Platelets are essential for normal hemostasis, but close regulation is required to avoid the destructive effects of either inappropriate platelet activation or excessive responses to injury . Here, we describe a novel complex comprising the scaffold protein, spinophilin (SPL), and the tyrosine phosphatase, SHP-1, and show that it can modulate platelet activation by sequestering RGS10 and RGS18, 2 members of the regulator of G protein signaling family. We also show that SPL/ RGS/SHP1 complexes are present in resting platelets where constitutive phosphorylation of SPL(Y398) creates an atypical binding site for SHP-1. Activation of the SHP-1 occurs on agonist-induced phosphorylation of SHP-1(Y536), triggering dephosphorylation and decay of the SPL/ RGS/SHP1 complex. Preventing SHP-1 activation blocks decay of the complex and produces a gain of function. Conversely, deleting spinophilin in mice inhibits platelet activation. It also attenuates the rise in platelet cAMP normally caused by endothelial prostacyclin (PGI 2 ). Thus, we propose that the role of the SPL/RGS/ SHP1 complex in platelets is time and context dependent. Before injury, the complex helps maintain the quiescence of circulating platelets by maximizing the impact of PGI 2 . After injury, the complex gradually releases RGS proteins, limiting platelet activation and providing a mechanism for temporal coordination of pro thrombotic and antithrombotic inputs. IntroductionPlatelet responses to most agonists are mediated by G proteincoupled receptors, giving rise to the intracellular events that trigger platelet aggregation and granule exocytosis. 1 It has been known for some time that signaling by G proteins in platelets is subject to regulation by extrinsic factors arising from endothelial cells, especially nitric oxide and prostacyclin (PGI 2 ). 2 However, intrinsic modulators of platelet activation also exist, including members of the RGS (regulator of G protein signaling) family, 3 proteins that suppress G protein signaling by accelerating the hydrolysis of GTP bound to active G ␣ . 4,5 In contrast to nitric oxide and PGI 2 , RGS proteins are thought to have their effect once activation has begun; hence, the gain of function that we observed when an RGSinsensitive variant of G i2␣ was introduced into platelets. 3 This inhibitory role for RGS proteins produces a potential conundrum: although preventing unwarranted platelet activation is desirable, preventing the rapid onset of the hemostatic response to injury is not. We have, therefore, sought the means by which the onset of signal suppression by RGS proteins can be delayed, allowing signaling to begin. That search brought us to spinophilin (SPL or neurabin-II), a 130-kDa scaffold protein originally identified in screens for brain proteins that can bind to the serine/ threonine phosphatase, PP1, 6 and F-actin, 7 and subsequently found to associate with other proteins as well, 8 including a limited set of G protein-coupled receptors and RGS proteins. [9][10][11][12] Prior evidence suggests that one reg...
Although much is known about extrinsic regulators of platelet function such as nitric oxide and prostaglandin I 2 (PGI 2 ), considerably less is known about intrinsic mechanisms that prevent overly robust platelet activation after vascular injury. Here we provide the first evidence that regulators of G-protein signaling (RGS) proteins serve this role in platelets, using mice with a G184S substitution in G i2␣ that blocks RGS/G i2 interactions to examine the consequences of lifting constraints on G i2 -dependent signaling with- IntroductionThe hemostatic response to injury in humans and other species with a closed, high-pressure circulatory system represents a balance between minimizing blood loss and avoiding vascular occlusion. How this balance is achieved is only partially understood. Hemostatic thrombi are formed by a combination of fibrin and platelets. While fibrin deposition is controlled by regulating production of thrombin and subsequently by protease inhibitors, platelet activation is controlled by limiting the availability of platelet agonists and by releasing inhibitors such as prostaglandin I 2 (PGI 2 ) and nitric oxide from endothelial cells. PGI 2 and nitric oxide prevent unwarranted platelet activation by raising basal cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels. Coming from a source outside of the platelet, they can be viewed as extrinsic regulators of platelet function. Here we asked whether platelet activation is also subject to autonomous (ie, intrinsic) regulation that limits the extent of platelet accumulation by limiting internal platelet signaling.Although there are multiple ways that intrinsic regulation could be accomplished, most platelet agonists activate members of the G protein-coupled receptor family, making G proteins a logic target for regulation. In general, G proteins remain active until hydrolysis of G ␣ -bound guanosine-5Ј-triphosphate (GTP) restores the resting state. The rate of inactivation is determined in part by members of the regulators of G-protein signaling (RGS) family, which accelerate GTP hydrolysis by G ␣ . [1][2][3][4] The 37 known RGS proteins have a 120 amino acid RGS domain that can interact with the switch region of activated G ␣ . 5,6 As many as 10 RGS proteins have been reported in platelets, although many of them solely at the level of RNA transcripts. [7][8][9][10][11][12] Nothing is known about the impact of these proteins on platelet activation in vitro or in vivo.Here we asked whether RGS proteins limit platelet accumulation during thrombus formation and, if so, whether this is through an effect on the initiation or the magnitude of the platelet response to agonists. Because of uncertainty about the full repertoire of RGS proteins expressed in platelets, the paucity of available RGS protein gene knockouts, and ambiguities about the specificity of RGS/G ␣ interactions, we studied mice in which a substitution (G184S) in the ␣ subunit of G i2 renders it resistant to accelerated turn-off by all RGS proteins. 13 T...
161 Platelets play an essential role in hemostasis, but excessive platelet responses to vascular injury or disease can be catastrophic. We have recently reported that members of the RGS protein family can modulate platelet responses to injury by limiting the duration of G protein-dependent signaling initiated by platelet agonists. Here we show that platelets contain a previously-unrecognized regulatory complex comprised of the 130 kDa scaffold protein, spinophilin (SPL) with at least two RGS proteins (RGS10 and RGS18) and the protein tyrosine phosphatase, SHP-1. In resting platelets, this complex is phosphorylated on spinophilin residues Y398 and Y483. Mutating both tyrosines to phenylalanine inhibits the binding of SHP-1 to spinophilin. Platelet activation by thrombin or thromboxane A2, but not ADP or collagen, stimulates a transient increase in spinophilin-associated SHP-1 tyrosine phosphatase activity and causes dephosphorylation and decay of the SPL/RGS/SHP-1 complex. Conversely, blocking SHP-1 phosphatase activity in platelets or omitting SHP-1 in transfected CHO cells inhibits dephosphorylation of spinophilin and prevents dissociation of the SPL/RGS/SHP-1 complex. While we have shown previously that inhibiting interactions between G proteins and RGS proteins produces a gain of function in platelets, knocking out spinophilin in mice inhibits platelet aggregation. This aggregation defect does not occur with all agonists, but is selective for those that are able to trigger decay and dephosphorylation of the SPL/RGS/SHP-1 complex. In addition to inhibiting platelet aggregation in vitro, the spinophilin knockout delays carotid artery occlusion in vivo following application of FeCl3 and reduces platelet accumulation following laser injury in cremaster muscle arterioles. Underlying these effects of the knockout is a decrease in Rap1 activation, an event that supports integrin activation, and attenuation of the cAMP increase otherwise caused by endothelial PGI2. Collectively, these observations show for the first time that a regulatory complex based on spinophilin helps to regulate platelet responses to injury and suggest that it does this by sequestering RGS proteins in resting platelets and releasing them after activation begins. Dissociation of the SPL/RGS complex is regulated by an agonist-induced increase in the activity of SHP-1 associated with spinophilin. Disclosures: No relevant conflicts of interest to declare.
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