Focus Article

The Coagulation Cascade
Guest Contributor: George J Broze, Jr, MD

Introduction


   Blood coagulation is p...
Coagulation Cascade


generation of thrombin proceeding through a single, "common" pathway (Figure 1).
The intrinsic pathw...
Coagulation Cascade


   The segregation of the known coagulation factors into the intrinsic and extrinsic
pathways and th...
Coagulation Cascade


showed that, in the presence of FX, an agent in the lipoprotein fraction of plasma
produced inhibiti...
Coagulation Cascade


Figure 2 Tissue Factor Pathway of Coagulation


          Vessel                   IX               ...
Coagulation Cascade


coagulation assays) occurs at low thrombin concentrations (~10 nM), long before the
bulk of the thro...
Coagulation Cascade


Factor XI and Thrombin-Activatable Fibrinolysis Inhibitor (TAFI)


   In the intrinsic pathway of co...
Coagulation Cascade


Important New Wrinkles


   Figures 1 and 2 only depict the proteins that are involved in the coagul...
Coagulation Cascade


References

1.    Broze GJ Jr, Girard TJ, Novotny WF: Perspectives in biochemistry: Regulation of co...
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Revision cascada de la coagulacion

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Revision cascada de la coagulacion

  1. 1. Focus Article The Coagulation Cascade Guest Contributor: George J Broze, Jr, MD Introduction Blood coagulation is part of the hemostatic response to injury and assists in maintaining the integrity of the vascular system. It involves a complex series of interactions between protease zymogens, enzymes, and cofactors that leads to the generation of thrombin and a fibrin clot. Owing to the influence of Schmidt and Morawitz, it was widely accepted in the first half of the 20th century that the initiating event in blood coagulation was the exposure of plasma to damaged tissues. The substance in tissues responsible for this induction of coagulation was initially called tissue thromboplastin and later tissue factor (factor III). Early studies suggesting an alternative pathway of coagulation that does not require tissue factor were rationalized to conform to the prevalent theory of Schmidt and Morawitz. Mounting evidence, in particular the observation that blood from hemophiliacs apparently clotted normally after the addition of tissue factor, however, eventually forced a reassessment of the coagulation mechanism. In 1964, the "cascade" theory of McFarlane and the "waterfall" theory of Davie and Ratnoff separated the known coagulation factors into two pathways, the intrinsic and the extrinsic, which converge at the activation of factor X (FX) with the subsequent
  2. 2. Coagulation Cascade generation of thrombin proceeding through a single, "common" pathway (Figure 1). The intrinsic pathway, in which exposure of the contact factors (factor XII [FXII], high- molecular weight kininogen, and prekallikrein) in plasma to a surface leads to the initiation of coagulation, appeared to be the pathway most important for hemostasis since it contained factors VIII (FVIII) and IX (FIX), whose deficiencies cause the severe bleeding of hemophilia. Tissue factor-mediated extrinsic coagulation was relegated to a lesser role. Figure 1 Cascade & Waterfall Hypothesis of Coagulation Surface HMWK Prekallikrein XII XIIa X XI XIa IX IXa VIIa VIIIa TF Xa Va Prothrombin Thrombin Fibrinogen Fibrin The cascade and waterfall hypothesis of blood coagulation. The intrinsic pathway of coagulation is initiated by exposure of the contact factors (factor XII, prekallikrein, high-molecular weight kininogen) to an appropriate surface with subsequent activation of factor XI by factor XIIa. The extrinsic pathway of coagulation is initiated by exposure of factor VIIa to tissue factor. The requirement for phospholipids and calcium ions in certain of the reactions is not indicated. Page 2 Broze (2002, updated 2005) CLOT-ED, Inc., Copyright 2005. All rights reserved.
  3. 3. Coagulation Cascade The segregation of the known coagulation factors into the intrinsic and extrinsic pathways and the availability of assays to test each pathway (partial thromboplastin time [aPTT] and prothrombin time [PT], respectively) proved invaluable in the diagnosis of hemorrhagic diseases. Today, these tests still provide the foundation of any coagulation evaluation. The cascade and waterfall hypotheses, however, failed to reflect hemostasis accurately. Individuals deficient in one of the contact factors required for the initiation of intrinsic coagulation are asymptomatic, whereas individuals deficient in factor VII bleed abnormally. Moreover, if the factor VII(a)/tissue factor (FVIIa/TF) complex can activate FX, albeit considerably less effectively than FIXa/FVIIIa, why do hemophiliacs bleed? An important clue to the resolution of this dilemma was provided in 1977 by Osterud and Rapaport who showed that FVIIa/TF can activate FIX of the intrinsic pathway as well as FX. Finally, the rediscovery of an endogenous inhibitor of the FVIIa/TF complex, now called tissue factor pathway inhibitor (TFPI), led to a reformulation of the coagulation cascade in which tissue factor is responsible for the initiation of coagulation, but subsequent amplification of the clotting process through the action of FVIII and FIX is absolutely required for sustained hemostasis 1,2. Tissue Factor Pathway Inhibitor (TFPI) In 1947, Thomas and Schneider independently observed that incubation of crude tissue thromboplastin with serum prevented the lethal disseminated intravascular coagulation that follows thromboplastin infusion in animals. Later, Hjort showed that the serum inhibitor recognized the FVIIa/TF complex rather than FVIIa or TF alone. About the same time, Biggs and her colleagues showed that coagulation was delayed and incomplete after the addition of low concentrations of TF to hemophilic plasma. This result contrasted with that of the standard PT assay in which relatively large quantities of TF are used to initiate coagulation, and in which plasma deficient in FVIII or FIX is indistinguishable from normal plasma. With the subsequent demonstration of an "intrinsic" pathway of coagulation, however, interest was diverted away form the possible connection between Biggs' work and the thromboplastin inhibitor. Finally, a report by Rapaport and colleagues revitalized interest in the inhibitor in 1985. They Page 3 Broze (2002, updated 2005) CLOT-ED, Inc., Copyright 2005. All rights reserved.
  4. 4. Coagulation Cascade showed that, in the presence of FX, an agent in the lipoprotein fraction of plasma produced inhibition of tissue factor-mediated coagulation. Other investigators soon confirmed this result and found that the properties of this inhibitor were identical to those of the FVIIa/TF inhibitor described by Hjort in 1957. TFPI directly inhibits FXa and, in a FXa-dependent fashion, produces feedback inhibition of the FVIIa/TF catalytic complex. The latter process involves the formation of a quaternary inhibitory complex that contains FVIIa/TF, FXa, and TFPI. The formation of this complex is frequently described as a two-step process in which TFPI first binds to soluble FXa and then the FXa-TFPI complex binds to FVIIa/TF. Instead, kinetic studies strongly suggest that TFPI interacts with FXa that has not yet been released from the FVIIa/TF or remains at the phospholipid surface in close proximity to FVIIa/TF3. As a result, FVIIa/TF inhibition by TFPI is extremely rapid and the concentration of active FXa and that escapes regulation is linearly dependent on the level of available of TF. A Revised Coagulation Cascade 1,2 The properties of TFPI led to a revised coagulation cascade (Figure 2). Coagulation ensues when damage to blood vessels allows the exposure of blood to the TF produced constitutively by cells beneath the endothelium. The FVII or FVIIa present in plasma then binds to TF and the FVIIa/TF complex activates limited quantities of FX and FIX. Some of the FXa generates low levels of thrombin sufficient to activate platelets and the critical coagulation cofactors FV and FVIII, but TFPI dampens the clotting process by producing FXa-dependent feedback inhibition of the FVIIa/TF complex. Persistent and amplified FXa and thrombin generation then proceeds through the actions of FIXa with its cofactor FVIIIa. Consistent with its regulatory role, inhibition of TFPI activity has been shown to ameliorate bleeding in an animal model of hemophilia 4. Page 4 Broze (2002, updated 2005) CLOT-ED, Inc., Copyright 2005. All rights reserved.
  5. 5. Coagulation Cascade Figure 2 Tissue Factor Pathway of Coagulation Vessel IX IX Injury VII a TF IX a TFPI XI a VIII a X X Throm bin XI Xa Va TAFI Throm bin Prothrom bin Throm bin TAFI a Fibrinogen FIBRIN Fibrinolysis Hemostasis is initiated when factor VII and factor VIIa in plasma gain access to tissue factor at a site of blood vessel injury. Limited quantities of factor IXa and factor Xa are produced before feedback inhibition of the factor VIIa/tissue complex mediated by TFPI. The subsequent generation of factor Xa and thrombin is then amplified through the action of factor VIIIa and factor IXa, the latter produced initially by factor VIIa/tissue factor and supplemented by factor XIa formed through the action of thrombin. Thrombin activation of TAFI, which is enhanced by the cofactor thrombomodulin (TM), inhibits fibrinolysis of the clot. Activated platelets provide a membrane surface critical for many of the reactions: for example, the activation of factor X by factors IXa/VIIIa; the activation of prothrombin by factors Xa/Va; and factor XI activation by thrombin. In this scheme, thrombin generation can be artificially separated into two phases: initiation and amplification/propagation (Figure 3). In normal blood it is FXa generation that determines the rate and extent of thrombin production 5. FVIIa/TF is predominantly responsible for the slow rate of FX activation at the initiation of coagulation. Enhanced FXa production through the actions of FIXa and FVIIIa leads to a rapid acceleration of thrombin generation and the amplification phase of coagulation. In hemophilia (FVIII or FIX deficiency), the duration of the initiation phase is extended and the amplification phase of coagulation is dramatically reduced when low levels of tissue factor are used to trigger coagulation. Note that initial fibrin formation (the end-point of clinical Page 5 Broze (2002, updated 2005) CLOT-ED, Inc., Copyright 2005. All rights reserved.
  6. 6. Coagulation Cascade coagulation assays) occurs at low thrombin concentrations (~10 nM), long before the bulk of the thrombin has been generated (arrows in Figure 3). Figure 3 Initiation & Amplification Phases of Coagulation Thrombin Activity Time Depiction of thrombin generation in normal and hemophilic blood following their exposure to low levels of tissue factor. Arrows denote time of clot formation The high rate and level of thrombin produced during the amplification phase of coagulation, however, is critical for the stability of the clot and hemostasis. This "extra" thrombin overcomes the effect of coagulation inhibitors, affects the structure of the developing fibrin network, and reduces the rate of subsequent fibrinolysis (see below). Depression of the amplification phase of coagulation appears to be responsible for the delayed hemorrhage that is the clinical hallmark of hemophilia. Page 6 Broze (2002, updated 2005) CLOT-ED, Inc., Copyright 2005. All rights reserved.
  7. 7. Coagulation Cascade Factor XI and Thrombin-Activatable Fibrinolysis Inhibitor (TAFI) In the intrinsic pathway of coagulation, activation of FXI by FXII of the contact system is responsible for the initiation of coagulation. In the revised scheme, it is FVIIa/TF that triggers coagulation suggesting that FXI functions later in the coagulation cascade. In 1991, two groups found that thrombin could activate FXI in a FXIIa- 6,7 independent manner . Importantly, Baglia and Walsh subsequently showed that this thrombin activation of FXI was dramatically enhanced at the surface of activated 8 platelets . FXIa generated at the wound can then produce additional FIXa to supplement that initially produced by FVIIa/TF and limited by TFPI. The model predicts that this ancillary FIXa would be most important at sites with limited TF exposure and/or high fibrinolytic activity. This is consistent with the phenotype of FXI deficiency: overall a moderate hemorrhagic diathesis, but with a substantial risk of bleeding following surgical manipulation in the mouth or the urinary tract. The thrombin generated during coagulation also inhibits fibrinolysis by activating (TAFI) 9. Clot lysis involves a positive feedback loop. Plasmin cleaves fibrin at sites following lysine residues. These exposed lysine residues serve to enhance the fibrin binding of plasminogen, which contains lysine-binding sites. As plasminogen bound to fibrin is activated to plasmin much more effectively by tPA and uPA than plasminogen in solution, fibrinolysis is enhanced. Activated TAFI (TAFIa) is a carboxypeptidase enzyme that proteolytically excises basic (lysine, arginine) residues from the end of polypeptides. By removing the C-terminal lysine residues from the polypeptides in the partially digested fibrin, TAFIa interferes with the positive feedback mechanism and inhibits fibrinolysis. The therapeutic inhibitors of fibrinolysis, ,ε-amino caproic acid (Amicar®) and tranexamic acid, are mimics of the amino acid lysine and produce a similar effect by binding to the lysine-binding sites in plasminogen. The premature lysis of hemostatic clots in hemophiliacs and individuals with factor XI deficiency appears to be due in part to inefficient TAFI activation 10,11. Page 7 Broze (2002, updated 2005) CLOT-ED, Inc., Copyright 2005. All rights reserved.
  8. 8. Coagulation Cascade Important New Wrinkles Figures 1 and 2 only depict the proteins that are involved in the coagulation. It is important to realize that many of the critical reactions in coagulation occur at the surface of cells. Although phospholipid vesicles are frequently used to provide the procoagulant surface for coagulation factor assembly in vitro, they do not completely replicate the results obtained with cells whose membranes contain a variety of additional constituents. Activated platelets, which accumulate at the site of a wound, appear to provide the optimal surface for coagulation reactions and inhibitors of platelet function affect thrombin generation (e.g. ref. 12). Based on an in vitro cell-based system, Hoffman, Monroe, Roberts, and their colleagues argue that a major reason for the bleeding in hemophilia is that FXa formed on the surface of a TF-bearing cell is rapidly inactivated by proteinase inhibitors as it attempts to transfer to the platelet. In contrast, FIXa produced by FVIIa/TF is much less susceptible to proteinase inhibitors, reaches the platelet surface, and there, with its cofactor, FVIIIa, generates FXa in a protected environment (see ref. 13 for review). Recent work by Nemerson and his colleagues suggests that TF plays a role not only in the initiation, but also in the propagation of coagulation (see ref. 14 for review). They have shown that circulating leukocytes and their shed microvesicles carrying TF bind to activated platelets at the site of vascular injury and enhance thrombus growth. This process involves an interaction between P-selectin on the platelet and CD15 (P-selectin ligand, sialyl Lewis x [sLex]) on the leukocyte-derived membrane (see ref. 15 for review). Apparently the circulating tissue factor is "encrypted" or at a level below a threshold needed to initiate coagulation. Interaction of microvesicles/leukocytes with activated platelets serves to concentrate and, perhaps, de-encrypt the tissue factor at the site of the developing thrombus. As the delivery of circulating microvesicles/leukocytes is dependent on flow rate, this process may be most important at sites of arterial injury. Page 8 Broze (2002, updated 2005) CLOT-ED, Inc., Copyright 2005. All rights reserved.
  9. 9. Coagulation Cascade References 1. Broze GJ Jr, Girard TJ, Novotny WF: Perspectives in biochemistry: Regulation of coagulation by a multivalent Kunitz-type inhibitor. Biochemistry 1990;29:7539-7546. 2. Broze GJ Jr. The role of tissue factor pathway inhibitor in a revised coagulation cascade. Sem. Hematol. 1992;29:159-169. 3. Baugh RJ, Broze GJ Jr, Krishnaswamy S. Regulation of extrinsic pathway factor Xa formation by tissue factor pathway inhibitor. J. Biol. Chem. 1998;273:4378-4386. 4. Erhardtsen E, Ezban M, Madsen MT, Diness V, Glazer S, Hedner U, Nordfang O. Blocking of tissue factor pathway inhibitor (TFPI) shortens the bleeding time in rabbits with antibody induced haemophilia A. Blood Coag Fibrinol 1995;6:388-394. 5. Rand MD, Lock JB, van't Veer C, Gaffney DP, Mann KG. Blood clotting in minimally altered whole blood. Blood 1996;88:3432-3445. 6. Naito 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;266:7353-7358. 7. Gailani D, Broze GJ Jr. Factor XI activation in a revised model of blood coagulation. Science 1991;253:909-912. 8. Baglia FA, Walsh PN. 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;37:2271-2281. 9. Bajzar L, Manuel R, Nesheim ME. Purification and characterization of TAFI. A thrombin-activatable fibrinolysis inhibitor. J Biol Chem 1995;270:14477-14484. 10. Broze GJ Jr, Higuchi DA. Coagulation-dependent inhibition of fibrinolysis: Role of carboxypeptidase-U and the premature lysis of clots from hemophilic plasma. Blood 1996;88:3815-3823. 11. Von dem Borne PA. Bajzar L. Meijers JC. Nesheim ME. Bouma BN. Thrombin-mediated activation of factor XI results in a thrombin-activatable fibrinolysis inhibitor-dependent inhibition of fibrinolysis. J Clin Invest 1997;99:2323-2327. 12. Butenas S, Cawthern KM, van't Veer C, DiLorenzo ME, Lock JB, Mann KG. Antiplatelet agents in tissue factor-induced blood coagulation. Blood 2001;97:2314-2322. 13. Hoffman M, Monroe DM. A cell-based model of hemostasis. Thromb Haemost 2001;83:958-965. 14. Rauch U, Nemerson Y. Tissue factor, the blood, and the arterial wall. Trends Cardiovasc Med 2000;10:139-143. 15. Vandendries ER, Furie BC, Furie B. Role of P-selectin and PSGL-1 in coagulation and thrombosis. Thromb Haemost 2004;92:459-466. Address for Correspondence George J Broze, Jr, MD Washington University School of Medicine, Division of Hematology, Box 8125 660 S. Euclid Avenue St Louis, MO 63110 T: (314) 362-8809, F: (314) 362-8813 e-mail: gbroze@im.wustl.edu Page 9 Broze (2002, updated 2005) CLOT-ED, Inc., Copyright 2005. All rights reserved.

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