HD1, a thrombin- and prothrombin-binding DNA aptamer, inhibits thrombin generation by attenuating prothrombin activation and thrombin feedback reactions.

HD1, a DNA aptamer, binds exosite 1 on thrombin and blocks its clotting activity. Because HD1 also binds prothrombin and inhibits its activation by prothrombinase, we hypothesised that HD1 would be a more potent inhibitor of coagulation than other exosite 1-directed ligands, such as Hir(54-65)(SO(3)(-)). Supporting this concept, the effect of HD1 on the prothrombin time and activated partial thromboplastin time was two-fold greater than that of Hir(54-65)(SO(3)(-)) even though both agents inhibited thrombin-mediated factor (F) V and FVIII activation to a similar extent. In thrombin generation assays, HD1 (a) delayed the lag time, (b) reduced peak thrombin concentration, and (c) decreased endogenous thrombin potential to a greater extent than Hir54-65(SO(3)(-)). To eliminate thrombin feedback, studies were repeated in FV- and/or FVIII-deficient plasma supplemented with FVa and/or FVIIIa. Only HD1 prolonged the lag time in FV- and FVIII-deficient plasma supplemented with FVa and FVIIIa. In contrast, HD1 and Hir54-65(SO(3)(-)) inhibited the lag time in FVIII-deficient plasma supplemented with FVIIIa and in normal plasma. The more potent anticoagulant properties of HD1, therefore, reflect its capacity to attenuate FV activation by thrombin and inhibit prothrombinase assembly. These findings identify prothrombin as a potential target for new anticoagulants.

Identification of surface epitopes of human coagulation factor Va that are important for interaction with activated protein C and heparin.

Inactivation of factor Va (FVa) by activated protein C (APC) is a key reaction in the down-regulation of thrombin formation. FVa inactivation by APC is correlated with a loss of FXa cofactor activity as a result of three proteolytic cleavages in the FVa heavy chain at Arg306, Arg506, and Arg679. Recently, we have shown that heparin specifically inhibits the APC-mediated cleavage at Arg506 and stimulates cleavage at Arg306. Three-dimensional molecular models of APC docked at the Arg306 and Arg506 cleavage sites in FVa have identified several FVa amino acids that may be important for FVa inactivation by APC in the absence and presence of heparin. Mutagenesis of Lys320, Arg321, and Arg400 to Ala resulted in an increased inactivation rate by APC at Arg306, which indicates the importance of these residues in the FVa-APC interaction. No heparin-mediated stimulation of Arg306 cleavage was observed for these mutants, and stimulation by protein S was similar to that of wild type FVa. With this, we have now demonstrated that a cluster of basic residues in FVa comprising Lys320, Arg321, and Arg400 is required for the heparin-mediated stimulation of cleavage at Arg306 by APC. Furthermore, mutations that were introduced near the Arg506 cleavage site had a significant but modest effect on the rate of APC-catalyzed FVa inactivation, suggesting an extended interaction surface between the FVa Arg506 site and APC.

Comparative analysis of prothrombin activators from the venom of Australian elapids.

A key component of the venom of many Australian snakes belonging to the elapid family is a toxin that is structurally and functionally similar to that of the mammalian prothrombinase complex. In mammals, this complex is responsible for the cleavage of prothrombin to thrombin and is composed of factor Xa in association with its cofactors calcium, phospholipids, and factor Va. The snake prothrombin activators have been classified on the basis of their requirement for cofactors for activity. The two major subgroups described in Australian elapid snakes, groups C and D, are differentiated by their requirement for mammalian coagulation factor Va. In this study, we describe the cloning, characterization, and comparative analysis of the factor X- and factor V-like components of the prothrombin activators from the venom glands of snakes possessing either group C or D prothrombin activators. The overall domain arrangement in these proteins was highly conserved between all elapids and with the corresponding mammalian clotting factors. The deduced protein sequence for the factor X-like protease precursor, identified in elapids containing either group C or D prothrombin activators, demonstrated a remarkable degree of relatedness to each other (80%-97%). The factor V-like component of the prothrombin activator, present only in snakes containing group C complexes, also showed a very high degree of homology (96%-98%). Expression of both the factor X- and factor V-like proteins determined by immunoblotting provided an additional means of separating these two groups at the molecular level. The molecular phylogenetic analysis described here represents a new approach for distinguishing group C and D snake prothrombin activators and correlates well with previous classifications.

Isolation and study of an acquired inhibitor of human coagulation factor V.

A coagulation Factor V inhibitor developed in a man 75 yr of age in association with an anaplastic malignancy and drug treatment (including the aminoglycoside antibiotic, gentamicin). The patient did not bleed abnormally, despite both surgical challenge and plasma Factor V activity of less than 1%. The inhibited plasma had grossly prolonged prothrombin and activated partial thromboplastin times, but a normal thrombin time. Mixing studies indicated progressive coagulation inhibition with normal plasma, but not with Factor V-deficient plasma, and reversal of coagulation inhibition by the addition of bovine Factor V to the patient's plasma. 1 ml of patient plasma inhibited the Factor V activity of 90 ml of normal human plasma. The inhibitor was isolated by sequential affinity chromatography on protein A-Sepharose and Factor V-Sepharose. The IgG isolate markedly inhibits the activity of prothrombinase assembled from purified Factors Xa and Va, calcium ion, and phospholipid vesicles, and partially inhibits prothrombinase assembled from purified Factor Xa, calcium ion, and normal platelets. The Factor V of platelets, however, appears relatively inaccessible to the antibody, inasmuch as platelets isolated from whole blood supplemented for 8 h with the antibody functioned normally with respect to platelet Factor V-mediated prothrombinase function. The absence of obvious hemorrhagic difficulties in the patient, the total inhibition of plasma Factor V by the inhibitor, and the apparent inaccessibility of platelet Factor V to the inhibitor specifically implicate platelet Factor V in the maintenance of hemostasis.

Protein S is essential for the activated protein C-catalyzed inactivation of platelet-associated factor Va.

The prothrombin-converting activity of Factor Xa was enhanced by thrombin-stimulated Factor V-deficient platelets and supplementary extraneous Factor Va, and also by thrombin-stimulated normal human platelets. Both extraneous Factor Va and intra-platelet Factor Va were equally inactivated by a gamma-carboxyglutamic acid-containing plasma protease, activated protein C. However, a relatively larger amount of activated protein C was required for efficient inactivation of platelet-associated Factor Va as compared with the amount of activated protein C needed for inactivation of phospholipid vesicle-associated Factor Va. Protein S, another gamma-carboxyglutamic acid-containing plasma protein, increased the rate of the inactivation of platelet-associated Factor Va about 25-fold. This stimulating effect was observed only slightly with the thrombin-modified protein S. Thus, it was concluded that protein S is essential for the process of inactivation of platelet-associated Factor Va by activated protein C.

Protein C.

The protein C anticoagulant pathway provides many new insights into control mechanisms for regulating coagulation. The observation that protein C deficiency is associated with thrombotic tendencies in the heterozygote (106-109) and early, lethal thrombosis in the homozygote (110, 111) points to the importance of the system as a major regulatory pathway. The complexity of the system has only recently begun to emerge. Thrombin activation of protein C at the endothelial cell surface requires not only the synthesis of thrombomodulin but the coupling of the receptor to a protein C binding site. It is reasonable to assume that an inherited or acquired deficiency in thrombomodulin might lead to thrombotic tendencies. This aspect of the system may explain, in part, the association between vascular disease and thrombosis. Once activated, protein C has an almost total dependence on protein S to express anticoagulant activity. (98) This suggests that deficiencies of protein S may also be associated with thrombotic tendencies. Protein S offers an additional intriguing property. Protein S, a regulatory protein of the coagulation system, is found both free and associated with C4BP, a regulatory protein of the complement system. The high affinity, very stable interaction between these components (85) suggests that the interaction is likely to be involved in regulation. (89) The importance of the interaction remains to be demonstrated, but clearly this is a potential direct link between major control proteins of the coagulation and complement system. Clinical studies suggest that protein C and/or thrombomodulin might be effective therapeutically. Certainly, protein C supplementation during the onset of oral anticoagulant therapy would be expected to circumvent the transient rapid decrease in protein C levels that may influence the early effectiveness of oral anticoagulants. (119) In addition to the systems clinical importance, protein C, its activation, and its function offer a variety of intriguing biochemical problems. For instance, how does thrombomodulin alter the specificity of thrombin? What is the protein C binding site on the cell surface, and what role does Factor Va or its degradation products play in the formation and regulation of this site? How does protein S facilitate activated protein C anticoagulant activity and what roles do membrane surfaces play in this system? What role does beta-hydroxyaspartic acid play in protein C activation and function? How does activated protein C influence fibrinolytic activity? The answers to these questions will undoubtedly add to our understanding of the fundamental mechanisms involved in regulating blood coagulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Pumpkin seed inhibitor of human factor XIIa (activated Hageman factor) and bovine trypsin.

A strong inhibitor of human Hageman factor fragment (HFf, beta-factor XIIa) and bovine trypsin was isolated from pumpkin (Cucurbita maxima) seed extracts by acetone fractionation, by chromatography on columns of diethyl-aminoethylcellulose and carboxylmethyl-Sephadex C-25, and by Sephadex G-50 gel filtration. Pumpkin seed Hageman factor inhibitor (PHFI) is unusual in its lack of inhibition of several other serine proteinases tested--human plasma, human urinary, and porcine pancreatic kallikreins, human alpha-thrombin, and bovine alpha-chymotrypsin. Human plasmin and bovine factor Xa are only weakly inhibited. PHFI also inhibits the HFf-dependent activation of plasma prekallikrein and clotting of plasma. Other properties of PHFI are a pI of 8.3, 29 amino acid residues, amino-terminal arginine, carboxyl-terminal glycine, 3 cystine residues, undetectable sulfhydryl groups and carbohydrate, and arginine at the reactive site. The minimum molecular weight of PHFI is 3268 by amino acid analysis. PHFI may be the smallest protein inhibitor of trypsin known.