Platelet factor 4 stimulates thrombomodulin protein C-activating cofactor activity. A structure-function analysis.
Thrombomodulin (TM) is an anionic (pI approximately 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 alpha-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 micrograms/ml. No such stimulation occurs in response to beta-thromboglobulin or thrombospondin, or when protein C lacking its gamma-carboxyglutamic acid (Gla) domain is the substrate. Heparin and chondroitin sulfates A and E reverse PF4 stimulation. PF4 minimally affects the Kd for THR but decreases 30-fold (from 8.3 to 0.3 microM) the Km 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.
Slungaard
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Department of Medicine, University of Minnesota, Minneapolis 55455.
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D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006025:Glycosaminoglycans / Q000494:pharmacology; D006801:Humans; D019007:P-Selectin; D010978:Platelet Factor 4 / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011486:Protein C / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D018180:Thrombomodulin / Q000235:genetics / Q000378:metabolism*
D002412:Cations; D006025:Glycosaminoglycans; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D011486:Protein C; D011994:Recombinant Proteins; D018180:Thrombomodulin; D010978:Platelet Factor 4; D013917:Thrombin; D002118:Calcium