Abstract
Thrombosis-associated pathologies, such as myocardial infarction and stroke, are major causes of morbidity and mortality worldwide. Because platelets are necessary for hemostasis and thrombosis, platelet directed therapies must balance inhibiting platelet function with bleeding risk. Glutamate receptor interacting protein 1 (GRIP1) is a large scaffolding protein that localizes and organizes interacting proteins in other cells, such as neurons. We have investigated the role of GRIP1 in platelet function to determine its role as a molecular scaffold in thrombus formation. Platelet-specific GRIP1-/- mice were used to determine the role of GRIP1 in platelets. GRIP1-/- mice had normal platelet counts, but a prolonged bleeding time and delayed thrombus formation in a FeCl3-induced vessel injury model. In vitro stimulation of WT and GRIP1-/- platelets with multiple agonists showed no difference in platelet activation. However, in vivo platelet rolling velocity after endothelial stimulation was significantly greater in GRIP1-/- platelets compared to WT platelets, indicating a potential platelet adhesion defect. Mass spectrometry analysis of GRIP1 platelet immunoprecipitation revealed enrichment of GRIP1 binding to GPIb-IX complex proteins. Western blots confirmed the mass spectrometry findings that GRIP1 interacts with GPIbα, GPIbβ, and 14-3-3. Additionally, in resting GRIP1-/- platelets, GPIbα and 14-3-3 have increased interaction compared to WT platelets. GRIP1 interactions with the GPIb-IX binding complex are necessary for normal platelet adhesion to a stimulated endothelium.
Highlights
Platelets contain many molecules and proteins most identified with neurons such as N-ethylmaleimide factor [1,2], serotonin [3,4], glutamate, and glutamate receptors [5,6,7,8]
Similar multi-organ defects have been reported in humans with glutamate receptor interacting protein 1 (GRIP1) mutations consistent with Fraser syndrome, a syndrome found with deletion of specific GRIP1 interacting proteins including Frem1 (Fras1 related extracellular matrix 1) and Fras1 (Fraser syndrome 1) [13,14,15]
We report that GRIP1 binds to the GPIb-IX complex and that deletion of GRIP1 from platelets causes altered platelet rolling behavior and decreased thrombosis
Summary
Platelets contain many molecules and proteins most identified with neurons such as N-ethylmaleimide factor [1,2], serotonin [3,4], glutamate, and glutamate receptors [5,6,7,8]. Glutamate receptors are trafficked and assembled into functional complexes by multiple accessory proteins including glutamate receptor interacting protein 1 (GRIP1) [9]. GRIP1 is a multi-PDZ domain scaffolding protein that forms homodimers that can bind and simultaneously organize and assemble protein complexes via PDZ (Post-synaptic density 95, discs large, and zonula occludens 1) domains [11]. Similar multi-organ defects have been reported in humans with GRIP1 mutations consistent with Fraser syndrome, a syndrome found with deletion of specific GRIP1 interacting proteins including Frem (Fras related extracellular matrix 1) and Fras (Fraser syndrome 1) [13,14,15]. Despite the multi-organ defects and hemorrhage in complete GRIP1-/- mice, GRIP1 expression and function has not been studied in most other cell types, including platelets
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