Amanda J. Unsworth

Platelet signaling: a complex interplay between inhibitory and activatory networks.

The role of platelets in hemostasis and thrombosis is dependent on a complex balance of activatory and inhibitory signaling pathways. Inhibitory signals released from the healthy vasculature suppress platelet activation in the absence of platelet receptor agonists. Activatory signals present at a site of injury initiate platelet activation and thrombus formation; subsequently, endogenous negative signaling regulators dampen activatory signals to control thrombus growth. Understanding the complex interplay between activatory and inhibitory signaling networks is an emerging challenge in the study of platelet biology, and necessitates a systematic approach to utilize experimental data effectively. In this review, we will explore the key points of platelet regulation and signaling that maintain platelets in a resting state, mediate activation to elicit thrombus formation, or provide negative feedback. Platelet signaling will be described in terms of key signaling molecules that are common to the pathways activated by platelet agonists and can be described as regulatory nodes for both positive and negative regulators.

Ibrutinib Inhibits Platelet Integrin αIIbβ3 Outside-In Signaling and Thrombus Stability But Not Adhesion to Collagen

Objective—Ibrutinib is an irreversible Bruton tyrosine kinase inhibitor approved for treatment of Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, and mantle cell lymphoma that increases the risk of bleeding among patients. Platelets from ibrutinib-treated patients exhibit deficiencies in collagen-evoked signaling in suspension; however, the significance of this observation and how it relates to bleeding risk is unclear, as platelets encounter immobile collagen in vivo. We sought to clarify the effects of ibrutinib on platelet function to better understand the mechanism underlying bleeding risk. Approach and Results—By comparing signaling in suspension and during adhesion to immobilized ligands, we found that the collagen signaling deficiency caused by ibrutinib is milder during adhesion to immobilized collagen. We also found that platelets in whole blood treated with ibrutinib adhered to collagen under arterial shear but formed unstable thrombi, suggesting that the collagen signaling deficiency caused by ibrutinib may not be the predominant cause of bleeding in vivo. However, clot retraction and signaling evoked by platelet adhesion to immobilized fibrinogen were also inhibited by ibrutinib, indicating that integrin &agr;IIb&bgr;3 outside-in signaling is also effected in addition to GPVI signaling. When ibrutinib was combined with the P2Y12 inhibitor, cangrelor, thrombus formation under arterial shear was inhibited additively. Conclusions—These findings suggest that (1) ibrutinib causes GPVI and integrin &agr;IIb&bgr;3 platelet signaling deficiencies that result in formation of unstable thrombi and may contribute toward bleeding observed in vivo and (2) combining ibrutinib with P2Y12 antagonists, which also inhibit thrombus stability, may have a detrimental effect on hemostasis.

Pharmacological actions of nobiletin in the modulation of platelet function

The discovery that flavonoids are capable of inhibiting platelet function has led to their investigation as potential antithrombotic agents. However, despite the range of studies on the antiplatelet properties of flavonoids, little is known about the mechanisms by which flavonoids inhibit platelet function. In this study, we aimed to explore the pharmacological effects of a polymethoxy flavonoid, nobiletin, in the modulation of platelet function.

Farnesoid X Receptor and Its Ligands Inhibit the Function of Platelets

Objective—Although initially seemingly paradoxical because of the lack of nucleus, platelets possess many transcription factors that regulate their function through DNA-independent mechanisms. These include the farnesoid X receptor (FXR), a member of the superfamily of ligand-activated transcription factors, that has been identified as a bile acid receptor. In this study, we show that FXR is present in human platelets and FXR ligands, GW4064 and 6&agr;-ethyl-chenodeoxycholic acid, modulate platelet activation nongenomically. Approach and Results—FXR ligands inhibited the activation of platelets in response to stimulation of collagen or thrombin receptors, resulting in diminished intracellular calcium mobilization, secretion, fibrinogen binding, and aggregation. Exposure to FXR ligands also reduced integrin &agr;IIb&bgr;3 outside-in signaling and thereby reduced the ability of platelets to spread and to stimulate clot retraction. FXR function in platelets was found to be associated with the modulation of cyclic guanosine monophosphate levels in platelets and associated downstream inhibitory signaling. Platelets from FXR-deficient mice were refractory to the actions of FXR agonists on platelet function and cyclic nucleotide signaling, firmly linking the nongenomic actions of these ligands to the FXR. Conclusions—This study provides support for the ability of FXR ligands to modulate platelet activation. The atheroprotective effects of GW4064, with its novel antiplatelet effects, indicate FXR as a potential target for the prevention of atherothrombotic disease.

Farnesoid X Receptor and Liver X Receptor Ligands Initiate Formation of Coated Platelets

Objectives— The liver X receptors (LXRs) and farnesoid X receptor (FXR) have been identified in human platelets. Ligands of these receptors have been shown to have nongenomic inhibitory effects on platelet activation by platelet agonists. This, however, seems contradictory with the platelet hyper-reactivity that is associated with several pathological conditions that are associated with increased circulating levels of molecules that are LXR and FXR ligands, such as hyperlipidemia, type 2 diabetes mellitus, and obesity. Approach and Results— We, therefore, investigated whether ligands for the LXR and FXR receptors were capable of priming platelets to the activated state without stimulation by platelet agonists. Treatment of platelets with ligands for LXR and FXR converted platelets to the procoagulant state, with increases in phosphatidylserine exposure, platelet swelling, reduced membrane integrity, depolarization of the mitochondrial membrane, and microparticle release observed. Additionally, platelets also displayed features associated with coated platelets such as P-selectin exposure, fibrinogen binding, fibrin generation that is supported by increased serine protease activity, and inhibition of integrin &agr;IIb&bgr;3. LXR and FXR ligand-induced formation of coated platelets was found to be dependent on both reactive oxygen species and intracellular calcium mobilization, and for FXR ligands, this process was found to be dependent on cyclophilin D. Conclusions— We conclude that treatment with LXR and FXR ligands initiates coated platelet formation, which is thought to support coagulation but results in desensitization to platelet stimuli through inhibition of &agr;IIb&bgr;3 consistent with their ability to inhibit platelet function and stable thrombus formation in vivo.

PPARγ agonists negatively regulate αIIbβ3 integrin outside-in signalling and platelet function through upregulation of protein kinase A activity

Essentials peroxisome proliferator‐activated receptor γ (PPARγ) agonists inhibit platelet function. PPARγ agonists negatively regulate outside‐in signaling via integrin αIIbβ3. PPARγ agonists disrupt the interaction of Gα13 with integrin β3. This is attributed to an upregulation of protein kinase A activity.

Severe platelet dysfunction in NHL patients receiving ibrutinib is absent in patients receiving acalabrutinib

The Bruton tyrosine kinase (Btk) inhibitor ibrutinib induces platelet dysfunction and causes increased risk of bleeding. Off-target inhibition of Tec is believed to contribute to platelet dysfunction and other side effects of ibrutinib. The second-generation Btk inhibitor acalabrutinib was developed with improved specificity for Btk over Tec. We investigated platelet function in patients with non-Hodgkin lymphoma (NHL) receiving ibrutinib or acalabrutinib by aggregometry and by measuring thrombus formation on collagen under arterial shear. Both patient groups had similarly dysfunctional aggregation responses to collagen and collagen-related peptide, and comparison with mechanistic experiments in which platelets from healthy donors were treated with the Btk inhibitors suggested that both drugs inhibit platelet Btk and Tec at physiological concentrations. Only ibrutinib caused dysfunctional thrombus formation, whereas size and morphology of thrombi following acalabrutinib treatment were of normal size and morphology. We found that ibrutinib but not acalabrutinib inhibited Src family kinases, which have a critical role in platelet adhesion to collagen that is likely to underpin unstable thrombus formation observed in ibrutinib patients. We found that platelet function was enhanced by increasing levels of von Willebrand factor (VWF) and factor VIII (FVIII) ex vivo by addition of intermediate purity FVIII (Haemate P) to blood from patients, resulting in consistently larger thrombi. We conclude that acalabrutinib avoids major platelet dysfunction associated with ibrutinib therapy, and platelet function may be enhanced in patients with B-cell NHL by increasing plasma VWF and FVIII.

RXR ligands negatively regulate thrombosis and hemostasis

Objective— Platelets have been found to express intracellular nuclear receptors including the retinoid X receptors (RXR&agr; and RXR&bgr;). Treatment of platelets with ligands of RXR has been shown to inhibit platelet responses to ADP and thromboxane A2; however, the effects on responses to other platelet agonists and the underlying mechanism have not been fully characterized. Approach and Results— The effect of 9-cis-retinoic acid, docosahexaenoic acid and methoprene acid on collagen receptor (glycoprotein VI [GPVI]) agonists and thrombin-stimulated platelet function; including aggregation, granule secretion, integrin activation, calcium mobilization, integrin &agr;IIb&bgr;3 outside-in signaling and thrombus formation in vitro and in vivo were determined. Treatment of platelets with RXR ligands resulted in attenuation of platelet functional responses after stimulation by GPVI agonists or thrombin and inhibition of integrin &agr;IIb&bgr;3 outside-in signaling. Treatment with 9-cis-retinoic acid caused inhibition of thrombus formation in vitro and an impairment of thrombosis and hemostasis in vivo. Both RXR ligands stimulated protein kinase A activation, measured by VASP S157 phosphorylation, that was found to be dependent on both cAMP and nuclear factor &kgr;-light-chain-enhancer of activated B cell activity. Conclusions— This study identifies a widespread, negative regulatory role for RXR in the regulation of platelet functional responses and thrombus formation and describes novel events that lead to the upregulation of protein kinase A, a known negative regulator of many aspects of platelet function. This mechanism may offer a possible explanation for the cardioprotective effects described in vivo after treatment with RXR ligands.

EphB2 regulates contact-dependent and contact-independent signaling to control platelet function.

The Eph kinases, EphA4 and EphB1, and their ligand, ephrinB1, have been previously reported to be present in platelets where they contribute to thrombus stability. Although thrombus formation allows for Eph-ephrin engagement and bidirectional signaling, the importance specifically of Eph kinase or ephrin signaling in regulating platelet function remained unidentified. In the present study, a genetic approach was used in mice to establish the contribution of signaling orchestrated by the cytoplasmic domain of EphB2 (a newly discovered Eph kinase in platelets) in platelet activation and thrombus formation. We conclude that EphB2 signaling is involved in the regulation of thrombus formation and clot retraction. Furthermore, the cytoplasmic tail of this Eph kinase regulates initial platelet activation in a contact-independent manner in the absence of Eph-ephrin ligation between platelets. Together, these data demonstrate that EphB2 signaling not only modulates platelet function within a thrombus but is also involved in the regulation of the function of isolated platelets in a contact-independent manner.

Non-genomic effects of nuclear receptors: insights from the anucleate platelet

Abstract Nuclear receptors (NRs) have the ability to elicit two different kinds of responses, genomic and non-genomic. Although genomic responses control gene expression by influencing the rate of transcription, non-genomic effects occur rapidly and independently of transcriptional regulation. Due to their anucleate nature and mechanistically well-characterized and rapid responses, platelets provide a model system for the study of any non-genomic effects of the NRs. Several NRs have been found to be present in human platelets, and multiple NR agonists have been shown to elicit anti-platelet effects by a variety of mechanisms. The non-genomic functions of NRs vary, including the regulation of kinase and phosphatase activity, ion channel function, intracellular calcium levels, and production of second messengers. Recently, the characterization of mechanisms and identification of novel binding partners of NRs have further strengthened the prospects of developing their ligands into potential therapeutics that offer cardio-protective properties in addition to their other defined genomic effects.