PROTEIN-C INHIBITOR SECRETED FROM ACTIVATED PLATELETS EFFICIENTLY INHIBITS ACTIVATED PROTEIN-C ON PHOSPHATIDYLETHANOLAMINE OF PLATELET MEMBRANE AND MICROVESICLES

Protein C inhibitor (PCI) was detected in human platelets (2.9 ng/10(9 ) cells) and megakaryocytic cells (1.5 ng/10(6) cells). PCI mRNA was a lso detected in both platelets and megakaryocytic cells using nested p olymerase chain reaction. PCI was found to be located in the alpha-gra nules of resting platelets. Approximately 30% of the total amount of P CI in platelets was released after stimulation with ADP, collagen, adr enalin, thrombin, or thrombin receptor-activating peptide. Secreted PC I was detected on the surface of activated platelets and phospholipid microvesicles. PCI secreted from thrombin receptor-activating peptide- stimulated platelets inhibited activated protein C (APC) efficiently. PCI significantly inhibited APC in the presence of phospholipid vesicl es prepared using rabbit brain cephalin (RBC) or a mixture of 40% phos phatidylethanolamine (PE), 20% phosphatidylserine (PS), and 40% phosph atidylcholine (PC) with a second order rate constant of 1.0 x 10(6) M- 1.min(-1). Of these phospholipids, PE was critical for this inhibition . The dissociation constants of the binding of APC or PCI to solid pha se phospholipids showed that APC binds more preferably to PE than to R BC or PS, and PCI to PE or RBC than to PS or PC. PCI binding to solid phase phospholipids depended on the presence of PE. RBC- or PE-bound P CI inhibited APC significantly but only weakly the gamma-carboxyglutam ic acid domainless APC. The gamma-carboxyglutamic acid fragment of pro tein C suppressed the PCI-mediated inhibition of APC on solid phase RB C or PE. Most of the APC PCI complex formed on solid phase RBC or PE w as released into the soluble phase. These findings suggest that PCI se creted from activated platelets binds preferably to PE of platelet mem brane and microvesicles and that it inhibits phospholipid-bound APC ef ficiently.