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Updated: Jun 25, 2026

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding
Published on: June 9, 2017
Rap1b is critical for glycoprotein VI-mediated but not ADP receptor-mediated alpha2beta1 activation
Z Wang1, S P Holly, M K Larson
1Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7260, USA.
Background:
The platelet alpha2beta1 integrin functions as both an adhesion and signaling receptor upon exposure to collagen. Recent studies have indicated that alpha2beta1 function can be activated via inside-out signaling, similar to the prototypical platelet integrin alphaIIbbeta3. However, signaling molecules that regulate alpha2beta1 activation in platelets are not well defined. A strong candidate molecule is the small GTPase Rap1b, the dominant platelet isoform of Rap1, which regulates alphaIIbbeta3 activation.
Objectives:
We hypothesized that Rap1b positively regulates alpha2beta1 during agonist-induced platelet activation.
Methods:
To test whether Rap1b activates alpha2beta1 downstream of glycoprotein (GP)VI or other platelet receptors, we stimulated platelets purified from Rap1b-/- or wild-type mice with diverse agonists and measured alpha2beta1 activation using fluorescein isothiocyanate-labeled monomeric collagen. We also examined the role of Rap1b in outside-in signaling pathways by analyzing adhesion and spreading of Rap1b-/- or wild-type platelets on monomeric, immobilized collagen. Finally, we monitored the activation status of related Rap GTPases to detect changes in signaling pathways potentially associated with Rap1b-mediated events.
Results:
Rap1b-/- platelets displayed comparable ADP-induced or thrombin-induced alpha2beta1 activation as wild-type platelets, but reduced convulxin-dependent alpha2beta1 activation. Rap1b-/- platelets exhibited increased spreading on immobilized collagen but similar adhesion to immobilized collagen compared to wild-type platelets. Rap1b-/- platelets also showed Rap1a and Rap2 activation upon agonist stimulation, possibly revealing functional compensation among Rap family members.
Conclusions:
Rap1b is required for maximal GPVI-induced but not ADP-induced activation of alpha2beta1 in murine platelets.
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