Inhibitory effects of human alpha2-macroglobulin and mouse serum on the PSGL-1 and glycoprotein Ib proteolysis by a snake venom metalloproteinase, triflamp.
Triflamp, a 28 kDa snake venom metalloproteinase purified from the venom of Trimeresurus flavoviridis, possesses the proteolytic activities toward P-selectin glycoprotein ligand-1 (PSGL-1), glycoprotein Ib (GPIb) and fibrinogen. In human whole blood preparation, however, triflamp (6 microg/ml) failed to cleave neutrophil PSGL-1 and platelet GPIb. Human alpha2-macroglobulin (alpha2M) was mainly responsible for the neutralization of the proteolytic effects of triflamp on PSGL-1, GPIb and fibrinogen. Human alpha2M neutralized triflamp at a stoichiometry about 1.1:1 (molar basis) determined by azocaseinolysis. SDS-PAGE analysis revealed that triflamp cleaved the bait-region of alpha2M. Western blot demonstrated that triflamp interacted with the C-terminal half-subunits of truncated alpha2M resulting in the formation of high-molecular-weight species of alpha2M-triflamp complexes. In the presence of competing nucleophile, 0.2 M methylamine, the proteolytic activity of triflamp was conserved. In vivo we found that mice neutrophils were resistant to the cleavage of PSGL-1 by triflamp. However, mouse PSGL-1 and GPIb were susceptible to be cleaved by triflamp in washed mouse neutrophil and platelet preparation, respectively. Similarly, mouse serum was also responsible for the inactivation of the proteolytic activity of triflamp. This study provides direct evidences for the reasonable explanation regarding the reduced proteolytic activity of triflamp toward its substrates in whole blood preparation and in vivo model.
Tseng
Yu-Lun
YL
Department of Pharmacology, College of Medicine, National Taiwan University, No.1. Sec. 1, Jen-Ai Rd, Taipei, Taiwan.
Wu
Wen-Bin
WB
Hsu
Chun-Chieh
CC
Peng
Hui-Chin
HC
Huang
Tur-Fu
TF
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002364:Caseins; D003435:Crotalid Venoms / Q000302:isolation & purification / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D045726:Metalloproteases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008744:Methylamines; D051379:Mice; D008810:Mice, Inbred C57BL; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D044967:Serum / Q000378:metabolism; D017841:Trimeresurus; D000511:alpha-Macroglobulins / Q000494:pharmacology*
D000911:Antibodies, Monoclonal; D002364:Caseins; D003435:Crotalid Venoms; D008562:Membrane Glycoproteins; D008744:Methylamines; C084882:P-selectin ligand protein; D019038:Platelet Glycoprotein GPIb-IX Complex; D000511:alpha-Macroglobulins; C026018:azocasein; D005340:Fibrinogen; C027451:methylamine; D045726:Metalloproteases