Platelet Factor 4 Binds to Glycanated Forms of Thrombomodulin and to Protein C

Abstract

Platelet factor 4 (PF4) is an abundant platelet ␣-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 thrombin⅐thrombomodulin complex by increasing its affinity for protein C 30-fold. This stimulatory effect requires presence of the ␥-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 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 thrombin⅐thrombomodulin complex for protein C and thereby promote the generation of activated protein C.

Novel Proteoglycan Linkage Tetrasaccharides of Human Urinary Soluble Thrombomodulin, SO4-3GlcAβ1–3Galβ1–3(±Siaα2–6)Galβ1–4Xyl

“…uTM does not contain chondroitin sulfate (2-4), although TM produced in cultured human endothelial cells and recombinant TMs were expressed in both cases with a high molecular weight TM containing chondroitin sulfate and a low molecular weight TM lacking this modification (10). Platelet factor 4 binds to the glycanated form of TM but not to the chondroitin sulfate-lacking TM (12). Acceleration of the inhibition of thrombin by antithrombin III by TM is dependent upon the presence of chondroitin sulfate linked to TM (10).…”

“…Recombinant TM expressed in Chinese hamster ovary cells contains chondroitin 4-sulfate (11). TM glycosylation in relation to biological activity has been discussed in several papers (12)(13)(14), but to understand the structure and biological activities of the glycoprotein, detailed knowledge of the sugar structures is essential. Recently, the structures of N-linked sugar chains have been reported (15).…”

Involvement of Human Natural Killer-1 (HNK-1) Sulfotransferase in the Biosynthesis of the GlcUA(3-O-sulfate)-Gal-Gal-Xyl Tetrasaccharide Found in α-Thrombomodulin from Human Urine

“…The structure of the nonradiolabeled reaction product was first characterized by DE MALDI-TOF MS to determine their molecular weights, from which the sugar composition and the number of O-sulfate groups present in the HNK-1ST reaction products were inferred. When the reaction product was analyzed, the molecular ion signal was observed as [M -3H ϩ 4Na ϩ SO 3 ] ϩ at m/z 1,272 (Fig. 1B), suggesting that a sulfate group was transferred to the linkage region.…”

“…In addition, platelet factor 4, which prevents the formation of a complex between heparin and antithrombin III, leading to blood coagulation, binds to ␤-TM but not to ␣-TM (3). The platelet factor 4/␤-TM complex displays anticoagulant activity by inactivating the clotting factors Va and VIIIa through limited proteolysis (3). Thus, the CS moiety of TM is essential for its biological functions.…”

“…Thrombomodulin (TM) 3 is an integral membrane glycoprotein expressed on the vascular endothelial cell surface, which occurs as both a chondroitin sulfate (CS) proteoglycan (PG) form (␤-TM) and a non-PG form without a CS chain (␣-TM) and hence is a part-time PG (1). TM binds to thrombin with high affinity and acts as a mediator of endothelial anticoagulant defenses.…”