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 μg/ml) failed to cleave neutrophil PSGL-1 and platelet GPIb. Human α2-macroglobulin (α2M) was mainly responsible for the neutralization of the proteolytic effects of triflamp on PSGL-1, GPIb and fibrinogen. Human α2M 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 α2M. Western blot demonstrated that triflamp interacted with the C-terminal half-subunits of truncated α2M resulting in the formation of high-molecular-weight species of α2M-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.