Glycoprotein Ib and glycoprotein IX in human platelets are acylated with palmitic acid through thioester linkages

The glycoprotein (GP) Ib-IX complex is a major component of the platelet membrane which mediates adhesion of platelets to exposed subendothelium. GP Ib is a heterodimer with a large α chain (M(r) = 135,000-145,000) and small β chain (M(r) = 22,000-27,000) linked by a disulfide bond(s). GP Ib is bound in a noncovalent 1:1 complex with GP IX (M(r) = 17,000-22,000). We labeled isolated human platelets with [³H] palmitate or surface-labeled platelet membrane glycoproteins with sodium periodate-[³H]sodium borohydride and immunoprecipitated the GP Ib-IX complex from radiolabeled platelet lysates using a mouse monoclonal antibody (SZ.1) which recognizes the intact complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitates from [³H]palmitate-labeled platelets revealed two radiolabeled bands under reducing conditions at 24 and 19 kDa and two bands under nonreducing conditions at 170 and 19 kDa. As demonstrated by the parallel analysis of immunoprecipitates from periodate-[³H]sodium borohydride-labeled platelets, the [³H]palmitate-labeled bands obtained under reducing conditions corresponded to GP Ibβ and GP IX and the ones obtained under nonreducing conditions to intact GP Ib and GP IX, respectively. Using alkaline methanolysis followed by high pressure liquid chromatography analysis of the methanolysis products, we demonstrated that the radioactivity associated with the GP Ib-IX complex from [³H]palmitate-labeled platelets was, in fact, covalently bound [³H]palmitate in ester linkage to protein. The protein-faty acid linkage was also disrupted by hydroxylamine at neutral pH. Thus, this study demonstrates that GP Ibβ and GP IX in human platelets are both fatty acid-acylated with palmitate through thioester linkages.