IDENTIFICATION OF 3 TYROSINE RESIDUES OF GLYCOPROTEIN IB-ALPHA WITH DISTINCT ROLES IN VON-WILLEBRAND-FACTOR AND ALPHA-THROMBIN BINDING

The interaction between von Willebrand factor (vWF) and the platelet m embrane glycoprotein (GP) Ib-IX-V complex is essential for platelet ad hesion at sites of vascular injury under high shear stress flow condit ions. Moreover, GP Ib-IX-V may contribute to the mechanisms of platele t activation through its high affinity binding of alpha-thrombin. Ther e are two distinct but partially overlapping regions of GP Ib alpha th ought to be involved in interacting with vWF (residues 251-279) and al pha-thrombin (residues 271-284); they share three tyrosine residues (p ositions 276, 278, and 279) that have recently been shown to be sulfat ed (Dong, J., Li, C. Q., and Lopez, J., A. (1994) Biochemistry 33, 139 46-13953). To define the functional role of these three residues, we h ave introduced selected mutations in a soluble recombinant GP Ib(alpha fragment (corresponding to the sequence 1-302 of the mature protein) that binds vWF and alpha-thrombin with the same attributes as intact G P Ib-IX-V complex. Fragments containing a single Tyr --> Phe substitut ion either at position 276 or 278 or 279 exhibited normal interaction with vWF but markedly reduced or absent binding of a-thrombin. GP Ib a lpha fragment with normal sequence but synthesized under sulfate free conditions also failed to bind alpha-thrombin and, in addition, had ma rkedly reduced interaction with vWF. The simultaneous substitution of three neighboring Asp residues with Asn at positions 272, 274, and 277 , a multiple mutation that may impair Tyr sulfation, also resulted in loss of binding of both ligands. These results define distinct structu ral features of GP Ib alpha selectively involved in supporting the int eraction with vWF or alpha-thrombin.