Rap1b is critical for glycoprotein VI-mediated but not ADP receptor-mediated alpha2beta1 activation.
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.


OBJECTIVE
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.
Wang
Z
Z
Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7260, USA.
Holly
S P
SP
Larson
M K
MK
Liu
J
J
Yuan
W
W
Chrzanowska-Wodnicka
M
M
White
G C
GC
Parise
L V
LV
D000818:Animals; D001792:Blood Platelets / Q000166:cytology / Q000378:metabolism; D048430:Cell Shape; D003094:Collagen; D038982:Integrin alpha2beta1 / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D010973:Platelet Adhesiveness; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D015398:Signal Transduction; D020668:rap GTP-Binding Proteins / Q000172:deficiency / Q000502:physiology*
D038982:Integrin alpha2beta1; D010980:Platelet Membrane Glycoproteins; D018048:Receptors, Purinergic P2; C098341:platelet membrane glycoprotein VI; D003094:Collagen; C492136:Rap1b protein, mouse; D020668:rap GTP-Binding Proteins