Early Metabolic Disruption and Predictive Biomarkers of Delayed-Cerebral Ischemia in Aneurysmal Subarachnoid Hemorrhage.

Delayed cerebral ischemia (DCI) following aneurysmal subarachnoid hemorrhage (aSAH) is a major cause of complications and death. Here, we set out to identify high-performance predictive biomarkers of DCI and its underlying metabolic disruptions using metabolomics and lipidomics approaches. This single-center prospective observational study enrolled 61 consecutive patients with severe aSAH; among them, 22 experienced a DCI. Nine patients without aSAH were included as validation controls. Blood and cerebrospinal fluid (CSF) were sampled within the first 24 h after admission. We identified a panel of 20 metabolites that, together, showed high predictive performance for DCI. This panel of metabolites included lactate, cotinine, salicylate, 6 phosphatidylcholines, and 4 sphingomyelins. The interplay of the metabolome and the lipidome found between CSF and plasma in our patients underscores that aSAH and its associated DCI complications can extend beyond cerebral implications, with a peripheral dimension as well. As an illustration, early biological disruptions that might explain the subsequent DCI found systemic hypoxia driven mainly by higher blood lactate, arginine, and proline metabolism likely associated with vascular NO and disrupted ceramide/sphingolipid metabolism. We conclude that targeting early peripheral hypoxia preceding DCI could provide an interesting strategy for the prevention of vascular dysfunction.

Granulocyte microvesicles with a high plasmin generation capacity promote clot lysis and improve outcome in septic shock.

Microvesicles (MVs) have previously been shown to exert profibrinolytic capacity, which is increased in patients with septic shock (SS) with a favorable outcome. We, therefore, hypothesized that the plasmin generation capacity (PGC) could confer to MVs a protective effect supported by their capacity to lyse a thrombus, and we investigated the mechanisms involved. Using an MV-PGC kinetic assay, ELISA, and flow cytometry, we found that granulocyte MVs (Gran-MVs) from SS patients display a heterogeneous PGC profile driven by the uPA (urokinase)/uPAR system. In vitro, these MVs lyse a thrombus according to their MV-PGC levels in a uPA/uPAR-dependent manner, as shown in a fluorescent clot lysis test and a lysis front retraction assay. Fibrinolytic activators conveyed by MVs contribute to approximately 30% of the plasma plasminogenolytic capacity of SS patients. In a murine model of SS, the injection of high PGC Gran-MVs significantly improved mouse survival and reduced the number of thrombi in vital organs. This was associated with a modification of the mouse coagulation and fibrinolysis properties toward a more fibrinolytic profile. Interestingly, mouse survival was not improved when soluble uPA was injected. Finally, using a multiplex array on plasma from SS patients, we found that neutrophil elastase correlates with the effect of high-PGC-capacity plasma and modulates the Gran-MV plasmin generation capacity by cleaving uPA-PAI-1 complexes. In conclusion, we show that the high PGC level displayed by Gran-MVs reduces thrombus formation and improves survival, conferring to Gran-MVs a protective role in a murine model of sepsis.

A rare coding mutation in the MAST2 gene causes venous thrombosis in a French family with unexplained thrombophilia: The Breizh MAST2 Arg89Gln variant.

Rare variants outside the classical coagulation cascade might cause inherited thrombosis. We aimed to identify the variant(s) causing venous thromboembolism (VTE) in a family with multiple relatives affected with unprovoked VTE and no thrombophilia defects. We identified by whole exome sequencing an extremely rare Arg to Gln variant (Arg89Gln) in the Microtubule Associated Serine/Threonine Kinase 2 (MAST2) gene that segregates with VTE in the family. Free-tissue factor pathway inhibitor (f-TFPI) plasma levels were significantly decreased in affected family members compared to healthy relatives. Conversely, plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in affected members than in healthy relatives. RNA sequencing analysis of RNA interference experimental data conducted in endothelial cells revealed that, of the 13,387 detected expressed genes, 2,354 have their level of expression modified by MAST2 knockdown, including SERPINE1 coding for PAI-1 and TFPI. In HEK293 cells overexpressing the MAST2 Gln89 variant, TFPI and SERPINE1 promoter activities were respectively lower and higher than in cells overexpressing the MAST2 wild type. This study identifies a novel thrombophilia-causing Arg89Gln variant in the MAST2 gene that is here proposed as a new molecular player in the etiology of VTE by interfering with hemostatic balance of endothelial cells.

Impaired adhesion of neutrophils expressing Slc44a2/HNA-3b to VWF protects against NETosis under venous shear rates.

Genome-wide association studies linked expression of the human neutrophil antigen 3b (HNA-3b) epitope on the Slc44a2 protein with a 30% decreased risk of venous thrombosis (VT) in humans. Slc44a2 is a ubiquitous transmembrane protein identified as a receptor for von Willebrand factor (VWF). To explain the link between Slc44a2 and VT, we wanted to determine how Slc44a2 expressing either HNA-3a or HNA-3b on neutrophils could modulate their adhesion and activation on VWF under flow. Transfected HEK293T cells or neutrophils homozygous for the HNA-3a- or HNA-3b-coding allele were purified from healthy donors and perfused in flow chambers coated with VWF at venous shear rates (100 s-1). HNA-3a expression was required for Slc44a2-mediated neutrophil adhesion to VWF at 100 s-1. This adhesion could occur independently of ß2 integrin and was enhanced when neutrophils were preactivated with lipopolysaccharide. Moreover, specific shear conditions with high neutrophil concentration could act as a "second hit," inducing the formation of neutrophil extracellular traps. Neutrophil mobilization was also measured by intravital microscopy in venules from SLC44A2-knockout and wild-type mice after histamine-induced endothelial degranulation. Mice lacking Slc44a2 showed a massive reduction in neutrophil recruitment in inflamed mesenteric venules. Our results show that Slc44a2/HNA-3a is important for the adhesion and activation of neutrophils in veins under inflammation and when submitted to specific shears. The fact that neutrophils expressing Slc44a2/HNA-3b have a different response on VWF in the conditions tested could thus explain the association between HNA-3b and a reduced risk for VT in humans.

APOLD1 loss causes endothelial dysfunction involving cell junctions, cytoskeletal architecture, and Weibel-Palade bodies, while disrupting hemostasis.

Vascular homeostasis is impaired in various diseases thereby contributing to the progression of their underlying pathologies. The endothelial immediate early gene Apolipoprotein L domain-containing 1 (APOLD1) helps to regulate endothelial function. However, its precise role in endothelial cell biology remains unclear. We have localized APOLD1 to endothelial cell contacts and to Weibel-Palade bodies (WPB) where it associates with von Willebrand factor (VWF) tubules. Silencing of APOLD1 in primary human endothelial cells disrupted the cell junction-cytoskeletal interface, thereby altering endothelial permeability accompanied by spontaneous release of WPB contents. This resulted in an increased presence of WPB cargoes, notably VWF and angiopoietin-2 in the extracellular medium. Autophagy flux, previously recognized as an essential mechanism for the regulated release of WPB, was impaired in the absence of APOLD1. In addition, we report APOLD1 as a candidate gene for a novel inherited bleeding disorder across three generations of a large family in which an atypical bleeding diathesis was associated with episodic impaired microcirculation. A dominant heterozygous nonsense APOLD1:p.R49* variant segregated to affected family members. Compromised vascular integrity resulting from an excess of plasma angiopoietin-2, and locally impaired availability of VWF may explain the unusual clinical profile of APOLD1:p.R49* patients. In summary, our findings identify APOLD1 as an important regulator of vascular homeostasis and raise the need to consider testing of endothelial cell function in patients with inherited bleeding disorders without apparent platelet or coagulation defects.

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.

Screening platelet function in blood donors.

BACKGROUND: Transfusion of defective platelets could contribute to the inefficiency of platelet transfusion in preventing or stopping bleeding. STUDY DESIGN AND METHODS: This single-center prospective study aimed to determine the prevalence of functional platelet abnormalities in a population of blood donors with a clinical history of bleeding diathesis or with history of hematoma (>4 cm) during blood donation. Donors with positive bleeding screening questionnaire were referred to the reference center for rare platelet diseases at La Timone University Hospital (Marseille) to confirm the bleeding tendency using a more extensive bleeding questionnaire (MCMDMscore) and to assess hemostasis, including a comprehensive platelet analysis. RESULTS: One hundred and ninety-five donors identified based on a history of hematoma and 2434 blood donors were included in the study. Eighty-eight donors (3.6%) had a bleeding score indicating a potential bleeding disorder. Five donors with a history of hematoma (2.5%) and 15 (17%) donors with a confirmed bleeding score underwent hemostatic analysis, including two men and 18 women with average age of 33.9 years. Minor hemostatic abnormalities were observed in three donors. Two donors exhibited accelerated fibrinolysis with reduced euglobulin lysis time and increased D-dimer levels in serum. Two donors had a platelet granule defect, without identification of genetic abnormality. CONCLUSION: The bleeding questionnaire proved to be a valuable tool to screen blood donors for potential platelet defects. Platelet dysfunction was rare in the blood donor population assessed. Additional studies are necessary to understand the clinical impact that the transfusion of platelets with qualitative defects has on recipients.

Perioperative Open-lung Approach, Regional Ventilation, and Lung Injury in Cardiac Surgery.

BACKGROUND: In the Protective Ventilation in Cardiac Surgery (PROVECS) randomized, controlled trial, an open-lung ventilation strategy did not improve postoperative respiratory outcomes after on-pump cardiac surgery. In this prespecified subanalysis, the authors aimed to assess the regional distribution of ventilation and plasma biomarkers of lung epithelial and endothelial injury produced by that strategy. METHODS: Perioperative open-lung ventilation consisted of recruitment maneuvers, positive end-expiratory pressure (PEEP) = 8 cm H2O, and low-tidal volume ventilation including during cardiopulmonary bypass. Control ventilation strategy was a low-PEEP (2 cm H2O) low-tidal volume approach. Electrical impedance tomography was used serially throughout the perioperative period (n = 56) to compute the dorsal fraction of ventilation (defined as the ratio of dorsal tidal impedance variation to global tidal impedance variation). Lung injury was assessed serially using biomarkers of epithelial (soluble form of the receptor for advanced glycation end-products, sRAGE) and endothelial (angiopoietin-2) lung injury (n = 30). RESULTS: Eighty-six patients (age = 64 ± 12 yr; EuroSCORE II = 1.65 ± 1.57%) undergoing elective on-pump cardiac surgery were studied. Induction of general anesthesia was associated with ventral redistribution of tidal volumes and higher dorsal fraction of ventilation in the open-lung than the control strategy (0.38 ± 0.07 vs. 0.30 ± 0.10; P = 0.004). No effect of the open-lung strategy on the dorsal fraction of ventilation was noted at the end of surgery after median sternotomy closure (open-lung = 0.37 ± 0.09 vs. control = 0.34 ± 0.11; P = 0.743) or in extubated patients at postoperative day 2 (open-lung = 0.63 ± 0.18 vs. control = 0.59 ± 0.11; P > 0.999). Open-lung ventilation was associated with increased intraoperative plasma sRAGE (7,677 ± 3,097 pg/ml vs. 6,125 ± 1,400 pg/ml; P = 0.037) and had no effect on angiopoietin-2 (P > 0.999). CONCLUSIONS: In cardiac surgery patients, open-lung ventilation provided larger dorsal lung ventilation early during surgery without a maintained benefit as compared with controls at the end of surgery and postoperative day 2 and was associated with higher intraoperative plasma concentration of sRAGE suggesting lung overdistension.

RasGRP2 Structure, Function and Genetic Variants in Platelet Pathophysiology.

RasGRP2 is calcium and diacylglycerol-regulated guanine nucleotide exchange factor I that activates Rap1, which is an essential signaling-knot in "inside-out" αIIbß3 integrin activation in platelets. Inherited platelet function disorder caused by variants of RASGRP2 represents a new congenital bleeding disorder referred to as platelet-type bleeding disorder-18 (BDPLT18). We review here the structure of RasGRP2 and its functions in the pathophysiology of platelets and of the other cellular types that express it. We will also examine the different pathogenic variants reported so far as well as strategies for the diagnosis and management of patients with BDPLT18.

Novel ACTN1 variants in cases of thrombocytopenia.

The ACTN1 gene has been implicated in inherited macrothrombocytopenia. To decipher the spectrum of variants and phenotype of ACTN1-related thrombocytopenia, we sequenced the ACTN1 gene in 272 cases of unexplained chronic or familial thrombocytopenia. We identified 15 rare, monoallelic, nonsynonymous and likely pathogenic ACTN1 variants in 20 index cases from 20 unrelated families. Thirty-one family members exhibited thrombocytopenia. Targeted sequencing was carried out on 12 affected relatives, which confirmed presence of the variant. Twenty-eight of 32 cases with monoallelic ACTN1 variants had mild to no bleeding complications. Eleven cases harbored 11 different unreported ACTN1 variants that were monoallelic and likely pathogenic. Nine variants were located in the α-actinin-1 (ACTN1) rod domain and were predicted to hinder dimer formation. These variants displayed a smaller increase in platelet size compared with variants located outside the rod domain. In vitro expression of the new ACTN1 variants induced actin network disorganization and led to increased thickness of actin fibers. These findings expand the repertoire of ACTN1 variants associated with thrombocytopenia and highlight the high frequency of ACTN1-related thrombocytopenia cases. The rod domain, like other ACTN1 functional domains, may be mutated resulting in actin disorganization in vitro and thrombocytopenia with normal platelet size in most cases.

Expanded repertoire of RASGRP2 variants responsible for platelet dysfunction and severe bleeding.

Heritable platelet function disorders (PFDs) are genetically heterogeneous and poorly characterized. Pathogenic variants in RASGRP2, which encodes calcium and diacylglycerol-regulated guanine exchange factor I (CalDAG-GEFI), have been reported previously in 3 pedigrees with bleeding and reduced platelet aggregation responses. To better define the phenotype associated with pathogenic RASGRP2 variants, we compared high-throughput sequencing and phenotype data from 2042 cases in pedigrees with unexplained bleeding or platelet disorders to data from 5422 controls. Eleven cases harbored 11 different, previously unreported RASGRP2 variants that were biallelic and likely pathogenic. The variants included 5 high-impact variants predicted to prevent CalDAG-GEFI expression and 6 missense variants affecting the CalDAG-GEFI CDC25 domain, which mediates Rap1 activation during platelet inside-out αIIbß3 signaling. Cases with biallelic RASGRP2 variants had abnormal mucocutaneous, surgical, and dental bleeding from childhood, requiring ≥1 blood or platelet transfusion in 78% of cases. Platelets displayed reduced aggregation in response to adenosine 5'-diphosphate and epinephrine, but variable aggregation defects with other agonists. There were no other consistent clinical or laboratory features. These data enable definition of human CalDAG-GEFI deficiency as a nonsyndromic, recessive PFD associated with a moderate or severe bleeding phenotype and complex defects in platelet aggregation.

Thromboxane-prostaglandin receptor antagonist, terutroban, prevents neurovascular events after subarachnoid haemorrhage: a nanoSPECT study in rats.

BACKGROUND: Several lipid metabolites in cerebrospinal fluid are correlated with poor outcomes in aneurysmal subarachnoid haemorrhage. Most of these metabolites bind to ubiquitous thromboxane-prostaglandin (TP) receptors, causing vasoconstriction and inflammation. Here, we evaluated terutroban (TBN), a specific TP receptor antagonist, for the prevention of post-haemorrhage blood-brain barrier disruption, neuronal apoptosis and delayed cerebral hypoperfusion. METHODS: The rat double subarachnoid haemorrhage model was produced by twice injecting (days 1 and 2) autologous blood into the cisterna magna. Seventy-eight male Sprague-Dawley rats were assigned to experimental groups. Rats exposed to subarachnoid haemorrhage were allocated to no treatment (SAH group) or TBN treatment by gastric gavage during the first 5 days after haemorrhage (SAH+TBN group). Control rats received artificial cerebrospinal fluid injections (CSF group). Sham-operated rats with or without TBN administration were also studied. Body weight and Garcia neurological scores were assessed on day 2 and day 5. We used nanoscale single-photon emission computed tomography (nanoSPECT) to measure brain uptake of three radiolabelled agents: 99mTechnetium-diethylenetriaminepentacetate (99mTc-DTPA), which indicated blood-brain barrier permeability on day 3, 99mTechnetium-annexin V-128 (99mTc-Anx-V128), which indicated apoptosis on day 4, and 99mTechnetium-hexamethylpropyleneamineoxime (99mTc-HMPAO), which indicated cerebral perfusion on day 5. Basilar artery narrowing was verified histologically, and cerebral TP receptor agonists were quantified. RESULTS: 99mTc-DTPA uptake unveiled blood-brain barrier disruption in the SAH group. TBN mitigated this disruption in the brainstem area. 99mTc-Anx-V128 uptake was increased in the SAH group and TBN diminished this effect in the cerebellum. 99mTc-HMPAO uptake revealed a global decreased perfusion on day 5 in the SAH group that was significantly counteracted by TBN. TBN also mitigated basilar artery vasoconstriction, neurological deficits (on day 2), body weight loss (on day 5) and cerebral production of vasoconstrictors such as Thromboxane B2 and Prostaglandin F2α. CONCLUSIONS: Based on in vivo nanoscale imaging, we demonstrated that TBN protected against blood-brain barrier disruption, exerted an anti-apoptotic effect and improved cerebral perfusion. Thus, TP receptor antagonists showed promising results in treating post-haemorrhage neurovascular events.

Anti-αIIb ß3 immunization in Glanzmann thrombasthenia: review of literature and treatment recommendations.

Glanzmann thrombasthenia (GT) is caused by inherited defects of the αIIb ß3 platelet glycoprotein. This bleeding disorder can be treated with platelet transfusion therapy, but some patients will be immunized and begin to form anti-human leucocyte antigen (HLA) and/or anti-αIIb ß3 antibodies. These antibodies can bind and interfere with the function of the transfused platelets, rendering treatment ineffective. However, platelet transfusion refractoriness attributable to HLA antibodies may be managed by the selection of compatible donors, although they are not always readily available, particularly in an emergency. Thus, anti-αIIb ß3 antibodies represent one of the most severe complications in GT. Both genetic and environmental factors may contribute to the risk of anti-αIIb ß3 development, but the underlying pathogenic mechanisms are still unknown. This review will summarize the current knowledge of the risk factors for development of anti-αIIb ß3 antibodies in patients with GT and discuss how these findings may influence the clinical management of patients.

The first intracellular loop of GLUT4 contains a retention motif.

Glucose transporter GLUT4 (also known as SLC2A4) plays a major role in glucose homeostasis and is efficiently retained intracellularly in adipocytes and myocytes. To simplify the analysis of its retention, here, various intracellular GLUT4 domains were fused individually to reporter molecules. Of the four short cytoplasmic loops of GLUT4, only the first nine-residue-long loop conferred intracellular retention of truncated forms of the transferrin receptor and CD4 in adipocytes. In contrast, the same loop of GLUT1 was without effect. The reporter molecules to which the first loop of GLUT4 was fused localized, unlike GLUT4, to the trans-Golgi network (TGN), possibly explaining why these molecules did not respond to insulin. The retention induced by the GLUT4 loop was specific to adipocytes as it did not induce retention in preadipocytes. Of the SQWLGRKRA sequence that constitutes this loop, mutation of either the tryptophan or lysine residue abrogated reporter retention. Mutation of these residues individually into alanine residues in the full-length GLUT4 molecule resulted in a decreased retention for GLUT4-W105A. We conclude that the first intracellular loop of GLUT4 contains the retention motif WLGRK, in which W105 plays a prominent role.

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

Mutations of the integrin αIIb/ß3 intracytoplasmic salt bridge cause macrothrombocytopenia and enlarged platelet α-granules.

Rare gain-of-function mutations within the ITGA2B or ITGB3 genes have been recognized to cause macrothrombocytopenia (MTP). Here we report three new families with autosomal dominant (AD) MTP, two harboring the same mutation of ITGA2B, αIIbR995W, and a third family with an ITGB3 mutation, ß3D723H. In silico analysis shows how the two mutated amino acids directly modify the salt bridge linking the intra-cytoplasmic part of αIIb to ß3 of the integrin αIIbß3. For all affected patients, the bleeding syndrome and MTP was mild to moderate. Platelet aggregation tended to be reduced but not absent. Electron microscopy associated with a morphometric analysis revealed large round platelets; a feature being the presence of abnormal large α-granules with some giant forms showing signs of fusion. Analysis of the maturation and development of megakaryocytes reveal no defect in their early maturation but abnormal proplatelet formation was observed with increased size of the tips. Interestingly, this study revealed that in addition to the classical phenotype of patients with αIIbß3 intracytoplasmic mutations there is an abnormal maturation of α-granules. It is now necessary to determine if this feature is a characteristic of all mutations disturbing the αIIb R995/ß3 D723 salt bridge.

Assessment of platelet function on the routine coagulation analyzer Sysmex CS-2000i.

BACKGROUND: Light transmission aggregometry (LTA) is considered as the gold standard for testing platelet function in the setting of both platelet disorders suspicion and response to antiplatelet therapy evaluation. LTA requires however specialized equipment, substantial blood sample volumes, is technically challenging and time-consuming. AIM: To evaluate an automated platelet aggregation method performed on a routine coagulation analyzer Sysmex CS-2000i. METHODS: 46 patients presenting a bleeding syndrome and 62 patients with acute coronary syndrome receiving dual antiplatelet therapy were studied in total. Platelet aggregations were performed on CS-2000i equipped with a dedicated software and on APACT-4004 (Elitech, France) as the reference instrument. Aggregation was measured by monitoring the changes in light absorbance occurring in response to ADP 2.5, 5 and 10µM, collagen 3.3 µg/mL, epinephrin 10µM, ristocetin 1.25 mg/mL and arachidonic acid 0.5 mg/mL in platelet rich plasma (PRP). PRP were tested simultaneously on both CS-2000i and APACT-4004 devices. Platelet stirred speed were 800 rpm for both instruments. RESULTS: Significant correlations were observed between CS-2000i and LTA after all stimulations (p< 0.001). Patients presenting a bleeding syndrome had similar aggregation profiles with both methods. A single patient presented a severe platelet disorder (Glanzmann Thrombasthenia) and its PRP showed defective aggregation in response to all agonists except ristocetin with both instruments. Finally, the inter-agreement rates for CS-2000i and APACT-4004 to detect low responders to thienopyridines or aspirine were strong (weighted kappa> 0.70). CONCLUSION: Platelet aggregation on the routine coagulation analyzer CS-2000i is an easily accessible, handy, reliable, standardized, and rapid tool to assess platelet function which allows to skirt most of the LTA limitations.

Benefit of switching dual antiplatelet therapy after acute coronary syndrome: the TOPIC (timing of platelet inhibition after acute coronary syndrome) randomized study.

AIMS: Newer P2Y12 blockers (prasugrel and ticagrelor) demonstrated significant ischaemic benefit over clopidogrel after acute coronary syndrome (ACS). However, both drugs are associated with an increase in bleeding complications. The objective of the present study was to evaluate the benefit of switching dual antiplatelet therapy (DAPT) from aspirin plus a newer P2Y12 blocker to aspirin plus clopidogrel 1 month after ACS. METHODS AND RESULTS: We performed an open-label, monocentric, and randomized trial. From March 2014 to April 2016, patients admitted with ACS requiring coronary intervention, on aspirin and a newer P2Y12 blocker and without adverse event at 1 month, were assigned to switch to aspirin and clopidogrel (switched DAPT) or continuation of their drug regimen (unchanged DAPT). The primary outcome was a composite of cardiovascular death, urgent revascularization, stroke and bleeding as defined by the Bleeding Academic Research Consortium (BARC) classification ≥2 at 1 year post ACS. Six hundred and forty six patients were randomized and 645 analysed, corresponding to 322 patients in the switched DAPT and 323 in the unchanged DAPT group. The primary endpoint occurred in 43 (13.4%) patients in the switched DAPT group and in 85 (26.3%) patients in the unchanged DAPT (HR 95%CI 0.48 (0.34-0.68), P < 0.01). No significant differences were reported on ischaemic endpoints, while BARC ≥ 2 bleeding occurred in 13 (4.0%) patients in the switched DAPT and in 48 (14.9%) in the unchanged DAPT group (HR 95%CI 0.30 (0.18-0.50), P < 0.01). CONCLUSION: A switched DAPT is superior to an unchanged DAPT strategy to prevent bleeding complications without increase in ischaemic events following ACS.

The Annexin A1 Receptor FPR2 Regulates the Endosomal Export of Influenza Virus.

The Formyl Peptide Receptor 2 (FPR2) is a novel promising target for the treatment of influenza. During viral infection, FPR2 is activated by annexinA1, which is present in the envelope of influenza viruses; this activation promotes virus replication. Here, we investigated whether blockage of FPR2 would affect the genome trafficking of influenza virus. We found that, upon infection and cell treatment with the specific FPR2 antagonist WRW4 or the anti-FPR2 monoclonal antibody, FN-1D6-AI, influenza viruses were blocked into endosomes. This effect was independent on the strain and was observed for H1N1 and H3N2 viruses. In addition, blocking FPR2signaling in alveolar lung A549 epithelial cells with the monoclonal anti-FPR2 antibody significantly inhibited virus replication. Altogether, these results show that FPR2signaling interferes with the endosomal trafficking of influenza viruses and provides, for the first time, the proof of concept that monoclonal antibodies directed against FPR2 inhibit virus replication. Antibodies-based therapeutics have emerged as attractive reagents in infectious diseases. Thus, this study suggests that the use of anti-FPR2 antibodies against influenza hold great promise for the future.

Hysteresis-like binding of coagulation factors X/Xa to procoagulant activated platelets and phospholipids results from multistep association and membrane-dependent multimerization.

Binding of coagulation factors X (fX) and Xa (fXa) to activated platelets is required for the formation of membrane-dependent enzymatic complexes of intrinsic tenase and prothrombinase. We carried out an in-depth characterization of fX/fXa binding to phospholipids and gel-filtered, thrombin-activated platelets. Flow cytometry, surface plasmon resonance, and computational modeling were used to investigate interactions of fX/fXa with the membranes. Confocal microscopy was employed to study fXa binding to platelet thrombi formed in flowing whole blood under arterial conditions. Binding of fX/fXa to either vesicles or procoagulant platelets did not follow a traditional one-step reversible binding model. Their dissociation was a two-step process resulting in a plateau that was up to 10-fold greater than the saturation value observed in the association experiments. Computational modeling and experimental evidence suggested that this was caused by a combination of two-step association (mainly for fX) and multimerization on the membrane (mainly for fXa). Importantly, fX formed multimers with fXa, thereby improving its retention. The same binding/dissociation hysteresis was observed for annexin V known to form trimers on the membranes. Experiments with platelets from gray syndrome patients showed that alpha-granular factor Va provided an additional high-affinity binding site for fXa that did not affect the hysteresis. Confocal microscopy observation of fXa binding to platelet thrombi in a flow chamber and its wash-out confirmed that this phenomenon persisted under physiologically relevant conditions. This suggests its possible role of "locking" coagulation factors on the membrane and preventing their inhibition in plasma and removal from thrombi by flow.