Alboserpin, a factor Xa inhibitor from the mosquito vector of yellow fever, binds heparin and membrane phospholipids and exhibits antithrombotic activity

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.     

Ligand Binding Shuttles Thrombin along a Continuum of Zymogen- and Proteinase-like States

The critical and multiple roles of thrombin in blood coagulation are regulated by ligands and cofactors. Zymogen activation imparts proteolytic activity to thrombin and also affects the binding of ligands to its two principal exosites. We have used the activation peptide fragment 1.2 (F12), a ligand for anion-binding exosite 2, to probe the zymogenicity of thrombin by isothermal titration calorimetry. We show that F12 binding is sensitive to subtle aspects of proteinase formation beyond simply reporting on zymogen cleavage. Large thermodynamic differences in F12 binding distinguish between a series of thrombin species poised along the transition of zymogen to proteinase. Active-site ligands transitioned a zymogen-like state to a proteinase-like state. Conversely, removal of Na⁺ converted proteinase-like thrombin to a more zymogen-like form. Thrombin mutants, with deformed x-ray structures, previously considered to be emblematic of specific regulated states of the enzyme, are instead shown to be variously zymogen-like and can be made proteinase-like by active-site ligation. Thermodynamic linkage between anion-binding exosite 2, the Na⁺-binding site, and the active site arises from interconversions of thrombin between a continuum of zymogen- and proteinase-like states. These interconversions, reciprocally regulated by different ligands, cast new light on the problem of thrombin allostery and provide a thermodynamic framework to explain the regulation of thrombin by different ligands.     

Replacing the Factor VIII C1 Domain with a Second C2 Domain Reduces Factor VIII Stability and Affinity for Factor IXa

Factor VIII (FVIII) consists of a heavy chain (A1(a1)A2(a2)B domains) and light chain ((a3)A3C1C2 domains). To gain insights into a role of the FVIII C domains, we eliminated the C1 domain by replacing it with the homologous C2 domain. FVIII stability of the mutant (FVIII_C2C2) as measured by thermal decay at 55 °C of FVIII activity was markedly reduced (∼11-fold), whereas the decay rate of FVIIIa due to A2 subunit dissociation was similar to WT FVIIIa. The binding affinity of FVIII_C2C2 for phospholipid membranes as measured by fluorescence resonance energy transfer was modestly lower (∼2.8-fold) than that for WT FVIII. Among several anti-FVIII antibodies tested (anti-C1 (GMA8011), anti-C2 (ESH4 and ESH8), and anti-A3 (2D2) antibody), only ESH4 inhibited membrane binding of both WT FVIII and FVIII_C2C2. FVIIIa cofactor activity measured in the presence of each of the above antibodies was examined by FXa generation assays. The activity of WT FVIIIa was inhibited by both GMA8011 and ESH4, whereas the activity of FVIII_C2C2 was inhibited by both the anti-C2 antibodies, ESH4 and ESH8. Interestingly, factor IXa (FIXa) binding affinity for WT FVIIIa was significantly reduced in the presence of GMA8011 (∼10-fold), whereas the anti-C2 antibodies reduced FIXa binding affinity of FVIII_C2C2 variant (∼4-fold). Together, the reduced stability plus impaired FIXa interaction of FVIII_C2C2 suggest that the C1 domain resides in close proximity to FIXa in the FXase complex and contributes a critical role to FVIII structure and function.