Roles of factor XI, platelets and tissue factor-initiated blood coagulation

Abstract

Since the first clear and comprehensive statement of the postulated sequence of enzymatic reactions that comprise the blood coagulation mechanism was published in 1964 [1Davie E.W. Ratnoff O.D. Waterfall sequence for intrinsic blood clotting.Science. 1964; 145: 1310-2Crossref PubMed Scopus (709) Google Scholar, 2MacFarlane R.G. An enzyme cascade in the blood clotting mechanism, and its function as a biochemical amplifier.Nature. 1964; 202: 498-9Crossref PubMed Scopus (696) Google Scholar], an enormous amount of new information has accumulated. This has required revisions of our concepts concerning the inter-relationships between the extrinsic and intrinsic pathways of blood coagulation, the role of platelets in these biochemical pathways, and the sequence of biochemical reactions that comprise the hemostatic mechanism. Thus, since 1964 virtually all the coagulation proteins have been purified and characterized from bovine and human plasmas, their genes have been cloned, sequenced, and characterized, and many gene knockouts have been evaluated in the mouse model. The structural biology of many of the coagulation proteins and their important regulatory proteins and receptors have been largely elucidated from X-ray crystallography and nuclear magnetic resonance spectroscopy. This has yielded important information essential for understanding the assembly of coagulation complexes on cellular receptors, and their regulation by plasma and cellular inhibitors, including serine protease inhibitors (serpins), the Kunitz-type protease inhibitors (kunins), and the important regulatory proteins of the protein C-thrombomodulin anticoagulant mechanism. Historically, the intrinsic pathway of blood coagulation, initiated by the assembly of the so-called contact proteins on negatively charged surfaces [3Colman R.W. Contact activation pathway: inflammatory, fibrinolytic, anticoagulant, antiadhesive and antiangiogenic activities.in: Colman RW Hirsh J Marder VJ Clowes AW George JN Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Lippincott Williams & Wilkins, 2001: 103-22Google Scholar], and the extrinsic pathway, initiated by the exposure of tissue factor (TF) at sites of vascular injury [4Morrissey J.H. Tissue factor and factor VII initiation of coagulation.in: Colman RW Hirsh J Marder VJ Clowes AW George JN Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Lippincott Williams & Wilkins, 2001: 89-102Google Scholar], were regarded as separate and distinct pathways for the generation of thrombin, which would then effect normal hemostasis. The concept that the extrinsic and intrinsic pathways were separate and alternative coagulation mechanisms to achieve the same result, i.e. the conversion of fibrinogen to crosslinked fibrin, was then re-examined in the light of clinical evidence that complete deficiencies of the contact activation proteins [factor (F)XII, high molecular weight kininogen and prekallikrein] were not associated with any abnormal predisposition to bleeding even after major trauma or surgery [3Colman R.W. Contact activation pathway: inflammatory, fibrinolytic, anticoagulant, antiadhesive and antiangiogenic activities.in: Colman RW Hirsh J Marder VJ Clowes AW George JN Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Lippincott Williams & Wilkins, 2001: 103-22Google Scholar] whereas, in contrast, FXI deficiency, although not usually associated with spontaneous hemorrhage, was clearly associated with excessive, and sometimes life-threatening, bleeding complications after surgery or trauma [5Asakai R. Chung D.W. Davie E.W. Seligsohn U. Factor XI deficiency in Ashkenazi Jews in Israel.N Engl J Med. 1991; 325: 153-8Crossref PubMed Google Scholar, 6Bolton-Maggs P.H. Young Wan-Yin B. McCraw A.H. Slack J. Kernoff P.B. Inheritance and bleeding in factor XI deficiency.Br J Haematol. 1988; 69: 521-8Crossref PubMed Google Scholar, 7Hu C.J. Baglia F.A. Mills D.C. Konkle B.A. Walsh P.N. Tissue-specific expression of functional platelet factor XI is independent of plasma factor XI expression.Blood. 1998; 91: 3800-7Crossref PubMed Google Scholar, 8Leiba H. Ramot B. Many A. Hereditary and coagulation studies in ten families with factor XI (plasma thromboplastin antecedent) deficiency.Br J Haematol. 1965; 11: 654-65Crossref PubMed Google Scholar, 9Ragni M.V. Sinha D. Seaman F. Lewis J.H. Spero J.A. Walsh P.N. Comparison of bleeding tendency, factor XI coagulant activity, and factor XI antigen in 25 factor XI-deficient kindreds.Blood. 1985; 65: 719-24Crossref PubMed Google Scholar, 10Rapaport S.I. Proctor R.R. Patch M.J. Yettra M. The mode of inheritance of PTA deficiency: evidence for the existence of a major PTA deficiency and a minor PTA deficiency.Blood. 1961; 18: 149-55Crossref PubMed Google Scholar, 11Rosenthal R.L. Dreskin O.H. Rosenthal N. New hemophilia-like disease caused by deficiency of a third plasma thromboplastin factor.Proc Soc Exp Biol Med. 1953; 82: 171-4Crossref PubMed Google Scholar, 12Sidi A. Seligsohn U. Jonas P. Many M. Factor XI deficiency. detection and management during urologic surgery.J Urol. 1978; 119: 528-30Crossref PubMed Google Scholar]. This discrepancy was difficult to reconcile with the in vitro observation that FXIIa was the only protease known to activate FXI. This suggests that the activation of FXI to the protease (FXIa) proceeds physiologically by a mechanism that is independent of FXII. A potential solution to this conundrum was addressed by the seminal studies of Naito and Fujikawa [13Naito K. Fujikawa K. Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces.J Biol Chem. 1991; 266: 7353-8Abstract Full Text PDF PubMed Google Scholar], and Gailani and Broze [14Gailani D. Broze Jr, G.J. Factor XI activation in a revised model of blood coagulation.Science. 1991; 253: 909-12Crossref PubMed Google Scholar], who simultaneously showed in 1991 that thrombin could activate FXI by cleaving it at the same scissile bond cleaved by FXIIa. However, the concentrations of thrombin required for FXI activation were physiologically too high (>10 nmol L−1), and the incubation times required for FXI activation were protracted unless dextran sulfate was included in the incubation mixture [13Naito K. Fujikawa K. Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces.J Biol Chem. 1991; 266: 7353-8Abstract Full Text PDF PubMed Google Scholar, 14Gailani D. Broze Jr, G.J. Factor XI activation in a revised model of blood coagulation.Science. 1991; 253: 909-12Crossref PubMed Google Scholar]. Subsequently, our laboratory provided evidence that platelets could bind FXI [15Baglia F.A. Jameson B.A. Walsh P.N. Identification and characterization of a binding site for platelets in the Apple 3 domain of coagulation factor XI.J Biol Chem. 1995; 270: 6734-40Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 16Greengard J.S. Heeb M.J. Ersdal E. Walsh P.N. Griffin J.H. Binding of coagulation factor XI to washed human platelets.Biochemistry. 1986; 25: 3884-90Crossref PubMed Google Scholar, 17Ho D.H. Baglia F.A. Walsh P.N. Factor XI binding to activated platelets is mediated by residues R (250), K (255), F (260), and Q (263) within the Apple 3 domain.Biochemistry. 2000; 39: 316-23Crossref PubMed Scopus (0) Google Scholar], and promote its activation at rates accelerated 5000- to 10 000-fold by thrombin at concentrations (<1 nmol L−1) achievable in vivo[18Baglia F.A. Walsh P.N. Prothrombin is a cofactor for the binding of factor XI to the platelet surface and for platelet-mediated factor XI activation by thrombin.Biochemistry. 1998; 37: 2271-81Crossref PubMed Scopus (92) Google Scholar, 19Baglia F.A. Walsh P.N. Thrombin-mediated feedback activation of factor XI on the activated platelet surface is preferred over contact activation by factor XIIa or factor XIa.J Biol Chem. 2000; 275: 20514-9Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. It has now been demonstrated that thrombin is the preferred activator of FXI on the platelet surface [19Baglia F.A. Walsh P.N. Thrombin-mediated feedback activation of factor XI on the activated platelet surface is preferred over contact activation by factor XIIa or factor XIa.J Biol Chem. 2000; 275: 20514-9Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar], which appears to occur on the platelet receptor glycoprotein Ib-IX-V [20Baglia F.A. Badellino K.O. Li C.Q. Lopez J.A. Walsh P.N. Factor XI binding to the platelet glycoprotein Ib-IX-V complex promotes factor XI activation by thrombin.J Biol Chem. 2002; 277: 1662-8Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar], within membrane microdomains referred to as lipid rafts, on activated platelets [21Baglia F.A. Shrimpton C.N. Lopez J.A. Walsh P.N. The glycoprotein Ib-IX-V complex mediates localization of factor XI to lipid rafts on the platelet membrane.J Biol Chem. 2003; 278: 21744-50Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar]. These observations support the concept that the initial event in normal hemostasis is the exposure of TF at sites of vascular injury, resulting in the activation of FX by FVIIa–TF [4Morrissey J.H. Tissue factor and factor VII initiation of coagulation.in: Colman RW Hirsh J Marder VJ Clowes AW George JN Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Lippincott Williams & Wilkins, 2001: 89-102Google Scholar]. However, once the first few molecules of FXa are formed, the enzymatic activity of the FVIIa–TF–FXa complex is rapidly shut down by TF pathway inhibitor. This results in the generation of only trace concentrations of thrombin, insufficient to cleave fibrinogen to fibrin, but sufficient to activate platelets, FXI, FVIII, and FV, thereby allowing the consolidation (or intrinsic) pathway of blood coagulation to generate sufficient quantities of thrombin to effect normal hemostasis [12Sidi A. Seligsohn U. Jonas P. Many M. Factor XI deficiency. detection and management during urologic surgery.J Urol. 1978; 119: 528-30Crossref PubMed Google Scholar, 13Naito K. Fujikawa K. Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces.J Biol Chem. 1991; 266: 7353-8Abstract Full Text PDF PubMed Google Scholar, 14Gailani D. Broze Jr, G.J. Factor XI activation in a revised model of blood coagulation.Science. 1991; 253: 909-12Crossref PubMed Google Scholar, 22Broze G.J.J. Miletich J.P. Isolation of the tissue factor inhibitor produced by HepG2 hepatoma cells.Proc Natl Acad Sci USA. 1987; 84: 1886-90Crossref PubMed Google Scholar, 23Broze G.J.J. Miletich J.P. Characterization of the inhibition of tissue factor in serum.Blood. 1987; 69: 150-5Crossref PubMed Google Scholar, 24Hubbard A.R. Jennings C.A. Inhibition of tissue thromboplastin-mediated blood coagulation.Thromb Res. 1986; 42: 489-98Abstract Full Text PDF PubMed Google Scholar, 25Hubbard A.R. Jennings C.A. Inhibition of the tissue factor-factor VII complex: involvement of factor Xa and lipoproteins.Thromb Res. 1987; 46: 527-37Abstract Full Text PDF PubMed Google Scholar, 26Morrison S.A. Jesty J. Tissue factor-dependent activation of tritium labeled factor IX and factor X in human plasma.Blood. 1984; 63: 1338-47Crossref PubMed Google Scholar, 27Rao L.V.M. Rapaport S.I. Studies on the mechanisms of inactivation of the extrinsic pathway of coagulation.Blood. 1987; 69: 645-51Crossref PubMed Google Scholar, 28Sanders N.L. Bajaj S.P. Zivelin A. Rapaport S.I. Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component.Blood. 1985; 66: 204-12Crossref PubMed Google Scholar]. A major issue integral to an understanding of the relative contributions of the extrinsic and intrinsic pathways to the generation of thrombin is the question of the normal mechanism of activation of coagulation FIX. Initially, it was demonstrated that FIX is activated by the protease FXIa by proteolytic cleavage at two scissile bonds, R145-V146 and R180-I181, to generate the active protease FIXa [29Anson D.S. Choo K.H. Rees D.J. Giannelli F. Gould K. Huddleston J.A. Brownlee G.G. The gene structure of human anti-haemophilic factor IX.Embo J. 1984; 3: 1053-60Crossref PubMed Scopus (0) Google Scholar, 30Di Scipio R.G. Kurachi K. Davie E.W. Activation of human factor IX (Christmas factor).J Clin Invest. 1978; 61: 1528-38Crossref PubMed Google Scholar, 31Fujikawa K. Legaz M.E. Kato H. Davie E.W. The mechanism of activation of bovine factor IX (Christmas factor) by bovine factor XIa (activated plasma thromboplastin antecedent).Biochemistry. 1974; 13: 4508-16Crossref PubMed Google Scholar, 32Jagadeeswaran P. Lavelle D.E. Kaul R. Mohandas T. Warren S.T. Isolation and characterization of human factor IX cDNA: identification of Taq I polymorphism and regional assignment.Somat Cell Mol Genet. 1984; 10: 465-73Crossref PubMed Scopus (0) Google Scholar, 33Jaye M. De La Salle H. Schamber F. Balland A. Kohli V. Findeli A. Tolstoshev P. Lecocq J.P. Isolation of a human anti-haemophilic factor IX cDNA clone using a unique 52-base synthetic oligonucleotide probe deduced from the amino acid sequence of bovine factor IX.Nucl Acids Res. 1983; 11: 2325-35Crossref PubMed Scopus (0) Google Scholar, 34Kurachi K. Davie E.W. Isolation and characterization of a cDNA coding for human factor IX.Proc Natl Acad Sci USA. 1982; 79: 6461-4Crossref PubMed Google Scholar, 35Österud B. Bouma B.N. Griffin J.H. Human blood coagulation factor IX. Purification, properties, and mechanism of activation by activated factor XI.J Biol Chem. 1978; 253: 5946-51Abstract Full Text PDF PubMed Google Scholar, 36Sinha D. Seaman F.S. Walsh P.N. Role of calcium ions and the heavy chain of factor XIa in the activation of human coagulation factor IX.Biochemistry. 1987; 26: 3768-75Crossref PubMed Google Scholar, 37Yoshitake S. Schach B.G. Foster D.C. Davie E.W. Kurachi K. Nucleotide sequence of the gene for human factor IX (antihemophilic factor B).Biochemistry. 1985; 24: 3736-50Crossref PubMed Google Scholar]. This mechanism appeared to be consistent with the view that the intrinsic and extrinsic pathways were separate mechanisms for the activation of FX and the generation of thrombin. However, a seminal and important observation made by Österud and Rapaport in 1977 [38Österud B. Rapaport S.I. Activation of factor IX by the reaction product of tissue factor and factor VII: additional pathway for initiating blood coagulation.Proc Natl Acad Sci USA. 1977; 74: 5260-4Crossref PubMed Google Scholar] was that FIX could also be activated by the FVIIa–TF complex by proteolytic cleavage of the same two scissile bonds cleaved by FXIa. In fact, the kinetics of FIX activation by FXIa and by FVIIa–TF were very similar, although the kinetics of FIX activation by FVIIa were highly dependent on the TF concentration [39Jesty J. Silverberg S.A. Kinetics of the tissue factor-dependent activation of coagulation factors IX and X in a bovine plasma system.J Biol Chem. 1979; 254: 12337-45Abstract Full Text PDF PubMed Google Scholar, 40Walsh P.N. Bradford H. Sinha D. Piperno J.R. Tuszynski G.P. Kinetics of the factor XIa catalyzed activation of human blood coagulation factor IX.J Clin Invest. 1984; 73: 1392-9Crossref PubMed Google Scholar, 41Zur M. Nemerson Y. Kinetics of factor IX activation via the extrinsic pathway dependence of Km on tissue factor.J Biol Chem. 1980; 255: 5703-7Abstract Full Text PDF PubMed Google Scholar]. Whereas these observations were initially interpreted as suggesting that the TF pathway could initiate the intrinsic pathway directly independent of the contact factor pathway, they did not explain the hemostatic defect observed in patients with FXI deficiency [5Asakai R. Chung D.W. Davie E.W. Seligsohn U. Factor XI deficiency in Ashkenazi Jews in Israel.N Engl J Med. 1991; 325: 153-8Crossref PubMed Google Scholar, 6Bolton-Maggs P.H. Young Wan-Yin B. McCraw A.H. Slack J. Kernoff P.B. Inheritance and bleeding in factor XI deficiency.Br J Haematol. 1988; 69: 521-8Crossref PubMed Google Scholar, 7Hu C.J. Baglia F.A. Mills D.C. Konkle B.A. Walsh P.N. Tissue-specific expression of functional platelet factor XI is independent of plasma factor XI expression.Blood. 1998; 91: 3800-7Crossref PubMed Google Scholar, 8Leiba H. Ramot B. Many A. Hereditary and coagulation studies in ten families with factor XI (plasma thromboplastin antecedent) deficiency.Br J Haematol. 1965; 11: 654-65Crossref PubMed Google Scholar, 9Ragni M.V. Sinha D. Seaman F. Lewis J.H. Spero J.A. Walsh P.N. Comparison of bleeding tendency, factor XI coagulant activity, and factor XI antigen in 25 factor XI-deficient kindreds.Blood. 1985; 65: 719-24Crossref PubMed Google Scholar, 10Rapaport S.I. Proctor R.R. Patch M.J. Yettra M. The mode of inheritance of PTA deficiency: evidence for the existence of a major PTA deficiency and a minor PTA deficiency.Blood. 1961; 18: 149-55Crossref PubMed Google Scholar, 11Rosenthal R.L. Dreskin O.H. Rosenthal N. New hemophilia-like disease caused by deficiency of a third plasma thromboplastin factor.Proc Soc Exp Biol Med. 1953; 82: 171-4Crossref PubMed Google Scholar, 12Sidi A. Seligsohn U. Jonas P. Many M. Factor XI deficiency. detection and management during urologic surgery.J Urol. 1978; 119: 528-30Crossref PubMed Google Scholar]. These interesting observations gave rise to two related important questions: (i) what is the relative contribution of FXIa vs. FVIIa-TF in the activation of FIX and the generation of thrombin during normal hemostasis?; and (ii) what is the preferred substrate for the FVIIa-TF complex, FIX or FX? These questions have been addressed in a series of well-designed and clearheaded investigations by a number of investigators, including a study presented in the present issue of the Journal of Thrombosis and Haemostasis by Butenas et al. The authors have presented a series of well-planned, meticulously conducted, and interesting experiments to investigate the influence of plasma and platelet FXI on thrombin generation initiated with relatively high concentrations (10 pmol L−1) of TF in a synthetic coagulation model, in the presence of either phospholipids or physiological concentrations of platelets, and physiological concentrations of vitamin K-dependent proteins or increased concentrations of these proteins up to 500%. The data clearly indicate that in the presence of phospholipids and in the absence of FXI, vitamin K-dependent proteins, particularly FIX and prothrombin, significantly prolong the initiation phase of thrombin generation and decrease maximum levels of thrombin. In experiments in the presence of platelets, either in the presence or absence of added physiological concentrations of FXI, an increase in vitamin K-dependent proteins had very little effect on the initiation phase of thrombin generation, indicating that FXI has no effect on thrombin generation at 10 pmol L−1 TF at physiological concentrations of vitamin K-dependent proteins. In contrast, platelets and plasma FXI are able to compensate for the inhibitory effects of elevated vitamin K-dependent proteins. It should be noted that in the experiments carried out with platelets, no specific platelet agonist was added. Since unactivated platelets are inert in supporting blood coagulation, a conclusion to be drawn from these observations is that the minute concentrations of thrombin generated by the addition of TF were sufficient to activate not only FXI, FVIII, and FV, but also platelets. Previous studies by these investigators and by other groups have shown that at low TF concentrations (<5 pmol L−1), the generation of thrombin was delayed and the maximum level of thrombin cleaved was reduced in FXI-deficient blood [42Cawthern K.M. Van't Veer C. Lock J.B. DiLorenzo M.E. Branda R.F. Mann K.G. Blood coagulation in hemophilia A and hemophilia C.Blood. 1998; 91: 4581-92Crossref PubMed Google Scholar, 43He R. Xiong S. He X. Liu F. Han J. Li J. He S. The role of factor XI in a dilute thromboplastin assay of extrinsic coagulation pathway.Thromb Haemost. 2001; 85: 1055-9Crossref PubMed Scopus (21) Google Scholar, 44Keularts I.M. Zivelin A. Seligsohn U. Hemker H.C. Beguin S. The role of factor XI in thrombin generation induced by low concentrations of tissue factor.Thromb Haemost. 2001; 85: 1060-5Crossref PubMed Scopus (77) Google Scholar, 45Butenas S. Van’t Veer C. Mann K.G. Evaluation of the initiation phase of blood coagulation using ultrasensitive assays for serine proteases.J Biol Chem. 1997; 272: 21527-33Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. This observation supports the essential role of FXI in hemostatic reactions, and is consistent with clinical observations, indicating that although spontaneous hemorrhage is rare in patients with FXI deficiency, about 50% of patients with FXI deficiency experience abnormal bleeding after trauma or surgical intervention [5Asakai R. Chung D.W. Davie E.W. Seligsohn U. Factor XI deficiency in Ashkenazi Jews in Israel.N Engl J Med. 1991; 325: 153-8Crossref PubMed Google Scholar, 6Bolton-Maggs P.H. Young Wan-Yin B. McCraw A.H. Slack J. Kernoff P.B. Inheritance and bleeding in factor XI deficiency.Br J Haematol. 1988; 69: 521-8Crossref PubMed Google Scholar, 7Hu C.J. Baglia F.A. Mills D.C. Konkle B.A. Walsh P.N. Tissue-specific expression of functional platelet factor XI is independent of plasma factor XI expression.Blood. 1998; 91: 3800-7Crossref PubMed Google Scholar, 8Leiba H. Ramot B. Many A. Hereditary and coagulation studies in ten families with factor XI (plasma thromboplastin antecedent) deficiency.Br J Haematol. 1965; 11: 654-65Crossref PubMed Google Scholar, 9Ragni M.V. Sinha D. Seaman F. Lewis J.H. Spero J.A. Walsh P.N. Comparison of bleeding tendency, factor XI coagulant activity, and factor XI antigen in 25 factor XI-deficient kindreds.Blood. 1985; 65: 719-24Crossref PubMed Google Scholar, 10Rapaport S.I. Proctor R.R. Patch M.J. Yettra M. The mode of inheritance of PTA deficiency: evidence for the existence of a major PTA deficiency and a minor PTA deficiency.Blood. 1961; 18: 149-55Crossref PubMed Google Scholar, 11Rosenthal R.L. Dreskin O.H. Rosenthal N. New hemophilia-like disease caused by deficiency of a third plasma thromboplastin factor.Proc Soc Exp Biol Med. 1953; 82: 171-4Crossref PubMed Google Scholar, 12Sidi A. Seligsohn U. Jonas P. Many M. Factor XI deficiency. detection and management during urologic surgery.J Urol. 1978; 119: 528-30Crossref PubMed Google Scholar]. The observation that levels of vitamin K-dependent proteins, particularly FIX and prothrombin, inhibit thrombin generation in the absence of FXI and platelets, and that the presence of FXI and platelets tend to obviate these inhibitory effects, suggests that the levels of FIX and prothrombin could regulate clot formation, especially in FXI deficiency. What is the explanation of this interesting phenomenon? Previously, Butanes et al. [46Butenas S. Van't Veer C. Mann K.G. ‘Normal’ thrombin generation.Blood. 1999; 94: 2169-78Crossref PubMed Google Scholar] have investigated the influence of alterations of blood coagulation factor levels to between 50% and 150% of their plasma levels for prothrombin, FX, FXI, FIX, FVII, FVIII, FV, protein C, protein S, antithrombin III, and TF pathway inhibitor in a synthetic plasma system, using either synthetic phospholipid vesicles or platelets with reactions initiated by a complex of FVIIa with TF (5 pmol L−1). Interestingly, increases in FIX concentrations to 150% of normal plasma levels resulted in decreased thrombin generation, whereas decreases to 50% resulted in enhanced thrombin generation. This was interpreted as a consequence of FIX as a competitive substrate with FX for FVIIa–TF. In marked contrast, studies from our laboratory have shown that zymogen FIX potentiates FIXa-catalyzed FX activation in the presence of platelets and FVIIIa [47London F.S. Walsh P.N. Zymogen factor IX potentiates factor IXa-catalyzed factor X activation.Biochemistry. 2000; 39: 9850-8Crossref Scopus (11) Google Scholar]. Thus, plasma levels of zymogen FIX specifically increased the affinity of FIXa for the intrinsic FX-activating complex on the surface of activated platelets [47London F.S. Walsh P.N. Zymogen factor IX potentiates factor IXa-catalyzed factor X activation.Biochemistry. 2000; 39: 9850-8Crossref Scopus (11) Google Scholar]. Therefore, it can be concluded that excess FIX decreases the generation of thrombin initiated by a TF-driven system by competing with FX for binding sites on TF–FVIIa. In contrast, zymogen FIX increases the flux of thrombin formed by the intrinsic pathway by potentiating FXa generation by the FIXa–FVIIIa complex on activated platelets. The observations presented by Butenas et al. in the present issue of the Journal of Thrombosis and Haemostasis and in previous communications are entirely consistent with those presented by Oliver et al. [48Oliver J.A. Monroe D.M. Roberts H.R. Hoffman M. Thrombin activates factor XI on activated platelets in the absence of factor XII.Arterioscler Thromb Vasc Biol. 1999; 19: 170-7Crossref PubMed Scopus (159) Google Scholar, 49Hoffman M. Monroe D.M. Oliver J.A. Roberts H.R. Factors IXa and Xa play distinct roles in tissue factor-dependent initiation of coagulation.Blood. 1995; 86: 1794-801Crossref PubMed Google Scholar], utilizing a synthetic coagulation model initiated by TF–FVIIa, indicating that thrombin activates FXI on activated platelets in the absence of FXII. They are also consistent with observations by von dem Borne et al. [50Von Dem Borne P.A. Meijers J.C. Bouma B.N. Feedback activation of factor XI by thrombin in plasma results in additional formation of thrombin that protects fibrin clots from fibrinolysis.Blood. 1995; 86: 3035-42Crossref PubMed Google Scholar], indicating that feedback activation of FXI by thrombin in plasma results in additional formation of thrombin, which protects fibrin clots from fibrinolysis. Thus, FXI plays a role in the downregulation of fibrinolysis, a consequence of an FXI-dependent burst in thrombin generation. This then results in the activation of thrombin activatable fibrinolysis inhibitor or procarboxypeptidase B, which inhibits the activation of plasminogen by removing from fibrin carboxyterminal lysines that are essential for plasminogen binding and activation [50Von Dem Borne P.A. Meijers J.C. Bouma B.N. Feedback activation of factor XI by thrombin in plasma results in additional formation of thrombin that protects fibrin clots from fibrinolysis.Blood. 1995; 86: 3035-42Crossref PubMed Google Scholar, 51Bajzar L. Manuel R. Nesheim M.E. Purification and characterization of TAFI, a thrombin-activatable fibrinolysis inhibitor.J Biol Chem. 1995; 270: 14477-84Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 52Hendriks D. Scharpe S. Van Sande M. Lommaert M.P. Characterization of a carboxypeptidase in human serum distinct from carboxypeptidase.Clin Chem Clin Biochem. 1989; 27: 277-85PubMed Google Scholar, 53Redlitz A. Tan A.K. Eaton D.L. Plow E.F. Plasma carboxypeptidases as regulators of the plasminogen system.J Clin Invest. 1995; 96: 2534-8Crossref PubMed Google Scholar, 54Tan A.K. Eaton D.L. Activation and characterization of procarboxypeptidase B from human plasma.Biochemistry. 1995; 34: 5811-6Crossref PubMed Google Scholar, 55Wang W. Hendriks D. Scharpe S. Carboxypeptidase U: a plasma carboxypeptidase with high affinity for plasminogen.J Biol Chem. 1994; 269: 15937-44Abstract Full Text PDF PubMed Google Scholar]. A final interesting point arising from the observations of Butenas et al. in this issue of the Journal is that in the presence of platelets compared with phospholipids, the lag time to the generation of thrombin was considerably shortened, and the rate of thrombin generation and the total yield of thrombin generated were significantly greater in the presence of platelets than in the presence of phospholipids, even in the absence of added FXI. Moreover, the inhibitory effects of supraphysiological concentrations of FIX and prothrombin were not observed when platelets were present, but were observed when phospholipids were present. These observations raise the interesting question of the role of platelet FXI in TF-mediated thrombin generation. Thus, the presence of platelet FXI coagulant activity and FXI antigen in well-washed platelet suspensions accounts for ∼0.5% of the FXI activity in normal plasma or ∼300 molecules per platelet of platelet FXI [56Komiyama Y. Nomura S. Murakami T. Masuda M. Egawa H. Murata K. Okubo S. Kokawa T. Yasunaga K. Purification and characterization of platelet-type factor XI from human platelets.Thromb Haemost. 1993; 69: 1238Google Scholar, 57Schiffman S. Rapaport S.I. Chong M.M. Platelets and initiation of intrinsic clotting.Br J Haematol. 1973; 24: 633-42Crossref PubMed Google Scholar, 58Schiffman S. Yeh C.H. Purification and characterization of platelet factor XI.Thromb Res. 1990; 60: 87-97Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 59Tuszynski G.P. Bevacqua S.J. Schmaier A.H. Colman R.W. Walsh P.N. Factor XI antigen and activity in human platelets.Blood. 1982; 59: 1148-56Crossref PubMed Google Scholar], which is enriched in the plasma membrane fraction of platelets [60Lipscomb M.S. Walsh P.N. Human platelets and factor XI. Localization in platelet membranes of factor XI-like activity and its functional distinction from plasma factor XI.J Clin Invest. 1979; 63: 1006-14Crossref PubMed Google Scholar]. Structural differences have been observed between platelet and plasma FXI [56Komiyama Y. Nomura S. Murakami T. Masuda M. Egawa H. Murata K. Okubo S. Kokawa T. Yasunaga K. Purification and characterization of platelet-type factor XI from human platelets.Thromb Haemost. 1993; 69: 1238Google Scholar, 58Schiffman S. Yeh C.H. Purification and characterization of platelet factor XI.Thromb Res. 1990; 60: 87-97Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 59Tuszynski G.P. Bevacqua S.J. Schmaier A.H. Colman R.W. Walsh P.N. Factor XI antigen and activity in human platelets.Blood. 1982; 59: 1148-56Crossref PubMed Google Scholar], and the presence of FXI mRNA amplified by RT-PCR from platelets and megakaryocytes [61Hsu T.C. Shore S.K. Seshsmma T. Bagasra O. Walsh P.N. Molecular cloning of platelet factor XI, an alternative splicing product of the plasma factor XI gene.J Biol Chem. 1998; 273: 13787-93Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 62Martincic D. Kravtsov V. Gailani D. Factor XI messenger RNA in human platelets.Blood. 1999; 94: 3397-404Crossref PubMed Google Scholar] confirms the view that platelet FXI might arise by expression of a megakaryocyte gene. Furthermore, platelet FXI can be identified by flow cytometry in the platelets of patients with severe deficiency of plasma FXI without bleeding complications even after trauma or major surgery [7Hu C.J. Baglia F.A. Mills D.C. Konkle B.A. Walsh P.N. Tissue-specific expression of functional platelet factor XI is independent of plasma factor XI expression.Blood. 1998; 91: 3800-7Crossref PubMed Google Scholar], suggesting that the presence of platelet FXI might account for the absence of hemostatic abnormalities in 50% of patients with plasma FXI deficiency [9Ragni M.V. Sinha D. Seaman F. Lewis J.H. Spero J.A. Walsh P.N. Comparison of bleeding tendency, factor XI coagulant activity, and factor XI antigen in 25 factor XI-deficient kindreds.Blood. 1985; 65: 719-24Crossref PubMed Google Scholar]. Thus, although the molecular nature of platelet FXI is controversial [61Hsu T.C. Shore S.K. Seshsmma T. Bagasra O. Walsh P.N. Molecular cloning of platelet factor XI, an alternative splicing product of the plasma factor XI gene.J Biol Chem. 1998; 273: 13787-93Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 62Martincic D. Kravtsov V. Gailani D. Factor XI messenger RNA in human platelets.Blood. 1999; 94: 3397-404Crossref PubMed Google Scholar], the present observations by Butenas et al. strongly suggest that platelet FXI might play an important role in the generation of thrombin initiated by the TF pathway. Thus, the observations that deficiencies of FXII, prekallikrein, and high molecular weight kininogen are not associated with hemostatic abnormalities, but that FXI deficiency produces abnormal bleeding complications in at least 50% of affected individuals [8Leiba H. Ramot B. Many A. Hereditary and coagulation studies in ten families with factor XI (plasma thromboplastin antecedent) deficiency.Br J Haematol. 1965; 11: 654-65Crossref PubMed Google Scholar, 9Ragni M.V. Sinha D. Seaman F. Lewis J.H. Spero J.A. Walsh P.N. Comparison of bleeding tendency, factor XI coagulant activity, and factor XI antigen in 25 factor XI-deficient kindreds.Blood. 1985; 65: 719-24Crossref PubMed Google Scholar, 10Rapaport S.I. Proctor R.R. Patch M.J. Yettra M. The mode of inheritance of PTA deficiency: evidence for the existence of a major PTA deficiency and a minor PTA deficiency.Blood. 1961; 18: 149-55Crossref PubMed Google Scholar, 63Rimon A. Schiffman S. Feinstein D.I. Rapaport S.I. Factor XI activity and factor XI antigen in homozygous and heterozygous factor XI deficiency.Blood. 1976; 48: 165-74Crossref PubMed Google Scholar, 64Rosenthal R.L. Dreskin O.H. Rosenthal N. Plasma thromboplastin antecedent (PTA) deficiency. Clinical coagulation, therapeutic and hereditary aspects of a new hemophilia-like disease.Blood. 1955; 10: 120-31Crossref PubMed Google Scholar], provides evidence that the more relevant pathway for activation of plasma FXI might be feedback activation by thrombin [13Naito K. Fujikawa K. Activation of human blood coagulation factor XI independent of factor XII. Factor XI is activated by thrombin and factor XIa in the presence of negatively charged surfaces.J Biol Chem. 1991; 266: 7353-8Abstract Full Text PDF PubMed Google Scholar, 14Gailani D. Broze Jr, G.J. Factor XI activation in a revised model of blood coagulation.Science. 1991; 253: 909-12Crossref PubMed Google Scholar, 18Baglia F.A. Walsh P.N. Prothrombin is a cofactor for the binding of factor XI to the platelet surface and for platelet-mediated factor XI activation by thrombin.Biochemistry. 1998; 37: 2271-81Crossref PubMed Scopus (92) Google Scholar]. Therefore, it is clear that FXI plays an important role in normal hemostasis. In addition, all three proteases cleave each monomer of FXI at the R369-I370 bond, generating the new amino-terminal sequence of the catalytic domain, I-V-G-G, which then activates the catalytic triad of the serine protease. On anionic surfaces, it has been postulated that a ternary complex is formed when FXI, in stoichiometric complex with HK in plasma, is adsorbed to the negatively charged surface where FXIIa recognizes it as a substrate [65Griffin J.H. Cochrane C.G. Mechanisms for the involvement of high molecular weight kininogen in surface-dependent reactions of Hageman factor.Proc Natl Acad Sci USA. 1976; 73: 2554-8Crossref PubMed Google Scholar, 66Meier H.L. Pierce J.V. Colman R.W. Kaplan A.P. Activation and function of human Hageman factor. The role of high molecular weight kininogen and prekallikrein.J Clin Invest. 1977; 60: 18-31Crossref PubMed Google Scholar, 67Ratnoff O.D. Davie E.W. Mallett D.L. Studies on the action of Hageman factor: evidence that activated Hageman factor in turn activates plasma thromboplastin antecedent.J Clin Invest. 1961; 40: 803-19Crossref PubMed Google Scholar, 68Thompson R.E. Mandle Jr, R. Kaplan A.P. Association of factor XI, high molecular weight kininogen in human plasma.J Clin Invest. 1977; 60: 1376-80Crossref PubMed Google Scholar, 69Wiggins R.C. Bouma B.N. Cochrane C.G. Griffin J.H. Role of high-molecular-weight kininogen in surface-binding and activation of coagulation factor XI and prekallikrein.Proc Natl Acad Sci USA. 1977; 74: 4636-40Crossref PubMed Google Scholar, 70Liu C.Y. Scott C.F. Bagdasarian A. Pierce J.V. Kaplan A.P. Colman R.W. Potentiation of the function of Hageman factor fragments by high molecular weight kininogen.J Clin Invest. 1977; 60: 7-17Crossref PubMed Google Scholar, 71Saito H. Ratnoff O.D. Marshall J.S. Pensky J. Partial purification of plasma thromboplastin antecedent (factor XI) and its activation by trypsin.J Clin Invest. 1973; 52: 850-61Crossref PubMed Google Scholar, 72Schiffman S. Lee P. Preparation, characterization, and activation of a highly purified factor XI: evidence that a hitherto unrecognized plasma activity participates in the interaction of factors XI and XII.Br J Haematol. 1974; 27: 101-14Crossref PubMed Google Scholar, 73Schiffman S. Markland Jr, F.S. Effect of intermediates of extrinsic clotting on purified factor XI: factor VII and/or thromboplastin.Thromb Res. 1975; 6: 273-9Abstract Full Text PDF PubMed Google Scholar]. The FXI homodimer is thereby cleaved to generate two disulfide-linked heavy chains (Mr 45 000–50 000) and two active-site-containing light chains (Mr 30 000–35 000) [74Bouma B.N. Griffin J.H. Human blood coagulation factor XI. Purification, properties, and mechanism of activation by activated factor XII.J Biol Chem. 1977; 252: 6432-7Abstract Full Text PDF PubMed Google Scholar, 75Kurachi K. Davie E.W. Activation of human factor XI (plasma thromboplastin antecedent) by factor XIIa (activated Hageman factor).Biochemistry. 1977; 16: 5831-9Crossref PubMed Google Scholar]. A very interesting issue addressed by a number of recent studies is the source of the TF that initiates the blood coagulation cascade and the cellular locus upon which coagulation proteases and cofactors are assembled, leading to the formation of the hemostatic thrombus. It has never made sense that coagulation reactions should be initiated on one cellular surface in the subendothelium, only to be propagated upon receptors exposed on the surface of activated platelets. How does the minute amount of FXa or thrombin generated by the TF pathway of subendothelial cells find its way to platelet receptors that promote the sequential activation of FXI, FIX, FX, and prothrombin, leading to sufficient thrombin generation to convert fibrinogen to fibrin? A plausible solution to this conundrum based on recent observations [76Falati S. Gross P. Merrill-Skoloff G. Furie B.C. Furie B. Real-time in vivo imaging of platelets, tissue factor and fibrin during arterial thrombus formation in the mouse.Nat Med. 2002; 8: 1175-81Crossref PubMed Scopus (544) Google Scholar, 77Giesen P.L. Rauch U. Bohrmann B. Kling D. Roque M. Fallon J.T. Badimon J.J. Himber J. Riederer M.A. Nemerson Y. Blood-borne tissue factor: another view of thrombosis.Proc Natl Acad Sci USA. 1999; 96: 2311-5Crossref PubMed Scopus (896) Google Scholar, 78Himber J. Kling D. Fallon J.T. Nemerson Y. Riederer M.A. In situ localization of tissue factor in human thrombi.Blood. 2002; 99: 4249-50Crossref PubMed Scopus (0) Google Scholar, 79Morrissey J.H. Tissue factor: in at the start… and the finish?.J Thromb Haemost. 2003; 1: 878-80Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 80Rauch U. Bonderman D. Bohrmann B. Badimon J.J. Himber J. Riederer M.A. Nemerson Y. Transfer of tissue factor from leukocytes to platelets is mediated by CD15 and tissue factor.Blood. 2000; 96: 170-5Crossref PubMed Google Scholar, 81Rauch U. Nemerson Y. Tissue factor, the blood, and the arterial wall.Trends Cardiovasc Med. 2000; 10: 139-43Crossref PubMed Scopus (0) Google Scholar] is that TF-bearing microparticles shed by monocytes (which synthesize TF) can fuse with platelet membranes via adhesive receptors, thereby transferring TF to platelets that do not normally express it. This interesting hypothesis is supported by the recent demonstration that plasma membrane-associated TF in monocytes is enriched in specialized membrane microdomains (i.e. lipid rafts) which are the source of membrane microparticles rich in phosphatidylserine and TF [82Conde I. Shrimpton C.N. Thiagarajan P. Lopez J.A. Tissue factor-bearing microparticles arise from monocyte lipid rafts and can fuse with activated platelets, consolidating all of the membrane-bound coagulation reactions on the platelet surface.J Thromb Haemost. 2003; 1 (Abstract OC146)Google Scholar]. These microparticles can bind to P-selectin on activated platelets via PSGL-1 in microparticles, thereby transferring TF by membrane fusion from microparticles to activated platelets. Confirmation of this interesting hypothesis will require the demonstration that the TF antigen so convincingly demonstrated in these studies is functionally active. In conclusion, a revised model of the sequence of coagulation events triggered by the exposure of TF at sites of vascular injury and leading to thrombin generation sufficient to cleave fibrinogen to cross-linked fibrin is presented in the accompanying figure. It should be noted that virtually all the enzymatic reactions that comprise the coagulation mechanism occur on cellular surfaces, including subendothelial vascular cells and monocytes, which expose TF at sites of vascular injury, and platelets, which are inert in exposing receptors for coagulation complexes until activated by binding to collagen or interaction with low concentrations of thrombin. Following the exposure of TF and the assembly of the FVIIa–FX–TF complex, FXa generation simultaneously allows TF pathway inhibitor to shut down the further generation of FXa while simultaneously generating small quantities of thrombin that can activate platelets, FXI, FVIII, and FV. Although the assembly of the TF–FVIIa–FIX complex can also give rise to FIXa and thereby generate FXa, it would appear that this pathway by itself is insufficient to trigger the consolidation pathway of blood coagulation, as evidenced by the bleeding complications that occur in 50% of patients with FXI deficiency. The small quantities of thrombin generated lead to the activation of FXI on the activated platelet surface in the presence either of prothrombin and calcium ions or of high molecular weight kininogen and zinc ions. Factor XIa and FIX can both bind to the activated platelet surface and give rise to sufficient quantities of FIXa to activate FX in the presence of FVIII on the activated platelet surface. Protease nexin II is secreted from platelet alpha-granules at sufficiently high concentrations to rapidly and effectively inhibit the activation of FIX by FXIa in solution, but FIX activation by platelet-bound FXIa proceeds in a protected environment unaffected by protease nexin II. The assembly of the FX-activating complex on the platelet surface results in a >20-million-fold acceleration of FX activation on the platelet surface compared with the solution-phase reaction. This in turn gives rise to FXa, which binds to activated platelets in the presence of FVa and activates prothrombin 200 000-fold more effectively than solution phase FXa. Consequently, sufficient quantities of thrombin are formed to convert fibrinogen to fibrin and to effect normal hemostasis.