Plasma prekallikrein (PK) complexes with its receptor, high molecular weight kininogen (HK), on human umbilical vein endothelial cells (HUVEC). When assembled on endothelial cells, PK is activated to plasma kallikrein independent of FXIIa by the serine protease prolylcarboxypeptidase (PRCP) (Km=9 nM). PRCP was shown to be a PK activator when isolated from HUVEC (JBC 277:17962, 2002) and produced as a recombinant protein (Blood 103:4554, 2004). To additionally confirm that human PRCP is a physiologic PK activator, PRCP was over-expressed in Chinese hamster ovary (CHO) cells. CHO cells were transfected with full-length PRCP under the control of a CMV promoter and CHO rPRCP was expressed as a fusion protein with C-terminal enhanced green fluorescent protein (eGFP). The presence of rPRCP in transfected CHO was detected by real time RT-PCR, immunoblot and immunoprecipitation. In CHO cells, PRCP mRNA and PK activation were 2-3 fold higher than controls. The increase in PRCP-induced PK activation on the transfected CHO cells paralleled the increase in PRCP antigen expression on these cells, as determined by anti-PRCP and anti-GFP antibodies. PK activation of the transfected cells was blocked by siRNA to PRCP. Anti-PRCP antibody and Z-Pro-Pro-aldehyde dimethyl acetate also blocked PK activation with IC₅₀ of 0.01 and 7.0 mM, respectively. The cellular localization of PRCP in intact cells using confocal microscopy and flow cytometry also confirmed the over-expression of PRCP on the external membrane. These investigations independently confirm that prolylcarboxypeptidase is expressed on cell membranes and its expression increases PK activation.