RAPID ACTIVATION OF PROTEIN-C BY FACTOR XA AND THROMBIN IN THE PRESENCE OF POLYANIONIC COMPOUNDS

A recent study indicated that negatively charged substances such as he parin and dextran sulfate accelerate thrombin activation of coagulatio n factor XI by a template mechanism. Because the serine proteinase of the natural anticoagulant pathway, activated protein C, can bind hepar in, it was reasonable to think that these compounds may also bind prot ein C (PC) and accelerate its activation by thrombin or other heparin binding plasma serine proteinases by a similar mechanism. To test this , PC activation by thrombin and factor Xa (fXa) was studied in the pre sence of these polysaccharides. With thrombin in the absence of thromb omodulin (TM), these polysaccharides markedly reduced the K-m for PC a nd Gla-domainless PC (GDPC) activation in the presence of Ca2+. With T M containing chondroitin sulfate, heparin did not influence PC activat ion by thrombin, but with TM lacking chondroitin sulfate, the characte ristic high-affinity PC interaction at low Ca2+ (similar to 50 to 100 mu mol/L) was largely eliminated by heparin. In EDTA, heparin enhanced thrombin activation of GDPC by reducing the K-m, but it inhibited PC activation by increasing the K-m. PC activation in EDTA was insensitiv e to the presence of heparin if the exosite 2 mutant, R93,97,101A thro mbin, was used for activation. These results suggest that, when the Gl a-domain of PC is not fully stabilized by Ca2+, it interacts with the anion binding exosite 2 of thrombin and that heparin binding to this s ite prevents this interaction. Additional studies indicated that, in t he presence of phospholipid vesicles, heparin and dextran sulfate dram atically accelerate PC activation by fXa by also reducing the K-m. Int erestingly, on phospholipids containing 40% phosphatidylethanolamine, the activation rate of near physiological PC concentrations (similar t o 80 nmol/L) by fXa in the presence of dextran sulfate was nearly comp arable to that observed by the thrombin-TM complex. The biochemical an d potential therapeutical ramifications of these findings are discusse d. (C) 1998 by The American Society of Hematology.