Platelet factor 4 binds to glycanated forms of thrombomodulin and to protein C - A potential mechanism for enhancing generation of activated protein C

被引:45
作者
Dudek, AZ
Pennell, CA
Decker, TD
Young, TA
Key, NS
Slungaard, A
机构
[1] UNIV MINNESOTA,DEPT INTERNAL MED,MINNEAPOLIS,MN 55455
[2] UNIV MINNESOTA,DEPT LAB MED & PATHOL,MINNEAPOLIS,MN 55455
[3] BERLEX BIOSCI,RICHMOND,CA
关键词
D O I
10.1074/jbc.272.50.31785
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Platelet factor 4 (PF4) is an abundant platelet alpha-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 gamma-carboxyglutamic acid (Gla) domain in protein C and is enhanced by the presence of a chondroitin sulfate glycosaminoglycan (GAG) domain on 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 binds 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 a K-D of 0.37 mu M. 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 and thereby promote the generation of activated protein C.
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页码:31785 / 31792
页数:8
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