OC 41.1 - Platelet αIIbβ3 integrin is produced in different covalent forms that have different functions

Background: The αIIbβ3 integrin receptor coordinates platelet adhesion, activation, and mechano-sensing in thrombosis and haemostasis. We identified covalent forms of αIIbβ3 integrin on healthy human platelets that differ from each other by the absence of specific disulfide bonds. A covalent form of the αIIb subunit missing the disulfide bond linking cysteines 490-545 comprises ~1 in 3 molecules on resting and activated platelets, whereas a covalent form of the β3 subunit missing the bond linking cysteines 177-184 comprises ~1 in 2 molecules. These covalent forms of αIIbβ3 are pre-determined, as they are also produced by megakaryoblasts and transfected cells.

Aims: To determine the function and significance of alternate covalent forms of αIIbβ3 integrin.

Methods: Distribution of the alternate αIIbβ3 forms on platelets was measured by co-immunoprecipitation and differential cysteine alkylation and mass spectrometry. The functions of the covalent forms were evaluated in cells expressing a mutant integrin with an ablated bond and by molecular dynamics (MD) simulations.

Results: The covalent form missing the αIIb Cys490-Cys545 bond functions in focal adhesions where it has extended residency time due to decreased clathrin-mediated internalisation. MD simulations revealed that absence of this bond propagates mechanical forces into the knee joint, resulting in a higher energy barrier for cycling between integrin conformations. The covalent form missing the β3 Cys177-Cys184 disulfide bond does not bind the integrin’s physiological ligands. MD simulations indicate that absence of the bond has allosteric effects on the βI-domain metal-binding sites.

Conclusion(s): Megakaryocytes produce several covalent forms of αIIbβ3 integrin that have different functions. The disulfide bonds described here are conserved in all 18 integrin α subunits and 7 β subunits, suggesting that covalent regulation is conserved across integrins. The prevalence of different covalent forms of αIIbβ3 integrin varies considerably amongst individuals, which could contribute to the variability in thrombotic propensity in humans.