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

Abstract

Summary. 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 KD 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 μg g−1 (P < 0.05) and activation at 0.25 μg g−1 (P < 0.001) in experimentally induced arterial thrombi in mice while increasing blood loss at 1 μg 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.

Atorvastatin Delays Murine Platelet Activation In Vivo Even in the Absence of Endothelial NO Synthase

“…This finding provides further validation for this highspeed, 2-color microscopy method for evaluating the effects of inhibitors on the markers of platelet activation in vivo. 24 The observation that atorvastatin delayed CD41 upregulation in eNOS-deficient mice, despite the fact that it only inhibited thrombin-and GYP-induced platelet activation, is consistent with previous observations that platelet activation in the laserinduced arteriolar model is thrombin dependent. 41,42 In conclusion, we have extended our understanding of how statins inhibit platelet activation.…”

“…24 Using the jugular vein cannulus, mice were given an infusion of 0.1 µg/g X488. Where indicated, an infusion of 1.3 µg/g of DyLight-647-tagged F ab fragments of rat anti-mouse CD41 (MWREG30, Emfret Analytics), a monoclonal antibody directed against α IIb that recognizes both the resting and activated form of this platelet-specific integrin, or 2 µg/g of DyLight-647-tagged antibody against P-selectin (RB40.34, Becton Dickinson) was also administered.…”

“…25 Fluorophore conjugation and F ab fragment preparation were performed according to the directions from the supplier (Pierce). After laser injury to cremaster arterioles, platelet accumulation and activation were measured and analyzed as previously described, 24 except that platelet activation was assessed on the basis of both CD41 upregulation and P-selectin surface expression. Three-dimensional movies were generated using Slidebook (Version 5, Intelligent Imaging Innovations, Denver, CO).…”

Annexins – insights from knockout mice

“…Mechanistically, the ability of AnxA5 to form two-dimensional crystal lattices on phosphatidylserine-rich membranes in a Ca 2+ -dependent manner in vitro seems most relevant (van Genderen et al, 2008;Rand et al, 2010). Hence, the AnxA5 KO animals could become a suitable model to further investigate therapeutic approaches to prevent inappropriate thrombogenesis causing infertility by the antiphospholipid syndrome in humans (Rand et al, 2012).…”

Analysis of procoagulant phosphatidylserine‐exposing platelets by imaging flow cytometry

“…A critical role has emerged for the procoagulant platelet subpopulation in regulating both normal hemostasis and pathological thrombus formation 1, 3. Indeed, we have previously demonstrated that PS exposure on activated platelets persists in vitro,13 and in vivo,14 and that blocking the procoagulant surface inhibits platelet‐mediated events in experimentally induced arterial thrombosis 15…”