Synthesis and ultrastructural localization of protein C inhibitor in humanplatelets and megakaryocytes

The occurrence of protein C inhibitor (PCI) in human platelets and megakary ocytes was analyzed. As judged from enzyme-linked immunosorbent assays (ELI SAs), PCI was present in platelets at a concentration of 160 ng/(2) x 10(9) cells. Its specific activity was 5 times higher than that of plasma PCI, C onsistently, mainly the 57-kD form (active PCI) and some high molecular wei ght (M-r,) forms, but no bands corresponding to cleaved PCI, were detected when platelet lysates were immunoprecipitated with monoclonal anti-PCI-IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis ( SDS-PAGE) and Western blotting. The localization of PCI in platelets was st udied by immunofluorescence histochemistry and immunotransmission electron microscopy: PCI was detected in a granules, in the open canalicular system, and on the plasma membrane. At these sites, colocalization with plasminoge n activator inhibitor-1 was seen. Studies were performed to clarify whether platelet PCI is endogenously synthesized or taken up from plasma. Internal ization of biotinylated-PCI was analyzed using platelets in suspension and gold-labeled streptavidin for visualization of incorporated biotin. Dose- a nd time-dependent uptake of PCI was found. PCI mRNA was detected in platele ts by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, as well as In megakaryocytes by in situ hybridization of human b one marrow cryosections, We therefore conclude that platelets contain a fun ctionally active PCI pool that is derived from both endogenous synthesis as well as internalization. (C) 1999 by The American Society of Hematology.