Alboserpin, a Factor Xa Inhibitor from the Mosquito Vector of Yellow Fever, Binds Heparin and Membrane Phospholipids and Exhibits Antithrombotic Activity

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 siteblocked FXa, thrombin, and 12 other enzymes. Alboserpin displays high affinity binding to heparin (K D ϳ 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.

Detection of Heparin in the Salivary Gland and Midgut of Aedes togoi

“…In a previous study [4], heparin exhibited a high affinity to alboserpin, which is one of the major anticoagulants in the salivary gland of Aedes albopictus . The binding between heparin and alboserpin interacts with phosphatidylcholine and phosphatidylethanolamine but not with phosphatidylserine and exhibits potent antithrombotic properties under in vivo conditions.…”

Serpins in arthropod biology

“…albopictus [100–102]. This serpin prevents coagulation in vitro and in vivo by inhibition of Factor Xa using an atypical mode of inhibition characterized by reversible interaction of proteinase and inhibitor at a 1:1 stoichiometry [100,101]. Alboserpin also binds phospholipids and heparin, and possibly other glycosaminoglycans.…”

“…Alboserpin also binds phospholipids and heparin, and possibly other glycosaminoglycans. While neither of these binding activities are required nor promote Factor Xa inhibition, they are likely to facilitate alboserpin localization to the site of injury and/or platelet aggregation [100]. However, alboserpin seems to be an evolutionary exception, and the saliva of mosquitoes does not contain a large panel of serpins.…”

The roles of serpins in mosquito immunology and physiology

“…Alboserpin, the ortholog of AFXa from Ae. albopictus is likewise present in female salivary glands, and is a functional FXa inhibitor (Calvo et al, 2011). Both SRPNs use an inhibitory mechanism that is divergent from the classical serpin suicide-substrate mechanism.…”