are indicated by solid circles; catalytic protease domains are represented by rounded rectangles.

Figure 9 Activation and regulation of fibrinolysis. Plasminogen is activated to plasmin primarily by tissue plasminogen activator (tPA) and urokinase, and to a lesser degree by kallikrein (KLK), factor XIIa (FXIIa), and factor XIa (FXIa). Plasminogen activator inhibitors 1 and 2 (PAI-1/2) inhibit tPA and urokinase. Plasmin catalyzes the proteolytic degradation of fibrin clot, but the enzyme is itself susceptible to inhibition by a2-antiplasmin, a2-macroglobulin, and protease nexin-1. Thrombin activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase that degrades the C-terminus of fibrin to prevent binding of plasmin.

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The Molecular Basis of Blood Coagulation

inhibited by a series of plasma proteins. The most prominent of these is a2-antiplasmin, a serpin family member that irreversibly neutralizes plasmin escaping the thrombus into circulation (Holmes et al., 1987). Other fluid phase inhibitors with unique mechanisms include a2-macroglobulin and protease nexin-1. Second, a distinct set of inhibitors regulates activation of plasminogen. The serpin plasminogen activator inhibitor-1 (PAI-1) is an abundant inhibitor of both tPA and urokinase; homozygous deficiency of PAI-1 results in a clinically significant bleeding diathesis in humans (Fay et al., 1992). Although plasminogen activator inhibitor (PAI-2) shows specificity for tPA in vitro and in mice (Erickson et al., 1990), the protein is not abundant except during pregnancy and its deficiency has yet to be described in humans, thus its importance to fibrinolysis in vivo remains unclear. Third, TAFI is a 60 000 molecular weight carboxypeptidase that actually digests the C-terminal basic residues of fibrin to eliminate the binding site for profibrinolytic factors such as tPA and plasmin (Eaton et al., 1991; Wang et al., 1994; Bajzar et al., 1995). Finally, a number of cell surface receptors bind plasminogen, plasmin, and its activators to modulate fibrinolysis. One subset of receptors, including glycoprotein IIb/IIIa and annexin A2, supports plasminogen activation. By contrast, the hepatic low density lipoprotein receptorrelated protein 1 and mannose receptor help to clear serpininactivated proteases from circulation.

Degradation of Fibrin Once activated, plasmin cleaves both fibrinogen and fibrin in a relatively well orchestrated sequence of proteolysis events yielding a set of well characterized degradation products (Nussenzweig et al., 1961; Marder et al., 1969). After cleavage of the C-terminus of the Aa chain from the D domain of fibrinogen, the N-terminus of the Bb chain is removed to release fibrinopeptide B (Mosesson et al., 1973); the resulting polypeptide is fragment X. Cleavage of X between a D and E domain yields fragments D and Y, the latter of which can be trimmed further to generate fragment E. Fibrin is divided similarly by plasmin, with a few notable exceptions. Fibrinopeptides A and B were already removed prior to fibrin oligomerization, so these are not available for action by plasmin. If fibrin molecules have been cross-linked to their neighbors due to the transaminase activity of factor XIII, then the liberated fragments D and E are covalently joined. This special fibrin degradation product is known as a D-dimer (Gaffney and Brasher, 1973; Pizzo et al., 1973; Dudek-Wojciechowska et al., 1973), an important clinical biomarker for diagnosis of increased fibrinolytic activity observed in thromboembolic disease and disseminated intravascular coagulation (Wells et al., 2003). Although the delicate balance between coagulation and fibrinolysis is perturbed in many inherited and acquired disorders of hemostasis, this balance can be restored in these patients using pharmacologic inhibitors of fibrinolysis such as ε-aminocaproic acid and tranexamic acid.

See also: Acquired Coagulopathy; Hemophilia; Other Congenital Coagulopathies; Platelets; von Willebrand Disease.

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