Adenosine diphosphate and its analogues bind to P2 receptors, which are expressed ubiquitously throughout the human body including on platelets, on vascular smooth muscle cells, and on endothelial cells. P2 receptors are divided into P2X and P2Y receptors. P2X receptors are ligand-gated ion channel receptors, and P2Y receptors are G-protein coupled receptors. Each subtype of P2X and P2Y receptors is classified by the relative potency of purine and pyrimidine nucleotides, as agonists, to induce intracellular calcium transients or smooth muscle contraction after genetic cloning. Seven P2X receptors (P2X₁, P2X₂, P2X₃, P2X₄, P2X₅, P2X₆, and P2X₇) and P2Y receptors (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, and P2Y₁₃) are cloned in mammalian cells.3) Platelets express P2X₁, P2Y₁, and P2Y₁₂ receptors.4-6)

Adenosine diphosphate binding to P2X₁ receptor results in rapid extracellular calcium influx, leading to alteration of platelet shape, but without platelet activation. ADP-stimulation of Gq-coupled P2Y₁ receptor activates phospholipase C (PLC) and induces a transient increase in intracellular calcium concentration resulting in platelet shape change and in weak, transient platelet aggregation. Stable and firm platelet aggregation requires ADP binding to P2Y₁₂ receptor and activation of GPIIb/IIIa receptors in the platelets membrane.7) Activation of Gi-coupled P2Y₁₂ receptor liberates the Gi protein subunits, α_Gi and βγ. The subunit α_Gi decreases the platelet cyclic adenosine monophosphate (cAMP) level through the inhibition of adenylyl cyclase. This decrease in cAMP production leads, in turn, to a reduction in the activation of protein kinase C (PKC), and, after complex signal transduction processes, activation of GP IIb/IIIa receptors (Fig. 1). The subunit βγ activates the phosphatidylinositol 3-kinase (PI-3K), which regulates protein kinase Akt and contributes to the activation of GP IIb/IIIa receptors.

The P2Y₁₂ receptor is inhibited by ticlopidine, clopidogrel, prasugrel, or ticagrelor, which are currently used for patients with acute coronary syndrome or coronary stents to prevent thrombotic complications.

Thrombin is the most potent activator of platelets. Thrombin formation is initiated by the exposure of tissue factor to the coagulation system after disruption of the vascular endothelium. Thrombin formation takes place on cellular surfaces, including the phospholipid membrane of activated platelets, which amplifies coagulation processes. Thrombin interacts with protease-activated receptors (PARs) on the surface of human platelets, which couple to G_q, G₁₂/G₁₃ and G_i family of heterotrimeric G proteins.8) Of the 4 PARs, human platelets express PAR1 and PAR4. PAR1 mediates human platelet activation at low thrombin concentrations, whereas PAR4 induces platelet activation only at high thrombin concentrations.9)

Thrombin binding of PARs initiates the cleavage of the extracellular domain of the receptor and exposure of a tethered ligand at the new end of the receptor. Signaling through either PAR uses PLC pathway and PKC pathway, leading to the activation of GP IIb/IIIa receptors.10) In addition, interaction of thrombin with PARs causes the production of TXA₂, the release of ADP, and generation of more thrombin on the platelet surface.

Vorapaxar is an oral reversible PAR1 antagonist. In a phase 2 trial, vorapaxar showed a trend toward lower incidence of major adverse cardiovascular events (MACE) without differences of major and minor bleeding in patients scheduled for angiography and possible coronary stenting.11) However, in a recent large randomized clinical trial, the addition of vorapaxar to standard therapy in patients with non-ST elevation acute coronary syndrome did not reduce MACE but significantly increased the risk of major bleeding, including intracranial hemorrhage.12)

Thromboxane A₂ is produced from arachidonic acid through conversion by cyclooxygenase-1 and thromboxane synthase. Like ADP, TXA₂ functions as a positive-feedback mediator during platelet activation. Human TXA₂/prostaglandin (TP) receptor exists in 2 isoforms, TPα and TPβ, and human platelets express both isoforms.13) TP receptor couples to G_q and G₁₂/G₁₃. TXA₂ stimulation of G_q proteins causes activation of PLC, resulting in accumulation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Stimulation of G₁₂/G₁₃ proteins activates Rho signaling. TP receptor-mediated platelet shape change is mainly dependent on G₁₂/G₁₃, while aggregation is dependent on G_q.14)

Recent studies have emphasized the role of the Rap family of small GTPases in inside-out signaling of integrin activation. Rap1b is the most abundant Ras family member in platelets.19) Rap1b deficiency in murine platelets showed reduced platelet aggregation, decreased activation of integrin αIIbβ3, and protection from arterial thrombosis.20) Rap proteins cycle between GDP-bound inactive form and GTP-bound active form. GTP binding to Rap is facilitated by guanine nucleotide exchange factors (GEFs). In platelets, Rap1b is controlled by Ca²⁺ and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), which contains binding sites for Ca²⁺ and DAG, and a GEF domain.21) The importance of CalDAG-GEFI was demonstrated in CalDAG-GEFI-deficient mice. Cal-DAG-GEFI deficiency resulted in impaired platelet aggregation responses to ADP or TXA₂, but not to collagen or thrombin.22) CalDAG-GEFI^-/- mice showed prolonged bleeding time and protection against experimental thrombosis.

Different agonist responses in CalDAG-GEFI-deficient platelets suggest the existence of a CalDAG-GEFI-independent mechanism of integrin αIIbβ3 activation. In murine platelets, PAR4 agonist-induced aggregation in the absence of CalDAG-GEFI required cosignaling through the α_Gi-coupled P2Y₁₂ receptor involving PKC. Therefore, PKC was identified as an alternative pathway leading to Rap1 and αIIbβ3 activation. It has been proposed that rapid, but reversible activation of Rap1 is mediated by CalDAG-GEFI, whereas PKC ensures sustained Rap1 activation through its central role in the release of ADP from dense granules.21)

Agonist activation of platelets is usually accompanied by an increase in the cytosolic Ca²⁺ concentration, which is required for integrin activation and releases of the second wave mediators, such as ADP or TXA₂. The source of Ca²⁺ can be either intracellular or extracellular. Agonists binding of platelet Gq coupled-receptors activates PLC, which in turn produces inositol 1,4,5-triphosphate (IP₃) from phosphoinositol biphosphate (PIP₂). The intracellular Ca²⁺ is released from the sarcoplasmatic reticulum (SR) by IP₃. Because of the limited Ca²⁺ store of the ER, the major source of an intracellular Ca²⁺ transient is store-operated Ca²⁺ entry (SOCE) of the extracellular Ca²⁺ through the activation of Ca²⁺ release activated calcium channels (CRAC), Orai1, in the platelets membrane, which are controlled by the depletion of SR Ca²⁺ stores.23) Recent studies identified stromal interaction molecule 1 (STIM1) as a key regulator of SOCE into platelets.24) STIM1 is a type I transmembrane protein containing a calcium binding EF hand motif, which is occupied by Ca²⁺ in resting platelets. After platelet activation, Ca²⁺ is released from the ER and STIM1 is no longer occupied by Ca²⁺, resulting in translocation of STIM1 within the platelets to allow SOCE by opening the Orai1. Platelets of STIM1 or Orai1-deficient mice showed defective SOCE, but largely intact agonist-induced integrin activation, demonstrating that this process can be initiated by relatively small rises in intracellular Ca²⁺ transients from the ER.24)25)

The Rap1 effector molecule Rap1-GTP-interacting adaptor molecule (RIAM) is a member of the MRL (Mig-10, RIAM, and lamellipodin) family of adaptor proteins.26) MRL proteins function as scaffolds to connect the membrane targeting sequences in Ras GTPases to talin-1. This interaction of MRL proteins with both Rap1-GTP and talin-1 mediates adhesion of β1 and β2 integrins.27) In living cells, RIAM over-expression stimulates talin recruitment to αIIbβ3 with integrin activation,19) but RIAM knockdown blocks talin recruitment to αIIbβ3 with integrin response.28) These findings suggest the important interaction of Rap1-GTP, RIAM and talin-1, resulting in the binding of talin-1 to integrin β cytoplasmic tails.29)

PI-3K, via phosphatidylinositol-dependent kinase-1, generates phosphoinositide products including Akt, which is a family of intracellular serine/threonine protein kinases (also called protein kinase B).30) Three isoforms of Akt, which possess more than 80% homology, have been identified: Akt1, Akt2, and Akt3. Platelets contain both the Akt1 and Akt2 isoforms. Platelets from Akt1-deficient mice exhibited defects in dense and α-granule secretion, reduced fibrinogen binding, and impaired aggregation in response to low concentrations of thrombin.31) Also, Akt2-deficient mice showed impaired platelet aggregation, fibrinogen binding, and granules secretion especially in response to low concentrations of thrombin and TXA₂, which activate the Gq-coupled receptors.30) The above findings showed that both Akt-1 and Akt-2 are important in low dose agonist-induced platelet activation and in platelet-dependent thrombus formation.

Several signaling molecules have been implicated as the downstream targets of PI-3K- and Akt-mediated platelet activation. These include glycogen synthase kinase (GSK)-3β, nitric oxide synthase (NOS) 3, and cAMP-dependent phosphodiesterase (PDE3A). GSK-3β suppresses platelet function and thrombosis in mice. Akt-mediated phosphorylation of GSK-3β inhibits its suppressive function of platelet activation.32) Akt-mediated stimulation of NOS3 induces protein kinase G-dependent degranulation of platelets.33) In addition, Akt has been implicated in activation of PDE3A, resulting in reduced platelet cAMP levels after thrombin stimulation.34)

Talin is one of several proteins that link the cytoplasmic domains of integrin β subunits to actin filaments. Binding of talin to cytoplasmic domains of β-integrins triggers a conformational change in the extracellular domains that increases its affinity for ligands.35)

Talin-1, a 270-kDa cytoplasmic protein, is composed of a globular N-terminal head domain and a flexible rod domain, which can be dissociated by the protease calpain 2. The head region of talin-1 contains a FERM (protein 4.1, ezrin, radixin, moesin) domain comprising 3 subdomains F1, F2, and F3, which has binding sites for the cytoplasmic domains of β-integrins, as well as for filamentous actin (F-actin). The talin rod contains an additional integrin-binding site, at least 2 actin-binding sites and several binding sites for vinculin.36)

The cytoplasmic domains of β-integrins are essential for integrin activation. The β3 tail interacts with a large number of cytosolic proteins to induce the conformational change of the extracellular domain. To identify structural features responsible for these interactions, nuclear magnetic resonance (NMR) studies about β3 transmembrane (TM) and cytoplasmic domains were performed.37)38) The results of these studies showed 3 α helices followed by a short unstructured C-terminus. The first helix, β3 TM helix, embedded in the lipid bilayer, but oriented at a tilted angle because the length of this helix is longer than that of a typical membrane bilayer. The β3 TM helix is followed by a hinge at residues H722-D723, the second helix (residues K725-A735), a hinge containing NPLY motif (residues 744-747), and the third helix at the C-terminal end.

In one suggested model for talin-mediated activation of integrins, the low-affinity αIIbβ3 is maintained through the interaction between α and β subunit at the TM and membrane-proximal domains. The TM helices specifically pack together and are in a long and tilted geometry. The membrane-proximal regions of αIIb and β3 cytoplasmic tails are believed to interact through a salt bridge between a conserved Arg in the αIIb tail and an Asp in the β3 tail, thereby keeping αIIbβ3 in the inactive state. The F3 subdomain of talin FERM domain contains a phosphotyrosine binding (PTB) domain, which interacts with NPLY motif of β3 tail. This interaction is proposed to induce αIIbβ3 activation by disrupting salt bridge formed between αIIb-Arg995 and β3-Asp723, and hence inducing a series of conformational changes that give rise to the affinity modulation of the extracellular domain.19)

The analysis of platelets from conditional talin-1-deficient mice (Tln^-/-, CreloxP) supported an essential function of talin-1 in platelet integrin activation.39)40) Talin-1-deficient platelets were unable to activate integrin αIIbβ3 in response to any tested agonists and consequently did not aggregate. Furthermore, they failed to spread on immobilized fibrinogen, suggesting that talin-1 is required for αIIbβ3-dependent outside-in signaling, as well as inside-out signaling. This complete lack of integrin function in Tln^-/-platelets resulted in defective hemostasis and thrombus formation in injured vessels in vivo.

Kindlins constitute a family of evolutionarily conserved cytoplasmic components of cell-extracellular matrix adhesions that bind to cytoplasmic tails of β-integrins directly and cooperate with talin in integrin activation. There are 3 mammalian kindlins: kindlin-1 (also known as kindlerin and FERMT1), kindlin-2 (also known as MIG-2) and kindlin-3 (also known as URP 2). They exhibit identical domain architecture and high sequence similarities. One of the important features of kindlins is that they contain a FERM domain, which binds to the cytoplasmic tail of β1 and β3 integrins. So far, 2 more kindling binding proteins other than β integrins have been identified: integrin-linked kinase (ILK) and migfilin.41)

It is proposed that the PTB site of the F3 domain in kindlin-3 directly interacts with the NITY motif (residues 756-759) located just after the 3rd helix of integrin β tails.35) Different binding regions of talin-1 and kindlin-3 allow either simultaneous or sequential binding of the 2 proteins to activate the integrin.16)

Platelets from fetal liver cell chimeric kindlin-3^-/- mice were unable to activate integrin αIIbβ3 in response to any tested agonist, despite unaltered expression of talin-1, and kindlin-3^-/- platelets did not spread on fibrinogen. This complete loss of integrin function resulted in a severe hemostatic defect and resistance to arterial thrombosis in kindlin-3^-/-chimeric mice.42)