Thrombin interaction with platelet GpIB: role of the heparin binding domain.
The platelet membrane glycoprotein Ib (GpIb) has a high affinity binding site for alpha-thrombin whose occupancy is thought to positively modulate the thrombin-induced platelet activation. In this study, aimed at further characterizing the thrombin-GpIb interaction, two thrombin anion exosites referred to as "heparin binding site" (HBS) and "fibrinogen recognition site" (FRS) were investigated as the possible domains involved in GpIb binding. The role of thrombin HBS was explored by performing binding measurements of 125I-alpha-thrombin to purified glycocalicin (GC), the extracytoplasmic portion of GpIb, in the presence of heparin as well as after chemical modifications of the thrombin heparin binding site (thrombin-HBS phosphopyridoxylation). These studies showed that a) thrombin binding to GC could be competitively inhibited by heparin and b) the equilibrium association constant for thrombin-GC interaction was reduced up to ten-fold by chemical modifications at the HBS. On the other hand, the role of FRS in the thrombin-GC interaction could be excluded by other experiments showing that GC in solution could not influence the interaction of alpha-thrombin with two substrates which bind to both the catalytic site and the fibrinogen recognition site: 1) the thrombin receptor peptide 38-60 (TR, L38-E60) and 2) the A alpha-chain of fibrinogen. Altogether these results demonstrated that GC interaction with thrombin involves the enzyme heparin binding site, whereas the fibrinogen recognition site does not play a significant role.
De Candia
E
E
Centro Ricerche Fisiopatologia dell'Emostasi, Istituto di Semeiotica Medica, Università Cattolica S. Cuore, Roma, Italia.
De Cristofaro
R
R
De Marco
L
L
Mazzucato
M
M
Picozzi
M
M
Landolfi
R
R
D001665:Binding Sites; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D016014:Linear Models; D010455:Peptides / Q000097:blood; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011732:Pyridoxal Phosphate; D013917:Thrombin / Q000378:metabolism*
D010455:Peptides; D019038:Platelet Glycoprotein GPIb-IX Complex; C023592:glycocalicin; D011732:Pyridoxal Phosphate; D006493:Heparin; D013917:Thrombin