Multicolor flow cytometry in clinical samples for platelet signaling assessment.

Background: Availability of multichannel cytometers and specific commercial antibodies makes flow cytometry a new option to simultaneously assess multiple intracellular platelet signaling pathways for clinical purposes, in small volume of blood or low platelet count. Objectives: To describe a multicolor flow cytometry with fluorescent barcoding technique for screening signaling pathways downstream membrane receptors of major platelet agonists (adenosine diphosphate, thrombin, thromboxane, and collagen). Methods: By comparison with immunoblotting, we first selected the target phosphoproteins, AKT, P38MAPK, LIMK, and SPL76; the times of stimulation; and phosphoflow barcoding conditions. We then performed a clinical study on whole blood of patients without evidence of blood platelet disorder on standard biological screening, consulting for trivial or occasionally provoked bleeds without familial antecedent (bleeding of unknown origin, n = 23) or type-1 von Willebrand disease (n = 9). In addition, we included a small group of patients with definite platelet disorders (Glanzmann thrombasthenia, δ-storage pool deficiency, and immune glycoprotein VI-related disease with granule secretion defect). Results: The range, kinetics, and distribution of fluorescence intensity were established for each agonist-target protein combination. Principal component analysis indicates a correlation in response to a target phosphoprotein (AKT and P38MAPK) to different agonists but no correlation in the response of different target phosphoproteins to the same agonist. The heterogeneity of individual responses in the whole population displayed was analyzed using clustering algorithm. Patients with platelet storage pool deficiency were positioned as lowest responders on the heatmap. Conclusion: In complement of functional tests, this study introduces a new approach for rapid platelet signaling profiling in clinical practice.

Evolution of platelet activation parameters during septic shock in intensive care unit.

During severe sepsis, platelet activation may induce disseminate microvascular thrombosis, which play a key role in critical organ failure. Crucially, most of the studies in this field have explored platelet-leukocyte interactions in animal models, or explored platelets under the spectrum of thrombocytopenia or disseminated intravascular coagulation and have not taken into account the complex interplay that might exist between platelets and leukocytes during human septic shock nor the kinetics of platelet activation. Here, we assessed platelet activation parameters at the admission of patients with sepsis to the intensive care unit (ICU) and 48 hours later. Twenty-two patients were enrolled in the study, thirteen (59.1%) of whom were thrombocytopenic. The control group was composed of twelve infection-free patients admitted during the study period. The activation parameters studied included platelet-leukocyte interactions, assessed by flow cytometry in whole blood, as well as membrane surface and soluble platelet activation markers measured by flow cytometry and dedicated ELISA kits. We also investigated platelet aggregation and secretion responses of patients with sepsis following stimulation, compared to controls. At admission, the level of circulating monocyte-platelet and neutrophil-platelet heterotypic aggregates was significantly higher in sepsis patients compared to controls and returned to a level comparable to controls or even below 48 hours later. Basal levels of CD62P and CD63 platelet membrane exposure at admission and 48 hours later were low and similar to controls. In contrast, plasma level of soluble GPVI and soluble CD40 ligand was significantly increased in septic patients, at the two times of analysis, reflecting previous platelet activation. Platelet aggregation and secretion responses induced by specific agonists were significantly decreased in septic conditions, particularly 48 hours after admission. Hence, we have observed for the first time that critically ill septic patients compared to controls have both an early and durable platelet activation while their circulating platelets are less responsive to different agonists.

Cholesterol-Rich Microdomains Contribute to PAR1 Signaling in Platelets Despite a Weak Localization of the Receptor in These Microdomains.

Platelet protease-activated receptor 1 (PAR1) is a cell surface G-protein-coupled receptor (GPCR) that acts as a thrombin receptor promoting platelet aggregation. Targeting the PAR1 pathway by vorapaxar, a PAR1 antagonist, leads to a reduction in ischemic events in cardiovascular patients with a history of myocardial infarction or with peripheral arterial disease. In platelets, specialized microdomains highly enriched in cholesterol act as modulators of the activity of several GPCRs and play a pivotal role in the signaling pathway. However, their involvement in platelet PAR1 function remains incompletely characterized. In this context, we aimed to investigate whether activation of PAR1 in human platelets requires its localization in the membrane cholesterol-rich microdomains. Using confocal microscopy, biochemical isolation, and proteomics approaches, we found that PAR1 was not localized in cholesterol-rich microdomains in resting platelets, and only a small fraction of the receptor relocated to the microdomains following its activation. Vorapaxar treatment increased the level of PAR1 at the platelet surface, possibly by reducing its endocytosis, while its colocalization with cholesterol-rich microdomains remained weak. Consistent with a cholesterol-dependent activation of Akt and p38 MAP kinase in thrombin receptor-activating peptide (TRAP)-activated platelets, the proteomic data of cholesterol-rich microdomains isolated from TRAP-activated platelets showed the recruitment of proteins contributing to these signaling pathways. In conclusion, contrary to endothelial cells, we found that PAR1 was only weakly present in cholesterol-rich microdomains in human platelets but used these microdomains for efficient activation of downstream signaling pathways following TRAP activation.

Effects of novel Btk and Syk inhibitors on platelet functions alone and in combination in vitro and in vivo.

BACKGROUND: Inhibitors of tyrosine kinases downstream of the B-cell receptor, such as Bruton's tyrosine kinase (Btk) or Spleen tyrosine kinase (Syk), used alone or in combination are new therapeutic options in the treatment of B-cell malignancies. A challenge in the development of second-generation Btk inhibitors is to limit their side effects such as the increased bleeding risk. Considering the pivotal role of Syk in immunoreceptor tyrosine-based activation motif mediated platelet signaling, the impact of inhibiting this kinase on platelet functions is also worth analyzing. OBJECTIVES: We investigated the effect of a novel Btk inhibitor, tirabrutinib, and a Syk inhibitor, entospletinib, alone and in combination on platelet signaling and functions in vitro and ex vivo. METHODS: Platelet aggregation, secretion, and signaling responses as well as thrombus growth under flow were analyzed in the presence of the inhibitors alone or in combination in vitro, at clinically relevant doses, and ex vivo in patients treated with these inhibitors in the context of a phase I trial. RESULTS: Although tirabrutinib alone had modest effects on platelet activation in vitro and ex vivo, entospletinib alone efficiently inhibited washed platelet aggregation in response to collagen. However, entospletinib weakly affected platelet activation in platelet-rich plasma, in whole blood and ex vivo. Importantly, the combination of tirabrutinib and entospletinib induced a significant decrease in platelet response to collagen in vitro and ex vivo correlating with mild bleedings reported in some of the treated patients. CONCLUSION: These new results should contribute to improve the safety of these targeted therapies.

Platelet Activation Is Limited during Transcatheter Aortic Valve Implantation in Patients on Aspirin Monotherapy and without per Procedural Clinical Complications.

Transcatheter aortic valve implantation (TAVI) is an established treatment option for symptomatic patients with severe aortic valve stenosis (AS). During and early after the procedure, both ischemic events (predominantly stroke) and bleedings remain prevalent. The optimal antithrombotic regimen is still debated. Single- versus dual-antiplatelet therapy is associated with a lower rate of severe bleeding, without difference in thrombotic complications. Although platelets have been empirically targeted, little is known on their contribution to these events primarily related to embolization of thrombotic material and tissue-derived debris from the wounded aortic valve and large vessels. The objective of this study was to assess local platelet activation in blood sampled in the ascending aorta immediately before and within minutes postimplantation. A series of 18 patients with AS on monotherapy with aspirin successfully underwent TAVI with the self-expandable Medtronic CoreValve by transfemoral route. No clinical thrombotic complication occurred at 30-day follow-up. Compared with patients with stable coronary artery disease unscathed of AS and similarly treated by low-dose aspirin, AS patients displayed a chronic state of platelet activation before TAVI, assessed in venous blood using various biomarkers. However, per procedure, in aortic blood, no change occurred between the two time points in the plasma levels of serotonin or 12-lipoxgenase products, or membrane exposure of granule markers CD62-P and CD63. Our results suggest that local acute platelet activation is limited during TAVI on monotherapy with aspirin.

Differences and similarities in the effects of ibrutinib and acalabrutinib on platelet functions.

While efficient at treating B-cell malignancies, Bruton tyrosine kinase (BTK) inhibitors are consistently reported to increase the risk of bleeding. Analyzing platelet aggregation response to collagen in platelet-rich plasma allowed us to identify two groups in the healthy population characterized by low or high sensitivity to ibrutinib in vitro Inhibition of drug efflux pumps induced a shift from ibrutinib low-sensitive platelets to high-sensitive ones. At a clinically relevant dose, acalabrutinib, a second-generation BTK inhibitor, did not affect maximal collagen-induced platelet aggregation in the ibrutinib low-sensitive group but did inhibit aggregation in a small fraction of the ibrutinib high-sensitive group. Consistently, acalabrutinib delayed aggregation, particularly in the ibrutinib high-sensitive group. In chronic lymphocytic leukemia patients, acalabrutinib inhibited maximal platelet aggregation only in the ibrutinib high-sensitive group. Acalabrutinib inhibited collagen-induced tyrosine-753 phosphorylation of phospholipase Cγ2 in both groups, but, in contrast to ibrutinib, did not affect Src-family kinases. Acalabrutinib affected thrombus growth under flow only in the ibrutinib high-sensitive group and potentiated the effect of cyclooxygenase and P2Y12 receptor blockers in both groups. Since the better profile of acalabrutinib was observed mainly in the ibrutinib low-sensitive group, replacement therapy in patients may not systematically reduce the risk of bleeding.

Platelet activation and prothrombotic properties in a mouse model of peritoneal sepsis.

Sepsis is associated with thrombocytopenia and microvascular thrombosis. Studies have described platelets implication in this pathology but their kinetics of activation and behavior remain poorly known. We show in a mouse model of peritonitis, the appearance of platelet-rich thrombi in organ microvessels and organ damage. Complementary methods are necessary to characterize platelet activation during sepsis as circulating soluble markers and platelet-monocyte aggregates revealed early platelet activation, while surface activation markers were detected at later stage. A microfluidic based ex-vivo thrombosis assay demonstrated that platelets from septic mice have a prothrombotic behavior at shear rate encountered in microvessels. Interestingly, we found that even though phosphoinositide-3-kinase ß-deficient platelet mice formed less thrombi in liver microcirculation, peritoneal sepsis activates a platelet alternative pathway to compensate the otherwise mandatory role of this lipid-kinase to form stable thrombi at high shear rate. Platelets are rapidly activated during sepsis. Thrombocytopenia can be attributed in part to platelet-rich thrombi formation in capillaries and platelet-leukocytes interactions. Platelets from septic mice have a prothrombotic phenotype at a shear rate encountered in arterioles. Further studies are necessary to unravel molecular mechanisms leading to this prothrombotic state of platelets in order to guide the development of future treatments of polymicrobial sepsis.

Non-autonomous role of Cdc42 in cell-cell communication during collective migration.

Collective cell migration is involved in numerous processes both physiological, such as embryonic development, and pathological such as metastasis. Compared to single cell migration, collective motion requires cell behaviour coordination through an as-yet poorly understood but critical cell-cell communication mechanism. Using Drosophila border cell migration, we show here that the small Rho GTPase Cdc42 regulates cell-cell communication. Indeed, we demonstrate that Cdc42 controls protrusion formation in a cell non-autonomous manner. Moreover, we found that the endocytic small GTPase Rab11, controls Cdc42 localisation to the periphery of migrating border cell clusters. Accordingly, over-expression of Cdc42 in border cells rescues the loss of Rab11 function. In addition, we showed that Cdc42 acts upstream of Moesin, a cytoskeletal regulator known to function downstream of rab11. Thus, our study positions Cdc42 as a new key player in cell-cell communication, acting downstream of Rab11.