Heparin binding of protein-C inhibitor--analysis of the effect of heparin on the interaction of protein-C inhibitor with tissue kallikrein.

The non-specific serine-protease inhibitor protein-C inhibitor (PCI) inactivates its target enzymes by forming stable 1:1 complexes. Heparin stimulates most PCI/protease reactions, but interferes with the inhibition of tissue kallikrein by PCI by a hitherto unknown mechanism. In this study we analyzed the inhibitory effect of heparin on the tissue-kallikrein-PCI interaction. Free PCI and tissue-kallikrein x PCI complexes but not free tissue kallikrein bound to heparin-Sepharose, implying that the inhibitory effect of heparin cannot be caused by a tissue-kallikrein-heparin interaction. Heparin did not dissociate tissue-kallikrein x PCI complexes, making it unlikely that in the presence of heparin PCI becomes a substrate for, rather than an inhibitor of, tissue kallikrein. However, heparin-bound PCI, which was able to form complexes with 125I-urokinase, did not form complexes with 125I-tissue-kallikrein. This suggests that the inhibitory effect of heparin is either based on the neutralization of positive charges in the PCI molecule, which might be required for the interaction of PCI with the acidic protease tissue kallikrein, or on a change in reactivity of PCI upon heparin binding, making heparin-bound PCI no longer a tissue-kallikrein inhibitor. Neutralization of basic amino acids in the PCI molecule by glutamic acid, which prevented in a dose-dependent way the inhibitory effect of heparin, did not have any effect on the tissue-kallikrein-PCI interaction. Therefore, direct involvement of basic amino acid residues present in the heparin-binding site of PCI in the tissue-kallikrein-PCI interaction can be excluded. Heparin binding might rather cause a change in reactivity of PCI (e.g. by inducing a conformational change or by steric interference), thereby preventing its interaction with tissue kallikrein.

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.

Inhibition of acrosin by protein C inhibitor and localization of protein C inhibitor to spermatozoa.

Protein C inhibitor (PCI) is synthesized by cells throughout the male reproductive tract and is present in high concentrations (220 micrograms/ml) in seminal plasma. Seminal plasma as well as the acrosome of spermatozoa are rich in possible target proteases for PCI. We analyzed the interaction of PCI with acrosin, a serine protease stored in its zymogen form in the acrosome of spermatozoa. Purified human PCI inhibited the amidolytic activity of purified boar acrosin with an apparent second-order rate constant of 3.7 x 10(4) M-1.s-1. Inhibition was paralleled by the degradation of PCI from its 57- to its 54-kDa form. Human PCI also inhibited the amidolytic activity of activated human sperm extracts and formed complexes with acrosin as determined by an enzyme-linked immunosorbent assay. Immunocytochemistry revealed that morphologically abnormal spermatozoa stained for PCI antigen, whereas morphologically normal spermatozoa were negative. In immunoelectron microscopy, PCI was exclusively localized in the immediate vicinity of disrupted acrosomal membranes of sperm heads. These data suggest that PCI might function as a scavenger of prematurely activated acrosin, thereby protecting intact surrounding cells and seminal plasma proteins from possible proteolytic damage.

Vitronectin modulates glycosaminoglycan dependent reactions of protein C inhibitor.

Protein C inhibitor (PCI), a glycosaminoglycan (GAG) dependent serine protease inhibitor, inhibits its target proteases by forming SDS-stable 1:1 complexes. GAGs alter target enzyme specificity of PCI in such a way that e.g. urokinase (uPA) is the preferred target enzyme in the presence of GAGs while in their absence preferentially tissue kallikrein (TK) complexes are formed. The effect of the GAG-binding adhesive glycoprotein vitronectin (Vn) on the GAG-stimulated inhibition of uPA by PCI was studied using an amidolytic assay. In the presence of heparin, Vn protected uPA from inhibition by PCI in a dose-dependent manner with respect to both, Vn- and heparin-concentration. Vn also was active when heparin was replaced by low-molecular weight heparin or heparan sulfate, respectively. In the absence of GAGs, Vn had no effect on the inhibition of uPA by PCI. In a similar system, Vn was far less effective in modifying the inhibitory function of heparin on the inhibition of TK by PCI. When equimolar concentrations of radiolabelled uPA and TK were incubated with PCI in the presence of heparin, only complexes of PCI with uPA were detectable. Addition of Vn reduced this complex formation, whereas, in contrast, complexes of PCI and TK appeared. These results indicate that Vn modulates both, the activity and specificity of PCI and suggest different structural heparin-requirements for the PCI/uPA versus PCI/TK interaction.

Inhibition of tissue kallikrein by protein C inhibitor. Evidence for identity of protein C inhibitor with the kallikrein binding protein.

We studied the inhibition of tissue kallikrein by protein C inhibitor (PCI), a relatively unspecific heparin-dependent serine protease inhibitor present in plasma and urine. PCI inhibited the amidolytic activity (cleavage of H-D-valyl-L-leucyl-arginine-p-nitroaniline) of urinary kallikrein with an apparent second order rate constant of 2.3 x 10(4) M-1 s-1 and formed stable complexes (85 kDa) with urinary kallikrein as judged from silver-stained sodium dodecyl sulfate-polyacrylamide gels. Complex formation was time-dependent and was paralleled by a decrease in the intensity of the main PCI protein band (Mr = 57,000) and an increase in the intensity of the lower Mr (54,000) PCI form (cleaved inhibitor). Heparin interfered with the inhibition of tissue kallikrein by PCI and with the formation of tissue kallikrein-PCI complexes in a dose-dependent fashion and completely abolished PCI-tissue kallikrein interaction at 300 micrograms/ml. This is in contrast to findings on the interaction of PCI with all other target proteases studied so far (i.e. stimulation of inhibition by heparin) but is similar to the reaction pattern of 125I-labeled tissue kallikrein with so called kallikrein binding protein described in serum and other systems. To study a possible relationship between PCI and this kallikrein binding protein we incubated 125I-labeled urinary kallikrein in serum and in PCI-immunodepleted serum in the absence and presence of heparin and analyzed complex formation using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In normal serum, formed complexes co-migrated with complexes of purified PCI and 125I-kallikrein and were less intense in the presence of heparin. No complex formation at all was seen in PCI-depleted serum. Our data indicate that PCI may be a physiologically important endogenous inhibitor of tissue kallikrein and provide evidence that PCI may be identical to the previously described kallikrein binding protein.

Possible identity of kallikrein binding protein with protein C inhibitor.

Protein C inhibitor (PCI) inhibits tissue kallikrein by forming stable 1:1 complexes (k1 = 2.3 x 10(4)M-1s-1). Heparin inhibits the tissue kallikrein/PCI-interaction and complex formation of 125I-tissue kallikrein in serum. 125I-tissue kallikrein complexes formed in plasma can be immunoprecipitated with monoclonal anti-PCI IgG suggesting that PCI might be identical to the kallikrein binding protein described previously (J. Chao et al. 1986, Biochem. J. 239, 325-331).