Alexander P. Bye

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.

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.

The chaperone protein HSP47: a platelet collagen binding protein that contributes to thrombosis and haemostasis

Essentials Heat shock protein 47 (HSP47), a collagen specific chaperone is present on the platelet surface. Collagen mediated platelet function was reduced following blockade or deletion of HSP47. GPVI receptor regulated signalling was reduced in HSP47 deficient platelets. Platelet HSP47 tethers to exposed collagen thus modulating thrombosis and hemostasis.

Cobimetinib and trametinib inhibit platelet MEK but do not cause platelet dysfunction

Abstract The MEK inhibitors cobimetinib and trametinib are used in combination with BRAF inhibitors to treat metastatic melanoma but increase rates of hemorrhage relative to BRAF inhibitors alone. Platelets express several members of the MAPK signalling cascade including MEK1 and MEK2 and ERK1 and ERK2 but their role in platelet function and haemostasis is ambiguous as previous reports have been contradictory. It is therefore unclear if MEK inhibitors might be causing platelet dysfunction and contributing to increased hemorrhage. In the present study we performed pharmacological characterisation of cobimetinib and trametinib in vitro to investigate potential for MEK inhibitors to cause platelet dysfunction. We report that whilst both cobimetinib and trametinib are potent inhibitors of platelet MEK activity, treatment with trametinib did not alter platelet function. Treatment with cobimetinib results in inhibition of platelet aggregation, integrin activation, alpha-granule secretion and adhesion but only at suprapharmacological concentrations. We identified that the inhibitory effects of high concentrations of cobimetinib are associated with off-target inhibition on Akt and PKC. Neither inhibitor caused any alteration in thrombus formation on collagen under flow conditions in vitro. Our findings demonstrate that platelets are able to function normally when MEK activity is fully inhibited, indicating MEK activity is dispensable for normal platelet function. We conclude that the MEK inhibitors cobimetinib and trametinib do not induce platelet dysfunction and are therefore unlikely to contribute to increased incidence of bleeding reported during MEK inhibitor therapy.

Move along, nothing to see here: Btk inhibitors stop platelets sticking to plaques

Platelets have evolved an intricate array of overlapping mechanisms to respond effectively to vessel damage by forming a clot to minimize blood loss. Unfortunately, these same responses may also be triggered by atherosclerotic lesions in diseased arteries. Vessels can become occluded when platelets form a thrombus at the site of a ruptured plaque, causing myocardial infarction (MI) or ischemic stroke, depending on where the thrombus forms or where a resulting embolus becomes lodged. Current therapy following MI or ischemic stroke involves treatment with antiplatelet drugs to reduce the risk of recurrent events by inhibiting one or more of the mechanisms that drive the formation of a platelet thrombus. However, the overlap between the mechanisms that drive pathological thrombus formation and those that facilitate hemostasis means that patients receiving antiplatelet medication are at higher risk of hemorrhage, including intracranial hemorrhage and gastrointestinal bleeding. The benefits of current antiplatelet therapy outweigh the risks for patients, but there is still a need for safer antiplatelet therapies that do not compromise hemostasis to better serve this patient population, which is large and growing globally. In order to develop new antiplatelet drugs, researchers must consider the similarities and differences between the pathological process of thrombosis and the physiological process of hemostasis. Platelets skim over the surfaces of vascular endothelial cells, scanning for collagen and other exposed subendothelial matrix proteins that serve as environmental cues indicating that vessel damage has occurred. The initial interaction with exposed collagen occurs via the plasma protein von Willebrand factor, which binds to collagen and undergoes conformational change at high shear rates (present in arteries), enabling the platelet protein glycoprotein (GP) Iba to bind to it. Collagen is also recognized directly by the platelet GPVI receptor and integrin a2b1, which, together, mediate adhesion to collagen and trigger a cascade of intracellular signaling events that ultimately help the aggregate to grow and become stable. Atherosclerotic plaques are rich in collagen and provide a strong environmental cue to platelets to adhere and form an aggregate within the lumen of the vessel. The intracellular signaling processes stimulated in both scenarios are similar, and result in the platelets synthesizing thromboxane A2 and secreting ADP, which, via paracrine and autocrine mechanisms, act as secondary mediators to recruit platelets to the growing aggregate and stabilize it. The antiplatelet drugs aspirin and P2Y12 inhibitors such as clopidogrel or ticagrelor target these positive feedback mechanisms to limit thrombus growth and prevent vessel occlusion. The success of these drugs in providing relatively safe platelet inhibition can be understood in the context of the ‘core’ and ‘shell’ model of thrombus formation, whereby a core of platelets is strongly activated by direct contact with collagen, and is surrounded by an outer shell of weakly activated platelets stimulated by secondary mediators. In this model, the core is required for hemostasis, whereas the shell is partially redundant but serves a pathological role in thrombosis by causing vessel occlusion. However, both aspirin and P2Y12 inhibitors are associated with increased rates of hemorrhage, suggesting that either these drugs are not sufficiently specific in targeting the shell, or the shell is also important for hemostasis. Although the environment of an atherosclerotic plaque and that of a damaged blood vessel have similarities, some in vitro studies have identified differences that could be exploited pharmacologically to provide platelet inhibition specifically in the context of atherothrombosis while sparing normal hemostasis. One such difference concerns the morphologically distinct forms of type I and type III collagen present in atherosclerotic plaques, which trigger Correspondence: Alexander Paul Bye, Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, Harborne Building, University of Reading, Whiteknights, Reading RG6 6AS, UK Tel.: +44 (0)118 378 4562 E-mail: a.bye@reading.ac.uk