Figure 6
Figure 6. Competition assays. Fluorescein-labeled C1C2 (C1C2*), C2 (C2*), or FVIIIa (FVIIIa*) were added to activated platelets. The bound, labeled proteins were competed by adding increasing amounts of unlabeled C2, C1C2, FVIII, or FVIIIa; alternatively, platelet binding was examined after preincubation with a monoclonal antibody, ESH4. (A) C1C2* competition by C1C2 or C2: 0.125 μM C1C2* binding was reduced from 88% (left) to 5% after a 10-fold molar excess of unlabeled C1C2 (center), but adding a 20-fold molar excess of unlabeled C2 reduced the percentage of labeled platelets to only 34% (right). (B) C1C2* competition by ESH4: 0.125 μM C1C2* binding was reduced from 92% to 21% by adding equimolar ESH4 and to 16% with a 5-fold molar excess of ESH4. (C) C1C2* or C2* competition by FVIII or FVIIIa: 0.25 μM C1C2* (left) or C2* (right) was competed by adding unlabeled FVIII () or FVIIIa (); controls are shown as (). Before competition, binding was near saturation for C1C2* and for C2*; however, note the different scales for the left and right panels, which indicate that binding of C2* to activated platelets was lower than for C1C2*. Data represent the mean and standard deviations (upper error bars shown) for measurements using 6 different platelet preparations. Representative data for one of the C1C2* competition experiments plotted in histogram and scattergram format are available in Figure S1A,B, available on the Blood website; see the Supplemental Figure link at the top of the online article. (D) FVIIIa* competition by FVIIIa, C1C2, or C2: 10 nM FVIIIa* (near-saturation binding, Figure 5) was competed by FVIIIa (left panel, ), C1C2 (center panel, ), or C2 (right panel, ) at molar ratios as indicated. Data represent the mean and standard deviations (upper error bars shown) for measurements using 5 (for right and center panels) and 4 (for left panel) different platelet preparations. Representative data from one of the FVIIIa*-C1C2 competition experiments plotted in histogram and scattergram format are available in Figure S1C,D.

Competition assays. Fluorescein-labeled C1C2 (C1C2*), C2 (C2*), or FVIIIa (FVIIIa*) were added to activated platelets. The bound, labeled proteins were competed by adding increasing amounts of unlabeled C2, C1C2, FVIII, or FVIIIa; alternatively, platelet binding was examined after preincubation with a monoclonal antibody, ESH4. (A) C1C2* competition by C1C2 or C2: 0.125 μM C1C2* binding was reduced from 88% (left) to 5% after a 10-fold molar excess of unlabeled C1C2 (center), but adding a 20-fold molar excess of unlabeled C2 reduced the percentage of labeled platelets to only 34% (right). (B) C1C2* competition by ESH4: 0.125 μM C1C2* binding was reduced from 92% to 21% by adding equimolar ESH4 and to 16% with a 5-fold molar excess of ESH4. (C) C1C2* or C2* competition by FVIII or FVIIIa: 0.25 μM C1C2* (left) or C2* (right) was competed by adding unlabeled FVIII () or FVIIIa (); controls are shown as (). Before competition, binding was near saturation for C1C2* and for C2*; however, note the different scales for the left and right panels, which indicate that binding of C2* to activated platelets was lower than for C1C2*. Data represent the mean and standard deviations (upper error bars shown) for measurements using 6 different platelet preparations. Representative data for one of the C1C2* competition experiments plotted in histogram and scattergram format are available in Figure S1A,B, available on the Blood website; see the Supplemental Figure link at the top of the online article. (D) FVIIIa* competition by FVIIIa, C1C2, or C2: 10 nM FVIIIa* (near-saturation binding, Figure 5) was competed by FVIIIa (left panel, ), C1C2 (center panel, ), or C2 (right panel, ) at molar ratios as indicated. Data represent the mean and standard deviations (upper error bars shown) for measurements using 5 (for right and center panels) and 4 (for left panel) different platelet preparations. Representative data from one of the FVIIIa*-C1C2 competition experiments plotted in histogram and scattergram format are available in Figure S1C,D.

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