Thiols in the alphaIIbbeta3 integrin are necessary for platelet aggregation.
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 P2Y(12) ADP receptor is involved in most types of platelet aggregation, we used the ADP scavenger apyrase and the P2Y(12) receptor antagonist 2-MeSAMP to examine thiol-dependent reactions in the absence of contributions from this receptor. We provide evidence for a non-P2Y(12) thiol-dependent reaction near the final alphaIIbbeta3-dependent events of aggregation. We then used 3-(N-maleimidylpropionyl)biocytin (MPB) and pCMBS to study thiols in alphaIIbbeta3. As previously reported, disruption of the receptor was required to obtain labelling of thiols with MPB. Specificity of labelling for thiols in the alphaIIb and beta3 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 alphaIIbbeta3 and blocked aggregation under physiological conditions. Similarly, pCMBS preferentially inhibited signalling-independent activation of alphaIIbbeta3 by Mn(2+). Our results suggest that the thiols in alphaIIbbeta3 that are blocked by pCMBS are important in the activation of this integrin.
Manickam
Nagaraj
N
Department of Medicine Division of Hematology, The University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
Sun
Xiuhua
X
Hakala
Kevin W
KW
Weintraub
Susan T
ST
Essex
David W
DW
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D001081:Apyrase / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D005434:Flow Cytometry; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008239:Lysine / Q000031:analogs & derivatives / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D010974:Platelet Aggregation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D008301:Maleimides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D013438:Sulfhydryl Compounds; D002731:4-Chloromercuribenzenesulfonate; C047272:3-(N-maleimidopropionyl)biocytin; D001081:Apyrase; D008239:Lysine