Inactivation of factor Va by plasmin.

Optimal rates of factor X (FX) activation require occupancy of receptors for factor IXa (FIXa), factor VIII (FVIII), and FX on the activated platelet surface. The presence of FVIII and FX increases 5-fold the affinity of FIXa for the surface of activated platelets, and the presence of FVIII or FVIIIa generates a high affinity, low capacity specific FX-binding site on activated platelets. We have now examined the effects of FX and active site-inhibited FIXa (EGR-FIXa) on the binding of both FVIII and FVIIIa to activated platelets and show the following: (a) von Willebrand factor inhibits FVIII binding (K(i) = 0.54 nM) but not FVIIIa binding; (b) thrombin and the thrombin receptor activation peptide (SFLLRN amide) are the most potent agonists required for FVIII-binding site expression, whereas ADP is inert; (c) FVa does not compete with FVIIIa or FVIII for functional platelet-binding sites; and (d) Annexin V is a potent inhibitor of FVIIIa binding (IC(50) = 10 nM) to activated platelets. The A2 domain of FVIII significantly increases the affinity and stoichiometry of FVIIIa binding to platelets and contributes to the stability of the FX-activating complex. Both FVIII and FVIIIa binding were specific, saturable, and reversible. FVIII binds to specific, high affinity receptors on activated platelets (n = 484 +/- 59; K(d) = 3.7 +/- 0.31 nM) and FVIIIa interacts with an additional 300-500 sites per platelet with enhanced affinity (K(d) = 1.5 +/- 0.11 nM). FVIIIa binding to activated platelets in the presence of FIXa and FX is closely coupled with rates of F-X activation. The presence of EGR-FIXa and FX increases both the number and the affinity of binding sites on activated platelets for both FVIII and FVIIIa, emphasizing the validity of a three-receptor model in the assembly of the F-X-activating complex on the platelet surface.

Bothrojaracin (BJC) is a 27 kDa snake venom protein from Bothrops jararaca that has been characterized as a potent ligand (KD = 75 nM) of human prothrombin (Monteiro RQ, Bock PE, Bianconi ML, Zingali RB, Protein Sci 2001; 10: 1897-904). BJC binds to the partially exposed anion-binding exosite I (proexosite I) forming a stable 1:1, non-covalent complex with the zymogen whereas no interaction with fragment 1 or 2 domains is observed. In addition, BJC interacts with thrombin through exosites I and II (KD = 0.7 nM), and influences but does not block the proteinase catalytic site. In the present work we studied the effect of BJC on human prothrombin activation by factor Xa in the absence or in the presence of its cofactors, factor Va and phospholipids. In the absence of phospholipids, BJC strongly inhibited (80%) the zymogen activation by factor Xa in the presence but not in the absence of factor Va, suggesting a specific interference in the cofactor activity. In the presence of phospholipid vesicles (75% phosphatidylcholine, 25% phosphatidylserine), BJC also inhibited (35%) prothrombin activation by factor Xa in the presence but not in the absence of factor Va. BJC showed a higher inhibitory effect (70%) towards thrombin formation by prothrombinase complex assembled on phospholipid vesicles composed by 95% phosphatidylcholine, 5% phosphatidylserine. Activation of prothrombin by platelet-assembled prothrombinase complex (factor Xa, factor Va and thrombin-activated platelets) showed that hirudin (SO3-) and BJC efficiently inhibit the thrombin formation (43% and 84%, respectively). Taken together, our results suggest that proexosite I blockage decreases the productive recognition of prothrombin as substrate by factor Xa-factor Va complex and prothrombinase complex. Furthermore, data obtained with human platelets suggest that proexosite I may play an important role in the physiological activation of prothrombin.

Factor Va was incubated with 1-azidopyrene, a fluorescent lipophilic probe, in the presence of phospholipid vesicles composed of various proportions of phosphatidylcholine (PC) and phosphatidylserine (PS). The majority of the label was associated with the light chain of factor Va. The light chain was found to be labeled in the presence of phospholipid vesicles containing either 100% PC or 100% PS. After cleavage by factor Xa and incubation with PC/PS vesicles composed of 75% PC and 25% PS, label was found both on the M(r) = 30,000 fragment, derived from the NH2-terminal portion of the bovine factor Va light chain (residues 1537-1752), and on the M(r) = 46,000/48,000 carboxyl-terminal fragment of the factor Va light chain (residues 1753-2183). The M(r) = 46,000/48,000 fragment incorporated 1-azidopyrene independent of the phospholipid composition, while label incorporation into the M(r) = 30,000 fragment required phospholipid vesicles containing PC. No labeling of the M(r) = 30,000 fragment was observed with phospholipid vesicles composed of 100% PS. The label incorporation into the two portions of the molecule was found to be independent of the ionic strength in the presence of phospholipid vesicles containing 75% PC and 25% PS. In contrast, the labeling of the M(r) = 46,000/48,000 fragment with phospholipid vesicles composed of 100% PS was ionic strength dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies into the Mechanism by Which Glycosaminoglycans Potentiate Protein C Activation by Factor Xa.

Measurement of procoagulant platelet subpopulations in whole blood: Development of an assay for population-based studies

It is believed that heparin accelerates factor Xa (FXa) inactivation by antithrombin (AT) by conformationally activating the inhibitor rather than by bridging AT and FXa in a ternary complex (template effect). This is derived from kinetic studies done in the absence of Ca2+ or in the presence of EDTA. To test the possibility that the anionic Gla domain of FXa, when not neutralized by Ca2+ ions, prevents heparin binding to FXa, the heparin and pentasaccharide dependence of FXa inactivation by AT in both the absence (100 microM EDTA) and presence of Ca2+ (2.5 mM) was studied using wild-type FXa and a FXa derivative that lacks the Gla domain (GDFXa). AT inactivated both FXa derivatives similarly in both the absence and presence of Ca2+ (k2 = 1.7-2.5 x 10(3) M-1 s-1). The active AT-binding pentasaccharide also accelerated the inactivation rates of both derivatives similarly in both the absence and presence of Ca2+ (k2 = 5.7-8.0 x 10(5) M-1 s-1). However, in the presence of an optimum concentration of heparin ( approximately 50 nM) the inactivation rate constant of FXa in the presence of Ca2+ (k2 = 4.4 x 10(7) M-1 s-1) was 13-fold higher than the rate constant in the absence of Ca2+ (k2 = 3.5 x 10(6) M-1 s-1). Heparin acceleration of GDFXa inactivation by AT was rapid and insensitive to the presence or absence of Ca2+ (k2 = 5.1-5.9 x 10(7) M-1 s-1). The additional cofactor effect of heparin with all FXa derivatives was a bell-shaped curve, which disappeared if the ionic strength of the reaction was increased to approximately 0.4. These results suggest that although the major effect of heparin in acceleration of FXa inactivation is through a heparin-induced conformational change in the reactive site loop of AT, the template effect of heparin, nevertheless, contributes significantly to rapid FXa inactivation at physiological Ca2+.

The capacity of inflammatory cell-derived matrix metalloproteinases (MMPs) to cleave tissue factor pathway inhibitor (TFPI) and alter its activity was investigated. MMP-7 (matrilysin) rapidly cleaved TFPI to a major 35-kDa product. In contrast, MMP-1 (collagenase-1), MMP-9 (gelatinase B), and MMP-12 (macrophage elastase) cleaved TFPI into several fragments including the 35-kDa band. However, rates of cleavage were most rapid for MMP-7 and MMP-9. NH₂-terminal amino acid sequencing revealed that MMP-12 cleaved TFPI at Lys²⁰-Leu²¹(close to Kunitz I domain and producing a 35-kDa band), Arg⁸³-Ile⁸⁴ (between Kunitz I and II domains), and Ser¹⁷⁴-Thr¹⁷⁵ (between Kunitz II and III domains). MMP-7 and MMP-9 cleaved TFPI at Lys²⁰-Leu²¹ with additional COOH-terminal processing. These MMPs did not cleave tissue factor (TF), factor VII, and factor Xa. Proteolytic cleavage by MMP-1, MMP-7, MMP-9, and MMP-12 resulted in considerable loss of TFPI activity. These observations indicate specific cleavage of TFPI by MMPs, which broadens their substrate profile. Co-localization of MMPs, TF, and TFPI in atherosclerotic tissues suggests that release of MMPs from inflammatory cell leukocytes may effect TF-mediated coagulation.

A Ca2+ Switch Triggers Formation of Inactive Factor Xa Dimers on Phosphatidylserine-Containing Membranes.

Activated Protein C (APC) is a plasma serine protease with antithrombotic function. APC acts as an anticoagulant by promoting the degradation of factors Va and VIIIa, thus inhibiting the formation of thrombin. Specific inhibition of APC has been proposed to benefit hemophilia therapy. We used chromogenic tripeptide substrate hydrolysis assay to screen a series of arginine and arginine-like containing small molecules to identify inhibitors of APC. Similar hydrolysis assays were used to determine selectivity against other serine proteases and blood-clotting enzymes. Molecular modeling was exploited to illustrate the binding of the most potent and selective inhibitor onto the putative binding site. We identified inhibitor 2 as a potent inhibitor with an IC50 value of 1.1 μM. The molecule demonstrated >100-fold selectivity against thrombin, factor XIa, and neutrophil elastase, >50-fold selectivity against factor XIIIa, 10-fold selectivity against factor Xa, and 8-fold selectivity against human plasmin. Molecular modeling reveals that inhibitor 2 binds to the active site of APC with the best-docked structure, indicating that one protonated amidino group establishes a salt bridge to the side chain carboxylate of Asp189 residue. Another inhibitor was identified, yet it was not as selective to APC. Importantly, inhibitor 2 demonstrates favorable physicochemical, pharmacokinetic, and drug-likeness properties. Inhibitor 2 is a selective and potent inhibitor of APC that serves as a powerful lead for the development of hemophilia therapy.

Thrombogenicity of small-diameter prosthetic grafts: Relative contributions of graft-associated thrombin and factor Xa

A simulation model for the production of thrombin in plasma is presented. Values of the reaction rate constants as determined in purified systems are used and the model is tested by comparison of simulations of factor Xa, factor Va and thrombin generation curves with experimental data obtained in thromboplastin-activated plasma. Simulations of the effect of hirudin indicate that factor V is predominantly activated by thrombin and not by factor Xa. The model predicts a threshold value for the factor Xa production which, if exceeded, results in explosive and complete activation of prothrombinase. The dependence of this threshold value on different negative feedback reactions, e.g. the inactivation of thrombin and factor Xa by antithrombin III (+ heparin), is investigated. The threshold value, for control plasma in the range of 1-10 pM total factor Xa production, can be raised two orders of magnitude by accelerated inactivation of factor Xa and prothrombinase but is hardly affected by a tenfold increase in the rate of thrombin inactivation or by increased production of activated protein C. This latter effect, however, results in a more gradual input-response relation between factor Xa input and the extent of prothrombinase activation.

The activation of porcine factor VIII:C by thrombin and by factor Xa was studied by a chromogenic substrate assay and by sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis of 125I-labeled factor VIII:C activation products. In the chromogenic assay, the kinetics of factor VIII:C dependent activation of factor X by factor IXa in the presence of calcium and phosphatidylserine/phosphatidylcholine vesicles were measured with N-benzoyl-L-isoleucyl-L-glutamylglycyl-L-arginine p-nitroanilide (S2222) as substrate. Substrate dependence of initial rates of the reaction at fixed factor IXa, factor VIII:C, lipid, and calcium obeyed Michaelis-Menten kinetics. At fixed factor IXa, factor X, lipid, and calcium the initial rates of the reaction varied linearly with lower factor VIII:C concentrations and plateaued at higher concentrations. The linear initial rate dependence formed the basis of a rapid, plasma-free assay of activated factor VIII:C. The activation of factor VIII:C by thrombin or factor Xa and the enzyme-independent rate of spontaneous inactivation were studied under conditions of excess enzyme. A model of the activation kinetics was developed and fit to the data by a nonlinear least-squares technique. From the model, the catalytic efficiencies (kcat/Km) of factor VIII:C activation by thrombin and factor Xa were 5.0 X 10(6) M-1 s-1 and 1.1 X 10(6) M-1 s-1, respectively. By comparison with published values of the catalytic efficiencies of several other coagulation enzymes for various substrates, both thrombin and factor Xa are efficient enzymes toward factor VIII:C.(ABSTRACT TRUNCATED AT 250 WORDS)

The inhibition of blood coagulation by activated protein C through the selective inactivation of activated factor V

Factors Influencing the Acceleration of Human Factor XIa Inactivation by Antithrombin III

Human coagulation FXa (Factor Xa) plays a key role in blood coagulation by activating prothrombin to thrombin on 'stimulated' platelet membranes in the presence of its cofactor FVa (Factor Va). PS (phosphatidylserine) exposure on activated platelet membranes promotes prothrombin activation by FXa by allosterically regulating FXa. To identify the structural basis of this allosteric regulation, we used FRET to monitor changes in FXa length in response to (i) soluble short-chain PS [C6PS (dicaproylphosphatidylserine)], (ii) PS membranes, and (iii) FVa in the presence of C6PS and membranes. We incorporated a FRET pair with donor (fluorescein) at the active site and acceptor (Alexa Fluor® 555) at the FXa N-terminus near the membrane. The results demonstrated that FXa structure changes upon binding of C6PS to two sites: a regulatory site at the N-terminus [identified previously as involving the Gla (γ-carboxyglutamic acid) and EGFN (N-terminus of epidermal growth factor) domains] and a presumptive protein-recognition site in the catalytic domain. Binding of C6PS to the regulatory site increased the interprobe distance by ~3 Å (1 Å=0.1 nm), whereas saturation of both sites increased the distance by a further ~6.4 Å. FXa binding to a membrane produced a smaller increase in length (~1.4 Å), indicating that FXa has a somewhat different structure on a membrane from when bound to C6PS in solution. However, when both FVa2 (a FVa glycoform) and either C6PS- or PS-containing membranes were bound to FXa, the overall change in length was comparable (~5.6-5.8 Å), indicating that C6PS- and PS-containing membranes in conjunction with FVa2 have comparable regulatory effects on FXa. We conclude that the similar functional regulation of FXa by C6PS or membranes in conjunction with FVa2 correlates with similar structural regulation. The results demonstrate the usefulness of FRET in analysing structure-function relationships in FXa and in the FXa·FVa2 complex.