The Discovery and Characterisation of Novel Covalent Forms of αIIbβ3 Integrin

The same protein can fulfill different functions depending on the time, place, and environment in which it functions. This is especially important for thrombotic proteins, which have different roles depending on the timing of thrombosis or haemostasis in the vasculature. αIIbβ3 integrin is the most abundant platelet receptor and coordinates platelet adhesion, activation, and mechano-sensing. Using differential cysteine alkylation and mass spectrometry, different covalent forms of platelet αIIbβ3 have been identified that are characterised by the absence of specific disulfide bonds in the integrin subunits. These disulfide bonds are not cleaved in the mature protein but rather never form during maturation of the receptor in megakaryocytes. The functions of two αIIbβ3 covalent forms have been characterised. A form missing the αIIb Cys490-Cys545 disulfide bond comprises about one in three integrin molecules on human platelets and functions selectively in focal adhesions, where it has extended residency time due to a decreased rate of internalisation and recycling. Molecular dynamics (MD) simulations revealed that absence of this bond propagates mechanical forces into the αIIbβ3 knee joint that result in a higher energy barrier for cycling between different integrin conformations. A covalent form missing the β3 Cys177-Cys184 disulfide bond comprises about one in two integrin molecules on platelets and does not bind the integrin’s physiological RGD ligands. MD simulations indicate that absence of the bond has long-range allosteric effects on the βI-domain metal-binding sites that are essential for RGD binding. The disulfide bonds are conserved in all 18 integrin α subunits and 7 of 8 β subunits and were found unformed in other integrins, suggesting that the covalent regulation is conserved. The prevalence of different covalent forms of αIIbβ3 integrin varies considerably amongst individuals, which could contribute to the variability in thrombotic propensity in humans