The interaction between von Willebrand factor (vWF) and the platelet membrane glycoprotein (GP) Ib-IX-V complex is essential for platelet adhesion at sites of vascular injury under high shear stress flow conditions. Moreover, GP Ib-IX-V may contribute to the mechanisms of platelet activation through its high affinity binding of α-thrombin. There are two distinct but partially overlapping regions of GP Ibα thought to be involved in interacting with vWF (residues 251-279) and α-thrombin (residues 271-284); they share three tyrosine residues (positions 276, 278, and 279) that have recently been shown to be sulfated (Dong, J., Li, C. Q., and Lopez, J. A. (1994) Biochemistry 33, 13946-13953). To define the functional role of these three residues, we have introduced selected mutations in a soluble recombinant GP Ibα fragment (corresponding to the sequence 1-302 of the mature protein) that binds vWF and α-thrombin with the same attributes as intact GP Ib-IX-V complex. Fragments containing a single Tyr → Phe substitution either at position 276 or 278 or 279 exhibited normal interaction with vWF but markedly reduced or absent binding of α-thrombin. GP Ibα fragment with normal sequence but synthesized under sulfate-free conditions also failed to bind α-thrombin and, in addition, had markedly 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 structural features of GP Ibα selectively involved in supporting the interaction with vWF or α-thrombin.
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