Association of laboratory test results with the bleeding history in patients with inherited platelet function disorders (the Bleeding Assesment Tool - LABoratory tests substudy): communication from the Platelet Physiology ISTH-SSC.

Background: In hemophilia and von Willebrand disease, the degree of alteration of laboratory assays correlates with bleeding manifestations. Few studies have assessed the predictive value for bleeding of laboratory assays in patients with inherited platelet function disorders (IPFDs). Objectives: To assess whether there is an association between platelet function assay results and bleeding history, as evaluated by the International Society on Thrombosis and Haemostasis (ISTH) bleeding assessment tool (BAT). Methods: Centers participating in the international ISTH-BAT validation study were asked to provide results of the diagnostic assays employed for the patients they enrolled, and the association with the individual patients' bleeding score (BS) was assessed. Results: Sixty-eight patients with 14 different IPFDs were included. Maximal amplitude of platelet aggregation was significantly lower in patients with a pathologic BS and correlated inversely with the BS, a finding largely driven by the subgroup of patients with Glanzmann thrombasthenia and CalDAG-GEFI deficiency; after their exclusion, TRAP-induced aggregation remained significantly lower in patients with a pathologic BS. Bleeding time was significantly more prolonged in patients with a high BS than in those with a normal BS (27.1 ± 6.2 minutes vs 15.1 ± 10.6 minutes; P < .01). Reduced α-granule content was significantly more common among patients with a pathologic BS than among those with a normal BS (80% vs 20%; P < .05). Receiver operating characteristic curve analysis revealed a significant discriminative ability of all the aforementioned tests for pathologic BS (P < .001), also after exclusion of patients with Glanzmann thrombasthenia and CalDAG-GEFI deficiency. Conclusion: This study shows that altered platelet laboratory assay results are associated with an abnormal ISTH-BAT BS in IPFD.

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.

Emergency management of patients with Glanzmann thrombasthenia: consensus recommendations from the French reference center for inherited platelet disorders.

Glanzmann thrombasthenia (GT) is a genetic bleeding disorder characterised by severely reduced/absent platelet aggregation in response to multiple physiological agonists. The severity of bleeding in GT varies markedly, as does the emergency situations and complications encountered in patients. A number of emergency situations may occur in the context of GT, including spontaneous or provoked bleeding, such as surgery or childbirth. While general management principles apply in each of these settings, specific considerations are essential for the management of GT to avoid escalating minor bleeding events. These recommendations have been developed from a literature review and consensus from experts of the French Network for Inherited Platelet Disorders, the French Society of Emergency Medicine, representatives of patients' associations, and Orphanet to aid decision making and optimise clinical care by non-GT expert health professionals who encounter emergency situations in patients with GT.

Multicenter evaluation of light transmission platelet aggregation reagents: communication from the ISTH SSC Subcommittee on Platelet Physiology.

BACKGROUND: Light transmission aggregation (LTA) is used widely by the clinical and research communities. Although it is a gold standard, there is a lack of interlaboratory harmonization. OBJECTIVES: The primary objective was to assess whether sources of activators (mainly adenosine diphosphate [ADP], collagen, arachidonic acid, epinephrine, and thrombin receptor activating peptide6) and ristocetin contribute to poor LTA reproducibility. The secondary objective was to evaluate interindividual variability of results to appreciate the distribution of normal values and consequently better interpret pathologic results. METHODS: An international multicenter study involving 28 laboratories in which we compared LTA results obtained with center-specific activators and a comparator that we supplied. RESULTS: We report variability in the potency (P) of activators in comparison with the comparator. Thrombin receptor activating peptide 6 (P, 1.32-2.68), arachidonic acid (P, 0.87-1.43), and epinephrine (P, 0.97-1.34) showed the greatest variability. ADP (P, 1.04-1.20) and ristocetin (P, 0.98-1.07) were the most consistent. The data highlighted clear interindividual variability, notably for ADP and epinephrine. Four profiles of responses were observed with ADP from high-responders, intermediate-responders, and low-responders. A fifth profile corresponding to nonresponders (5% of the individuals) was observed with epinephrine. CONCLUSION: Based on these data, the establishment and adoption of simple standardization principles should mitigate variability due to activator sources. The observation of huge interindividual variability for certain concentrations of activators should lead to a cautious interpretation before reporting a result as abnormal. Confidence can be taken from the fact that difference between sources is not exacerbated in patients treated with antiplatelet agents.

Single-cell analysis of megakaryopoiesis in peripheral CD34+ cells: insights into ETV6-related thrombocytopenia.

BACKGROUND: Germline mutations in the ETV6 transcription factor gene are responsible for familial thrombocytopenia and leukemia predisposition syndrome. Although previous studies have shown that ETV6 plays an important role in megakaryocyte (MK) maturation and platelet formation, the mechanisms by which ETV6 dysfunction promotes thrombocytopenia remain unclear. OBJECTIVES: To decipher the transcriptional mechanisms and gene regulatory network linking ETV6 germline mutations and thrombocytopenia. METHODS: Presuming that ETV6 mutations result in selective effects at a particular cell stage, we applied single-cell RNA sequencing to understand gene expression changes during megakaryopoiesis in peripheral CD34+ cells from healthy controls and patients with ETV6-related thrombocytopenia. RESULTS: Analysis of gene expression and regulon activity revealed distinct clusters partitioned into 7 major cell stages: hematopoietic stem/progenitor cells, common-myeloid progenitors (CMPs), MK-primed CMPs, granulocyte-monocyte progenitors, MK-erythroid progenitors (MEPs), progenitor MKs/mature MKs, and platelet-like particles. We observed a differentiation trajectory in which MEPs developed directly from hematopoietic stem/progenitor cells and bypassed the CMP stage. ETV6 deficiency led to the development of aberrant cells as early as the MEP stage, which intensified at the progenitor MK/mature MK stage, with a highly deregulated core "ribosome biogenesis" pathway. Indeed, increased translation levels have been documented in patient CD34+-derived MKs with overexpression of ribosomal protein S6 and phosphorylated ribosomal protein S6 in both CD34+-derived MKs and platelets. Treatment of patient MKs with the ribosomal biogenesis inhibitor CX-5461 resulted in an increase in platelet-like particles. CONCLUSION: These findings provide novel insight into both megakaryopoiesis and the link among ETV6, translation, and platelet production.

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.

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.

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.

GATA1 pathogenic variants disrupt MYH10 silencing during megakaryopoiesis.

BACKGROUND: GATA1 is an essential transcription factor for both polyploidization and megakaryocyte (MK) differentiation. The polyploidization defect observed in GATA1 variant carriers is not well understood. OBJECTIVE: To extensively phenotype two pedigrees displaying different variants in the GATA1 gene and determine if GATA1 controls MYH10 expression levels, a key modulator of MK polyploidization. METHOD: A total of 146 unrelated propositi with constitutional thrombocytopenia were screened on a multigene panel. We described the genotype-phenotype correlation in GATA1 variant carriers and investigated the effect of these novel variants on MYH10 transcription using luciferase constructs. RESULTS: The clinical profile associated with the p.L268M variant localized in the C terminal zinc finger was unusual in that the patient displayed bleeding and severe platelet aggregation defects without early-onset thrombocytopenia. p.N206I localized in the N terminal zinc finger was associated, on the other hand, with severe thrombocytopenia (15G/L) in early life. High MYH10 levels were evidenced in platelets of GATA1 variant carriers. Analysis of MKs anti-GATA1 chromatin immunoprecipitation-sequencing data revealed two GATA1 binding sites, located in the 3' untranslated region and in intron 8 of the MYH10 gene. Luciferase reporter assays showed their respective role in the regulation of MYH10 gene expression. Both GATA1 variants significantly alter intron 8 driven MYH10 transcription. CONCLUSION: The discovery of an association between MYH10 and GATA1 is a novel one. Overall, this study suggests that impaired MYH10 silencing via an intronic regulatory element is the most likely cause of GATA1-related polyploidization defect.

The ISTH bleeding assessment tool as predictor of bleeding events in inherited platelet disorders: Communication from the ISTH SSC Subcommittee on Platelet Physiology.

BACKGROUND: The ISTH Bleeding Assessment Tool (ISTH-BAT) has been validated for clinical screening of suspected von Willebrand disease (VWD) and for bleeding prediction. Recently it has been validated for subjects with inherited platelet disorders (IPD) (BAT-VAL study). OBJECTIVES: To determine whether the ISTH-BAT bleeding score (BS) predicts subsequent bleeding events requiring treatment in IPD patients. METHODS: Patients with IPD, type 1 VWD (VWD-1) and age- and sex-matched healthy controls enrolled in the BAT-VAL study were prospectively followed-up for 2 years and bleeding episodes requiring treatment were recorded. RESULTS: Of the 1098 subjects initially enrolled, 955 were followed-up and 124 suffered hemorrhages during follow-up, 60% of whom had inherited platelet function disorders (IPFD). Total number of events was significantly higher in IPFD (n = 235) than VWD-1 (n = 52) or inherited thrombocytopenia (IT; n = 20). Events requiring transfusions were 66% in IPFD, 5.7% in VWD-1, and 3% in IT. Baseline BS was significantly higher in IPFD patients with a bleeding event at follow-up than in those without (p < .01) and the percentage of subjects suffering a bleeding event increased proportionally to baseline BS quartile. A significant association between the BS and the chance of suffering severe bleeding was found in the overall, IPFD, and VWD-1 populations. Similar results were obtained for the pediatric population. CONCLUSIONS: Inherited platelet function disorder patients with high BS at enrollment are more likely to suffer from bleeding events requiring treatment at follow-up. Moreover, the higher the baseline BS quartile the greater the incidence of subsequent events, suggesting that independently from diagnosis a high BS is associated with a greater risk of subsequent hemorrhage.

Severe thrombophilia in a factor V-deficient patient homozygous for the Ala2086Asp mutation (FV Besançon).

BACKGROUND: Coagulation factor V (FV), present in plasma and platelets, has both pro- and anticoagulant functions. OBJECTIVE: We investigated an FV-deficient patient (FV:C 3%, FV:Ag 4%) paradoxically presenting with recurrent venous thrombosis (11 events) instead of bleeding. METHODS/RESULTS: Thrombophilia screening revealed only heterozygosity for the F2 20210G>A mutation. Although thrombin generation in the patient's platelet-poor plasma was suggestive of a hypocoagulable state, thrombin generation in the patient's platelet-rich plasma (PRP) was higher than in control PRP and extremely resistant to activated protein C (APC). This was partially attributable to the complete abolition of the APC-cofactor activity of FV and a marked reduction of plasma tissue factor pathway inhibitor antigen and activity. The patient was homozygous for a novel missense mutation (Ala2086Asp, FVBesançon ) that favors a "closed conformation" of the C2 domain, predicting impaired binding of FV(a) to phospholipids. Recombinant FVBesançon was hardly secreted, indicating that this mutation is responsible for the patient's FV deficiency. Model system experiments performed using highly diluted plasma as a source of FV showed that, compared with normal FVa, FVaBesançon has slightly (≤1.5-fold) unfavorable kinetic parameters (Km , Vmax ) of prothrombin activation, but also a lower rate of APC-catalyzed inactivation in the presence of protein S. CONCLUSIONS: FVBesançon induces a hypercoagulable state via quantitative (markedly decreased FV level) and qualitative (phospholipid-binding defect) effects that affect anticoagulant pathways (anticoagulant activities of FV, FVa inactivation, tissue factor pathway inhibitor α level) more strongly than the prothrombinase activity of FVa. A possible specific role of platelet FV cannot be excluded.

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.

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.

On-Ticagrelor Platelet Reactivity and Clinical Outcome in Patients Undergoing Percutaneous Coronary Intervention for Acute Coronary Syndrome.

BACKGROUND: A strong association between on-thienopyridine platelet reactivity (PR) and the risk of both thrombotic and bleeding events in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI) has been demonstrated. However, no study has analyzed the relationship between on-ticagrelor PR and clinical outcome in this clinical setting. OBJECTIVES: We aimed to investigate the relationship between on-ticagrelor PR, assessed by the vasodilator-stimulated phosphoprotein (VASP) index, and clinical outcome in patients with ACS undergoing PCI. METHODS: We performed a prospective, multicenter, observational study of patients undergoing PCI for ACS. PR was measured using the VASP index following ticagrelor loading dose. The primary study endpoint was the rate of Bleeding Academic Research Consortium (BARC) type ≥2 at 1 year. The key secondary endpoint was the rate of major adverse cardiovascular events (MACE) defined as the composite of cardiovascular death, myocardial infarction, stroke, and urgent revascularization. RESULTS: We included 570 ACS patients, among whom 33.9% had ST-elevation myocardial infarction. BARC type ≥2 bleeding occurred in 10.9% and MACE in 13.8%. PR was not associated with BARC ≥2 or with MACE (p = 0.12 and p = 0.56, respectively). No relationship between PR and outcomes was observed, neither when PR was analyzed quantitatively nor when it was analyzed qualitatively (low on-treatment PR [LTPR] vs. no LTPR). CONCLUSION: On-ticagrelor PR measured by the VASP was not associated with bleeding or thrombotic events in ACS patients undergoing PCI. PR measured by the VASP should not be used as a surrogate endpoint in studies on ticagrelor.

A Combination of Single Nucleotide Polymorphisms is Associated with the Interindividual Variability of Cholesterol Bioavailability in Healthy Adult Males.

SCOPE: Cholesterol bioavailability displays a high interindividual variability, partly due to genetic factors. Existing studies have focused on single nucleotide polymorphisms (SNPs) analyzed individually, which only explained a minor fraction of the variability of this complex phenotype. The aim is to identify a combination of SNPs associated with a significant part of the variability in cholesterol bioavailability. METHODS AND RESULTS: Thirty-nine healthy adult males are given a standard test snack containing 80 mg heptadeuterated (D7) cholesterol. The plasma D7-cholesterol concentration is measured at equilibrium 40 h after test snack intake. The D7-cholesterol response (D7-cholesterol/total cholesterol concentration) exhibits a relatively high interindividual variability (CV = 32%). The association of exonic SNPs in candidate genes (188 genes involved in or related to cholesterol metabolism) with the plasma D7-cholesterol response is assessed by univariate statistics followed by partial least squares regression. A significant model (p-value after cross-validation ANOVA = 1.64 × 10-7 ) that includes 8 SNPs (SOAT2-rs9658625, DNAH11-rs11768670, LIPC-rs690, MVK-rs2287218, GPAM-rs10787428, APOE-rs7412, CBS-rs234706, and WRN-rs1801196) explains 59.7% of the variance in cholesterol bioavailability (adjusted R²). CONCLUSION: Here a combination of SNPs is significantly associated with the variability in dietary cholesterol bioavailability in healthy adult males.

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.

Laboratory Techniques Used to Diagnose Constitutional Platelet Dysfunction.

Platelets play a major role in primary hemostasis, where activated platelets form plugs to stop hemorrhaging in response to vessel injuries. Defects in any step of the platelet activation process can cause a variety of platelet dysfunction conditions associated with bleeding. To make an accurate diagnosis, constitutional platelet dysfunction (CPDF) should be considered once von Willebrand disease and drug intake are ruled out. CPDF may be associated with thrombocytopenia or a genetic syndrome. CPDF diagnosis is complex, as no single test enables the analysis of all aspects of platelet function. Furthermore, the available tests lack standardization, and repeat tests must be performed in specialized laboratories especially for mild and moderate forms of the disease. In this review, we provide an overview of the laboratory tests used to diagnose CPDF, with a focus on light transmission platelet aggregation (LTA), flow cytometry (FC), and granules assessment. Global tests, mainly represented by LTA, are often initially performed to investigate the consequences of platelet activation on platelet aggregation in a single step. Global test results should be confirmed by additional analytical tests. FC represents an accurate, simple, and reliable test to analyze abnormalities in platelet receptors, and granule content and release. This technique may also be used to investigate platelet function by comparing resting- and activated-state platelet populations. Assessment of granule content and release also requires additional specialized analytical tests. High-throughput sequencing has become increasingly useful to diagnose CPDF. Advanced tests or external research laboratory techniques may also be beneficial in some cases.

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.

Strengths and Weaknesses of Light Transmission Aggregometry in Diagnosing Hereditary Platelet Function Disorders.

Hereditary defects in platelet function are responsible for sometimes severe mucocutaneous hemorrhages. They are a heterogeneous group of abnormalities whose first-line diagnosis typically involves interpreting the results of in vitro light transmission aggregometry (LTA) traces. Interpretation of LTA is challenging. LTA is usually performed in specialized laboratories with expertise in platelet pathophysiology. This review updates knowledge on LTA, describing the various platelet aggregation profiles typical of hereditary platelet disorders to guide the physician in the diagnosis of functional platelet disorders.