Inhibition of Thrombin in Plasma by Heparin or Arginine

Abstract

The inhibition of plasmatic thrombin is of clinical importance in a broad range of diseases. To obtain reliable data the assay system should be as similar to physiology as possible. Using a newly developed physiologic assay system for fibrinogen/thrombin interaction (the FIFTA), the inhibition of plasmatic thrombin by heparin or by arginine was studied. The standard fibrinogen functional turbidimetric assay (FIFTA) was performed, varying heparin or arginine concentrations and varying the time point the inhibitor was added to the FIFTA. Plasmatic heparin concentrations equal to or greater than 0.63 IU/mL completely inhibit thrombin in the assay system described. The IC(50) is 0.1 IU/mL heparin. Heparin can only inhibit fibrin generation within the first 2 minutes at room temperature (RT=23 degrees C). The 50% inhibitory time point, that is, the time point that a 10 IU/mL final concentration of heparin results in 50% inhibition of FIFTA, is 30 seconds at RT. A final arginine concentration of at least 125 mM in the first 100 seconds of the FIFTA reaction at RT completely inhibits turbidity increase. Half-maximal turbidity increase occurs at 63 mM arginine. Final arginine concentrations of at least 250 mM completely inhibit turbidity increase, when arginine acts in the first 4 minutes (RT) of the thrombin/ fibrinogen interaction. A final arginine concentration of 477 mM added at the 12-minute or 30-minute thrombin/ fibrinogen reaction time point decreases the resulting turbidity by 50% after an additional 30 minutes at RT. Pathologic disseminated intravascular coagulation occurs in a multitude of diseases; in common is always the generation of thrombin either by the contact phase or by the tissue factor phase of coagulation. Such pathologically elevated thrombin activity in blood or blood products must be prevented or inhibited. This study demonstrates the efficiency of two physiologic thrombin inhibitors: heparin and arginine.