Laura D. Healy

The BCR-ABL inhibitor ponatinib inhibits platelet immunoreceptor tyrosine-based activation motif (ITAM) signaling, platelet activation and aggregate formation under shear

BACKGROUND Treatment of chronic myelogenous leukemia (CML) with the BCR-ABL tyrosine kinase inhibitor (TKI) imatinib significantly improves patient outcomes. As some patients are unresponsive to imatinib, next generation BCR-ABL inhibitors such as nilotinib have been developed to treat patients with imatinib-resistant CML. The use of some BCR-ABL inhibitors has been associated with bleeding diathesis, and these inhibitors have been shown to inhibit platelet functions, which may explain the hemostasis impairment. Surprisingly, a new TKI, ponatinib, has been associated with a high incidence of severe acute ischemic cardiovascular events. The mechanism of this unexpected adverse effect remains undefined. OBJECTIVE AND METHODS This study used biochemical and functional assays to evaluate whether ponatinib was different from the other BCR-ABL inhibitors with respect to platelet activation, spreading, and aggregation. RESULTS AND CONCLUSIONS Our results show that ponatinib, similar to other TKIs, acts as a platelet antagonist. Ponatinib inhibited platelet activation, spreading, granule secretion, and aggregation, likely through broad spectrum inhibition of platelet tyrosine kinase signaling, and also inhibited platelet aggregate formation in whole blood under shear. As our results indicate that pobatinib inhibits platelet function, the adverse cardiovascular events observed in patients taking ponatinib may be the result of the effect of ponatinib on other organs or cell types, or disease-specific processes, such as BCR-ABL+cells undergoing apoptosis in response to chemotherapy, or drug-induced adverse effects on the integrity of the vascular endothelium in ponatinib-treated patients.

Histone deacetylase 6-mediated deacetylation of α-tubulin coordinates cytoskeletal and signaling events during platelet activation

The tubulin cytoskeleton plays a key role in maintaining the characteristic quiescent discoid shape of resting platelets. Upon activation, platelets undergo a dramatic change in shape; however, little is known of how the microtubule system contributes to regulating platelet shape and function. Here we investigated the role of the covalent modification of α-tubulin by acetylation in the regulation of platelet physiology during activation. Superresolution microscopy analysis of the platelet tubulin cytoskeleton showed that the marginal band together with an interconnected web of finer tubulin structures collapsed upon platelet activation with the glycoprotein VI (GPVI)-agonist collagen-related peptide (CRP). Western blot analysis revealed that α-tubulin was acetylated in resting platelets and deacetylated during platelet activation. Tubacin, a specific inhibitor of the tubulin deacetylase HDAC6, prevented tubulin deacetylation upon platelet activation with CRP. Inhibition of HDAC6 upregulated tubulin acetylation and disrupted the organization of the platelet microtubule marginal band without significantly affecting platelet volume changes in response to CRP stimulation. HDAC6 inhibitors also inhibited platelet aggregation in response to CRP and blocked platelet signaling events upstream of platelet Rho GTPase activation. Together, these findings support a role for acetylation signaling in controlling the resting structure of the platelet tubulin marginal band as well as in the coordination of signaling systems that drive platelet cytoskeletal changes and aggregation.

Oral administration of Bruton's tyrosine kinase inhibitors impairs GPVI-mediated platelet function

The Tec family kinase Brutons tyrosine kinase (Btk) plays an important signaling role downstream of immunoreceptor tyrosine-based activation motifs in hematopoietic cells. Mutations in Btk are involved in impaired B-cell maturation in X-linked agammaglobulinemia, and Btk has been investigated for its role in platelet activation via activation of the effector protein phospholipase Cγ2 downstream of the platelet membrane glycoprotein VI (GPVI). Because of its role in hematopoietic cell signaling, Btk has become a target in the treatment of chronic lymphocytic leukemia and mantle cell lymphoma; the covalent Btk inhibitor ibrutinib was recently approved by the US Food and Drug Administration for treatment of these conditions. Antihemostatic events have been reported in some patients taking ibrutinib, although the mechanism of these events remains unknown. We sought to determine the effects of Btk inhibition on platelet function in a series of in vitro studies of platelet activation, spreading, and aggregation. Our results show that irreversible inhibition of Btk with two ibrutinib analogs in vitro decreased human platelet activation, phosphorylation of Btk, P-selectin exposure, spreading on fibrinogen, and aggregation under shear flow conditions. Short-term studies of ibrutinib analogs administered in vivo also showed abrogation of platelet aggregation in vitro, but without measurable effects on plasma clotting times or on bleeding in vivo. Taken together, our results suggest that inhibition of Btk significantly decreased GPVI-mediated platelet activation, spreading, and aggregation in vitro; however, prolonged bleeding was not observed in a model of bleeding.

Activated protein C inhibits neutrophil extracellular trap formation in vitro and activation in vivo

Activated protein C (APC) is a multifunctional serine protease with anticoagulant, cytoprotective, and anti-inflammatory activities. In addition to the cytoprotective effects of APC on endothelial cells, podocytes, and neurons, APC cleaves and detoxifies extracellular histones, a major component of neutrophil extracellular traps (NETs). NETs promote pathogen clearance but also can lead to thrombosis; the pathways that negatively regulate NETosis are largely unknown. Thus, we studied whether APC is capable of directly inhibiting NETosis via receptor-mediated cell signaling mechanisms. Here, by quantifying extracellular DNA or myeloperoxidase, we demonstrate that APC binds human leukocytes and prevents activated platelet supernatant or phorbol 12-myristate 13-acetate (PMA) from inducing NETosis. Of note, APC proteolytic activity was required for inhibiting NETosis. Moreover, antibodies against the neutrophil receptors endothelial protein C receptor (EPCR), protease-activated receptor 3 (PAR3), and macrophage-1 antigen (Mac-1) blocked APC inhibition of NETosis. Select mutations in the Gla and protease domains of recombinant APC caused a loss of NETosis. Interestingly, pretreatment of neutrophils with APC prior to induction of NETosis inhibited platelet adhesion to NETs. Lastly, in a nonhuman primate model of Escherichia coli-induced sepsis, pretreatment of animals with APC abrogated release of myeloperoxidase from neutrophils, a marker of neutrophil activation. These findings suggest that the anti-inflammatory function of APC at therapeutic concentrations may include the inhibition of NETosis in an EPCR-, PAR3-, and Mac-1-dependent manner, providing additional mechanistic insight into the diverse functions of neutrophils and APC in disease states including sepsis.

Colocalization of neutrophils, extracellular DNA and coagulation factors during NETosis: Development and utility of an immunofluorescence-based microscopy platform.

BACKGROUND Neutrophils, the most populous innate immune cell type, are the first responders to sites of infection and inflammation. Neutrophils can release their DNA to form extracellular traps (NETs), webs of DNA and granular proteases that contribute to pathogen clearance and promote thrombus formation. At present, the study of NETs is in part limited to the qualitative analysis of fluorescence microscopy-based images, thus quantification of the interactions between NETs and coagulation factors remains ill-defined. AIM Develop a quantitative method to measure the spatial distribution of DNA and colocalization of coagulation factor binding to neutrophils and NETs utilizing fluorescence-based microscopy. APPROACH Human neutrophils were purified from peripheral blood, bound to fibronectin and treated with the PKC-activator phorbol myristate acetate (PMA) to induce neutrophil activation and NETs formation. Samples were incubated with purified coagulation factors or plasma before staining with a DNA-binding dye and coagulation factor-specific antibodies. The spatial distribution of DNA and coagulation factors was imaged via fluorescence microscopy and quantified via a custom-built MATLAB-based image analysis algorithm. The algorithm first established global thresholding parameters on a training set of fluorescence image data and then systematically quantified intensity profiles across treatment conditions. Quantitative comparison of treatment conditions was enabled through the normalization of fluorescent intensities using the number of cells per image to determine the percent and area of DNA and coagulation factor binding per cell. RESULTS Upon stimulation with PMA, NETs formation resulted in an increase in the area of DNA per cell. The coagulation factor fibrinogen bound to both the neutrophil cell body as well as NETs, while prothrombin, FX and FVIIa binding was restricted to the neutrophil cell body. The Gla domain of FX was required to mediate FX-neutrophil binding. Activated protein C (APC), but not Gla-less APC, bound to neutrophil cell bodies and NETs in a punctate manner. Neither FXIIa nor FXIa were found to bind either neutrophil cell bodies or NETs. Fibrinogen binding was dependent on extracellular DNA, while FX and APC required phosphatidylserine exposure for binding to activated neutrophils. CONCLUSIONS We have developed a quantitative measurement platform to define the spatial localization of fluorescently-labeled coagulation factor binding to neutrophils and extracellular DNA during NETosis.

Regulation of immune cell signaling by activated protein C

Innate immune cells are an essential part of the host defense response, promoting inflammation through release of proinflammatory cytokines or formation of neutrophil extracellular traps. While these processes are important for defense against infectious agents or injury, aberrant activation potentiates pathologic inflammatory disease. Thus, understanding regulatory mechanisms that limit neutrophil extracellular traps formation and cytokine release is of therapeutic interest for targeting pathologic diseases. Activated protein C is an endogenous serine protease with anticoagulant activity as well as anti‐inflammatory and cytoprotective functions, the latter of which are mediated through binding cell surface receptors and inducing intracellular signaling. In this review, we discuss certain leukocyte functions, namely neutrophil extracellular traps formation and cytokine release, and the inhibition of these processes by activated protein C.