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

BACKGROUND: Von Willebrand disease type 2N (VWD2N) is usually perceived as a mild bleeding disorder that can be treated by desmopressin (DDAVP). However, VWD2N-patients can be compound heterozygous or homozygous for different variants, p.Arg854Gln (R854Q) being the most frequent causative one. There is limited data about the impact of 2N-variants on VWD2N phenotype and DDAVP-response. OBJECTIVES: To describe the phenotype of VWD2N, including DDAVP-response, according to genotype. PATIENTS/METHODS: VWD2N-patients with a complete genotype/phenotype characterization by the French reference center for VWD, including MCMDM-1VWD bleeding score (BS) were eligible to the study. Results of 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 one 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 to R854QHmz-ones (p<0.001 and p<0.0001 respectively). R854QNeg-patients were diagnosed earlier due to bleeding symptoms and had a higher BS than R854QPos-patients (p<0.001). In DDAVP-trial, FVIII:C survival was lower in VWD type 2N than in type 1. 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.

Key Drivers of Coagulation Factor Use in Von Willebrand Disease During Hospitalization: An Overview of the French BERHLINGO Cohort.

BACKGROUND: Von Willebrand disease (VWD) is the most common inherited bleeding disorder. However, studies of hospitalisation patterns with replacement treatment are scarce. OBJECTIVES: The aim of this study was to investigate the current therapeutic management of VWD and determine the key drivers of coagulation factor uses in patients during hospitalisation. METHODS: Hopscotch-WILL was a multi-centric retrospective study conducted over a 48-month period in any patients with VWD. The data were collected from the BERHLINGO Research Database and the French Hospital database. RESULTS: A total of 988 patients were included; 153 patients (15%) were hospitalised during 293 stays requiring treatment with von Willebrand factor (VWF) concentrates-pure or in association with Factor VIII (FVIII). Their median basal concentrations of VWF and FVIII were significantly lower than in untreated patients: VWF antigen < 30 IU/dL, VWF activity < 20 IU/dL and FVIII:C < 40 IU/dL. The median (interquartile range) concentrate consumption was similar between highly purified VWF or VWF combined with FVIII (72 [110] vs 57 [89] IU/kg/stay, p = 0.154). The use of VWF was highly heterogeneous by VWD type; type 3 had a particularly high impact on VWF consumption in non-surgical situations. The main admissions were for ear/nose/throat, hepato-gastroenterology, and trauma/orthopaedic conditions, besides gynaecological-obstetric causes in women. CONCLUSIONS: The use of VWF concentrates is mostly influenced by low basal levels of VWF and FVIII, but also by VWD type or the cause for hospitalisation. These results could inform future studies of newly released recombinant VWF.

Efficacy and safety of turoctocog alfa in patients with hemophilia A requiring surgical procedures: A multicentre retrospective study.

BACKGROUND: Turoctocog alfa is a recombinant Factor VIII used in patients with hemophilia A. The aim is to assess the real-life evidence of turoctocog alfa in surgery. STUDY DESIGN AND METHODS: Data were extracted from a national database. RESULTS: Turoctocog alfa was used for 86 surgeries (49 major and 37 minor) in 56 patients. The results are expressed as medians (interquartile range). Six (10.7%) patients had severe hemophilia A, four (7.1%) moderate, and 46 (82.2%) mild. For patients who underwent major surgeries, basal plasma FVIII coagulant activity (FVIII:C) levels were 15 IU.dL-1 (8-22). Eight (5-14) infusions were given, at a preoperative loading dose of 40.0 (35.0-45.5) IU.kg-1 and a total dose of 253.3 (125.0-507.0) IU.kg-1 . In patients who underwent minor surgeries, basal FVIII:C levels were 18 IU.dL-1 (9-31). Two (1-3) infusions were required, at a preoperative loading dose of 34.0 (28.8-38.5) IU.kg-1 and a total dose of 73.7 (37.6-122.1) IU.kg-1 . The overall clinical efficacy was judged excellent/good in 77 procedures (89.5%) and fair/poor in nine (10.5%). The fair/poor efficacy concerned seven patients (six mild hemophilia and one severe), for four urological surgeries, two dermatological procedures, one heart surgery, one ear-nose-throat procedure, and one dental avulsion in the patient with severe hemophilia. Three out of those seven patients received antiplatelet therapy. No thromboembolic events, anti-FVIII antibodies, or adverse events were reported. DISCUSSION: The efficacy and safety of turoctocog alfa were confirmed for the management of surgery in patients with hemophilia A. No adverse events were observed and overall efficacy was good.

Prothrombin consumption as an indicator of hemorrhagic phenotype in mild platelet function disorders.

BACKGROUND: The bleeding risk of patients with mild platelet function disorders is difficult to assess and their phenotype remains ill-explored. AIM: This study was designed to establish a comprehensive biological phenotype of patients with mild platelet function disorders. METHODS: Twenty patients were included with persistent abnormal light transmission aggregometry (LTA). The ISTH bleeding assessment tool (ISTH-BAT) was assessed to identify laboratory analyses associated with an abnormal hemorrhagic score. RESULTS: The majority of patients had defects that might affect Gαi protein signaling pathways or minor abnormalities. No LTA nor flow cytometry parameters were associated with an above-normal hemorrhagic score. However, prothrombin consumption, which corresponds to the ratio of serum residual factor II to plasma residual factor II, was significantly higher (p = .006) in the abnormal ISTH-BAT group (mean = 14%, SD = 6) compared with the normal ISTH-BAT group (mean = 8%, SD 4). Prothrombin consumption was significantly associated with ISTH-BAT score (r = .5287, IC 95% 0.0986-0.7924, p = .0165). CONCLUSION: In this group of patients, there was an association between a pathological bleeding score and increased prothrombin consumption. This test could be used as an additional indicator of platelet function abnormality liable to be related to bleeding risk.

Biological, clinical features and modelling of heterozygous variants of glycoprotein Ib platelet subunit alpha (GP1BA) and glycoprotein Ib platelet subunit beta (GP1BB) genes responsible for constitutional thrombocytopenia.

Constitutional thrombocytopenias are rare disorders, often difficult to discriminate from acquired thrombocytopenias. More than 80 genes have been described as being at the origin of these diseases. Among them, several variants of the glycoprotein Ib platelet subunit alpha (GP1BA) and glycoprotein Ib platelet subunit beta (GP1BB) genes, coding for the GpIb-IX-V glycoprotein complex, have been reported in the literature. The study reported here aimed at describing newly identified monoallelic anomalies affecting the GP1BA and GP1BB genes on a clinical, biological and molecular level. In a cohort of nine patients with macrothrombocytopenia, eight heterozygous variants of the GP1BA or GP1BB genes were identified. Five of them had never been described in the heterozygous state. Computer modelling disclosed structure/function relationships of these five variants.

FVIII regulates the molecular profile of endothelial cells: functional impact on the blood barrier and macrophage behavior.

Hemophilia A is an inherited X-linked recessive bleeding disorder caused by deficient activity of blood coagulation factor VIII (FVIII). In addition, hemophilia patients show associated diseases including osteopenia, altered inflammation and vascular fragility which may represent the consequence of recurrent bleeding or may be related to the direct FVIII deficiency. Nowadays, recombinant FVIII is proposed to treat hemophilia patients with no circulating FVIII inhibitor. Initially described as a coenzyme to factor IXa for initiating thrombin generation, there is emerging evidence that FVIII is involved in multiple biological systems, including bone, vascular and immune systems. The present study investigated: (i) the functional activities of recombinant human FVIII (rFVIII) on endothelial cells, and (ii) the impact of rFVIII activities on the functional interactions of human monocytes and endothelial cells. We then investigated whether rFVIII had a direct effect on the adhesion of monocytes to the endothelium under physiological flow conditions. We observed that direct biological activities for rFVIII in endothelial cells were characterized by: (i) a decrease in endothelial cell adhesion to the underlying extracellular matrix; (ii) regulation of the transcriptomic and protein profiles of endothelial cells; (iii) an increase in the vascular tubes formed and vascular permeability in vitro; and (iv) an increase in monocyte adhesion activated endothelium and transendothelial migration. By regulating vascular permeability plus leukocyte adhesion and transendothelial migration, the present work highlights new biological functions for FVIII.

FVIII at the crossroad of coagulation, bone and immune biology: Emerging evidence of biological activities beyond hemostasis.

Hemophilia A is an X-linked hereditary disorder that results from deficient coagulation factor VIII (FVIII) activity, leading to spontaneous bleeding episodes, particularly in joints and muscles. FVIII deficiency has been associated with altered bone remodeling, dysregulated macrophage polarization, and inflammatory processes that are associated with the neoformation of abnormal blood vessels. Treatment based on FVIII replacement can lead to the development of inhibitors that render FVIII concentrate infusion ineffective. In this context, hemophilia has entered a new therapeutic era with the development of new drugs, such as emicizumab, that seek to restore the hemostatic balance by bypassing pathologically acquired antibodies. We discuss the potential extrahemostatic functions of FVIII that may be crucial for defining future therapies in hemophilia.

Platelet features allow to differentiate immune thrombocytopenia from inherited thrombocytopenia.

Immune thrombocytopenia (ITP) is an acquired bleeding disorder, for which no specific diagnostic test exists. Inherited thrombocytopenia (IT) can mimic ITP and lead to unappropriated management with significant morbidity. Here, in small cohorts of these two disorders, we explored whether platelet sialylation and platelet activation could allow to discriminate the two conditions. We also aimed to confirm the value of immature platelet counts in this discrimination. Platelet sialylation and the expression level of P-selectin were assessed by multiparameter flow cytometry. Immature platelets were estimated on a Sysmex XN 9000 analyzer. No significant difference in platelet sialylation was observed between ITP and IT. Contrarily, platelet activation was significantly higher in ITP patients (p = 0.008). The immature platelet fraction, as previously demonstrated, was significantly lower in the ITP group compared to the IT group (p = 0.014). That statistical significance was achieved in this small pilot study suggests that the two easily available assays of immature platelet count and P-selectin expression could help physicians to reach the proper diagnosis in complex cases of thrombocytopenia.

Heparin Anti-Xa Activity, a Readily Available Unique Test to Quantify Apixaban, Rivaroxaban, Fondaparinux, and Danaparoid Levels.

BACKGROUND: Despite their usefulness in perioperative and acute care settings, factor-Xa inhibitor-specific assays are scarcely available, contrary to heparin anti-Xa assay. We assessed whether the heparin anti-Xa assay can (1) be used as a screening test to rule out apixaban, rivaroxaban, fondaparinux, and danaparoid levels that contraindicate invasive procedures according to current guidelines (>30 ng·mL-1, >30 ng·mL-1, >0.1 µg·mL-1, and >0.1 IU·mL-1, respectively), (2) quantify the anticoagulant level if found significant, that is, if it exceeded the abovementioned threshold. METHODS: In the derivation cohort then in the validation cohort, via receiver operating characteristics (ROC) curve analysis, we evaluated the ability of heparin anti-Xa assay to detect levels of factor-Xa inhibitors above or below the abovementioned safety thresholds recommended for an invasive procedure (screening test). Among samples with relevant levels of factor-Xa inhibitor, we determined the conversion factor linking the measured level and heparin anti-Xa activity in a derivation cohort. In a validation cohort, the estimated level of each factor-Xa inhibitor was thus inferred from heparin anti-Xa activity. The agreement between measured and estimated levels of factor-Xa inhibitors was assessed. RESULTS: Among 989 (355 patients) and 756 blood samples (420 patients) in the derivation and validation cohort, there was a strong linear relationship between heparin anti-Xa activities and factor-Xa inhibitors measured level (r = 0.99 [95% confidence interval {CI}, 0.99-0.99]). In the derivation cohort, heparin anti-Xa activity ≤0.2, ≤0.3, <0.1, <0.1 IU·mL-1 reliably ruled out a relevant level of apixaban, rivaroxaban, fondaparinux, and danaparoid, respectively (area under the ROC curve ≥0.99). In the validation cohort, these cutoffs yielded excellent classification accuracy (≥96%). If this screening test indicated relevant level of factor-Xa inhibitor, estimated and measured levels closely agreed (Lin's correlation coefficient close to its maximal value: 95% CI, 0.99-0.99). More than 96% of the estimated levels fell into the predefined range of acceptability (ie, 80%-120% of the measured level). CONCLUSIONS: A unique simple test already widely used to assay heparin was also useful for quantifying these 4 other anticoagulants. Both clinical and economic impacts of these findings should be assessed in a specific study.

Platelet immunophenotyping in health and inherited bleeding disorders, a review and practical hints.

Inherited platelet function disorders are rare hemorrhagic diseases. The gold standard for their exploration is optical aggregometry; however, investigations by flow cytometry (FCM) are being increasingly used. In this review, the physiology of platelets is first recalled, setting the stage for the compartments of platelets that can be apprehended by specific and appropriate labeling. As this requires some pre-analytical precautions and specific analytical settings, a second part focuses on these characteristic aspects, based on literature and on the authors' experience in the field, for qualitative or quantitative explorations. Membrane labeling with antibodies to CD42a or CD41, respectively, useful to assess the genetic-related defects of Glanzmann thrombocytopenia and Bernard Soulier syndrome are then described. Platelet degranulation disorders are detailed in the next section, as they can be explored, upon platelet activation, by measuring the expression of surface P-Selectin (CD62P) or CD63. Mepacrin uptake and release after activation is another test allowing to explore the function of dense granules. Finally, the flip-flop anomaly related to Scott syndrome is depicted. Tables summarizing possible FCM assays, and characteristic histograms are provided as reference for flow laboratories interested in developing platelet exploration.