Abstract
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 also detected in both platelets and megakaryocytic cells using nested polymerase chain reaction. PCI was found to be located in the alpha-granules of resting platelets. Approximately 30% of the total amount of PCI in platelets was released after stimulation with ADP, collagen, adrenalin, thrombin, or thrombin receptor-activating peptide. Secreted PCI 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 vesicles prepared using rabbit brain cephalin (RBC) or a mixture of 40% phosphatidylethanolamine (PE), 20% phosphatidylserine (PS), and 40% phosphatidylcholine (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 phase phospholipids showed that APC binds more preferably to PE than to RBC 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 PCI inhibited APC significantly but only weakly the gamma-carboxyglutamic acid domainless APC. The gamma-carboxyglutamic acid fragment of protein C suppressed the PCI-mediated inhibition of APC on solid phase RBC or PE. Most of the APC.PCI complex formed on solid phase RBC or PE was released into the soluble phase. These findings suggest that PCI secreted from activated platelets binds preferably to PE of platelet membrane and microvesicles and that it inhibits phospholipid-bound APC efficiently.
Highlights
Protein C inhibitor (PCI),1 a member of the plasma serine protease inhibitor family, was originally identified in plasma as an inhibitor of activated protein C (APC), the main proteolytic enzyme of the protein C anticoagulant pathway [1,2,3]
PCI is the main inhibitor of APC, but when its plasma concentration becomes limited in conditions associated with increased activation of protein C, ␣1-antitrypsin and ␣2macroglobulin form complexes with APC [8, 9]
To determine the amount of APC1⁄7PCI complex bound to solid phase rabbit brain cephalin (RBC), PE, or heparin, 100 l of Trisbuffered saline containing 0.5% Triton X-100 was added to RBC, PE, or heparin-coated wells and incubated for 15 min with agitation
Summary
Materials—Sepharose CL-2B was obtained from LKB Biotechnology. Bovine serum albumin (BSA), rabbit brain cephalin (RBC), phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and heparin (160 units/mg) were from Sigma.
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