Crosslinking of platelet glycoprotein Ib by N-succinimidyl(4-azidophenyldithio)propionate and 3,3'-dithiobis(sulfosuccinimidyl propionate).
To examine the relationship between glycoprotein Ib and other proteins in the platelet membrane and the interaction of this protein with thrombin, platelets were crosslinked by two cleavable reagents, SADP (N-succinimidyl(4-azidophenyldithio)propionate) and DTSSP (3,3'-dithiobis(sulfosuccinimidyl propionate]. Two-dimensional, unreduced-reduced sodium dodecyl sulphate (SDS)-polyacrylamide electrophoresis and staining by silver or wheat germ agglutinin-conjugated peroxidase, after protein transfer to nitrocellulose, demonstrated that SADP intramolecularly crosslinked glycoprotein Ib and formed intermolecular complexes of glycorprotein IIb and some high molecular weight proteins. DTSSP intermolecularly crosslinked glycoprotein Ib, glycoprotein IIb, and other high molecular weight proteins. With a low concentration of 125I-labeled TLCK-thrombin (6 nM), crosslinking with SADP yielded a 200 000 Da complex containing radioactive-labeled thrombin, and high TLCK-thrombin concentration (0.1 microM) gave the complex and a 167 000 band. alpha- and TLCK-thrombin crosslinking with DTSSP also yielded the 200 000 complex formed by reaction with SADP or DTSSP was markedly reduced by preincubation of platelets with excess unlabeled TLCK-thrombin and had a pI similar to glycoprotein Ib. These results suggest that glycoprotein Ib is one of the proteins composing the high affinity receptor for thrombin.
D001386:Azides; D001792:Blood Platelets / Q000378:metabolism / Q000648:ultrastructure; D003432:Cross-Linking Reagents; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D013388:Succinimides; D013917:Thrombin / Q000378:metabolism
D001386:Azides; D003432:Cross-Linking Reagents; D006023:Glycoproteins; D008565:Membrane Proteins; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D013388:Succinimides; C023592:glycocalicin; C033169:3-((4-azidophenyl)dithio)propionic N-hydroxysuccinimide; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate); D013917:Thrombin