PLATELET-FACTOR-4 BINDS TO GLYCANATED FORMS OF THROMBOMODULIN AND TO PROTEIN-C - A POTENTIAL MECHANISM FOR ENHANCING GENERATION OF ACTIVATED PROTEIN-C

Platelet factor 4 (PF4) is an abundant platelet alpha-granule heparin- binding protein. We have previously shown that PF4 accelerates up to 2 5-fold the proteolytic conversion of protein C to activated protein C by the thrombin thrombomodulin complex by increasing its affinity for protein C 30-fold. This stimulatory effect requires presence of the ga mma-carboxyglutamic acid (Gla) domain in protein C and is enhanced by the presence of a chondroitin sulfate glycosaminoglycan (GAG) domain o n thrombomodulin. We hypothesized that cationic PF4 binds to both prot ein C and thrombomodulin through these anionic domains. Qualitative SD S-polyacrylamide gel electrophoresis analysis of avidin extracts of so lutions containing biotinylated PF4 and candidate ligands shows that P F4 binds to GAG+ but not GAG- forms of thrombomodulin and native but n ot Gla-domainless protein C. Quantitative analysis using the surface p lasmon resonance-based BIAcore(TM) biosensor system confirms the extre mely high affinity of PF4 for heparin (K-D = 4 nM) and shows that PF4 binds to GAG+ thrombomodulin with a K-D of 31 nM and to protein C with a K-D of 0.37 mu M. In contrast, PF4 had no measurable interaction wi th GAG- thrombomodulin or Gla-domainless protein C. Western blot analy sis of normal human plasma extracted with biotinylated PF4 demonstrate s PF4 binding to protein C in a physiologic context. Thus, PF4 binds w ith relative specificity and high affinity to the GAG-domain of thromb omodulin and the Gla domain of protein C, These interactions may enhan ce the affinity of the thrombin thrombomodulin complex for protein C a nd thereby promote the generation of activated protein C.