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.

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.

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 α _IIb β ₃ outside-in signaling is also effected in addition to GPVI signaling. When ibrutinib was combined with the P2Y ₁₂ inhibitor, cangrelor, thrombus formation under arterial shear was inhibited additively. Conclusions—: These findings suggest that (1) ibrutinib causes GPVI and integrin α _IIb β ₃ platelet signaling deficiencies that result in formation of unstable thrombi and may contribute toward bleeding observed in vivo and (2) combining ibrutinib with P2Y ₁₂ antagonists, which also inhibit thrombus stability, may have a detrimental effect on hemostasis.

Quercetin, a dietary flavonoid, has been reported to possess antiplatelet activity. However, its extensive metabolism following ingestion has resulted in difficulty elucidating precise mechanisms of action. In this study, we aimed to characterize the antiplatelet mechanisms of two methylated metabolites of quercetin—isorhamnetin and tamarixetin—and explore potential interactions with aspirin. Isorhamnetin and tamarixetin inhibited human platelet aggregation, and suppressed activatory processes including granule secretion, integrin αIIbβ3 function, calcium mobilization, and spleen tyrosine kinase (Syk)/linker for activation of T cells (LAT) phosphorylation downstream of glycoprotein VI with similar potency to quercetin. All three flavonoids attenuated thrombus formation in an in vitro microfluidic model, and isoquercetin, a 3-O-glucoside of quercetin, inhibited thrombosis in a murine laser injury model. Isorhamnetin, tamarixetin, and quercetin enhanced the antiplatelet effects of aspirin more-than-additively in a plate-based aggregometry assay, reducing aspirin IC₅₀ values by an order of magnitude, with this synergy maintained in a whole blood test of platelet function. Our data provide mechanistic evidence for the antiplatelet activity of two quercetin metabolites, isorhamnetin and tamarixetin, and suggest a potential antithrombotic role for these flavonoids. In combination with their interactions with aspirin, this may represent a novel avenue of investigation for the development of new antithrombotic strategies and management of current therapies.

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.

Background and Purpose: 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.Experimental Approach: The ability of nobiletin to modulate platelet function was explored by using a range ofin vitroandin vivoexperimental approaches. Aggregation, dense granule secretion and spreading assays were performed using washed platelets. Fibrinogen binding, α‐granule secretion and calcium mobilization assays were performed using platelet‐rich plasma and whole blood was used in impedance aggregometry and thrombus formation experiments. The effect of nobiletinin vivowas assessed by measuring tail bleeding time usingC57BL/6 mice.Key Results: Nobiletin was shown to suppress a range of well‐established activatory mechanisms, including platelet aggregation, granule secretion, integrin modulation, calcium mobilization and thrombus formation. Nobiletin extended bleeding time in mice and reduced the phosphorylation ofPKB(Akt) andPLCγ2 within the collagen receptor (glycoproteinVI)‐stimulated pathway, in addition to increasing the levels of cGMPand phosphorylation of vasodilator‐stimulated phosphoprotein, a protein whose activity is associated with inhibitory cyclic nucleotide signalling.Conclusions and Implications: This study provides insight into the underlying molecular mechanisms through which nobiletin modulates haemostasis and thrombus formation. Therefore, nobiletin may represent a potential antithrombotic agent of dietary origins.