In this issue of Blood Advances, Wraith et al1 showed that the immunoreceptor tyrosine-based activation motif (ITAM)–containing Fc receptor γ-chain (FcRγ) interacts closely with CD36 on the platelet surface and is necessary for platelet activation downstream of CD36 binding to its ligand oxidized low-density lipoprotein (oxLDL). Using human platelets, mouse genetic models, and sophisticated assays of platelet function, they showed that CD36-mediated FcRγ phosphorylation was necessary for oxLDL-mediated human platelet hyperactivity ex vivo and for enhanced thrombosis in a mouse in vivo model of acute arterial injury. The article builds on previous work from this group and others defining elements of a CD36-mediated signaling pathway in platelets2 that promotes platelet activation and arterial thrombosis in the setting of chronic inflammatory disease, such as hyperlipidemia, atherosclerosis, and diabetes.2-4 It fills an important mechanistic gap in the field by identifying FcRγ as an adapter protein linking CD36 ligand engagement and src-family kinase activation with downstream effectors, including the tyrosine kinase syk, necessary for platelet activation.
Platelet hyperreactivity, defined generally as enhanced sensitivity to activation by classical agonists such as thrombin, collagen, and adenosine 5′-diphosphate, plays an important role in the increased risk of arterial thrombosis associated with many chronic diseases. Research over the past 2 decades studying platelets from patients with chronic diseases or from mice engineered to develop chronic diseases has identified specific platelet surface receptors and signaling pathways that are activated in these settings but not in platelets from healthy individuals. For example, activation of the atypical redox-sensitive MAP kinase ERK5, not previously associated with platelet activation, was shown to trigger platelet hyperactivity and infarct expansion in mouse models of atherosclerosis5 and acute myocardial infarction with ischemia/reperfusion injury.6
The altered vascular environment in chronic disease settings can generate agonists for platelet receptors, such as scavenger receptors, Toll-like receptors, Fc receptors, and Tyro3/Axl/Mer (TAM) family receptors, not typically considered part of classical hemostasis. These receptors recognize products of both the innate and adaptive immune systems, as well as danger-associated molecular patterns (DAMPs) produced during inflammation, hyperglycemia, hyperlipidemia, and tissue injury. The platelet scavenger receptor CD36 recognizes several of these DAMPs, including oxLDL,2 glycated proteins,3 cell-derived microvesicles,7 and S100A proinflammatory peptides released by activated neutrophils and platelets.8 CD36 engagement with its ligands increases the sensitivity of platelets to classical agonists and promotes arterial thrombosis in mouse models of diabetes, hyperlipidemia, and chronic systemic inflammation. Mechanistically, CD36 signals when engaged with its ligands by assembling a multiprotein complex of membrane and intracellular proteins4 that activate specific src-family kinases lyn and fyn. Downstream of this step, specific MAP kinases including JNK and ERK5,6 syk tyrosine kinase, Rho family GTPases, and phospholipase γ2 become activated. These signals feed forward to enhance platelet activation by canonical G-protein–coupled receptors and the collagen receptor GP6, in part through generation of intracellular reactive oxygen species (ROS),6 while at the same time inhibiting cyclic adenosine and cyclic guanosine monophosphate-mediated platelet pacifying pathways.9
The importance of the Wraith et al study is its identification of FcRγ phosphorylation and syk activation as critical proximal steps in the CD36 platelet activation pathway. CD36 itself does not contain obvious scaffolding or kinase domains, so it is possible that the FcRγ ITAM domain serves the important function of initiating assembly of the multiprotein signalosome. Because syk inhibitors are clinically available, it would be feasible to test whether syk inhibition ameliorates all downstream signaling in oxLDL-stimulated platelets, including ERK5 activation and ROS generation, and whether syk inhibition in settings of chronic inflammation could lower the incidence of adverse cardiovascular events in humans at high thrombotic risk. Genetic studies in human populations suggest that platelet CD36 levels associate with such risk, and that CD36 deficiency is not associated with increased bleeding risk, so the pathway is an attractive target.10 The murine thrombosis model used by Wraith et al, however, involved injection of oxLDL into the circulation of mice before subjecting them to arterial injury by FeCl3. This is a model of acute atherosclerotic plaque rupture, not chronic atherosclerosis or chronic inflammation, so testing the role of FcRγ in these settings, which are more amenable to interventions, remains to be done.
Several important gaps remain in our understanding of CD36 prothrombotic signaling. FcRγ phosphorylation and syk activation occurred seconds after ligand engagement and required prior activation of src-family kinases fyn and lyn.1 How CD36 activates these proximal kinases remains unknown. Studies in macrophages and adipocytes suggest that ligand-dependent association of CD36 with the sodium/potassium ATPase, a known regulator of src kinase activation, may trigger their activation,11 but this has not been studied in platelets. Other studies showed that CD36 localizes in cholesterol-rich membrane microdomains, perhaps with tetraspannin proteins such as CD9, and receptor clustering within these domains might be necessary for signal initiation.12 An intriguing finding of Wraith et al is the unusual stoichiometry of the platelet CD36/FcRγ interaction. CD36 is very abundant in platelets with ∼7000 to 15 000 copies per cell, whereas there are only 1500 copies of FcRγ per platelet. FcRγ also associates with other highly abundant membrane proteins, including GP6 and integrins. Coprecipitation studies by Wraith et al indeed showed that only 8% of platelet FcRγ (∼120 molecules per cell) was physically associated with CD36; that is, ∼1% of CD36. How such a small pool of CD36 and FcRγ can generate such powerful downstream action remains to be determined.
Platelet reactivity is regulated by a tightly controlled balance of intracellular kinase pathways, calcium and ROS concentrations, and cyclic nucleotide levels. The studies of Wraith et al support the hypothesis that perturbation of this balance, for example, by CD36 signaling in states of hyperlipidemia and chronic inflammation, can push platelets into a hyperactive state and promote pathological thrombosis. The studies also have translational potential beyond platelet activation. CD36 is expressed on monocyte/macrophages, adipocytes, B- and T-cell subsets, some cancer stem cells, endothelial cells, and cardiac myocytes, where it mediates many important physiological and pathological functions, including fatty acid trafficking, atherogenesis, metabolic reprogramming, and immunometabolism.13 Many of these cells also express ITAM-containing adapter proteins, such as FcRγ, ZAP70, and/or the T-cell receptor ζ-chain. Whether CD36 signaling in these cells is regulated by ITAMs is another important and unanswered question.
Conflict-of-interest disclosure: R.L.S. is supported by the National Institutes of Health grant R01HL164460 and their family has an equity interest in Pfizer.
