Figure 3.
AB002 catalytically converts protein C to APC on activated platelet aggregates and inhibits thrombin-mediated TAFI activation. (A) Washed human platelets were activated and aggregated by collagen and treated with 50 nM AB002 or S195A-WE and 100 nM protein C. Platelets treated with protein C only and thrombin mutants incubated with protein C in the absence of platelets were prepared at the same time. Supernatants were collected after 1 hour and APC measured. Data are shown as the mean ± 1 standard deviation for 4 independent experiments (n = 4 donors). *P < .05 compared with platelet-only control and #P < .05 compared with platelet-free AB002-only control (repeated measures ANOVA and Holm-Sidak 2-tailed post hoc tests). (B) Washed human platelets were activated by collagen and pretreated with blocking reagents for 1 hour: 40 µg/mL anti-TM or anti-endothelial protein C receptor (EPCR), or 20 µg/mL recombinant human receptor-associated protein (rhRAP). Anti-CD31 (40 µg/mL) was used as a negative control for antibody blocking. Pretreated platelets were incubated with 50 nM AB002 and 100 nM protein C or protein C alone, as indicated. Supernatants were collected after 1 hour and APC measured. Data are shown as the mean ± 1 standard deviation for 4 independent experiments (n = 4 donors). *P < .05 compared with platelet-only control and #P < .05 compared with platelets with AB002 control (repeated measures ANOVA and Holm-Sidak 2-tailed post hoc tests). (C) WT α-thrombin (WT) or AB002 were added to HEPES buffered saline (HBS) with or without TM, purified TAFI was added, and samples incubated at room temperature for the indicated times before evaluating the activation of TAFI. Samples containing 500 nM purified TAFI and 150 nM WT with or without 15 nM TM (lanes 1-4) or 150 nM AB002 with or without 15 nM TM (lanes 5-12) were subjected to automated western blot analysis. (D) AB002 was added to 5 nM WT and 5 nM TM in increasing concentrations from 5 to 200 nM and TAFI activity measured. The activity of WT in the absence of AB002 was set to 100%. (E) A standard aPTT reaction was performed in a KC4 coagulometer using either healthy or TAFI-deficient platelet-poor plasma; at the end of the incubation time, 2 µg/mL tPA with or without 10 µg/mL AB002 was added, and the reaction was recalcified to initiate clot formation. aPTT was measured and time to complete lysis of the clot was monitored. Horizontal bars denote mean of all data points plus or minus SEM. *P < .05 (independent Student t test). (F) Putative mechanism by which low doses of AB002 can rapidly interrupt developing thrombi through its ability to locally activate protein C on the platelet-rich thrombus luminal surface and by competitive inhibition of TAFI activation (TAFIa).

AB002 catalytically converts protein C to APC on activated platelet aggregates and inhibits thrombin-mediated TAFI activation. (A) Washed human platelets were activated and aggregated by collagen and treated with 50 nM AB002 or S195A-WE and 100 nM protein C. Platelets treated with protein C only and thrombin mutants incubated with protein C in the absence of platelets were prepared at the same time. Supernatants were collected after 1 hour and APC measured. Data are shown as the mean ± 1 standard deviation for 4 independent experiments (n = 4 donors). *P < .05 compared with platelet-only control and #P < .05 compared with platelet-free AB002-only control (repeated measures ANOVA and Holm-Sidak 2-tailed post hoc tests). (B) Washed human platelets were activated by collagen and pretreated with blocking reagents for 1 hour: 40 µg/mL anti-TM or anti-endothelial protein C receptor (EPCR), or 20 µg/mL recombinant human receptor-associated protein (rhRAP). Anti-CD31 (40 µg/mL) was used as a negative control for antibody blocking. Pretreated platelets were incubated with 50 nM AB002 and 100 nM protein C or protein C alone, as indicated. Supernatants were collected after 1 hour and APC measured. Data are shown as the mean ± 1 standard deviation for 4 independent experiments (n = 4 donors). *P < .05 compared with platelet-only control and #P < .05 compared with platelets with AB002 control (repeated measures ANOVA and Holm-Sidak 2-tailed post hoc tests). (C) WT α-thrombin (WT) or AB002 were added to HEPES buffered saline (HBS) with or without TM, purified TAFI was added, and samples incubated at room temperature for the indicated times before evaluating the activation of TAFI. Samples containing 500 nM purified TAFI and 150 nM WT with or without 15 nM TM (lanes 1-4) or 150 nM AB002 with or without 15 nM TM (lanes 5-12) were subjected to automated western blot analysis. (D) AB002 was added to 5 nM WT and 5 nM TM in increasing concentrations from 5 to 200 nM and TAFI activity measured. The activity of WT in the absence of AB002 was set to 100%. (E) A standard aPTT reaction was performed in a KC4 coagulometer using either healthy or TAFI-deficient platelet-poor plasma; at the end of the incubation time, 2 µg/mL tPA with or without 10 µg/mL AB002 was added, and the reaction was recalcified to initiate clot formation. aPTT was measured and time to complete lysis of the clot was monitored. Horizontal bars denote mean of all data points plus or minus SEM. *P < .05 (independent Student t test). (F) Putative mechanism by which low doses of AB002 can rapidly interrupt developing thrombi through its ability to locally activate protein C on the platelet-rich thrombus luminal surface and by competitive inhibition of TAFI activation (TAFIa).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal