Identification and characterization of prolylcarboxypeptidase as an endothelial cell prekallikrein activator.
Our recent investigations have postulated a human umbilical vein endothelial cell (HUVEC)-associated prekallikrein activator (PKA). When prekallikrein (PK) assembles on high molecular weight kininogen on HUVEC, PK is activated to kallikrein. PKA was found in the 15,800 x g pellet of HUVEC lysates using an assay that measures PK activation only when bound to high molecular weight kininogen linked to microtiter plates. Sequential DEAE, wheat germ lectin affinity, and hydroxyapatite chromatography resulted in four protein bands on SDS-PAGE. One protein in the 73-kDa band was identified by amino acid sequencing as prolylcarboxypeptidase (PRCP). On gel filtration, PKA activity was a single homogenous peak identical in migration to the 73-kDa immunoblot of PRCP. Anti-PRCP inhibits PKA activity and PK activation on HUVEC. Purified PKA was blocked by diisopropyl fluorophosphate (1 mm), phenylmethylsulfonyl fluoride (3 mm), leupeptin (100 microm), antipain (IC(50) = 2 microm), HgCl(2) (IC(50) = 500 microm), Z-Pro-Pro-aldehyde-dimethyl acetate (IC(50) = 1 microm), and corn trypsin inhibitor (IC(50) = 40 nm). PKA did not correct the coagulant defect in factor XII deficient plasma, was purified from HUVEC cultured in factor XII-deficient serum, was not detected by antibody to factor XII, did not activate FXI, and was not inhibited by a neutralizing antibody to FXII. Angiotensin II (IC(50) = 2 microm) or bradykinin (IC(50) = 100 microm), but not angiotensin II-(1-7) or bradykinin(1-5), and the prolyl oligopeptidase inhibitor Fmoc-Ala-Pyr-CN (IC(50) = 50 nm) also blocked purified PKA activation of PK. The K(m) of PK activation by PRCP is 6.7 nm. PRCP antigen is present on the membrane of fixed but not permeabilized HUVEC. PRCP appears to be a HUVEC-associated PK activator.
Shariat-Madar
Zia
Z
Division of Hematology and Oncology, Departments of Internal Medicine and Pathology, University of Michigan, Ann Arbor, Michigan 48109-0640, USA.
Mahdi
Fakhri
F
Schmaier
Alvin H
AH
D000804:Angiotensin II / Q000494:pharmacology; D002268:Carboxypeptidases / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology*; D015956:Factor XIIa / Q000378:metabolism*; D006801:Humans; D007531:Isoflurophate / Q000378:metabolism; D007610:Kallikreins / Q000378:metabolism; D018613:Microscopy, Confocal; D008970:Molecular Weight; D011288:Prekallikrein / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000804:Angiotensin II; D007531:Isoflurophate; D011288:Prekallikrein; D002268:Carboxypeptidases; C022448:lysosomal Pro-X carboxypeptidase; D007610:Kallikreins; D015956:Factor XIIa