THROMBIN INTERACTION WITH PLATELET GPIB - ROLE OF THE HEPARIN-BINDINGDOMAIN

The platelet membrane glycoprotein Ib (GpIb) has a high affinity bindi ng site for alpha-thrombin whose occupancy is thought to positively mo dulate 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 ''fib rinogen recognition site'' (FRS) were investigated as the possible dom ains involved in GpIb binding. The role of thrombin HBS was explored b y performing binding measurements of I-125-alpha-thrombin to purified glycocalicin (GC), the extracytoplasmic portion of GpIb, in the presen ce of heparin as well as after chemical modifications of the thrombin heparin binding site (thrombin-HBS phosphopyridoxylation). These studi es showed that a) thrombin binding to GC could be competitively inhibi t ed by heparin and b) the equilibrium association constant for thromb in-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 int eraction could be excluded by other experiments showing that GC in sol ution could not influence the interaction of cr-thrombin with two subs trates which bind to both the catalytic site and the fibrinogen recogn ition site: 1) the thrombin receptor peptide 38-60 (TR, L38-E60) and 2 ) the A alpha-chain of fibrinogen. Altogether these results demonstrat ed that GC interaction with thrombin involves the enzyme heparin bindi ng site, whereas the fibrinogen recognition site does nor play a signi ficant role.