S protein modulates the heparin-catalyzed inhibition of thrombin by antithrombin III. Evidence for a direct interaction of S protein with heparin

Eur J Biochem. 1986 May 2;156(3):645-50. doi: 10.1111/j.1432-1033.1986.tb09626.x.

Abstract

The interference of S protein with the heparin-catalyzed inhibition of thrombin by antithrombin III was studied in a purified system and in plasma. The effect of S protein to counteract heparin activity was documented by kinetic analysis of the initial phase of the inhibition reaction. Addition of S protein induced a concentration-dependent reduction of the inhibition rate, reflected in a decrease of the apparent pseudo-first-order rate constant by a factor of 5-8 in the presence of a twofold molar excess of S protein over antithrombin III. A non-competitive interaction of S protein with the thrombin--antithrombin-III--heparin inhibition reaction with Ki = 0.6 microM was found. While the association constant of thrombin--antithrombin III in the presence of 0.05 U/ml heparin amounted to 2.5 X 10(8) M-1, an approximately 200-fold decrease of this value was observed in the presence of S protein. The fast formation of the covalent complex between thrombin and antithrombin III in the presence of heparin was impaired as a result of the presence of S protein, as was shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In the absence of heparin the inhibition of thrombin by antithrombin III alone was not influenced by S protein. The heparin-counteracting activity of S protein was found to be mainly expressed in the range of 0.01-0.1 U/ml heparin, thereby shifting the point of 50% inhibition of thrombin from 0.003 U/ml to 0.1 U/ml heparin with a second-order rate constant of k2 = 1.4 X 10(6) M-1. A direct interaction of S protein with heparin was demonstrated by crossed immunoelectrophoresis with purified proteins as well as in plasma and serum. The analysis of plasma and serum by crossed immunoelectrophoresis against rabbit anti-(human S protein) serum revealed an additional cathodal peak in the serum sample, resulting from the interaction of S protein with serum components. These findings not only indicate a direct interaction of S protein with heparin in the onset of the inhibition of thrombin by antithrombin-III--heparin, but also a contribution of S protein during enzyme-inhibitor complex formation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antithrombin III / physiology*
  • Blood Coagulation Tests
  • Catalysis
  • Glycoproteins / physiology*
  • Heparin / physiology*
  • Heparin Antagonists*
  • Humans
  • Hydrogen-Ion Concentration
  • Immunoelectrophoresis, Two-Dimensional
  • Kinetics
  • Membrane Glycoproteins*
  • Protein Binding
  • Thrombin / antagonists & inhibitors*

Substances

  • Glycoproteins
  • Heparin Antagonists
  • Membrane Glycoproteins
  • S-protein (control)
  • Antithrombin III
  • Heparin
  • Thrombin