Platelet Signal Transduction

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Platelet Signal Transduction Robert H. Lee*, Lucia Stefanini† and Wolfgang Bergmeier ‡ *

McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States, †Department of Internal Medicine and Medical Specialties, Sapienza University of Rome, Rome, Italy, ‡McAllister Heart Institute, Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

INTRODUCTION 329 MAJOR STIMULATORY AND INHIBITORY RECEPTORS EXPRESSED ON THE PLATELET SURFACE 329 G Protein-Coupled Receptors (GPCRs) 330 (hem)ITAM and ITIM Receptors 331 SECOND MESSENGERS 332 Phospholipase C 332 Calcium Signaling 333 Diacylglycerol Signaling 334 PI3K and 3-Phosphorylated Phosphoinositide Signaling 334 Cyclic Nucleotide Signaling 336 SIGNAL INTEGRATORS 337 Kinases 337 RAP GTPase Signaling and Platelet Integrin Activation 339 SUMMARY 341 REFERENCES 342

INTRODUCTION Platelets play a critical role in the maintenance of vascular integrity at sites of mechanical trauma (hemostasis). To make contact with the damaged vascular wall, platelets depend on a unique surface receptor, the glycoprotein (GP) Ib-V-IX complex (see Chapter 10). The interaction of the GPIbα subunit with von Willebrand factor (VWF) deposited in the extracellular matrix (ECM) facilitates a transient interaction (tethering) and thus deceleration of quiescent platelets. Once engaged, platelets sense and respond to components of the exposed ECM, e.g., collagen, and/or locally generated soluble agonists, e.g., thrombin. Collagen triggers platelet activation via the GPVI-Fc receptor γ (FcRγ)-chain complex, the latter containing an immunoreceptor tyrosine-based activation motif (ITAM) consensus sequence (YxxI/Lx(6–12)YxxI/L, where x is any amino acid) in its cytoplasmic tail. Thrombin activates platelets via cleavage of protease-activated receptors (PARs; see section “G Protein-Coupled Receptors (GPCRs)”) and activation of heterotrimeric G proteins. Both ITAM and GPCR signaling lead to the activation of phospholipase C (PLC) and the generation of the second messengers calcium (Ca2+) and diacylglycerol (DAG) (Fig. 18.1). Kinases and small GTPases then integrate these signals, triggering major platelet responses including granule secretion, generation and release of lipid mediators, and the activation of cell surface integrin adhesion receptors. The interaction of activated integrins, especially the main platelet integrin αIIbβ3, with immobilized ligands exposed in the ECM or with multivalent plasma ligands, e.g., fibrinogen, is arguably the most critical step in platelet plug formation. Platelets. https://doi.org/10.1016/B978-0-12-813456-6.00018-7 Copyright © 2019 Elsevier Inc. All rights reserved.

Consequently, alterations in the kinetics and/or duration of integrin activation can lead to bleeding or thrombocytopenia/thrombosis. In this chapter, we will discuss the signaling responses most critical for hemostatic plug formation. On the surface, the platelet signaling machinery looks similar to that of any other cell type. Instructed by their unique environment, however, the signaling pathways in platelets evolved to be extremely sensitive and fast responding towards changes in the vasculature. At the same time, strong negative regulators ensure (1) that platelets remain in a quiescent state while in circulation, and (2) that thrombus formation is a selflimiting process. The following sections will review the main players (proteins, lipids, ions), both positive and negative, required for efficient platelet integrin activation and adhesion during hemostatic plug formation. The signaling machinery required for other important cellular responses, such as granule secretion and cytoskeletal rearrangements, are discussed in detail in other chapters (for example Chapters 3, 10–15 and 19). In this chapter, we give special emphasis to GPCRs, the second messengers Ca2+, DAG, and phosphatidylinositol3, 4, 5-triphosphate (PI(3,4,5)P3), kinases, and the small GTPase RAP1, as we believe that these molecules are central to platelet adaptation to their unique environment and to the challenge of forming a three-dimensional hemostatic plug under flow. Outside the scope of this chapter, there is also exciting literature on how platelets help secure vascular integrity in other situations of challenge, such as at sites of inflammation, where much smaller holes in the endothelial lining, generated by transmigrating inflammatory cells, require plugging by single platelets. As shown by us and others, vascular integrity in inflammation and development is strongly dependent on platelet ITAM, but not GPCR signaling. What is less clear is the cellular response(s) by which platelets secure vascular integrity in inflammation and support endothelial barrier function. See Chapters 11, 16, and 28 for more details.

MAJOR STIMULATORY AND INHIBITORY RECEPTORS EXPRESSED ON THE PLATELET SURFACE The platelet surface is covered by glycoproteins that mediate a variety of cellular processes, including cell-cell and cell-matrix adhesion, interplay with the coagulation system, and cellular activation. Chapter 9 gives an overview of the most important platelet surface agonist and adhesion receptors. Chapters 10– 15 summarize the state-of-the-art regarding stimulatory and inhibitory receptors expressed on the platelet surface. Chapters 11, 13, and 14 provide an in-depth discussion on platelet hem (ITAM), PAR, and P2 receptors, respectively. Chapters 10 and 12 discuss signaling associated with the two major adhesion receptors on the cell surface, the GPIb-IX-V complex and integrin αIIbβ3. Inhibitory signaling is the focus of Chapter 15. For the purpose of this chapter, we will briefly summarize the most

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Fig. 18.1 The platelet signaling machinery. Overview of the major signaling nodes that control platelet activation. The main platelet agonist receptors (green boxes) are, from left to right, the Gq-coupled receptors for thrombin (protease-activated receptors 1 and 4, PAR1/4), thromboxane A2 (TPα/β), and adenosine diphosphate (P2Y1), the (hem)ITAM-coupled receptors for collagen (glycoprotein VI-Fc receptor γ (FcRγ)chain complex, GPVI-FcRγ), IgG immune-complexes (FcγRIIA, CD32A) and podoplanin (CLEC2), the Gi (Gz)-coupled receptors for adenosine diphosphate (P2Y12) and epinephrine (alpha-2A adrenergic receptor, α2A), the Gs-coupled receptors for prostaglandin I2 (IP), adenosine (A2) and pituitary AC-activating and vasoactive intestinal peptides (VPAC1), and the intracellular receptor for nitric oxide (soluble guanylyl cyclase, sGC). These receptors activate (black arrow) or inhibit (red blocking symbol) effectors (blue boxes) that generate second messengers (blue fonts). Phospholipase C (PLCβ and PLCγ) promotes calcium (Ca2+) mobilization and generation of diacylglycerol (DAG). Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) generates phosphatidylinositol-3,4,5-trisphosphate (PIP3). Adenylyl cyclase generates cyclic adenosine monophosphate (cAMP), while cyclic guanosine monophosphate (cGMP) is synthetized directly by the intracellular receptor sGC. Signal integrators (yellow boxes), such as the small GTPase RAP1 and the kinases protein kinase C (PKC), protein kinase B (PKB, AKT), protein kinase A (PKA) and protein kinase G (PKG), convert these signals into cellular responses (red box) resulting in platelet activation.

critical information on the expression and the proximal signaling of the two major stimulatory and inhibitory receptor types, GPCRs and (hem)ITAM/ITIM receptors.

G Protein-Coupled Receptors (GPCRs) GPCRs are seven transmembrane receptors that mediate cellular activation via the engagement of heterotrimeric G proteins, made up of α, β and γ subunits. The Gα families (Gq/11, G12/13, Gi, Gs) are grouped based on their structure and downstream effectors (Fig 18.2): adenylyl cyclase (AC)-stimulating Gs proteins, AC-inhibiting Gi proteins,1 PLC-linked Gq proteins,2,3 and the RHOGEF-activating G12/13 proteins.4 In nonstimulated cells, GDP-bound Gα is in a complex with Gβγ. This interaction is important as it localizes Gα to the membrane,5 prevents spontaneous GTP binding and activation of Gα,6 and prevents signaling by the β/γ-complex, which has been increasingly recognized as a critical component of GPCR signaling.7 When engaged, the GPCR serves as a guanine nucleotide exchange factor (GEF) and induces the exchange of GDP for GTP in the Gα-subunit.8 The GTP-bound Gα-subunit and the Gβγ unit separate and interact with their respective downstream effectors (Fig. 18.2). Inactivation of the system requires GTPase-activating proteins (GAPs), which increase the hydrolysis rate of the γ-phosphate in GTP. The prototypic GAPs for G proteins are Regulator of G-protein Signaling (RGS) proteins; platelets express several members of this family, including RGS2, 10, and 18.9 During the process of hemostatic plug formation, the primary agonist, thrombin, and the second wave mediators, adenosine diphosphate (ADP) and thromboxane (Tx)A2, activate PARs, the ADP receptors (P2Y1 and P2Y12), and the TxA2 receptors (TPα and TPβ), respectively.10–12 GPCRs with less wellcharacterized contributions to hemostatic plug formation include the serotonin receptor, 5-HT2A, the adrenergic receptor, α2A, and multiple chemokine receptors (see Chapter 9). Platelets also express GPCRs that inhibit cellular activation, most

Fig. 18.2 The G protein cycle. G protein-coupled receptors (GPCRs) are seven-transmembrane proteins coupled to heterotrimeric G proteins, which function through a binary switching mechanism that is driven by the binding of guanosine trisphosphate (GTP). In nonstimulated conditions (red), the receptors associate with an inactive heterotrimeric complex, consisting of a Gα subunit loaded with guanosine diphosphate (GDP) and bound to the Gβ and Gγ subunits. When GPCRs are activated by external stimuli (green), they act as guanine nucleotide exchange factors (GEFs) for the heterotrimeric G proteins, catalyzing the release of GDP by the Gα subunit. GDP is immediately replaced with GTP which is more abundant in the cytosol. The complex dissociates into Gα/GTP and Gβγ subunits that are then capable of activating or inhibiting a wide range of effectors. The most notable effectors in platelets and, in parenthesis, the specific G proteins that control them, are listed. The regulators of G protein signaling (RGS) proteins function as GTPase-activating proteins (GAPs) to accelerate the intrinsic GTPase activity of the Gα subunit, which is the off reaction that terminates GPCR signaling.

notably the prostacyclin receptor, IP. Human PAR1 as well as mouse and human PAR4 couple to Gq13–15 and G12/13 proteins to induce PLC and RHO-GEF/RHOA activation, respectively.16 The TxA2 receptors also couple to both Gq and G12/13.17,18 In

Platelet Signal Transduction

contrast, P2Y1 couples specifically to Gq, and P2Y12 specifically activates Gi.19 A connection between PAR1 and Gi was also reported,20 but this finding needs further confirmation. Gq-mediated activation of PLC is critical for the generation of the second messengers, Ca2+ and DAG, and the nearimmediate activation of integrin receptors required for platelet adhesion under shear stress conditions (see the section on “Phospholipase C”). Thrombin induces fast but reversible PLC activation via cleavage of PAR1, and sustained activation via cleavage of PAR4. The TPα/β and P2Y1 receptors are less powerful but provide important feedback activation of PLCβ. Gi activation downstream of P2Y12 leads to the inhibition of AC mediated by Giα, and Gβγ-mediated activation of phosphoinositide 3-kinase (PI3K). AC inhibition ensures low levels of cyclic adenosine monophosphate (cAMP) and thus minimal negative feedback on platelet activation via protein kinase A (PKA) signaling (see the sections on “Cyclic Nucleotide Signaling” and “Protein Kinase A/Protein Kinase G (PKA/G)”). PI3K signaling leads to the inhibition of the RAP1-GAP, RASA3,21 and the activation of AKT. The coupling of PAR1, PAR4, and TP to G12/13 is critical for enhanced myosin light chain (MLC)-dependent rearrangement of the cytoskeleton and platelet shape change16 (Fig. 18.3). In platelets, G13α is the predominant isoform, both in humans and mice.22–24 Once activated, G13α couples to p115RHO-GEF,25 which activates RHOA and Rho-associated kinase (ROCK). ROCK then phosphorylates and inhibits MLC phosphatase (MYPT), leading to increased MLC phosphorylation and activity.16 While RHOA and ROCK are critical for cytoskeletal changes downstream of thrombin and TxA2 receptors, ADP does not directly trigger this response. Instead, increased levels of cytoplasmic Ca2+, triggered via P2Y1 receptor signaling, leads to the activation of MLC kinase (MLCK) and phosphorylation of MLC.26 It is important to note that integrins also signal (outside-in) via G13α to RHOA, a mechanism that is important during platelet spreading.27–29 The endothelium plays an important role in preventing unwanted platelet activation (see Chapter 17). Healthy endothelial cells release nitric oxide (NO) and prostacyclin

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(prostaglandin I2, PGI2),30 which cause the activation of soluble NO-sensitive guanylyl cyclase (sGC) and AC, respectively. While NO is membrane-permeable, PGI2 binds to the Gscoupled GPCR receptor, IP, to induce AC activation. Increased levels of cGMP and cAMP lead to the activation of PKG and PKA, protein kinases that have inhibitory activity in platelets31 by phosphorylating and thus inactivating multiple substrates implicated in G protein activation, Ca2+ mobilization and actin cytoskeleton remodeling. Degradation of cGMP and cAMP by phosphodiesterases limits the extent of PKG/A signaling.31

(hem)ITAM and ITIM Receptors Several platelet receptors contain immunoreceptor tyrosinebased activation motifs (ITAMs) or immunoreceptor tyrosine-based inhibitory motifs (ITIMs), conserved sequences that are best known for their role in signal transduction in immune cells. Platelets express three (hem)ITAM-coupled receptors: (1) the ITAM-containing Fc receptor γ chain (FcRγ), which associates with GPVI, a receptor for collagen, laminin, and fibrin in the damaged vascular wall, (2) the ITAMcontaining FcγRIIA, a receptor for immune complexes, and (3) CLEC-2, a hemITAM-containing receptor for podoplanin expressed on select cell types such as podocytes, lymphatic endothelial cells and type I alveolar cells.32,33 Critical to the signaling activity of (hem)ITAM receptors are the cytosolic YxxL motifs which, when phosphorylated, serve as docking sites for SH2 domain containing signaling molecules.34,35 Both the FcRγ chain and FcγRIIA contain a pair of cytosolic YxxL motifs, separated by 6–12 residues, which facilitate the binding of the non-receptor tyrosine kinase, spleen tyrosine kinase (SYK).33,36 CLEC-2 contains only one YxxL motif in its cytoplasmic tail and thus exists as a homodimer on the cell surface to enable SYK binding to two phosphorylated hemITAM motifs.37 SYK is critical for the formation of a signalosome consisting of various adapter and effector proteins, including linker for activation of T cells (LAT),38,39 growth factor receptor–bound protein-2 (GRB2), growth

Fig. 18.3 G13 signaling. Recent studies indicate that G13α is coupled to both GPCRs and integrins at different stages of platelet activation. (Left panel) GPCR-mediated signaling. Downstream of the thrombin receptors, PAR1/4, and the thromboxane A2 receptors (TPα/β), G13α stimulates shape change by promoting the activation of the small-GTPase RHOA and its downstream effector Rho-activated kinase (ROCK). In turn, ROCK promotes actomyosin contraction by activating LIM kinase (LIMK), that blocks the actin-severing protein COFILIN, and by inhibiting the myosin light chain phosphatase (MLCP), which enables increased myosin light chain phosphorylation (pMLC). At high enough agonist concentrations Gqα-coupled receptors (PAR1/4, TPα/β, P2Y1) can induce shape change independently of G13α signaling by promoting calcium (Ca2+)-dependent activation of the myosin light chain kinase (MLCK), myosin light chain phosphorylation (pMLC) and acto-myosin contraction. (Right panel) Integrin-mediated signaling. During the early stages of outside-in signaling, integrins associate with G13α, which stimulates c-SRC-mediated inhibition of RHOA and enables RAC1dependent spreading. In the late stages of outside-in signaling, G13α dissociates from the integrin tail, thus enabling RHOA/ROCK-dependent actomyosin contraction necessary for clot retraction in synergy with RAC1/MLCK signaling.

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factor receptor–bound protein-2 adaptor downstream of SHC (GADS), and SH2 containing leukocyte protein of 76 kDa (SLP-76).40 Both LAT and SLP-76 contribute to the recruitment of phospholipase Cγ2 (PLCγ2),41 the enzyme required for the generation of the second messengers Ca2+ and DAG. Genetic deficiency in mice of any of the proteins mentioned above leads to severely impaired ITAM signaling in platelets. In addition to this core group of proteins, other adapters and effectors also contribute to effective (hem)ITAM signaling, including signal transducer and activator of transcription 3 (STAT3),42 the small GTPase RAC143,44 and its exchange factors VAV1 and VAV3,45–47 the tyrosine kinases Bruton’s tyrosine kinase (BTK),48 and TEC,49 or various phosphatidylinositol-3 (PI3) kinase isoforms.50 The signaling events downstream of PLCγ2 will be discussed later in this chapter. ITAM receptors indirectly engage signaling pathways leading to cytoskeletal rearrangements and shape change or inhibition of adenylyl cyclase by inducing the release of the second wave mediators, TxA2 and ADP. The contribution of platelet (hem)ITAM signaling to thrombosis and hemostasis has been determined using transgenic mice and specific inhibitors against individual components of the signalosome (see Chapter 11).33 In summary, these studies suggest that ITAM signaling plays a minor role for platelet adhesion at sites of vascular injury when compared with signaling via platelet GPCRs. More recent work, however, identified a key role for (hem)ITAM receptors in immune complexmediated thrombocytopenia and thrombosis, vascular integrity at sites of inflammation, and vascular development.32 Platelet endothelial cell adhesion molecule 1 (PECAM-1, CD31) is a critical platelet surface receptor containing two ITIM sequences in its cytoplasmic tail. Sequential serine and tyrosine phosphorylation of both ITIM sequences (S/I/V/LxYxxI/V/L) is

required for inhibitory signaling by PECAM-1. Once phosphorylated, PECAM-1/SHP-2 complexes sequester the p85 regulatory subunit of PI3K away from its participation with the ITAM signalosome.51 Consistent with this inhibitory activity, loss of PECAM-1 function renders platelets more responsive to stimulation by ITAM-coupled agonist receptors, both in vitro and in vivo.52–54 G6B is another important ITIM receptor on the platelet surface. Like PECAM, G6B contains a pair of ITIM domains that are phosphorylated upon cellular activation. Interestingly, G6B knockout mice exhibit macrothrombocytopenia, impaired platelet function, and an increased tendency to bleed.55 The thrombocytopenia is in part due to increased platelet turnover, consistent with G6B being an inhibitor of platelet function. Megakaryocytes in G6B
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mice show several defects, including impaired proplatelet release and increased shedding of cell surface receptors such as GPVI and GPIb, again consistent with G6B being an inhibitor of megakaryocyte function. The increased receptor shedding is a contributing factor to impaired platelet function, but intracellular signaling is also defective in these cells. At this point, it is not clear why intracellular signaling is downmodulated in these cells. The inhibitory properties of both PECAM-1 and G6B are discussed at length in Chapter 15.

SECOND MESSENGERS Phospholipase C Phospholipases (PLCs) are membrane-associated enzymes that catalyze the hydrolysis of phosphatidylinositol 4,5bisphosphate (PI(4,5)P2) to inositol 1,4,5-trisphosphate (IP3) and DAG (Fig. 18.4). IP3 is freely diffusible and binds to IP3operated calcium channels, leading to the release of calcium ions (Ca2+) from intracellular stores into the cytosol. DAG remains

Fig. 18.4 Phosphoinositide signaling. Phosphatidylinositol-4,5-bisphosphate (PIP2), which is synthesized by the phosphatidylinositol-4phosphate-5-kinase type I (PIP5KI), is the substrate of phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K). Gq-coupled receptors stimulate PLCβ and PI3Kβ. The Gi-coupled receptor for adenosine diphosphate (P2Y12) stimulates PI3Kβ and PI3Kγ. ITAM-coupled receptors and integrins stimulate PLCγ and PI3Kβ. PLCs catalyze the hydrolysis of PIP2 to inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 is freely diffusible and binds to IP3-operated calcium channels, leading to the increase of cytoplasmic levels of calcium ions (Ca2+), which mediate various cellular responses including the stimulation of CalDAG-GEFI-mediated RAP1 activation and consequent integrin activation. DAG remains membraneassociated and activates protein kinase C (PKC), which is critical for granule secretion and may be contributing directly to sustained RAP1 activation through a yet unknown pathway. DAG also activates diacylglycerol kinases (DGK), thus providing negative feedback for its own signaling. PI3Ks phosphorylate PIP2 to generate phosphatidylinositol-3,4,5-trisphosphate (PIP3) at the plasma membrane. PIP3 levels are reduced by lipid phosphatases (PTEN; Phosphatase and tensin homolog; SHIP, SH2 domain-containing inositol 50 -phosphatase). Downstream of ITAM-coupled receptors and integrins, PIP3 recruits PLCγ and the protein-tyrosine kinases BTK and TEC, which in turn activate PLCγ by phosphorylation. Downstream of P2Y12, PIP3 inhibits the RAP1 GTPase-activating protein, RASA3, thus promoting sustained integrin activation. Mainly downstream of GPCRs, PIP3 contributes to platelet activation through PDK-dependent activation of AKT and inhibition of GSK3, an established platelet inhibitor. Moreover, PIP3 may be contributing to integrin-mediated adhesion by binding the component of the integrin activation complex, KINDLIN3.

Platelet Signal Transduction

membrane-associated and activates various proteins, most importantly protein kinase C (PKC). PLCs represent a critical signaling node in platelet activation as they are stimulated downstream of most agonist receptors and, in turn, affect the levels of three second messengers: Ca2+, DAG, and phosphoinositides. Platelets express members of two major PLC subfamilies, PLCβ and PLCγ, which have complementary functions in platelet biology. The PLCβ isoforms, in particular PLCβ2 and PLCβ3,56 are activated by Gq-coupled GPCRs, while PLCγ257–59 and PLCγ159 are activated by signaling involving tyrosine kinase cascades downstream of (hem)ITAM-coupled and integrin receptors. Based on expression profiling and functional studies, PLCγ2 is the dominant PLCγ isoform in platelets.59,60 Stimulation of PLCβ induces a cytosolic Ca2+ increase with high amplitude and fast reversion, while PLCγ2 induces a slower but more sustained Ca2+ mobilization.61 At sites of vascular injury, the relative importance of PLCβ and PLCγ for platelet adhesion depends on the type and severity of the lesion. In models of arterial thrombosis and classical hemostasis, the Gq/PLCβ signaling module is critical to ensure thrombus formation,56,62 while PLCγ2 is largely dispensable.62 Consistently, a partial reduction in PLCβ2 expression is sufficient to cause a prolonged bleeding diathesis in a human patient.63 On the other end, (hem)ITAM/ PLCγ2, but not GPCR/PLCβ, signaling is required to support vascular integrity at sites of inflammation64 or during lymphatic vascular development,65 situations where adhesion of single platelets is required to prevent blood loss from very small lesions in the vasculature.66

Calcium Signaling Many platelet responses are regulated by calcium, which is why cytoplasmic concentrations of free Ca2+ ions, i.e., the biologically active form of calcium, must be tightly controlled. Under

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resting conditions, platelets maintain very low levels of cytosolic Ca2+ (50 nM).67 Large amounts of free Ca2+ are sequestered in membrane-bound intracellular stores, most notably the dense tubular system (DTS) and the acidic stores,68 such as lysosomes and dense granules (Fig. 18.5). The most prominent intracellular store is the DTS, which is the equivalent of the endoplasmic reticulum of other cell types and of the sarcoplasmic reticulum found in muscle cells. The DTS contains an estimated concentration of 250 μM Ca2+.69 The plasma level of ionized calcium is in the range of 1.3–1.5 mM. In order to maintain such steep gradients between the cytosol, the intracellular stores and the extracellular space, Ca2+ is actively pumped across the plasma membrane and into the stores. Ca2+ efflux across the plasma membrane is mediated by two types of energy-driven pumps, the Plasma Membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger (NCX). Both are implicated in maintaining low Ca2+ levels in resting platelets, and in regulating the local reuptake of Ca2+ in agonist-stimulated platelets in order to shape subcellular microdomains, where selective Ca2+ effectors are activated.70,71 PMCA activity is upregulated both by cAMP, which is high in resting platelets (see the section on “Cyclic Nucleotide Signaling”), and by Ca2+/calmodulin complexes, which increase upon platelet stimulation. PMCA is inhibited by FAK-dependent tyrosine phosphorylation and by calpaindependent proteolysis, which occur in the late stages of platelet activation.70 As a result, pharmacological inhibition of PMCA or genetic deletion of Pmca4, the main isoform expressed in platelets, increases resting levels of cytosolic Ca2+ and dampens agonist-induced aggregation and secretion, but increases clot retraction and spreading.71 Similarly, the predominant Na+/Ca2+ exchanger expressed in platelets, NCX3, has been implicated in the export of Ca2+ in both resting and thrombin-stimulated platelets.72 The loading of Ca2+ into

Fig. 18.5 Calcium signaling. Several mechanisms (blue boxes) are in place to control the cytoplasmic Ca2+ concentrations in resting ([Ca2+]R) and activated ([Ca2+]A) platelets: (1) Ca2+ sequestration and (2) Ca2+ release from the dense tubular system (DTS) and the acidic stores, (3) Ca2+ efflux to and Ca2+ influx from the extracellular space, i.e., plasma. (1) Ca2+ sequestration into intracellular stores is mediated by Sarco-Endoplasmic Reticulum Ca2+ ATPases (SERCA) and, to the acidic Ca2+ stores only, through H+/Ca2+ exchangers coupled to the vacuolar H+-ATPases. (2) Ca2+ release from the DTS is mediated by IP3 receptors (IP3R); Ca2+ release from the acidic stores is regulated by the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP). (3) Ca2+ efflux across the plasma membrane is mediated by the Plasma Membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger (NCX). (4) The main mechanism of Ca2+ influx from the extracellular space is store-operated Ca2+-entry (SOCE), which occurs through plasma membrane Ca2+ channels composed of ORAI1 subunits, upon activation by the Ca2+ sensor, stromal interaction molecule 1 (STIM1). Additionally, extracellular Ca2+ enters through P2X1, a receptor-operated Ca2+ channel stimulated by ATP, and TRPC6 (transient receptor potential canonical 6), stimulated by diacylglycerol (DAG).

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intracellular stores is mediated by Sarco-Endoplasmic Reticulum Ca2+ ATPases (SERCA).73,74 Additionally, pharmacological studies suggest that the acidic Ca2+ stores contain H+/Ca2 + exchangers coupled to the vacuolar H+-ATPases.69 Inhibition of SERCAs and H+/Ca2+ exchangers additively increases agonist-evoked cytosolic Ca2+ elevation, but with different kinetics,69 suggesting specialized functions for the two types of pumps. Adding to the complexity of Ca2+ homeostasis, there is evidence that the concentration of cytosolic free Ca2+ is also regulated by buffering proteins,75 which are most likely important to prevent toxic Ca2+ accumulations and to modulate the spatiotemporal dynamics of Ca2+ signals. However, although many potential buffering proteins have been identified in the platelet proteome,22 very little is known about their importance for proper platelet function. Upon platelet stimulation, the cytosolic Ca2+ concentration rapidly increases by at least one order of magnitude (0.2–1 μM) through two main mechanisms: 1) the release of Ca2+ from intracellular stores and 2) the entry of Ca2+ through the plasma membrane from the extracellular space (Fig. 18.5). The main mechanism allowing the release of Ca2+ from intracellular stores involves IP3 receptors (IP3R), which act as Ca2+ channels upon IP3 binding. Following agonist-induced PLC activation, the cytoplasmic IP3 concentration rises sharply by several fold and peaks within 2–30 s after agonist delivery.76–78 Stimulation of IP3Rs evokes a rapid rise in cytosolic Ca2+ concentration that mirrors that of IP3. This IP3-mediated Ca2+ spike is not only rapid but also transient, since IP3 is degraded by phosphatases within 30–60 s after agonist stimulation.79 However, other mechanisms are in place to shape the duration and the amplitude of the Ca2+ flux. Upon stimulation, a smaller pool of Ca2+ is also released from the acidic stores through the action of the second messenger nicotinic acid adenine dinucleotide phosphate (NAADP).80,81 However, the Ca2+ channels involved and the role of this Ca2+ flux in platelet physiology are still not well understood. Sustained cytosolic Ca2+ elevation is largely dependent on the entry of Ca2+ across the plasma membrane. The main mechanism of Ca2+ influx from the extracellular space is storeoperated Ca2+-entry (SOCE), which is evoked upon depletion of the intracellular Ca2+ stores.82 SOCE occurs through plasma membrane Ca2+ channels composed of ORAI1 subunits, which are activated by the Ca2+ sensor, stromal interaction molecule 1 (STIM1). The closely related proteins STIM2, ORAI2 and ORAI3 are not detectable by platelet proteomic profiling,22 and Stim2
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platelets show unaltered Ca2+ responses.83 STIM1 is a single transmembrane protein that senses Ca2+ on the luminal side of the DTS via a single functional EF hand domain. Upon agonist-induced store release, Ca2+ dissociation from the EF hand evokes a conformational change that enables STIM1 to interact with ORAI1 at DTS-plasma membrane junctions and to induce the opening of ORAI1 channels. The importance of SOCE in platelet biology is emphasized by the fact that several human diseases caused by gain or loss of function mutations in Stim1 and Orai1 affect platelet activity and/or number.84 In mice, expression of a Stim1 mutant unable to bind Ca2+ leads to increased baseline Ca2+ levels, preactivation and premature clearance of circulating platelets.85 On the other hand, lack of either Stim1 or Orai1 or expression of a loss-of-function mutant of Orai1 (Orai1R93W) in platelets (using bone marrow chimeras to circumvent the perinatal lethality of germline mutant mice) abolishes SOCE almost completely in response to all platelet agonists.83, 86, 87 Interestingly, platelets from Stim1
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chimeras also show a defect in Ca2+ mobilization from intracellular stores, suggesting that STIM1 is also important for proper store refilling. When studying integrin activation and secretion, Stim1 and Orai1 mutant platelets exhibit only a mild defect when

stimulated via GPCRs. The more pronounced dependence on SOCE observed for ITAM-dependent platelet activation is consistent with a weaker Ca2+-store release downstream of PLCγ2 when compared to PLCβ, and the fact that thrombin is able to evoke alternative SOCE-independent routes of Ca2+ influx.83 Prolonged high cytoplasmic Ca2+ levels, facilitated by Ca2+ influx, are a requirement for the platelet procoagulant response, i.e., the ability of activated platelets to express phosphatidylserine (PS) on the cell surface. Stim1 and Orai1 mutant platelets show marked defects in PS exposure, both under static and flow conditions.83,88 In vivo, impaired SOCE leads to a partial protection from arterial thrombosis and ischemic stroke and a very modest prolongation of tail bleeding time.87 Receptor- and second messenger-operated Ca2+ channels represent other important mechanisms of extracellular Ca2+ entry in platelets. The only receptor-operated Ca2+ channel expressed in human platelets is P2X1,89 a purinergic receptor stimulated by ATP, a short-lived paracrine agonist released by activated platelets or by damaged erythrocytes and endothelial cells. P2X1 receptors are critical to amplify the Ca2+ signal in response to low doses of agonists and to facilitate platelet adhesion in the presence of high shear stress conditions in vitro and in vivo.90 Additionally, platelets express low levels of second messenger-operated channels of the transient receptor potential canonical subfamily (TRPC).91 Genetic evidence with Trpc6 knockout mice demonstrates that TRPC6 is the main receptor mediating Ca2+ entry following DAG formation by PLCs,92 suggesting an important crosstalk between these two second messengers.

Diacylglycerol Signaling PLCs produce DAG through the same reaction that releases IP3 (Fig. 18.4). Its signaling function depends on its ability to bind and activate C1 domain-containing proteins. The most wellestablished effectors of DAG are Ser/Thr kinases of the protein kinase C family, which translate this static signal into the phosphorylation of a variety of substrates (see the section on “Protein Kinase C (PKC)”). Human platelets express several diacylglycerol kinases (DGKs),93 enzymes responsible for terminating DAG signaling. Genetic evidence suggests that loss of Dgke (diacylglycerol kinase ε) may be associated with the pro-thrombotic state of patients with atypical hemolyticuremic syndrome.94 Pharmacological inhibition of DGKs impairs SOCE-independent Ca2+ entry, thus confirming the role of DAG in regulating second messenger-operated Ca2+ channels.95

PI3K and 3-Phosphorylated Phosphoinositide Signaling The phosphorylated forms of phosphatidylinositol (PI), known as phosphoinositides, are crucial components of platelet intracellular signaling mechanisms, although they account for only 10%–15% of membrane phospholipids. Because of their signaling function, PIs are preferentially localized to the cytosolic leaflet of cellular membranes. The most abundant PI present in membranes is phosphatidylinositol-4,5bisphosphate (PI(4,5)P2), which is mainly synthesized from phosphatidylinositol-4-phosphate by the lipid kinase activity of phosphatidylinositol-4-phosphate-5-kinase type I (PIP5KI).96 PI(4,5)P2 has two critical functions in platelet signaling: it is the substrate of phospholipase C (PLC) and phosphoinositide 3-kinase (PI3K), enzymes that generate second messengers, and it serves as an anionic docking site for proteins implicated in integrin activation and membrane-cytoskeleton interactions, such as TALIN97,98 (Fig. 18.4). Interestingly,

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studies in mice deficient in specific PIP5KI isoforms suggest that separate pools of PI(4,5)P2 exist within the platelet membrane; PI(4,5)P2 produced by PIP5KI-γ is important for the anchoring of the membrane to the underlying cytoskeleton,99 while PI(4,5)P2 synthetized by PIP5KI-α and PIP5KI-β is used as a substrate for second messenger generation.100 PI3Ks are a large family of lipid kinases that phosphorylate the 30 -OH position of the inositol ring of phosphatidylinositol, generating 3-phosphorylated PI second messengers.101 All 3-phosphorylated PIs function by recruiting signaling proteins containing PI-binding domains, such as PX, FYVE or pleckstrin homology (PH) domains, to cellular membranes. The best characterized members of this family are class I PI3Ks that, upon cellular stimulation, phosphorylate PI(4,5)P2 to generate phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) at the plasma membrane (Fig. 18.4). Class IA PI3Ks are heterodimeric proteins, which are further classified based on their subunit composition. They are composed of a catalytic subunit (p110α, p110β or p110δ) associated with a SH2containing regulatory subunit (five variants among which p85α is the most abundant), which binds to phosphorylated tyrosine residues in a YxxM motif (x is any amino acid). Such Y(P)xxM motifs are typical of receptor tyrosine kinases and in platelets are found on the adaptor molecule LAT following stimulation of integrins or (hem)ITAM-coupled receptors. Moreover, p110β is also activated by Gβγ subunits downstream of GPCRs.102,103 Class IB PI3K (PI3Kγ) consists of a p110γ catalytic subunit associated with a regulatory subunit (p84), which is activated by binding to Gβγ subunits following GPCR stimulation. The most highly expressed PI3Ks in platelets are PI3Kβ (p110β/p85α) and PI3Kγ (p110γ/p84). Studies with p110β knockout or kinase-dead mice and with the highly selective inhibitor TGX-221 concur that PI3Kβ is the predominant platelet PI3K activated downstream of GPCRs, ITAM-coupled receptors and integrins. Its catalytic activity is critical for 1) the efficient activation of PLCγ2 downstream of (hem)ITAMcoupled and integrin receptors,104,105 2) the sustained upregulation of integrin αIIbβ3 affinity downstream of the ADP receptor P2Y12,104,106 and 3) the increase of integrin αIIbβ3 avidity and its linkage to the underlying cytoskeleton.107 In vivo studies demonstrate that the key function of PI3Kβ signaling is to promote irreversible integrin activation and thrombus stability.106 For this reason, PI3Kβ has been proposed as a potential target for future anti-thrombotic therapies. PI3Kγ is activated by the Gβγ subunit of the ADP receptor P2Y12 and it is mainly involved, together with PI3Kβ, in mediating P2Y12-dependent sustained integrin αIIbβ3 activation108–110 through its effect on the RAP1 regulator RASA3.21 PI3Kα (p110α/p85α) is much less abundant, but it shares with PI3Kβ a role in GPVI proximal signaling events.105,111 Moreover, PI3Kα alone mediates the potentiating effect of insulin like growth factor 1 (IGF-1) on platelet aggregation induced by low doses of other agonists.112 However, PI3Kα is not implicated in the sustained regulation of integrin affinity and avidity. PI3Kδ (p110δ/p85α) expression is very low and its deficiency has a very modest effect on platelet function.113 The platelet responses mediated by class I PI3Ks are primarily the consequence of PH-domain containing proteins binding to PI(3,4,5)P3. To achieve optimal (hem)ITAM and integrin signaling, the main PH-domain containing proteins recruited to PI(3,4,5)P3-microdomains are PLCγ2 and the protein-tyrosine kinases BTK and TEC, which in turn activate PLCγ2 by phosphorylation.114 Downstream of P2Y12, PI3Kβ, and PI3Kγ mediate sustained integrin activation primarily by inhibiting the RAP1-GAP, RASA3 (see the section on “RAP1 Regulators”). Another well-established effector of PI(3,4,5)P3

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is the serine/threonine kinase AKT/PKB. In fact, AKT phosphorylation is the most widely used read-out for PI3K-dependent PI (3,4,5)P3 generation in platelets; however, the role of AKT in integrin signaling is still unclear. Lastly, it is not known how PI3K affects integrin avidity and the linkage between high affinity integrins with the contractile cytoskeleton. It is notable that KINDLIN3, the component of the integrin activation complex that is believed to control avidity, has an atypical FERM domain comprising a PH domain that binds with high affinity to PI(3,4,5)P3115 and that is critical for KINDLIN to synergize with TALIN in integrin activation.116,117 A recent study employed mass spectrometry to identify additional PI(3,4,5) P3-binding proteins in platelets,118 including several regulators of the cytoskeleton and of small GTPase signaling. Future studies will have to clarify how these novel PI(3,4,5)P3 binding partners contribute to the PI(3,4,5)P3 signalosome and to platelet function. Importantly, platelets also express lipid phosphatases (PTEN, SHIP1, SHIP2) that, by reducing the level of PI (3,4,5)P3, downregulate PI3K-dependent platelet activation. PTEN is a ubiquitous PI(3,4,5)P3 3-phosphatase that is weakly expressed in platelets. However, its deficiency in mouse platelets leads to a significantly shortened bleeding time and increased sensitivity to collagen-induced activation and aggregation.119 SHIP1 is a 5-phosphatase120,121 highly expressed in hematopoietic cells, whereas the closely related enzyme SHIP2 is ubiquitous. Studies with mouse platelets suggest that SHIP1, rather than SHIP2, plays a major role in controlling the overall PI(3,4,5)P3 levels in response to thrombin or collagen activation.122 However, these results may need to be revisited, since only heterozygous SHIP2 mice were examined to circumvent the perinatal lethality of SHIP2 deficiency.123 Interestingly, SHIP1 deficiency does not affect integrin inside-out activation,124 suggesting that other phosphatases may be regulating the specific pool of PI(3,4,5)P3 that controls RASA3 function and RAP1 activation. It is generally accepted that SHIP1 is an important regulator of integrin outside-in signaling, but it is not exactly clear how, since one study reports enhanced platelet spreading and platelet adhesion under flow ex vivo,125 while another study reports that clot retraction, thrombus growth and hemostasis are impaired.124 This conflicting data could be due to the fact that SHIP1 deficiency leads to a strong increase in PI(3,4,5)P3 levels, and concomitant decrease of PI(3,4)P2,122,125 another lipid which is normally produced in large amounts in platelets after integrin engagement.126,127 Platelets also express class II PI3Kα and β as well as class III PI3K, which do not use PI(4,5)P2 as a substrate, but synthesize phosphatidylinositol-3-phosphate (PI(3)P) and phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) (only class II). However, studies on the function of these enzymes in platelets are still limited. Two independent groups using different mouse models128,129 have shown that the class II PI3KC2α is important to control platelet membrane structure and elasticity, and one of the two observed a contribution of this enzyme in maintaining basal, but not agonist-induced, levels of PI(3) P.129 On the contrary, VPS34, the sole class III phosphoinositide 3-kinase, was recently shown to raise agonist-induced, but not basal, PI(3)P levels in platelets. Counterintuitively, genetic or pharmacological blockade of platelet VPS34 increases the intensity and timing of platelet secretion but impairs thrombus growth under arterial wall shear rate.130 Moreover, inhibitor studies suggest a role for this enzyme in regulating autophagy.131 These interesting findings underline the importance of phosphoinositides other than PI(4,5)P2 and PI(3,4,5)P3 in platelet signaling and demand further studies to decipher their complex interplay with currently known pathways.

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Cyclic Nucleotide Signaling Another important class of second messengers in platelets are cyclic nucleotides, which are typically elevated in quiescent circulating platelets and low in active, pro-adhesive platelets. In fact, while in most cells cyclic adenosine 3’,5’-monophosphate (cAMP) has a positive effect on cell function, in platelets it negatively regulates activation. Similarly, cyclic guanosine 30 ,50 monophosphate (cGMP) dampens platelet responsiveness, often in synergy with cAMP signaling, although there are some controversies over its ability to amplify platelet activation in response to low doses of agonists (see the section on “Protein Kinase A/Protein Kinase G (PKA/G)”). The predominant signaling pathways that induce the production of cyclic nucleotides are evoked by prostaglandin I2 (PGI2) and nitric oxide (NO), which are short-lived platelet antagonists released by the intact and healthy endothelium to maintain patrolling platelets in a resting state (Chapter 17 and Fig. 18.6). PGI2 increases intracellular cAMP levels by binding the IP receptor,132 a Gs-coupled GPCR on the platelet membrane that stimulates adenylyl cyclase (AC) activity. NO, a neutral oxide, can freely diffuse into the cytoplasm, where it activates soluble guanylyl cyclase (sGC), resulting in the conversion of GTP into cyclic GMP (cGMP).133 Other receptors that increase cAMP levels through Gs-mediated AC stimulation are the A2A and A2B receptors for adenosine,134 the product of the sequential hydrolysis of ADP by CD39 and CD73 on the extracellular surface of endothelial cells, and the VPAC1 receptors for pituitary AC-activating and vasoactive intestinal peptides.135

When an injury occurs, endothelial-mediated inhibitory mechanisms must be rapidly reversed to ensure hemostatic plug formation. PGI2 and NO have a very short half-life, thus their concentration drops in proximity of the damaged endothelium. Simultaneously, agonist-stimulated platelets reduce the threshold of platelet activation by counteracting cyclic nucleotide signaling with two synergistic strategies.136 Agonists, such as ADP or epinephrine, stimulate the Gi/z-coupled receptors P2Y12 and α2-adrenergic receptor, respectively, which inhibit AC-mediated cAMP synthesis. In parallel, platelets express several phosphodiesterases (PDE2A, PDE3A, and PDE5A) that, by catalyzing the hydrolysis of cAMP and cGMP to inactive 50 -AMP and 50 -GMP, terminate their signaling. PDE2A and PDE3A mainly regulate cAMP in platelets,137,138 whereas PDE5A specifically degrades cGMP. Pharmacological inhibition of PDE2A results in increased basal cAMP.138 PDE3A deficiency leads to increased cAMP levels in both resting and PGI2-stimulated platelets and protects against collagen/epinephrine-induced pulmonary thrombosis and death.139 PDE3A is activated by PKC-mediated and possibly also AKTmediated phosphorylation in thrombin stimulated platelets. In addition, PDE3A and PDE5A are activated by PKA- or PKG-mediated phosphorylation that provides negative feedback on cyclic nucleotide levels. PKA-mediated phosphorylation of PDE3A on Ser312 is inversely regulated by ADP and PGI2.140 The downstream signaling evoked by cAMP and cGMP negatively regulates all aspects of platelet activation, from Ca2+ mobilization to G protein activation and cytoskeleton

Fig. 18.6 Cyclic nucleotide signaling. Gs-coupled receptors for prostaglandin I2 (IP), adenosine (A2) and pituitary AC-activating and vasoactive intestinal peptides (VPAC1) stimulate cAMP synthesis by activating AC, while the intracellular sGC for nitric oxide (NO) generates cGMP. von Willebrand factor (VWF), thrombin and collagen are probably also able to activate sGC, although to a much lesser extent than NO. Cyclic nucleotide levels are downregulated by the Gi-coupled receptor for ADP, P2Y12, which inhibits AC synthesis of cAMP, and by phosphodiesterases (PDE2A, PDE3A, PDE5A) that hydrolyze cAMP and cGMP to inactive 50 -AMP and 50 -GMP. The main effectors of cAMP and cGMP are the cAMP-dependent protein kinase (protein kinase A, PKA) and the cGMP-dependent protein kinase (protein kinase G, PKG), respectively. PKA and PKG phosphorylate common substrates, which have been grouped according to function (grey box). Substrate phosphorylation results in inhibition of GPCR signaling, Ca2+ mobilization, RAP1 signaling, RAC1/RHOA signaling and cytoskeletal dynamics. PKA and PKG also phosphorylate PDE3A and PDE5A, thereby providing negative feedback on cyclic nucleotide levels. Abbreviations: TP, thromboxane A2 receptor; RGS18, regulator of G protein signaling 18; IP3R, inositol 1,4,5-trisphosphate receptor; IRAG, inositol trisphosphate receptor-associated cGMP-kinase substrate; PMCA, plasma membrane calcium ATPase; STIM1, stromal interaction molecule 1; CalDAG-GEFI, calcium and diacylglycerol-regulated guanine nucleotide exchange factor I; RAP1GAP2, RAP1 GTPase-activating protein 2; ARHGEF6, RAC/CDC42 guanine nucleotide exchange factor 6; ARHGAP17, RHO GTPase activating protein 17; TRPC6, transient receptor potential channel 6; VASP, vasodilator-stimulated phosphoprotein; LASP, Lim and SH3 domain protein; HSP27, heat shock protein 27.

Platelet Signal Transduction

remodeling (Fig. 18.6). The main effectors of cAMP and cGMP in platelets are protein kinase A (PKA) and protein kinase G (PKG), respectively. Both kinases mediate platelet inhibition by phosphorylating multiple substrates (see the section on “Protein Kinase A/Protein Kinase G (PKA/G)”). Consistently, platelet function is markedly affected by genetic and pharmacological approaches affecting cyclic nucleotide metabolism. IP-R knockout mice display increased thrombotic tendency.141 Loss of Gsα signaling leads to platelet hyperreactivity in mice and humans.142,143 In contrast, a gain-of-function mutation in Gsα leads to increased susceptibility to bleeding144 and cAMP-elevating and/or cGMP-elevating agents, such as PDE inhibitors145 or prostacyclin mimetics, have shown clinical benefit as platelet inhibitors. Interestingly, there is also evidence that some agonists can induce a modest sGC-dependent increase in cGMP levels146,147 and that platelet-specific sGC deficiency reduces platelet activation upon weak stimulation.148 However, systemic deficiency of sGC strongly reduces the bleeding time in mice,149 and defective platelet sGC function has been described in prothrombotic patients with ischemic heart disease, heart failure, and diabetes.150 Thus, the physiological role of this modest NO-independent cGMP elevation during platelet activation remains to be clarified.

SIGNAL INTEGRATORS Kinases Protein Kinase C (PKC) Members of the PKC family are important effectors of the second messengers Ca2+ and DAG. They are grouped into conventional, novel and atypical PKCs.151 Conventional PKC (cPKC) isoforms contain a DAG-sensitive C1 domain and a Ca2+-responsive C2 domain. Novel PKC (nPKC) isoforms also contain a DAG-sensitive C1 domain, but they lack Ca2+ responsiveness in their C2-like domain. Atypical PKC (aPKC)

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isoforms lack both DAG and Ca2+ responsiveness and depend on alternative routes for membrane translocation and activation.152 Platelets express the cPKC isoforms PKCα and PKCβ; the nPKC isoforms PKCδ, PKCθ, PKCη, and PKCε (expressed in mouse platelets only); and the aPKC isoforms PKCι/λ and PKCζ. The specific function of individual PKC isoforms has been methodically explored in mouse and human platelets using both genetic and pharmacological approaches. However, challenges remain in the interpretation and integration of existing literature on platelet PKC signaling, as some of the reported results are contradictory and so-called isoform-specific PKC inhibitors were reported to have off-target effects.153,154 Despite these issues, some dogmas of platelet PKC signaling have been revealed (Fig. 18.7). cPKCs: While human platelets express similar levels of PKCα and β,22 mouse platelets predominantly express PKCα with minimal apparent expression of the β isoform.24 PKCα unquestionably plays a positive regulatory role in platelet granule release.155–158 Genetic deletion of PKCα in mice impairs both α- and δ-granule release and δ-granule biogenesis, defects that negatively impact αIIbβ3 inside-out activation.156 Surprisingly, loss of PKCα is partially compensated by low level expression of PKCβ, or other PKC isoforms, as PKCα-deficient mice have a normal hemostatic response time in the tail bleed assay despite a reduction in platelet adhesion both under flow in vitro and in cremaster arterioles in vivo.156 PKCβ also has a specific role in integrin outside-in signaling in platelets.159 Following integrin ligand binding, PKCβ associates with the integrin β3 tail to mediate platelet spreading on fibrinogen.159 The disparate functions of PKCα and PKCβ are in part due to the reliance on different regulators, such as Receptor for Activated C Kinase-1 (RACK1), which shows specificity for the PKCβ isoform.160 In human platelets, inhibition of cPKCs was reported to preferentially inhibit GPVI/ITAM-mediated δ-granule release,161 but it was later discovered that the utilized inhibitor, Go6976, is not cPKC-specific as it also impairs SYK/PLCγ2 signaling upstream of PKC.162 Furthermore, PKCα
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platelets have a similarly impaired response to both CRP and

Fig. 18.7 PKC signaling. Protein kinase Cs (PKCs) are critical signal integrators that control a wide range of platelet responses (red boxes). Downstream of either GPCR/PLCβ or GPVI/PLCγ, classical PKCs (PKCα, PKCβ) are activated by calcium ions (Ca2+) and diacylglycerol (DAG) and have a positive role in platelet activation, mainly in the regulation of granule secretion. Novel PKCs, such as PKCδ and PKCθ, are activated solely by DAG and have a positive role (black arrow) downstream of Gq-coupled receptors and a negative role (blocking symbol) downstream of the collagen receptor GPVI. PKCε, which is expressed in mouse, but not human platelets, has been implicated in the negative regulation of ADP-dependent platelet activation. PKCη seems to be implicated in ADP-induced thromboxane (Tx)A2 formation. Abbreviations: RACK1, Receptor of activated protein C kinase 1; VASP, vasodilator-stimulated phosphoprotein; ERK, extracellular signal–regulated kinases.

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thrombin.156 Thus, determining the absolute role of cPKC isoforms in human platelets will depend on the development of verified, isoform-specific inhibitors. nPKCs: In contrast to the significant positive role of cPKCs in platelet aggregation, nPKC isoforms can positively or negatively regulate platelet function, although some discrepancies in the literature exist. Both human and murine platelets express PKCδ, PKCθ, and PKCη, but PKCε is expressed only in murine platelets.163 Platelets deficient in PKCδ exhibit increased GPVI-induced aggregation and thrombus growth on collagen.155,164,165 PKCδ also restrains filopodia formation, which is dependent on direct interaction with vasodilator-stimulated phosphoprotein (VASP), suggesting a mechanism by which PKCδ limits thrombus propagation on collagen.165 PKCδ was also shown to be a negative regulator of dense granule release and TxA2 formation in response to GPVI stimulation.164 In contrast to collagen signaling, PKCδ seems to positively regulate thrombin signaling.164 However, no change in FeCl3-induced thrombus formation was observed in PKCδ
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mice.164 To study PKCδ function in human platelets, many groups have used the δ isoform-specific inhibitor rottlerin. Although many of the findings in rottlerin-treated human platelets mirror those in PKCδ
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mouse platelets,155,161 the specificity of this inhibitor has been called into question.153 PKCθ also has a critical negative regulatory role in the platelet response to collagen binding.155,166–168 PKCθ
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platelets exhibit enhanced GPVI-induced secretion166–168 and increased thrombus propagation on collagen under flow conditions.155,166 In platelets activated through GPVI, Ca2+ influx is also enhanced and sustained in the absence of PKCθ; thus, PKCθ acts to suppress phosphatidylserine (PS) exposure and switching of platelets to a procoagulant phenotype.155,168 Interestingly, PKCθ has also been observed to positively regulate GPVI-mediated platelet activation,169 although only at high concentrations of the GPVI agonist CRP (see response to167). In contrast to the response to collagen, PKCθ is important for enhancement of thrombin signaling, which may explain the increased tail bleeding time and decreased FeCl3induced thrombosis observed in PKCθ
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mice.169,170 Finally, PKCθ also plays a role in platelet outside-in signaling, as PKCθdeficient platelets exhibit impaired spreading on fibrinogen.171 PKCε plays a minor, dose-dependent role in platelet function. PKCε-deficient murine platelets are characterized by impaired aggregation and secretion in response to low and medium doses of GPVI agonists, but a normal response at high doses of agonists.163,172 On the contrary, PKCε negatively regulates ADP-induced secretion, Ca2+ influx and TxA2 formation.172,173 However, no defect in adhesion and thrombus formation on collagen under flow is observed in PKCε
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platelets.163,172 While one group reported reduced time to hemostasis in the tail bleed assay and more rapid FeCl3induced thrombosis,173 another observed no difference in blood loss in the tail bleed assay.172 Lastly, little is known about PKCη function in platelets, but inhibition of PKCη with an isoform-specific RACK peptide specifically inhibited ADPinduced TxA2 formation in human platelets.174 aPKCs: Limited data is available regarding aPKC isoforms in platelet function. In mice lacking PKCι/λ in the megakaryocyte lineage, no defect in in vitro platelet function or hemostasis/ thrombosis was observed.175 Platelet function in PKCζdeficient mice has not yet been determined.

Protein Kinase A/Protein Kinase G (PKA/G) The second messengers cAMP and cGMP act through their effectors, protein kinase A (PKA) and protein kinase G (PKG), to suppress inappropriate platelet activation in circulation (Fig. 18.6). PKA and PKG, like PKC, are members of the

larger AGC group of protein kinases. The production of cyclic nucleotides in platelets is driven by the endothelial cell-derived soluble mediators, prostacyclin (PGI2) and nitric oxide (NO). Upon activation by cAMP/cGMP, PKA and PKG phosphorylate a number of target proteins at all levels of platelet activation, some common to both PKA and PKG and some unique to each. PKA and PKG also activate PDE3A and PDE5A, respectively, thus providing negative feedback for their own activation by restricting levels of cyclic nucleotides. At the receptor level, PKA phosphorylates the TxA2 receptor (TPα) on a site which could lead to receptor desensitization,176,177 and the GPCR regulator RGS18,178 causing a more effective inhibition of Gi and Gq signaling. At the second messenger level, both PKA and PKG prevent increases in cytosolic Ca2+ concentrations.179 Early studies demonstrated that PKAmediated phosphorylation increases the activity of PMCA,180 responsible for transporting Ca2+ from the cytosol to the extracellular space. In parallel, PKA and PKG phosphorylate IP3Rs to block Ca2+ release from intracellular stores.181,182 PKG can also inhibit IP3R activation by phosphorylation of the IP3R binding protein, IRAG.183 In addition, a recent phospho-proteomic analysis of platelets treated with Iloprost, the synthetic analogue of PGI2, identified altered phosphorylation patterns in TRPC6, STIM1, and ORAI1,176 all of which mediate platelet Ca2+ influx. At the small-GTPase level, PKA inhibits RAP1 signaling through phosphorylation of CalDAG-GEFI.176, 184, 185 RAP1B is also phosphorylated by PKA,186,187 although in platelets this does not seem to affect GTP loading.187 In other cell types, RAP1B phosphorylation by PKA actually sustains signaling to ERK through prolonged interaction with B-RAF.188 PKG can also inhibit RAP1B activation,189 although direct phosphorylation of CalDAG-GEFI/RAP1B by PKG has not yet been demonstrated. Additionally, PKA and PKG phosphorylate the RAP-GAP, RAP1GAP2,190 but the physiological relevance of this event still needs to be determined. Similar to RAP1, PKA and PKG simultaneously inhibit ARHGEF6 and stimulate ARHGAP17 leading to attenuated RAC1 activation.191 In addition, RHOA signaling is inhibited indirectly by PKA-induced phosphorylation of Gα13192 and directly through phosphorylation of RHOA, which reduces its association with downstream effectors.193,194 PKA and PKG inhibit cytoskeletal dynamics not only by controlling small GTPase activation but also by directly phosphorylating actin binding proteins, such as the vasodilator-stimulated phosphoprotein (VASP). Phosphorylation of VASP inhibits platelet integrin activation and aggregation but does not affect Ca2+ flux or granule secretion.195,196 Of clinical relevance, VASP phosphorylation is counteracted by P2Y12 activation, and the phosphorylation status of VASP is used as a marker for on-treatment platelet reactivity in patients receiving P2Y12 inhibitors (Chapter 36).197 Other actin regulatory proteins that are targets for PKA phosphorylation include heat shock protein 27 (HSP27), Lim and SH3 domain protein (LASP) and filamin-A.198–200 Phosphorylation of HSP27 impairs actin polymerization, LASP phosphorylation reduces LASP binding to F-actin, and phosphorylation of filamin-A protects it from calpain-mediated degradation. However, the importance of these phosphorylation events in hindering platelet activation is not completely understood. PKA also plays an important role in the regulation of platelet apoptosis and lifespan. Basal PKA activity is downregulated in platelets from patients with immune thrombocytopenia purpura (ITP), sepsis, and diabetes as assessed by phosphorylation of GPIbβ.201 Mice lacking PKA are thrombocytopenic with decreased platelet lifespan, suggesting PKA is also critical for platelet survival in circulation under physiological conditions.201 In contrast, the anti-cancer agents ABT-737 and thymoquinone (TQ), both known to cause thrombocytopenia, were shown to activate PKA and induce platelet apoptosis,

Platelet Signal Transduction

independent of cyclic nucleotide levels or changes in VASP phosphorylation.202 PKA may therefore be regulating platelet apoptosis pathways by multiple mechanisms. NO-mediated sGC/PKG activity inhibits dual-agonist induced platelet procoagulant response (PS exposure, mitochondrial membrane depolarization)203 but the impact on platelet lifespan has yet to be determined.

Mitogen-Activated Protein Kinases (MAPKs) MAPKs are Ser/Thr kinases that play an important role for proliferation, migration, differentiation, and apoptosis in eukaryotic cells. The family can be divided into several subfamilies: extracellular signal-regulated kinases 1 & 2 (ERK1/2), p38 MAP kinases, c-Jun amino-terminal kinases (JNK), ERK5, and the atypical MAPKs ERK3 and ERK7.204 The activity of MAPKs is regulated by the sequential activation of other Ser/Thr kinases, triggered by RAS and RHO GTPases. Platelets express several members of the MAPK family, including ERK1/2, p38α, JNK1, and ERK5.205,206 It is well documented that ERK, p38, and JNK MAPKs are activated following platelet stimulation with physiological agonists such as thrombin, collagen, ADP, and TxA2. Our understanding of how MAPKs affect platelet function is largely dependent on experiments with inhibitors, many of which have been shown to have off-target effects. Thus, the existing literature has to be interpreted with caution. Irrespective of this limitation, MAPKs seem to be very important for the activation of cytosolic phospholipase A2 (cPLA2),207 an enzyme critical for the generation of the second wave mediator, TxA2. At this point it is not entirely clear whether MAPKs directly or indirectly (via TxA2 generation) affect other cellular functions, such as integrin activation and granule secretion. We will not discuss MAPKs in more detail, as this chapter focuses largely on the signaling events regulating integrin activation. Instead we would like to refer the reader to an excellent review written on this topic by Bryckaert and colleagues.205

RAP GTPase Signaling and Platelet Integrin Activation The near-immediate inside-out activation of αIIbβ3 integrin is critical for platelet adhesion to the damaged vascular wall as well as for platelet-platelet cohesion. Critical for this process is the assembly of the integrin activation complex, consisting of the small GTPase RAP1 and the adapter proteins TALIN and KINDLIN3, at the cytoplasmic tail of the β3 subunit. TALIN1 binds the integrin β-subunit cytoplasmic tail via its FERM domain (“head” domain), disrupting the close association of the α and β integrin tails and triggering conformational changes for increased ligand affinity; the KINDLIN head domain, highly homologous with the TALIN head, also binds the β-subunit tail and functions in a cooperative manner to promote integrin activation.208 Platelets from mice deficient in αIIbβ3 integrin, TALIN1 or KINDLIN3 do not aggregate in response to cellular stimulation, irrespective of the agonist.209–212 A similar platelet phenotype is observed in patients with Glanzmann’s thrombasthenia (GT) or leukocyte adhesion deficiency type 3 (LADIII), which harbor mutations in αIIbβ3 integrin and KINDLIN3, respectively.213 Patients with mutations in TALIN1 have not been identified to date.

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(Ras-related protein 1, previously called KREV-1/SMG21) as a very highly expressed protein in human platelets.187,214,215 RAP1 functions as a rapid molecular switch, cycling between a GDP-bound “off” state and a GTP-bound “on” state. This cycling is mediated by opposing regulators. GEFs turn RAP1 “on” by facilitating exchange of GDP for GTP, while GAPs turn RAP1 “off” by driving GTP hydrolysis.216 Functional studies in human platelets further demonstrated that RAP1 is activated within seconds upon cellular stimulation.217 This first phase of RAP1 activation is dependent on an increase in cytoplasmic Ca2+ but is independent of platelet aggregation. Cellular stimulation also leads to sustained RAP1 activation, mediated by the PKC signaling pathway.218 This late phase of RAP1 activation is dependent on ADP signaling through P2Y12, the βγ subunit of Gi heterotrimeric proteins, PI3K and its lipid product PI (3,4,5)P3.109,110,219,220 Together, these biochemical studies in human platelets demonstrate a strong temporal correlation between integrin activation and RAP1-GTP formation, the latter controlled by synergistic signaling pathways involving Ca2+ mobilization and PKC/P2Y12/PI3K signaling (Fig. 18.8). Platelets express both RAP1 isoforms, RAP1A and RAP1B, which show 90% homology in their amino acid sequences.221 RAP1B accounts for 60% and 90% of RAP1 protein in human and mouse platelets, respectively.22,24 The first genetic evidence for a role of RAP1 in αIIbβ3 affinity modulation came from studies in transduced murine megakaryocytes. Fibrinogen binding was enhanced by expression of a constitutively active RAP1B (V12) variant and inhibited by expression of RAP1GAP.222 Germline deletion of Rap1b in mice leads to high embryonic/perinatal lethality, likely caused by endothelial cell dysfunction.223 The surviving mice exhibit a moderate platelet aggregation defect and prolonged bleeding after challenge. Mice deficient in Rap1a exhibit functional defects in some myeloid cells, but not platelets.224 To determine the specific role of RAP1A and RAP1B in platelet function, we deleted both isoforms in the megakaryocyte lineage.225 These studies demonstrate redundancy between RAP1A and RAP1B in the inside-out activation of β1 and β3 integrins. In contrast, RAP1B is the major driver of RAC1-dependent granule secretion. Mice deficient in both RAP1A and RAP1B are characterized by markedly impaired hemostasis and strong protection from carotid artery thrombosis, a phenotype similar to that of mice lacking TALIN.225 Thus, RAP1 signaling is critical for platelet function, with both isoforms providing significant contributions. Platelets also express low levels of all three RAP2 isoforms, RAP2A, RAP2B, and RAP2C,22,24 which share 60% sequence homology with RAP1. In cell types other than platelets, RAP1 and RAP2 proteins control distinct cellular responses by connecting with unique downstream effectors.226 At this point, little is known about the contribution of RAP2 to platelet function. RAP2 undergoes nucleotide exchange in platelets following agonist treatment,227 regulated by CalDAG-GEFI and P2Y12 signaling.44,227 Basal RAP2-GTP levels are much higher than that of RAP1-GTP, potentially due to lower sensitivity of RAP2 to GAP activity.228 Importantly, the kinetics of RAP2-GTP formation and αIIbβ3 integrin activation do not correlate in stimulated platelets, even in the absence of RAP1, strongly suggesting that RAP2 is important for cellular functions other than integrin affinity regulation in platelets.225

RAP1 Regulators RAP GTPases The contribution of RAP1 to integrin inside-out activation in platelets has been the focus of intense studies over the last three decades. Early studies identified the small GTPase RAP1

Genetic studies also helped identify the main regulators of RAP1 in platelets. Eto et al. used embryonic stem cell-derived megakaryocytes to demonstrate an important role for CalDAG-GEFI (calcium and diacylglycerol-regulated guanine

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Fig. 18.8 RAP GTPase signaling. RAP GTPases are critical signaling integrators regulating platelet activation. RAP1 activation in platelets is tightly controlled by the antagonistic balance between the calcium-sensitive guanine nucleotide exchange factor, CalDAG-GEFI (calcium and diacylglycerolregulated guanine nucleotide exchange factor I), and the GTPase activating protein, RASA3 (RAS P21 Protein Activator 3). In quiescent platelets, active RASA3 restrains unwanted RAP signaling to ensure platelet homeostasis. Additionally, signaling of the Gs-coupled prostaglandin I2 receptor (IP) ensures cAMP/PKA-mediated inhibition of CaLDAG-GEFI and activation of RAP1GAP2 (RAP1 GTPase-activating protein 2), a RAP-GAP expressed at low copy numbers in platelets. At sites of vascular injury, platelet stimulation through ITAM-coupled and G protein–coupled receptors leads to the activation of PLCγ and PLCβ, respectively. PLCs convert PIP2 to DAG and IP3. IP3 stimulates a near-immediate rise in cytosolic Ca2+ concentrations, which trigger rapid CalDAG-GEFI-dependent RAP1 activation. DAG leads to PKC activation, which contributes to granule secretion and to activation of RAP, through a yet unknown pathway (black dotted line). CalDAG-GEFI signaling eventually subsides and signaling via the Gi-coupled receptor for ADP, P2Y12, is required to ensure sustained RAP activation. P2Y12 inactivates RASA3 by signaling through phosphoinositide 3-kinase (PI3K) and its lipid product, PIP3. Additionally, P2Y12 inhibits cAMP/PKA signaling. Once active, RAP GTPases drive platelet activation at sites of vascular injury by switching on multiple platelet responses, including TALIN-mediated integrin activation, TxA2 generation, probably though MAPK (mitogen-activated protein kinase) signaling, and RAC1-regulated granule secretion. Ligand binding to active integrins induces outside-in signaling pathways and promotes spreading and clot retraction via the small GTPases RAC1 and RHOA. Integrin stimulation of PLCγ and the signaling mediated by autocrine agonists released from active platelets evoke positive feedback loops (grey dotted line) that further support sustained RAP activation.

nucleotide exchange factor I; encoded for by the gene RASGRP2) in RAP1-mediated inside-out activation of αIIbβ3 integrin.229 Consistent with this finding, germline deletion of Caldaggef1 led to strongly impaired RAP1 signaling in platelets and impaired hemostasis in mice.230 Caldaggef1
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platelets exhibit a partially impaired aggregation response to various agonists, including TxA2, collagen, and thrombin. Aggregation is fully impaired when platelets are stimulated with ADP or calcium ionophore, but normal in response to phorbol ester stimulation.230 Importantly, aggregation of Caldaggef1
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platelets occurs with a delay, leading to reduced adhesion and thrombus formation under flow conditions, both ex vivo and in vivo. Caldaggef1
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mice are protected from experimental thrombosis but also exhibit a marked defect in hemostasis.230,231 Delayed aggregation of Caldaggef1
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platelets is mediated by the PKC/P2Y12/PI3K pathway.231,232 Thus, CalDAG-GEFI is critical for the near-immediate, Ca2+-dependent activation of RAP1 in stimulated platelets. Proteomics studies have confirmed that CalDAG-GEFI is a major RAP-GEF expressed in human and murine platelets.22,24 In fact, CalDAG-GEFI is the only known RAP-GEF expressed at a high copy number in platelets (10,000–30,000 copies per cell). CalDAG-GEFI is a multidomain protein consisting of a N-terminal catalytic and a C-terminal regulatory domain. The latter contains a pair of EF hands and a C1-like domain. The EF hands bind Ca2+ with very high affinity (KD < 100nM), consistent with its role in Ca2+-dependent RAP1 activation.233,234 Very recent biochemical and biophysical studies demonstrate that Ca2+ binding to the EF hands is required to release

autoinhibition in CalDAG-GEFI.235 The contribution of the C1-like domain to CalDAG-GEFI function has not been investigated. Mutations in RASGRP2 have recently been identified in various patients with a platelet function disorder. These patients suffer from a moderate to severe bleeding diathesis, and patient platelets exhibit functional defects similar to those described for platelets from Caldaggef1
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mice.236–242 Interestingly, the reported severity of bleeding varies from patient to patient, suggesting that some mutations are more detrimental than others. Consistent with this conclusion, mice expressing low levels of CalDAG-GEFI also bleed very little, even though platelet function is strongly impaired.243 Moreover, these mice are protected from experimental thrombosis, suggesting that CalDAG-GEFI could serve as a novel target for anti-platelet therapy. Proteomics and transcriptomics studies identified RASA3 (RAS P21 Protein Activator 3, also known as GAP1-IP4BP) as the most highly expressed RAP1-GAP in human and murine platelets,22,24 consistent with earlier biochemical work demonstrating high expression of RASA3 in the plasma membrane of human platelets.244 Functional studies on the role of RASA3 in platelet biology, however, are difficult as germline or megakaryocyte/platelet-specific deletion of Rasa3 leads to embryonic/perinatal lethality due to vascular mixing.21,245 Rasa3 was also identified as the causative gene in thrombocytopenic mice derived from a forward genetic screen for modifiers of blood cell counts (H794L, hlb [Mouse “Heart, Lung, Blood and Sleep” Disorders Center, The Jackson Laboratory]). Rasa3hlb/hlb and Rasa3 knockout mice present with a robust

Platelet Signal Transduction

macrothrombocytopenia, with peripheral platelet counts that are less than 5% of that in control mice.21 Platelets from Rasa3hlb/hlb mice exhibit increased basal RAP1-GTP levels and enhanced RAP1 activation following activation with ADP, increased expression of activated αIIbβ3 integrin on circulating platelets, and rapid platelet turnover. Concomitant deletion of Caldaggef1 leads to improved platelet survival and a normal peripheral platelet count in RASA3 mutant mice.21 Thus, RASA3 is an important antagonist of Ca2+/CalDAG-GEFI signaling in circulating platelets. Negative feedback by RASA3, however, would be a liability for platelet adhesion and hemostatic plug formation at sites of vascular injury. To circumvent this problem, RASA3 activity is downmodulated as part of PKC/P2Y12/PI3K signaling, and loss of RASA3 is analogous to constitutive P2Y12/RAP1 signaling.21 These findings have important clinical implications as they suggest that P2Y12 inhibitors affect hemostatic and thrombotic plug formation mainly via their inhibitory effect on RASA3 inactivation and sustained RAP1 activation. RASA3 depends on a unique PH/BTK domain for membrane localization. It is likely that PI3K signaling affects RASA3 activity by changing the membrane microenvironment, but the exact molecular mechanisms underlying RASA3 activity regulation are currently unclear. Overall, these studies have identified RAP1 as the primary molecular switch regulating the platelet adhesive state under flow; RASA3 is required to restrain RAP1 activation and maintain platelets in a quiescent state, while CalDAG-GEFI mediates the rapid and sensitive response required for hemostatic plug formation. Platelets also express low levels of other RAP1-GEFs and GAPs, including C3G, Epac1, PDZ-GEFI, and RAP1GAP2.22,24 The role of C3G was investigated in a transgenic model with overexpression of wild-type or GEF domain-deficient C3G driven by the PF4 promoter.246 C3G-Tg mice exhibit enhanced platelet activation and shortened tail bleeding times compared to control mice. These studies, however, do not address the question if and how low levels of endogenous platelet C3G affect RAP1 activation and integrin-mediated platelet aggregation. Epac1-deficient mice have a bleeding phenotype that, at least in part, may be caused by a platelet function defect.247 However, the described phenotype does not include a RAP1

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activation defect, and thus does not support an important role of this exchange factor in platelet RAP1 signaling. RAP1GAP2 can be detected on the protein level in human platelets.190,248 The phosphorylation state of RAP1GAP2 is affected by activating and inhibitory signaling in platelets, suggesting that it contributes to platelet function. Additional studies are required to determine whether RAP1-GEFs and -GAPs other than CalDAG-GEFI and RASA3 are important for platelet function.

SUMMARY Platelets have evolved a powerful yet self-limiting signaling system that facilitates cellular adhesion under shear stress conditions. The key aspects of this system are best described as a 1–2 punch response, in which two independent signaling mechanisms are required to initiate and to sustain integrinmediated platelet adhesion. Central to the 1–2 punch response are GPCRs and small G proteins, proteins that are regulated by a simple on-off switch mechanism (Fig. 18.9). GPCRs are cell surface receptors that respond to soluble agonists such as thrombin and ADP. They couple to heterotrimeric G proteins and thus can rapidly cycle between a GDP-bound “off” and a GTP-bound “on” state. They are key to hemostatic plug formation for several reasons: (1) soluble agonists can penetrate into the outer layers of a growing thrombus, i.e., areas where ECM components are not available, (2) they facilitate the nearimmediate activation of intracellular signaling pathways required for platelet adhesion under flow conditions, and (3) they provide an elegant system to fine-tune the strength and duration of the activation response, as key agonists trigger the activation of multiple GPCRs. For example, the initial response to thrombin in human platelets is mediated by PAR1, a GPCR that is highly sensitive to agonist stimulation and that induces a rapid but reversible response. To sustain the signal and thus mediate firm adhesion, platelets also express PAR4, a GPCR with lower affinity for thrombin that mediates a slower but more sustained response.249 ADP also activates platelets through two surface receptors, P2Y1 and P2Y12. P2Y1 is critical to initiate a response rapidly,250 while P2Y12 is required to sustain the signal.110 GPCR signaling is antagonized by RGS proteins, which limit platelet activation

Fig. 18.9 Key role for G proteins in platelet activation. (Left panel) GPCRs. G protein-coupled receptors (GPCRs) for thrombin (PARs) or adenosine diphosphate (P2Y receptors) promote platelet activation at sites of injury. (1) PAR1 and P2Y1 are high affinity receptors that initiate the response. (2) PAR4 and P2Y12 are critical for a sustained response. The regulators of G protein signaling (RGS) proteins inhibit GPCR signaling by facilitating GTP hydrolysis. (Right panel) Small GTPases. The small GTPase RAP1 is a critical signal integrator leading to integrin inside-out activation. (1) RAP1 signaling is initiated by calcium (Ca2+) binding to CalDAG-GEFI and inhibited by the GTPase-activating protein, RASA3. (2) Sustained Rap1 signaling requires RASA3 inhibition downstream of the platelet ADP receptor, P2Y12. Abbreviations: DAG, diacylglycerol; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PKC, protein kinase C.

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in circulation and at sites of vascular injury by increasing the rate of GTP hydrolysis in heterotrimeric G proteins. The small GTPase RAP1 is another intracellular switch, which operates downstream of PLCs and immediately upstream of the integrin receptors. Its main activator, CalDAG-GEFI, responds to small increases in the cytoplasmic Ca2+ concentration, thereby mediating the rapid but reversible activation of integrin receptors. Counteracting this activation is the RAP-GAP, RASA3. Sustained RAP1 signaling requires the inactivation of RASA3, mediated by phosphoinositide 3-kinase (PI3K)/PI(3,4,5)P3 signaling downstream of P2Y12. Consistent with their key role in hemostatic/thrombotic plug formation, GPCRs are the target of various antiplatelet therapies,251 and bleeding is a frequent complication in patients with mutations in GPCRs or CalDAGGEFI.242,252,253

18. 19. 20. 21.

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