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

Aster E. Pijning, Mitchell T. Blyth, Michelle L. Coote, Freda Passam, Joyce Chiu, Philip J. Hogg

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

The αIIbβ3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbβ3 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-C545 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 αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb 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 αIIbβ3 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 α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

Original languageEnglish
Pages (from-to)1359-1372
Number of pages14
JournalBlood
Volume138
Issue number15
DOIs
Publication statusPublished - 14 Oct 2021
Externally publishedYes

Keywords

  • platelet
  • adhesion
  • thrombosis
  • hemostasis
  • cysteine
  • alkylation
  • spectrometry

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