Activation Of Human Prothrombin Research Articles

Activation of human prothrombin by a procoagulant fraction from the venom of Echis carinatus. Identification of a high molecular weight intermediate with thrombin activity.

In the presence of a procoagulant fraction (Echis carinatus procoagulant) isolated from the venom of the saw-scaled viper Echis carinatus sochureki, purified human prothrombin (P1) is completely converted to thrombin. The first step is the removal of an NH2-terminal peptide (F1) representing approximately one-third of the prothrombin molecule. The remaining peptide (P2) is then cleaved by the action of E.c. procoagulant to yield a two-chain, disulfide-bridged protein (P'2) which has the same molecular weight as P2. P'2 has enzymic (thrombin) activity, as evidence by incorporation of radiolabeled diisopropylphosphate into its heavy chain (TB), hydrolysis of p-toluenesulfonylarginine methyl ester, and clotting of fibrinogen. Relative to thrombin, its esterolytic activity greatly exceeds its clot-promoting activity. Examination of the polypeptide chains obtained by reducing P'2 has shown that its larger chain (TB) is indistinguishable from the heavy chain of thrombin. Its other chain (F2TA) consists of the light chain (TA) of thrombin bound by peptide linkage to the protion of the prothrombin molecule which had been adjacent to F1. Removal of this portion (F2) is catalyzed by thrombin (and, evidently, by P'2), but not by the E.c. procoagulant. When F2 is removed from P'2, the remaining two-chian protein is indistinguishable from thrombin by any of the criteria applied--molecular weight, subunit chain composition, or enzymic activity. Polyacrylamide gel electrophoresis was carried out in sodium dodecyl sulfate before and after disulfide reduction of samples generated in the presence and in the absence of diisopropylphosphorofluoridate, which inhibits thrombin but not the E.c. procoagulant. Such experiments showed that thrombin (and probably P'2), as well as E.c. procoagulant, catalyzes the release of F1. Furthermore, thrombin brings about the cleavage of F1 to yield a two-chain, disulfidebridged protein (F'1). These observations, particularly those made in the course of characterizine P'2, have led to the conclusion that cleavage of the peptide bond connecting the TA and TB portions of the prothrombin molecule (or its derivatives) produces a serine active center and, hence, a molecule possessing thrombin activity. This cleavage is catalyzed by the E.c. procoagulant but not by thrombon itself.

Activation of human prothrombin by highly purified human factors V and X-a in presence of human antithrombin.

In this communication we describe the first method for isolating human Factor V. The final product contains no other coagulation components as judged by functional assays and is physically homogeneous as shown by isofocusing gel electrophoresis. In addition, we present a means for obtaining intrinsically activated human Factor X-a. This preparation is usually homogeneous as judged by isofocusing gel electrophoresis. However, on occasion, an additional minor electrophoretic species with Factor X-a activity is observed. Furthermore, we describe the use of isoelectric focusing in sucrose density gradients to free human prothrombin from contamination by coagulation factors and other components. These homogeneous human proteins are employed to examine the conversion of prothrombin to thrombin in the presence and absence of human antithrombin. The latter component is responsible for virtually all of the plasm's capacity to neutralize Factor X-a and thrombin. In the absence of antithrombin, prothrombin (67,800) is converted to the precursor P-2 (51,600) and the fragment F-a (19,500). Subsquently, P-2 is cleaved to form the precursor P-3 (37,000), and the fragment F-b (11,500). Finally, P3 IS proteolyzed to form the heavy chain T-h (29,500) and the light chain T-L (6,500) of active thrombin. In the presence of antithrombin, an additional prothrombin conversion pathway is observed in which the zymogen is directly cleaved to form P-3 and F-A + B (30,000) prior to thrombin generation. Trace amounts of free thrombin remain uninhibited by antithrombin and could bias the zymogen activation pathway. Hirudin is known to neutralized thrombin instantaneoulsly. We demonstrate that the purified leech protein also binds to P-3 and prevents thrombin formation. When hirudin is added to activation mixtures at concentrations sufficient to virtually suppress P-3 conversion to thrombin, molecular species from both activation pathways are observed. Thus two human prothrombin conversion sequences appear to be initiated by Factor X-3 and may be of physiological significance.

Cleavage of arginyl bonds of human prothrombin during activation by intrinsic prothrombin activator

Abstract As determined by carboxypeptidase digestion, complete activation of human prothrombin by intrinsic prothrombin activator exposed only C-terminal arginine, amounting to 2.9 moles per mole of prothrombin and indicating cleavage of three arginyl bonds during activation.

Isolation and characterization of multiple forms of human thrombin

Established procedures for the isolation of human prothrombin (1) and thrombin (2) have been modified so that large quantities of human thrombin could be isolated in a rapid, yet facile, manner. Thrombins derived from the activation of human prothrombin with thromboplastin or venom from the Australian Taipan (Oxyuranus Scutellatus Scutellatus) have been isolated and characterized with respect to their molecular weights and isoelectric points. Multiple active human thrombin components were found. Molecular weights of proteins found in these preparations conformed to the molecular weights reported for bovine thrombins. Activity studies with the synthetic substrate Benzoyl-Phe-Val-Arg-p-nitro-anilide and inhibitors fibrinopeptides A and B, have been performed. The majority of coagulant and esterase activity of the human thrombin preparations was associated with a protein component with an isoelectric point of 7,1. Coagulant, but no esterase activity, was found at isoelectric point 6, 7. Additional minor peaks of both activities had isoelectric points of 5,6 and 6,2, which are the sole isoelectric points reported previously for bovine thrombin. Results of the characterization studies show that thromboplastin and taipan venom activation produce very similar thrombins.

Two Forms of Human Thrombin: ISOLATION AND CHARACTERIZATION

Abstract When preparations of human prothrombin are rapidly activated to thrombin (EC 3.4.4.13) in the presence of a partially purified enzyme from Taipan snake venom (Oxyuranus scutellatus scutellatus), the thrombin activity isolated from the activation mixture by ion exchange chromatography is associated with a single molecule of molecular weight of approximately 39,000. The subunit structure of this blood-clotting enzyme was examined by sodium dodecyl sulfate electrophoresis and was found to consist of two polypeptide chains connected by disulfide bonds, of molecular weight of approximately 32,000 and 7,000. NH2-terminal amino acid analysis by the 5-dimethylaminonaphthalene-1-sulfonyl procedure indicated that the larger chain contained NH2-terminal isoleucine while the smaller chain contained NH2-terminal threonine. With time, the enzyme self-digests itself without change in the clotting activity of the system to produce a smaller thrombin molecule of molecular weight of approximately 26,000, which consists of a 19,000 and a 7,000 molecular weight peptide with NH2-terminal isoleucine and threonine, respectively. The other peptide, which is inactive against fibrinogen, is split from the larger chain of the parent molecule presumably because of autolysis. It was found to have a molecular weight of approximately 11,000 and to contain a single NH2-terminal glycine. These structural changes were confirmed by isolation of the various thrombin peptides by gel filtration in 10% acetic acid. Slow and incomplete activation of human prothrombin in the presence of concentrated citrate solution or activation with a human Factor Xa preparation results in the isolation of a mixture of both thrombin molecules from the activation systems, including the inactive thrombin peptide. These molecules showed the same sequence of structural changes due to autolysis without a corresponding change in clotting activity as did the venom-derived enzyme. Thrombin preparations containing a mixture of the larger and smaller molecules, particularly those in which the smaller molecule predominates, have a significantly higher clotting activity than preparations containing only the larger molecule. No evidence was obtained for the existence of a three-chain polypeptide human thrombin molecule as has been reported by others for the bovine enzyme. Since preliminary observations seemed to indicate that under our conditions of study bovine and horse thrombin preparations contain similar species of molecules as those described above for human thrombin, methods of preparation of prothrombin or activation (or both) employed by others may account for this apparent discrepancy. These studies suggest that the thrombin derived from human, bovine, and possibly horse prothrombin appear quite similar in size, NH2-terminal amino acids, and subunit structure, their relative composition in a prothrombin activation mixture being dependent only upon the rate or yield (or both) of thrombin generated from its zymogen, rather than the source of activators.

Esterase and Clotting Activities Derived from Citrate Activation of Human Prothrombin

Abstract When human prothrombin is activated in 25% (weight per volume) sodium citrate, the apparent clotting and esterase activities of thrombin develop at equal rates and reach maximum levels in about 16 hours. Following this, the esterase activity remains stable, but over 90% of the fibrinogen-clotting activity disappears from the system during the next 500 hours. Chromatography of the 16-hour system yields a single protein peak containing 40% of the protein and all of the clotting and esterase activity of the activation system. Chromatography of the 500-hour system yields two protein peaks, the major one having essentially only esterase activity and a minor one which has principally clotting activity. This results in almost a 2-fold purification of the esterase fraction over that obtained by chromatography of the 16-hour system. Comparisons were made between the preparations having both clotting and esterase activity and those having only esterase or clotting activity. With respect to the elution volume on gel filtration, electrophoresis, and sedimentation properties, no differences were observed. All preparations behaved as essentially single components having an estimated molecular weight of 30,000 g, with no evidence of dissociation to enzymatically active smaller molecules. Titration experiments with phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate showed a striking difference between the preparations, however. In addition, the clotting preparation had a profound affect on the citrate activation of prothrombin, while the esterase preparation did not. The evidence thus obtained indicates that thrombin is similar to certain other estero-proteolytic enzyme systems in which the molecule can be modified so as to alter its relative activity toward ester and protein substrates. This is discussed in relation to various interpretations concerning the nature of the active center on the thrombin molecule, as well as the implications these findings may have on the factors believed necessary for the inception of blood coagulation.

Chromatographic analysis of the activation of human prothrombin with human thrombokinase.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTChromatographic Analysis of the Activation of Human Prothrombin with Human Thrombokinase*D. L. Aronson and D. MénachéCite this: Biochemistry 1966, 5, 8, 2635–2640Publication Date (Print):August 1, 1966Publication History Published online1 May 2002Published inissue 1 August 1966https://doi.org/10.1021/bi00872a022RIGHTS & PERMISSIONSArticle Views30Altmetric-Citations51LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (459 KB) Get e-Alerts Get e-Alerts