High-Affinity, Specific Factor IXa Binding to Platelets Is Mediated in Part by Residues 3-11

Abstract

To identify the amino acids in the Gla domain that mediate factor IXa binding to human platelets, we have used chimeric molecules and point mutations in the Gla domain of recombinant factor IX, based on molecular modeling using the coordinates of the Gla domain of bovine prothrombin, which reveals two surface structures whose sequences differ among factor IX, factor X, and factor VII. Binding to thrombin-activated platelets of factor IXa in the presence of factor VIIIa (2 units/mL) and factor X (1.5 microM) revealed a stoichiometry of approximately 550 sites per platelet with a Kd of approximately 0.65 nM compared with a Kd of approximately 2.5 nM in the absence of factor VIIIa and factor X. In contrast, mutations of factor IX to factor X residues at positions 4 and 5 or at positions 9, 10, and 11 results in decreases in the number of sites and affinity of factor IXa binding in the presence or absence of factor VIIIa and factor X. A chimera consisting of the Gla domain of factor VII with factor IX residues at positions 33, 34, 35, 39, and 40 displayed abnormal factor IXa binding and a decreased Vmax and a normal Km for factor X activation, and the replacement of amino acid residues 3-10 with those of factor IX restored normal binding and factor X activation kinetics to this chimeric protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombin Activation of Factor XI on Activated Platelets Requires the Interaction of Factor XI and Platelet Glycoprotein Ibα with Thrombin Anion-binding Exosites I and II, Respectively

“…In a current model of FXI activation, the circulating FXI dimer is complexed with either HMWK (17)(18)(19) or prothrombin (18,20), both of which bind to the A1 domain of FXI (21).…”

Mutations associated with hemophilia A in the 558-565 loop of the factor VIIIa A2 subunit alter the catalytic activity of the factor Xase complex

“…FVIIIa binds to phospholipid via the C2 domain 33,34 and FIXa binds to the phospholipid surface via the amino-terminal 3-11 residues of the Gla domain. 35,36 This association facilitates the orientation and interaction of the proteins and has the effect of increasing the affinity of the FVIIIa-FIXa interaction. [37][38][39] The FVIIIa-FIXa interaction in the absence of phospholipid is less efficient, with the rate enhancement component due to the orientation effect of phospholipid removed.…”

Relocating the Active Site of Activated Protein C Eliminates the Need for Its Protein S Cofactor

“…Consistent with this view, exchanging the Gla domain of factor VIIa for that of APC had no affect on APC plasma anticoagulant activity (18). On the other hand, replacing the Gla domain of factor IXa with that of factor VIIa decreased the V max for factor X activation (19). Furthermore, the membrane binding affinities of vitamin K-dependent plasma proteins differ (e.g.…”