Synthesis and ultrastructural localization of protein C inhibitor in human platelets and megakaryocytes.
The occurrence of protein C inhibitor (PCI) in human platelets and megakaryocytes was analyzed. As judged from enzyme-linked immunosorbent assays (ELISAs), 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. Consistently, mainly the 57-kD form (active PCI) and some high molecular weight (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 studied by immunofluorescence histochemistry and immunotransmission electron microscopy: PCI was detected in alpha granules, in the open canalicular system, and on the plasma membrane. At these sites, colocalization with plasminogen activator inhibitor-1 was seen. Studies were performed to clarify whether platelet PCI is endogenously synthesized or taken up from plasma. Internalization of biotinylated-PCI was analyzed using platelets in suspension and gold-labeled streptavidin for visualization of incorporated biotin. Dose- and time-dependent uptake of PCI was found. PCI mRNA was detected in platelets by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, as well as in megakaryocytes by in situ hybridization of human bone marrow cryosections. We therefore conclude that platelets contain a functionally active PCI pool that is derived from both endogenous synthesis as well as internalization.
Prendes
M J
MJ
Department of Vascular Biology and Thrombosis Research, Department of Histology and Embryology, University of Vienna, Vienna, Austria.
Bielek
E
E
Zechmeister-Machhart
M
M
Vanyek-Zavadil
E
E
Carroll
V A
VA
Breuss
J
J
Binder
B R
BR
Geiger
M
M
D001792:Blood Platelets / Q000378:metabolism* / Q000648:ultrastructure; D002462:Cell Membrane / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008533:Megakaryocytes / Q000378:metabolism* / Q000648:ultrastructure; D016253:Microscopy, Immunoelectron; D018046:Protein C Inhibitor / Q000096:biosynthesis*
D018046:Protein C Inhibitor