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Platelets are small anucleate cells that play a key role in thrombosis and hemostasis. Our group previously identified apolipoprotein A-IV (apoA-IV) as an endogenous inhibitor of thrombosis by competitive blockade of the αIIbß3 integrin on platelets. ApoA-IV inhibition of platelets was dependent on the N-terminal D5/D13 residues, and enhanced with absence of the C-terminus, suggesting it sterically hinders its N-terminal platelet binding site. The C-terminus is also the site of common apoA-IV polymorphisms apoA-IV-1a (T347S) and apoA-IV-2 (Q360H). Interestingly, both are linked with an increased risk of cardiovascular disease, however, the underlying mechanism remains unclear. Here, we generated recombinant apoA-IV and found that the Q360H or T347S polymorphisms dampened its inhibition of platelet aggregation in human platelet-rich plasma and gel-filtered platelets, reduced its inhibition of platelet spreading, and its inhibition of P-selectin on activated platelets. Using an ex vivo thrombosis assay, we found that Q360H and T347S attenuated its inhibition of thrombosis at both high (1800s-1) and low (300s-1) shear rates. We then demonstrate a conserved monomer-dimer distribution among apoA-IV WT, Q360H, and T347S and use protein structure modelling software to show Q360H and T347S enhance C-terminal steric hindrance over the N-terminal platelet-binding site. These data provide critical insight into increased cardiovascular risk for individuals with Q360H or T347S polymorphisms.
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Apolipoproteínas A , Plaquetas , Agregación Plaquetaria , Trombosis , Humanos , Trombosis/genética , Trombosis/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/genética , Plaquetas/metabolismo , Plaquetas/efectos de los fármacos , Polimorfismo Genético , Apoproteína(a)/genética , Apoproteína(a)/metabolismo , Apoproteína(a)/química , Selectina-P/genética , Selectina-P/metabolismoRESUMEN
Background: Constitutive inflammation and hemostatic activation have been identified as key contributors to the pathophysiology of sickle cell disease (SCD), leading to clinical consequences such as vaso-occlusive crises and stroke. Patients with hemoglobin SS (HbSS) and hemoglobin SC (HbSC) genotypes are reported to have different symptoms, as do patients in steady-state and crisis situations. Differences among these groups remain unclear in pediatric patients. Objectives: To compare hemostatic activity in HbSS and HbSC pediatric patients during steady state, in crisis, and in clinical follow-up and compare HbSS and HbSC patients with normal healthy children. Methods: Whole-blood coagulation assay thromboelastography (TEG) was used to assess hemostatic activity. In parallel, flow cytometry was used to assess procoagulant surface expression of platelets and red blood cells. Results: TEG results indicated no significant differences in clotting onset (R time), clot maximum amplitude, or maximum rate of thrombus generation among steady-state, crisis, and follow-up subgroups of HbSS and HbSC patients. TEG parameters did not differ significantly between HbSC patients and healthy children, while HbSS patients showed significantly shorter R time and greater maximum amplitude and maximum rate of thrombus generation, all indicative of a constitutive hypercoagulable state. Flow cytometry results did not detect increased platelet integrin αIIbß3 activation or red blood cell procoagulant surface expression in SCD patients compared with unaffected children. Conclusion: Our results indicate that pediatric SCD patients with the HbSS genotype have constitutively activated hemostasis relative to HbSC patients and healthy children. It remains to be determined how treatments that improve clinical outcomes in SCD patients affect this constitutively hypercoagulable state.
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Clinical manifestations and laboratory parameters of haemostasis were investigated in 23 children with newly diagnosed immune thrombocytopenia (ITP) before and after intravenous immunoglobulin (IVIg) treatment. ITP patients with platelet counts of less than 20 × 109 /L and mild bleeding symptoms, graded by a standardized bleeding score (BS), were compared with healthy children with normal platelet counts and children with chemotherapy-related thrombocytopenia. Markers of platelet activation and platelet apoptosis in the absence and presence of platelet activators were analysed by flow cytometry; thrombin generation in plasma was determined. ITP patients at diagnosis presented with increased proportions of platelets expressing CD62P and CD63 and activated caspases, and with decreased thrombin generation. Thrombin-induced activation of platelets was reduced in ITP compared with controls, while increased proportions of platelets with activated caspases were observed. Children with a higher BS had lower proportions of CD62P-expressing platelets compared with those with a lower BS. IVIg treatment increased the number of reticulated platelets, the platelet count to more than 20 × 109 /L and improved bleeding in all patients. Decreased thrombin-induced platelet activation, as well as thrombin generation, were ameliorated. Our results indicate that IVIg treatment helps to counteract diminished platelet function and coagulation in children with newly diagnosed ITP.
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Púrpura Trombocitopénica Idiopática , Trombocitopenia , Humanos , Niño , Plaquetas/fisiología , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Inmunoglobulinas Intravenosas/farmacología , Inmunoglobulinas Intravenosas/uso terapéutico , Trombina , Hemorragia/tratamiento farmacológico , CaspasasRESUMEN
Background: Severe hemophilia A (SHA) patients vary in severity of bleeding, arthropathy, and requirements for replacement factor VIII (FVIII). Baseline hemostatic activity assays using calibrated automated thrombography (CAT) and thromboelastography (TEG) may offer insights into the physiological basis of clinical heterogeneity. Objectives: Use CAT and TEG to measure baseline hemostatic activity in a cohort of 30 pediatric SHA patients with available clinical data. Determine effect of contact activation inhibition with corn trypsin inhibitor (CTI). Assess heterogeneity among patients for baseline hemostatic activity and examine correlations between assay results and clinical parameters including FVIII dosing regimen, von Willebrand factor level, and Pettersson arthropathy score. Methods: SHA blood after FVIII washout was subjected to TEG, and platelet-rich (PRP) and platelet-poor plasma was used for CAT assays. Varying concentrations of tissue factor (TF) were used. Statistical analysis examined relationships between assay results, and clinical parameters. Results: CTI treatment was required to obtain TEG and CAT results representative of baseline hemostatic activity. Weak activity was observed in assays with low TF concentrations (0.5-2 pM), and most but not all samples approached normal activity levels at high TF concentrations (10-20 pM). A significant positive correlation was observed between results of TEG and CAT-PRP assays. Correlations were not detected between hemostatic assay results and clinical parameters. Conclusions: In vitro hemostatic assay results of samples containing platelets showed concordance. Assay results were not predictive of FVIII requirements or correlated with other clinical parameters. SHA patient heterogeneity is influenced by factors other than baseline hemostatic activity.
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Approximately 20% of vascular patients treated with acetyl salicylic acid (i.e., aspirin) demonstrate less than expected platelet inhibition - putting them at a four-fold increased risk of adverse cardiovascular events. Low-dose rivaroxaban (2.5 mg twice daily) in combination with low-dose aspirin has been shown to reduce adverse cardiovascular and limb events when compared to aspirin alone. In this study, light transmission aggregometry was used to measure arachidonic acid-induced platelet aggregation to evaluate the potential of combining low-dose rivaroxaban and aspirin in attenuating or overcoming aspirin non-sensitivity. In the discovery phase, 83 patients with peripheral arterial disease (PAD) taking 81 mg aspirin daily were recruited from the outpatient vascular surgery clinic at St Michael's Hospital between January to September 2021. 19 (23%) were determined to be non-sensitive to aspirin. After ex-vivo addition of 2.5 mg dosage equivalent of rivaroxaban, aspirin non-sensitivity was overcome in 11 (58%) of these 19 patients. In the validation phase, 58 patients with cardiovascular risk factors who were not previously prescribed aspirin were recruited. In this group, ex-vivo addition of 2.5 mg dosage equivalent of rivaroxaban significantly reduced arachidonic acid-induced platelet aggregation in the presence of aspirin. These results demonstrate the potential for low-dose rivaroxaban to overcome aspirin non-sensitivity in patients with PAD. Further studies are needed to evaluate and confirm these findings.
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Persons with mild hemophilia A (HA) may use intranasal desmopressin prior to sports participation. Desmopressin is expensive and can cause vomiting, headache, palpitation, and occasionally seizures. Our group has previously documented a 2.3-fold increase in factor VIII activity (FVIII:C) in adolescents with mild HA after moderate-intensity aerobic exercise. Herein, we report principal findings of a randomized trial of intranasal desmopressin vs a standardized, moderate-intensity aerobic exercise regimen in adolescents with mild HA. Our primary objective was to compare the change in FVIII:C associated with these 2 interventions. We also examined changes in hemostatic parameters arising from their sequential administration. The study was conducted simultaneously at the Hospital for Sick Children, Canada, and Nationwide Children's Hospital, USA. Thirty-two eligible male adolescents (mean age ± standard deviation: 16.1 ± 2.6 years) with mild HA (mean baseline FVIII:C: 27.9% ± 18.4%) were randomized to 1 of 4 study arms (desmopressin followed by exercise, desmopressin alone, exercise followed by desmopressin, and exercise alone). Blood work was obtained at baseline and at 3 subsequent time-points. Participants randomized to exercise cycled on an ergometer for approximately 12 minutes, with the final 3 minutes at 85% of their predicted maximum heart rate. Standard weight-based dosing of desmopressin was used. Mean immediate increase in FVIII:C was 1.7-fold with exercise compared with 1.9-fold with desmopressin (noninferiority, P = .04). Exercise-induced improvement in hemostatic parameters including FVIII:C was brief compared with more sustained improvements seen with desmopressin. More than 60% of participants randomized to receive both exercise and desmopressin achieved normal (>50%) FVIII:C, 75 and 135 minutes into the study protocol.
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Desamino Arginina Vasopresina , Terapia por Ejercicio , Hemofilia A , Hemostáticos , Adolescente , Desamino Arginina Vasopresina/uso terapéutico , Factor VIII/uso terapéutico , Hemofilia A/tratamiento farmacológico , Hemostáticos/uso terapéutico , Humanos , MasculinoRESUMEN
BACKGROUND: Aspirin is a key antiplatelet therapy for the prevention of thrombotic events in patients with cardiovascular disease. Studies suggest that ≈20% of patients with cardiac disease suffer from aspirin nonsensitivity, a phenomenon characterized by the inability of 81 mg aspirin to inhibit platelet aggregation and/or prevent adverse cardiovascular events. OBJECTIVES: To investigate aspirin nonsensitivity in patients with vascular disease and assess the consequences of aspirin nonsensitivity. METHODS: One hundred fifty patients presenting to St. Michael's Hospital's outpatient clinics with evidence of vascular disease (peripheral arterial disease or carotid artery stenosis) and a previous prescription of 81 mg of aspirin were recruited in this study. Light transmission aggregometry with arachidonic acid induction was used to determine sensitivity to aspirin. Patients with a maximum aggregation ≥20% in response to arachidonic acid were considered aspirin nonsensitive, as per previous studies. RESULTS: Of the 150 patients recruited, 36 patients (24%) were nonsensitive to 81 mg of aspirin. Of these 36 nonsensitive patients, 30 patients provided a urine sample for urine salicyluric acid analysis (a major metabolite of aspirin). Urine analysis demonstrated that 14 patients were compliant and 16 were noncompliant with their aspirin therapy. Major adverse cardiovascular events and major adverse limb events were significantly higher in the nonsensitive patients compared to sensitive patients (hazard ratio, 3.68; P < 0.001). CONCLUSION: These data highlight the high prevalence of aspirin nonsensitivity and noncompliance in patients with vascular disease and emphasizes the urgent need for improved medical management options for this patient population.
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Aspirin (ASA) therapy is proven to be effective in preventing adverse cardiovascular events; however, up to 30% of patients are non-sensitive to their prescribed ASA dosage. In this pilot study, we demonstrated, for the first time, how ASA non-sensitivity can be diagnosed using Plateletworks®, a point-of-care platelet function test. Patients prescribed 81 mg of ASA were recruited in a series of two successive phases-a discovery phase and a validation phase. In the discovery phase, a total of 60 patients were recruited to establish a cut-off point (COP) for ASA non-sensitivity using Plateletworks®. Each sample was simultaneously cross-referenced with a light transmission aggregometer (LTA). Our findings demonstrated that >52% maximal platelet aggregation using Plateletworks® had a sensitivity, specificity, and likelihood ratio of 80%, 70%, and 2.67, respectively, in predicting ASA non-sensitivity. This COP was validated in a secondary cohort of 40 patients prescribed 81 mg of ASA using Plateletworks® and LTA. Our data demonstrated that our established COP had a 91% sensitivity and 69% specificity in identifying ASA non-sensitivity using Plateletworks®. In summary, Plateletworks® is a point-of-care platelet function test that can appropriately diagnose ASA non-sensitive patients with a sensitivity exceeding 80%.
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OBJECTIVES: To prospectively quantify bleeding severity and elaborate hemorrhagic symptoms in children with 22q11.2 deletion syndrome (22q11DS) using 2 validated bleeding assessment tools (BATs), namely the Pediatric Bleeding Questionnaire and the International Society on Thrombosis and Hemostasis BAT (ISTH-BAT). We also sought to compare subjects' bleeding scores to unaffected first-degree family members. STUDY DESIGN: Children with 22q11DS and unaffected first-degree family members were recruited for the study. Two validated BATs were administered by a pediatric hematologist. Additional clinical and laboratory data were abstracted from patient medical records. Standard descriptive and nonparametric statistical methods were used. RESULTS: In total, 29 eligible subjects and controls were assessed. Median age (range) of subjects and controls was 8 (5-17) years and 38 (9-56) years, respectively. In total, 17 of 29 subjects had a positive bleeding score on ISTH-BAT compared with 1 of 29 control patients (P < .0001). Median ISTH-BAT score in subjects was 3 (0-12), compared with 2 (0-6) in control patients (P = .022). Median Pediatric Bleeding Questionnaire score in subjects was 2 (-1 to 12). The most frequent bleeding symptoms reported in subjects with 22q11DS were epistaxis (69%) and bruising (52%). Eighteen subjects had been surgically challenged, and 6 were noted to have increased perioperative hemorrhage. CONCLUSIONS: Children with 22q11DS have increased bleeding scores compared with their first-degree unaffected relatives. The majority of the bleeding symptoms described were mucocutaneous.
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Síndrome de Deleción 22q11/complicaciones , Hemorragia/etiología , Síndrome de Deleción 22q11/sangre , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Preescolar , Estudios Transversales , Familia , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Índice de Severidad de la Enfermedad , Encuestas y Cuestionarios , Adulto JovenRESUMEN
Standard pharmacokinetic (PK) assessments are demanding for persons with hemophilia A, requiring a 72-hour washout and 5 to 11 timed blood samples. A no-washout, single-clinic visit, sparse sampling population PK (PPK) protocol is an attractive alternative. Here, we compared PK parameters obtained with a traditional washout, 6-sampling time point PPK protocol with a no-washout, single-clinic visit, reverse 2-sampling time point PPK protocol in persons with severe hemophilia A (SHA) receiving ADVATE. A total of 39 inhibitor-negative males with SHA (factor VIII activity [FVIII:C] < 2%) were enrolled in a prospective sequential design PK study. Participants completed a washout, 6-sampling time point PPK protocol as well as a no-washout, reverse 2-sampling time point protocol, with samples taken during a single 3-hour clinic visit 24 hours post home infusion of FVIII and then 3 hours post infusion in clinic. FVIII:C levels were analyzed by one-stage and chromogenic assays; blood group and von Willebrand factor antigen (VWF:Ag) were determined; and PK parameters were analyzed using the ADVATE myPKFiT dosing tool. There was moderate to almost perfect agreement for the PK parameters obtained with the 2- and the 6- point PPK protocols using a one-stage FVIII:C assay and a substantial to almost perfect agreement using a chromogenic FVIII:C assay. Significant associations between specific PK parameters and blood group and VWF:Ag were observed. The no-washout, single-clinic visit, reverse 2-sampling time point PPK protocol can be used in the routine clinical setting since it demonstrates sufficient accuracy compared with the more demanding and less practical washout, 6-sampling time point PPK protocol in persons with SHA receiving ADVATE.
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Coagulación Sanguínea/efectos de los fármacos , Coagulantes/farmacocinética , Monitoreo de Drogas , Factor VIII/farmacocinética , Hemofilia A/tratamiento farmacológico , Adolescente , Adulto , Anciano , Atención Ambulatoria , Australia , Canadá , Niño , Preescolar , Protocolos Clínicos , Coagulantes/administración & dosificación , Coagulantes/sangre , República Checa , Factor VIII/administración & dosificación , Hemofilia A/sangre , Hemofilia A/diagnóstico , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , Valor Predictivo de las Pruebas , Estudios Prospectivos , Adulto JovenRESUMEN
Acetylsalicylic acid (ASA), also known as aspirin, appears to be ineffective in inhibiting platelet aggregation in 20-30% of patients. Light transmission aggregometry (LTA) is a gold standard platelet function assay. In this pilot study, we used LTA to personalize ASA therapy ex vivo in atherosclerotic patients. Patients were recruited who were on 81 mg ASA, presenting to ambulatory clinics at St. Michael's Hospital (n = 64), with evidence of atherosclerotic disease defined as clinical symptoms and diagnostic findings indicative of symptomatic peripheral arterial disease (PAD), with an ankle brachial index (ABI) of <0.9 (n = 52) or had diagnostic features of asymptomatic carotid arterial stenosis (CAS), with >50% stenosis of internal carotid artery on duplex ultrasound (n = 12). ASA compliance was assessed via multisegmented injection-capillary electrophoresis-mass spectrometry based on measuring the predominant urinary ASA metabolite, salicyluric acid. LTA with arachidonic acid was used to test for ASA sensitivity. Escalating ASA dosages of 162 mg and 325 mg were investigated ex vivo for ASA dose personalization. Of the 64 atherosclerotic patients recruited, 8 patients (13%) were non-compliant with ASA. Of ASA compliant patients (n = 56), 9 patients (14%) were non-sensitive to their 81 mg ASA dosage. Personalizing ASA therapy in 81 mg ASA non-sensitive patients with escalating dosages of ASA demonstrated that 6 patients became sensitive to a dosage equivalent to 162 mg ASA and 3 patients became sensitive to a dosage equivalent to 325 mg ASA. We were able to personalize ASA dosage ex vivo in all ASA non-sensitive patients with escalating dosages of ASA within 1 h of testing.
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Background and Objectives: Aspirin (acetylsalicylic acid-ASA) is a first-line antiplatelet therapy provided to patients with coronary artery disease (CAD). However, it has been demonstrated that 20-30% of these patients are non-sensitive to their ASA therapy. ASA non-sensitivity is a phenomenon where low-dose ASA (81-325 mg) does not completely inhibit arachidonic-acid-induced platelet aggregation, putting patients at risk of adverse cardio-thrombotic events. Ticagrelor is a P2Y12 receptor inhibitor and alternative antiplatelet that has been approved to reduce the risk of stroke, myocardial infarction, and overall cardiovascular-related death. In this study, we aimed to identify ASA non-sensitive patients and evaluate if they would be sensitive to ticagrelor. Materials and Methods: For this pilot study, thirty-eight patients with CAD taking 81 mg ASA were recruited. Blood samples were collected from each patient and platelet rich plasma (PRP) from each sample was isolated. Light-transmission aggregometry (LTA) was used to determine baseline ASA sensitivity in each patient using 0.5 mg/mL arachidonic acid as a platelet agonist. Patients with ≥20% maximal platelet aggregation after activation were considered ASA non-sensitive. Fresh PRP samples from all patients were then spiked with a clinical dosage of ticagrelor (3 µM-approximately equivalent to a loading dose of 180 mg ticagrelor). Sensitivity was determined using LTA and 5 µM ADP as a platelet agonist. Patients with ≥46% maximal platelet aggregation were considered ticagrelor non-sensitive. Results: Of the 38 CAD patients taking 81 mg ASA, 32% (12/38) were non-sensitive to their 81 mg ASA therapy. All 38 of the recruited patients (100%) were sensitive to ticagrelor ex vivo. In conclusion, we were able to identify ASA non-sensitivity using LTA and determine that ASA non-sensitive patients were sensitive to ticagrelor. Conclusions: Our results suggest that ticagrelor is a promising alternative therapy for patients who are non-sensitive to ASA.
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Aspirina , Inhibidores de Agregación Plaquetaria , Aspirina/uso terapéutico , Humanos , Proyectos Piloto , Agregación Plaquetaria , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Ticagrelor/farmacología , Ticagrelor/uso terapéuticoRESUMEN
OBJECTIVE: Peripheral artery disease patients have been shown to be more susceptible to thrombotic events compared to non-peripheral artery disease patients. Therefore, the aim of this study was to investigate the coagulation profile in peripheral artery disease patients with chronic limb threatening ischemia, moderate peripheral artery disease patients with claudication, and non-peripheral artery disease controls. METHODS: Chronic limb threatening ischemia patients were matched to peripheral artery disease patients with claudication and non-peripheral artery disease controls in a 1:1:1 ratio. Each patient had their cytokines, markers of thrombin generation, coagulation factors, natural anti-coagulants, fibrinolysis, and endothelial injury markers assessed. RESULTS: Markers of thrombin activation, thrombin Fragments F1 + 2 (Frag 1 + 2), and thrombin-anti-thrombin complex were found to be significantly elevated in all peripheral artery disease and chronic limb threatening ischemia patients relative to non-peripheral artery disease controls. Similarly, relative to non-peripheral artery disease controls, inflammatory markers including C-reactive protein, soluble platelet factor 4, and neutrophil gelatinase-associated lipocalin were also found to be significantly upregulated in chronic limb threatening ischemia patients, but not in peripheral artery disease patients with claudication. Furthermore, our data demonstrated significant increases in markers of endothelial injury in chronic limb threatening ischemia patients relative to non-peripheral artery disease controls. Finally, decreases in natural anti-coagulants (protein C and protein S) and coagulation factors FIX, FXI, and FXII were also observed in chronic limb threatening ischemia patients when compared with non-peripheral artery disease controls. CONCLUSIONS: Our data suggest that in relation to non-peripheral artery disease controls, chronic limb threatening ischemia patients are more hypercoagulable. However, peripheral artery disease patients with claudication appear to have similar levels of circulating procoagulant markers as non-peripheral artery disease patients. This may explain the increased risk of thrombotic events observed in chronic limb threatening ischemia patients.
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Coagulación Sanguínea , Claudicación Intermitente/sangre , Isquemia/sangre , Enfermedad Arterial Periférica/sangre , Anciano , Antitrombina III , Biomarcadores/sangre , Factores de Coagulación Sanguínea/análisis , Estudios de Casos y Controles , Enfermedad Crónica , Femenino , Humanos , Mediadores de Inflamación/sangre , Claudicación Intermitente/diagnóstico , Isquemia/diagnóstico , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/sangre , Péptido Hidrolasas/sangre , Enfermedad Arterial Periférica/diagnóstico , Proyectos Piloto , ProtrombinaRESUMEN
The physiological heterogeneity of platelets leads to diverse responses and the formation of discrete subpopulations upon platelet stimulation. Procoagulant platelets are an example of such subpopulations, a key characteristic of which is exposure either of the anionic aminophospholipid phosphatidylserine (PS) or of tissue factor on the activated platelet surface. This review focuses on the former, in which PS exposure on a subpopulation of platelets facilitates assembly of the intrinsic tenase and prothrombinase complexes, thereby accelerating thrombin generation on the activated platelet surface, contributing importantly to the hemostatic process. Mechanisms involved in platelet PS exposure, and accompanying events, induced by physiologically relevant agonists are considered then contrasted with PS exposure resulting from intrinsic pathway-mediated apoptosis in platelets. Pathologies of PS exposure, both inherited and acquired, are described. A consideration of platelet PS exposure as an antithrombotic target concludes the review.
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Immune thrombocytopenia (ITP) is an autoimmune disease which leads to accelerated platelet clearance. We investigated the plasma cytokine, chemokine and growth factor signatures and their clinical significance in pediatric ITP patients during acute, chronic and follow-up stages as well as the effects of intravenous immunoglobulin (IVIg) treatment, by using the Multiplex technology. In acute ITP before and/or after IVIg treatment we found significantly increased plasma levels of the pro- (tumour necrosis factor-α (TNF-α), interleukin IL-15) and anti- (IL-1 receptor antagonist (Ra), IL-10 and the growth factor interferon γ-induced protein (IP-10)) inflammatory cytokines, compared to healthy controls. Except for IL1-Ra, these cytokines decreased to normal levels in chronic patients. In contrast, growth-regulated α protein (GRO) and soluble CD40 ligand (sCD40L), known as platelet-derived molecules, were found to be significantly decreased in acute and increased in chronic ITP patients compared to healthy controls. GRO levels positively correlated with the platelet counts in the follow-up and chronic cohort. Monocyte counts showed a significant positive correlation only with IP-10 levels in acute ITP after IVIg treatment and follow-up patients. Expression levels of mRNAs for macrophage inflammatory protein MIP1-ß, IL-1Ra and GRO determined in peripheral blood mononuclear cells (PBMCs) were significantly reduced in both acute and chronic ITP compared to controls. Our findings suggest that the different clinical presentation of acute and chronic pediatric ITP and to a lesser extent the IVIg treatment effects are characterized overall by a counterbalanced cytokine, chemokine and growth factor pattern response that might exert a pathogenic role in this disease.
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BACKGROUND: Bleeding assessment is part of the diagnostic workup of von Willebrand disease (VWD). Bleeding assessment tools (BATs) have standardized obtaining this information but have been criticized because they are time consuming. OBJECTIVE: To use our legacy data to determine which questions from BATs are the strongest predictors of a VWD diagnosis. PATIENTS/METHODS: Bleeding score data from 3 different BATs were used. Patients aged <12 years were excluded. Questions on BATs relate to different bleeding symptoms, and each symptom is scored by severity. Scores for each symptom were sorted based on whether they indicated clinically significant bleeding, and nonsignificant scores were set as the reference category. Multivariable logistic regression was used to determine the symptoms that were the strongest predictors of a laboratory-confirmed VWD diagnosis. RESULTS: A total of 927 participants were included; 144 (16%) were patients with VWD, and 783 (84%) were healthy controls. The top 3 symptoms for which a clinically significant positive response increased the likelihood of VWD were hemarthrosis (odds ratio [OR], 19.2; 95% confidence interval [CI], 3.7-100.4), postsurgical bleeding (OR, 15.2; 95% CI, 5.9-38.9), and menorrhagia (OR, 10.3; 95% CI, 4.9-21.9). With each increase in number of bleeding symptom categories with clinically significant scores, subjects had a stepwise increase in odds of a VWD diagnosis. CONCLUSIONS: Our results suggest that most of the bleeding symptoms on BATs are significant predictors of VWD, and there is value in assessing multiple bleeding symptoms when eliciting a bleeding history. Certain bleeding symptoms are more useful predictors than others. Future BAT revisions may consider adding a relative weighting to each symptom.
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Essentials The PK parameters of Eloctate vs Adynovate were compared using one-stage and chromogenic assays in 25 boys (12-18 years). The FVIII levels were taken at 3, 24, 48, and 72 hours following a dose of either FVIII; levels analyzed by WAPPS PK program. The PK profiles (half-life, clearance, and time to 5%, 3%, and 1%) were not statistically different for the two EHL FVIIIs. The significant interpatient variability in PK is mainly related to VWF levels (and blood group). BACKGROUND: A head-to-head comparison of the pharmokinetcs (PK) of extended half-life (EHL) factor VIII (FVIII) concentrates in the same subjects has not been reported. Recently, boys (ages 12-18 years) with hemophilia A in Canada were required to switch from Eloctate to Adynovate. OBJECTIVES: Compare the PK profiles of Eloctate vs Adynovate in the same boys. METHODS: Boys switching from Eloctate to Adynovate prophylaxis had FVIII levels sampled at 3, 24, 48, and 72 hours following a regular prophylactic infusion of Eloctate and then 1-3 months later, of Adynovate. Testing was done by one-stage assay (OSA) and chromogenic assay (CA). The PK parameters were determined with the Web Accessible Population Pharmacokinetic Service (WAPPS)-Hemo PK tool. RESULTS: Twenty-five boys (mean age 15.3 years; range: 12.1-18.4; 9 O blood group) underwent switching. Mean (range) terminal half-lives with the OSA were 16.1 hours (10.4 to 23.4; Eloctate) and 16.7 hours (11.0 to 23.6; Adynovate) (NS). With the CA, these were 18.0 hours (12.0 to 25.5; Eloctate) and 16.0 hours (10.3 to 22.9; Adynovate) (P = 0.001). There were no significant differences between the two EHL-FVIIIs in clearance, area under the concentration vs time curve (AUC), Vss, or time for FVIII levels to drop to 5%, 3%, and 1%. At the 72-h time point, mean observed FVIII levels following a mean dose of 39.3 IU/kg of Eloctate were 4.4% (OSA) and 4.4% (CA). For Adynovate, these were 5.1% (OSA) and 5.3% (CA) following similar doses. There was considerable interpatient variation in PK, mainly explained by differences in blood group/von Willebrand factor (VWF) levels. CONCLUSIONS: Eloctate and Adynovate have almost identical PK parameters. When switching from one to another no prophylaxis regimen change is needed.
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Factor VIII/farmacocinética , Hemofilia A/tratamiento farmacológico , Hemostáticos/farmacocinética , Proteínas Recombinantes de Fusión/farmacocinética , Sistema del Grupo Sanguíneo ABO , Adolescente , Niño , Sustitución de Medicamentos , Factor VIII/administración & dosificación , Semivida , Hemofilia A/sangre , Hemofilia A/diagnóstico , Hemostáticos/administración & dosificación , Humanos , Fragmentos Fc de Inmunoglobulinas/administración & dosificación , Masculino , Tasa de Depuración Metabólica , Ontario , Proteínas Recombinantes de Fusión/administración & dosificación , Índice de Severidad de la Enfermedad , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: In the pediatric population, pathologic bleeding is often challenging to identify. The pediatric bleeding questionnaire (PBQ) was developed as a screening tool for von Willebrand disease (VWD) but was designed to be self-completed by children above 12 years of age. The study objective was to determine whether a modified Self-PBQ could be completed by 8- to 12-year-old children with adult assistance. PROCEDURE: The initial phase involved seven children who underwent cognitive debriefing to identify problems in the questionnaire, resulting in modifications to wording and response options. In phase 2, children completed the modified Self-PBQ independently or with assistance from their parent at five Canadian treatment centers. Parents filled out the Self-PBQ separately to serve as a comparison. Bleeding scores derived from the child self-report were compared to those of the parent proxy. RESULTS: Twenty-nine out of 31 patient/parent pairs successfully completed the Self-PBQ. Child and parent scores demonstrated a high level of agreement with an intraclass correlation (ICC) of 0.825. In the age subgroup analysis, the ICC was 0.834 and 0.824 for the 8- to 9-year-old and 10- to 12-year-old groups, respectively. The ICC was also determined in children with type 1 VWD (ICC = 0.829) versus those with more severe bleeding disorders (ICC = 0.802). Thus, age and disease severity had no significant effect on degree of agreement. CONCLUSIONS: Our study shows that agreement was maintained even in younger children aged 8-9 years and in children with varying bleeding phenotypes. This supports the administration of the modified Self-PBQ to 8- to 12-year-old children.
Asunto(s)
Hemorragia/diagnóstico , Tamizaje Masivo/métodos , Instituciones Académicas/estadística & datos numéricos , Autoinforme , Enfermedades de von Willebrand/diagnóstico , Niño , Femenino , Estudios de Seguimiento , Hemorragia/complicaciones , Humanos , Masculino , Pronóstico , Encuestas y Cuestionarios , Enfermedades de von Willebrand/complicacionesRESUMEN
BACKGROUND: Upon platelet activation, a subpopulation of procoagulant platelets is formed, characterized by the exposure of the anionic aminophospholipid phosphatidylserine (PS) on the surface membrane. OBJECTIVE: To evaluate procoagulant PS-exposing platelets by imaging flow cytometry. METHODS: Platelet ultrastructure was examined by transmission electron microscopy, and a comprehensive analysis of procoagulant platelets was performed using imaging flow cytometry; platelets were fluorescently labeled for the markers glycoprotein (GP)IX, activated integrin αIIbß3, CD62P, and PS exposure. RESULTS: A subpopulation of platelets stimulated in suspension by the physiological agonists thrombin+collagen, and all platelets stimulated by the calcium ionophore A23187, had a distinct round morphology. These platelets were PS-exposing, larger in size, had an increased circularity index, and had reduced internal complexity compared with non-PS-exposing platelets. They expressed CD62P and αIIbß3 in an inactive conformation on the surface, and demonstrated depolarized inner mitochondrial membranes. For the first time, using imaging flow cytometry, a large proportion of PS-exposing platelets possessing platelet-associated extracellular vesicles (EVs) was observed, which demonstrated heterogeneous platelet marker expression that was different from free released EVs. CONCLUSIONS: Innovative imaging flow cytometry allowed detailed fluorescence-based, quantitative morphometric analysis of PS-exposing platelets; in becoming procoagulant, platelets undergo remarkable morphological changes, transforming into spherical "balloons," almost devoid of their normal internal architecture. Almost all PS-exposing platelets have associated EVs that are not detectable by traditional flow cytometry. While their functions have yet to be fully elucidated, the heterogeneity of platelet-associated and released EVs suggests that they may contribute to different aspects of hemostasis and of thrombosis.
RESUMEN
Platelet αIIbß3 integrin and its ligands are essential for thrombosis and hemostasis, and play key roles in myocardial infarction and stroke. Here we show that apolipoprotein A-IV (apoA-IV) can be isolated from human blood plasma using platelet ß3 integrin-coated beads. Binding of apoA-IV to platelets requires activation of αIIbß3 integrin, and the direct apoA-IV-αIIbß3 interaction can be detected using a single-molecule Biomembrane Force Probe. We identify that aspartic acids 5 and 13 at the N-terminus of apoA-IV are required for binding to αIIbß3 integrin, which is additionally modulated by apoA-IV C-terminus via intra-molecular interactions. ApoA-IV inhibits platelet aggregation and postprandial platelet hyperactivity. Human apoA-IV plasma levels show a circadian rhythm that negatively correlates with platelet aggregation and cardiovascular events. Thus, we identify apoA-IV as a novel ligand of αIIbß3 integrin and an endogenous inhibitor of thrombosis, establishing a link between lipoprotein metabolism and cardiovascular diseases.