General Considerations of Coagulation Proteins

Home , Factor IX, Factor VII, Factor X, Intrinsic factor, Sialoglycoprotein, Tissue factor

DAVID GREEN, M.D., Ph.D.*

Atherosclerosis Program, Rehabilitation Institute of Chicago, Section of Hematology, Department of Medicine, and Northwestern University Medical School, Chicago, IL 60611.

ABSTRACT

The coagulation system is part of the continuum of host response to injury and is thus intimately involved with the kinin, complement and fibrinolytic systems. In fact, as these multiple interrelationships have un folded, it has become difficult to define components as belonging to just one system. With this limitation in mind, an attempt has been made to present the biochemistry and physiology of those factors which appear to have a dominant role in the coagulation system. Coagulation proteins in general are single chain glycoprotein molecules. The reactions which lead to their activation are usually dependent on the presence of an appropriate surface, which often is a phospholipid micelle. Large molecular weight cofactors are bound to the surface, frequently by calcium, and act to induce a favorable conformational change in the reacting molecules. These mole cules are typically serine proteases which remove small peptides from the clotting factors, converting the single chain species to two chain molecules with active site exposed. The sequence of activation is defined by the enzymes and substrates involved and eventuates in fibrin formation. Mul tiple alternative pathways and control mechanisms exist throughout the normal sequence to limit coagulation to the area of injury and to prevent interference with the systemic circulation.

Introduction RatnofP4 eloquently indicates in an arti cle aptly entitled: “A Tangled Web. The Blood coagulation forms part of a con Interdependence of Mechanisms of tinuum of body response to injury. Acti Blood Clotting, Fibrinolysis, Immunity vation of blood coagulation occurs in and Inflammation,” the four systems are concert with the awakening of the kinin, extensively interrelated. Therefore, it is complement and fibrinolytic systems. As very difficult to arrive at a definition of a coagulation protein. For example, pro * Reprint requests: David Green, M.D., Ph.D., Rehabilitation Institute of Chicago, 345 East longed coagulation times as measured by Superior Street, Chicago, IL 60611. the partial thromboplastin time test are 95 96 GREEN observed in plasma deficient in prekal- factor, prekallikrein and high molecular likrein, and yet patients with this disor weight kininogen. The contemporary der may not suffer from a hemorrhagic version shown has not been accepted as diathesis. Is prekallikrein a coagulation official, but is simply included as a guide protein? By informal convention, it is now to the discussion of the coagulation pro listed among the clotting factors. teins which follows. Some years ago, the International Committee on Blood Clotting Factors as Biochemistry and Physiology signed Roman numerals to the then rec of the Coagulation Proteins ognized coagulation proteins. The official numbers and common synonyms are F ib r in o g e n (F a c t o r I) shown in table I. Adjacent to this list is a contemporary version which includes Fibrinogen is perhaps the best charac coagulation proteins characterized since terized of the coagulation proteins. It has the original list was formulated. The a molecular weight of approximately major modifications include (1) the rec 340.000 and consists of three pairs of ognition of specific platelet coagulant ac polypeptide chains called the A a, B ¡3 tivities, which now could be assigned and y chains.29 These are arranged in the under the previously unused Roman form of a dimer, linked by symmetrical numeral VI; (2) the indication that factor disulfide bonds.104 The A a chain has a VIII probably represents a complex of molecular weight of 64,000; the B /3 chain proteins designated anti-hemophilic fac 57.000 with approximately 480 resi tor, von Willebrand factor and the factor dues115; and the y chain 48,000 with 409 VIII related antigen; (3) and the multiple residues, the complete sequence of proteins involved in the initiation of which has been determined.46 Approxi coagulation, assigned under Roman mately 3 to 5 percent of the molecule is numeral XII and designated Hageman carbohydrate.11 A detailed description of fibrinogen structure has recently been published by Mosesson and Finlayson.78

T A B L E I Fibrinogen is synthesized by the

The Coagulation Proteins hepatocyte30 and has a plasma half-life of between 3.2 and 4.5 days.3'72, 107 It is Official Number Synonym Contemporary version catabolized at the rate of 31 to 46 mg per kilogram per day. However, the site of I Fibrinogen I (Fibrinogen) fibrinogen catabolism has not been iden II Prothrombin II (Prothrombin) III Tissue Thromboplastin III (Tissue Factor) tified and the routes of excretion or IV Calcium IV (Calcium) V Labile Factor V (Labile Factor) reutilization of the molecule are un --- VI :PF-j (Platelet coagulant activities) known. An important, unanswered ques VI:PF4 VII Stable Factor VII {Stable Factor) tion is what percentage of fibrinogen is VIII Antihemophilic Factor VIII:AHF (Antihemophilic Factor) normally converted to fibrin in the non VIII :VWF (von Willebrand Factor) bleeding state. VIII:RAg (Related-Antigen) IX Christmas Factor IX (Christmas Factor) X Stuart-Prower Factor X (Stuart-Prower Factor) P r o t h r o m b in (F a c t o r II) XI Plasma Thromboplastin XI (Plasma Thromboplastin Antecedent Antecedent) XII Hageman Factor XII:HF (Hageman Factor) Prothrombin has a molecular weight of XIIsPK (Prekallikrein, Fletcher) 68.000 and consists of a single polypep XII:HMWK (High Molecular Weight Kininogen) tide chain of 522 amino acid residues. It XIII Fibrin Stabilizing Factor XIII (Fibrin Stabilizing Factor) is a glycoprotein containing hexose, hex- osamine and sialic acid.58 A characteristic The use of the suffix "a" after any factor indicates its activated form. feature is a calcium binding region in the GENERAL CONSIDERATIONS OF COAGULATION PROTEINS 97 a helical portion of the molecule.112 This cent carbohydrate, mainly sialic acid, binding is due to the presence of hexoses and other neutral sugars.42 A •y-carboxyglutamic acid residues, incor heavy (125,000) and light (64,000) chain porated into the molecule through the ac have been identified and are present in a tion of vitamin K.105 Prothrombin is 1:2 ratio.21 Factor V is synthesized by the synthesized by the liver4'86 and has a liver36,86 and has a plasma half-life of ap plasma half-life of 2.8 to 4.4 days.54,103 proximately 20 hours.99 Shapiro and Martinez103 estimated a synthetic rate of 2.5 mg per kilogram per day. The sites of catabolism and excretion P l a t e l e t C o m p o n e n t s are unknown. Platelet factor 3, a lipoprotein compo nent of the platelet membrane, consists T is s u e F a c t o r (F a c t o r III) principally of phosphatidyl serine and Tissue factor is a complex material phosphatidyl ethanolamine.70,71 Platelet consisting of approximately 49 percent factor 4 has been extensively charac protein, 42 percent phospholipid and terized and has a molecular weight of 7 percent cholesterol.81 Phosphatidyl 9600. It consists of a single polypeptide ethanolamine appears to be the principal chain of 92 amino acid residues having lipid component.80 The protein compo two disulfide bridges.113 As it occurs in nent has a molecular weight of approxi the platelet, it is probably a tetramer.76 Other platelet coagulant activities have mately 55,000.8 Tissue factor activity is been identified83'111 but not as com mainly found in the lung, brain and pletely characterized. placenta117 and may also be recovered from cultured skin fibrinoblasts40 It is principally localized to the microsomal F a c t o r VII fractions.118 Factor VII is a glycoprotein with an apparent molecular weight of 60,000.57 It C a l c iu m (F a c t o r IV) is a single chain polypeptide with an ac According to Tullis,109 calcium in a cessible enzymatic site; however, as will concentration of 0.5 x 10-3 M is a prere be discussed subsequently, the form of quisite for each phase of hemostasis. A the molecule active in clotting reactions simple experiment indicates the essential has two chains. The amino acid sequence role of calcium in coagulation. If fresh of the molecule shows considerable simi whole blood is diluted 1:20 in imidazole larity to that of prothrombin and the light buffer, pH 7.4, it will not clot. However, chain of factor X.56 It also is synthesized by if the buffer contains calcium, 5 mEq per the liver and vitamin K is required for the liter, clotting occurs in 2 to 3 minutes formation of the complete molecule. Fac (unpublished observation). This experi tor VII has a half-life of only 100 to 300 ment indicates that calcium concentra min.47 tion is the limiting factor in coagulation, in vitro. F a c t o r VIII Factor VIII is a glycoprotein with a F a c t o r V molecularweight in excess ofl millionand Factor V is a large protein with a consists of a series of subunits held to molecular weight in excess of 200,000.27 gether by disulfide bonds.59,101 V III: AHF Its activity is labile but can be stabilized (anti-hemophilic factor) can be separated by divalent cations and reducing from V III: VWF (vonWillebrand factor) by agents.2'9 It contains from 10 to 20 per cryoprecipitation,48 Polyelectrolytes,65 IM 98 GREEN

NaCl116 and 0.25 M CaCl2.90 The VIII: F a c t o r XI AHF formed by CaCl2 treatment has subunits of 30,000 to 100,000 molecular Current evidence indicates that factor weight73 while the V III: VWF has 200,000 XI is a protein of molecular weight molecular weight subunits.59 It has not yet 175.000.108 It normally exists in the circula tion complexed with high molecular been resolved as to whether VIII: VWF weight kininogen. It can be absorbed onto and V III: AHF are themselves subunits of barium salts, aluminum hydroxide gels, a single protein or represent separate mol ecules which are chemically bonded and celite, and other surfaces.109 Its activity is circulate as a protein complex. It has been heat stable and appears to increase with established that the various subunits ofthe storage. It is probably synthesized by the parent complex differ in molecular size liver93 and has a plasma half-life of40 to 84 and degree of aggregation.120 hours.84 The site(s) of synthesis of VIII: AHF are F a c t o r XII presently unknown, but the liver undoub tedly contributes to the plasma pool of Hageman factor is a sialoglycoprotein V III: AHF.69 V III: VWF is synthesized by with a molecular weight of 80,000.26 In endothelial cells52 and perhaps also by plasma it exists as a single polypeptide megakaryocytes.43 While the half-life of chain.17 It absorbs strongly to negatively V III: AHF is 12 hours,92 that of VIII: VWF charged surfaces.85 The half-life is 48 to 52 is probably of the order of 16 to 24 hours.14 hours.28 Prekallikrein (Fletcher factor) is a Radio-iodinated VIII accumulates pre protein with a subunit molecular weight of dominantly in the liver and, with time, the 85.000.13 High molecular weight kinino free isotope appears in the urine.39 gen is a glycoprotein with molecular weight 160,000.100 It contains bradykinin F a c t o r IX and a glycoprotein of 110 amino acid resi dues . The glycopeptide consists of a heavy Factor IX is a glycoprotein of molecular and light chain joined by a single disulfide weight 55,000.109 It is a single chain mole bridge.50,108 cule, with the N-terminal portion showing homology with prothrombin and the light F a c t o r XIII chain of factor X.32 Like prothrombin and factors VII and X, the molecule is synthe Factor XIII (fibrin stabilizing factor) is a sized in the liver and has the vitamin K glycoprotein with a molecular weight of dependent y-carboxyglutamic acid resi between 156,000 and 195,000,61 and con dues necessary for calcium binding.105 The sists of two subunits of molecular weight plasma half-life is approximately 20 to 24 81,00062,74 The site of synthesis is uncer hours ,12 tain; the half-life is approximately 12 days.75

F a c t o r X Activation of the Coagulation Factor X is a glycoprotein of molecular Proteins weight 55,00051 It is composed of a heavy chain (38,000) and a light chain (17,000), As previously indicated, the majority of joined by disulfide bonds.33 The heavy coagulation factors are single-chain chain carries the carbohydrate near its glycoproteins. These circulate in an inac N-terminal region. The protein is synth tive form. They are activated by serine esized by the liver and requires vitamin K. proteases, which cleave peptide bonds The plasma half-life is in the range of32 to and convert the single chain molecules 48 hours.12,97 into two-chain structures joined by disul GENERAL CONSIDERATIONS OF COAGULATION PROTEINS 99 fide bonds. This alteration in architecture AHF reacts with thrombin to form a exposes active sites, so that a previously product which has coagulant and serine inactive coagulation protein is now itself esterase activity.110 However, it has not an active serine protease, capable of ac been established whether or not activa tivating additional coagulation factors. tion of factor VIII is required for the next For each activated factor, there is a spe step in the clotting reaction—the forma cific coagulation protein that conforms tion of a complex between factor VIII and to its active site and can be activated. Thus, factor IXa.49,88 This complex, in the pres the clotting factors are activated in se ence of platelet phospholipid and cal quence and, since each activated factor has cium ions, activates factor X by cleaving a the capability of activating more than one peptide from the amino terminal end of inactive factor, a small initial stimulus can the heavy chain of factor X.24 The acti have wide-spread ramifications. This is vated factor has a molecular weight of the biological amplification theory origi 44,000 and is a serine esterase, inhibit- nally proposed by Macfarlane.66 able by DFP and soybean trypsin in The coagulation cascade is typically hibitor.60, 64- The role of phospholipid in turned on by exposure of plasma to a vas the activation of factor X is uncertain; it cular surface which has either been dam may provide a micellar surface for the in aged or denuded of endothelium.28 Cir teraction of the several components.5 culating factor XII: Hageman factor There are several other pathways lead becomes bound to the surface. Griffin41 ing to factor X activation. The most im suggests that surface binding greatly in portant of these involves tissue factor and creases the susceptibility of Hageman factor VII. These two factors form a com factor to proteolytic activation by kalli- plex which has proteolytic activity, in- krein in the presence of surface bound hibitable by DFP and soybean trypsin high molecular weight kininogen. There inhibitor.82 It has not been possible to is a reciprocal activation of prekallikrein demonstrate an activated factor VII in and Hageman factor under these cir this complex; however, single chain fac cumstances. Activated Hageman factor is tor VII can be converted into a two-chain inhibited by DFP,6 suggesting it is a molecule by factor Xa, thrombin, Hage serine protease. The activated form con man factor fragments and plasmin, and in sists of a heavy chain (50,000) and a light this form its coagulant activity is greatly chain (28,000).19 The surface-bound acti enhanced.7 The tissue factor-factor VII vated Hageman factor activates factor complex, with calcium ions as cofactor, XI95 which also is complexed to high converts factor X to factor Xa. Factor X molecular weight kininogen.108 Activated may also be activated directly by Rus factor XI has esterase as well as coagulant sell’s viper venom or trypsin.34,119 activity, and is inhibited by DFP.55 The next reaction in the main sequence Activated factor XI (factor XIa) acti involves the conversion of prothrombin vates factor IX in a calcium-dependent to thrombin by a complex consisting of reaction.96 The activated factor IX con factor Xa, factor V, calcium and phos tains two disulfide-linked polypeptide pholipid. Like factor VIII, factor V can chains of 27,000 and 16,000 molecular also be activated by thrombin20,114 but weight. The active site on the factor IX never acquires enzymatic properties. molecule exposed by this step resides on Rather, factor V appears to be a regulator the 27,000 polypeptide87 and is a serine.23 protein.25 Factor V is a calcium metallo- The next series of reactions involve fac protein, and through its calcium ion is able tor IXa, factor VIII, factor X, calcium, to bind to phospholipid.21 This may give phospholipid, and thrombin. Factor VIII: the molecule the appropriate orientation 1 0 0 GREEN to enhance the activity of factor Xa.53 The aggregation to form fibrin; the polymeri latter splits specific peptide bonds in zation process is accelerated by cal prothrombin, converting it from a single cium.10 The fibrin polymer is soluble in chain polypeptide of molecular weight agents such as 1 percent monochloroacetic 72,000, to a two-chain structure of acid102 and 5 M urea. Furthermore, it is molecular weight 39,000 (thrombin).68,81 very susceptible to lysis by the fibrinoly The two chains of thrombin, are desig tic enzyme, plasmin.35,38 Stabilization of nated the A and B chains, and have 49 the polymer occurs when adjacent y and 260 amino acid residues, respec chains15 and, more slowly, adjacent a tively.67 Thrombin is a serine protease, chains102 are cross-linked by activated sensitive to DFP.37 factor XIII. Factor XIII is activated by Thrombin acts as a proteolytic enzyme thrombin, which splits an arginyl-glycine on fibrinogen, splitting arginyl-glycyl bond on the a-subunit,106 and calcium, bonds at the N-terminal amino acid of the which induces the dissociation of the two A a and B /3 chains. The resulting free subunits of the molecule16 and exposes peptides are designated fibrinopeptides the active site on the a-subunit.22 This A and B, and the parent molecule fibrin catalyzes cross-linking, which is a trans monomer. The monomer undergoes amidation reaction involving the forma-

Figure 1. Sequence of activation of the clotting factors. Through surface contact, factor XII be comes activated in a proc ess which is augmented by surface-bound HMW kininogen. Xlla activates | Lipid- -T issue Factor prekallikrein, which in turn accelerates the acti vation of XII. Xlla acti vates XI. The next step, activation of IX, depends on the ability of IX to bind to phospholipid. The complex of IXa and VIII : C activates X in the intrinsic system. In the ex trinsic system, the initial step is the activation of VII by binding to tissue factor. Vila then activates X. Xa converts phospholipid-bound prothrombin to thrombin in the pres ence of V, which acts as a regulator protein. Throm bin splits fibrinopeptides A and B from fibrinogen giving rise to fibrin monomer. Aggregation and cross-linking lead to the production of stable fibrin. GENERAL CONSIDERATIONS OF COAGULATION PROTEINS 1 0 1 tion of peptide bonds between e-amino in the anti-thrombin III exposing addi lysine and -y-glutamyl residues of adja tional arginines. A second plasma in cent fibrin chains.63,91 These reactions hibitor is a 2 macroglobulin, which is also involving factor Xllla and fibrin polymer an inhibitor of serine esterases.44 Several are the final steps in the formation of a additional inhibitors of importance in hemostatically effective clot. The entire se clude the inactivator of C-l esterase, quence is shown schematically in figure 1. which inhibits activated Hageman factor, kallikrein, and XIa31; a ( anti-trypsin45; Modulation of the Coagulation and a 2 anti-plasmin.77,79 Other controls Reactions serving to limit clot formation include the diluting effect of rapid blood flow, the It is obvious that with such a complex clearance of activated intermediates by system, alternative pathways and exten the liver, the enzymatic destruction of sive controls must be present. Since pa certain intermediates (principally factors V and VIII) by activated products, and tients with deficiencies of either factor XII : Hageman factor, prekallikrein or the consumption and absortion of various high molecular weight kininogen may factors by the clot itself. All these mecha nisms serve to contain the coagulation not have a hemorrhagic diathesis, alterna tive routes for activation of the coagula process, so that it serves only to impede blood loss and form a framework for tis tion sequence must exist. One of these is through the factor VII-tissue factor path sue repair and not affect the systemic cir culation. way, but the importance of this should not be overemphasized, since deficien cies of factor XI, IX and VIII, all appar Acknowledgment ently bypassed by the factor VII-tissue The author is grateful to Dr. I. Cohen for his care factor activation of factor X, are associated ful review of the manuscript. with clinically important bleeding. These same considerations must influence our assessment of the recently described pathway involving the activation of factor References IX by factor VII and tissue factor.89 Simi larly, platelet components appear to have 1. Ab ild g a a rd , U.: Purification of two progres sive antithrombins of human plasma. Scand. J. important roles in many of the reactions Clin. Lab. Invest. 19 -.190-195, 1967. involving clotting factors; yet, the bleed 2. Aok i, N., H a rm iso n, C. R., and Seeg e rs, W. ing problems of thrombocytopenic pa H.: Properties of bovine Ac-globulin concen trates and methods of preparation. Can. J. tients are related more to impaired vascu Biochem. Physiol. 41 ¡2409-2421, 1963. lar hemostasis than to defective intrinsic 3. Amris, A. and Amris, C. J.: Turnover and dis coagulation. tribution of 131 iodine-labeled human fibrino gen. Thromb. Diath. HaemorrhJi ¡404-422, Key inhibitors regulate many of the 1964. coagulation reactions. The principle one 4. Barnhart, M. I.: Cellular site for prothrom is anti-thrombin III, a glycoprotein with a bin synthesis. Amer. J. Physiol. i99:360-366, 1960. molecular weight of 65,00g.1 This in 5. Barton, P. G.: Sequence theories of blood hibitor complexes with factors VII, IXa, coagulation re-evaluated with reference to XIa and thrombin.98 Enzymatic activity is lipid-protein interactions. Nature 2J5:1508- 1509, 1967. inhibited by the binding of arginine resi 6. Becker, E. L.: Inactivation of Hageman factor dues on the anti-thrombin III to active by diisopropyl-fluorophosphate (DFP). J. site serines of the activated clotting fac Lab. Clin. Med. 56¡136-138, 1960. 7. Bennett, B.: Coagulation pathways: Interre tors; the effect is potentiated by heparin, lationships and control mechanisms. Semi which produces conformational changes nars in Hematology. Miescher, P. A. and Jaffe, 1 0 2 GREEN

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