Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

Abstract

Thrombin-stimulated platelets expose very little phosphatidylserine (PS) but express binding sites for factor VIII (fVIII), casting doubt on the role of exposed PS as the determinant of binding sites. We previously reported that fVIII binding sites are increased three- to sixfold when soluble fibrin (SF) binds the αIIbβ3 integrin. This study focuses on the hypothesis that platelet-bound SF is the major source of fVIII binding sites. Less than 10% of fVIII was displaced from thrombin-stimulated platelets by lactadherin, a PS-binding protein, and an fVIII mutant defective in PS-dependent binding retained platelet affinity. Therefore, PS is not the determinant of most binding sites. FVIII bound immobilized SF and paralleled platelet binding in affinity, dependence on separation from von Willebrand factor, and mediation by the C2 domain. SF also enhanced activity of fVIII in the factor Xase complex by two- to fourfold. Monoclonal antibody (mAb) ESH8, against the fVIII C2 domain, inhibited binding of fVIII to SF and platelets but not to PS-containing vesicles. Similarly, mAb ESH4 against the C2 domain, inhibited >90% of platelet-dependent fVIII activity vs 35% of vesicle-supported activity. These results imply that platelet-bound SF is a component of functional fVIII binding sites.

Figure 1

Binding of fVIII to platelets and competition for fVIII binding sites. (A) fVIII-fluor, at various concentrations was mixed with platelets (1 × 10⁸/mL) stimulated with 10 µM A23187 + 1 u/mL thrombin for 10 minutes. The sample was diluted 10-fold and fVIII-fluor was added for 10 minutes. Samples were further diluted to 1 × 10⁶ platelets/mL and bound fVIII-fluor evaluated by flow cytometry. fVIII-fluor binding increased in a saturable manner. (B) fVIII-fluor (4 nM) was mixed with various concentrations of lactadherin prior to mixing with platelets stimulated by A23187 and thrombin. Lactadherin competed for at least 98% of binding sites indicating that PS is a critical determinant of most sites. (C) Platelets were stimulated with 1 u/mL thrombin alone for 1 minute prior to the addition of 3 u/mL hirudin. fVIII-fluor (4 nM) mixed with unlabeled fVIII or lactadherin at the indicated concentration was added to the platelets for 10 minutes prior to dilution and reading. Unlabeled fVIII competed for >95% of binding sites whereas lactadherin did not compete significantly. The fluorescence signal was corrected for the signal of unstimulated platelets with fVIII-fluor (supplemental Figure 2). Results are mean ± standard error of the mean (SEM) from 2 experiments for each panel.

Figure 2

Binding of fVIII-4Ala, with defective phospholipid affinity, to platelets. (A) fVIII-4Ala in complex with GMA8021-fluor were mixed with platelets stimulated as in Figure 1A. fVIII-4Ala had a 99% reduction in binding for A23187 + thrombin-stimulated platelets but a 55% reduction in binding to thrombin-stimulated platelets (B). Efficacy of fVIII or fVIII-4Ala in the factor Xase activity was evaluated in the presence of platelets stimulated with A23187 and thrombin (A23187) or by thrombin or PLVs with 4% or 20% PS content. The fVIII or fVIII-4Ala concentrations were 0.1 nM (A23187, 20% PS) or 1 nM (thrombin, 4% PS) mixed with factor IXa, 0.5 nM and factor X, 150 nM. Stimulated platelets, 1 × 10⁸/mL, or 20 μM PLVs (4:20:76 PS:PE:PC or 20:20:60 PS:PE:PC, extruded) were added with the simultaneous addition of 0.2 u/mL thrombin and 1.5 mM Ca⁺⁺. The reaction was stopped after 5 minutes by the addition of EDTA, and factor Xa was measured with chromogenic substrate S-2765. fVIII-4Ala supported ∼5% of the activity on A23187 + thrombin-stimulated platelets and ∼12% residual activity on thrombin-stimulated platelets. In contrast, fVIII-4Ala supported <1% residual activity on PLVs and activity was not significantly above background. Results in (A) are mean ± standard deviation (SD) for 4 measurements with the same fVIII-4Ala or fVIII concentrations between 1 to 8 nM and for (B) are mean ± SD from 2 experiments (A23187) or 3 experiments (thrombin, 4% PS and 20% PS) each performed in duplicate. PC, phosphatidylcholine; PE, phosphatidylethanolamine.

Figure 3

Binding of fVIII to fibrin. (A) Various concentrations of fVIII-fluor were incubated with SF immobilized on antifibrinogen Superose beads. After 10 minutes, bound fVIII was evaluated by flow cytometry. Displayed results represent mean ± range of duplicates. Saturable binding of fVIII-fluor was observed. (B) Various concentrations of VWF were incubated with 4 nM fVIII-fluor for 15 minutes prior to mixing with immobilized fibrin–anti-fibrinogen Superose beads. VWF inhibited fVIII binding to fibrin. (C) Various concentrations of fVIII-C2 were incubated with fVIII-fluor prior to mixing with fibrin–antifibrinogen Superose beads. Experiments were performed in tris-buffered saline containing 0.02 M NaCl. fVIII-C2 competed with fVIII-fluor for binding to immobilized fibrin. Displayed data are corrected for measured background fluorescence with control beads lacking fibrin. Displayed results are mean ± SEM for 3 such experiments; also representative of 6 additional experiments with slightly different conditions (A), 2 experiments (B), and 2 experiments (C).

Figure 4

Effect of fibrin on function of fVIII. (A) Various concentrations of fibrinogen were mixed with fVIII (0.5 nM), factor IXa (0.5 nM), factor X (150 nM), and PLVs (50 μM) (4:20:76 PS:PE:PC, extruded) prior to the simultaneous addition of 0.2 u/mL thrombin and 1.5 mM Ca⁺⁺. The reaction was stopped after 5 minutes by the addition of EDTA, and factor Xa was measured with chromogenic substrate S-2765. Fibrin increased activity ∼3.5-fold and the half-maximal effect was observed at ∼5 µg/mL. The smooth line represents a nonlinear least squares curve fit assuming a single, saturable fibrin-binding site (B). The experiment in (A) was repeated with PLVs of various PS content and 0.5 nM fVIII. All vesicles were extruded and had 20% PE with the balance as PC. Activity is displayed as the V_max / V₀ ratio with increasing fibrin from the respective curve fits (as in Figure 4A). Phospholipid concentrations were 1% PS (200 µM), 2% PS (100 µM), 4% PS (50 µM), and 8% and 16% PS (10 µM). Results are mean ± SD from 6 experiments (A) and from 2 experiments performed in duplicate for each vesicle type (B).

Figure 5

Effect of anti-fVIII mAbs on fVIII binding to fibrin. (A) fVIII-fluor, 4 nM, was incubated with 0.75 µg/mL ESH4 or 0.75 µg/mL ESH8 for 1 hour prior to mixing with fibrin–antifibrin–Superose or control beads lacking fibrin. ESH4 and ESH8 decreased binding to below control levels (*), observed when fVIII-fluor was incubated with control Superose beads. (B) fVIII was incubated with 10 μg/mL ESH4 or ESH8 for 1 hour at 23°C prior to the addition of factor IXa, factor X, thrombin, PLV, and Ca⁺⁺ as noted in Figure 4A description. In the absence of antibodies, the addition of 10 µg/mL fibrin increased Xase activity about twofold. Fibrin did not increase activity in the presence of ESH4 or ESH8 above the levels observed in the absence of fibrin. Results are from a single experiment representative of 4 experiments (A) and are mean ± SD for 4 experiments (B).

Figure 6

Effect of ESH4 and ESH8 on fVIII binding to thrombin-stimulated platelets. (A) fVIII-fluor was incubated with ESH8 for 1 hour on ice prior to mixing with platelets stimulated by thrombin as noted in Figure 1C description. The fVIII-fluor/antibody mix was added at a final fVIII concentration of 8 nM and allowed to bind for 10 minutes prior to dilution and reading. ESH8 decreased bound fVIII by ∼50%. (B) The same procedure was followed using ESH4. ESH4 inhibited ∼80% of fVIII binding. (C) ESH8 or ESH4, 10 µg/mL, was mixed with fVIII for 60 minutes prior to mixing with platelets (1 × 10⁸/mL), factor IXa, factor X, Ca⁺⁺, and thrombin. ESH8 inhibited 84% and ESH4 inhibited 78% of activity, respectively. To obtain average aggregate values, factor Xase activity was normalized to the value in the absence of ESH4 or ESH8 for each experiment. All data are corrected for the signal obtained from unstimulated platelets and normalized to facilitate comparison. Results are mean ± SEM for 3 experiments (A) and 4 experiments (B). (C) Mean ± SEM from 4 experiments, each performed in duplicate.

Figure 7

Effect of ESH4 and ESH8 on platelet-supported activity of fVIII. (A) Inhibition of 1 unit/mL fVIII activity by ESH4 and ESH8 was evaluated against an fVIII concentration curve in reconstituted platelet-rich plasma. Clotting was initiated by the simultaneous addition of factor XIa, thrombin receptor activation peptides, and Ca⁺⁺. A log-linear plot demonstrates sensitivity to fVIII. ESH4 or ESH8 was incubated with fVIII in the presence of 10% fVIII-deficient plasma for 1 hour prior to mixing with additional fVIII-deficient plasma and initiation of clotting. ESH4 inhibited activity to a greater extent than ESH8. Results are mean ± SEM for triplicates, representative of 3 experiments performed in full and 5 performed with fewer fVIII concentrations. (B) Bar graph comparing residual fVIII activity in various plasma-based activities. aPTT and chromogenic assay results represents mean ± SEM using commercial aPTT and chromogenic reagents. Inhibition was also evaluated in reconstituted plasma and platelet-rich plasma lacking VWF (aPTT [-VWF]), (act platelet [-VWF]). Results are from 4 experiments (aPTT, chromogenic); activated platelets mean ± SD for 3 experiments, activated platelets without VWF (1 experiment). (C) Bar graph comparing residual fVIII activity in the presence of 10 µg/mL ESH8. aPTT without VWF (aPTT [-VWF]). Values on activated platelets are mean ± SEM for 2 experiments and 1 experiment for plasma lacking VWF.