Competition for factor VIII– and factor V–binding sites by lactadherin or annexin V.

(A) First, 4 nM fluorescein-labeled factor VIII was mixed with the indicated concentration of competitor, lactadherin (▴) or annexin V (●), in the presence of 1.5 mM Ca⁺⁺. Lipospheres were added, and bound factor VIII was evaluated by flow cytometry after 10 minutes. Liposphere membrane composition of PS-PE-PC was 4:20:76. The inhibition curve was modeled under the assumption that theK_d of factor VIII with lipospheres was 4.8 nM; the K_i (×) for lactadherin, 0.5 nM.16 (B) First, 4 nM fluorescein-labeled factor V was mixed with lactadherin (▴) or annexin V (●), and bound factor V was evaluated under the same conditions as those in panel A. The inhibition curve was modeled under the assumption that theK_d of factor V with lipospheres was 4.3 nM; theK_i (×) for lactadherin, 1.0 nM.16(C) Lactadherin (▴) or annexin V (●) was mixed with 0.1 nM factor IXa, 1 nM factor VIII, and 100 nM factor X, prior to the addition of 1 μM sonicated vesicles with 1.5 mM Ca⁺⁺ and thrombin. The reaction was stopped after 5 minutes, and factor Xa was measured with chromogenic substrate S-2765 in a kinetic microplate reader. Lactadherin was an effective competitor for binding sites of factor VIII and factor V and an inhibitor of the factor Xase complex. Results displayed are from a single experiment representative of at least 2 experiments for all conditions.