Effect of phosphopyridoxylation on thrombin interaction with platelet glycoprotein Ib.
The purpose of this study was to determine the effect of chemical modification of lysyl residues on thrombin interaction with platelet membrane proteins. Modification of lysyl residues by pyridoxal-5'-phosphate affected two different sites on thrombin and resulted in a greatly decreased binding to platelets. Using a crosslinking bifunctional reagent [bis(sulphosuccinimidyl) suberate (BS3)], we show that modified thrombin retained the ability to form high molecular mass (greater than or equal to 400 kDa) complexes with yet unidentified platelet proteins and to bind to platelet protease nexin I, but had lost the ability to bind to platelet glycoprotein Ib (GPIb). As previously reported by others, heparin protected one of the two sites from phosphopyridoxylation. In contrast modified thrombin, heparin-protected modified thrombin retained the ability to bind to GPIb, indicating that the lysyl residue(s) protected by heparin from the modification are essential for GPIb binding. While unprotected modified thrombin failed to bind hirudin, heparin-protected modified thrombin retained its ability to bind the carboxy-terminal hirudin peptide H54-65. Tritium-labelling of the modified lysyl residues and degradation of modified thrombins by CNBr or trypsin confirmed that the lysyl residue(s) protected by heparin and essential for GPIb binding are located in the thrombin binding domain for the carboxyl-terminal tail of hirudin, within the sequence 18-73 of the thrombin B chain.
Ternisien
C
C
Laboratoire de Recherche sur l'Hémostase et la Thrombose, Faculté Xavier Bichat, Paris, France.
Jandrot-Perrus
M
M
Huisse
M G
MG
Guillin
M C
MC
D001665:Binding Sites; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D006493:Heparin / Q000494:pharmacology; D006629:Hirudins / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008970:Molecular Weight; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology; D014316:Tritium; D014357:Trypsin
D003432:Cross-Linking Reagents; D006629:Hirudins; D010980:Platelet Membrane Glycoproteins; D014316:Tritium; D011732:Pyridoxal Phosphate; D006493:Heparin; D014357:Trypsin; D013917:Thrombin; D008239:Lysine; D003488:Cyanogen Bromide