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Formation, characterization and detection of a ternary complex between S protein, thrombin and antithrombin III in serum.

ABSTRACT: S protein, a plasma glycoprotein with Mr 78,000, has been shown to interfere with the heparin-catalysed inhibition of thrombin by antithrombin III. This interaction was further evaluated in the present study. Native human blood was replaced by either radiolabelled antithrombin III or radiolabelled prothrombin in the reaction mixture, which was incubated at 37 degrees C. At various time intervals the serum formed from the incubated blood was withdrawn and analysed by crossed immunoelectrophoresis against anti-(S protein) serum in the second dimension. Increasing quantities of radioactivity originating both from antithrombin III and from thrombin were precipitated in a cathodal shoulder to the S protein peak. This observation indicated the formation of a ternary S protein-thrombin-antithrombin III (STAT) complex in serum. This complex could also be observed by the same technique after incubation of purified thrombin in the presence of antithrombin III and S protein. Complex-formation was independent of the presence of heparin and did not require Ca2+ ions. Owing to the association of S protein with the thrombin-antithrombin III (TAT) complex, the STAT complex assembled in vitro exhibited a higher Mr than the TAT complex as judged by polyacrylamide-gradient-gel electrophoresis in the absence of SDS. Both the serum-originated STAT complex and the STAT complex assembled from purified components sedimented faster than the single components and showed comparable apparent sedimentation coefficients in the range 11-14 S, corresponding to a mean Mr of 350,000. The STAT complex could be detected in serum at a dilution of 1:3200 by a sensitive immuno-radiometric assay employing affinity-purified IgG against S protein. These results indicate that S protein, in addition to its role as a heparin-neutralizing factor, becomes incorporated into the nascent TAT complex or can bind to preformed TAT complex during the clotting process.

SUBMITTER: Preissner KT

PROVIDER: S-EPMC1147820 | biostudies-other | 1987 Apr

REPOSITORIES: biostudies-other

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Project description:The kinetics of inhibition of human and bovine alpha-thrombin and human factor Xa by antithrombin III were examined under pseudo-first-order conditions as a function of the concentration of pentosan polysulphate [a fully sulphated (beta 1-4)-linked D-xylopyranose with a single laterally positioned 4-O-methyl-alpha-D-glucuronic acid]. Double-reciprocal plots of the observed first-order rate constant against concentration of pentosan polysulphate gave straight lines, intercepts on the axes giving values for maximum increase in second-order rate constant (by calculation) and apparent dissociation constant. These values were: for human alpha-thrombin 1.52 X 10(7) M-1 . min-1 and 3.6 microM respectively, for bovine alpha-thrombin 6.56 X 10(6) M-1 . min-1 and 0.16 microM and for factor Xa 6.86 X 106 M-1 . min-1 and 20 microM. In the presence of pentosan polysulphate the dissociation constant for the initial complex of antithrombin III and thrombin was shown to be reduced from approx. 2 X 10(-3) M to 61 X 10(-6) M without apparent change in the limiting rate constant of 750 min-1. An oligosaccharide (primarily 8-10 saccharide units) prepared from heparin and with high affinity for antithrombin III but low potency in the thrombin-antithrombin III interaction did not diminish the rate of interaction catalysed by pentosan polysulphate. The catalysis was shown to be due to a weak electrostatic interaction, since it was completely reversed by concentrations of NaCl greater than 0.3 M. It is concluded that the mechanism is independent of the heparin high-affinity binding site on antithrombin III and is probably due to binding of the high-charge-density polysaccharide to the proteinase. It is calculated that the acceleration in rate achieved, although lower than that of heparin, approaches that required to be of physiological significance and may be of importance in the anticoagulation role of antithrombin III at sites of high charge density which may occur in vivo.

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Formation, characterization and detection of a ternary complex between S protein, thrombin and antithrombin III in serum.

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