New Concepts and Mechanisms of Platelet Activation Signaling

Abstract

Upon blood vessel injury, platelets are exposed to adhesive proteins in the vascular wall and soluble agonists, which initiate platelet activation, leading to formation of hemostatic thrombi. Pathological activation of platelets can induce occlusive thrombosis, resulting in ischemic events such as heart attack and stroke, which are leading causes of death globally. Platelet activation requires intracellular signal transduction initiated by platelet receptors for adhesion proteins and soluble agonists. Whereas many platelet activation signaling pathways have been established for many years, significant recent progress reveals much more complex and sophisticated signaling and amplification networks. With the discovery of new receptor signaling pathways and regulatory networks, some of the long-standing concepts of platelet signaling have been challenged. This review provides an overview of the new developments and concepts in platelet activation signaling.

Platelet activation signaling plays a critical role in the function of platelets in hemostasis and thrombosis, and has been the subject of intensive investigations for many years. With the explosive increases in our knowledge recently, a much more complex and sophisticated picture of platelet signaling and amplification networks has emerged, and some long-standing concepts of platelet activation are now challenged. Classically, platelet activation is induced by collagen or soluble platelet agonists that bind G-protein-coupled receptors, leading to the activation of platelet adhesion receptors, mainly the integrin α_IIbβ₃, which mediates platelet adhesion and aggregation. However, various platelet adhesion receptors, which were previously regarded as “passive,” are increasingly recognized to be important in mediating and amplifying platelet activation signals. Increasing numbers of ligand-receptor pairs, which were known to be critical for mediating immune or inflammatory responses in leukocytes, are now shown to play important roles in platelet activation, in the role of platelets in immune or inflammatory responses, and in the coupling between thrombosis and inflammation. Importantly, it is now increasingly evident that a long-established negative regulator of platelet activation, the nitric oxide (NO)-cyclic guanylyl monophosphate (cGMP) pathway, in fact plays biphasic roles in platelet activation and is important in mediating platelet activation induced by several receptor signaling pathways, including recently discovered platelet pattern-recognition receptor signaling pathways. In this review, we will provide an overview and update to the concepts and mechanisms of platelet activation signaling.

With the recent advances and complexity of the platelet signaling network in mind, we divide platelet activation signaling into the following phases: 1) divergent early receptor signaling induced by not only classic soluble platelet agonists but also adhesion receptor ligands and inflammatory stimuli; 2) convergence of early signaling pathways on common intermediates and signal amplification networks; 3) inside-out signaling leading to activation of the main platelet adhesion receptor, the integrin α_IIbβ₃, which mediates stable platelet adhesion and aggregation; and 4) integrin outside-in signaling, which greatly amplifies platelet activation and thrombus size. Accordingly, we will review each of these phases in the following sections.

Receptor Signaling Pathways Leading to Platelet Activation

Adhesion Receptor-Mediated Platelet Activation Signaling

GPIb-IX signaling.

Under flow conditions, particularly at high flow shear rates, the initial adhesion of platelets to the blood vessel wall requires the interaction between immobilized VWF on the surface of endothelium or in the subendothelial matrix with its platelet receptor, the glycoprotein (GP) Ib-IX-V complex. The ligand binding subunit of the GPIb-IX-V receptor complex, GPIbα, contains binding sites for the A1 domain of VWF in its NH₂-terminal domain (42, 93, 143, 148, 215). VWF and GPIbα respond to increasing shear force by undergoing conformational changes that increase their affinity for one another (135, 151, 186), forming shear-resistant “catch bonds” or “flex bonds” that allow platelet adhesion under shear stress (93, 108, 135, 232). The cytoplasmic domain of GPIbα is anchored to actin filaments, which underlie the platelet plasma membrane, through interaction with filamin A. This interaction is critical for maintaining membrane structure and platelet shape, and is also an important structural reenforcement for resisting shear force during platelet adhesion (40).

Despite its ability to resist shear, GPIb-IX-mediated platelet adhesion to VWF is transient in nature. Stable adhesion of platelets initiated by VWF/GPIb-IX binding requires the activation of another VWF receptor, integrin α_IIbβ₃. In fact, it is increasingly evident that VWF binding to GPIb-IX under shear triggers platelet activation signals, leading to integrin activation and integrin-dependent stable platelet adhesion (53, 125, 183). A region within the extracellular juxtamembrane stalk of GPIbα, called the mechanosensitive domain (MSD) (Ala⁴¹⁷-Phe⁴⁸³), was shown to unfold and extend when subject to VWF-dependent pulling force (247). Another region in the leucine-rich repeat domain (LRRD) has also been shown to unfold by VWF-mediated pulling force (101). The unfolding of LRRD and MSD plays distinct roles in VWF binding-dependent transduction of pulling force, triggering intracellular signaling as indicated by calcium elevation (100). The mechanical force-signaling coupling appears to require the interaction between GPIb cytoplasmic domain, with an intracellular molecule ζ form of 14-3-3 protein (100).

GPIb-IX also binds thrombin and is important for low dose thrombin-induced platelet activation (78, 95, 242). This function of GPIb-IX requires the thrombin binding sites around three sulfated tyrosine residues (Y²⁷⁶DYY) (27, 48, 55, 143) in the ligand-binding domain of GPIbα, which is distinct from the VWF binding site (27, 47, 48, 55, 143). It is important to note that, under conditions where thrombosis is induced by limited vascular injury (such as the laser-induced arterial injury model), only low concentrations of thrombin are detectable, which, however, are critical for platelet thrombus formation in vivo (57).

GPIb-IX signaling induced by either VWF or thrombin both require the binding of 14-3-3ζ to the cytoplasmic domain of GPIbα (44, 57, 80), a Src family kinase (SFK) (Lyn and possibly Src)-Rac1 signaling pathway (51, 53, 239), and downstream activation of the phosphoinositide 3-kinase (PI3K)-Akt and cGMP-dependent protein kinase (PKG) pathway (127, 128, 239, 240), mitogen-activated protein kinase (MAPK) (ERK1/2 and p38) pathway (69, 126, 128), and LIM kinase 1 (LIMK1) pathway (57, 59). Several other proteins such as Bruton's tyrosine kinase (Btk) (132) and ADAP (102) have also been shown to be important, although it remains to be established whether this is because they play roles in the amplification pathways, such as the ITAM pathway, or the integrin signaling pathway, in which these proteins are known to be important (103, 176). It is interesting that the role of LIMK1 in stimulating VWF and low-dose thrombin-induced platelet activation is specific for the GPIb-IX signaling pathway, because LIMK1 appears to play a negative role in platelet activation induced by the GPIb-IX-independent platelet agonists. LIMK1 promotes VWF-stimulated activation of cytosolic phospholipase A2 (cPLA₂) and consequent TXA₂ production (59) (FIGURE 1).

FIGURE 1.

Signaling pathways of the major platelet receptors for adhesive ligands leading to integrin α_IIbβ₃ activation and important roles of ITAM pathway

sGC, soluble guanylyl cyclase; NOS, NO synthase; SFK, src family kinase; LEC, lymphatic endothelial cell; SLP-76, SH2 domain-containing leukocyte phosphoprotein of 76 kDa; Btk, Bruton's tyrosine kinase; Rap1b, RAS-related protein 1b; MAPKs, mitogen-activated protein kinases; TXA₂, thromboxane A₂.

ITAM signaling in platelet activation triggered during platelet adhesion.

The immunoreceptor tyrosine-based activation motif (ITAM) is a protein sequence motif in the cytoplasmic domain of certain immunoreceptors containing two tyrosine residues within a conserved YxxL/IX_6-12 YxxL/I sequence (where x is any amino acid), and it plays an important role in receptor signaling leading to activation of leukocytes (10, 22, 222). ITAM receptors expressed in human platelets include the Fc receptor γ chain (FcRγ), a subunit of certain Ig Fc receptors critically important for their signal transduction, and Fcγ receptor IIa (FcγRIIa), an IgG Fc receptor important for human platelet response to antibody-antigen complexes and aggregated IgG (not expressed in mouse platelets) (180, 225). ITAM receptors are not only critical in signal transduction of immunoreceptors in platelets and leukocytes, they are also important for signal transduction during platelet adhesion (23, 62, 209). FcRγ is reportedly associated with GPVI and GPIb-IX (62), and FcγRIIa with GPIb-IX and integrin α_IIbβ₃ (202, 248). CLEC2, a platelet podoplanin receptor important for platelet adhesion to lymphatic vessels, has a single ITAM-like YxxL motif known as a hemITAM.

gpvi.

The role of ITAM in platelet adhesion signaling is best exemplified in the collagen receptor GPVI (104, 106, 155), which plays a critical role in robust platelet activation induced by collagen. GPVI forms a noncovalent complex with FcRγ (60, 73, 209) and is reportedly associated with certain SFK, such as Lyn and Fyn, through the intracellular proline-rich domain of GPVI (60, 185). This complex is necessary for transmitting signals induced by collagen binding to the extracellular immunoglobulin-like domains of GPVI (15, 162, 175, 228). Upon collagen-induced clustering of GPVI, ITAM in the associated FcRγ is tyrosine phosphorylated by SFK (60, 177). However, the role of Lyn in GPVI remains controversial, with some studies suggesting that Lyn plays a negative regulatory role in GPVI-mediated ITAM signaling (177), probably via its role in the regulatory immunoreceptor tyrosine-based inhibition motif (ITIM), such as those mediated via PECAM-1 and paired immunoglobulin-like receptor B, which counteracts ITAM signaling (64, 150). Tyrosine phosphorylation of ITAM enables binding of the tyrosine kinase Syk (10, 60, 72, 94, 175, 177) through its dual Src homology 2 (SH2) domains (92), enabling its activation. Interestingly, NOX-dependent reactive oxygen species production has been shown to stimulate GPVI-induced Syk activation, possibly by inhibition of protein tyrosine phosphatases (49). Activated Syk initiates a cascade of events, involving adapter molecules and kinases: SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) (33, 79), linker for activated T-cells (LAT) (171), Grb2 (56), Gads (91), Tec family kinases (Btk and Tec) (12, 176), and PI3K (20, 25, 220), which leads to translocation to the plasma membrane, phosphorylation, and activation of phospholipase C-γ2 (PLCγ2) (20, 175, 205, 218, 221, 222). PLCγ2 hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP₂) into 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃). DAG and IP₃ activate protein kinase C and release calcium into the cytosol from intracellular stores, respectively, promoting thromboxane production and granule secretion, inside-out signaling, and integrin activation (120, 221, 222) (FIGURE 1).

clec-2.

The platelet receptor for the transmembrane glycoprotein podoplanin is CLEC-2. In laboratories, the snake venom protein rhodocytin was used to stimulate CLEC-2 signaling (203, 204). The CLEC-2-mediated platelet adhesion to podoplanin-expressing cells (e.g., lymphatic endothelial cells and pericytes) is important for the development and separation of lymphatic vessels from blood vessels (184, 210), in maintaining the integrity of the blood-lymphatic vessel junction, in preventing blood cell flow into lymphatics (86), and possibly also in playing a role in maintaining vascular integrity during inflammation (21, 85). Thus deletion of podoplanin or CLEC-2 in mice causes blood-lymphatic mixing (16, 66, 184, 204, 210). The hemITAM-dependent CLEC-2/PDPN signaling pathway involves many of the same signaling molecules used by full ITAM receptors and appears to require the binding of Syk SH2 domain to the phosphorylated CLEC-2 hemITAM motif (92). However, one report suggests that Syk activation in CLEC-2 signaling involves PI3K and Tec family kinase-mediated Syk phosphorylation, whereas, in GPVI-mediated signaling, PI3K and Tec family kinases are activated downstream of Syk (142). The mechanisms of ITAM or hemITAM motif phosphorylation may also be different. CLEC-2 hemITAM phosphorylation appears to require both Syk and SFK (191). In contrast, GPVI ITAM phosphorylation is mediated by SFK (60, 177) (FIGURE 1).

GPIb-IX and integrin α_IIbβ₃ are also reportedly associated with ITAM receptors (62, 202, 248). However, it appears that their primary signaling does not require the ITAM signaling pathway (102, 133, 141, 241) but that ITAM signaling plays important roles in the amplification of the signaling initiated by these receptors, leading to significantly greater platelet responses (132, 228, 248). This notion is supported by the data that GPIb-IX-triggered, integrin-dependent stable platelet adhesion to VWF and integrin-mediated platelet spreading are not inhibited by inhibitors of Syk (240), a key enzyme in the ITAM pathway, unlike the ITAM-dependent secondary platelet secretion and aggregation induced via GPIb-IX and integrin signaling pathways. Interestingly, association of Syk with filamin A, which also interacts with GPIbα cytoplasmic domain, reportedly regulates ITAM signaling induced by GPVI (63).

Platelet Activation Signaling Mediated by Pattern Recognition Receptors

Pathogen-associated molecular patterns (PAMPs) are molecules, such as DNA, RNA, glycoproteins, and lipopolysaccharides (LPS), produced by microorganisms. Damage-associated molecular patterns (DAMPs) are molecules [oxidized lipid derivatives, DNA fragments, and proteins, such as high-mobility group box 1 (HMGB1)] released in response to tissue damage. PAMPs and DAMPs are recognized by pattern recognition receptors, such as Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors, and the receptor for advanced glycation end products (RAGE), and serve as initiating signals for innate immunity and non-infectious inflammatory responses. Members of TLRs and NOD recently have been shown to promote platelet activation. Platelets express several TLRs, including TLR1, TLR2, TLR4, TLR6, and TLR9 (7, 11, 35, 194, 243). [TLR1 and TLR6 have only been reported in human but not in mouse platelets (194).] TLR2 has been shown to be critical for platelet activation induced by a synthetic TLR1-/TLR2-specific agonist, Pam3CSK4 (19), and oxidized lipid derivatives such as carboxyalkylpyrrole-phosphatidylethanolamine (18). TLR9 mediates platelet activation signals induced by carboxy(alkylpyrrole) protein adducts generated during oxidative stress (170). TLR4 is a major platelet receptor for LPS and mediates signals leading to platelet granule secretion in response to LPS (7), and also mediates platelet activation and secretion induced by HMGB1, a DNA-binding protein, which is released by monocytes and macrophages during inflammation (159) and is also stored in platelet granules and released during platelet activation (216). Signaling of TLR2, TLR4, and TLR9 in platelets is dependent on MyD88. Data published by different groups have shown that TLR4 mediates platelet activation signaling by MyD88-dependent activation of the cGMP-PKG pathway (216, 243). The cGMP pathway was not investigated in the TLR2 and TLR9 signaling pathways, although these receptors have been shown to involve SFK and PI3K/AKT, which are known activators of the NO-cGMP pathway, as well as the ITAM pathway (18, 19, 170). Platelets also express NOD2 (but not NOD1), which is a cytosolic receptor recognizing muramyl dipeptide (MDP) found on all bacteria (246). MDP-NOD2 interaction triggers platelet activation via MyD88- and MyD88-dependent activation of MAPK and the NO-cGMP-PKG signaling pathway, which potentiates platelet responses, including aggregation, clot retraction, and thrombosis (246) (FIGURE 2).

FIGURE 2.

Reported signaling pathways of pattern recognition receptors in platelets

Note that TLR receptors all require MyD88 for signaling. LPS, lipopolysaccharide; MDP, muramyl dipeptide; HMGB1, high-mobility group box 1; CAP-PE, carboxyalkylpyrrole-phosphatidylethanolamine; TLR, toll-like receptor; PKG, cGMP-dependent protein kinase; RIP2, receptor-interacting protein-2; TIRAP, toll/interleukin-1 receptor domain-containing adapter protein; Myd88, myeloid differentiation factor 88. The question marks represent unknown or hypothesized pathways requiring further investigation.

Platelet activation induced by pattern recognition receptors is likely to serve as a mechanism for thrombotic response to microbial infection and tissue damage in parallel to innate immunity and inflammation. Conversely, platelet storage of DAMPs, such as HMGB1, and their release during platelet activation is likely to exacerbate inflammation. The importance of platelet activation and thrombosis in inflammation and infection has been suggested in sepsis and during the development of atherosclerosis (21, 39, 131).

In addition to the pattern-recognition receptors, certain oxidized lipid species may stimulate platelets via glycoprotein IV, which involves the MAPK kinase signaling pathway (29). The activation of platelets during immune and inflammatory response may also be stimulated by other thrombotic and inflammatory stimuli, such as antigen-antibody complexes via the FcγRIIA-ITAM pathway, as well as thrombin or platelet-activating factor via G-protein-coupled pathways.

G-Protein-Coupled Receptor-Mediated Platelet Activation Signaling Induced by Soluble Platelet Agonists

Platelets can be activated by many soluble agonists either released from the sites of blood vessel injury and inflammation or from platelets during platelet activation. These agonists activate platelets by binding to their respective receptors on the platelet membrane, most of which are G-protein-coupled receptors (GPCR). GPCRs are a family of membrane proteins with seven transmembrane domains, which upon activation by their ligands elicit intracellular signaling by activating the heterotrimeric guanine nucleotide-binding proteins (G proteins) (166). Heterotrimeric G proteins consist of an α-subunit in complex with the tightly associated β- and γ-subunits. In the inactive state, in which the G-protein complex is receptor-associated, the α-subunit is bound to guanosine diphosphate (GDP) and complexed with βγ-subunits. Agonist binding to GPCRs induces conformational changes that activate the exchange of GDP for guanosine triphosphate (GTP) on the Gα-subunit (113, 214). Consequently, the GTP-bound Gα-subunit dissociates from the βγ-subunits and from the receptor. The GTP-bound α-subunit and the βγ-subunit then interact with their respective downstream effector proteins, propagating signals. The GTP-bound Gα signal propagation is then terminated by GTP hydrolysis, which is mediated by intrinsic GTPase activity of Gα-subunits and greatly accelerated upon binding of a regulator of G-protein signaling (RGS) domain within members of the RGS protein family. Based on primary sequence homology of the Gα-subunit and their effects on downstream targets, heterotrimeric G proteins are grouped into four classes: Gα_i/o/z, Gα_s, Gα_q/11, and Gα_12/13 (169).

Gα_q.

Upon activation of Gα_q-coupled GPCRs, Gα_q dissociates from the receptor, and binds and activates PLC β isozymes that catalyze hydrolysis of PIP₂ to form second messengers IP₃ and DAG. Gα_q-null platelets are defective in IP₃ release and cannot mobilize calcium in response to GPCR agonist stimulation (168). Gα_q is critical for platelet aggregation in response to most platelet GPCR agonists (thrombin, ADP, 5HT, PAF, and TXA₂) (166) and is indirectly important in platelet response to adhesion proteins such as collagen (168), which require the amplification signaling induced by TXA₂, ADP, etc. for optimal platelet activation (30).

Gα_12/13.

A classical pathway of Gα_12/13 signaling is the binding of GTP-Gα₁₃ (or Gα₁₂) to the guanine nucleotide exchange factors (GEF) for small GTPase RhoA, including p115RhoGEF, LARG, and GEF-H1, activating RhoA and Rho kinase (ROCK), subsequently promoting phosphorylation of myosin light chain (MLC) and actin-myosin-dependent contraction (74, 110, 152, 153, 167, 206). Platelets express both Gα₁₂ and Gα₁₃; however, only Gα₁₃ is required for low-dose thrombin- or U46619 (a TXA₂ analog)-induced platelet aggregation and secretion. Gα₁₃-null platelets also show defects in thrombin and U46619-induced platelet shape change, which is consistent with defects in RhoA activation and MLC phosphorylation, granule secretion, and clot retraction (13, 70, 99, 123, 172). RhoA may not be directly required for integrin activation and platelet aggregation per se, and RhoA-null platelets reportedly displayed enhanced aggregation responses to collagen and CRP(174). Recently, it was shown that GTP-bound Gα₁₃ directly interacts with the cytoplasmic domain of the integrin β₃-subunit, and this interaction is critically important in integrin outside-in signaling (77). Interestingly, Gα₁₃ binding to β₃ potently inhibits RhoA activation, providing a mechanism for dynamically regulating RhoA and RhoA-dependent contractility (see below) (77).

Gα_i and Gα_s.

A typical Gα_i-coupled receptor is the ADP receptor P₂Y₁₂, and the typical Gα_s-coupled receptor is the prostaglandin I₂ receptor PTGIR (IP receptor). Gα_i/o and Gα_s both bind to adenylyl cyclase, with Gα_i inhibiting but Gαs stimulating the function of adenylyl cyclase to synthesize cAMP (166, 233). cAMP, by stimulating cAMP-dependent protein kinase (PKA), plays a critical role in keeping platelets in a resting state, which is the mechanism by which PGI₂ inhibits platelet activation. In contrast, Gα_i, by inhibiting synthesis of the potent platelet inhibitory second messenger cAMP, is critically important in platelet activation (2, 14, 17, 179, 195). Platelets express several members of the Gα_i family, including Gα_i2, Gα_i3, and Gα_z (166). Defects in one isoform, such as Gα_i2, cause only a partial defect in agonist-induced inhibition of cAMP synthesis (52, 96, 233). Gα_i stimulates platelet activation not only by regulating cAMP levels but also via cAMP-independent mechanisms (136, 233). In particular, upon Gα_i activation, dissociated Gβγ has been shown to interact with and activate PI3Kγ and possibly β-isoforms of PI3K (116, 140, 208, 212), which is important in G_i signaling (25, 37, 88, 105, 130, 187). The Gα_i pathway also plays roles in activation of the small GTP-binding RAS-related protein 1b (Rap1b) as well as Rap1b regulator RASA3 (32, 136, 137, 196, 197, 227), and in activating SFKs (230).

Synergy Between Different Receptor Pathways of Platelet Activation

Several platelet agonists have more than one platelet receptor, and optimal platelet activation induced by these agonists often requires synergy or cooperativity between different receptor pathways. ADP, which is released from platelet-dense granules or damaged cells and tissues (26), requires both Gα_q-coupled P₂Y₁ and Gα_i-coupled P₂Y₁₂ signaling for platelet activation (166). Platelet activation becomes defective when any of these two receptors or their associated G proteins are blocked or deficient (61, 90, 97). Thus it appears that cooperativity of the G_i and G_q pathway is necessary for ADP-induced platelet activation. Thromboxane A₂ (TXA₂) has a single receptor (TP) (207), which, however, is coupled to both Gα_q and Gα_12/13, and both classes of G proteins are important for optimal platelet response (153), particularly at low doses, suggesting synergy between two different pathways. Thrombin-induced platelet activation involves even more complex receptor and signaling pathways. Thrombin has at least three receptors on the platelet surface: PAR1, PAR4, and GPIb-IX in human platelets, and PAR3, PAR4, and GPIb-IX in mouse platelets (38, 45), wherein PAR3 was suggested to be a dock for efficient cleavage and signaling via PAR4 (160). Upon thrombin stimulation, PAR1 has been suggested to form heterodimers with PAR4 that enhance cleavage of PAR4 (8). In addition, PAR4 and P2Y12 have been reported to dimerize, and their interaction promotes PAR signaling, leading to AKT activation (107). PAR1 and PAR4 are both coupled to Gα_q, Gα₁₃ and possibly Gα_i pathways (controversial, indirect coupling was reported in some studies) (38, 109). Thrombin binds and cleaves these GPCRs to expose a new NH₂-terminal sequence, which serves as an internal ligand to interact and activate receptor signaling (217). The role of non-GPCR receptor, GPIb-IX, in thrombin-induced platelet activation has been controversial. Whereas some investigators proposed that GPIb-IX serves as a thrombin dock that promotes thrombin cleavage of PARs (46), other investigators suggested that GPIb-IX transmitted platelet activation signals independent of PARs (3, 178). However, recent work indicates that GPIb-IX is neither a passive thrombin dock nor a PAR-independent thrombin receptor, but that mutual cooperativity between thrombin-induced GPIb-IX signaling and PAR signaling is required for optimal platelet response (57). The synergy of these different receptors greatly enhances platelet sensitivity to low concentrations of thrombin, which is important for arterial thrombosis (57) (FIGURE 3).

FIGURE 3.

Synergy among multiple thrombin receptor signaling pathways

P115 RhoGEF, p115 Rho guanine nucleotide exchange factor; PKA, cAMP-dependent protein kinase; AC, adenylyl cyclase; Rap1b, RAS-related protein 1b; DAG, diacylglycerol; IP₃, inositol 1,4,5-trisphosphate; ROS, reactive oxygen species; NOX, NADPH oxidase.

Signal Amplification Networks

Whereas receptors for many different platelet agonists and adhesive proteins transmit platelet activation signals, these divergent receptor signaling pathways converge onto common signaling pathways that greatly amplify the initial receptor signals leading to robust platelet responses.

Phospholipase c, Calcium Elevation, and Diacylglycerol

Phospholipase C (PLC) is like a hub upon which numerous platelet-activation signaling pathways converge. Platelets express at least three distinct families of PLC: PLCβ, PLCγ, and PLCδ. In human platelets, PLCγ2, PLCβ2, and PLCβ3 appear to be the predominantly expressed PLC family members (122). PLCβ and PLCγ are activated via different mechanisms (182). The former is activated by binding Gα_q and the latter by Syk-mediated protein tyrosine phosphorylation. This difference in regulation is imparted by a COOH-terminal coil-coil domain found in PLCβ that is not present in PLCγ (182). By contrast, PLCγ contains SH2, SH3, and split PH domains that are not found in PLCβ (182). However, recent work suggests that Gα_q-coupled agonists, such as thrombin, also induced phosphorylation and activation of PLCγ through a ROS-dependent signaling pathway (50). Conversely, ITAM agonists, such as collagen, induce release of GPCR agonists, such as ADP and TXA₂, thus indirectly activating PLCβ via the G_q pathway (161, 198). Among its main functions, PLC catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃) (182). IP₃ binds the IP₃ receptor, resulting in Ca²⁺ release from the dense tubular system into the cytosol, which is a major mechanism of calcium elevation (213). IP₃-receptor-mediated Ca²⁺ release and subsequent depletion of intracellular Ca²⁺ stores further stimulates store-operated calcium entry (SOCE) (213), causing sustained intracellular Ca²⁺ elevation. Elevation of intracellular calcium plays a central role in platelet activation induced by all agonists, wherein it is critical to many intracellular signaling events, including integrin inside-out signaling, and activation of many calcium-dependent proteins including PKC, calpain, and calmodulin. Elevation of intracellular calcium is required for almost all platelet functions, including stable platelet adhesion, shape change, aggregation, secretion, exposure of procoagulant activity, and clot retraction. DAG activates conventional (α,β) and novel (δ, θ, η, ε) protein kinase C (PKC) isoforms (83), which are also important for integrin activation and platelet granule secretion. Elevations in intracellular Ca²⁺ concentrations also lead to activation of the Ca²⁺- and DAG-regulated guanine nucleotide exchange factor I (CalDAG-GEFI) (41, 198). It is possible that DAG may also be involved in CalDAG-GEFI activation, although CalDAG-GEFI has an atypical low-affinity binding site for DAG (43). CalDAG-GEFI is critical for activation of the small GTPase, RAS-related protein 1b (Rap1b), which is important for granule secretion and for inside-out signaling leading to integrin activation.

Phosphoinositide 3-Kinase (PI3K)-Akt Pathway

The PI3K-Akt signaling pathway is an important signaling pathway inducing platelet granule release and is important for platelet activation and stable platelet adhesion induced by GPIb-IX signaling (127, 128, 239, 240), integrin outside-in signaling, and ITAM signaling (118, 154, 165). This pathway, however, is not required for primary platelet activation induced by GPCR agonists but serves to amplify GPCR signaling. Among different phosphoinositide species, phosphatidylinositol 3,4,5-triphosphate (PIP₃) is mainly converted from phosphatidylinositol 4,5-bisphosphate (PIP₂) by class I phosphoinositide 3-kinase (PI3K) that phosphorylates the 3 position on the PIP₂ inositol ring. Agonist stimulation induces PI3K activation and thus generation of PIP₃, which mediates membrane translocation of 3-phosphoinositide-dependent kinase 1 (PDK1) and Akt isoforms via the PIP₃-binding PH domains in these proteins, allowing PDK1-mediated phosphorylation and activation of Akt. All three isoforms of Akt are expressed in platelets, and knockout of any one of them reduces low-dose thrombin- and U46619-induced platelet secretion and the secretion-dependent second-wave aggregation (28, 165, 226). Knockout of Akt1 or Akt2 also diminishes VWF-/GPIb-IX-induced platelet activation (240), but only deletion of Akt1 significantly affects collagen-induced platelet secretion and aggregation (28). Akt isoforms, predominantly Akt3, are also important in integrin outside-in signaling promoting platelet spreading. Two Akt effectors have been shown to mediate the PI3K-Akt signaling, leading to granule secretion and platelet activation: nitric oxide (NO) synthase (NOS) and GSK3β. Platelets express endothelial NOS, which is phosphorylated and activated by Akt, and stimulates the cGMP pathway leading to granule secretion and GPIb-IX-dependent platelet activation (see below). GSK3β, however, plays differential roles in different platelet activation pathways: it plays an inhibitory role in thrombin-induced platelet activation and integrin outside-in signaling, which is relieved by Akt-mediated GSK3β phosphorylation and inhibition, but appears to promote collagen-induced platelet activation (165). Thus the role of Akt in collagen-induced platelet activation is unlikely to be mediated by GSK3β, whereas the role of Akt in integrin outside-in signaling is mediated mainly via its phosphorylation and inhibition of GSK3β (118, 154, 164, 165), which has been shown to negatively regulate platelet spreading, clot retraction, and thrombus stability (118, 124). However, the mechanism of GSK3β action is unknown. Recent studies also suggest an important role for class II PI3KC2α in platelet morphology and thrombus stability under flow (158, 211). In addition, class III PI3K vps34 appears to play a role in platelet autophagy (65). Lipid phosphatases, such as PTEN, hydrolyze PIP₃ and thus inhibit the PI3K-Akt and cGMP signaling pathway (224).

New Concept in Platelet cGMP Signaling

Cyclic guanosine monophosphate (cGMP) is synthesized by guanylyl cyclases (GC) in many cell types, including platelets, and plays an important role in activating the cGMP-dependent protein kinases (PKG). Despite the early evidence that platelet agonists stimulate cGMP elevation and that exogenous cGMP analogs may have a stimulatory effect on platelets, it has been a textbook concept since the 1980s that the NO-cGMP pathway is a major negative regulator of platelet activation (84). This is mainly due to the finding that soluble guanylyl cyclase (sGC) is activated by NO and that NO donor compounds, which dramatically elevate cGMP levels in platelets, inhibit platelet activation. However, despite development of many compounds that mimic or elevate cGMP since the 1980s, none has emerged as an effective anti-platelet drug. More recently, this concept has been challenged by the discovery of the biphasic roles of the NO-cGMP-PKG signaling pathway in regulating platelet activation: low concentrations of NO and cGMP endogenously synthesized during platelet activation promote platelet activation and significantly increase platelet sensitivity to low concentrations of platelet agonists, including GPIb-IX agonists, ITAM receptor agonists, and GPCR agonists (127, 199, 239, 244). Deficiency in endothelial NOS, inducible NOS, sGC, and PKG, as well as pharmacological inhibitors of the NOS/cGMP/PKG pathway have been shown to inhibit platelet activation in response to low-dose agonists, highlighting the importance of this pathway in stimulating platelet activation (127, 144, 145, 199, 239, 244), Furthermore, the important stimulatory roles of the cGMP-PKG pathway in the platelet activation signaling of pattern recognition receptors have been reported by different groups (216, 243, 246). Only high concentrations of NO (such as that provided by NO donors) and cGMP analogs showed an inhibitory effect on platelet activation, mainly via cGMP-dependent elevation of cAMP and activation of cAMP-dependent protein kinase (244), although PKG may also play a role in the inhibitory phase (147). The biphasic role of the cGMP pathway in platelet activation is likely to be an important mechanism for self-limiting stimulation of platelet response to low-level thrombotic and inflammatory stimuli.

Eicosanoids Pathways

During platelet activation, platelets synthesize biologically active lipid species, called eicosanoids, functioning as second messengers. Eicosanoid synthesis requires calcium-dependent activation of cytosolic phospholipase A2 (PLA₂), which hydrolyzes membrane phospholipids to release arachidonic acid (AA). AA is metabolized via the cyclooxygenase and lipoxygenase pathways (5). Cyclooxygenases (COX), which are inhibited by aspirin, initiate the conversion of AA into prostaglandins (PG) (87, 237). Among several PGs synthesized in platelets, thromboxane A2 (TXA₂) is a potent platelet agonist and plays an important role in augmenting platelet activation and promoting thrombus formation by binding to its G_q-/G₁₃-coupled platelet receptor. Lipoxygenases initiate the conversion of AA into oxylipins, among which, 12-hydroxyeicosatetraenoic acid (12-HETE) has been reported both to potentiate platelet activation induced by PAR agonists, FcγRIIa cross linking, or GPVI agonists (34, 89, 236, 238), and to inhibit platelet activation induced by collagen (189, 237) and ADP (98). Whereas the reasons for this controversy remain unclear, 12-LOX-null mice reportedly prolongs tail bleeding times (236), and a 12-LOX inhibitor, ML355, inhibits 12-HETE production, platelet aggregation, and secretion induced by FcγRIIa cross linking, as well as by PAR4 agonist peptide-induced platelet activation (138, 238).

Granule Secretion as a Signal Amplification Mechanism

One of the most efficient common mechanisms of signal amplification during platelet activation is secretion of platelet granule contents. Platelets contain three major secretory organelles: dense (δ) granules that contain small molecules (e.g., divalent cations, ADP, ATP, serotonin, and polyphosphates); alpha (α) granules that contain adhesion proteins (e.g., VWF, fibrinogen), growth factors (e.g., platelet-derived growth factor, vascular endothelial growth factors), coagulation factors (e.g., factor V, factor XI), and chemokines (e.g., platelet factor 4, neutrophil-activating peptide-2); and lysosomes that contain degradative enzymes (e.g., cathepsin D, β-hexosaminidase) (76, 114). Secreted granule contents not only enhance platelet activation signaling but also activate and recruit circulating resting platelets, greatly facilitating formation and growth of a thrombus. In addition, granule secretion contributes to physiological vascular remodeling, inflammation, and wound repair, and pathological atherosclerosis and cancer metastasis (54, 71, 75, 117, 131, 146, 188). A critical mechanism for release of granule contents is fusion of granule membranes with plasma membranes on the platelet surface or in the open canalicular system; this fusion enables release of granules into the extracellular milieu. Contraction of platelets then forces out granule cargo. Secretion of granule cargo requires highly regulated and coordinated interactions between N-ethylmaleimide-sensitive fusion protein (NSF), soluble NSF-attachment protein (SNAP), and soluble NSF-attachment protein receptors (SNAREs), which are reviewed elsewhere (76, 114). The granule secretion process may be stimulated by elevations in intracellular Ca²⁺ and phosphorylation of SNARE proteins by PKC, IKK, and PKG (181). Whereas almost all platelet agonists stimulate platelet granule secretion, it has been long recognized that “strong” agonists such as collagen and thrombin induce granule secretion independent of platelet aggregation. By contrast, the “weak” agonists, such as ADP, or low concentrations of “strong” agonists induce secretion in an aggregation-dependent manner, which requires integrin outside signaling. Several platelet signaling pathways are important in promoting agonist-stimulated platelet granule secretion: 1) calcium elevation (201); 2) PKC signaling; 3) SFK signaling (31, 60, 129, 177, 185, 190, 239); 4) PI3K/AKT and NO/cGMP/PKG signaling pathways (127, 128, 239, 240); 5) MAPK signaling (4, 24, 115, 126); 6) CalDAG-GEFI and Rap1b signaling (41, 197, 227, 245); and 7) small RhoGTPase Rac1 and RhoA signaling (6, 51, 149, 174). The mechanisms by which these pathways promote secretion remain unclear and are the subject of reviews elsewhere (75, 76, 114).

Integrin Inside-Out Signaling

Integrins play key roles in platelet adhesion and aggregation. Platelet integrins include collagen receptor α₂β₁, fibronectin receptor α₅β₁, laminin receptor α₆β₁, and vitronectin receptor αvβ₃ (36, 125). The most abundant and important platelet integrin is the integrin α_IIbβ₃, which serves as a receptor for fibrinogen, VWF, vitronectin, and several other matrix adhesive proteins, and is required for stable platelet adhesion to the vascular wall and platelet aggregation. In circulating platelets, integrin α_IIbβ₃ is expressed in a “resting” form, wherein it is suggested to be in a bent conformation with a low affinity for ligands. After agonist stimulation, intracellular signals induce the extracellular domain of α_IIbβ₃ to transform into a suggested extended conformation with a high affinity for ligands (58). This process is known as inside-out signaling. A key step of inside-out signaling is the binding of cytosolic adapter protein talin to a site on the β₃ cytoplasmic tail containing an NPXY motif, which is thought to facilitate a secondary interaction with a membrane proximal binding site on β₃ (223) (FIGURE 4). These interactions were suggested to disrupt inner and outer membrane clasps held between α- and β-chain cytoplasmic and transmembrane regions triggering α_IIbβ₃ activation. Deletion of talin1 or mutation of the talin binding site on the β₃ cytoplasmic tail impairs agonist-induced integrin activation (163, 173) and platelet aggregation (163). In addition to talin, kindlins are also important in inside-out signaling. Kindlins bind to a β₃ COOH-terminal NXXY motif distinct from the talin binding site (157). Several studies suggested that the binding of talin and kindlins cooperates to induce full integrin activation (111, 234). How kindlins induce integrin activation is still a subject of debate. There have been reports suggesting that kindlins facilitate talin binding (139). Others suggest that kindlins, unlike talin, is dispensable for integrin binding to a monovalent ligand (fibronectin tenth type III repeat) but is required for binding to multivalent ligand fibrinogen, possibly by promoting clustering of integrins (156, 234, 235).

The mechanism responsible for inducing talin and kindlin binding to integrins is not totally clear. There have been reports suggesting that the small GTPase Rap1 plays an important role and hypothesizing that Rap1 GTP-interacting adaptor molecule (RIAM) may be the Rap1 effector responsible for inducing talin binding (82, 121, 219). However, although RIAM is important for leukocyte integrin function (112), RIAM deficiency does not affect platelet integrin activation and function (200).

Integrin Outside-In Signaling

Ligand binding to integrins not only mediates platelet adhesion and aggregation, but it also induces intracellular signals. This process is known as outside-in signaling, which, in the early phase, induces stabilization of platelet adhesion, platelet spreading, granule secretion, and amplification of platelet aggregation, which is important for growth of an occlusive intravascular thrombus (58, 125, 192), and, in the later phase, stimulates clot retraction (FIGURE 4). The early phase of outside-in signaling requires the binding of heterotrimeric G protein Gα₁₃ to a conserved EXE motif on the β₃ cytoplasmic tail (77, 193). A role for kindlin-3 was also reported for outside-in signaling (157). Integrin ligation induces Gα₁₃ binding and talin dissociation from β3 (193). Gα₁₃ binding is important for integrin-mediated c-Src activation (77). Activation of the β₃ COOH-terminal-associated c-Src (9) is required for transmitting outside-in signaling via phosphorylation of β₃ cytoplasmic NXXT motifs (1, 119), inhibition of RhoA, activation of the PI3K/Akt pathway, and Rac1 as well as Rap1b, leading to platelet spreading (67, 81, 245) (FIGURE 4). Src is also important in integrin-dependent activation of the ITAM signaling pathway (190, 248). All these events lead to integrin-dependent granule secretion and greatly amplified thrombus growth. In the late phase, calpain cleavage of the COOH-terminal Src binding site in β₃ abolishes Src-dependent inhibition of RhoA, resulting in activation of RhoA- and platelet-mediated clot retraction (68, 229). Interestingly, talin becomes re-associated with β₃ cytoplasmic domain during the late phase of outside-in signaling and is important for clot retraction (77, 193). Recently, vacuolar protein sorting-associated protein 33B (VPS33B), previously shown to be important in megakaryocyte and platelet α-granule biogenesis (134), was found to bind to the β₃ tail. VPS33B-null platelets display defective outside-in signaling and endocytosis of fibrinogen (231).

FIGURE 4.

Integrin signaling pathways

A: integrin inside-out signaling. A key final step of inside-out signaling is the binding of cytosolic adapter protein talin and kindlins. Question marks indicate that the mechanisms remain unclear and require further investigation. B: early phase integrin outside-in signaling, which requires the interaction between Gα₁₃ and Src with the β₃ cytoplasmic domain. Talin dissociation occurs during the early phase outside-in signaling. C: late-phase integrin outside-in signaling, which requires talin reassociation and calpain cleavage of Src binding site in the β₃ tail.

Conclusions and Perspectives

With significant recent advances in platelet physiology, the intricate platelet activation signaling networks continue to grow in complexity and sophistication. Recent advances also necessitate the revision of some textbook concepts in platelets such as the roles of NO and cGMP in platelet activation. Notably, pattern recognition receptors important for immune response and inflammation utilize the cGMP pathway to transmit platelet activation signals, suggesting a link between inflammation and thrombosis via this self-regulated signal pathway promoting platelet activation and secretion. These advances are largely attributed to widespread use of targeted gene deletion/mutation techniques in mice combined with pharmacological innovations, which not only have generated numerous insights into the mechanisms of platelet activation, but also have created new models and avenues for future research and pharmaceutical development. One important goal of studying platelet activation signaling is to provide targets for developing new drugs that can be used to more precisely treat thrombosis and hemorrhage. In this regard, the discovery of selectively targeting of integrin outside-in signaling without perturbing the ligand binding to integrin α_IIbβ₃ allows selective inhibition of occlusive thrombosis without apparent effect on hemostasis in mouse models (193), which is in contrast to the adverse effects of hemorrhage associated with current anti-platelet drugs. Clearly, more precise understanding of the complex platelet activation signaling pathways should help us in developing new generations of platelet drugs in treating thrombotic, inflammatory, and hemorrhagic disorders. After all, the precision of our understanding in mechanisms of health and disease determines how precise medicine can be.