This chapter summarizes current ideas about the intracellular signaling that drives platelet responses to vascular injury. After a brief overview of platelet activation intended to place the signaling pathways into context, the first section considers the early events of platelet activation leading up to integrin activation and platelet aggregation. The focus is on the G protein-mediated events utilized by agonists such as thrombin and ADP, and the tyrosine kinase-based signaling triggered by collagen. The second section considers the events that occur after integrin engagement, some of which are dependent on close physical contact between platelets. A third section addresses the regulatory events that help to avoid unprovoked or excessive platelet activation, after which the final section briefly considers individual variations in platelet reactivity and the role of platelet signaling in the innate immune response and embryonic development.

Renewal of nongermative epithelia is poorly understood. The novel mitogen “lacritin” is apically secreted by several nongermative epithelia. We tested 17 different cell types and discovered that lacritin is preferentially mitogenic or prosecretory for those types that normally contact lacritin during its glandular outward flow. Mitogenesis is dependent on lacritin's C-terminal domain, which can form an α-helix with a hydrophobic face, as per VEGF's and PTHLP's respective dimerization or receptor-binding domain. Lacritin targets downstream NFATC1 and mTOR. The use of inhibitors or siRNA suggests that lacritin mitogenic signaling involves Gαi or Gαo–PKCα-PLC–Ca2+–calcineurin–NFATC1 and Gαi or Gαo–PKCα-PLC–phospholipase D (PLD)–mTOR in a bell-shaped, dose-dependent manner requiring the Ca2+ sensor STIM1, but not TRPC1. This pathway suggests the placement of transiently dephosphorylated and perinuclear Golgi–translocated PKCα upstream of both Ca2+ mobilization and PLD activation in a complex with PLCγ2. Outward flow of lacritin from secretory cells through ducts may generate a proliferative/secretory field as a different unit of cellular renewal in nongermative epithelia where luminal structures predominate.

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...

An optimal platelet response to injury can be defined as one in which blood loss is restrained and haemostasis is achieved without the penalty of further tissue damage caused by unwarranted vascular occlusion. This brief review considers some of the ways in which thrombus growth and stability can be regulated so that an optimal platelet response can be achieved in vivo. Three related topics are considered. The first focuses on intracellular mechanisms that regulate the early events of platelet activation downstream of G protein coupled receptors for agonists such as thrombin, thromboxane A2 and ADP. The second considers the ways in which signalling events that are dependent on stable contacts between platelets can influence the state of platelet activation and thus affect thrombus growth and stability. The third focuses on the changes that are experienced by platelets as they move from their normal environment in freely-flowing plasma to a very different environment within the growing haemostatic plug, an environment in which the narrowing gaps and junctions between platelets not only facilitate communication, but also increasingly limit both the penetration of plasma and the exodus of platelet-derived bioactive molecules.

Cell surface heparan sulfate (HS) proteoglycans are carbohydrate-rich regulators of cell migratory, mitogenic, secretory, and inflammatory activity that bind and present soluble heparin-binding growth factors (e.g., fibroblast growth factor, Wnt, Hh, transforming growth factor β, amphiregulin, and hepatocyte growth factor) to their respective signaling receptors. We demonstrate that the deglycanated core protein of syndecan-1 (SDC1) and not HS chains nor SDC2 or -4, appears to target the epithelial selective prosecretory mitogen lacritin. An important and novel step in this mechanism is that binding necessitates prior partial or complete removal of HS chains by endogenous heparanase. This limits lacritin activity to sites where heparanase appears to predominate, such as sites of exocrine cell migration, secretion, renewal, and inflammation. Binding is mutually specified by lacritin's C-terminal mitogenic domain and SDC1's N terminus. Heparanase modification of the latter transforms a widely expressed HS proteoglycan into a highly selective surface-binding protein. This novel example of cell specification through extracellular modification of an HS proteoglycan has broad implications in development, homeostasis, and disease.

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...

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Key points GRK6 regulates the hemostatic response by limiting platelet activation via thrombin and adenosine 5′-diphosphate. GRK6 regulates the hemostatic response by reducing PAR1/4- and P2Y12-dependent signaling.