Thrombin-activated Factor Research Articles

Thrombotic Disorders

The three main elements in the pathophysiology of thrombosis are endothelial injury, a decrease in blood flow, and an imbalance between procoagulant and anticoagulant factors. The latter element can be either hereditary (e.g., antithrombin deficiency) or acquired (e.g., antiphospholipid syndrome). This review details the assessment of patients with thrombotic disorders, hereditary and acquired hypercoagulable states, and the management of venous thromboembolism. Figures show how the degradation of thrombin-activated factor V Leiden by activated protein C (APC) is significantly slower than that of normal activated factor V (factor Va), leading to enhanced thrombin generation; how normal factor V serves as a cofactor of APC in the inhibition of factor VIIIa, whereas factor V Leiden has a poor cofactor function; and how IgG antibodies recognize platelet factor 4–heparin complexes in heparin-induced thrombocytopenia. Tables list inherited and acquired hypercoagulable states, questions for assessing thrombosis, screening tests for patients with suspected hypercoagulable states, clinical features that suggest thrombophilia, frequency and relative risk of venous thrombosis in selected hypercoagulable states, proposed clinical and laboratory criteria for antiphospholipid syndrome, the classification of antiphospholipid antibodies, the 4Ts scoring system for heparin-induced thrombocytopenia, and general guidelines for the management of patients with venous thromboembolism. This review contains 2 highly rendered figures, 9 tables, and 168 references.

Thrombotic Disorders

The three main elements in the pathophysiology of thrombosis are endothelial injury, a decrease in blood flow, and an imbalance between procoagulant and anticoagulant factors. The latter element can be either hereditary (e.g., antithrombin deficiency) or acquired (e.g., antiphospholipid syndrome). This review details the assessment of patients with thrombotic disorders, hereditary and acquired hypercoagulable states, and the management of venous thromboembolism. Figures show how the degradation of thrombin-activated factor V Leiden by activated protein C (APC) is significantly slower than that of normal activated factor V (factor Va), leading to enhanced thrombin generation; how normal factor V serves as a cofactor of APC in the inhibition of factor VIIIa, whereas factor V Leiden has a poor cofactor function; and how IgG antibodies recognize platelet factor 4–heparin complexes in heparin-induced thrombocytopenia. Tables list inherited and acquired hypercoagulable states, questions for assessing thrombosis, screening tests for patients with suspected hypercoagulable states, clinical features that suggest thrombophilia, frequency and relative risk of venous thrombosis in selected hypercoagulable states, proposed clinical and laboratory criteria for antiphospholipid syndrome, the classification of antiphospholipid antibodies, the 4Ts scoring system for heparin-induced thrombocytopenia, and general guidelines for the management of patients with venous thromboembolism. This review contains 2 highly rendered figures, 9 tables, and 168 references.

Fibrin(ogen) as a Therapeutic Target: Opportunities and Challenges.

Fibrinogen is one of the key molecular players in haemostasis. Thrombin-mediated release of fibrinopeptides from fibrinogen converts this soluble protein into a network of fibrin fibres that form a building block for blood clots. Thrombin-activated factor XIII further crosslinks the fibrin fibres and incorporates antifibrinolytic proteins into the network, thus stabilising the clot. The conversion of fibrinogen to fibrin also exposes binding sites for fibrinolytic proteins to limit clot formation and avoid unwanted extension of the fibrin fibres. Altered clot structure and/or incorporation of antifibrinolytic proteins into fibrin networks disturbs the delicate equilibrium between clot formation and lysis, resulting in either unstable clots (predisposing to bleeding events) or persistent clots that are resistant to lysis (increasing risk of thrombosis). In this review, we discuss the factors responsible for alterations in fibrin(ogen) that can modulate clot stability, in turn predisposing to abnormal haemostasis. We also explore the mechanistic pathways that may allow the use of fibrinogen as a potential therapeutic target to treat vascular thrombosis or bleeding disorders. Better understanding of fibrinogen function will help to devise future effective and safe therapies to modulate thrombosis and bleeding risk, while maintaining the fine balance between clot formation and lysis.

Our panel of experts highlight the most important research articles across the spectrum of topics relevant to the field of regenerative medicine.

Enzymatically crosslinked microporous hydrogel scaffolds that form in situ promote dermal healing Evaluation of: Griffin DR, Weaver WM, Scumpia PO, DiCarlo D, Segura T. Accelerated wound healing by injectable microporous gel scaffolds assembled from annealed building blocks. Nat. Mater. 14, 737–744 (2015). Tunable scaffolds that respond to environmental factors could enable enhanced tissue integration and wound healing. To achieve this goal, scaffold designs have incorporated biomimetic functionality, including thrombin-activated factor XIII (FXIIIa) crosslinking [1], matrix metalloproteinase responsiveness [2] and mechanical matching [3]. Scaffolds that can alter their properties in situ in response to environmental stimuli are desired to achieve changes in stiffness, shape, release and degradation that can lead to improved tissue regeneration. In this study, Griffin et al. describe a bottom-up strategy to fabricating scaffolds that accelerate wound healing [4]. They use a microfluidic approach to form hydrogel microparticle (microgel) building blocks that can establish interconnected network structures upon FXIIIa-mediated crosslinking. These microporous annealed particles (MAPs) incorporate reactive multiarmed poly(ethylene)glycol-vinyl sulfone, the commonly used RGD cell adhesion peptide and the K and Q noncanonical transglutaminase peptide substrates with cysteine-terminated matrix metalloproteinase-sensitive peptides that allow for cell-controlled material degradation. Scaffolds are annealed by FXIIIa addition and can be injected into wound sites for in situ solidification and shape-conformation. These MAPs are mechanically robust and can be tuned to achieve storage moduli from 10 to 1000 Pa, which fall within the stiffness range of soft tissues. In vitro studies demonstrate that MAP scaffolds enable 3D cellular network formation and cell proliferation and survival after 6 days. In vivo studies in SKH1-HR and Balb/c epidermal wound healing models demonstrate integration of MAP scaffolds after 24 h, significant wound closure as compared with nonporous chemically matched controls after 5 days or physically matched control scaffolds up to 7 days. 5 days following injection of MAP scaffolds, keratin-5, keratin-14 and CD49f staining revealed re-epithelialization, and PECAM-1, NG2 and PDGFR-β staining indicated that the scaffolds promoted complex vascular development. MAP scaffolds were also able to sustain normal hair follicle formation when compared with normal tissue. The scaffolds promoted less CD11b cell infiltration as compared with nonporous controls after 5 days, which suggests that MAP scaffolds cause a lower immune response. Taken together, MAP scaffolds promote accelerated wound healing by utilizing natural enzymatic mechanisms and mimicking favorable mechanical and degradation properties to facilitate cell infiltration and proliferation. The study may have benefited from evaluating wound closure, dermal regeneration and Our panel of experts highlight the most important research articles across the spectrum of topics relevant to the field of regenerative medicine

Factor XIII Transglutaminase Supports the Resolution of Mucosal Damage in Experimental Colitis

The thrombin-activated transglutaminase factor XIII (FXIII) that covalently crosslinks and stablizes provisional fibrin matrices is also thought to support endothelial and epithelial barrier function and to control inflammatory processes. Here, gene-targeted mice lacking the FXIII catalytic A subunit were employed to directly test the hypothesis that FXIII limits colonic pathologies associated with experimental colitis. Wildtype (WT) and FXIII-/- mice were found to be comparable in their initial development of mucosal damage following exposure to dextran sulfate sodium (DSS) challenge. However, unlike FXIII-sufficient mice, FXIII-deficient cohorts failed to efficiently resolve colonic inflammatory pathologies and mucosal damage following withdrawal of DSS. Consistent with prior evidence of ongoing coagulation factor activation and consumption in individuals with active colitis, plasma FXIII levels were markedly decreased in colitis-challenged WT mice. Treatment of colitis-challenged mice with recombinant human FXIII-A zymogen significantly mitigated weight loss, intestinal bleeding, and diarrhea, regardless of whether cohorts were FXIII-sufficient or were genetically devoid of FXIII. Similarly, both qualitative and quantitative microscopic analyses of colonic tissues revealed that exogenous FXIII improved the resolution of multiple colitis disease parameters in both FXIII-/- and WT mice. The most striking differences were seen in the resolution of mucosal ulceration, the most severe histopathological manifestation of DSS-induced colitis. These findings directly demonstrate that FXIII is a significant determinant of mucosal healing and clinical outcome following inflammatory colitis induced mucosal injury and provide a proof-of-principle that clinical interventions supporting FXIII activity may be a means to limit colitis pathology and improve resolution of mucosal damage.

Amino Acid Region 1000–1008 of Factor V Is a Dynamic Regulator for the Emergence of Procoagulant Activity

Single chain factor V (fV) circulates as an Mr 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000-1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg(709)/Arg(1018)/Arg(1545)) mutated to glutamine (fV(Q3)), a mutant fV molecule with region 1000-1008 deleted (fV(ΔB9)), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV(ΔB9/Q3)). The recombinant molecules along with wild type fV (fV(WT)) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV(Q3) was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, KD(app) values for fV(ΔB9) (0.9 nM), fVa(ΔB9) (0.4 nM), and fV(ΔB9/Q3) (0.7 nM) were similar to the affinity of fVa(WT) for fXa (0.3 nM). Two-stage clotting assays revealed that although fV(Q3) was deficient in its clotting activity, fV(ΔB9/Q3) had clotting activity comparable with fVa(WT). The kcat value of prothrombinase assembled with fV(ΔB9/Q3) was minimally affected, whereas the Km value of the reaction was increased 57-fold compared with the Km value obtained with prothrombinase assembled with fVa(WT). These findings strongly suggest that amino acid region 1000-1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences.

Experimental approach to improve endothelial barrier function in myocardium

Recently, we showed that activated factor XIII (FXIIIa) has a direct influence on permeability (P) of cultured endothelial monolayer. Clinical investigation on children operated on for congenital heart disease has demonstrated a distinct correlation between decrease of endothelial barrier function (edema formation) and reduced FXIII activity. Our experiments investigated whether significant effects of FXIII could also be shown in a full-organ model. The effect of FXIII on endothelial barrier function was studied by determining the passage of trypan blue-labeled albumin in a model of cultured monolayers of porcine aortic endothelial cells and changes in myocardial water content of saline-perfused rat hearts in a Langendorff-circuit. The plasma FXIIIa (1 U/ml) reduced albumin permeability of aortic endothelial monolayers within 20 minutes from a basal value of 5.9±0.4×10−6 cm/second by 30±7% whereas the nonactivated plasma FXIII had no effect on permeability. Reduction of permeability to the same extent (by 34±9%) could also be obtained with a thrombin-activated recombinant factor XIII A subunit (rhu FXIII; 1 U/ml) in this culture model. The effect of factor XIII A* on permeability was dose dependent with maximum effect at 5 U/ml (52±11% reduction of permeability compared with control), and existed even if cells had hyperpermeability stress (KCN/2-DG). Activated rhuFXIII prevented the increase in myocardial water content in anoxic-reperfused rat hearts (448±24 vs 517±29 ml/100g dry weight). The data of the present study show that FXIII has a stabilizing effect at the site of endothelial cells not only in cultured monolayers but also in a model of the anoxic perfused rat heart.

Solidification mechanisms of chitosan–glycerol phosphate/blood implant for articular cartilage repair

Chitosan-glycerol phosphate (chitosan-GP) is a unique polymer solution that is mixed with whole blood and solidified over microfractured or drilled articular cartilage defects in order to elicit a more hyaline repair cartilage. For clinical ease-of-use, a faster in situ solidification is preferred. Therefore, we investigated the mechanisms underlying chitosan-GP/blood implant solidification. In vitro solidification of chitosan-GP/blood mixtures, with or without added clotting factors, was evaluated by thromboelastography. Serum was analyzed for the onset of thrombin, platelet, and FXIII activation. In vivo solidification of chitosan-GP/blood mixtures, with and without clotting factors, was evaluated in microdrilled cartilage defects of adult rabbits (N=41 defects). Chitosan-GP/blood clots solidified in an atypical biphasic manner, with higher initial viscosity and minor platelet activation followed by the development of clot tensile strength concomitant with thrombin generation, burst platelet and FXIII activation. Whole blood and chitosan-GP/blood clots developed a similar final clot tensile strength, while polymer-blood clots showed a unique, sustained platelet factor release and greater resistance to lysis by tissue plasminogen activator. Thrombin, tissue factor (TF), and recombinant human activated factor VII (rhFVIIa) accelerated chitosan-GP/blood solidification in vitro (P<0.05). Pre-application of thrombin or rhFVIIa+TF to the surface of drilled cartilage defects accelerated implant solidification in vivo (P<0.05). Chitosan-GP/blood implants solidify through coagulation mechanisms involving thrombin generation, platelet activation and fibrin polymerization, leading to a dual fibrin-polysaccharide clot scaffold that resists lysis and is physically more stable than normal blood clots. Clotting factors have the potential to enhance the practical use, the residency, and therapeutic activity of polymer-blood implants.

Depolymerized Holothurian Glycosaminoglycan Inhibits Plasma Thrombin Generation Via Interaction with the Factor IXa Heparin-Binding Exosite

Depolymerized holothurian glycosaminoglycan (DHG) is a fucosylated chrondroitin sulfate that possesses antithrombin-independent antithrombotic properties in rodent thrombosis and dog hemodialysis models. DHG demonstrates significantly less bleeding in template or tail transection assays than therapeutically equivalent doses of heparins. Several potential in vitro mechanisms have been described for DHG, including acceleration of thrombin inhibition by heparin cofactor II (HCII), inhibition of factor VIII activation by thrombin, and inhibition of factor X activation by the intrinsic tenase complex (factor IXa-factor VIIIa). The relevant mechanism(s) for inhibition of tissue factor (TF) induced plasma thrombin generation by DHG were examined in HCII or mock-immunodepleted, and factor-deficient human plasmas, using selected recombinant factor IX(a) with mutations in the heparin-binding exosite. Plasma thrombin generation was detected by fluorogenic substrate cleavage in the presence of corn trypin inhibitor to block contact activation, and compared to a standard curve generated with α2-macroglobulin-thrombin complex. The dose-dependent decrease in velocity index, a parameter reflecting the rate of thrombin generation between lag phase and peak thrombin concentration, was used to compare DHG potency. When triggered by 0.2 pM TF, the EC50 for inhibition of thrombin generation by DHG was 0.16 ± 0.01 μM in both HCII-depleted and mock-depleted plasma, suggesting that DHG acts independently of HCII. When triggered by excess (4 pM) TF, plasma thrombin generation was independent of factors VIII and IX. Under these conditions, the EC50 for DHG inhibition of thrombin generation was increased 13-fold in mock-depleted plasma (2.02 ± 0.09 μM) and 28-fold in HCII-depleted plasma (4.31 ± 0.23 μM). These results suggest that components of the intrinsic tenase complex contribute to inhibition of plasma thrombin generation by DHG, and HCII contributes only at high tissue factor concentrations. In the presence of 0.2 pM TF, Western blotting under nonreducing conditions showed preservation of the prothrombin/meizothrombin band and delayed/reduced thrombin generation in the presence of 0.5 μM DHG, confirming that the inhibition involves reduced prothrombin activation rather than accelerated thrombin inhibition. When triggered by 0.2 pM TF in factor VIII-deficient plasma supplemented with 700 pM factor VIII or thrombin-activated factor VIIIa, the EC50 for inhibition by DHG was 0.41 ± 0.02 μM and 0.44 ± 0.05 μM, respectively. Similarly, the EC50 for DHG inhibition of thrombin generation in factor IX deficient plasma supplemented with 0.2 pM TF and 100% plasma-derived factor IX (90 nM), or 100 pM plasma-derived factor IXa alone, was 0.36 ± 0.01 μM and 0.34 ± 0.02 μM, respectively. Thus, activation of factors VIII and IX do not contribute significantly to the inhibition mechanism for DHG. The contribution of intrinsic tenase activity to DHG inhibition of plasma thrombin generation was assessed using recombinant factor IX(a) mutants with moderate (R170A) or marked (R233A) reductions in heparin affinity. Factor IX deficient plasma was supplemented with 0.2 pM TF and 100% recombinant factor IX, or 100 pM factor IXa, with increasing concentrations of DHG. Similar to plasma-derived factor IX(a), DHG demonstrated an EC50 of 0.38 ± 0.01 μM for inhibition of thrombin generation in the presence of factor IX(a) wild type (WT) zymogen or protease. In the presence of factor IX(a) R170A, the EC50 for DHG was 0.86 ± 0.06 μM and 1.02 ± 0.02 μM, respectively, a 2–3 fold increase relative to WT (P ≤ 0.01). For factor IX(a) R233A, the EC50 for DHG was 3.55 ± 0.47 μM for zymogen and 2.98 ± 0.64 μM for protease, an 8–9 fold increase relative to WT (P ≤ 0.01). Thus, mutations in the factor IXa heparin-binding exosite induced resistance to DHG inhibition of thrombin generation as follows: factor IX(a) R233A> R170A> WT. These findings are consistent with the common mechanism for intrinsic tenase inhibition demonstrated for heparin and DHG in purified systems, and establish the factor IXa heparin-binding exosite as the relevant molecular target for inhibition of plasma thrombin generation by DHG. This antithrombin-independent mechanism likely mediates the antithrombotic efficacy of DHG and related glycosaminoglycans, and may represent a novel therapeutic target with lower bleeding risk.

Evidence that alpha2-antiplasmin becomes covalently ligated to plasma fibrinogen in the circulation: a new role for plasma factor XIII in fibrinolysis regulation.

Plasma alpha2-antiplasmin (alpha2AP) is a rapid and effective inhibitor of the fibrinolytic enzyme plasmin. Congenital alpha2AP deficiency results in a severe hemorrhagic disorder due to accelerated fibrinolysis. It is well established that in the presence of thrombin-activated factor XIII (FXIIIa), alpha2AP becomes covalently ligated to the distal alpha chains of fibrin or fibrinogen at lysine 303 (two potential sites per molecule). Some time ago we showed that alpha2AP is covalently linked to plasma fibrinogen . That singular observation led to our hypothesis that native plasma factor XIII (FXIII), which is known to catalyze covalent cross-linking of fibrinogen in the presence of calcium ions, can also incorporate alpha2AP into fibrinogen in the circulation. We now provide evidence that FXIII incorporates I 125-labelled alpha2AP into the Aalpha-chain sites on fibrinogen or fibrin. We also measured the content of alpha2AP in isolated plasma fibrinogen fractions by ELISA and found that substantial amounts were present (1.2-1.8 moles per mole fibrinogen). We propose that alpha2AP becomes ligated to fibrinogen while in the circulation through the action of FXIII, and that its immediate presence in plasma fibrinogen contributes to regulation of in vivo fibrinolysis.

Variegin, a Novel Fast and Tight Binding Thrombin Inhibitor from the Tropical Bont Tick

Tick saliva contains potent antihemostatic molecules that help ticks obtain their enormous blood meal during prolonged feeding. We isolated thrombin inhibitors present in the salivary gland extract from partially fed female Amblyomma variegatum, the tropical bont tick, and characterized the most potent, variegin, one of the smallest (32 residues) thrombin inhibitors found in nature. Full-length variegin and two truncated variants were chemically synthesized. Despite its small size and flexible structure, variegin binds thrombin with strong affinity (K(i) approximately 10.4 pM) and high specificity. Results using the truncated variants indicated that the seven residues at the N terminus affected the binding kinetics; when removed, the binding characteristics changed from fast to slow. Further, the thrombin active site binding moiety of variegin is in the region of residues 8-14, and the exosite-I binding moiety is within residues 15-32. Our results show that variegin is structurally and functionally similar to the rationally designed thrombin inhibitor, hirulog. However, compared with hirulog, variegin is a more potent inhibitor, and its inhibitory activity is largely retained after cleavage by thrombin.

Plasticity of Mammalian Myotubes Upon Stimulation with a Thrombin-activated Serum Factor

Salamanders display unique regeneration abilities among adult vertebrates. An intriguing feature of salamander regeneration is the dedifferentiation of cells, such as myofibers and myotubes at the injury site, a process that involves cell cycle re-entry from the differentiated state. A thrombin-activated serum factor that is distinct from conventional growth factors is known to cause S-phase re-entry in salamander myotubes. While mammalian myotubes do not re-enter S-phase upon serum stimulation, an upregulation of some immediate early genes such as jun and fos has been observed. Until now, it was unknown whether this transcriptional response was stimulated by conventional growth factors or by the thrombin-activated serum factor. By measuring transcriptional activity in individually purified C2C12 mouse myotubes using quantitative reverse transcription polymerase chain reactions, we show that a set of immediate early genes are activated in response to the thrombin-activated serum factor in a distinct manner from the growth factors PDGF, FGF and EGF. A partially purified fraction of the thrombin activated serum factor elicited stronger upregulation of a broader set of genes compared to individual growth factors and additionally caused downregulation of E2F6. Despite this robust transcriptional response in mammalian myotubes, we did not detect a large-scale change in histone H3K9 di-methylation or S-phase, a feature that characterizes salamander serum-stimulated myotubes. Our results indicate that mammalian myotubes have retained responsiveness to the thrombin-activated serum factor, but full re-entry into S-phase is prevented by factors downstream of the immediate early genes.

A1 Subunit-mediated Regulation of Thrombin-activated Factor VIII A2 Subunit Dissociation

Factor VIII (fVIII) is the plasma protein that is missing or deficient in hemophilia A. In contrast, elevated levels of fVIII are associated with an increased risk of arterial and venous thrombosis. fVIII is activated by thrombin to form a non-covalently linked A1/A2/A3-C1-C2 heterotrimer. At physiological concentrations, fVIIIa decays as a result of A2 subunit dissociation, which may help regulate the balance between hemostasis and thrombosis. A2 subunit dissociation is faster in human fVIIIa than in porcine fVIIIa, which may represent an evolutionary adaptation associated with the development of the upright posture and venous stasis in the lower extremities. To investigate the basis for the different decay kinetics of human and porcine fVIIIa, hybrid fVIII molecules representing all possible combinations of human and porcine A domains were isolated. The kinetics of fVIIIa decay were measured and fit to a model describing a reversible bimolecular reaction in which the dissociation rate constant, k, and dissociation constant, Kd, were the fitted parameters. Substitution of the porcine A1 domain into human fVIIIa produced a dissociation rate constant indistinguishable from porcine fVIIIa. Subsequently, substitution of the second cupredoxin-like A1 subdomain resulted in a dissociation rate constant similar to porcine fVIIIa, whereas substitution of the first cupredoxin-like A1 subdomain resulted in a dissociation rate constant intermediate between human and porcine fVIIIa. We propose that cupredoxin-like A1 subdomains in fVIII contain inter-species differences that are a result of selective pressure on the dissociation rate constant.

Structural Requirements for Expression of Factor Va Activity

Thrombin activated factor Va (factor VIIa, residues 1-709 and 1546-2196) has an apparent dissociation constant (Kd,app) for factor Xa within prothrombinase of approximately 0.5 nM. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp697, Asp1509, and Asp1514 to produce a molecule (factor VNN) that is composed of a Mr 100,000 heavy chain (amino acid residues 1-696) and a Mr 80,000 light chain (amino acid residues 1509/1514-2196). Factor VNN, has a Kd,app for factor Xa of 4 nm and reduced clotting activity. Cleavage of factor VIIa by NN at Asp697 results in a cofactor that loses approximately 60-80% of its clotting activity. An enzyme from Russell's viper venom (RVV) cleaves human factor V at Arg1018 and Arg1545 to produce a Mr 150,000 heavy chain and Mr 74,000 light chain (factor VRVV, residues 1-1018 and 1546-2196). The RVV species has affinity for factor Xa and clotting activity similar to the thrombin-activated factor Va. Cleavage of factor VNN at Arg1545 by alpha-thrombin (factor VNN/IIa) or RVV (factor VNN/RVV) leads to enhanced affinity of the cofactor for factor Xa (Kd,app approximately 0.5 nM). A synthetic peptide containing the last 13 residues from the heavy chain of factor Va (amino acid sequence 697-709, D13R) was found to be a competitive inhibitor of prothrombinase with respect to prothrombin. The peptide was also found to specifically interact with thrombin-agarose. These data demonstrate that 1) cleavage at Arg1545 and formation of the light chain of factor VIIa is essential for high affinity binding and function of factor Xa within prothrombinase and 2) a binding site for prothrombin is contributed by amino acid residues 697-709 of the heavy chain of the cofactor.

The structure and biological features of fibrinogen and fibrin.

Fibrinogen and fibrin play important, overlapping roles in blood clotting, fibrinolysis, cellular and matrix interactions, inflammation, wound healing, and neoplasia. These events are regulated to a large extent by fibrin formation itself and by complementary interactions between specific binding sites on fibrin(ogen) and extrinsic molecules including proenzymes, clotting factors, enzyme inhibitors, and cell receptors. Fibrinogen is comprised of two sets of three polypeptide chains termed A alpha, B beta, and gamma, that are joined by disulfide bridging within the N-terminal E domain. The molecules are elongated 45-nm structures consisting of two outer D domains, each connected to a central E domain by a coiled-coil segment. These domains contain constitutive binding sites that participate in fibrinogen conversion to fibrin, fibrin assembly, crosslinking, and platelet interactions (e.g., thrombin substrate, Da, Db, gamma XL, D:D, alpha C, gamma A chain platelet receptor) as well as sites that are available after fibrinopeptide cleavage (e.g., E domain low affinity non-substrate thrombin binding site); or that become exposed as a consequence of the polymerization process (e.g., tPA-dependent plasminogen activation). A constitutive plasma factor XIII binding site and a high affinity non-substrate thrombin binding site are located on variant gamma' chains that comprise a minor proportion of the gamma chain population. Initiation of fibrin assembly by thrombin-mediated cleavage of fibrinopeptide A from A alpha chains exposes two EA polymerization sites, and subsequent fibrinopeptide B cleavage exposes two EB polymerization sites that can also interact with platelets, fibroblasts, and endothelial cells. Fibrin generation leads to end-to-middle intermolecular Da to EA associations, resulting in linear double-stranded fibrils and equilaterally branched trimolecular fibril junctions. Side-to-side fibril convergence results in bilateral network branches and multistranded thick fiber cables. Concomitantly, factor XIII or thrombin-activated factor XIIIa introduce intermolecular covalent epsilon-(gamma glutamyl)lysine bonds into these polymers, first creating gamma dimers between properly aligned C-terminal gamma XL sites, which are positioned transversely between the two strands of each fibrin fibril. Later, crosslinks form mainly between complementary sites on alpha chains (forming alpha-polymers), and even more slowly among gamma dimers to create higher order crosslinked gamma trimers and tetramers, to complete the mature network structure.

Comparison of the properties of phospholipid surfaces formed on HPA and L1 biosensor chips for the binding of the coagulation factor VIII

Binding of a coagulation factor VIII to phosphatidylserine-containing membranes is critical for exerting its cofactor activity. The use of surface plasmon resonance allows studying factor VIII interaction with immobilized phospholipids. In the present study we compared factor VIII-binding properties of phospholipid surfaces immobilized on L1 and HPA Biacore chips in the form of a flexible bilayer and rigid monolayer, respectively. We demonstrated that immobilized phospholipid surfaces with physiological contents of PS and PE formed on L1 but not on HPA chip closely mimic intact phospholipid vesicles in their factor VIII and thrombin-activated factor VIII (factor VIIIa) binding properties.

Hemophilia A mutations associated with 1-stage/2-stage activity discrepancy disrupt protein-protein interactions within the triplicated A domains of thrombin-activated factor VIIIa

AbstractThrombin-activated factor VIII (FVIIIa) is a heterotrimer with the A2 subunit (amino acid residues 373-740) in a weak ionic interaction with the A1 and A3-C1-C2 subunits. Dissociation of the A2 subunit correlates with inactivation of FVIIIa. Patients with hemophilia A have been described whose plasmas display a discrepancy between their FVIII activities, where the 1-stage activity assay displays greater activity than the 2-stage activity assay. The molecular basis for one of these mutations, ARG531HIS, is an increased rate of A2 subunit dissociation. Examination of a homology model of the A domains of FVIII predicted ARG531 to lie at the interface of the A1 and A2 subunits and stabilize their interaction. Indeed, patients with mutations either directly contacting ARG531 (ALA284GLU, ALA284PRO) or closely adjacent to the A1-A2 interface in the tightly packed hydrophobic core (SER289LEU) have the same phenotype of 1-stage/2-stage discrepancy. TheALA284GLU andSER289LEU mutations in FVIII were produced by transfection of COS-1 monkey cells. Compared to FVIII wild-type both mutants had reduced specific activity by 1-stage clotting activity and at least a 2-fold lower activity by 2-stage analysis (COAMATIC), similar to the reported clinical data. Analysis of immunoaffinity purified ALA284GLU andSER289LEU proteins in an optical biosensor demonstrated that A2 dissociation was 3-fold faster for both FVIIIa mutants compared to FVIIIa wild-type. Therefore, these mutations within the A1 subunit of FVIIIa introduce a similar destabilization of the FVIIIa heterotrimer compared to the ARG531HISmutation within the A2 subunit and support that these residues stabilize the A domain interface of FVIIIa.

Partial glycosylation at asparagine-2181 of the second C-type domain of human factor V modulates assembly of the prothrombinase complex.

Thrombin-activated factor Va exists as two isoforms, factor Va(1) and factor Va(2), which differ in the size of their light chains and their affinity for biological membranes. The heterogeneity of the light chain remained following incubation of factor Va with N-glycanase. However, we found that the factor V C2 domain, which contains a single potential glycosylation site at Asn-2181, was partially glycosylated when expressed in COS cells. To confirm the structural basis for factor Va(1) and factor Va(2), we mutated Asn-2181 to glutamine (N2181Q) and expressed this mutant using a B domain deletion construct (rHFV des B) in COS cells. Thrombin activation of N2181Q released a light chain with mobility identical to that of factor Va(2) on SDS-PAGE. The functional properties of purified N2181Q were similar to those of factor Va(2) in prothrombinase assays carried out in the presence of limiting concentrations of phosphatidylserine. The binding of human factor Va(1) and factor Va(2) to 75:25 POPC/POPS vesicles was also investigated in equilibrium binding assays using proteins containing a fluorescein-labeled heavy chain. The affinity of human factor Va(2) binding to POPC/POPS vesicles was approximately 3-fold higher than that of factor Va(1). These results indicate that partial glycosylation of factor V at asparagine-2181 is the structural basis of the light chain doublet and that the presence of this oligosaccharide reduces the affinity of factor Va for biological membranes.

Proteolysis of blood coagulation factor VIII by the factor VIIa-tissue factor complex: generation of an inactive factor VIII cofactor.

Activation of factor VIII by thrombin occurs via limited proteolysis at R372, R740, and R1689. The resultant active factor VIIIa molecule consists of three noncovalently associated subunits: A1-a1, A2-a2, and A3-C1-C2 (50, 45, and 73 kDa respectively). Further proteolysis of factor VIIIa at R336 and R562 by activated protein C subsequently inactivates this cofactor. We now find that the factor VIIa-tissue factor complex (VIIa-TF/PL), the trigger of blood coagulation with restricted substrate specificity, can also catalyze limited proteolysis of factor VIII. Proteolysis of factor VIII was observed at 10 sites, producing 2 major fragments (47 and 45 kDa) recognized by an anti-factor VIII A2 domain antibody. Time courses indicated the slow conversion of the large fragment to 45 kDa, followed by further degradation into at least two smaller fragments. N-Terminal sequencing along with time courses of proteolysis indicated that VIIa-TF/PL cleaved factor VIII first at R740, followed by concomitant cleavage at R336 and R372. Although cleavage of the light chain at R1689 was observed, the majority remained uncleaved after 17 h. Consistent with this, only a transient 2-fold increase in factor VIII clotting activity was observed. Thus, heavy chain cleavage of factor VIII by VIIa-TF/PL produces an inactive factor VIII cofactor no longer capable of activation by thrombin. In addition, VIIa-TF/PL was found to inactivate thrombin-activated factor VIII. We hypothesize that these proteolyses may constitute an alternative pathway to regulate coagulation under certain conditions. In addition, the ability of VIIa-TF/PL to cleave factor VIII at 10 sites greatly expands the known protein substrate sequences recognized by this enzyme-cofactor complex.

Mechanisms that regulate the anticoagulant function of coagulation factor V

Coagulation factor V is composed of domains A1-A2-B-A3-C1-C2 and is activated by thrombin through proteolytic cleavage at Arg 709, Arg 1018 and Arg 1545. Upon thrombin activation, the B-domain is released and the active factor Va is formed by the heavy (A1-A2) and light chains (A3-C1-C2). Factor Va functions as an essential cofactor to factor Xa in the conversion of prothrombin to thrombin during coagulation. Recently tit was shown that coagulation factor V, apart from being a precursor form to the procoagulant factor Va, also has anticoagulant properties, as it functions as a cofactor to activated protein C (APC). APC is a member of the anticoagulant pathway and downregulates the coagulation process through proteolytic inactivation of factors VIII/VIIIa and factors V/Va. In a factor VIIIa degradation assay, the APC-mediated inactivation of factor VIIIa is potentiated by the synergistic cofactors protein S and factor V. Protein S alone has little cofactor activity, whereas in the presence of factor V it is dramatically enhanced. This study provides insights into the molecular mechanisms that regulate the anticoagulant activity of factor V. Thrombin cleavage of factor V occurs in a sequential order. The thrombin cleavage site Arg 1545 is kinetically less favored than the other two sites, and cleavage at this site is the last to occur during thrombin activation of factor V. As a consequence of this, different activation intermediates exist that express different levels of procoagulant activity. The anticoagulant activities of these intermediates have now been studied. It was found that factor V could be cleaved by thrombin at both Arg 709 and Arg 1018 and still work fully as a cofactor to APC, whereas cleavage at Arg 1545 completely abolished the anticoagulant activity of factor V. This suggests that the APC cofactor function of factor V depends on the B-domain remaining attached to the A3 domain. This study further shows that APC converts coagulation factor V into a member of the anticoagulant pathway by cleaving factor V in the A2 domain at Arg 506. By cleavage of factor V, APC not only produces an anticoagulant cofactor, but at the same time eliminates the pool of procoagulant factor V, since APC cleaved factor V will have no future as a cofactor in the coagulation. The unique way by which APC and thrombin, through proteolytic cleavage, can convert factor V into either an anticoagulant or a procoagulant adds to the intriguing mechanisms that balance the procoagulant and anticoagulant forces.