Cyclic nucleotide-dependent inhibitory signaling interweaves with activating pathways to determine platelet responses

Abstract

Platelets are regulated by extracellular cues that impact on intracellular signaling. The endothelium releases prostacyclin and nitric oxide which stimulate the synthesis of cyclic nucleotides cAMP and cGMP leading to platelet inhibition. Other inhibitory mechanisms involve immunoreceptor tyrosine-based inhibition motif-containing receptors, intracellular receptors and receptor desensitization. Inhibitory cyclic nucleotide pathways are traditionally thought to represent a passive background system keeping platelets in a quiescent state. In contrast, cyclic nucleotides are increasingly seen to be dynamically involved in most aspects of platelet regulation. This review focuses on crosstalk between activating and cyclic nucleotide-mediated inhibitory pathways highlighting emerging new hub structures and signaling mechanisms. In particular, interactions of plasma membrane receptors like P2Y12 and GPIb/IX/V with the cyclic nucleotide system are described. Furthermore, differential regulation of the RGS18 complex, second messengers, protein kinases, and phosphatases are presented, and control over small G-proteins by guanine-nucleotide exchange factors and GTPase-activating proteins are outlined. Possible clinical implications of signaling crosstalk are discussed.

Keywords: 14‐3‐3 protein; G‐protein; RGS18; cyclic AMP; kinase; protein kinase A.

Figure 1

Prostacyclin signaling. Prostacyclin (PGI ₂) is released by healthy endothelial cells. The prostacyclin receptor on platelets couples to the stimulatory heterotrimeric Gs protein complex. Prostacylin binding leads to a conformational change in the receptor activating its guanine‐nucleotide exchange factor (GEF) activity towards Gαs resulting in the exchange of GDP by GTP. Gαs‐GTP binds to and activates the transmembrane protein adenylate cyclase (AC) to synthesize cAMP from ATP. The second messenger cAMP has only one major target in platelets which is the cAMP‐dependent protein kinase (PKA) family. cAMP binding to the regulatory subunits of PKA leads to activation of the catalytic subunits and to the phosphorylation of numerous substrate proteins resulting in a profound inhibition of most platelet functions

Figure 2

Nitric oxide signaling. Nitric oxide (NO) is released by healthy endothelial cells. NO is a small gaseous molecule that diffuses through the plasma membrane of platelets. The main target of NO in platelets is the soluble guanylate cyclase (sGC) which is stimulated by NO binding to synthesize the second messenger cGMP from GTP. The sole target of cGMP in platelets is the Iβ isoform of cGMP‐dependent protein kinase (PKG). cGMP binding to PKG Iβ activates the kinase domain to phosphorylate many substrate proteins resulting in a profound inhibition of most platelet functions

Figure 3

Gi signaling. The P2Y12 receptor for ADP is coupled to an inhibitory heterotrimeric Gi protein complex composed of Gαi and Gβγ subunits. ADP binding leads to exchange of GDP by GTP on the Gαi subunit and to the dissocation of the Gβγ subunit. The Gαi‐GTP subunit binds to and inhibits the function of adenylate cyclase (AC) resulting in a reduction of prostacyclin and cAMP/PKA signaling and diminished platelet inhibition. The Gβγ subunit binds to and activates phosphatidylinositol 3‐kinase β and γ (PI3K) to synthesize the membrane lipid phosphatidylinositol 3,4,5‐trisphosphate (PIP ₃) from phosphatidylinositol 4,5‐bisphosphate. PIP ₃ serves as docking site to recruit and activate the kinase Akt which phosphorylates substrate proteins contributing to platelet activation

Figure 4

Regulation of Gq signaling by RGS18. Binding of the platelet activators thrombin and thromboxane A2 (TXA ₂) to their respective receptors induces the formation of active Gαq‐GTP which binds to and activates phospholipase Cβ (PLCβ) leading to the generation of inositol‐3‐phosphate (IP ₃) and diacylglycerol (DAG) from phosphatidylinositol 4,5‐bisphosphate (PIP ₂). IP ₃ triggers the release of calcium ions (Ca²⁺) from intracellular stores which are essential for platelet activation. DAG activates protein kinase C (PKC) isoforms which phosphorylate many proteins supporting platelet activation. Regulator of G‐protein signaling 18 (RGS18) is a GTPase‐activating protein (GAP) which accelerates the intrinsic GTPase activity of Gαq. RGS18 enables the hydrolysis of GTP bound to Gαq resulting in the formation of inactive Gαq‐GDP and in the termination of Gq signaling. Platelet activators like thrombin and TXA ₂ inhibit RGS18 thus support Gq signaling, whereas platelet inhibitors stimulate RGS18 function to stop Gq signaling

Figure 5

Activation/inactivation cycle of RGS18. Regulator of G‐protein signaling 18 (RGS18) terminates Gq signaling and is regulated by both platelet activating and inhibitory pathways. In freshly isolated resting platelets RGS18 is found in a complex with the adapter protein spinophilin, the tyrosine phosphatase SHP‐1 and the phospho‐serine/threonine binding protein 14‐3‐3γ attached to phosphorylated serine 218 of RGS18 (transition state on the way towards inactivation, transition i). Platelet activators like thrombin and TXA ₂ induce the phosphorylation of serine 49 of RGS18 generating a second 14‐3‐3 binding site leading to enhanced 14‐3‐3 binding. Simultaneously, SHP‐1 is activated and detaches from spinophilin (involving de‐phosphorylation of tyrosine residues on spinophilin), the serine/threonine phosphatase PP1 binds to spinophilin instead, and spinophilin dissociates from RGS18. In this state the RGS18 complex is inactive. Platelet inhibitors like prostacylin and nitric oxide induce the phosphorylation of serine 216 on RGS18 and serine 94 on spinophilin which lead to the activation of PP1 and detachment of spinophilin from RGS18 (transition state on the way towards activation, transition a). Active PP1 removes phosphate groups from serines 49 and 218 of RGS18 leading to the loss of 14‐3‐3 binding. This state of the RGS18 complex is characterized by free catalytically active RGS18 which can hydrolyse Gαq‐GTP to form inactive Gαq‐GDP (Figure 4). Phosphorylation sites linked to platelet inhibition are highlighted in red, whereas sites linked to platelet activation are marked in green

Figure 6

PDE3A signaling. Upon binding of active Gαs‐GTP adenylate cyclase (AC) is stimulated to synthesize cAMP from ATP. cAMP is required for the activation of cAMP‐dependent protein kinase (PKA). cAMP levels can be reduced by phosphodiesterase PDE3A which specifically degrades cAMP to AMP. PDE3A is regulated by multiple phosphorylation events. For example, platelet activation leads to activation of protein kinase C isoforms (PKC, Figure 4) which phosphorylate PDE3A on serine 428 leading to attachment of the phospho‐serine/threonine binding protein 14‐3‐3 and to stimulation of PDE3A function. In this way platelet activation interferes with inhibitory cAMP pathways

Figure 7

Regulation of Rap1 by GEFs and GAPs. Integrin αIIbß3 is a major platelet integrin required for platelet aggregation. The small G‐protein Rap1 is a positive regulator of integrin αIIbß3 activation and cycles between an inactive, GDP‐bound and an active, GTP‐bound state. Formation of Rap1‐GTP and integrin activation are enabled by the calcium ion (Ca²⁺) dependent guanine‐nucleotide exchange factor CalDAG‐GEFI, whereas hydrolysis of Rap1‐GTP to inactive Rap1‐GDP requires the GTPase‐activating proteins (GAPs) Rap1GAP2 and RASA3. Rap1GAP2 interacts with the phospho‐serine/threonine binding protein 14‐3‐3 through phosphorylated serine 9 on Rap1GAP2. During platelet activation levels of free intracellular Ca²⁺ rise (Figure 4) leading to the activation of CalDAG‐GEFI, increased Rap1‐GTP formation and integrin activation. In contrast, cyclic nucleotide‐dependent inhibitory pathways on one hand suppress intracellular Ca²⁺ levels, thus indirectly inhibit CalDAG‐GEFI activation, and on the other hand result in phosphorylation of CalDAG‐GEFI on serines 116, 117 and 587 inhibiting its activity. Simultaneously, cyclic nucleotide‐dependent inhibitory pathways induce Rap1GAP2 phosphorylation on serine 7 leading to an inhibition of 14‐3‐3 binding, activation of Rap1GAP2, reduced Rap1‐GTP levels and lowered Rap1 activity. In contrast, platelet activation leads to phosphorylation of Rap1GAP2 on serine 9 resulting in enhanced 14‐3‐3 binding and inhibition of Rap1GAP2. In parallel, αIIbβ3‐mediated outside‐in signaling leads to phosphatidylinositol 3‐kinase (PI3K) activation, which in turn reduces the GAP activity of RASA3. Platelet activators also suppress the phosphorylation of the inhibitory serine 587 site on CalDAG‐GEFI. The consequence of Rap1GAP2 and RASA3 inhibition, along with CalDAG‐GEFI stimulation is enhanced Rap1 activity. Phosphorylation sites linked to platelet inhibition are highlighted in red, whereas sites linked to platelet activation are marked in green