Identification of amino acid residues essential for heparin binding by the A1 domain of human von Willebrand factor.
Platelet adhesion is mediated by von Willebrand factor (VWF) that binds platelet glycoprotein Ib (GPIb). Previous observations suggested that heparin competitively inhibits the binding of VWF to GPIb and may down-regulate platelet adhesion. We performed charged-to-alanine scanning mutagenesis of domain A1 and studied dose-dependent binding to heparin-Sepharose beads. Mutations at Lys1362 and Arg1395, at which the GPIb binding was markedly decreased, showed 41% and 42% binding, respectively. Clustered mutations in the segments 1332KDRKR1336 and 1405KKKK1408, which have been proposed as heparin binding sequences, showed 72% and 52% binding, respectively. However, single alanine substitutions within these clusters showed normal binding. Our findings suggest that heparin may inhibit the binding of VWF to GPIb by interacting with GPIb binding and interpret why some hemorrhagic complications of heparin therapy are not predictable based on techniques for monitoring the conventional anticoagulant effects of heparin.
Adachi
Tatsuya
T
Department of Hematology, Nagoya University Graduate School of Medicine, Nagoya, Japan.
Matsushita
Tadashi
T
Dong
Zhengyu
Z
Katsumi
Akira
A
Nakayama
Takayuki
T
Kojima
Tetsuhito
T
Saito
Hidehiko
H
Sadler
J Evan
JE
Naoe
Tomoki
T
D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry*; D001665:Binding Sites; D003198:Computer Simulation; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry*; D006801:Humans; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010980:Platelet Membrane Glycoproteins / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry; D014841:von Willebrand Factor / Q000737:chemistry*
D000596:Amino Acids; D016326:Extracellular Matrix Proteins; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; C460760:VWA1 protein, human; D014841:von Willebrand Factor; C028254:von Willebrand factor receptor; D006493:Heparin