Bothrojaracin, a Proexosite I Ligand, Inhibits Factor Va-Accelerated Prothrombin Activation

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.

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

The Regulatory Function of Amino Acid Region 659–663 of Factor Va on Prothrombinase during Thrombin Formation.

The antiphospholipid syndrome (APS) is associated with production of autoantibodies with lupus anticoagulant (LA) activity. These antibodies cause prolongation of in vitro clotting tests by inhibition of the conversion of prothrombin to thrombin in the presence of anionic phospholipid (PL). The extent to which this inhibition reflects antibody binding to, or functional inhibition of, phospholipids alone, prothrombin alone, or a prothrombin-phospholipid complex is pertinent to our understanding of the pathophysiology of this syndrome. Immunoglobulin fractions (IgG) from 18 patients with LA activity were tested for inhibitory activity against prothrombin activation by either factor Xa, in a purified prothrombinase system, or by purified fractions of snake venoms (E. carinatus, E. multisquamatus) which cleave prothrombin at the same initial site as the prothrombinase complex but do not require anionic phospholipid as a cofactor. Parallel testing of the same IgG samples for prothrombin binding by immunoassay was performed. Although all IgG samples inhibited the prothrombinase reaction, only three exhibited any inhibition of venom protease prothrombin activation in either the presence or absence of PL. Only one sample exhibited prothrombin binding by Western blot. These results suggest that lupus anticoagulant antibodies are heterogenous and that many, if not most, of the autoantibody populations responsible for LA activity impair prothrombin activation by interaction either with phospholipid alone or with a restricted range of prothrombin-phospholipid epitopes expressed by prothrombin only as part of the intact prothrombin-prothrombinase complex.

Anticoagulant mechanism of the coagulation factor IX/factor X-binding protein (IX/X-bp) isolated from the venom of Trimeresurus flavoviridis was investigated. IX/X-bp had no effect on the amidase activity of factor Xa measured with a synthetic peptide substrate Boc-Leu-Gly-Arg-pNA. Prothrombin activation by factor Xa without cofactors, such as factor Va and phospholipids, was only slightly influenced by IX/X-bp. However, prothrombin activation by factor Xa in the presence of factor Va resulted in IX/X-bp inhibiting the increase of k(cat) of thrombin formation through inhibition of interaction between factor Xa and factor Va. IX/X-bp also inhibited the decrease of K(m) for thrombin formation through interaction with phospholipids. Thus, IX/X-bp appears to act as an anticoagulant protein by inhibiting the interaction between factor Xa and its cofactors in the prothrombinase complex by binding to the Gla domain of factor Xa.

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

After the platelet release reaction, factor Xa binds to a receptor on the platelet surface with high affinity, Ka 5.2 × 1o9 M−1. Binding of the factor Xa substrate, prothrombin to the platelet surface has not yet been possible to demonstrate. The interaction between prothrombin and platelets was therefore studied by determination of the structural requirements on the prothrombin molecule for activation by platelet-bound factor Xa. A comparison was made between the rates of activation of normal prothrombin, prethrombin 1, prethrombin 2, and acarboxyprothrombin (prothrombin lacking γ-carboxyglutamic acid residues) in the presence of factor Xa and isolated platelets. Both the vitamin-K-dependent fragment 1 of prothrombin and fragment 2 which interacts with factor V were found to be required for rapid prothrombin activation. Acarboxyprothrombin and prethrombin 1 were slowly activated to thrombin by factor Xa on the platelet surface after the platelet release reaction. In the same system prethrombin 2 was activated more slowly to thrombin. In the presence of fragment 2, which binds noncovalently to prethrombin 2, the activation rate was enhanced and equal to that of prethrombin 1 supporting the conclusion that factor V is part of the platelet receptor. The cleavage products of prothrombin by factor Xa in the presence of platelets were found to be the same as those previously described when the activation was catalyzed by the prothrombinase complex. The coefficient for proteolytic efficiency, Kcat/Km, was found to be approximately 50 times higher in the presence of prothrombin than in the presence of acarboxyprothrombin. We conclude that prothrombin interacts with a specific platelet structure consisting of phospholipid and an activated form of factor V exposed on the platelet surface as a result of the release reaction.

Factor Va Cofactor Activity Is Dependent on the Interaction of Prothrombin with the Membrane Surface.

The kringle 2 domain of prothrombin has been shown to interact with factor Va during the activation of prothrombin by the prothrombinase complex composed of factor Xa, factor Va, negatively charged phospholipids and Ca2+ ions. However, contradictory results have been reported about the role of the kringle 1 domain of prothrombin during the assembly of the prothrombinase complex. In an attempt to clarify the role of the kringle 1 domain of prothrombin, its effect on the activation of prothrombin by the prothrombinase complex and its direct binding to human factor Va were assessed. Comparative evaluation with the effects caused by other prothrombin structural components [a fragment 1 (gamma-carboxyglutamic acid and kringle 1 domains), a kringle 2 domain and a catalytic protease domain] was also performed. In the presence of factor Va, each kringle 1 and kringle 2 fragment significantly inhibited the factor Xa-catalysed prothrombin activation in the absence of phospholipids. However, in the absence of both factor Va and phospholipids, kringle 2 fragment, but not kringle 1 fragment, inhibited prothrombin activation. Evaluation of the molecular interaction of the kringle domains with factor Va in assays with solid-phase phospholipid vesicles showed that each kringle 1 and kringle 2 fragment inhibited the prothrombinase complex activity. Assessment of the direct binding of prothrombin and each kringle domain of prothrombin with factor Va by fluorescence polarization showed that prothrombin, kringle 1 and kringle 2 fragments bind directly to factor Va with dissociation constants of 1.9+/-0.1, 2.3+/-0.1 and 2.0+/-0.4 microM (means+/-S.D.) respectively. These findings suggest that both kringle 1 and 2 domains of prothrombin interact with factor Va during the assembly of the prothrombinase complex.

17] Meizothrombin: Active intermediate formed during prothrombinase-catalyzed activation of prothrombin

The inactivation of Factor Va by plasmin was studied in the presence and absence of phospholipid vesicles and calcium ions. The cleavage patterns of bovine Factor Va and its isolated subunits were analyzed using polyacrylamide gel electrophoresis, and the progress of inactivation was monitored by clotting assays and measurements of prothrombin activation using 5-dimethylaminonaphthalene-1-sulfonylarginine-N-(3-ethyl-1,5-penta nediyl)amide. In addition, the ability of prothrombin and Factor Xa to protect Factor Va from inactivation by human plasmin was examined. The data presented indicate that the cofactor Factor Va is inactivated rapidly upon its interaction with human plasmin. The rate of inactivation is significantly enhanced in the presence of phospholipid vesicles, suggesting that the inactivation process is a membrane-bound phenomenon. The isolated D component (heavy chain of factor Va) was found to be slowly degraded by human plasmin, giving rise to cleavage products different from those obtained with activated protein C and Factor Xa. However, the 48- and 30-kDa fragments obtained from human plasmin degradation of component E (light chain of Factor Va) appear to be similar to those obtained following the proteolysis of the same subunit by activated protein C and Factor Xa.

Vascular endothelial cells derived from adult bovine aorta (ABAE) treated with factor Xa and calcium were found to activate prothrombin. In contrast, nonvascular cells (human foreskin fibroblasts, bovine corneal endothelial cells, or human fetal lung cells) had either no or very little effect on prothrombin activation. In the presence of 6 X 10(5) ABAE cells, 20 ng of factor Xa converted 90 micrograms of prothrombin into 80 units of thrombin after 45 min at 37 degrees C. Exogenous factor V was not required for prothrombin activation, but thrombin generation was enhanced 2- to 4-fold by the addition of factor V (500-2,500 ng/ml). Treatment of ABAE cells with anti-bovine factor V IgG markedly inhibited prothrombin activation by factor Xa and calcium. In cells grown in serum-free medium for 3 months, the amount of factor V activity was equivalent to that found in cells grown with serum, which suggests that these cells probably synthesize factor V. Sparse ABAE cells increased prothrombin activation by factor Xa 6-fold compared to activation in confluent cells. Although previous thrombin treatment of ABAE cells did not enhance prothrombin activation, addition of dansyl arginine-4-ethyl piperidine amide markedly inhibited activation of 125I-labeled prothrombin by factor Xa, indicating that thrombin formation is necessary for optimal prothrombin activation. These data indicate that aortic endothelium may provide a physiologically important surface for activation of prothrombin as well as a mechanism for optimal formation of clots at sites of vascular injury.

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

Thrombin activated bovine factor V is composed of two polypeptide chains with molecular weights 94,000 and 80,000. The two polypeptide chains are complexed via Ca2+ions.Factor Va enhances the rate of thrombin formation by drastically increasing the Vmax of the prothrombin activation. We have undertaken a study of the interactions of factor Va with the different components of the prothrombinase complex (e.g. factor Xa and prothrombin), in order to get more insight in the mode of action of factor Va.Our kinetic experiments in solution show that the functional enzyme in the prothrombinase complex is a equimolar complex of factor Va and factor Xa. The dissociationconstant, as determined over a wide range of prothrombin concentrations, has a value of 3×10-9M.For the stimulating effect of factor Va on the prothrombin activation by factor Xa in solution, the presence of Ca2+ions is required. The dissociationconstant of the Va-Xa complex was found to be independent of the Ca2+ concentration. In order to reveal whether an interaction between Ca2+ and γ- carboxyglutamic acid residues is responsible for the observed Ca2+ requirement, identical experiments were carried out with decarboxyfactor Xa and decarboxyprothrombin. The isolated polypeptide chains of factor Va have, in the presence or absence of factor Va, no effect on the kinetic parameters of the prothrombin activation. This let us conclude that there is no interaction between factor Xa and the separate polypeptide chains of factor Va.The affinity of factor Xa for negatively charged phospholipid or stimulated bloodplatelets is greatly enhanced by the presence of factor Va. Our Kd value measured for the Xa-Va complex in combination with reported dissociationconstants of factor Xa-phospholipid and Factor Va-phospholipid complexes give a quantitative explanation for the above mentioned effect of factor Va on the binding of factor Xa to phospholipid membranes.

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)