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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 (PGI2). 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 PGI2. After injury, the complex gradually releases RGS proteins, limiting platelet activation and providing a mechanism for temporal coordination of pro thrombotic and antithrombotic inputs.

Abstract 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.

Proteins that are expressed on the platelet surface can participate in contact-dependent signaling events which modulate thrombus formation or, after being shed from the platelet surface, serve as bioactive messengers that affect the function of nearby cells. Here we show for the first time that platelets express the class IV semaphorin known as sema4D or CD100, and that platelet activation causes the regulated shedding of the sema4D extracellular domain in a biologically-active form. Sema4D is a glycosylated 150 kDa disulfide-linked homodimer that has previously been implicated in interactions between T-cells and B-cells. Platelet activation by collagen, thrombin or PMA causes a transient increase in sema4D surface expression peaking at 15 min, followed by a complete loss of expression over 30–60 minutes. These events are accompanied by the release of the sema4D exodomain as a 130 kDa fragment, leaving a 25–30 kDa transmembrane and cytoplasmic domain fragment that is retained by the platelets. The cleavage event required to produce these fragments is inhibited by metalloprotease inhibitors and abolished in platelets from chimeric mice lacking the metalloprotease known as ADAM17 or TACE (TNF alpha converting enzyme). Incubation of recombinant sema4D with ADAM17 identified a single cleavage site just outside the predicted transmembrane domain. Western blots show that human platelets express ADAM17 in both its immature (zymogen) and mature (active) forms, and indicate that at least some of the ADAM17 is located on the platelet surface. ADAM17-dependent cleavage of sema4D does not require platelet aggregation, but the rate is accelerated when aggregation is allowed to occur and slowed when aggregation is prevented. Under both sets of conditions, cleavage of sema4D occurs to a greater extent and more rapidly than the ADAM17-dependent cleavage of GP Ib alpha, suggesting that there is a hierarchy of proteolytic events when platelets are activated. In terms of biological impact, the shedding of sema4D following platelet activation raises the possibility that the soluble extracellular domain of sema4D serves as a bioactive messenger. Two receptors for sema4D have been identified previously: CD72, which is present on lymphocytes where it regulates the activity of the tyrosine phosphatase, SHP-1, and plexin-B1, which is expressed in endothelial cells. Western blots suggest that both of these receptors are expressed on human platelets and show that SHP-1 is associated with CD72 in resting, but not activated platelets. Taken together, these results demonstrate that sema4D undergoes regulated ADAM17-dependent shedding when platelets are activated, and suggest that this results in the production of a bioactive form of the molecule that can affect responses in nearby platelets, lymphocytes and endothelial cells at sites of thrombosis.