Abstract
Blood platelets are required for normal wound healing, but they are also involved in thrombotic diseases, which are usually managed with anticoagulant drugs. Here, using genetic engineering, we coupled the disintegrin protein echistatin, which specifically binds to the platelet integrin αIIbβ3 receptor, to annexin V, which binds platelet membrane-associated phosphatidylserine (PS), to create the bifunctional antithrombotic molecule recombinant echistatin-annexin V fusion protein (r-EchAV). Lipid binding and plasma coagulation studies revealed that r-EchAV dose-dependently binds PS and delays plasma clotting time. Moreover, r-EchAV inhibited ADP-induced platelet aggregation in a dose-dependent manner and exhibited potent antiplatelet aggregation effects. r-EchAV significantly prolonged activated partial thromboplastin time, suggesting that it primarily affects the in vivo coagulation pathway. Flow cytometry results indicated that r-EchAV could effectively bind to the platelet αIIbβ3 receptor, indicating that r-EchAV retains echistatin's receptor-recognition region. In vivo experiments in mice disclosed that r-EchAV significantly prolongs bleeding time, indicating a significant anticoagulant effect in vivo resulting from the joint binding of r-EchAV to both PS and the αIIbβ3 receptor. We also report optimization of the r-EchAV production steps and its purification for high purity and yield. Our findings indicate that r-EchAV retains the active structural regions of echistatin and annexin V and that the whole molecule exhibits multitarget-binding ability arising from the dual functions of echistatin and annexin V. Therefore, r-EchAV represents a new class of anticoagulant that specifically targets the anionic membrane-associated coagulation enzyme complexes at thrombogenesis sites and may be a potentially useful antithrombotic agent.
Highlights
Blood platelets are required for normal wound healing, but they are involved in thrombotic diseases, which are usually managed with anticoagulant drugs
Using genetic engineering, we coupled the disintegrin protein echistatin, which binds to the platelet integrin ␣IIb3 receptor, to annexin V, which binds platelet membrane-associated phosphatidylserine (PS), to create the bifunctional antithrombotic molecule recombinant echistatin–annexin V fusion protein (r-EchAV)
Our findings indicate that r-EchAV retains the active structural regions of echistatin and annexin V and that the whole molecule exhibits multitarget-binding ability arising from the dual functions of echistatin and annexin V
Summary
An ␣IIb3- and phosphatidylserine (PS)-binding recombinant fusion protein promotes PS-dependent anticoagulation and integrin-dependent antithrombosis. Using genetic engineering, we coupled the disintegrin protein echistatin, which binds to the platelet integrin ␣IIb3 receptor, to annexin V, which binds platelet membrane-associated phosphatidylserine (PS), to create the bifunctional antithrombotic molecule recombinant echistatin–annexin V fusion protein (r-EchAV). To improve the efficacy of the drug while not increasing the amount of drug used and reducing drug use costs, we attempted to couple echistatin (ECH), which binds to the platelet ␣IIb3 integrin receptor, with annexin V (ANV), which binds to the platelet membrane PS molecule, by genetic engineering techniques to form an efficient bifunctional recombinant antithrombotic molecule r-EchAV. ECH can efficiently bind to the integrin receptor ␣IIb3 exposed on the surface of activated platelets, inhibiting platelet aggregation efficiently to achieve an antithrombotic effect; ANV binds a large number of PS molecules valgus to the surface of activated platelets in a calcium-dependent manner. The preparation process and physicochemical characteristics of r-EchAV were studied and elaborated
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