Total inhibition of high shear stress induced platelet aggregation by homodimeric von Willebrand factor A1-loop fragments.
Under high shear stress, the binding of von Willebrand factor (VWF) A1-loop domain to platelet glycoprotein (GP) Ib alpha occurs as the earliest event in thrombus formation. Therefore recombinant VWF A1-loop fragments could be of therapeutic use in blocking this interaction as competing ligands. We have prepared three homodimeric VWF A1-loop fragments [315 kD Fr III (a homodimer of amino acid [aa] residues 1-1365 of the subunit), 220 kD Fr (a homodimer of aa residues 1-708 of the subunit), and 116 kD Fr (a homodimer of aa residues 449-728 of the subunit) and two monomeric fragments [39/34 kD Fr (a monomer of aa residues 480/481-718 of the subunit] and His-rVWF465-728 (a monomer of aa residues 465-728 of the subunit)], and assessed their potency as inhibitors of botrocetin-induced VWF binding to GPIb alpha and high shear stress induced platelet aggregation mediated by intact VWE All these fragments completely inhibited botrocetin-induced VWF binding to GPIb alpha at a final concentration of 40-200 microM. The homodimeric A1-loop fragments also totally inhibited high shear stress induced platelet aggregation at a final concentration of 0.45-2.0 microM in the following order: 315 kD Fr > or = 220 kD Fr >> 116 kD Fr. In contrast, the monomeric A1-loop fragments were only partial inhibitors of high shear stress induced platelet aggregation even at a final concentration of 20 microM, and their IC50s were 13-39 times higher than those of the homodimers. These results indicate that the homodimeric structure of the A1-loop fragment is important for optimal molecular interaction with GPIb alpha under high shear stress.
Miura
S
S
Department of Blood Transfusion Medicine, Nara Medical University, Kashihara, Japan.
Sakurai
Y
Y
Takatsuka
H
H
Yoshioka
A
A
Matsumoto
M
M
Yagi
H
H
Kokubo
T
T
Ikeda
Y
Y
Matsui
T
T
Titani
K
K
Fujimura
Y
Y
D006801:Humans; D008666:Metalloendopeptidases / Q000494:pharmacology; D010446:Peptide Fragments; D010974:Platelet Aggregation / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D013314:Stress, Mechanical; D014841:von Willebrand Factor / Q000378:metabolism*
D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D019038:Platelet Glycoprotein GPIb-IX Complex; D014841:von Willebrand Factor; D008666:Metalloendopeptidases