Identification of a region in the cytoplasmic domain of the platelet membrane glycoprotein Ib-IX complex that binds to purified actin-binding protein.

R.K Andrews,J.E Fox

doi:10.1016/s0021-9258(19)37004-8

Abstract

The platelet membrane glycoprotein (GP) Ib-IX complex is a major site of attachment of the platelet membrane skeleton to the plasma membrane. This association is mediated by the interaction of actin-binding protein with the GP Ib-IX complex. The aim of the present work was to identify domains on the GP Ib-IX complex that interact with actin-binding protein. Synthetic peptides corresponding to sequences of the GP Ib alpha-chain and beta-chain cytoplasmic domains were analyzed for their ability to bind to purified actin-binding protein. Two overlapping peptides encompassing a sequence (Thr-536-Phe-568) from the central region of the cytoplasmic domain of GP Ib alpha were the most effective in binding 125I-actin-binding protein, as assessed by a microtiter well approach and peptide affinity chromatography. One of the active peptides (Thr-536-Leu-554) was chosen to evaluate the likelihood that the central region of the cytoplasmic domain of GP Ib alpha is involved in binding of the intact complex to actin-binding protein. This peptide could be specifically cross-linked to purified actin-binding protein in solution. Rabbit polyclonal antibody against this peptide inhibited the binding of purified actin-binding protein to the purified GP Ib-IX complex. Finally, as in intact platelets, the calpain-induced hydrolytic fragments of purified actin-binding protein (M(r) = 200,000 and M(r) = 91,000) showed little binding to the GP Ib alpha peptide. Taken together, these results provided evidence that a region between Thr-536 and Phe-568 of the cytoplasmic domain of GP Ib alpha participates in the interaction of the GP Ib-IX complex with actin-binding protein.

Highlights

Complex binds von Willebrandfactor, an interaction that by a microtiter well approach and peptide affinity mediates the adhesion of platelets to a damaged blood vessel chromatography
Recent work from our laboratory has demonstrated that purified platelet actin-binding protein binds directly to the cytoplasmic domain of the purified GP Ib-IX complex [22]
T o search for the binding site for actin-binding protein on the GP Ib-IX complex, synthetic peptides encompassing the major hydrophilic sequences of the cytoplasmic region of GP Ib, (Fig. 1) and the 34 amino acids that compose the entire cytoplasmic domain of G P Ib, were analyzed for their ability to bind to actin-binding protein

Summary

RESULTS

Several lines of evidence have shown that the GP Ib-IX complex associates withactin-bindingproteininplatelets [18,19,20,21]. Purified '251-actin-binding protein bound preferentially to two overlapping peptides, Thr536-Leu-554 and Arg-550-Phe-568, from the central region of the cytoplasmic domain of G P Ib,(Fig. 2). Either platelet lysates or purified lZ5I-actin-bindingprotein dues Trp-570-Ala-588 (Fig. 6C) and essentially none to pepwas passed over the peptide affinity columns, the columns tide Ala-582-Thr-600 from the carboxyl-terminalend of GP were washed, and bound protein was subsequently removed Ib, (data not shown). Several other proteins columns bound to thetwo overlappingpeptides downstream from the platelet lysateswere selectively boundto thecolumn of this region (Thr-536-Leu-554 (Fig.6B) andArg-550-Phe- containing active peptide.

InputJ

Coomassie Blue

DISCUSSION