Thrombin-mediated hemolytic activity as modulated by C5 convertase. (A) Thrombin-mediated hemolytic activity was compared with C5 convertase hemolytic activity. In the terminal pathway assay, a standard curve was generated by measuring lysis of chicken erythrocytes after addition of a range of concentrations of purified C5b,6 and excess C7, C8, and C9. Percent lysis is expressed relative to 100% lysis in H2O. To measure enzyme-induced hemolysis, reactions containing C5 (400nM) and C6 (600nM) incubated alone, with C5 convertase (CVF,Bb; 100nM) or with thrombin (400nM), were diluted 2000-fold, 8000-fold, and 2000-fold, respectively, to measure hemolytic activity in the terminal pathway assay. (B) Thrombin-mediated hemolytic activity in the presence of C5 convertase was measured. The ability of thrombin to augment C5 convertase hemolytic activity was assessed in reactions as noted above but with a lower concentration of thrombin (20nM) and in the presence or absence of C5 convertase (CVF,Bb; 100nM) or trypsin (20nM). Reactions were either subjected to SDS-PAGE or diluted 160 000-fold for the terminal pathway hemolytic assay. Data are mean ± SD; n = 3. *P < .05.
