Effects of annexin V, anti-PS mAb, anti-TF mAb, and anti-FVII mAb on the procoagulant activities of SkM, CM, and PC/PS vesicles. (A-B) Annexin V (A) and anti-PS mAb 11.31 (B) were tested for inhibition of prothrombinase activity supported by SkM, CM, or PC/PS LUVs (PC/PS, 80%/20% w/w). Each inhibitor was incubated with FVa (2 nM, final) and FXa (0.24 nM, final) in Tris buffered saline (TBS) containing 0.5% bovine serum albumin (TBSA) and 5 mM CaCl₂ in the presence of SkM (2.5 nM final), CM (2.5 nM final), or PC/PS vesicles (10 nM final). Addition of prothrombin (0.75 µM final) at room temperature initiated thrombin generation for 5 minutes that was quenched by EDTA (10 mM final). Thrombin activity was quantified by hydrolysis of the thrombin substrate (H-D-Phe-Pip-Arg-pNA). (C) Anti-PS mAb 11.31 was tested for inhibition of the prothrombinase activity supported by different sizes of PC/PS (80%/20% w/w) vesicles, as previously described. Large LMVs were prepared in TBS by vortexing a dried lipid suspension until all lipid was suspended.¹⁰  SUVs were prepared by sonication as described.^1-3  LUVs were prepared by extruding LMV suspensions 20 times back and forth (total of 10 passes through the membrane) through a 0.1-μm polycarbonate membrane with a Mini-Extruder (Avanti Polar Lipids).¹⁰  (D) The effects of SkM, CM, or PC/PS LUV (PC/PS, 80%/20% w/w) on the initial rate of prothrombin (solid circles) or DG-prothrombin (open circles) activation by FXa and FVa were determined. Various concentrations of SkM, CM, or PC/PS (80%/20%) LUVs were incubated with FVa (2.4 nM final) and FXa (0.2 nM final) in TBSA plus 5 mM CaCl₂. Thrombin generation was initiated by the addition of prothrombin or DG-prothrombin (0.75 μM final) and, after 10 minutes, was quenched by adding EDTA (10 mM final). The rate of thrombin formation was quantified by hydrolysis of thrombin fluorogenetic substrate. (E) FX activation assays were used to determine TF-like activity in SkM and CM preparations. Various concentrations of human or rabbit TF (Innovin, Dade or Pacific Hemostasis Thromboplastin-DS), SkM, or CM were mixed with FVIIa (0.5 nM final) in TBSA 5 mM CaCl₂, and the addition of FX (0.25 μM final) initiated the reaction. After 10 minutes at room temperature, the addition of EDTA (10 mM final) quenched the reaction, and the rate of FXa formation was quantified by hydrolysis of an FXa chromogenic substrate (BIOPHEN CS11 [22]; Aniara, West Chester, OH). (F) The inhibitory effect of anti-FVII mAb on FX activation by FVIIa with human or rabbit TF was determined. Anti-FVII mAb 3G12¹⁵  (20 µg/mL final) was preincubated with FVIIa for 10 minutes at room temperature. Then TF (1.5 pM) with FX (final 250 nM) plus 5 mM CaCl₂ was added and, after 10 minutes the reaction was quenched with EDTA (10 mM final). The rate of FXa formation was quantified by FXa activity (BIOPHEN CS11 [22]). (G) The effects of anti-FVII mAb or anti-TF mAb 3A5 on FX activation by FVIIa in the presence of rabbit TF, SkM, or CM was determined. Preincubation of anti-FVII mAb 3G12 (20 µg/mL, final) with FVIIa (0.5 nM final) for 10 minutes at room temperature was followed by the addition of FX (250 nM final) with rabbit TF (1.5 pM) or SkM (300 nM) or CM (50 nM) to start the reaction. After 10 minutes, the reaction was quenched by adding EDTA (10 mM final) and FXa activity (BIOPHEN CS11 [22]) was determined. For the anti-TF mAb experiment, preincubation of anti-TF mAb (RB TF 7-3A5)¹⁶  (20 µg/mL) with rabbit TF (1.5 pM) or SkM (300 nM) or CM (50 nM) for 10 minutes at room temperature was followed by the addition of FVIIa (0.5 nM final) and then FX (final 250 nM) in TBSA plus 5 mM CaCl₂ to start the reaction. After 10 minutes, the reaction was quenched by adding EDTA (10 mM final) and FXa activity (BIOPHEN CS11 [22]) was determined. The 100% value was defined as FXa generated in the absence of added mAb. (H-J) The effects of anti-TF 3A5 mAb and anti-FVII 3G12 mAb on the procoagulant activity of SkM and CM in modified APTT plasma clotting assays that were previously described for the procoagulant activity of myosins.⁴  After preincubating SkM (H-I) or CM (J) or vehicle control buffer aliquots with anti-TF mAb (50 µg/mL) (H) or anti-FVII mAb (20 µg/mL) (I-J) for 10 minutes, the procoagulant activity was determined in the APTT assays. Each study included clotting assays for SkM and CM performed with (dashed lines) or without (solid lines) the addition of PC/PS (80%/20%) LUVs (1.0 µM final). For all studies in Figure 1 using SkM and CM, as for our published studies,^1-5,17  the SkM preparation was dissolved and then dialyzed in Tris buffer (pH 7.4) containing 0.6 M NaCl at 4°C. After dialysis, particles causing turbidity were removed by high speed centrifugation (21 130g for 1 minute), which removed visible aggregates.