Thiols in the αIIbβ3 integrin are necessary for platelet aggregation

Abstract

Summary 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.

Vascular thiol isomerases

“…Ancillary studies will assess the PDI inhibitory activity in plasma to determine whether adequate plasma concentrations of isoquercetin or its metabolites are reached. Although inhibition of PDI does not affect standard coagulation studies, it does have some effect on platelet aggregation 41,85 and platelet-dependent thrombin generation. 86 Successful completion of the CAT-IQ trial will provide proof-of-principle for inhibition of PDI in primary prophylaxis of thrombosis and allow us to develop methods to monitor PDI-targeted therapies.…”

Redox Control of Platelet Function

“…Using tail bleeding times as another measure of platelet function the anti-PDI antibody RL90 in appropriate doses completely inhibited thrombus formation. It is likely that PDI has a role in activation of the aIIbb3 fibrinogen receptor in vivo that is similar to its role in vitro (82,179). Free thiols in aIIbb3 are required for platelet aggregation (179) and potential sites of PDI regulation of aIIbb3 are the free thiols localized to the disulfide knot of the b3 subunit between residues 400 and 650 (289).…”

Disulfide Bonds as Regulators of Integrin Function in Thrombosis and Hemostasis

“…Earlier, the general concept has been that all cysteines in aIIbb3 are engaged in disulfide bonds (5,13,93). More recently, studies revealed that both aIIb and b3 contain thiols that can be labeled by membrane-impermeable thiol-modifying agents (37,39,78,147), implying that some cysteines in aIIbb3 might be presented as free thiols. Using the maleimide reagent 1-biotinamido-4-[4¢-(maleimidomethyl)-cyclohexanecarbox amido]butane (biotin-BMCC), both subunits of purified aIIbb3 were labeled but only the b3 subunit was labeled in intact platelets.…”

“…Since EDTA does not, by itself, generate new thiols, the increased labeling suggests that cryptic thiols which are sterically hindered from the labeling reagent were exposed after disruption of the integrin. Using 4-(chloromercuri) benzenesulfonic acid (pCMBS), labeling of aIIb and b3 was obtained even without disruption of the aIIbb3 complex (78). The b3 subunit of avb3 was also labeled by MPB after stimulation of endothelial cells and activation of avb3 with Mn 2 + , suggesting that disulfide bond reduction and thiol formation in the b3 subunit is a general mechanism for both aIIbb3 and avb3 activation (120).…”

“…On the other hand, blockade of free thiols by various thiol-modifying agents, including biotin-BMCC (147), Nethylmaleimide (81), dithiobisnitrobenzoic acid (70,89), and pCMBS (70,78), inhibited aIIbb3 activation and platelet aggregation induced by physiological agonists, Mn2 + , activating antibodies and peptides, or DTT, showing that free thiols are necessary for aIIbb3 activation by various signalingdependent and -independent pathways. The observation that these reagents can block DTT activation of aIIbb3 implies that the reduction of disulfide bonds is not, by itself, sufficient to induce aIIbb3 activation, but rather requires an additional thiol/disulfide exchange step.…”