PMC

Chimpanzees received a bolus infusion (over 30seconds) with fXa and PCPS at varying doses. Plasma TAFIa levels were measured over a time course of 0–90 minutes. Mean TAFIa concentrations for at least 2 measurements of the same sample are shown plotted against the time the plasma sample was taken.

Comparison of thermal stabilities of TAFIa variants at 37 °C. The residual TAFIa activities were measured over time. The lines represent the nonlinear regression of the data to the exponential decay function.

Thrombin-activatable fibrinolysis inhibitor (TAFI) isoforms in plasma. Thrombin/thrombomodulin or plasmin can activate TAFI (zymogen, 60 kDa), yielding TAFIa (active form, 35 kDa). Subsequently, TAFIa is thermally inactivated (triangle) to TAFIai (shaded). All 3 TAFI isoforms, TAFI, TAFIa, and TAFIai, are found in plasma. TAFIa/ai-specific ELISA measured only TAFIa and TAFIai, not TAFI.

Ang1 and Ang2 inhibit TM-dependent formation of APC and TAFIa. (a) HUVECs were incubated with protein C and thrombin plus either 1 μM of Fc control protein or the indicated concentrations of native Ang1 or Ang2 for 60 minutes. Cell supernatants were then assayed for APC levels. The graph depicts the relative amounts of APC formation (compared to APC formation in the presence of the Fc control protein) as a function of angiopoietin concentration. The error bars represent the SD, n = 3. (b) HUVECs were incubated with protein C and thrombin plus either Fc control protein (0.5 μM), native Ang1 (0.5 μM) or PF4 (10 μg/ml, ~1.3 μM) for 60 minutes and cell supernatants were then assayed for APC levels. The graph depicts the relative amounts of APC generated (the amount of APC generated in the presence of Fc control protein was assigned a value of 1.0). The error bars represent the SD, n = 3. Relative levels of APC in the Ang1 and the PF4 treatment groups were significantly different from the control group (***P < 0.001, one-way ANOVA with Tukey’s multiple comparisons test). (c) HUVECs were incubated with protein C and thrombin plus either 1  μM of Fc control protein or the indicated concentrations of native Ang1 or Ang2 for 60 minutes. Cell supernatants were then assayed for TAFIa levels. The graph depicts the relative amounts of TAFIa generated (the amount of TAFIa generated in the presence of Fc control protein was assigned a value of 1.0). The error bars represent the SD, n = 3. Relative levels of TAFIa in the Ang1 100 nM and the Ang2 100 nM treatment groups were significantly different from the control group (***P < 0.001, one-way ANOVA with Tukey’s multiple comparisons test). (d,e) COS cells overexpressing TM were incubated with the indicated concentrations of native Ang1 or Ang2 for 30 minutes, and then assayed for APC (d) or TAFIa (e) formation upon addition of thrombin plus protein C or TAFI. The graphs depict relative APC or TAFIa formation as a function of angiopoietin concentration (the amounts formed in the absence of angiopoietin were assigned a value of 1.0).