Search

The binding of thrombin to fibrin is thought to be an important mechanism by which thrombi exhibit procoagulant activity; however, the extent to which other procoagulants are associated with thrombi has not been previously defined. This study was designed to determine whether clotting factors other than thrombin are bound to whole-blood clots and can thereby contribute to significant procoagulant activity. Clots formed in vitro from human blood exhibited minimal thrombin activity when incubated in plasma depleted of vitamin K-dependent factors by barium-citrate adsorption, as indicated by increases in the concentration of fibrinopeptide A (FPA), a marker of fibrin formation, to 72 nM after 30 min. Incubation of clots in barium-absorbed plasma repleted with 0.9 microM human prothrombin under the same conditions resulted in marked increases in the concentration of FPA (> 1,000 nM) and clotting by 30 min. The increases in FPA were attributable to activation of the added prothrombin by clot-associated Factor Xa, judging from concomitant increases in the concentration of prothrombin fragment 1.2. Similar results were obtained with thrombi induced in the axillary arteries of dogs by vascular injury and incubated with plasma in vitro. Activation of prothrombin was inhibited in a dose-dependent manner by tick anticoagulant peptide, a direct inhibitor of Factor Xa, at concentrations of 0.5-5.0 microM. Clot-associated Factor Xa activity was resistant to inhibition by anti-thrombin III, judging from the lack of inhibition of prothrombin activation during incubation of clots in plasma containing heparin pentasaccharide, an anti-thrombin III-mediated inhibitor of Factor Xa. Thus, the activity of Factor Xa appears to be an important determinant of the procoagulant activity of whole-blood clots and arterial thrombi, and is resistant to inhibition by anti-thrombin III-dependent inhibitors.

Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on Western blotting stained with an apparent intensity 5-8% that of Factor VII. This glycoprotein, tentatively called VII*, has a molecular weight 4,500 D less than Factor VII, lacks detectable Factor VII functional activity, does not bind to barium citrate, and is not recognized by a monoclonal antibody that recognizes Factor VII but not alpha-chymotrypsin-treated Factor VII. VII* was not proteolytically produced from Factor VII during in vitro coagulation or after infusion of human Factor VII into rabbits. As determined by Western blotting, the human hepatoma cell line, HepG2, cultured in the presence of vitamin K, secreted relatively greater levels of VII* in proportion to VII (75%) than that found in human plasma. Warfarin treatment of HepG2 cells decreased the quantity of VII secreted by 77%, whereas it only inhibited the secretion of VII* by 14%. Immunologic studies of the plasmas from a patient on chronic warfarin therapy and an individual given a short course of high dose warfarin therapy corroborated the in vitro synthetic studies obtained with HepG2 cells. The data are consistent with the production of VII* by posttranslational, proteolytic, modification of VII, that, at least in the HepG2 cells studied, occurs intracellularly. However, other mechanisms for the production of VII*, in particular, alternative RNA splicing of the transcript from a single gene, cannot be excluded.

Human platelets have binding sites for plasma coagulation Factor X(a) that are available only after the platelet release reaction. Platelets from 15 normal donors bound 216+/-52 (SD) molecules of Factor X(a) per platelet. The association of Factor X(a) with its platelet surface receptor results in a 300,000-fold increase in the catalytic activity of Factor X(a) in forming thrombin from prothrombin. The turnover number for platelet-bound Factor X(a) was 1,850+/-460 mol thrombin/ml per min per mol Factor X(a) in experiments with platelets from 15 normal donors. Platelets from five patients with varying degrees of Factor V deficiency were investigated to determine whether or not coagulation Factor V participates in either aspect of the Factor X(a)-platelet interaction. The binding of Factor X(a) to platelets and the accompanying increase in rate of thrombin formation were either reduced in parallel or absent in each case with values ranging from 0 to 45% of control values. The apparent affinity of Factor X(a) from Factor V-deficient patients was normal when platelet binding was detected. The supernate from thrombin-treated control platelets, which contains Factor V activity, corrected the Factor X(a) binding deficiency of the platelets from three patients tested. Immunoreactive Factor V determined with an homologous antibody corresponded to the functional Factor V activity of platelets from one patient with Factor V deficiency, suggesting that the patient's platelets have a decreased amount of normal Factor V. The ability of platelets from the patients to bind Factor X(a) and increase the rate of thrombin formation correlated with the severity of each patient's bleeding disorder better than the plasma level of Factor V. The results indicate that Factor V is required for the Factor X(a)-platelet interaction and that thrombin formation at the platelet surface is important in normal hemostasis.