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This Article Full Text Full Text (PDF) All Versions of this Article: 289/6/H2697    most recent 00715.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (4) Citing Articles via Google Scholar Google Scholar Articles by Shariat-Madar, Z. Articles by Schmaier, A. H. Search for Related Content PubMed PubMed Citation Articles by Shariat-Madar, Z. Articles by Schmaier, A. H.

Overexpression of prolylcarboxypeptidase enhances plasma prekallikrein activation on Chinese hamster ovary cells

¹Hematology/Oncology Division, Department of Internal Medicine, and ²Department of Pathology, University of Michigan, Ann Arbor, Michigan

Submitted 5 July 2005 ; accepted in final form 25 July 2005

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 factor XIIa by the serine protease prolylcarboxypeptidase (PRCP, K_m = 9 nM). PRCP was shown to be a PK activator when isolated from HUVEC (J Biol Chem 277: 17962–17969, 2002) and produced as a recombinant protein (Blood 103: 4554–4561, 2004). To additionally confirm that human PRCP is a physiological PK activator, PRCP was overexpressed in Chinese hamster ovary (CHO) cells. CHO cells were transfected with full-length PRCP under the control of a cytomegalovirus promoter, and CHO recombinant PRCP was expressed as a fusion protein with COOH-terminal enhanced green fluorescence protein (EGFP). The presence of recombinant PRCP in transfected CHO cells was detected by real-time RT-PCR, immunoblot, and immunoprecipitation. PRCP mRNA and PK activation were two- to threefold higher in transfected than in control CHO cells. The increase in PRCP-induced PK activation in the transfected CHO cells paralleled the increase in PRCP antigen expression, as determined by anti-PRCP and anti-green fluorescence protein antibodies. PK activation of the transfected cells was blocked by small interfering RNA to PRCP. Anti-PRCP antibody and Z-Pro-Pro-aldehyde dimethyl acetate also blocked PK activation (IC₅₀ = 0.01 and 7.0 mM, respectively). Localization of PRCP in intact cells observed via confocal microscopy and flow cytometry also confirmed overexpression of PRCP on the external membrane. These investigations independently confirm that PRCP is expressed on cell membranes and that PRCP expression increases PK activation.

plasma kallikrein-kinin system; high-molecular-weight kininogen

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