Abstract
The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors. Recombinant Alboserpin displays stoichiometric, competitive, reversible and tight binding to FXa (picomolar range). Binding is highly specific and is not detectable for FX, catalytic site-blocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K(D) ~ 20 nM), but no change in FXa inhibition was observed in the presence of the cofactor, implying that bridging mechanisms did not take place. Notably, Alboserpin was also found to interact with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine. Further, annexin V (in the absence of Ca(2+)) or heparin outcompetes Alboserpin for binding to phospholipid vesicles, suggesting a common binding site. Consistent with its activity, Alboserpin blocks prothrombinase activity and increases both prothrombin time and activated partial thromboplastin time in vitro or ex vivo. Furthermore, Alboserpin prevents thrombus formation provoked by ferric chloride injury of the carotid artery and increases bleeding in a dose-dependent manner. Alboserpin emerges as an atypical serpin that targets FXa and displays unique phospholipid specificity. It conceivably uses heparin and phosphatidylcholine/phosphatidylethanolamine as anchors to increase protein localization and effective concentration at sites of injury, cell activation, or inflammation.
Highlights
To acquire a blood meal, hematophagous arthropods usually break the host’s skin stratum corneum (1, 2)
The molecular mechanism of factor Xa (FXa) inhibition by Alboserpin, the major salivary gland anticoagulant from the mosquito and yellow fever vector Aedes albopictus, has been characterized. cDNA of Alboserpin predicts a 45-kDa protein that belongs to the serpin family of protease inhibitors
The clotting cascade can be activated by the intrinsic or extrinsic pathways, both converging to the activation of factor X (FX)2 to FXa, which converts prothrombin to thrombin; the latter cleaves fibrinogen to produce fibrin (6)
Summary
To acquire a blood meal, hematophagous arthropods usually break the host’s skin stratum corneum (1, 2). In order to counteract host response to injury, salivary glands (SGs) of blood-sucking arthropods express a number of inhibitors of blood coagulation, platelet aggregation, host immunity, inflammation, angiogenesis, neutrophil function, wound healing, and vasodilation (1, 8 –16). Inhibitors targeting FVIIa/tissue factor, thrombin, FXa, FIXa, FXIIa, and high molecular weight kininogen have been reported (17–20). The only serine protease inhibitor (serpin) described so far and found to target FXa was identified in the SG of the mosquito Aedes aegypti (22). Serpins have been identified in species of all kingdoms These protease inhibitors act to control proteolytic activity in a myriad of contexts, including coagulation, fibrinolysis, and complement activation. They can be found in the intracellular compartment and perform biologic functions that do not necessarily require their protease-inhibitory function.
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