Gene Therapy with Fidanacogene Elaparvovec in Adults with Hemophilia B.

BACKGROUND: Fidanacogene elaparvovec, an adeno-associated virus (AAV) gene-therapy vector for hemophilia B containing a high-activity human factor IX variant (FIX-R338L/FIX-Padua), was associated with sustained factor IX activity in a phase 1-2a study. METHODS: We conducted a phase 3 open-label study of fidanacogene elaparvovec at a dose of 5×1011 vector genome copies per kilogram of body weight. Men 18 to 65 years of age with hemophilia B and a factor IX level of 2% or less were eligible for screening if they had received at least 6 months of therapy with prophylactic factor IX concentrate. The primary end point, tested for noninferiority, was the annualized bleeding rate (treated and untreated bleeding episodes) from week 12 to month 15 after treatment with fidanacogene elaparvovec as compared with the prophylaxis lead-in period. Superiority, additional efficacy end points, and safety were also assessed. RESULTS: Of 316 men who underwent screening for the lead-in study, 204 (64.6%) were not eligible; 188 (59.5%) of those were ineligible owing to the presence of anti-AAV neutralizing antibodies. Of the 45 participants who received fidanacogene elaparvovec, 44 completed at least 15 months of follow-up. The annualized rate of bleeding for all bleeding episodes decreased by 71%, from 4.42 (95% confidence interval [CI], 1.80 to 7.05) at baseline to 1.28 (95% CI, 0.57 to 1.98) after gene therapy, a treatment difference of -3.15 episodes (95% CI, -5.46 to -0.83; P = 0.008). This result shows the noninferiority and superiority of fidanacogene elaparvovec to prophylaxis. At 15 months, the mean factor IX activity was 26.9% (median, 22.9%; range, 1.9 to 119.0) by one-stage SynthASil assay. A total of 28 participants (62%) received glucocorticoids for increased aminotransferase levels or decreased factor IX levels (or both) starting between 11 and 123 days. No infusion-related serious adverse events, thrombotic events, development of factor IX inhibitors, or malignant conditions were observed. CONCLUSIONS: Fidanacogene elaparvovec was superior to prophylaxis for the treatment of participants with hemophilia B, leading to reduced bleeding and stable factor IX expression. (Funded by Pfizer; BENEGENE-2 ClinicalTrials.gov number, NCT03861273.).

Cooperation Between Platelet ß1 and ß3 Integrins in the Arrest of Bleeding Under Inflammatory Conditions in Mice-Brief Report.

BACKGROUND: Platelets prevent bleeding in a variety of inflammatory settings, the adhesion receptors and activation pathways involved being highly context-dependent and functionally redundant. In some situations, platelets recruited to inflammatory sites act independently of aggregation. The mechanisms underlying stable platelet adhesion in inflamed microvessels remain incompletely understood, in particular, whether and if so, how ß1 and ß3 integrins are involved. METHODS: The impact of isolated or combined platelet deficiency in ß1 and ß3 integrins on inflammation-associated hemostasis was investigated in 3 models of acute inflammation: immune complex-based cutaneous reverse passive Arthus reaction, intranasal lipopolysaccharide-induced lung inflammation, and cerebral ischemia-reperfusion following transient (2-hour) occlusion of the middle cerebral artery. RESULTS: Mice with platelet-directed inactivation of Itgb1 (PF4Cre-ß1-/-) displayed no bleeding in any of the inflammation models, while mice defective in platelet Itgb3 (PF4Cre-ß3-/-) exhibited bleeding in all 3 models. Remarkably, the bleeding phenotype of PF4Cre-ß3-/- mice was exacerbated in the reverse passive Arthus model by the concomitant deletion of ß1 integrins, PF4Cre-ß1-/-/ß3-/- animals presenting increased bleeding. Intravital microscopy in reverse passive Arthus experiments highlighted a major defect in the adhesion of PF4Cre-ß1-/-/ß3-/- platelets to inflamed microvessels. Unlike PF4Cre-ß1-/- and PF4Cre-ß3-/- mice, PF4Cre-ß1-/-/ß3-/- animals developed early hemorrhagic transformation 6 hours after transient middle cerebral artery occlusion. PF4Cre-ß1-/-/ß3-/- mice displayed no more bleeding in lipopolysaccharide-induced lung inflammation than PF4Cre-ß3-/- animals. CONCLUSIONS: Altogether, these results show that the requirement for and degree of functional redundancy between platelet ß1 and ß3 integrins in inflammation-associated hemostasis vary with the inflammatory situation.

Sensitivity and specificity of strategies to identify patients with hemostasis abnormalities leading to an increased risk of bleeding before scheduled intervention: the Hemorisk study.

BACKGROUND: Preoperative identification of patients with hemostasis abnormalities leading to an increased bleeding risk is based on routine hemostasis tests: prothrombin time (PT), activated partial thromboplastin time (APTT), and platelet count. Because of their low predictive performance, guidelines recommend replacing them with structured bleeding risk questionnaires, but none is validated in this population. OBJECTIVES: To assess the diagnostic accuracy of 3 strategies, performed at the preanesthesia visit before scheduled interventions, and to identify patients with hemostasis abnormalities leading to an increased bleeding risk METHODS: A multicenter study was performed in 7 French academic hospitals, involving patients scheduled for surgical intervention, without antiplatelet/anticoagulant treatment. The 3 strategies consisted of 1-a structured screening questionnaire; 2-PT, APTT, and platelet count ordered in selected patients; and 3-systematic PT, APTT, and platelet count. The reference standard comprised von Willebrand factor activity/antigen, factor (F)VIII, FIX, FXI, platelet function analyzer, and, when required, FII, FV, FX, and FVII and hemostasis consultation. RESULTS: Eighteen (1.2%) of 1484 patients had a hemostasis abnormality leading to an increased bleeding risk according to reference standard. In the overall cohort, sensitivity of the questionnaire-based strategy was 50% (95% CI, 26%-74%; specificity, 87% [95% CI, 85%-88%]); sensitivity was 0% (95% CI, 0%-41%) in men vs 82% (95% CI, 48%-98%) in women. For selective routine tests, sensitivity was 33% (95% CI, 13%-59%) and specificity 97% (95% CI, 96%-98%). Corresponding values for systematic routine tests were 44% (95% CI, 22%-69%) and 93% (95% CI, 91%-94%). CONCLUSION: Sensitivity was low for all 3 strategies investigated. The structured screening questionnaire had clinically acceptable diagnostic accuracy only in women.

Antithrombin III Levels and Outcomes Among Patients With Trauma.

Importance: Patients with trauma exhibit a complex balance of coagulopathy manifested by both bleeding and thrombosis. Antithrombin III is a plasma protein that functions as an important regulator of coagulation. Previous studies have found a high incidence of antithrombin III deficiency among patients with trauma. Objective: To assess whether changes in antithrombin III activity are associated with thrombohemorrhagic complications among patients with trauma. Design, Setting, and Participants: This cohort study was conducted from December 2, 2015, to March 24, 2017, at a level I trauma center. A total of 292 patients with trauma were followed up from their arrival through 6 days from admission. Data, including quantification of antithrombin III activity, were collected for these patients. Thromboprophylaxis strategy; hemorrhage, deep vein thrombosis (DVT), and pulmonary embolism screenings; and follow-up evaluations were conducted per institutional protocols. Data analyses were performed from September 28, 2023, to June 4, 2024. Main Outcomes and Measures: The primary study outcome measurements were associations between antithrombin III levels and outcomes among patients with trauma, including ventilator-free days, hospital-free days, intensive care unit (ICU)-free days, hemorrhage, venous thromboembolic events, and mortality. Results: The 292 patients had a mean (SD) age of 54.4 (19.0) years and included 211 men (72.2%). Patients with an antithrombin III deficiency had fewer mean (SD) ventilator-free days (27.8 [5.1] vs 29.6 [1.4]; P = .0003), hospital-free days (20.3 [8.2] vs 24.0 [5.7]; P = 1.37 × 10-6), and ICU-free days (25.7 [4.9] vs 27.7 [2.3]; P = 9.38 × 10-6) compared with patients without a deficiency. Antithrombin III deficiency was also associated with greater rates of progressive intracranial hemorrhage (21.1% [28 of 133] vs 6.3% [10 of 159]; P = .0003) and thrombocytopenia (24.8% [33 of 133] vs 5.0% [8 of 159]; P = 1.94 × 10-6). Although antithrombin III deficiency was not significantly associated with DVT, patients who developed a DVT had a more precipitous decrease in antithrombin III levels that were significantly lower than patients who did not develop a DVT. Conclusions and Relevance: In this cohort study of patients with trauma, antithrombin III deficiency was associated with greater injury severity, increased hemorrhage, and increased mortality, as well as fewer ventilator-free, hospital-free, and ICU-free days. Although this was an associative study, these data suggest that antithrombin III levels may be useful in the risk assessment of patients with trauma.

Utility of thromboelastography with platelet mapping for monitoring platelet transfusion in qualitative platelet disorders.

BACKGROUND: Patients with pathogenic variants in RASGRP2 (inherited platelet disorder (IPD)-18) exhibit normal platelet counts but impaired platelet aggregation and αIIbß3 activation. Moderate-to-severe bleeding episodes require patients to be transfused with platelets and/or pro-hemostatic agents. We recently demonstrated that hemostatic efficacy of transfused platelets is limited by dysfunctional endogenous platelets in a mouse model of IPD-18 (Rasgrp2-/- mice), as dysfunctional platelets were recruited to the forming hemostatic plug but did not participate in clot contraction. Thus, higher amounts of transfused platelets were required to outcompete these dysfunctional cells and to reverse bleeding. OBJECTIVE: We here studied the usefulness of thromboelastography with platelet mapping (TEG-PM) for ex vivo monitoring of the hemostatic potential in Rasgrp2-/- mice transfused with various amounts of wild-type (WT) platelets. METHODS: Whole blood (WB) samples from WT and Rasgrp2-/- mice were tested in TEG-PM and parameters for clot formation and contraction (K time, α-angle, maximum amplitude [MA]) were measured. RESULTS: Rasgrp2-/- WB samples did not contract in TEG-PM, consistent with a critical role of this protein in αIIbß3 activation. Addition of WT platelets improved TEG parameters in a ratio-dependent manner, consistent with our recent in vivo studies showing impaired hemostasis at a 5:1, but not at a 2:1 ratio of mutant to WT platelets. K and α values were identified as better predictors of transfusion efficacy than MA, the most platelet-dependent TEG parameter. CONCLUSION: This proof-of-concept study supports the use of TEG-PM to monitor platelet transfusion ratios and hemostatic potential in IPD-18 and potentially other platelet disorders.

Type 2N von Willebrand disease: genotype drives different bleeding phenotypes and treatment needs.

BACKGROUND: Type 2 Normandy von Willebrand disease (VWD2N) is usually perceived as a mild bleeding disorder that can be treated with desmopressin (DDAVP). However, VWD2N patients can be compound heterozygous or homozygous for different variants, with p.Arg854Gln (R854Q) being the most frequent causative one. There are limited data about the impact of 2N variants on VWD2N phenotype and DDAVP response. OBJECTIVES: This study aims to describe the phenotype of VWD2N, including DDAVP response, according to genotype. METHODS: VWD2N patients with a complete genotype/phenotype characterization by the French reference center for VWD, including MCMDM-1VWD bleeding score, were eligible to be included in the study. Results of the DDAVP trial were also collected. RESULTS: A total of 123 VWD2N patients from the French registry were included in this study. Results were stratified according to the presence (R854QPos, n = 114) or absence (R854QNeg, n = 9) of at least 1 R854Q allele. Three R854QPos subgroups were further individualized: patients homozygous (R854QHmz, n = 55), compound heterozygous for R854Q and a null allele (R854Q/3, n = 48), or compound heterozygous for R854Q and another 2N variant (R854Q/2N, n = 11). FVIII: C levels were significantly lower in R854QNeg and R854Q/3 patients compared with R854QHmz ones (P < .001 and P < .0001, respectively). R854QNeg patients were diagnosed earlier due to bleeding symptoms and had a higher bleeding score than R854QPos patients (P < .001). In DDAVP trial, FVIII:C survival was lower in VWD type 2N than in type 1 patients. R854QPos patients had a heterogeneous DDAVP response, which was best predicted by baseline FVIII:C level. CONCLUSION: The heterogeneous genetic background of VWD2N drives different bleeding phenotypes and response patterns to DDAVP, underlining the clinical relevance of DDAVP trial to identify patients potentially eligible to alternative therapeutic options.

Characterization of recombinant factor IX fusion proteins enabling subcutaneous administration.

BACKGROUND: The X-linked bleeding disorder hemophilia B, caused by mutation(s) in the coagulation factor (F)IX gene, leads to partial or total loss of its function, requiring lifelong FIX replacement therapy. Although new recombinant FIX (rIX) therapeutics like albumin fusion proteins (rIX-FP) enable longer plasma half-life and thus less frequent administration, the complexity of intravenous (i.v.) injection remains. OBJECTIVES: The study aimed to characterize rIX-FP variants with anticipated enhanced specific activity, which would leverage rIX-FP's superior pharmacokinetic profile with beneficial characteristics for subcutaneous (s.c.) administration. METHODS: Two rIX-FP variants, R338L ("Padua variant") and R338L/E410K, were characterized in vitro. Pharmacokinetic profiles of FIX antigen and activity levels were evaluated in FIX-deficient mice after i.v. and s.c. administration of these variants (dosing based on antigen levels). The efficacy of the most promising variant was tested after i.v. and s.c. administration (dosing based on activity) in a tail clip bleeding model. A marketed wild-type (WT) rIX-FP product served as the comparator. RESULTS: Both rIX-FP variants showed a 4- to 5-fold increase in specific activity in vitro compared with rIX(WT)-FP, while FXIa-mediated activation was the fastest for rIX(WT)-FP and rIX(R338L)-FP. Compared with rIX(WT)-FP and rIX(R338L/E410K)-FP, rIX(R338L)-FP exhibited higher FIX activity exposure after i.v. and s.c. administration and demonstrated comparable efficacy with rIX(WT)-FP in reducing bleeding time and blood loss in FIX-deficient mice requiring ∼4 times lower protein amount. CONCLUSION: rIX(R338L)-FP was shown to be a promising candidate for s.c. administration, exhibiting increased specific activity combined with higher activity-based exposure and indicating efficacy at a lower protein dose.

Low Remnant Cholesterol and In-Hospital Bleeding Risk After Ischemic Stroke or Transient Ischemic Attack.

BACKGROUND: Bleeding risk brought by intensive lipid-lowering therapy and low low-density lipoprotein cholesterol is concerning, while evidence regarding the relationship between remnant cholesterol and bleeding is frightening. This study aimed to investigate the association between remnant cholesterol at admission and an in-hospital bleeding event after acute ischemic stroke or transient ischemic attack (TIA). METHODS AND RESULTS: A total of 3222 eligible patients admitted to Shanghai Huashan Hospital between 2015 and 2021 with complete lipid data were analyzed. Patients were classified into low (<20.0 mg/dL), moderate (20.0-29.9 mg/dL), and high (≥30 mg/dL) groups by remnant cholesterol. The mean age of patients was 63.0± 13.1 years, including 2301 (71.4%) men and 651 (20.2%) with TIA. The median (interquartile range) of remnant cholesterol was 18.6 (13.5-25.9) mg/dL. After adjustment for confounding variables, patients with low remnant cholesterol had a higher risk of bleeding events (odds ratio, 2.56 [95% CI, 1.12-6.67]) than those with moderate remnant cholesterol. The high remnant cholesterol group was not significantly associated with bleeding risk. Combined assessment of low-density lipoprotein cholesterol and remnant cholesterol further identified patients with the highest risk of bleeding events. CONCLUSIONS: Low remnant cholesterol levels were associated with bleeding events during the acute stage of ischemic stroke and TIA. The assessment of remnant cholesterol could inform the bleeding risk during hospitalization both for patients and physicians in clinical practice.

International Society on Thrombosis and Haemostasis clinical practice guideline for treatment of congenital hemophilia A and B based on the Grading of Recommendations Assessment, Development, and Evaluation methodology.

BACKGROUND: Hemophilia is a rare congenital bleeding disorder that results from complete or partial deficiency of blood coagulation factor (F)VIII (hemophilia A) or FIX (hemophilia B) due to pathogenic variants in their coding genes. Hemophilia requires complex management. To date, there is no evidence-based clinical practice guideline on hemophilia treatment based on the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach. OBJECTIVES: This evidence-based clinical practice guideline from the International Society on Thrombosis and Haemostasis aims to provide an overview of evidence and support patients, caregivers, hematologists, pediatricians, other clinicians, researchers, and stakeholders in treatment decisions about congenital hemophilia A and B. METHODS: The International Society on Thrombosis and Haemostasis formed a multidisciplinary guideline panel of physicians and patients with global representation, balanced to minimize potential bias from conflicts of interest. The panel prioritized a set of clinical questions and outcomes according to their importance for clinicians and patients. A methodological team supported the guideline development process, including searching for evidence and performing systematic reviews. The GRADE approach was used, including GRADE Evidence to Decision frameworks. The recommendations were subject to public comment. RESULTS: The panel selected 13 questions, of which 11 addressed the treatment of hemophilia A and 2 the treatment of hemophilia B. Specifically, the panel addressed questions on prophylactic and episodic treatment with FVIII concentrates, bypassing agents, and nonfactor therapy (emicizumab) for hemophilia A (with and without inhibitors) as well as immune tolerance induction for hemophilia A. For hemophilia B, the panel addressed questions on prophylactic and episodic treatment of bleeding events with FIX concentrates. Agreement was reached for all 13 recommendations, of which 7 (54%) were based on evidence from randomized clinical trials, 3 (23%) on observational studies, and 3 (23%) on indirect comparisons. CONCLUSION: Strong recommendations were issued for prophylactic over episodic treatment for severe and moderately severe hemophilia A and B. Only conditional recommendations were issued for the remaining questions. Future research should focus on direct treatment comparisons and the treatment of hemophilia B with and without inhibitors. Future updates of this guideline will provide an updated evidence synthesis on the current questions and focus on new FVIII and FIX concentrates, novel nonfactor therapies, and gene therapy for severe and nonsevere hemophilia A and B.

Bleeding phenotype according to factor level in 825 children with nonsevere hemophilia: data from the PedNet cohort.

BACKGROUND: Information on bleeding phenotype in nonsevere hemophilia may be used to determine target factor levels for prophylaxis or gene therapy in severe hemophilia. OBJECTIVES: To assess the association between endogenous factor level and bleeding phenotype in children with nonsevere (factor [F]VIII/FIX activity 1%-25%) hemophilia A (HA) and B without prophylaxis. METHODS: Data on annualized bleeding rate (ABR), annualized joint bleeding rate (AJBR), and onset of bleeding were extracted from the international PedNet cohort including children born since 2000. Mean ABR and AJBR were modeled and compared according to FVIII/FIX endogenous activity (1%-2%, 3%-5%, 6%-10%, 11%-15%, 16%-20%, and 21%-25%) using negative binomial regression. Onset of bleeding was analyzed using Kaplan-Meier survival curves. RESULTS: Eight hundred twenty-five children (40% with moderate hemophilia; 87% with HA) with median follow-up of 7.4 years/child were included. The median age at onset of bleeding and median bleeding rates changed with increasing endogenous activity. From endogenous FVIII 1% to 2% to 21% to 25%, the age at onset of bleeding changed from a median of 1.4 to 14.2 years, ABR from 1.6 to 0.1/y, and AJBR from 0.5 to 0.0/y. From endogenous FIX 1% to 2% to 16% to 25%, the onset of bleeding changed from a median of 1.7 to 6.1 years, ABR from 0.5 to 0.1/y, and AJBR from 0.1 to 0.0/y. The negative correlation between AJBR and factor level was most strongly pronounced up to a factor level of 6% in HA and hemophilia B. CONCLUSION: Endogenous factor activity of >5% was identified as a threshold to significantly lower joint bleeding rate, while FVIII levels >15% and FIX levels >10% were sufficient to achieve the goal of 0 bleeds in this pediatric cohort.

Examining variability in the diagnosis and management of people with bleeding disorders of unknown cause: communication from the ISTH SSC Subcommittee on von Willebrand Factor.

BACKGROUND: Bleeding disorder of unknown cause (BDUC) is characterized by a bleeding phenotype in the setting of normal hemostatic testing. No standardized diagnostic criteria or treatment algorithms exist for people with BDUC. To address the unmet need, the International Society on Thrombosis and Haemostasis von Willebrand Factor Scientific Subcommittee performed a real-world survey aimed at addressing knowledge gaps, developing consensus pathways, and ultimately improving care. OBJECTIVES: We sought to determine current international clinical practices in the investigation, registration, and treatment of people with BDUC internationally. METHODS: An online structured survey was conducted of healthcare providers who managed patients with bleeding disorders using the ISTH RedCap tool. RESULTS: Two hundred sixteen respondents from 39 countries were included in the final analysis. The clinical assessment of those with a possible bleeding disorder varied, with only 55% excluding hypermobility but high levels (80%) of bleeding assessment tool usage. In hemostatic testing, only the prothrombin time and activated partial thromboplastin time tests gained universal support. Tranexamic acid was favored for prophylaxis for minor (71%)/major (59%) surgeries and pregnancy (58%), but advice on the treatment if bleeding occurred was heterogeneous. The management of heavy menstrual bleeding in women despite combined oral contraceptive pill use also proved challenging, with healthcare providers selecting multiple alternative strategies. CONCLUSION: Significant variation exists in the recognition, registration, and management of people with BDUC worldwide. This survey emphasizes the need for consensus pathways to diagnose and treat BDUC to standardize and improve care for patients internationally.

Changes in the hematobiochemical, acid-base and blood gas elements as well as biomarkers of inflammation and bone metabolism in donkeys (Equus asinus) with acute bleeding.

Background: Acute hemorrhage is fatal in equines with a complication of severe hypovolemic shock that causes a sudden death in such cases. Aim: This study was designed to report the influences of acute bleeding in conscious non-sedated donkeys (Equus asinus) on the hematobiochemical variables, acid-base, blood gas elements, and markers of inflammation and bone metabolism. Methods: Eight healthy donkeys were used where a total of 900 ml of whole blood was collected. Five blood samples were collected from each animal: just before collection of blood (T0); (2) 30 (T1), 60 (T2), 120 (T3), and 240 minutes (T4) later. The blood panels including total white blood cells, lymphocytes, neutrophils, red blood cell counts (RBCs), HCT, hemoglobin (Hg), and RBCs indices were measured. Biochemical parameters and electrolytes were evaluated. The activity of aspartate aminotransferase (AST), creatine kinase (CK), and γ-glutamyl transferase (GGT) were also determined. Complete acid-base and blood gas panels were assessed. Serum amyloid A (SAA), haptoglobin (Hp), osteocalcin (OC), bone alkaline phosphatase (b-ALP), and pyridinoline cross-links (PYD) were measured. Results: The RBCs, Hg, and HCT increased significantly at points T1, T2, and T3 compared to T0. The concentrations of total proteins and albumin decreased significantly at points T3 and T4. The blood urea nitrogen concentrations increased significantly at T4. Creatinine concentrations increased significantly at T2 and T3. The AST, GGT, and CK decreased significantly. On the other hand, glucose increased significantly at T3 and T4. The pH decreased significantly at points T1, T2, T3, and T4. The PCO2 increased significantly at T3 and T4. The BE, HCO3, and TCO2 values decreased significantly at T2, T3, and T4. Contrary, the AG increased significantly at points T3 and T4. The potassium increased significantly at T1-T4 and chloride decreased significantly at T3 and T4. Lactate showed significant increases at T1-T4. The SAA, Hp, OC, b-ALP, and PYD did not differ significantly at T1-T4. Conclusion: In conscious non-sedated donkeys, induced bleeding resulted in significant changes in the hematobiochemical elements, the acid-base status, and blood gas and electrolyte parameters. However, it did not change the markers of inflammation and bone metabolism.

Deletion of tissue factor pathway inhibitor isoform beta or gamma, but not alpha, improves clotting in hemophilic mice.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) regulates tissue factor-triggered coagulation. Humans and mice express transcripts encoding for multidistributed (endothelial, platelet, and plasma) 3-Kunitz domain TFPIα and endothelial membrane-anchored 2-Kunitz TFPIß. Mice express a third transcript, γ, that encodes plasma lipoprotein-associated 2-Kunitz TFPI. In humans, proteolysis of α and/or ß produces plasma lipoprotein-associated 2-Kunitz TFPI at lower levels. In clinical trials, monoclonal antibodies that target all TFPI isoforms extend coagulation and correct bleeding in hemophilic patients but with some thrombosis risks. OBJECTIVES: To determine the impact of TFPI isoform-specific deletions on promoting clotting in hemophilic mice. METHODS: Engineered TFPI isoform-specific, hemophilic (factor VIII-null) mice were evaluated for clotting. RESULTS: Mice expressing any single TFPI isoform were healthy. Thrombin generation assays identified TFPIγ as the dominant anticoagulation isoform in mouse plasma. Hemostasis was assessed by serial bleeding times from a tail vein laceration. Repeatedly, after a clot forms, it was manually disrupted; the number of clots/disruptions occurring over a 15-minute period were reported. C57BL/6 and hemophilic mice clot on average 25.6 vs 5.4 times, respectively. On a hemophilia background, TFPIß or TFPIγ-specific deletion improved clotting to 14.6 and 15.2 times, respectively (P < .0001). TFPIα-specific deletion was without impact, clotting 5.1 times. Heterozygous deletion of TFPIß was effective, clotting 11.8 times (P < .0001). Heterozygous deletion of TFPIα or TFPIγ alone was ineffective, clotting 3.0 and 6.1 times, respectively, but heterozygous TFPIαγ deletion improved clotting to 11.2 times (P < .001). CONCLUSION: In hemophilic mice, endothelial TFPIß and plasma γ-derived 2-Kunitz TFPI individually contribute more to bleeding than total TFPIα.

Positive antiphospholipid antibodies increase the risk of ischemic stroke in patients with atrial fibrillation.

BACKGROUND: Antiphospholipid antibodies (aPL), including lupus anticoagulant, antibodies against ß2 glycoprotein I (anti-ß2GPI), and anticardiolipin (aCL) antibodies are associated with ischemic stroke (IS). Their prevalence and clinical relevance in atrial fibrillation (AF) remain unclear. OBJECTIVES: To assess whether aPL are associated with increased risk of IS in AF patients despite anticoagulation. METHODS: We conducted a post hoc analysis of aPL using blood samples from 243 consecutive AF patients enrolled in a cohort study. Markers of a prothrombotic state, including endogenous thrombin potential, fibrin clot permeability, and lysis time, were measured at baseline. During a median follow-up of 52 months, IS/transient ischemic attack and major bleeding were recorded. RESULTS: We observed aPL at a moderate or high titer in 51 (21%) patients, including 17 (7%) with anti-ß2GPI, 19 (7.8%) with aCL antibodies, and 37 (15.2%) with lupus anticoagulant. aPL-positive patients were more likely to have prior stroke (P = .01) and be active smokers (P = .03), along with increased endogenous thrombin potential (P = .02), without any changes in fibrin clot properties. Anti-ß2GPI (hazard ratio, 4.38; 95% CI, 1.58-12.19) and aCL (hazard ratio, 4.70; 95% CI, 1.80-12.30) at a moderate or high titer were associated with IS during follow-up (n = 20; 1.9% per year). There were 23 major bleedings (2.1% per year) and 20 deaths (1.9% per year), which were not associated with aPLs. CONCLUSION: Our study showed a relatively high prevalence of aPL positivity in AF patients, which was linked to an increased risk of IS/transient ischemic attack. This suggests that screening for aPL might help optimize anticoagulant therapy in such patients.

Acquired von Willebrand syndrome during extracorporeal membrane oxygenation support: a comprehensive review of current evidence: communication from the ISTH SSC on perioperative and critical care thrombosis and hemostasis.

During extracorporeal membrane oxygenation (ECMO) support, the high shear stress in the ECMO circuit results in increased proteolysis of von Willebrand factor (VWF), loss of VWF high-molecular-weight multimers, and impaired ability to bind to platelets and collagen. These structural changes in VWF are consistent with acquired von Willebrand syndrome (AVWS) type 2A and may contribute to the bleeding diathesis frequently observed in ECMO patients. We performed a systematic review of all clinical studies evaluating the prevalence and associated outcomes of AVWS in ECMO patients. Our findings suggest that almost all ECMO patients develop partial or complete loss of VWF high-molecular-weight multimers within a few hours of device implantation. The AVWS persists as long as the patient is supported by ECMO. Weaning from ECMO rapidly and completely resolves the AVWS. Nevertheless, few studies have reported bleeding outcomes in ECMO patients with AVWS, and the extent to which AVWS contributes to the bleeding diathesis during ECMO support cannot be determined by current evidence. Data supporting the use of VWF concentrates to prevent bleeding complications in ECMO patients remain limited.

Prothrombin and activated partial thromboplastin times, thromboelastography, hematocrit, and platelet count in a feline hemorrhage/over-resuscitation model using lactated Ringer's solution or 6% tetrastarch 130/0.4.

OBJECTIVE: To describe and compare prothrombin time (PT), activated partial thromboplastin time (aPTT), thromboelastography (TEG), HCT, and platelet count measurements in a hemorrhage/over-resuscitation model. DESIGN: Randomized crossover study. SETTING: University teaching hospital. ANIMALS: Six cats. INTERVENTIONS: Anesthetized cats underwent 3 treatments at 2-month intervals. The treatments were as follows: NHR-no controlled hemorrhage and sham resuscitation; LRS-controlled hemorrhage and lactated Ringer's solution (LRS) for resuscitation; and Voluven-controlled hemorrhage and 6% tetrastarch 130/0.4 for resuscitation. The LRS and Voluven were administered at 60 and 20 mL/kg/h, respectively, for 120 minutes. Blood samples were drawn for PT, aPTT, TEG, HCT, and platelet count measurements at a healthy check (T - 7d), after controlled hemorrhage (T0), at 60 and 120 minutes of resuscitation (T60 and T120), and at 24 hours after completion of resuscitation (T24h). Data were analyzed using a general linear mixed model approach (significance was P < 0.05). MEASUREMENTS AND MAIN RESULTS: Total median blood loss (controlled hemorrhage and blood sampling from T0 to T120) at T120 was 11.4, 31.0, and 30.8 mL/kg for NHR, LRS, and Voluven, respectively. PT and aPTT during LRS and Voluven were prolonged at T60 and T120 compared to NHR (P < 0.001). On TEG, the reaction time, kinetic time, and alpha-angle were within reference intervals for cats at all time points in all treatments, while maximum amplitude was less than the reference interval (40 mm) at T0, T60, and T120 during Voluven and at T60 and T120 during LRS compared to NHR (both P < 0.001). The HCT and platelet count were significantly lower at T60 and T120 during LRS and Voluven compared to NHR (P < 0.001). CONCLUSIONS: Hypocoagulopathy was observed during hemorrhage and liberal fluid resuscitation. Prolongation of PT and aPPT and decreased clot strength may have been caused by hemodilution and platelet loss.

Predictors of bleeding and thrombotic events among patients admitted to the hospital with COVID-19 and elevated D-dimer: insights from the ACTION randomized clinical trial.

Therapeutic anticoagulation showed inconsistent results in hospitalized patients with COVID-19 and selection of the best patients to use this strategy still a challenge balancing the risk of thrombotic and hemorrhagic outcomes. The present post-hoc analysis of the ACTION trial evaluated the variables independently associated with both bleeding events (major bleeding or clinically relevant non-major bleeding) and the composite outcomes thrombotic events (venous thromboembolism, myocardial infarction, stroke, systemic embolism, or major adverse limb events). Variables were assessed one by one with independent logistic regressions and final models were chosen based on Akaike information criteria. The model for bleeding events showed an area under the curve of 0.63 (95% confidence interval [CI] 0.53 to 0.73), while the model for thrombotic events had an area under the curve of 0.72 (95% CI 0.65 to 0.79). Non-invasive respiratory support was associated with thrombotic but not bleeding events, while invasive ventilation was associated with both outcomes (Odds Ratio of 7.03 [95 CI% 1.95 to 25.18] for thrombotic and 3.14 [95% CI 1.11 to 8.84] for bleeding events). Beyond respiratory support, creatinine level (Odds Ratio [OR] 1.01 95% CI 1.00 to 1.02 for every 1.0 mg/dL) and history of coronary disease (OR 3.67; 95% CI 1.32 to 10.29) were also independently associated to the risk of thrombotic events. Non-invasive respiratory support, history of coronary disease, and creatinine level may help to identify hospitalized COVID-19 patients at higher risk of thrombotic complications.ClinicalTrials.gov: NCT04394377.

The historical origins of modern international normalized ratio targets.

Prothrombin time (PT) and its derivative international normalized ratio (INR) are frequently ordered to assess the coagulation system. Plasma transfusion to treat incidentally abnormal PT/INR is a common practice with low biological plausibility and without credible evidence, yet INR targets appear in major clinical guidelines and account for the majority of plasma use at many institutions. In this article, we review the historical origins of INR targets. We recount historical milestones in the development of the PT, discovery of vitamin K antagonists (VKAs), motivation for INR standardization, and justification for INR targets in patients receiving VKA therapy. Next, we summarize evidence for INR testing to assess bleeding risk in patients not on VKA therapy and plasma transfusion for treating mildly abnormal INR to prevent bleeding in these patients. We conclude with a discussion of the parallels in misunderstanding of historic PT and present-day INR testing with lessons from the past that might help rationalize plasma transfusion in the future.

Bleeding risk in hemophilia A and B carriers: comparison of factor levels determined using chronometric and chromogenic assays.

BACKGROUND: Predicting the bleeding risk in hemophilia A and B carriers (HAC, HBC) is challenging. OBJECTIVE: The objectives of this study were to describe the bleeding phenotype in HAC and HBC using the standardized Tosetto bleeding score (BS); to determine whether the BS correlates better with factor levels measured with a chromogenic assay than with factor levels measured with chronometric and thrombin generation assays; and to compare the results in HAC and HBC. METHODS: This ambispective, noninterventional study included obligate and sporadic HAC and HBC followed at a hemophilia treatment center between 1995 and 2019. RESULTS AND CONCLUSION: The median BS (3, range 0-21 vs. 3.5, range 0-15, P  = ns, respectively) and the abnormal BS rate (35.6% vs. 38.2%, P  = ns) were not significantly different in 104 HAC and 34 HBC (mean age: 38 years, 6-80 years). However, some differences were identified. The risk of factor deficiency was higher in HBC than HAC. Specifically, Factor VIII activity (FVIII):C/Factor IX activity (FIX):C level was low (<40 IU/dl) in 18.3% (chronometric assay) and 17.5% (chromogenic assay) of HAC and in 47% and 72.2% of HBC ( P  < 0.001). Moreover, the FIX:C level thresholds of 39.5 IU/dl (chronometric assay) and of 33.5 IU/dl (chromogenic assay) were associated with very good sensitivity (92% and 100%, respectively) and specificity (80% for both) for bleeding risk prediction in HBC. Conversely, no FVIII:C level threshold could be identified for HAC, probably due to FVIII:C level variations throughout life.