Figure 4
Figure 4. Western blot analysis of limiting (A) and excess (B) TF triggered thrombin generation in mock-depleted plasma with or without DHG. Thrombin generation was initiated with 0.2 (A) or 4 (B) pM human TF, 8.3 μM PC:PS vesicles, and 40 μg/mL CTI (plasma concentrations) in mock-depleted plasma in the absence and presence of 0.5 μM or 2.5 μM DHG. Individual reactions were quenched over time with loading buffer containing 5 M urea and analyzed by SDS-PAGE under nonreducing conditions as described in “The time course of plasma thrombin generation by Western blot analysis.” Proteins transferred to Immobilon-P were detected with a polyclonal sheep anti-human thrombin primary antibody, followed by a peroxidase-conjugated affinity purified donkey anti-sheep immunoglobulin G (IgG), and subsequent development of signal with chemiluminescent substrate. Prothrombin indicates prothrombin/meizothrombin band. TAT indicates thrombin-antithrombin complex, and HCII-thrombin indicates heparin cofactor II-thrombin complex.

Western blot analysis of limiting (A) and excess (B) TF triggered thrombin generation in mock-depleted plasma with or without DHG. Thrombin generation was initiated with 0.2 (A) or 4 (B) pM human TF, 8.3 μM PC:PS vesicles, and 40 μg/mL CTI (plasma concentrations) in mock-depleted plasma in the absence and presence of 0.5 μM or 2.5 μM DHG. Individual reactions were quenched over time with loading buffer containing 5 M urea and analyzed by SDS-PAGE under nonreducing conditions as described in “The time course of plasma thrombin generation by Western blot analysis.” Proteins transferred to Immobilon-P were detected with a polyclonal sheep anti-human thrombin primary antibody, followed by a peroxidase-conjugated affinity purified donkey anti-sheep immunoglobulin G (IgG), and subsequent development of signal with chemiluminescent substrate. Prothrombin indicates prothrombin/meizothrombin band. TAT indicates thrombin-antithrombin complex, and HCII-thrombin indicates heparin cofactor II-thrombin complex.

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