Eduardo Novoa

Relationship of “new” vitamin K-dependent protein C and “old” autoprothrombin II-A

Abstract Vitamin K-dependent Protein C recently isolated by Stenflo ( J. Biol. Chem. 251, 355, 1976) was found to be related to autoprothrombin II-A by comparing amino acid analysis, molecular weight, immunochemical properties, and inhibitory activity in blood coagulation. Our purified Protein C converted to autoprothrombin II-A with purified thrombin in association with loss of a small peptide(s). Protein C or autoprothrombin II-A served as cofactors for epinephrine induced platelet aggregation in platelet rich plasma taken from dogs heavily dosed with warfarin. It is suggested that the designation Factor XIV be assigned to Protein C and Factor XIVa to autoprothrombin II-A with the following meaning: Factor XIV Thrombin Factor XIVa

Mechanisms of α-thrombin and β-thrombin-E formation: Use of ecarin for isolation of meizothrombin 1

Abstract Purified Ecarin from Echis carinatus venom converted purified bovine prethrombin 1 to meizothrombin 1. This involved only the breaking of an Arg-Ile bond. Meizothrombin 1 was purified. It was free of α-thrombin and was a single component by polyacrylamide gel electrophoresis. It had 14.5 times more esterase activity than proteolytic activity on fibrinogen. Like α-thrombin it degraded purified prothrombin to prethrombin 1. Meizothrombin 1 itself converted to α-thrombin plus prothrombin fragment 2 by autolysis, and also with purified Factor Xa. In the latter case, Factor V plus phospholipids or sodium taurocholate plus calcium ions accelerated the reaction. The amount of Factor Xa required in the presence of accessories was far less than needed for prothrombin as the substrate. The α-thrombin which formed converted to β-thrombin-E by autolysis. Purified meizothrombin 1 was dried from the frozen state without loss of activity, and the activity was stable at −20°C in 50% glycerol solution containing 0.05M phosphate buffer pH 6.5 and benzamidine 0.001M. A likely sequence for thrombin formation by Factor Xa begins with the production of meizothrombin possessing strong esterase and weak coagulant activity. Next, meizothrombin is converted to α-thrombin plus prothrombin fragment 1.2.

Preparation of bovine prethrombin 2: Use of Acutin and activation with prothrombinase or ecarin

Abstract The thrombin-like enzyme isolated from Agkistrodon acutus venom has been called Acutin. It degrades purified bovine prothrombin in several steps. A bond at Lys44-Tyr45 is broken with formation of prothrombin intermediate D. Prethrombin 1 forms and the digestion stops at the level of prethrombin 2. The latter is then easily isolated. By activating prethrombin 2 with Factor Xa alone at a 40:1 molar ratio, very little α-thrombin forms. To obtain α-thrombin from prethrombin 2 it is necessary to supply an enzyme system consisting of Factor Xa, phospholipid, calcium ions, Ac-globulin and, in addition, prothrombin fragment 1 and prothrombin fragment 2. Either fragment alone is not sufficient. It is proposed that this enzyme system be called prothrombinase. The amount of fragments 1 and 2 can be much less than the proportion that naturally occurs in prothrombin. Ecarin generated very little α-thrombin from prethrombin 2, but the yield was 100% when purified prothrombin fragment 2 was also added at a molar ratio of 1:1.

Effects of prothrombin fragments on thrombin, on thrombin formation, and separation from Ac-globulin (factor V)

Abstract Bovine prothrombin fragment 2 (profragment 2) and prethrombin 1 were found in normal oxalated bovine plasma after extensive adsorption with barium carbonate. This implies continuous physiological utilization of prothrombin. Ac-globulin-profragment 2 complex was isolated from plasma by the method of Dombrose and Seegers. This purified complex dissociated at pH 7.2 in a buffer consisting of 0.15 M NaCl in 0.15 M phosphate buffer. The two proteins were separated by ultrafiltration. The profragment 2 passed through the filter while the Ac-globulin (Factor V) was retained in pure form. Purified bovine profragment 2 enhanced esterase activity of thrombin and retarded the clotting function, thus producing a large esterase/coagulation ratio in prethrombin 1 activation mixtures. Inhibition of the thrombin-fibrinogen reaction by profragment 2 must be taken into account in some assays for prothrombin or prethrombin 1. If profragment 2 is not bound in the activation mixture of a prothrombin assay, a low thrombin titer may be recorded. Contrary to a previous conclusion reached in this laboratory, there is no enzyme with only esterase activity in the following degradation sequence: Prothrombin → Prethrombin 1 → Prethrombin 2 → Thrombin. The esterase enhancement of thrombin and its coagulation inhibition by profragment 2 were neutralized by additions of purified Ac-globulin or by specific antibodies to profragment 2. The procoagulant or anticoagulant properties of purified bovine prothrombin fragment 1 (profragment 1) were studied. For that purpose, purified bovine profragment 1 was incubated with purified Ac-globulin, purified autoprothrombin C (Factor Xa), crude “cephalin,” and calcium ions. Due to profragment 1, the procoagulant function of the mixture diminished when tested against purified prothrombin as substrate, but the procoagulant function increased when tested against purified prethrombin 1 as substrate. Thus, depending upon conditions, profragment 1 can function as a procoagulant or as an anticoagulant.

Blood coagulation as a cybernetic system: Control of autoprothrombin C (factor Xa) formation☆

Abstract The procoagulants of the intrinsic system are very powerful, but under physiological conditions seem to be weak because of their presence in functionally low concentrations. Autoprothrombin C (Factor Xa) activity generated rapidly from purified autoprothrombin III (Factor X) in a reaction mixture that contained optimum amounts of purified Factor VIII, a platelet factor 3 preparation (PF-3), a Factor IXa preparation, and 0.01 M calcium ions at pH 7.2. The initial generation of Auto-C was very rapid. Lowering the concentration of any one of the procoagulants below the optimum reduced the yield of Auto-C. In this system, Auto-C generation is governed by apparent stoichiometric reactions. At the completion of the reaction, very little Factor VIII activity remained and was apparently destroyed by Auto-C. Factor IXa generated some Auto-C without either PF-3 or Factor VIII. To meet the phospholipoprotein requirement, it was possible to substitute phosphatidylserine for PF-3, but not phosphatidylcholine or phosphatidylethanolamine. This enlarges on reports in the literature, where it is recorded that the latter two function with the protein of thromboplastin in Auto-C generation, and phosphatidylserine functions in thrombin generation. For the acceleration or initiation of blood coagulation, plasmatic factors as well as platelet materials are needed.

Immunological aspects of some vitamin K-dependent factors and preparation of depleted plasmas.

Abstract The specificity of the immune response elicited in rabbits to purified bovine prothrombin, autoprothrombin III (Factor X), Factor IX, and Protein C was studied. The immunizing proteins were homogeneous on polyacrylamide gel electrophoresis. By immunodiffusion analysis antigenic similarities were not detected between them in spite of known stretches of amino acid sequence homology. Immunoelectrophoresis of bovine plasma and each purified protein gave identical single precipitin lines with the homologous antisera. This experiment established that the antibodies prepared against each one of the purified vitamin K-dependent factors studied reacted with a single component in bovine plasma. Immunologic crossreactions were found between human, bovine, dog, rat and chicken prothrombin, Protein C, Factor X, and Factor IX. They were not species specific. Purified bovine prothrombin has at least four antigenic determinant sites. Incubation of the purified proteins with suitable proteolytic enzymes yielded fragments and intermediate products which were purified and tested for their potential capacity to react with antibodies in the native antisera. In immunoelectrophoresis tests both prothrombin fragments 1 and 2 and the intermediate product prethrombin 1 gave immunoprecipitates which were separately identified by their differing mobilities, while the enzyme portion, thrombin, did not react with antithrombin serum. In addition to prothrombin, bovine plasma contained prethrombin 1, prothrombin fragments 1 and 2, as well as prethrombin 2 and/or thrombin. Autoprothrombin III m (Factor Xβ) and autoprothrombin C (Factor Xa) crossreacted with anti-autoprothrombin III and the active form of Protein C. Gamma globulins of antisera to prothrombin, to autoprothrombin III, to Factor IX, and to Protein C were fractionated by using saturation ammonium sulfate solution and coupled to activated Agarose A-15m beads. The insolubilized antibodies were used to absorb the antigen from the plasma. Each depleted plasma served as test material for a specific clotting factor. The prolonged prothrombin time of the prothrombin depleted plasma was normalized by additions of purified prothrombin, but not by purified prethrombin 1. The prothrombin time of autoprothrombin III depleted plasma was shortened by fresh plasma, purified autoprothrombin III, and by purified autoprothrombin III m (Factor Xβ). In the case of Factor IX depleted plasma, the partial thromboplastin time was used. The depleted bovine plasmas are suitable for assay purposes. They serve the same role as the corresponding deficient plasma in bioassay coagulation tests.

Bovine antithrombin-III: Miscellaneous observations

Abstract Bovine antithrombin-III was purified by two procedures. With the ultracentrifuge we found S 20,w = 4.18 − 0.015X. It was found to have an NH 2 -terminal histidine residue, neutral sugars, sialic acid and disulfide bonds. It formed a complex with purified bovine thrombin in a 1:1 molar proportion with residual quantities of either component remaining. It formed a 1:1 molar complex with purified Factor Xa. In the ultracentrifuge this complex sedimented as a single component with S 20,w = 4.65 − 0.034X. Antibodies were used to deplete plasma of antithrombin-III. The depleted plasma progressively inactivated purified thrombin.