Diannexin, an annexin A5 homodimer, binds phosphatidylserine with high affinity and is a potent inhibitor of platelet-mediated events during thrombus formation

Background: Shielding of procoagulant phosphatidylserine (PS) with annexin A5 attenuates thrombosis, but annexin A5 (35.7 kDa) is rapidly cleared from the circulation. In contrast, Diannexin, a 73.1 kDa homodimer of annexin A5, has an extended half-life. Objectives: To quantify the affinity of Diannexin for PS, examine its interaction with activated platelets and determine its effects on platelet-mediated events during thrombus formation. Methods: The affinities of Diannexin and annexin A5 for PS-containing lipid bilayers were compared using surface plasmon resonance, and binding to activated platelets was assessed by flow cytometry. Calibrated automated thrombography and thromboelastography were employed to study the effects of Diannexin on thrombin generation and platelet-fibrin clot formation, respectively, whereas intravital videomicroscopy was used to examine its effect on platelet accumulation and activation after laser-induced injury to murine cremaster arterioles, and a tail tip bleeding model was used to explore its effects on hemostasis. Results: Diannexin and annexin A5 bind PS with K-D values of 0.6 and 5 nM, respectively, and both bind to the same subpopulation of PS-exposing platelets. Diannexin inhibited thrombin generation and platelet-fibrin clot formation in vitro at 10 nM (P < 0.05-0.001 compared with control), and reduced platelet accumulation at 1 mu g g(-1) (P < 0.05) and activation at 0.25 mu g g(-1) (P < 0.001) in experimentally induced arterial thrombi in mice while increasing blood loss at 1 mu g g(-1) (P < 0.01). Conclusions: Diannexin binds to PS with high affinity and is a potent inhibitor of platelet-mediated events during thrombus formation.