Platelet factor 4 (PF4) is an abundant platelet α-granule heparin- binding protein. We have previously shown that PF4 accelerates up to 25-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 γ-carboxyglutamic acid (Gla) domain in protein C and is enhanced by the presence of a chondroitin sulfate glycosaminoglycan (GAG) domain of thrombomodulin. We hypothesized that cationic PF4 binds to both protein C and thrombomodulin through these anionic domains. Qualitative SDS-polyacrylamide gel electrophoresis analysis of avidin extracts of solutions containing biotinylated PF4 and candidate ligands shows that PF4 bins to GAG+ but not GAG- forms of thrombomodulin and native but not Gla-domainless protein C. Quantitative analysis using the surface plasmon resonance-based BIAcore(TM) biosensor system confirms the extremely 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 K(D) of 0.37 μM. In contrast, PF4 had no measurable interaction with GAG- thrombomodulin or Gla-domainless protein C. Western blot analysis of normal human plasma extracted with biotinylated PF4 demonstrates PF4 binding to protein C in a physiologic context. Thus, PF4 binds with relative specificity and high affinity to the GAG- domain of thrombomodulin and the Gla domain of protein C. These interactions may enhance the affinity of the thrombin-thrombomodulin complex for protein C an thereby promote the generation of activated protein C.