An alternate covalent form of platelet αIIbβ3 integrin that resides in focal adhesions and has altered function

The alpha IIb beta 3 integrin receptor coordinates platelet adhesion, activation, and mechano-sensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the allb subunit linking cysteines 490 and 545 that is missing in similar to 1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of alpha IIb beta 3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-0545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the alpha llb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of alpha IIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the alpha IIb beta 3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin alpha subunits, and the disulfide bond in the alpha V and alpha 2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

Automated summary

Research has shown that the αIIbβ3 integrin receptor is produced in various covalent forms with different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin alpha subunits, suggesting that similar forms and functions of integrin are also conserved in cultured cells.