PLATELET FACTOR-4 STIMULATES THROMBOMODULIN PROTEIN-C ACTIVATING COFACTOR ACTIVITY - A STRUCTURE-FUNCTION ANALYSIS

Thrombomodulin (TM) is an anionic (pI approximate to 4) protein cofact or that promotes thrombin (THR) cleavage of protein C to generate acti vated protein C (APC), a potent anticoagulant. We find that the cation ic platelet alpha-granule protein platelet factor 4 (PF4) stimulates 4 -25-fold the cofactor activity of rabbit TM and two differentially gly canated versions of an extracellular domain human TM polypeptide in wh ich the glycosaminoglycan (GAG) is either present (GAG+ TM) or absent (GAG- TM) with an ED(50) of 3.3-10 mu g/ml. No such stimulation occurs in response to beta-thromboglobulin or thrombospondin, or when protei n C lacking its gamma-carboxyglutamic acid (Gla) domain is the substra te. Heparin and chondroitin sulfates A and E reverse PF4 stimulation. PF4 minimally affects the K-d for THR but decreases 30-fold (from 8.3 to 0.3 mu M) the K-m for protein C of APC generation by GAG+ TM. PF4 a lso strikingly transforms the [Ca2+] dependence profile of rabbit and GAG+ TM to resemble that of GAG- TM. A potential explanation for this is that PF4, like Ca2+, induces heparin-reversible alterations in nati ve (but not Gla-domainless) protein C conformation as assessed by auto fluorescence emission analysis. We conclude that PF4 stimulates TM APC generation by interacting electrostatically with both the TM GAG and the protein C Gla domain to enhance markedly the affinity of the THR.T M complex for protein C. By this mechanism, PF4 may play a previously unsuspected role in the physiologic regulation of clotting.