PF4 is an abundant cationic platelet a-granule protein that is released during aggregation and binds heparin strongly. We have shown that PF4 accelerates up to 25-fold the anticoagulant activation of protein C by the thrombin-thrombomodulin (TM) complex (JBC 269:25549, 1994). This acceleration requires the presence of the anionic v-carboxyglutamic acid (Ola) domain of protein C and is greatly enhanced by the presence of an anionic, chondroitin sulfate-like glycosaminoglycan (GAG) domain on TM. To explain these findings, we hypothesized that cationic PF4 binds and forms a complex with both the GAG domain of TM and the Gla domain of protein C. To detect such putative complexes, we used streptavidin beads to extract solutions containing biotinylated PF4 (bPF4) and either 1) GAG+ or GAGrecombinant isoforms of extracellular domain TM; or 2) native or Gla domainless protein C. Analysis of these extracts by silver-stained SDS/PAGE showed large amounts of GAG+ TM and native protein C, but no GAG- TM, and barely detectable amounts of Gla domainless protein C. To help elucidate the potential physiologic importance of these interactions, we quantitated their avidity by surface plasmon resonance. Candidate ligands were perfused across a dextran-streptavidin surface upon which bPF4 had been immobilized. Perfusion with GAG+ but not GAG- TM resulted in a rapid and prominent binding (association) interaction; subsequent perfusion with buffer alone revealed slow dissociation. Kinetic analyses of the association and dissociation phases showed a ka of 5 x 104 M~ V and a kd of 1.5 x 10'4 s"', yielding an equilibrium dissociation constant (Krj) of 3 x 10"-4 M. Native protein C also bound bPF4 strongly with a KD of 7 x 10'7 M. In sinking contrast, Gla domainless protein C showed no detectable binding. Thus, PF4 binds both to TM through ihe GAG moiety and to protein C through the Gla domain and may by this mechanism induce conformational changes that favor the activation of protein C by the thrombin-TM complex. The unexpectedly high affinity of these PF4 interactions further supports their physiologic relevance. Moreover, given the strict conservation of Gla domains, our findings predict that PF4 will also interact with other vitamin K-dependent clotting factors and may thereby play a hitherto unsuspected role in the regulation of clotting.
|Original language||English (US)|
|Journal||Journal of Investigative Medicine|
|State||Published - Jan 1 1996|