Non-canonical CXCR7 signaling mechanisms in the regulation of the second messengers cGMP and cAMP in platelets and vascular cells
G protein coupled signaling pathways mediate platelet response to a variety of platelet activating stimulus that engage these receptors on the platelets surface. These GPCRs include those that trigger either an activatory or an inhibitory signaling cascade so as to substantiate thrombotic or anti-thrombotic responses. Therefore, these GPCRs are important pharmacological targets and such receptor agonists (e.g. IP agonist iloprost) and antagonist (e.g. P2Y12 antagonist clopidogrel, ticagrelor) are used In clinical practice to prevent recurrent thrombotic and thromboischemic complications like myocardial Infarction.
The Gαi-coupled CXCR4 and the non-canonical CXCR7/ACKR3 are the two cognate receptors for the chemokine CXCL12/SDF-1α, the chemokine like cytokine macrophage migration inhibitory factor MIF, while the chemokine CXCL11/ITAC exclusively engages CXCR7 on platelets. Surface expression of both CXCR4 and CXCR7 is significantly enhanced on circulating platelets from coronary artery disease (CAD) patients offering us a readily available drug target. It is essential to consider that the expression of CXCR4 and CXCR7 is not limited to platelets but also other vascular (e.g. endothelial cells) and circulating (e.g. monocytes, lymphocytes, neutrophils) cells.
The functional dichotomy of CXCR4 and CXCR7 in platelet physiology is evident from the involvement of CXCR4 in mediating pro-thrombotic while the engagement of CXCR7 in mediating an anti-thrombotic and anti-thrombo-inflammatory influence. Employing a pharmacological CXCR7-agonist we have demonstrated the anti-thrombotic potential of platelet CXCR7 in antagonizing both CXCR4 and other canonical GPCR mediated activation of platelets.
The non-canonical GPCR CXCR7 usually does not signal through G proteins but rather uses β-arrestin biased signaling. However, recently, experimental evidences have emerged that demonstrate a crosstalk between CXCR7 instigated non-canonical and Gαs-coupled prostacyclin (IP) receptor mediated canonical signaling in platelets that exert an inhibitory effect on platelet hyper-reactivity. This opens an intriguing and uncharted area of research on potential interactions between canonical and non-canonical signaling cascades in platelets, and may reveal therapeutic targets that can be exploited pharmaceutically to regulate thrombotic and thrombo-inflammatory adversities without increasing bleeding risk.
CXCR7 surface expression on platelets is significantly enhanced in coronary artery disease patients, and is positively associated with functional recovery and improved prognosis following myocardial infarction (MI). CXCR7 stimulation by the physiological ligands CXCL11, CXCL12 and MIF substantiates platelet survival without interfering with the functions of CXCR4 in primary cells. Since MIF inhibits the externalization of the procoagulant phosphatidylserine, it exerts an anti-thrombotic effect through CXCR7 on platelets. Ubiquitous deletion of CXCR7 aggravates atherosclerosis, while genetic ablation of CXCR7 specifically from cardiomyocytes and endothelium contributes to the severity of myocardial infarction and mortality in murine models. Pharmacological CXCR7 agonist administration to hyperlipidemic mice reduces atheroprogression, and improves functional recovery following MI demonstrating the therapeutic potential of targeting CXCR7. Our objective is to validate the relevance of CXCR7 as a potential therapeutic target in regulating thrombotic and thrombo-inflammatory responses in diverse pathophysiological scenarios where platelet functions are altered or affected. However, therapeutic targeting of CXCR7 will have to consider several aspects besides the usual just pharmacologic, pharmacokinetic and regulatory toxicological measures, before its translational implementation as an anti-platelet therapy in clinical practice. This will require further in-depth research.
Since ACKR3/CXCR7 elicits both cell- and organ-specific functional response, any therapeutic strategy interfering with or promoting CXCR7 activity will require a thorough functional characterization of anticipated cellular targets (e.g. endothelial cells) under the influence of a CXCR7-agonist, to prevent undesirable off-target deleterious effects other than the one intended as an anti-platelet agent. Moreover, a pharmacological CXCR7-agonist intended as an anti-platelet agent will have to be validated for several platelet functions, their interaction with circulatory and vascular cells, and those in the organs (e.g., myocardium) that are infiltrated by activated platelets, to fully apprehend its scope for therapeutic application in cardiovascular pathologies. Additionally, a detailed mechanistic insight into the anti-platelet mode of action is needed to convincingly attribute the observed anti-thrombotic benefits to be specifically mediated through ACKR3/CXCR7 and not as a pleiotropic effect.
Our recent experiments revealed that the potent pharmacological CXCR7-agonist VUF11207 controls thrombotic and thrombo-inflammatory platelet functions, platelet response post MI and arterial injury. Like the structurally related β-arrestin-coupled GPCR rhodopsin in the eye, VUF11207-activated CXCR7 also increases inhibitory cGMP levels in platelets. In guanylyl cyclase (GC)-deficient mice, antithrombotic effects of the CXCR7 agonist are markedly decreased, proving that platelet inhibition critically depends on cGMP. However, the mechanistic basis underlying activation of GC following CXCR7 stimulation is yet unexplored. The increment in platelet inhibitory cAMP levels downstream of CXCR7 ligation is executed through a crosstalk between noncanonical CXCR7 and canonical IP-receptor as stated. However, the interplay and crosstalk between cAMP and cGMP mediated signaling pathways downstream of CXCR7 ligation needs to be unraveled.
We aim to disclose the unknown molecular interacting partners in the non-canonical signaling cascade, by which physiological and pharmacological CXCR7 agonists increase cGMP production, and consequently stabilize cAMP levels in platelets and endothelial cells. In later stages of the project, we will analyze the role of CXCR7 in affecting cGMP and cAMP levels and thrombo-inflammation in vivo and validate the therapeutic efficacy of pharmacological CXCR7 agonists in venous thrombosis, which is primarily governed by vascular and thrombo-inflammatory complications. Thus, the planned investigations will uncover the role and therapeutic potential of non-canonical CXCR7 signaling in vascular pathophysiology.
The functional dichotomy of platelet CXCR4 and ACKR3/CXCR7 CXCR4 is a Gαi-coupled GPCR which may exert its influence on platelet biogenesis (megakaryopoiesis), and pro-thrombotic response. CXCL12/SDF1αengages CXCR4 on platelets to trigger intraplatelet-calcium mobilization, the platelet activatory signaling pathway involving PI3K, Akt, which promotes platelet activation, aggregation, and a pro-thrombotic response. Moreover, Gαi-coupled CXCR4 induces a canonical signaling cascade following ligation by CXCL12/SDF-1α that imposes an inhibitory effect on AC and impedes the generation of cAMP. On the contrary, ACKR3/CXCR7 is an atypical GPCR, which does not engage a G protein but mostly β-arrestin. Physiological and pharmacological ligands of ACKR3/CXCR7 promote platelet survival. ACKR3/CXCR7-ligation by a pharmacological agonist (VUF11207) counteracts calcium mobilization induced by platelet-activating stimuli. ACKR3/CXCR7-ligation triggers the platelet inhibitory signaling cascade involving AC-cAMP-PKA, while counteracting activatory signaling mediators to impose an inhibition on platelet pro-thrombotic response (red arrows denote an inhibitory effect on the process).
Non-canonical ACKR3/CXCR7 co-ordinates with canonical Gαs-coupled IP-receptor to impose platelet inhibition.
ACKR3/CXCR7-ligation by a pharmacological agonist modulates the platelet lipidome, leading to an increased generation of anti-platelet lipid DGLA.12-LOX metabolizes DGLA into 12-HETrE, its anti-platelet oxylipin derivative. 12-HETrE released in the platelet microenvironment engages the Gαs-coupled IP receptor on platelets. This triggers the platelet inhibitory AC-cAMP-PKA signaling cascade, as adenylyl cyclase (AC) is activated to elevate cAMP levels and triggers the downstream cyclic nucleotide dependent protein kinase A (PKA).