Lowell E. McCoy

Preparation and properties of thrombin.

Abstract Purified bovine prothrombin preparations were converted to thrombin in 25% sodium citrate solution, and the thrombin was isolated by chromatography on Amberlite IRC-50 according to previously published procedures. In the ultracentrifuge So20 w = 3.7. Following a second chromatography on Amberlite IRC-50, 75 amino acid residues, predominately acidic, were deleted and 183 residues remained with the thrombin. The isoelectric point shifted from 5.75 to 6.2, and the specific activity increased from 4,200 to 8,230 U/mg protein. Acetylation also removed a postulated acidic moiety from the 3.7S thrombin. Ultracentrifugation of the twice chromatographed thrombin gave So20,w = 3.2. During centrifugation molecular association and aggregation occurred. The 3.2S thrombin contained 1.7% sialic acid, 5.02% orcinolreactive carbohydrate, 0.69% glucosamine, and 0.46% galactosamine. Molecular weight for only amino acids was 21,100, and with sialic acid and carbohydrate added it was 22,900. By thin-layer gel filtration we found 22,500, and with the same technique 33,000 for 3.7S thrombin and 40,000 for prethrombin. With TAMe as substrate Km for 3.7S thrombin = 2.97 × 10−4; 3.2S thrombin = 9.5 × 10−5, and for acetylated thrombin = 4.85 × 10−4. It was concluded that thrombin has two polypeptide chains. The N-termini are: threonine and isoleucine.

Purification and some properties of autoprothrombin II-A: An anticoagulant perhaps also related to fibrinolysis☆

Abstract Methods were developed for the assay and isolation of autoprothrombin II-A. The purified protein retarded blood coagulation in vitro as well as in vivo and induced fibrinolysis. It is tentatively concluded that the protein originated from prothrombin and possessed inhibitor-activator qualities respectively as anticoagulant and fibrinolytic agent. To obtain Auto II-A, concentrated prothrombin complex was digested with purified thrombin. Chromatography on a DEAE-cellulose column separated prethrombin, Auto II-A, and autoprothrombin III (Factor X). The inhibitor-activator was then purified by using a Sephadex G-100 or G-100 superfine column. A single component was indicated by polyacrylamide gel electrophoresis and by ultracentrifugation. The equation for S 20,w 0 was 3.9 + 0.0114X. Isoelectric point = pH 3.9. It is a competitive inhibitor of autoprothrombin C (Factor X a ). N-terminal amino acids were Ile and Gly. An amino acid analysis was compared with a new amino acid analysis of purified prothrombin. The latter had 518 amino acid residues per mole. The protein contained sialic acid, neutral sugars, and amino sugars. Serum from rabbits immunized with purified prothrombin crossreacted with purified Auto II-A. A working theory is proposed as follows: Thrombin functions in a negative feedback system which retards thrombin formation due to the production of prethrombin and Auto II-A. Platelet factor 3 also produced prethrombin and Auto II-A. This feedback system could be the molecular basis of latent coagulation and fibrinolysis. In accelerated coagulation, the latent coagulation sequences are by-passed with massive formation of fibrin.

Isolation and some properties of thrombin-E and other prothrombin derivatives

Abstract Multiple forms of thrombin zymogen occur as prothombin complex, prothrombin, abnormal prothrombin, prethrombin, and prethrombin-E. Prothrombin less PR fragment represents prethrombin and is the stage beyond which degradation of prothrombin does not readily go with thrombin. By removing O fragment from prethrombin, esterase activity develops, due to a structure called prethrombin-E. This enzyme is a single chain structure with molecular weight, amino acid composition and carbohydrate content the same as thrombin. By breaking an Arg-Ile bond in prethrombin-E with autoprothrombin C, thrombin forms and consists of the A chain held to the B chain by a disulfide bond. It has esterase and proteolytic activity, and basically classical thrombin occurs only in this one form. Commonly, thrombin preparations consist of thrombin and thrombin degraded to thrombin-E and B1 chain. During autolysis at 4° C and pH 8.0, proteolytic activity of thrombin is lost while the specific esterase activity increases. The B1 portion of B chain splits off at an Arg-Lys bond. It precipitates in pure form leaving in solution thrombin-E which has B2 chain attached to A chain by a disulfide bond, and has only esterase activity. Breaking the disulfide bond of thrombin-E enables the isolation of A and B2 chains. Either thrombin or autoprothrombin C can remove PR fragment from prothrombin. R fragment was also isolated and probably contains all the carbohydrate of prothrombin which is not associated with B1 chain of thrombin. Thrombin-E is free of carbohydrate. O fragment retarded fibrin formation by thrombin. It enhanced the esterase activity of thrombin. An unidentified procoagulant, probably a form of autoprothrombin C, was closely associated with O fragment, but was removed. The prothrombin activation sequence described by Seegers and Landaburu in 1957 has been confirmed; namely, esterase activity → esterase + proteolytic activity → esterase activity. The respective associated structures are prethrombin-E → thrombin → thrombin-E; and in these, the condition of B1 chain is as follows: bound, free at NH 2 -terminal end, and absent. In addition, an antecedent in the form of prethrombin is easily obtained as a degradation product of prothrombin. The fragments align in prothrombin as follows: P + R + O + A chain + B1 chain + B2 chain. Digestion of prothrombin with thrombin stops with PR removal which is the prethrombin stage, but continues if prothrombin is first denatured. Likewise, autolysis of thrombin stops at the thrombin-E stage (A chain + B2 chain), but if thrombin-E is denatured, thrombin can degrade it further.

Tissue extract thromboplastin: Quantitation, fractionation and characterization of protein components

Abstract Bovine brain and lung tissue thromboplastins were extracted with absolute ethanol into an ethanol soluble lipid fraction and an insoluble protein fraction. The lipid fraction functioned as a partial thromboplastin, while the protein fraction was devoid of activity. The protein fraction was further purified by deoxycholate extraction and column chromatography on Bio-Gel A-1.5 m in the presence of deoxycholate. Through this procedure, residual lipids in the protein fraction were removed. The sedimentation coefficient in the presence of 0.1% SDS was 2.9. The molecular weight as determined by SDS gel electrophoresis was 56,000. Protein aggregates with molecular weights greater than 1.5 million occurred in the absence of the detergents or bile salts. The purified protein, after addition of phospholipid, regained clotting activity. Recombination of the protein and the lipid fractions was achieved in ethanol. Recombination was also achieved in imidazole buffered saline at pH 7.2 in the presence of 0.025% deoxycholate. The recombined material had full thromboplastic activity. The protein components isolated from brain and lung tissue had similar biochemical and biological characteristics. Combining lung lipids with brain protein and brain lipids with lung protein gave essentially the same results observed when the recombined protein and lipids were derived from the same tissue source.

Preparation of bovine prothrombin complex and purified prothrombin

Abstract A simplified procedure is described for the large-scale purification of bovine prothrombin complex. The protein was adsorbed on barium carbonate, eluted with 3.5% sodium citrate solution, and fractionated with ammonium sulfate. Additionally, prothrombin was separated from autoprothrombin III (Factor X) and further purified by DEAE-cellulose chromatography. Prothrombin in the latter preparation was then obtained in pure form by two sequential filtrations through a Sephadex G-100 column. The specific activity was 3800 u/mg protein. The N-terminal amino acid was alanine. By ultracentrifugation, a single component was indicated. The equation for S 20,w o was 4.75 + 0.0196X. Eight mg% is a revised figure for the concentration of prothrombin in normal bovine plasma.

Further observations on the purification and properties of autoprothrombin III (Factor X)

Abstract Autoprothrombin III (Factor X) was separated from purified bovine prothrombin complex by chromatography on DEAE-cellulose and filtration through a Sephadex G-100 column. It had the same amino acid composition and approximately the same sedimentation characteristics in the ultracentrifuge as the first product obtained in this laboratory. The sedimentation constant was 3.58S. The product was studied by disc gel electrophoresis over a wide pH range. It was free of autoprothrombin C (Factor Xa), was stable, and had no tendency to activate spontaneously. On the basis of 27 Sephadex filtration experiments, the molecular weight was 81,900 ± 2,770. For autoprothrombin C, it was 53,500; by another method, it was 58,000. The N-terminal amino acids were alanine and serine, while those for autoprothrombin C were glycine and serine. Peptide maps for the zymogen and enzyme were similar except there were fewer peptides on the map of the latter. Based on a specific activity of 1,200 u/mg and a plasma concentration of 30 u/ml, the concentration of autoprothrombin III in bovine plasma most likely ranges from 2–3 mg%.

Component analysis of the fast photoelectric signal from model bacteriorhodopsin membranes: Part II. Effect of fluorescamine treatment

Abstract This report investigates the effect of fluorescamine treatment of bacteriorhodopsin on the fast photoelectric signal. Such a treatment was performed either before or after membrane reconstitution. The data so obtained are subject to component analysis as outlined in the first of the two companion papers. The analysis shows that the fluorescamine treatment inhibits the B2 component but leaves the B1 component intact. The result justifies our protocol of separating the signal into the B1 and the B2 components. The result is also consistent with the assignment of B1 to a molecular process located in the buried chromophore-binding region and the assignment of B2 to an interfacial proton binding process taking place at the cytoplasmic surface of the membrane.

The coagulation of blood: Preliminary survey of thrombin and autoprothrombin zymogen structure☆

Abstract Numerous facts are consistent with the conclusion that bovine thrombin zymogen is a dimer of thrombin and is easily dissociated from autoprothrombin III (Stuart factor, F-X, autoprothrombin, prothrombokinase). The latter comprises about 4.4% of the total protein in our preparation. When thrombin forms, at least two peptides are removed. Prethrombin has one N-terminal amino acid in common with thrombin. Autoprothrombin C has an amino acid composition and specific activity similar to autoprothrombin III, but has only half the molecular weight. A likely possibility is that autoprothrombin III is a dimer of autoprothrombin C. Alternatively it could represent one mole of autoprothrombin C and one of F-VII.

Phospholipid requirements of tissue thromboplastin in blood coagulation

Abstract The lipid portion of tissue thromboplastins from bovine brain and lung, analyzed by silica gel thin-layer chromatography, consisted primarily of phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin with trace amounts of phosphatidylserine, phosphatidylinositol, and lysophosphatidylcholine. The protein portion was purified by a procedure that involved organic solvent extraction, deoxycholate solubilization, and column chromatography on Bio-Gel A-1.5 m. Purified phospholipid was recombined with the purified thromboplastin protein and its activity in the formation of autoprothrombin C (Factor Xa) from autoprothrombin III (Factor X) was determined. Phosphatidylethanolamine and phosphatidylcholine were active in this test. Phosphatidylinositol and phosphatidylserine were inactive in autoprothrombin C generation but were active in thrombin generation.

Laboratory analysis of clotting - factor components concentrated for therapeutic use

Abstract A laboratory study was made of Factor IX preparations used or produced for therapeutic use. They contained large amounts of prothrombin and autoprothrombin III (F-X). In solution, ready for use, prothrombin and autoprothrombin III remained at the original concentration for about 3 weeks at 4° C without developing thrombin or autoprothrombin C (F-X a ) activity. One of the products was dissolved in 2.5 M glycine solution at 4° C. Autoprothrombin C activity developed and all the prothrombin converted to thrombin over a period of 70 days. The thrombin activity generated after the prothrombin was modified. The Factor IX activity in these preparations was easily measured by the platelet cofactor assay. It was not as stable as the prothrombin or autoprothrombin III. The same conclusion about stability was reached from prothrombin utilization experiments with plasma from a hemophilia B subject. The Factor IX preparations which were studied are suitable reagents for the assay of platelet factor 3. Purified bovine prothrombin complex prepared in this laboratory contained very little platelet cofactor (Factor VIII) activity nor did it contain platelet cofactor II (Factor IX) activity. For this reason, the purified bovine prothrombin complex is a satisfactory reagent for the assay of Factor VIII concentrates. Platelet cofactors have a synergistic procoagulant effect with platelet factor 3 in the formation of autoprothrombin C from its precursor.