Recombinant prolylcarboxypeptidase activates plasma prekallikrein

The serine protease prolylcarboxypeptidase (PRCP) isolated from endothelial cells (HUVEC) is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into S2 Schneider insect cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high molecular weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, angiotensin II, bradykinin, anti-PRCP, DFP, PMSF, Z-Pro-Pro-aldehyde-dimethyl acetate, but not by 1 mM EDTA, bradykinin 1-5, or angiotensin 1-7. Corn trypsin inhibitor binds to kallikrein to prevent rPRCP activation, but does not directly inhibit the active site of either enzyme. Unlike factor XIIa, ability of PRCP to activate PK is blocked by angiotensin II and not neutralizing antibody to factor XIIa. PRCP antigen is on HUVEC membranes on flow cytometry and laser scanning confocal microscopy. PRCP antigen does not co-localize with LAMP1 on non-permeabilized HUVEC, but partially co-localizes in permeabilized cells. PRCP co-localizes with all of the HK receptors, gC1qR, uPAR, and cytokeratin 1 antigen, on non-permeabilized HUVEC. PRCP activity and antigen expression on cultured HUVEC are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed PK activating enzyme in the intravascular compartment.