Skip to main content
PMC home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Feb 1.
Published in final edited form as: Transfus Apher Sci. 2008 Feb 20;38(1):15–23. doi: 10.1016/j.transci.2007.12.005

Thrombin Generation, Fibrin Clot Formation and Hemostasis

Alisa S Wolberg 1, Robert A Campbell 1
PMCID: PMC2408736  NIHMSID: NIHMS51663  PMID: 18282807

Abstract

Hemostatic clot formation entails thrombin-mediated cleavage of fibrinogen to fibrin. Previous in vitro studies have shown that the thrombin concentration present during clot formation dictates the ultimate fibrin structure. In most prior studies of fibrin structure, clotting was initiated by adding thrombin to a solution of fibrinogen; however, clot formation in vivo occurs in an environment in which the concentration of free thrombin changes over the reaction course. These changes depend on local cellular properties and available concentrations of pro- and anti-coagulants. Recent studies suggest that abnormal thrombin generation patterns produce abnormally structured clots associated with an increased risk of bleeding or thrombosis. Further studies of fibrin formation during in situ thrombin generation are needed to understand fibrin clot formation in vivo.

Keywords: thrombin, fibrinogen, fibrin, plasmin, fibrinolysis, clot structure, hemophilia, thrombosis

Fibrinogen and fibrin clot formation

Fibrinogen is a 450 Å, 340 kDa trinodular protein present at high (2 – 4 mg/mL) concentrations in plasma. Fibrinogen consists of pairs of three different disulfide-linked polypeptide chains: Aα, Bβ, and γ. These six polypeptide chains are arranged with their N-termini converged in a central “E” domain of the molecule. The C-termini of the Bβ and γ chains extend outward into distal “D” domains. The C-termini of the Aα chains are globular and situated near the central E domain of fibrinogen where they interact intra-molecularly.1 Comprehensive reviews on the biochemistry of fibrinogen and fibrin structure have been published.2–4

Mechanisms of fibrin production have been elucidated in studies conducted by adding exogenous thrombin to purified fibrinogen. These studies have shown that thrombin removes N-terminal peptides from the fibrinogen Aαl and Bβ chains, causing the spontaneous formation of half-staggered, double-stranded protofibrils followed by thickening of protofibril chains.5 Initial formation of protofibrils occurs during a “lag” phase in which no turbidity increase is detected. Subsequent lateral aggregation of fibrin protofibrils causes a turbidity increase. The magnitude of the turbidity increase relates to the structure of the formed clot; formation of thicker fibers causes a greater increase in final turbidity.6,7 Fibrin formation can be followed spectrophotometrically 8,9; while, fibrin structure can be determined using electron or confocal microscopy.10–15

The thrombin concentration influences fibrin clot formation, structure, and stability

Fibrin clot structure depends on a number of variables, including pH, ionic strength, and concentrations of calcium, fibrinogen, and thrombin present during gelation.5,8,10,16–21 In particular, the thrombin concentration influences both the fiber thickness and density of the fibrin clot. Low thrombin concentrations produce very turbid, highly permeable fibrin clots that are composed of thick, loosely-woven fibrin strands (Figure 1). Higher thrombin concentrations produce relatively non-turbid, less permeable clots that are composed of a dense network of relatively thin fibrin strands (Figure 1).11,21–23 The differential structures of these clots has been shown to impart varying degrees of stability (viscoelastic properties and resistance to fibrinolysis) on the clot.

Figure 1.

Figure 1

Open in a new tab

The thrombin concentration present at the time of gelation dictates the fibrin clot structure. Scanning electron (A an B) and laser confocal (C and D) micrographs of fibrin clots formed by adding thrombin [2.5 nM (A and C), or 10 nM (B and D)] to purified fibrinogen (1 mg/mL) in 20 mM HEPES (pH 7.4), 150 mM NaCl. Scanning electron micrographs are at 76,030 × (micron bar is 200 nm). Laser confocal micrographs are at 63 X (micron bar is 10 μm).

Individual fibers have significant extensibility and elasticity 24,25; however, the potential influence of fiber thickness on the properties of individual fibers is not yet known. In contrast, the influence of clot structure on viscoelastic properties has been extensively examined. These studies have shown the relationship between fibrin structure and clot stiffness to be highly complex, depending on fiber thickness, length, density, and degree of branching, and extent of cross-linking (reviewed in 21,26). Generally, increased thrombin concentrations are associated with the formation of stiffer clots 27; however, Ryan et al. observed that clots formed in the presence of thrombin concentrations above 0.25 U/mL (composed of thin fibers) are less stiff (smaller G′) compared to clots formed with lower thrombin concentrations (thicker fibrin fibers).21 The differences in findings from these studies may relate to differences in the experimental conditions (fibrinogen, thrombin and calcium concentrations, ionic strength, and pH).

Because fibrin functions as both a cofactor and a substrate for the fibrinolytic enzyme plasmin, the fibrin structure influences the clot’s susceptibility to fibrinolysis.12,28–30 Compared to thick fibers, thin fibrin fibers support a slower rate of tPA-mediated plasmin generation, reducing the rate of fibrinolysis.31 An elegant study by Collet et al. (2000) showed that while thin fibers are lysed more quickly than thick fibers, clots composed of thin fibers are more resistant to fibrinolysis than clots composed of thick fibrin fibers.12 These studies indicate that clots formed in even “simple” purified systems with single thrombin concentrations exhibit complex functionality.

Normal thrombin generation in situ

In vivo, thrombin generation is an intricate and highly dynamic process. Coagulation involves specific interactions between cellular surfaces and plasma-derived zymogens and cofactors. The reactions are typically described in three sequential phases: initiation, amplification, and propagation. During the initiation phase, active tissue factor is exposed to plasma, permitting formation of a complex between factor VIIa and tissue factor. The factor VIIa/tissue factor complex activates factors IX and X. During the amplification phase, the factor VIIa/tissue factor-activated factor Xa promotes small-scale thrombin generation, which activates cofactors VIII, V and platelets. Phosphatidylserine exposed on activated platelets provides binding sites for procoagulant zymogens and enzymes, including factors IXa, VIIIa, X, Xa, Va, and II.32–35 During the propagation phase, intrinsic activities supported by the activated platelet surface, produce a large-scale thrombin burst that catalyzes the conversion of fibrinogen to fibrin.36,37 Thrombin produced during these phases also activates the transglutaminase factor XIII, which catalyzes formation of covalent bonds between γ– chains and between γ– and Aα– chains.38 Cross-linking increases the elasticity of individual fibers in the clot24, as well as the overall viscoelasticity of the fibrin clot.16 Ultimately, fibrin promotes the generation of plasmin to degrade the clot, and re-establish a blood supply for new cells proliferating at the site.

During a coagulation reaction, the concentration of free thrombin present can range from less than 1 nM to greater than 500 nM. When thrombin generation is followed using chromogenic or fluorogenic substrates, a characteristic pattern emerges.39–42 This pattern correlates with the phases of coagulation described above. The small amounts of thrombin produced during the initiation/amplification phases are not detectable in amidolytic assays (lower limit of detection ~1 nM), resulting in a “lag” phase in the thrombin generation profile. The large burst of thrombin generation that occurs in the propagation phase on the activated platelet surface produces a “maximum rate of increase” in detectable free thrombin. When the rates of thrombin production and thrombin inhibition reach equilibrium, the thrombin generation profile exhibits a “peak.” Finally, the drop in thrombin production allows the amount of free thrombin to decrease, which is seen as a return to baseline in the thrombin generation profile. The total amount of free thrombin measured during the reaction course is referred to as the “area under the curve” (AUC) or “endogenous thrombin potential” (ETP).

The turbidimetric pattern of fibrin clot formation during in situ thrombin generation resembles the pattern seen after exogenous thrombin is added to purified fibrinogen. A lag phase, followed by a rapid increase in turbidity indicates clot formation in both situations. However, the “lag” phase of clot formation during in situ thrombin generation is more complex, reflecting not only protofibril formation, but also the time required to generate thrombin on the tissue factor-bearing cell surface, activate cofactors and platelets, and commence thrombin generation on the surface of activated platelets.

The biochemical environment required for in situ thrombin generation can profoundly affect fibrin formation. Assembly of procoagulant complexes and generation of thrombin requires calcium, which shortens the onset of clotting and produces thicker fibrin fibers than are seen in the absence of calcium.18 Additionally, several plasma proteins can directly or indirectly affect thrombin activity and fibrin formation. Antithrombin inhibits free thrombin, effectively lowering the apparent thrombin concentration, and resulting in prolonged times to gelation and thicker fibrin fibers.43–47 Albumin, γglobulin, and hemoglobin shorten the onset of fibrin clot formation, a phenomenon hypothesized to be caused by their influence on macromolecular interactions in the clotting solution.45,48 Further, these proteins cause significant differences between magnetic birefringence curves of fibrin polymerization in recalcified plasma and those of pure fibrinogen and thrombin solutions.45 Cells can influence local fibrin structure via direct interactions between integrins and fibrin(ogen). Cellular integrins organize fibrin into tighter bundles near the cell surface than are seen more distally within the clot. Furthermore, cell-associated fibrin is more resistant to fibrinolysis than distally-located fibrin.49–51 Cells also release intracellular stores of soluble proteins that influence clot formation and stability, including factor XIII, plasminogen activator inhibitor-1 (PAI-1), and fibrinogen.49,52–55

Effect of the thrombin generation pattern and location on fibrin clot formation and structure

Differences in cellular procoagulant activity and plasma factor levels can alter the relative influences of extrinsic and intrinsic activities during coagulation.13,14,41,56–61 Variations in these activities produce different patterns of thrombin generation, causing variations in the concentration of thrombin present during protofibril and fiber formation. Fibrinopeptide release may occur under low, medium, or high thrombin concentrations, resulting in significantly different kinetics of fibrinopeptide release and fibrin polymerization compared to assays in which a single thrombin concentration catalyzes the release of all fibrinopeptides. Since fibrinopeptide release dictates protofibril formation and lateral aggregation, clots produced during in situ thrombin generation contain considerably heterogeneous fibrin structures.13,14,45,62,63 Thus, the composition of a given clot may be quite specific to the circumstances under which it formed. Additionally, following formation of the initial clot, fibrin-bound thrombin released during clot lysis can modulate subsequent platelet procoagulant activity and fibrin deposition.64–66

It is currently hypothesized that extrinsic activities (on the tissue factor-bearing cell) and intrinsic activities (on the surface of activated platelets) play specific, independent roles during different stages of thrombin generation and fibrin formation.67–70 Low thrombin concentrations (less than 1 nM) are sufficient to trigger the onset of fibrin formation, and can be rapidly produced via extrinsic activities on surface of tissue factor-bearing cells. Thus, the onset of clot formation depends on the nature and procoagulant properties of the tissue factor-bearing cells. Since different tissue factor-bearing cells support different levels of procoagulant activity, they differ in their ability to initiate fibrin formation.68,71–73 Interestingly, Ovanesov et al. (2005) showed that the rate of thrombin generation on tissue factor-bearing cells does not influence the rate of propagation of fibrin into the plasma milieu.68 Rather, propagation of fibrin away from the site of initiation depends on the plasma composition and procoagulant activity of the surrounding milieu.68 These findings suggest that thrombin generated on the surface of platelets during the propagation phase dictates that rate of fibrin clot formation away from the initiating cells.67–70

Fibrin formation during cell-mediated thrombin generation likely includes a complex spatial component as well. It is likely that this physical progression of procoagulant activity from the tissue factor-bearing cell (low levels of thrombin generation) to the activated platelet surface (rapid burst of thrombin generation) induces the formation of a thrombin gradient in space. Given the direct effects of thrombin concentration on fibrin formation, this gradient may therefore, cause the formation of a range of fiber thicknesses across a region of growing thrombus.

Pathologic effects of altered thrombin generation on fibrin clot formation, structure and stability

Many studies have shown that the fibrin structure strongly influences the mechanical and fibrinolytic stability of the clot. Current thinking suggests that these differences in stability have significant physiologic implications. Dense, highly stable clots are associated with thrombosis; while, loosely-woven, unstable clots are associated with bleeding disorders.12,28–30,74–78 We and others have correlated abnormal patterns of thrombin generation with the formation of abnormal fibrin clots. We hypothesize that the altered thrombin generation pattern contributes to thrombotic or bleeding complications via its influence on fibrin structure and stability.

Hyperprothrombinemia

The G20210A mutation in the 3′ untranslated region of prothrombin causes increased plasma levels of prothrombin. Several studies have identified an increased risk of venous thrombosis in individuals with this mutation.79–81 In in vitro assays, elevated prothrombin levels increase the maximal rate, peak and area under the curve of thrombin generation.13,41,59,82 We have shown that elevated (pro)thrombin levels trigger the formation of densely-packed fibrin clots composed of thin fibrin fibers compared to normal clots.13 Increased thrombin generation in these individuals also increases activation of the thrombin-activatable fibrinolysis inhibitor (TAFI) in vitro.83 Activated TAFI downregulates fibrinolysis by cleaving C-terminal lysine residues from fibrin and reducing the number of tPA and plasminogen binding sites on fibrin. It has been suggested that the combination of abnormal structure and increased TAFI activation reduces the rate of fibrinolysis and contributes to the increased risk of thrombosis in these individuals.

Hemophilia and recombinant factor VIIa

The bleeding disorders hemophilia A or B result from deficiency in factors VIII or IX activities, respectively. Hemophilic plasma exhibits relatively normal extrinsic (initiating) activities on tissue factor-bearing cells, but reduced intrinsic (propagating) activities on platelets. Skin biopsies of wounds in hemophilic patients show a thin peripheral layer of fibrin deposited around a central region containing relatively few fibrin fibers.84 These findings are consistent with normal, low level thrombin and fibrin generation on and near tissue factor-bearing cells at the periphery of the wound, but deficient thrombin propagation and fibrin formation into the central region of the wound. In vitro assays modeling hemophilic conditions demonstrate delayed onset of measurable thrombin generation, as well as a reduced maximum rate and peak of thrombin generation.14,41,42,85 These reduced rates and levels of thrombin generation decrease the rate of fibrinopeptide release and delay fibrin formation85–87 Additionally, in vitro assays indicate that hemophilic clots are composed of abnormally thick fibrin fibers and have increased porosity compared to normal clots.14,88,89 The fiber composition of hemophilic clots decreases their mechanical stability and makes them highly susceptible to fibrinolysis.14,90,91 In vitro studies suggest that reduced thrombin levels limit TAFI activation, leaving clots more vulnerable to fibrinolysis.90,92,93 The physiologic importance of TAFI in clot stability is unclear, however, since TAFI-deficient animals exhibit minimal to no bleeding.94–96

Recombinant factor VIIa (rFVIIa, NovoSeven) is approved for the treatment of severe bleeds in hemophilic patients with inhibitory antibodies, and has demonstrated efficacy under these conditions and in patients with acquired hemophilia.97 By binding to the surface of activated platelets, rFVIIa reestablishes platelet tenase activity and restores thrombin generation on the platelet surface.98 RFVIIa increases the maximum thrombin generation rate and shortens the time to reach the peak thrombin level in hemophilic conditions.14 In vitro assays of hemophilic plasma and whole blood have shown that rFVIIa activity shortens the onset of clot formation and enables the formation of clots with thinner, more normal fibrin fibers, significantly improving the clot structure, porosity, and stability.14,87,89,91 By increasing the rate of fibrin formation, rFVIIa improves fibrin clot formation during a fibrinolytic challenge.14 RFVIIa may further protect the clot by increasing TAFI activation.93 Recently, several “superactive” analogs of rFVIIa have been described and tested in vitro and in animal models. These analogs demonstrate similar procoagulant and anti-fibrinolytic properties as rFVIIa, but at significantly lower concentrations.91,99,100

Conclusions

In sum, current studies suggest that the thrombin generation pattern determines biochemical features of the resulting clot, including its rate of formation, structure, and mechanical and fibrinolytic stability. Understanding the relationships between parameters of in situ thrombin generation and clot formation is essential for determining requirements of hemostatic clot formation and developing novel hemostatic and anti-thrombotic agents.

Figure 2.

Figure 2

Open in a new tab

Conceptual model of cellular tissue factor (TF)-initiated in situ thrombin generation. Please see text for details.

Acknowledgments

The authors thank Dr. C. Robert Bagnell Jr. (UNC Microscopy Services Laboratory) for his assistance with the scanning electron and confocal microscopy imaging.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Medved LV, Gorkun OV, Privalov PL. Structural organization of C-terminal parts of fibrinogen A alpha-chains. FEBS Lett. 1983;160:291–295. doi: 10.1016/0014-5793(83)80985-5. [DOI] [PubMed] [Google Scholar]
  • 2.Weisel JW. Fibrinogen and fibrin. Adv Protein Chem. 2005;70:247–299. doi: 10.1016/S0065-3233(05)70008-5. [DOI] [PubMed] [Google Scholar]
  • 3.Lord ST. Fibrinogen and fibrin: scaffold proteins in hemostasis. Curr Opin Hematol. 2007;14:236–241. doi: 10.1097/MOH.0b013e3280dce58c. [DOI] [PubMed] [Google Scholar]
  • 4.Weisel JW. Structure of fibrin: impact on clot stability. J Thromb Haemost. 2007;5(Suppl 1):116–124. doi: 10.1111/j.1538-7836.2007.02504.x. [DOI] [PubMed] [Google Scholar]
  • 5.Ferry JD, Morrison PR. Preparation and properties of serum and plasma proteins. VIII. The conversion of human fibrinogen to fibrin under various conditions. J Am Chem Soc. 1947;69:388–400. doi: 10.1021/ja01194a066. [DOI] [PubMed] [Google Scholar]
  • 6.Carr ME, Shen LL, Hermans J. Mass-length ratio of fibrin fibers from gel permeation and light scattering. Biopolymers. 1977;16:1–15. doi: 10.1002/bip.1977.360160102. [DOI] [PubMed] [Google Scholar]
  • 7.Wolberg AS, Gabriel DA, Hoffman M. Analyzing fibrin clot structure using a microplate reader. Blood Coagul Fibrin. 2002;13:533–539. doi: 10.1097/00001721-200209000-00008. [DOI] [PubMed] [Google Scholar]
  • 8.Carr ME, Hermans J. Size and density of fibrin fibers from turbidity. Macromolecules. 1978;11:46–50. doi: 10.1021/ma60061a009. [DOI] [PubMed] [Google Scholar]
  • 9.Hantgan RR, Hermans J. Assembly of fibrin. A light scattering study. J Biol Chem. 1979;254:11272–11281. [PubMed] [Google Scholar]
  • 10.Blomback B, Carlsson K, Hessel B, Liljeborg A, Procyk R, Aslund N. Native fibrin gel networks observed by 3D microscopy, permeation and turbidity. Biochim Biophys Acta. 1989;997:96–110. doi: 10.1016/0167-4838(89)90140-4. [DOI] [PubMed] [Google Scholar]
  • 11.Weisel JW, Nagaswami C. Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled. Biophys J. 1992;63:111–128. doi: 10.1016/S0006-3495(92)81594-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Collet JP, Park D, Lesty C, et al. Influence of fibrin network conformation and fibrin fiber diameter on fibrinolysis speed: dynamic and structural approaches by confocal microscopy. Arterio Thromb Vasc Biol. 2000;20:1354–1361. doi: 10.1161/01.atv.20.5.1354. [DOI] [PubMed] [Google Scholar]
  • 13.Wolberg AS, Monroe DM, Roberts HR, Hoffman M. Elevated prothrombin results in clots with an altered fiber structure: a possible mechanism of the increased thrombotic risk. Blood. 2003;101:3008–3013. doi: 10.1182/blood-2002-08-2527. [DOI] [PubMed] [Google Scholar]
  • 14.Wolberg AS, Allen GA, Monroe DM, Hedner U, Roberts HR, Hoffman M. High dose factor VIIa enhances clot stability in a model of hemophilia B. Brit J Haematol. 2005;131:645–655. doi: 10.1111/j.1365-2141.2005.05820.x. [DOI] [PubMed] [Google Scholar]
  • 15.Hartmann A, Boukamp P, Friedl P. Confocal reflection imaging of 3D fibrin polymers. Blood Cells Mol Dis. 2006;36:191–193. doi: 10.1016/j.bcmd.2005.12.033. [DOI] [PubMed] [Google Scholar]
  • 16.Glover CJ, McIntire LV, Brown CH, 3rd, Natelson EA. Rheological properties of fibrin clots. Effects of fibrinogen concentration, Factor XIII deficiency, and Factor XIII inhibition. J Lab Clin Med. 1975;86:644–656. [PubMed] [Google Scholar]
  • 17.Nair CH, Shah GA, Dhall DP. Effect of temperature, pH and ionic strength and composition on fibrin network structure and its development. Thromb Res. 1986;42:809–816. doi: 10.1016/0049-3848(86)90117-9. [DOI] [PubMed] [Google Scholar]
  • 18.Carr ME, Gabriel DA, McDonagh J. Influence of Ca2+ on the structure of reptilase-derived and thrombin-derived fibrin gels. Biochem J. 1986;239:513–516. doi: 10.1042/bj2390513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Blomback B, Carlsson K, Fatah K, Hessel B, Procyk R. Fibrin in human plasma: gel architectures governed by rate and nature of fibrinogen activation. Thromb Res. 1994;75:521–538. doi: 10.1016/0049-3848(94)90227-5. [DOI] [PubMed] [Google Scholar]
  • 20.Baradet TC, Haselgrove JC, Weisel JW. Three-dimensional reconstruction of fibrin clot networks from stereoscopic intermediate voltage electron microscope images and analysis of branching. Biophysical Journal. 1995;68:1551–1560. doi: 10.1016/S0006-3495(95)80327-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Ryan EA, Mockros LF, Weisel JW, Lorand L. Structural origins of fibrin clot rheology. Biophys J. 1999;77:2813–2826. doi: 10.1016/S0006-3495(99)77113-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Shah GA, Nair CH, Dhall DP. Physiological studies on fibrin network structure. Thromb Res. 1985;40:181–188. doi: 10.1016/0049-3848(85)90328-7. [DOI] [PubMed] [Google Scholar]
  • 23.Blomback B, Banerjee D, Carlsson K, et al. Native fibrin gel networks and factors influencing their formation in health and disease. Adv Exp Med Biol. 1990;281:1–23. doi: 10.1007/978-1-4615-3806-6_1. [DOI] [PubMed] [Google Scholar]
  • 24.Liu W, Jawerth LM, Sparks EA, et al. Fibrin fibers have extraordinary extensibillity and elasticity. Science. 2006;313:634. doi: 10.1126/science.1127317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Brown AE, Litvinov RI, Discher DE, Weisel JW. Forced Unfolding of Coiled-Coils in Fibrinogen by Single-Molecule AFM. Biophys J. 2007;92:L39–41. doi: 10.1529/biophysj.106.101261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Weisel JW. The mechanical properties of fibrin for basic scientists and clinicians. Biophys Chem. 2004;112:267–276. doi: 10.1016/j.bpc.2004.07.029. [DOI] [PubMed] [Google Scholar]
  • 27.Kaibara M, Fukada E. The influence of the concentration of thrombin on the dynamic viscoelasticity of clotting blood and fibrinogen-thrombin systems. Biorheology. 1971;8:139–147. doi: 10.3233/bir-1971-83-404. [DOI] [PubMed] [Google Scholar]
  • 28.Marchi R, Lundberg U, Grimbergen J, et al. Fibrinogen Caracas V, an abnormal fibrinogen with an Aalpha 532 Ser-->Cys substitution associated with thrombosis. Thromb Haemost. 2000;84:263–270. [PubMed] [Google Scholar]
  • 29.Collet J-P, Soria J, Mirshahi M, et al. Dusart syndrome: a new concept of the relationship between fibrin clot architecture and fibrin clot degradability: hypofibrinolysis related to an abnormal clot structure. Blood. 1993;82:2462–2469. [PubMed] [Google Scholar]
  • 30.Collet J-P, Woodhead JL, Soria J, et al. Fibrinogen Dusart: electron microscopy of molecules, fibers and clots, and viscoelastic properties of clots. Biophysical J. 1996;70:500–510. doi: 10.1016/S0006-3495(96)79596-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Gabriel DA, Muga K, Boothroyd EM. The effect of fibrin structure on fibrinolysis. J Biol Chem. 1992;267:24259–24263. [PubMed] [Google Scholar]
  • 32.Tracy PB, Peterson JM, Nesheim ME, McDuffie FC, Mann KG. Interaction of coagulation factor V and factor Va with platelets. J Biol Chem. 1979;254:10354–10361. [PubMed] [Google Scholar]
  • 33.Nesheim ME, Pittman DD, Wang JH, Slonosky D, Giles AR, Kaufman RJ. The binding of 35S-labeled recombinant factor VIII to activated and unactivated human platelets. J Biol Chem. 1988;263:16467–16470. [PubMed] [Google Scholar]
  • 34.Ahmad SS, Rawala-Sheikh R, Walsh PN. Comparative interactions of factor IX and factor IXa with human platelets. J Biol Chem. 1989;264:3244–3251. [PubMed] [Google Scholar]
  • 35.Rawala-Sheikh R, Ahmad SS, Ashby B, Walsh PN. Kinetics of coagulation factor X activation by platelet-bound factor IXa. Biochemistry. 1990;29:2606–2611. doi: 10.1021/bi00462a025. [DOI] [PubMed] [Google Scholar]
  • 36.Monroe DM, Hoffman M, Roberts HR. Transmission of a procoagulant signal from tissue factor-bearing cells to platelets. Blood Coag Fibrinol. 1996;7:459–464. doi: 10.1097/00001721-199606000-00005. [DOI] [PubMed] [Google Scholar]
  • 37.Hoffman M, Monroe DM, Oliver JA, Roberts HR. Factors IXa and Xa play distinct roles in tissue factor-dependent initiation of coagulation. Blood. 1995;86:1794–1801. [PubMed] [Google Scholar]
  • 38.Weisel JW, Francis CW, Nagaswami C, Marder VJ. Determination of the topology of factor XIIIa-induced fibrin gamma-chain cross-links by electron microscopy of ligated fragments. Journal of Biological Chemistry. 1993;268:26618–26624. [PubMed] [Google Scholar]
  • 39.van ‘t Veer C, Golden NJ, Kalafatis M, Simioni P, Bertina RM, Mann KG. An in vitro analysis of the combination of hemophilia A and factor V(LEIDEN) Blood. 1997;90:3067–3072. [PubMed] [Google Scholar]
  • 40.Al Dieri R, Peyvandi F, Santagostino E, et al. The thrombogram in rare inherited coagulation disorders: its relation to clinical bleeding. Thromb Haemost. 2002;88:576–582. [PubMed] [Google Scholar]
  • 41.Allen GA, Wolberg AS, Oliver JA, Hoffman M, Roberts HR, Monroe DM. Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system. J Thromb Haemost. 2004;2:402–413. doi: 10.1111/j.1538-7933.2003.00617.x. [DOI] [PubMed] [Google Scholar]
  • 42.Dargaud Y, Beguin S, Lienhart A, et al. Evaluation of thrombin generating capacity in plasma from patients with haemophilia A and B. Thromb Haemost. 2005;93:475–480. doi: 10.1160/TH04-10-0706. [DOI] [PubMed] [Google Scholar]
  • 43.Shah GA, Nair CH, Dhall DP. Comparison of fibrin networks in plasma and fibrinogen solution. Thromb Res. 1987;45:257–264. doi: 10.1016/0049-3848(87)90193-9. [DOI] [PubMed] [Google Scholar]
  • 44.Carr ME. Fibrin formed in plasma is composed of fibers more massive than those formed from purified fibrinogen. Thrombosis and Haemostasis. 1988;59:535–539. [PubMed] [Google Scholar]
  • 45.Torbet J. Fibrin assembly in human plasma and fibrinogen/albumin mixtures. Biochemistry. 1986;25:5309–5314. doi: 10.1021/bi00366a048. [DOI] [PubMed] [Google Scholar]
  • 46.Naski MC, Shafer JA. A kinetic model for the alpha-thrombin-catalyzed conversion of plasma levels of fibrinogen to fibrin in the presence of antithrombin III. J Biol Chem. 1991;266:13003–13010. [PubMed] [Google Scholar]
  • 47.Elgue G, Sanchez J, Fatah K, Olsson P, Blomback B. The effect of plasma antithrombin concentration on thrombin generation and fibrin gel structure. Thromb Res. 1994;75:203–212. doi: 10.1016/0049-3848(94)90069-8. [DOI] [PubMed] [Google Scholar]
  • 48.Wilf J, Gladner JA, Minton AP. Acceleration of fibrin gel formation by unrelated proteins. Thromb Res. 1985;37:681–688. doi: 10.1016/0049-3848(85)90197-5. [DOI] [PubMed] [Google Scholar]
  • 49.Braaten JV, Handt S, Jerome WG, Kirkpatrick J, Lewis JC, Hantgan RR. Regulation of fibrinolysis by platelet-released plasminogen activator inhibitor 1: light scattering and ultrastructural examination of lysis of a model platelet-fibrin thrombus. Blood. 1993;81:1290–1299. [PubMed] [Google Scholar]
  • 50.Braaten JV, Jerome WG, Hantgan RR. Uncoupling fibrin from integrin receptors hastens fibrinolysis at the platelet-fibrin interface. Blood. 1994;83:982–993. [PubMed] [Google Scholar]
  • 51.Jerome WG, Handt S, Hantgan RR. Endothelial cells organize fibrin clots into structures that are more resistant to lysis. Microsc Microanal. 2005;11:268–277. doi: 10.1017/S143192760505052X. [DOI] [PubMed] [Google Scholar]
  • 52.Pham TD, Kaplan KL, Butler VP. Immunoelectron microscopic localization of platelet factor 4 and fibrinogen in the granules of human platelets. J Histochem Cytochem. 1983;31:905–910. doi: 10.1177/31.7.6343481. [DOI] [PubMed] [Google Scholar]
  • 53.Reed GL, Matsueda GR, Haber E. Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the crosslinking of alpha 2-antiplasmin to fibrin [see comments] Thrombosis and Haemostasis. 1992;68:315–320. [PubMed] [Google Scholar]
  • 54.Handt S, Jerome WG, Tietze L, Hantgan RR. Plasminogen activator inhibitor-1 secretion of endothelial cells increases fibrinolytic resistance of an in vitro fibrin clot: evidence for a key role of endothelial cells in thrombolytic resistance. Blood. 1996;87:4204–4213. [PubMed] [Google Scholar]
  • 55.Mosnier LO, Buijtenhuijs P, Marx PF, Meijers JC, Bouma BN. Identification of thrombin activatable fibrinolysis inhibitor (TAFI) in human platelets. Blood. 2003;101:4844–4846. doi: 10.1182/blood-2002-09-2944. [DOI] [PubMed] [Google Scholar]
  • 56.Xi M, Beguin S, Hemker HC. The relative importance of the factors II, VII, IX and X for the prothrombinase activity in plasma of orally anticoagulated patients. Thromb Haemost. 1989;62:788–791. [PubMed] [Google Scholar]
  • 57.Xi M, Beguin S, Hemker HC. Importance of factor-IX-dependent prothrombinase formation--the Josso pathway--in clotting plasma. Haemostasis. 1989;19:301–308. doi: 10.1159/000216034. [DOI] [PubMed] [Google Scholar]
  • 58.Sumner WT, Monroe DM, Hoffman M. Variability in platelet procoagulant activity in healthy volunteers. Thromb Res. 1996;81:533–543. doi: 10.1016/0049-3848(96)00028-x. [DOI] [PubMed] [Google Scholar]
  • 59.Butenas S, van’t Veer C, Mann KG. "Normal" thrombin generation. Blood. 1999;94:2169–2178. [PubMed] [Google Scholar]
  • 60.Kempton CL, Hoffman M, Roberts HR, Monroe DM. Platelet heterogeneity: variation in coagulation complexes on platelet subpopulations. Arterioscler Thromb Vasc Biol. 2005;25:861–866. doi: 10.1161/01.ATV.0000155987.26583.9b. [DOI] [PubMed] [Google Scholar]
  • 61.Aird WC. Spatial and temporal dynamics of the endothelium. J Thromb Haemost. 2005;3:1392–1406. doi: 10.1111/j.1538-7836.2005.01328.x. [DOI] [PubMed] [Google Scholar]
  • 62.Torbet J. The thrombin activation pathway modulates the assembly, structure and lysis of human plasma clots in vitro. Thromb Haemost. 1995;73:785–792. [PubMed] [Google Scholar]
  • 63.von dem Borne PA, Meijers JC, Bouma BN. Feedback activation of factor XI by thrombin in plasma results in additional formation of thrombin that protects fibrin clots from fibrinolysis. Blood. 1995;86:3035–3042. [PubMed] [Google Scholar]
  • 64.Kumar R, Beguin S, Hemker HC. The effect of fibrin clots and clot-bound thrombin on the development of platelet procoagulant activity. Thromb Haemost. 1995;74:962–968. [PubMed] [Google Scholar]
  • 65.Weitz JI, Hudoba M, Massel D, Maraganore J, Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest. 1990;86:385–391. doi: 10.1172/JCI114723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Weitz JI, Leslie B, Hudoba M. Thrombin binds to soluble fibrin degradation products where it is protected from inhibition by heparin-antithrombin but susceptible to inactivation by antithrombin-independent inhibitors. Circulation. 1998;97:544–552. doi: 10.1161/01.cir.97.6.544. [DOI] [PubMed] [Google Scholar]
  • 67.Ovanesov MV, Krasotkina JV, Ul’yanova LI, et al. Hemophilia A and B are associated with abnormal spatial dynamics of clot growth. Biochim Biophys Acta. 2002;1572:45–57. doi: 10.1016/s0304-4165(02)00278-7. [DOI] [PubMed] [Google Scholar]
  • 68.Ovanesov MV, Ananyeva NM, Panteleev MA, Ataullakhanov FI, Saenko EL. Initiation and propagation of coagulation from tissue factor-bearing cell monolayers to plasma: initiator cells do not regulate spatial growth rate. J Thromb Haemost. 2005;3:321–331. doi: 10.1111/j.1538-7836.2005.01128.x. [DOI] [PubMed] [Google Scholar]
  • 69.Panteleev MA, Ananyeva NM, Greco NJ, Ataullakhanov FI, Saenko EL. Factor VIIIa regulates substrate delivery to the intrinsic factor X-activating complex. Febs J. 2006;273:374–387. doi: 10.1111/j.1742-4658.2005.05070.x. [DOI] [PubMed] [Google Scholar]
  • 70.Panteleev MA, Ovanesov MV, Kireev DA, et al. Spatial propagation and localization of blood coagulation are regulated by intrinsic and protein C pathways, respectively. Biophys J. 2006;90:1489–1500. doi: 10.1529/biophysj.105.069062. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Rodgers GM, Greenberg CS, Shuman MA. Characterization of the effects of cultured vascular cells on the activation of blood coagulation. Blood. 1983;61:1155–1162. [PubMed] [Google Scholar]
  • 72.Rodgers GM, Broze GJ, Jr, Shuman MA. The number of receptors for factor VII correlates with the ability of cultured cells to initiate coagulation. Blood. 1984;63:434–438. [PubMed] [Google Scholar]
  • 73.Zwaginga JJ, de Boer HC, MJ IJ, et al. Thrombogenicity of vascular cells. Comparison between endothelial cells isolated from different sources and smooth muscle cells and fibroblasts. Arteriosclerosis. 1990;10:437–448. doi: 10.1161/01.atv.10.3.437. [DOI] [PubMed] [Google Scholar]
  • 74.Carr ME, Jr, Dent RM, Carr SL. Abnormal fibrin structure and inhibition of fibrinolysis in patients with multiple myeloma. J Lab Clin Med. 1996;128:83–88. doi: 10.1016/s0022-2143(96)90116-x. [DOI] [PubMed] [Google Scholar]
  • 75.Collet JP, Mishal Z, Lesty C, et al. Abnormal fibrin clot architecture in nephrotic patients is related to hypofibrinolysis: influence of plasma biochemical modifications: a possible mechanism for the high thrombotic tendency? Thromb Haemost. 1999;82:1482–1489. [PubMed] [Google Scholar]
  • 76.Sugo T, Nakamikawa C, Yoshida N, et al. End-linked homodimers in fibrinogen Osaka VI with a B beta-chain extension lead to fragile clot structure. Blood. 2000;96:3779–3785. [PubMed] [Google Scholar]
  • 77.Dunn EJ, Ariens RA, Grant PJ. The influence of type 2 diabetes on fibrin structure and function. Diabetologia. 2005;48:1198–1206. doi: 10.1007/s00125-005-1742-2. [DOI] [PubMed] [Google Scholar]
  • 78.Dunn EJ, Philippou H, Ariens RA, Grant PJ. Molecular mechanisms involved in the resistance of fibrin to clot lysis by plasmin in subjects with type 2 diabetes mellitus. Diabetologia. 2006;49:1071–1080. doi: 10.1007/s00125-006-0197-4. [DOI] [PubMed] [Google Scholar]
  • 79.Poort SR, Rosendaal FR, Reitsma PH, Bertina RM. A common genetic variation in the 3′-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood. 1996;88:3698–3703. [PubMed] [Google Scholar]
  • 80.Rosendaal FR, Siscovick DS, Schwartz SM, Psaty BM, Raghunathan TE, Vos HL. A common prothrombin variant (20210 G to A) increases the risk of myocardial infarction in young women. Blood. 1997;90:1747–1750. [PubMed] [Google Scholar]
  • 81.Giordano P, De Lucia D, Coppola B, Iolascon A. Homozygous prothrombin gene mutation and ischemic cerebrovascular disease: a case report. Acta Haematologica. 1999;102:101–103. doi: 10.1159/000040979. [DOI] [PubMed] [Google Scholar]
  • 82.Kyrle PA, Mannhalter C, Beguin S, et al. Clinical studies and thrombin generation in patients homozygous or heterozygous for the G20210A mutation in the prothrombin gene. Arterio Thromb Vasc Biol. 1998;18:1287–1291. doi: 10.1161/01.atv.18.8.1287. [DOI] [PubMed] [Google Scholar]
  • 83.Colucci M, Binetti BM, Tripodi A, Chantarangkul V, Semeraro N. Hyperprothrombinemia associated with prothrombin G20210A mutation inhibits plasma fibrinolysis through a TAFI-mediated mechanism. Blood. 2004;103:2157–2161. doi: 10.1182/blood-2003-06-2169. [DOI] [PubMed] [Google Scholar]
  • 84.Sixma JJ, van den Berg A. The haemostatic plug in haemophilia A: a morphological study of haemostatic plug formation in bleeding time skin wounds of patients with severe haemophilia A. Br J Haematol. 1984;58:741–753. doi: 10.1111/j.1365-2141.1984.tb06121.x. [DOI] [PubMed] [Google Scholar]
  • 85.Cawthern KM, van’t Veer C, Lock JB, Di Lorenzo ME, Branda RF, Mann KG. Blood coagulation in hemophilia A and hemophilia C. Blood. 1998;91:4581–4592. [PubMed] [Google Scholar]
  • 86.Englert JM, Moen JL, Wolberg AS. Altered fibrinopeptide release produces thicker fibrin fibers and an abnormal fibrin clot structure in an in vitro model of hemophilia B. International Fibrinogen Workshop. 2004 [Google Scholar]
  • 87.Sorensen B, Johansen P, Christiansen K, Woelke M, Ingerslev J. Whole blood coagulation thrombelastographic profiles employing minimal tissue factor activation. J Thromb Haemost. 2003;1:551–558. doi: 10.1046/j.1538-7836.2003.00075.x. [DOI] [PubMed] [Google Scholar]
  • 88.Bettigole RE, Hampton JW, Bird RM. Abnormal plasma clots in hemophilia. Thromb Diath Haemorrh. 1964;12:331–337. [PubMed] [Google Scholar]
  • 89.He S, Blomback M, Jacobsson Ekman G, Hedner U. The role of recombinant factor VIIa (FVIIa) in fibrin structure in the absence of FVIII/FIX. J Thromb Haemost. 2003;1:1215–1219. doi: 10.1046/j.1538-7836.2003.00242.x. [DOI] [PubMed] [Google Scholar]
  • 90.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. [PubMed] [Google Scholar]
  • 91.Sorensen B, Persson E, Ingerslev J. Factor VIIa analogue (V158D/E296V/M298Q-FVIIa) normalises clot formation in whole blood from patients with severe haemophilia A. Br J Haematol. 2007;137:158–165. doi: 10.1111/j.1365-2141.2007.06534.x. [DOI] [PubMed] [Google Scholar]
  • 92.Mosnier LO, Lisman T, van den Berg HM, Nieuwenhuis HK, Meijers JC, Bouma BN. The defective down regulation of fibrinolysis in haemophilia A can be restored by increasing the TAFI plasma concentration. Thromb Haemost. 2001;86:1035–1039. [PubMed] [Google Scholar]
  • 93.Lisman T, Mosnier LO, Thierry Lambert T, et al. Inhibition of fibrinolysis by recombinant factor VIIa in plasma from patients with severe hemophilia A. Blood. 2002;99:175–179. doi: 10.1182/blood.v99.1.175. [DOI] [PubMed] [Google Scholar]
  • 94.Nagashima M, Werner M, Wang M, et al. An inhibitor of activated thrombin-activatable fibrinolysis inhibitor potentiates tissue-type plasminogen activator-induced thrombolysis in a rabbit jugular vein thrombolysis model. Thromb Res. 2000;98:333–342. doi: 10.1016/s0049-3848(00)00184-5. [DOI] [PubMed] [Google Scholar]
  • 95.Nagashima M, Yin ZF, Broze GJ, Jr, Morser J. Thrombin-activatable fibrinolysis inhibitor (TAFI) deficient mice. Front Biosci. 2002;7:d556–568. doi: 10.2741/A794. [DOI] [PubMed] [Google Scholar]
  • 96.Barrow JC, Nantermet PG, Stauffer SR, et al. Synthesis and evaluation of imidazole acetic acid inhibitors of activated thrombin-activatable fibrinolysis inhibitor as novel antithrombotics. J Med Chem. 2003;46:5294–5297. doi: 10.1021/jm034141y. [DOI] [PubMed] [Google Scholar]
  • 97.Negrier C, Hay CR. The treatment of bleeding in hemophilic patients with inhibitors with recombinant factor VIIa. Semin Thromb Hemost. 2000;26:407–412. doi: 10.1055/s-2000-8460. [DOI] [PubMed] [Google Scholar]
  • 98.Monroe DM, Hoffman M, Oliver JA, Roberts HR. Platelet activity of high-dose factor VIIa is independent of tissue factor. Brit J Haematol. 1997;99:542–547. doi: 10.1046/j.1365-2141.1997.4463256.x. [DOI] [PubMed] [Google Scholar]
  • 99.Lisman T, de Groot PG, Lambert T, Rojkjaer R, Persson E. Enhanced in vitro procoagulant and antifibrinolytic potential of superactive variants of recombinant factor VIIa in severe hemophilia A. J Thromb Haemost. 2003;1:2175–2178. doi: 10.1046/j.1538-7836.2003.00444.x. [DOI] [PubMed] [Google Scholar]
  • 100.Allen GA, Persson E, Campbell RA, Ezban M, Hedner U, Wolberg AS. A variant of recombinant factor VIIa with enhanced procoagulant and antifibrinolytic activities in an in vitro model of hemophilia. Arterioscler Thromb Vasc Biol. 2007;27:683–689. doi: 10.1161/01.ATV.0000257204.82396.2b. [DOI] [PubMed] [Google Scholar]

RESOURCES