Walter H. Seegers

Studies on the Antithrombin and Heparin Co-factor Activities of a Fraction Adsorbed from Plasma by Aluminum Hydroxide

A chemical method has been devised for the quantitative determination of plasma antithrombin. A concentrate of antithrombin has been prepared from plasma. It also contains heparin cofactor activity. The two distinct activities could not be separated and may quite possibly be the properties of a single substance. When the concentrate is combined with heparin the capacity of the concentrate to destroy thrombin is not increased but there is interference with the thrombin fibrinogen interaction. In a mixture of the concentrate and thrombin there is simultaneous destruction of antithrombin, and thrombin activities. Small amounts of the concentrate significantly prolong the clotting time of blood.

The amino acid composition of bovine prothrombin

Abstract The full amino acid composition of bovine prothrombin has been measured by using ion-exchange chromatography. In addition, other methods were used for the estimation of tyrosine, tryptophan, phenylalanine, and amide ammonia. Cysteine-cystine were estimated as cysteic acid. Eighteen amino acids and hexosamine are represented, glutamic acid, aspartic acid, and arginine being present in highest weight percentage. There is no resemblance between the known amino acid composition of serum albumin and that of prothrombin, though the two proteins are closely related with regard to their physicochemical characteristics in many respects.

Differentiation of Certain Platelet Factors Related to Blood Coagulation

Study of the platelets from the viewpoint of the blood coagulation mechanisms seems likely to be helpful for increasing our knowledge of thrombosis, and bleeding tendencies in thrombasthenias and thrombopathias. A logical approach to their study is the differentiation of their properties and where possible to ascribe those properties to individual components. Platelet factors 1, 3 and 4 are considered from that viewpoint. The first accelerates the formation of thrombin in a manner analogous to serum Ac-globulin. Factor 3 is required for the development of threone activity, and factor 4 has antiheparin properties.

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.

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

Autoprothrombin II-A: Thrombin removal and mechanism of induction of fibrinolysis☆

Abstract Traces of thrombin were removed from purified preparations of autoprothrombin II-A. This thrombin-free protein, when infused intravenously into a dog, retarded blood coagulation, stopped autoprothrombin III (Factor X) utilization, and induced plasmin activity. There were no associated changes in heart rate, respiratory rate or blood pressure. In experiments with purified components, it was found that autoprothrombin II-A possessed only weak ability to directly activate either bovine or canine plasminogen to plasmin, but did depress the function of plasmin inhibitors. Autoprothrombin II-A thus induced fibrinolysis by an indirect mechanism. There is clearly a pathway from the coagulation system to fibrinolysis. Autoprothrombin II-A retarded the activity of soybean trypsin inhibitor.

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.

Isolation and some properties of prethrombin and autoprothrombin III

Abstract Purified bovine prothrombin preparations were dissociated by digestion with thrombin. The reaction mixture was separated to obtain an inhibitor, the precursor of autoprothrombin C called autoprothrombin III, and the precursor of thrombin called prethrombin. Prethrombin was purified and found to have an isoelectric point at pH 5.5 and in the ultracentrifuge s020,w = 3.9. It contained 75 more amino acid residues than purified thrombin. Autoprothrombin III was also purified. The observed isoelectric point was pH 4.7, and s020,w = 3.4. After the separation and purification of prethrombin and autoprothrombin III, it was possible to study the activation requirements of each one independently of the other. Autoprothrombin III readily converted to autoprothrombin C in the presence of tissue thromboplastin and calcium ions, but other suitable conditions for activation were also found. The formation of autoprothrombin C was observed to occur slowly in the presence of calcium ions and platelet cofactor I. It is therefore concluded that rapid clotting of blood with tissue extracts as compared with platelets plus plasma is due to the vastly greater quantity of autoprothrombin C produced with tissue extracts than with platelets plus plasma. The conversion of prethrombin to thrombin required only autoprothrombin C; however, the reaction was accelerated by the simultaneous addition of purified Ac-globulin, lipids, and calcium ions. A sequence in blood coagulation occurs in three major steps; namely, (a) the formation of autoprothrombin C, (b) the formation of thrombin, and (c) the formation of fibrin. Purified autoprothrombin III corrected the prolonged one-stage prothrombin time of Stuart plasma and also of “factor VII” deficient plasma.

Some activation characteristics of the prethrombin subunit of prothrombin

Abstract Prethrombin, autoprothrombin III and an inhibitor were separated from purified prothrombin by degradation with thrombin and subsequent chromatography. Prethrombin was refractory to activation with tissue thromboplastin, purified Ac-globulin and calcium ions. Prethrombin activated in physiological saline solutions with only trypsin or purified autoprothrombin C. In 25% sodium citrate solution activation was very slow. This was, however, accelerated with either autoprothrombin C or autoprothrombin III, but not with purified thrombin. The specific activity and amino acid composition of prethrombin preparations was found to be about the same as that of purified thrombin. The autoprothrombin III fraction yielded autoprothrombin C with trypsin, Russells viper venom, and with tissue extracts plus calcium ions, but not in 25% sodium citrate solution.