Background: Background. Tetraspanins are transmembrane proteins involved in cellular events such as adhesion, motility, and proliferation. CD9 (Tspan-29) is highly expressed on the platelet surface. Previously, it was determined that CD9 and the platelet integrin, GPIIb-IIIa form noncovalent complexes that are dependent upon the GPIIb-IIIa conformation state.

Aims: The aim of this study was to further define how CD9 and its integrin partner, GPIIb-IIIa, regulate platelet function via common and unique protein interactomes.

Methods: We characterized CD9-dependent platelet phenotypes using Fab fragments of a well-characterized anti-CD9 mAb (mFab7). Platelet adhesion and spreading were examined on matrices fibrinogen (FG), fibronectin (FN), fibrin (Fb) or Fb-FN. Platelet activation, GPIIb-IIIa -mediated aggregation response and aggregate stability (LTA) were measured after mFab7 ligation or addition of a recombinant CD9 protein fragment. Protein interactomes were characterized by immunoprecipitation (IP) by 10E5 (anti-GPIIb-IIIa) or mAb7 (n=3) followed by shotgun mass spectrometry.

Results: Confocal microscopy showed that CD9 was highly localized in platelet-platelet contact regions. mFab7 pre-treatment increased platelet spreading and LTA responses to threshold levels of agonists ADP or collagen by 40% (p< 0.01) and 20% (p< 0.05), respectively. Consistent with platelet activation, fibrinogen binding, CD62p and CD63 expression were significantly increased
(p< 0.01). Interactome analyses identified 24 proteins common to both CD9 and GPIIb-IIIa. Six unique protein interaction partners for CD9 included tetraspanin-9, CD63 and Rho family GTPase-activating proteins (p ≤ 0.001) whereas eight unique GPIIb-IIIa partners included cytoskeleton organization regulators (p ≤ 0.01) and complexes involved in early stage hemostasis (p ≤ 0.001).

Conclusions: CD9 localizes to platelet-platelet contacts, modulates GPIIb-IIIa functional status and facilitates platelet responses to ligands upon platelet activation. Such interactions are likely controlled by co-complex members that serve as hubs for the crosstalk of different signaling events. Our findings provide new molecular insights contributing toward a better understanding of platelet signaling and function that might improve therapeutic strategies.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/tetraspanin-cd9-regulates-gpiib-iiia-mediated-platelet-function-identification-of-unique-protein-interactomes/