RhoA and the function of platelet integrin alphaIIbbeta3.
Integrins respond to "inside-out" signals, which enable them to bind adhesive ligands, and ligand binding initiates "outside-in" signals that mediate anchorage-dependent cellular responses. RhoA is a GTPase that regulates certain actin rearrangements and transcriptional events. It has also been implicated in integrin signaling, but the exact relationship is not understood. To examine this further, platelets were incubated with C3 exoenzyme to adenine diphosphate (ADP)-ribosylate and inactivate RhoA, and the function of integrin alphaIIbbeta3 was studied. Despite inactivation of >/= 90% of RhoA, platelets exhibited normal inside-out signaling, as monitored by agonist-induced binding of a fibrinogen-mimetic anti-alphaIIbbeta3 antibody and normal fibrinogen-dependent aggregation. On the other hand, RhoA inactivation decreased the adhesion of agonist-stimulated platelets to fibrinogen (P < .04) and the formation of vinculin-rich focal adhesions in platelets that did adhere (P < .001). These effects were selective because fibrin clot retraction, a response also dependent on alphaIIbbeta3 and actin contractility, was unaffected by C3, as was the content of F-actin in resting or agonist-stimulated platelets. Similar results were obtained in a Chinese hamster ovary (CHO) cell model system of alphaIIbbeta3: C3 exoenzyme (or overexpression of dominant-negative N19RhoA) failed to influence integrin activation state, but it blocked the formation of focal adhesions in cells spread on fibrinogen. These studies establish that RhoA plays a highly selective role in alphaIIbbeta3 signaling, and they identify a subset of responses to integrin ligation that may be uniquely dependent on the actin rearrangements regulated by this GTPase.
Leng
L
L
Department of Vascular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Kashiwagi
H
H
Ren
X D
XD
Shattil
S J
SJ
D036002:ADP Ribose Transferases / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D001905:Botulinum Toxins; D016466:CHO Cells; D003018:Clot Retraction; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism / Q000502:physiology*; D011485:Protein Binding; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein
D000199:Actins; D008024:Ligands; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate