Platelet factor 4 stimulates thrombomodulin protein C-activating cofactor activity: A structure-function analysis

Thrombomodulin (TM) is an anionic (pI ≈ 4) protein cofactor that promotes thrombin (THR) cleavage of protein C to generate activated protein C (APC), a potent anticoagulant. We find that the cationic platelet of-granule protein platelet factor 4 (PF4) stimulates 4-25-fold the cofactor activity of rabbit TM and two differentially glycanated versions of an extracellular domain human TM polypeptide in which the glycosaminoglycan (GAG) is either present (GAG+ TM) or absent (GAG- TM) with an ED₅₀ of 3.3-10 μg/ml. No such stimulation occurs in response to β-thromboglobulin or thrombospondin, or when protein C lacking its γ-carboxyglutamic acid (Gla) domain is the substrate. 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 μM) the K_m for protein C of APC generation by GAG+ TM. PF4 also 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 native (but not Gla-domainless) protein C conformation as assessed by autofluorescence 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·TM complex for protein C. By this mechanism, PF4 may play a previously unsuspected role in the physiologic regulation of clotting.