Binding of retinoic acid by the inhibitory serpin protein C inhibitor.

The serpin superfamily includes inhibitors of serine proteases and noninhibitory members with other functions (e.g. the hormone precursor angiotensinogen and the hormone carriers corticosteroid-binding globulin and thyroxine-binding globulin). It is not known whether inhibitory serpins have additional, noninhibitory functions. We studied binding of (3)H-labeled hydrophobic hormones (estradiol, progesterone, testosterone, cortisol, aldosterone, and all-trans-retinoic acid) to the inhibitory serpins antithrombin III, heparin cofactor II, plasminogen activator inhibitor-1, and protein C inhibitor (PCI). All-trans-[(3)H]retinoic acid bound in a specific dose-dependent and time-dependent way to PCI (apparent K(d) = 2.43 microm, 0.8 binding sites per molecule of PCI). We did not observe binding of other hormones to serpins. Intact and protease-cleaved PCI bound retinoic acid equally well, and retinoic acid did not influence inhibition of tissue kallikrein by PCI. Gel filtration confirmed binding of retinoic acid to PCI in purified systems and suggested that PCI may also function as a retinoic acid-binding protein in seminal plasma. Therefore, our present data, together with the fact that PCI is abundantly expressed in tissues requiring retinoic acid for differentiation processes (e.g. the male reproductive tract, epithelia in various organs), suggest an additional biological role for PCI as a retinoic acid-binding and/or delivering serpin.

Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility.

Protein C inhibitor (PCI) is a nonspecific, heparin-binding serpin (serine protease inhibitor) that inactivates many plasmatic and extravascular serine proteases by forming stable 1:1 complexes. Proteases inhibited by PCI include the anticoagulant activated protein C, the plasminogen activator urokinase, and the sperm protease acrosin. In humans PCI circulates as a plasma protein but is also present at high concentrations in organs of the male reproductive tract. The biological role of PCI has not been defined so far. However, the colocalization of high concentrations of PCI together with several of its target proteases in the male reproductive tract suggests a role of PCI in reproduction. We generated mice lacking PCI by homologous recombination. Here we show that PCI(-/-) mice are apparently healthy but that males of this genotype are infertile. Infertility was apparently caused by abnormal spermatogenesis due to destruction of the Sertoli cell barrier, perhaps due to unopposed proteolytic activity. The resulting sperm are malformed and are morphologically similar to abnormal sperm seen in some cases of human male infertility. This animal model might therefore be useful for analyzing the molecular bases of these human conditions.

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.

Molecular cloning and sequence analysis of the mouse protein C inhibitor gene.

The gene encoding mouse protein C inhibitor (mPCI) was isolated and its nucleotide sequence determined. Alignment of the genomic sequence with that of a cDNA obtained from mouse testis revealed that the mPCI gene (like the human counterpart) is composed of five exons and four introns with highly conserved exon/intron boundaries. It encodes a pre-polypeptide of 405 amino acids, which shows 63% identity with human PCI (hPCI). The putative reactive site is identical to that of hPCI from P5 to P3', suggesting a similar protease specificity. Also the putative heparin binding sites and 'hinge' regions are highly homologous in mouse and hPCI.

Protein C inhibitor (PCI).

PCI is a non-specific serpin that inhibits several proteases of the coagulation and fibrinolytic systems as well as plasma- and tissue kallikreins and the sperm protease acrosin. The precise physiological role of PCI has not been defined yet. Heparin stimulates most PCI/protease reactions, but interferes with the tissue kallikrein/PCI-interaction. Thereby heparin not only regulates PCI-activity but also its specificity in systems containing two or more of its target proteases. This effect is not restricted to heparin, but is also seen with other glycosaminoglycans (GAGs) and large, negatively charged molecules. PCI also binds to GAGs present on the surface of epithelial kidney cells, and GAGs isolated from these cells have a similar effect on PCI activity as heparin. Studies analyzing the role of PCI as an acrosin inhibitor revealed that endogenous PCI is immunocytochemically localized to disrupted acrosomal membranes of morphologically abnormal sperms, while intact sperms are negative for PCI-antigen. In a mouse in vitro fertilization model human PCI inhibited sperm/egg binding and decreased the fertilization rate. Northern blotting of human and mouse mRNA using human and mouse PCI-cDNA probes revealed that in the mouse PCI is exclusively synthesized in the genital tract (testis, seminal vesicle, ovary), while in humans PCI is additionally synthesized in many other organs (e.g., liver, pancreas, heart). Therefore PCI might regulate enzymes involved in fertilization (e.g. acrosin) in both species. Other proteases (e.g., tissue kallikrein) are possibly regulated in a species specific manner by different inhibitors.