High-affinity, specific factor IXa binding to platelets is mediated in part by residues 3-11.
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)
Ahmad
S S
SS
Sol Sherry Thrombosis Research Center, Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Rawala-Sheikh
R
R
Cheung
W F
WF
Jameson
B A
BA
Stafford
D W
DW
Walsh
P N
PN
D015055:1-Carboxyglutamic Acid / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism*; D002417:Cattle; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D015949:Factor IXa / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015944:Factor VIIIa / Q000378:metabolism*; D005170:Factor X / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; D015055:1-Carboxyglutamic Acid; D015944:Factor VIIIa; D005170:Factor X; D015949:Factor IXa; D013917:Thrombin