Sulfhydryl groups of platelet surface proteins are important in platelet aggregation. While p-chloromercuribenzene sulphonate (pCMBS) has been used in most studies on platelet surface thiols, the specific thiol-proteins that pCMBS reacts with to inhibit aggregation have not been well defined. Since the thiol-containing P2Y12 ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y12 receptor antagonist 2-MeSAMP to examine thiol-dependent reactions in the absence of contributions from this receptor. We provide evidence for a non-P2Y12 thiol-dependent reaction near the final αIIbβ3-dependent events of aggregation. We then used 3-(N-maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in αIIbβ3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the αIIb and β3 subunits was confirmed by identification of the purified proteins by mass spectrometry and by inhibition of labelling with 5,5′-dithiobis-(2-nitrobenzoic acid). In contrast to MPB, pCMBS preferentially reacted with thiols in αIIbβ3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling-independent activation of αIIbβ3 by Mn2+. Our results suggest that the thiols in αIIbβ3 that are blocked by pCMBS are important in the activation of this integrin.
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