Conjugation of von Willebrand factor-binding domain of platelet glycoprotein Ib alpha to size-controlled albumin microspheres.
Albumin microspheres (AMS), of which the average diameter is 240 +/- 10 nm, were prepared by pH control and heat treatment. Cytochrome c and rGPIb alpha; a water-soluble fragment of the alpha chain of a recombinant platelet glycoprotein (GP)Ib containing a von Willebrand factor (vWf)-binding site were selected as a receptor protein. Cytochrome c was used as a probe protein for monitoring. Onto the surface of the AMS and those proteins, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was reacted through the amide linkage to obtain PD-AMS, PD-cytochrome c, and PD-rGPIb alpha, respectively. The latter two were further reduced to SH-cytochrome c and SH-rGPIb alpha by dithiothreitol and conjugated with PD-AMS by a thiol-disulfide exchange reaction. The resulting AMS contain cytochrome c or rGPIb alpha of about 25,000 or 2500 molecules, respectively. The addition of ristocetin to the rGPIb alpha-AMS in the presence of vWf caused specific aggregation. Furthermore, the rGPIb alpha-AMS enhanced the ristocetin induced platelet aggregation in a low platelet concentration (4.0 x 10(7)/mL).
Takeoka
S
S
Department of Polymer Chemistry, Advanced Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555, Japan.
Teramura
Y
Y
Ohkawa
H
H
Ikeda
Y
Y
Tsuchida
E
E
D001792:Blood Platelets / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003574:Cytochrome c Group / Q000737:chemistry; D016503:Drug Delivery Systems; D006801:Humans; D008863:Microspheres; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D014841:von Willebrand Factor / Q000737:chemistry*
D003432:Cross-Linking Reagents; D003574:Cytochrome c Group; D019038:Platelet Glycoprotein GPIb-IX Complex; D011994:Recombinant Proteins; D012709:Serum Albumin; D014841:von Willebrand Factor