title: Platelet factor 4 binds to glycanated forms of thrombomodulin and to protein C. A potential mechanism for enhancing generation of activated protein C.
abstract: 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 BIAcoreTM biosensor system confirms the extremely high affinity of PF4 for heparin (KD = 4 nM) and shows that PF4 binds to GAG+ thrombomodulin with a KD of 31 nM and to protein C with a KD of 0.37 microM. 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.
authors.affiliation: Department of Internal Medicine, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: A Z
authors.initials: AZ
authors.lastname: Dudek
authors.forename: C A
authors.initials: CA
authors.lastname: Pennell
authors.forename: T D
authors.initials: TD
authors.lastname: Decker
authors.forename: T A
authors.initials: TA
authors.lastname: Young
authors.forename: N S
authors.initials: NS
authors.lastname: Key
authors.forename: A
authors.initials: A
authors.lastname: Slungaard
mesh_terms: D001665:Binding Sites; D015374:Biosensing Techniques; D006493:Heparin / Q000378:metabolism; D006801:Humans; D008024:Ligands; D010978:Platelet Factor 4 / Q000378:metabolism*; D011485:Protein Binding; D011486:Protein C / Q000378:metabolism*; D055672:Static Electricity; D018180:Thrombomodulin / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D008024:Ligands; D011486:Protein C; D018180:Thrombomodulin; D010978:Platelet Factor 4; D006493:Heparin