The spectrum of neutralizing and non-neutralizing anti-FVIII antibodies in a nationwide cohort of 788 persons with hemophilia A.

Objectives: Anti-factor VIII (FVIII) antibodies have been reported to exhibit both neutralizing and non-neutralizing characteristics. This is the first study investigating the full spectrum of FVIII-specific antibodies, including non-neutralizing antibodies, very-low titer inhibitors, and inhibitors, in a large nationwide population of persons with hemophilia A of all severities. Methods: All persons with hemophilia A (mild (FVIII > 5-40 IU/dL)/moderate [FVIII 1-5 IU/dL)/severe (FVIII < 1 IU/dL)] with an available plasma sample who participated in the sixth Hemophilia in the Netherlands study between 2018 and 2019 were included. The presence of anti-FVIII antibodies of the immunoglobulin A, M, and G isotypes and IgG subclasses, along with antibody titer levels, were assessed using direct-binding ELISAs. FVIII specificity was assessed using a competition-based ELISA approach. The inhibitor status was determined using the Nijmegen ultra-sensitive Bethesda assay (NusBA) and the Nijmegen Bethesda assay (NBA). Results: In total, 788 persons with hemophilia A (336 (42.6%) mild, 123 (15.6%) moderate, 329 (41.8%) severe hemophilia) were included. The median age was 45 years (IQR 24-60), and the majority (50.9%) had over 150 exposure days to FVIII concentrates. Within our population, 144 (18.3%) individuals had non-neutralizing FVIII-specific antibodies, 10 (1.3%) had very low-titer inhibitors (NusBA positive; NBA negative), and 13 (1.6%) had inhibitors (both NusBA and NBA positive). IgG1 was the most abundant FVIII-specific antibody subclass, and the highest titer levels were found for IgG4. In individuals without a reported history of inhibitor development, no clear differences were observed in antibody patterns between those who were minimally or highly exposed to FVIII concentrates. IgG4 subclass antibodies were only observed in persons with a reported history of FVIII inhibitor or in those with a currently detected (very low-titer) inhibitor. Conclusion: In this cross-sectional study, we identified non-neutralizing antibodies in a relatively large proportion of persons with hemophilia A. In contrast, in our population, consisting of persons highly exposed to FVIII concentrates, (very low-titer) inhibitors were detected only in a small proportion of persons, reflecting a well-tolerized population. Hence, our findings suggest that only a small subpopulation of non-neutralizing FVIII-specific antibodies is associated with clinically relevant inhibitors.

Pharmacokinetic-Pharmacodynamic Modelling in Hemophilia A: Relating Thrombin and Plasmin Generation to Factor VIII Activity After Administration of a VWF/FVIII Concentrate.

BACKGROUND: Hemophilia A patients are treated with factor (F) VIII prophylactically to prevent bleeding. In general, dosage and frequency are based on pharmacokinetic measurements. Ideally, an alternative dose adjustment can be based on the hemostatic potential, measured with a thrombin generation assay (TGA), like the Nijmegen hemostasis assay. OBJECTIVE: The objective of this study was to investigate the predicted performance of a previously developed pharmacokinetic-pharmacodynamic model for FVIII replacement therapy, relating FVIII dose and FVIII activity levels with thrombin and plasmin generation parameters. METHODS: Pharmacokinetic and pharmacodynamic measurements were obtained from 29 severe hemophilia A patients treated with pdVWF/FVIII concentrate (Haemate P®). The predictive performance of the previously developed pharmacokinetic-pharmacodynamic model was evaluated using nonlinear mixed-effects modeling (NONMEM). When predictions of FVIII activity or TGA parameters were inadequate [median prediction error (MPE) > 20%], a new model was developed. RESULTS: The original pharmacokinetic model underestimated clearance and was refined based on a two-compartment model. The pharmacodynamic model displays no bias in the observed normalized thrombin peak height and normalized thrombin potential (MPE of 6.83% and 7.46%). After re-estimating pharmacodynamic parameters, EC50 and Emax values were relatively comparable between the original model and this group. Prediction of normalized plasmin peak height was inaccurate (MPE 58.9%). CONCLUSION: Our predictive performance displayed adequate thrombin pharmacodynamic predictions of the original model, but a new pharmacokinetic model was required. The pharmacodynamic model is not factor specific and applicable to multiple factor concentrates. A prospective study is needed to validate the impact of the FVIII dosing pharmacodynamic model on bleeding reduction in patients.

Patients with moderate hemophilia A and B with a severe bleeding phenotype have an increased burden of disease.

BACKGROUND: Patients with moderate hemophilia express varying bleeding phenotypes. OBJECTIVES: To assess the burden of disease in patients with moderate hemophilia and a mild or severe phenotype incorporating the thrombin generation profile. METHODS: This sub-study of the 6th Hemophilia in the Netherlands study, analyzed data of adults with moderate hemophilia A or B. Patient characteristics and information on bleeding tendency, joint status, and quality of life were obtained from electronic patient files and self-reported questionnaires. A severe bleeding phenotype was defined as an annual bleeding rate ≥5, an annual joint bleeding rate ≥3, and/or the use of secondary/tertiary prophylaxis, and a mild phenotype vice versa. TG was measured with the Nijmegen Hemostasis Assay. RESULTS: This study included 116 patients: 21% had a severe phenotype of whom 46% used prophylaxis. Patients with a severe phenotype treated on demand reported a higher median annual bleeding rate (7), annual joint bleeding rate (3), and more frequently an impaired joint (77%) than patients with a severe phenotype on prophylaxis (2; 0; 70%) or patients with a mild phenotype (0; 0; 47%). Furthermore, patients with a severe phenotype treated on demand experienced a more decreased quality of life. Despite similar factor activity levels, patients with a severe phenotype had a lower thrombin peak height and thrombin potential (0.7%; 0.06%) than patients with a mild phenotype (21.3%; 46.8%). CONCLUSION: Patients with moderate hemophilia and a severe phenotype treated on demand displayed a high burden of disease as well as a low thrombin generation profile advocating them toward more intensive prophylactic treatment.

Synthesis and Evaluation of Glycosyl Luciferins.

Measuring glycosidase activity is important to monitor any aberrations in carbohydrate hydrolase activity, but also for the screening of potential glycosidase inhibitors. To this end, synthetic substrates are needed which provide an enzyme-dependent read-out upon hydrolysis by the glycosidase. Herein, we present two new routes for the synthesis of caged luminescent carbohydrates, which can be used for determining glycosidase activity with a luminescent reporter molecule. The substrates were validated with glycosidase and revealed a clear linear range and enzyme-dependent signal upon the in situ generation of the luciferin moiety from the corresponding nitrile precursors. Besides, we showed that these compounds could directly be synthesized from unprotected glycosyl-α-fluorides in a two-step procedure with yields up to 75 %. The intermediate methyl imidate appeared a key intermediate which also reacted with d-cysteine to give the corresponding d-luciferin substrate rendering this a highly attractive method for synthesizing glycosyl luciferins in good yields.

Solid-Phase Synthesis of Caged Luminescent Peptides via Side Chain Anchoring.

The synthesis of caged luminescent peptide substrates remains challenging, especially when libraries of the substrates are required. Most currently available synthetic methods rely on a solution-phase approach, which is less suited for parallel synthesis purposes. We herein present a solid-phase peptide synthesis (SPPS) method for the synthesis of caged aminoluciferin peptides via side chain anchoring of the P1 residue. After the synthesis of a preliminary test library consisting of 40 compounds, the synthetic method was validated and optimized for up to >100 g of resin. Subsequently, two separate larger peptide libraries were synthesized either having a P1 = lysine or arginine residue containing in total 719 novel peptide substrates. The use of a more stable caged nitrile precursor instead of caged aminoluciferin rendered our parallel synthetic approach completely suitable for SPPS and serine protease profiling was demonstrated using late-stage aminoluciferin generation.

The Nijmegen ultra-sensitive Bethesda Assay detects very low-titer factor VIII inhibitors in patients with congenital and acquired hemophilia A.

BACKGROUND: An inhibitor can develop in congenital hemophilia A (HA) patients against exogenous infused factor (F)VIII, whereas in acquired HA (AHA) inhibitors initially develop against endogenous FVIII. Inhibitors can be detected with the Nijmegen Bethesda Assay (NBA), which has an international cut-off level of 0.60 Nijmegen Bethesda Units/mL (NBU/mL). Thereby, very low-titer inhibitors may remain undetected. AIM: To describe the design and validation of the Nijmegen ultra-sensitive Bethesda Assay (NusBA) for the detection of very low-titer inhibitors. METHODS: The NusBA is a modification of the NBA in which the ratio of patient plasma to normal pooled plasma is changed from 1:1 to 9:1. Analytical validation was performed according to the CLSI EP10 guideline in order to determine trueness and reproducibility. Clinical validation was performed in two cohorts of congenital HA patients (82 adults) with pharmacokinetic data and four AHA patients. The limit of quantitation (LOQ) was determined by measuring plasma samples spiked with inhibitor levels in the low range (0.05-0.80 NBU/mL). RESULTS: The LOQ for the NusBA was 0.10 NusBU/mL, with a coefficient of variation of 24.2 %. Seven (8.5 %) congenital HA patients had a positive NusBA result, of which only one was detected with the NBA. There was no correlation between NusBA and FVIII half-life. In three of the AHA patients the NusBA remained positive, when the NBA became negative. DISCUSSION: The NusBA is able to detect very low-titer FVIII inhibitors of ≥0.10 NBU/mL. Thereby, it may have added value in early inhibitor detection and therapy adjustments in patients with congenital HA and AHA.

High prevalence of heavy menstrual bleeding in women with rare bleeding disorders in the Netherlands: retrospective data from the RBiN study.

BACKGROUND: Heavy menstrual bleeding (HMB) is associated with a reduced quality of life and limitations in social and physical functioning. Data on HMB in women with rare bleeding disorders (RBDs), including coagulation factor deficiencies and fibrinolytic disorders, are scarce. OBJECTIVES: To analyze the prevalence, severity, and treatment of HMB in Dutch women with an RBD. METHODS: The Rare Bleeding Disorders in the Netherlands (RBiN) study included 263 patients with an RBD from all 6 hemophilia treatment centers (October 2017-November 2019). In this analysis, data of 111 women aged ≥16 years were studied. According to the International Society on Thrombosis and Haemostasis bleeding assessment tool, HMB symptoms were scored from 0 (no/trivial) to 4 (severe symptoms requiring medical intervention). HMB was defined as a score ≥1. Age at RBD diagnosis was extracted from patient files. RESULTS: HMB was reported by 80% of women (89/111) and was more prevalent in women with a fibrinolytic disorder (33/35; 94%) than in women with a coagulation factor deficiency (56/76; 74%) (P = .011). Of the 89 women with HMB, 82% (n = 73) ever required treatment. Multiple treatment modalities were frequently used, both in severe and mild deficiencies. Hormonal treatment was mostly used (n = 64; 88%), while antifibrinolytics were prescribed less frequently (n = 18; 25%). In women with HMB since menarche (n = 61; 69%), median age at RBD diagnosis was 28 years (IQR, 14-41). CONCLUSION: HMB is common in women with RBDs. Women with mild deficiencies also frequently reported HMB. Only a minority of women were treated with hemostatic agents. A significant diagnostic delay was observed after the onset of HMB symptoms.

In patients with hemophilia, a decreased thrombin generation profile is associated with a severe bleeding phenotype.

Background: Heterogeneity in clinical bleeding phenotype has been observed in hemophilia patients with similar FVIII or FIX activity levels. Thrombin generation and plasmin generation, as a global hemostasis assay, may contribute to a better prediction of which patients are at an increased risk of bleeding. Objectives: The objective of this study was to describe the association between clinical bleeding phenotype and thrombin generation and plasmin generation profiles in patients with hemophilia. Methods: The Nijmegen Hemostasis Assay, which simultaneously measures thrombin and plasmin generation, was performed in plasma samples of patients with hemophilia participating in the sixth Hemophilia in the Netherlands study (HiN6). Patients receiving prophylaxis underwent a washout period. A severe clinical bleeding phenotype was defined as a self-reported annual bleeding rate of ≥5, a self-reported annual joint bleeding rate of ≥3, or the use of secondary/tertiary prophylaxis. Results: In total, 446 patients, with a median age of 44 years, were included in this substudy. Thrombin generation and plasmin generation parameters differed between patients with hemophilia and healthy individuals. The median thrombin peak height was 1.0 nM, 25.9 nM, 47.1 nM, and 143.9 nM in patients with severe, moderate, and mild hemophilia and healthy individuals, respectively. A severe bleeding phenotype was observed in patients with a thrombin peak height of <49% and a thrombin potential of <72% compared to healthy individuals, and was independent of the hemophilia severity. The median thrombin peak height was 0.70% in patients with a severe clinical bleeding phenotype and 30.3% in patients with a mild clinical bleeding phenotype. The median thrombin potentials for these patients were 0.06% and 59.3%, respectively. Conclusion: A decreased thrombin generation profile is associated with a severe clinical bleeding phenotype in patients with hemophilia. Thrombin generation in combination with bleeding severity may be a better tool to personalize prophylactic replacement therapy irrespective of hemophilia severity.

High prevalence of postpartum hemorrhage in women with rare bleeding disorders in the Netherlands: retrospective data from the RBiN study.

BACKGROUND: Women with rare bleeding disorders (RBDs), including coagulation factor deficiencies and fibrinolytic disorders, may have a higher risk of postpartum hemorrhage (PPH). Information on this patient category is lacking in the existing PPH guidelines because data on PPH in patients with RBDs are scarce. OBJECTIVE: To describe the prevalence of PPH in women with an RBD and evaluate the use of peripartum hemostatic prophylaxis. METHODS: In the Rare Bleeding Disorders in the Netherlands (RBiN) study, patients with RBDs (n = 263) were included from all 6 Dutch hemophilia treatment centers. Patient-reported information on delivery, peripartum hemostatic prophylaxis, and occurrence of PPH was collected retrospectively. If available, information about the precise volume of postpartum blood loss was extracted from electronic patient files. PPH was defined as blood loss ≥500 mL (World Health Organization guideline). RESULTS: A total of 244 pregnancies, including 193 livebirths, were reported by 85 women. A considerable proportion of these women experienced PPH, ranging from 30% in factor V deficiency to 100% in hyperfibrinolysis. Overall, PPH was reported in 44% of deliveries performed with and 53% of deliveries performed without administration of peripartum hemostatic prophylaxis. Blood loss was significantly higher in deliveries without administration of hemostatic prophylaxis (median 1000 mL) compared to deliveries with administration of prophylaxis (median 400 mL) (p = 0.011). Patients with relatively mild deficiencies also frequently experienced PPH when peripartum hemostatic prophylaxis was omitted. CONCLUSION: PPH is common in rare coagulation factor deficiencies, both severe and mild, and fibrinolytic disorders, especially when peripartum prophylactic hemostatic treatment was not administered. The use of prophylactic hemostatic treatment was associated with less postpartum blood loss.

Activity Sensing of Coagulation and Fibrinolytic Proteases.

The blood coagulation cascade is a complex physiological process involving the action of multiple coupled enzymes, cofactors, and substrates, ultimately leading to clot formation. Serine proteases have a crucial role, and aberrations in their activity can lead to life-threatening bleeding disorders and thrombosis. This review summarizes the essential proteases involved in blood coagulation and fibrinolysis, the endogenous peptide sequences they recognize and hydrolyze, and synthetic peptide probes based on these sequences to measure their activity. The information in this review can contribute to developing novel anticoagulant therapies and specific substrates for point-of-care diagnosis of coagulation pathologies.

Thrombin generation assays to personalize treatment in bleeding and thrombotic diseases.

Treatment of bleeding and thrombotic disorders is highly standardized and based on evidence-based medicine guidelines. These evidence-based treatment schemes are well accepted but may lead to either insufficient treatment or over-dosing, because the individuals' hemostatic properties are not taken into account. This can potentially introduce bleeding or thrombotic complications in individual patients. With the incorporation of pharmacokinetic (PK) and pharmacodynamic (PK-PD) parameters, based on global assays such as thrombin generation assays (TGAs), a more personalized approach can be applied to treat either bleeding or thrombotic disorders. In this review, we will discuss the recent literature about the technical aspects of TGAs and the relation to diagnosis and management of bleeding and thrombotic disorders. In patients with bleeding disorders, such as hemophilia A or factor VII deficiency, TGAs can be used to identify patients with a more severe bleeding phenotype and also in the management with non-replacement therapy and/or bypassing therapy. These assays have also a role in patients with venous thrombo-embolism, but the usage of TGAs in patients with arterial thrombosis is less clear. However, there is a potential role for TGAs in the monitoring of (long-term) antithrombotic therapy, for example with the use of direct oral anticoagulants. Finally this review will discuss controversies, limitations and knowledge gaps in relation to the introduction of TGAs to personalize medicine in daily medical practice.

Luminescent Assay for the Screening of SARS-CoV-2 MPro Inhibitors.

Since the outbreak of SARS-CoV-2 in December 2019 millions of infections have been reported globally. The viral chymotrypsin-like main protease (MPro ) exhibits a crucial role in viral replication and represents a relevant target for antiviral drug development. In order to screen potential MPro inhibitors we developed a luminescent assay using a peptide based probe containing a cleavage site specific for MPro . This assay was validated showing IC50 values similar to those reported in the literature for known MPro inhibitors and can be used to screen new inhibitors.

Importance of Genotyping in von Willebrand Disease to Elucidate Pathogenic Mechanisms and Variability in Phenotype.

Genotyping is not routinely performed at diagnosis of von Willebrand disease (VWD). Therefore, the association between genetic variants and pathogenic mechanism or the clinical and laboratory phenotype is unknown in most patients, especially in type 1 VWD. To investigate whether genotyping adds to a better understanding of the pathogenic mechanisms and variability in phenotype, we analyzed the VWF gene in 390 well-defined VWD patients, included in the WiN study. A VWF gene variant was found in 155 patients (61.5%) with type 1, 122 patients (98.4%) with type 2, and 14 patients (100%) with type 3 VWD. Forty-eight variants were novel. For each VWF gene variant, the pathogenic mechanisms associated with reduced VWF levels was investigated using the FVIII:C/VWF:Ag and VWFpp/VWF:Ag ratios. In type 1 VWD, reduced synthesis or secretion of VWF was most frequently found in patients with nonsense variants, frameshift variants, and deletions, whereas rapid clearance of VWF was mainly found in patients with missense variants. Furthermore, type 1 VWD patients with and without a VWF gene variant were clearly distinct in their clinical features such as age of diagnosis, laboratory phenotype, and bleeding phenotype. In type 2 VWD, 81% of variants were associated with an increased clearance of VWF. To conclude, we identified the pathogenic mechanisms associated with various VWF gene variants in type 1, 2, and 3 VWD patients. Additionally, major differences in the phenotype of type 1 VWD patients with and without a variant were observed, which may be of importance for clinical management.

Diagnosis of rare bleeding disorders.

Rare bleeding disorders result in significant morbidity but are globally underdiagnosed. Advances in genomic testing and specialist laboratory assays have greatly increased the diagnostic armamentarium. This has resulted in the discovery of new genetic causes for rare diseases and a better understanding of the underlying molecular pathology.

Desmopressin response depends on the presence and type of genetic variants in patients with type 1 and type 2 von Willebrand disease.

Patients with type 1 and type 2 von Willebrand disease (VWD) can be treated with desmopressin. Although a previous study has shown that the location of the causative VWF gene variant is associated with desmopressin response in type 1 VWD, the association between variants in the VWF gene and desmopressin response is not yet fully understood. Our primary aim was to compare desmopressin response in type 1 VWD patients with and without a VWF gene variant. Secondly, we investigated whether desmopressin response depends on specific VWF gene variants in type 1 and type 2 VWD. We included 250 patients from the Willebrand in the Netherlands study: 72 type 1 without a VWF gene variant, 108 type 1 with a variant, 45 type 2A, 16 type 2M, and 9 type 2N patients. VWF gene was analyzed with ion semiconductor sequencing and Multiplex Ligation-dependent Probe Amplification. Complete response to desmopressin was observed in all type 1 VWD patients without a variant, 64.3% of type 1 patients with a variant, and 31.3% of type 2 patients (P < .001). Despite a large interindividual variability in desmopressin response, patients with the same variant had comparable desmopressin responses. For instance, in 6 type 1 patients with exon 4 to 5 deletion, mean VWF activity at 1 hour after desmopressin was 0.81 IU/mL, with a coefficient of variation of 22.9%. In conclusion, all type 1 VWD patients without a VWF gene variant respond to desmopressin. In type 1 and type 2 VWD patients with a VWF variant, desmopressin response highly depends on the VWF gene variants.

Fibrinolytic assays in bleeding of unknown cause: Improvement in diagnostic yield.

Introduction: Analysis of fibrinolytic disorders is challenging and may potentially lead to underdiagnosis of patients with an increased bleeding tendency. Aim: To compare clinical characteristics, laboratory measurements, and treatment modalities in a monocenter cohort of patients in whom fibrinolytic studies were performed. Methods: Retrospective study of patients in whom fibrinolytic studies were performed between January 2016 and February 2020 in the Hemophilia Treatment Center, Nijmegen-Eindhoven-Maastricht, the Netherlands. Plasminogen activator inhibitor type 1 (PAI-1) antigen and activity level, α2-antiplasmin activity, tissue plasminogen activator, and euglobulin clot lysis time (ECLT) before and after venous compression were determined in all patients. Data of bleeding assessment tool (BAT) score, clinical characteristics, results of primary and secondary hemostasis assays, and general treatment plans were collected. Results: In total, 160 patients were included: 97 (61%) without and 63 (39%) with a laboratory-based fibrinolytic disorder. Mean BAT score did not differ between the groups (9.3 vs 9.8, respectively). The presumptive fibrinolytic disorders were distributed as follows: 34 patients had an increased ECLT ratio or low baseline ECLT, 25 patients had low PAI-1 antigen and activity level, and four patients had both. The majority of these patients were treated with tranexamic acid monotherapy (60%) with only 40% additional treatment options, whereas 80% of patients without a presumptive fibrinolytic disorder had multiple treatment modalities. Discussion: Analysis of fibrinolytic disorders in selected patients has a high diagnostic yield. General incorporation of fibrinolytic analysis in the diagnostic workup of patients with bleeding of unknown cause can improve diagnosis and management of their bleeding episodes.

Treatment of patients with rare bleeding disorders in the Netherlands: Real-life data from the RBiN study.

BACKGROUND: Patients with rare inherited bleeding disorders (RBDs) exhibit hemorrhagic symptoms, varying in type and severity, often requiring only on-demand treatment. Prolonged bleeding after invasive procedures is common. Adequate peri-procedural therapy may reduce this bleeding risk. OBJECTIVE: To describe general treatment plans of RBD patients and evaluate the use of peri-procedural hemostatic therapy. METHODS: In the Rare Bleeding Disorders in the Netherlands (RBiN) study, RBD patients from all six Dutch Hemophilia Treatment Centers were included. General treatment plans were extracted from patient files. Patients with a dental or surgical procedure in their history were interviewed about use of peri-procedural treatment and bleeding complications. RESULTS: Two-hundred sixty-three patients with a rare coagulation factor deficiency or fibrinolytic disorder were included. Eighty-four percent had a documented general treatment plan. General treatment plans of patients with the same RBD were heterogeneous, particularly in factor XI deficiency. Overall, 308 dental and 408 surgical procedures were reported. Bleeding occurred in 50% of dental and 53% of surgical procedures performed without hemostatic treatment and in 28% of dental and 19% of surgical procedures performed with hemostatic treatment. Not only patients with severe RBDs, but also patients with mild deficiencies, experienced increased bleeding without proper hemostatic treatment. CONCLUSION: Large heterogeneity in general treatment plans of RBD patients was found. Bleeding after invasive procedures was reported frequently, both before and after RBD diagnosis, irrespective of factor activity levels and particularly when peri-procedural treatment was omitted. Improved guidelines should include uniform recommendations for most appropriate hemostatic products per RBD and emphasize the relevance of individual bleeding history.

Von Willebrand disease type 2M: Correlation between genotype and phenotype.

BACKGROUND: An appropriate clinical diagnosis of von Willebrand disease (VWD) can be challenging because of a variable bleeding pattern and laboratory phenotype. Genotyping is a powerful diagnostic tool and may have an essential role in the diagnostic field of VWD. OBJECTIVES: To unravel the clinical and laboratory heterogeneity of genetically confirmed VWD type 2M patients and to investigate their relationship. METHODS: Patients with a confirmed VWD type 2M genetic variant in the A1 or A3 domain of von Willebrand factor (VWF) and normal or only slightly aberrant VWF multimers were selected from all subjects genotyped at the Radboud university medical center because of a high suspicion of VWD. Bleeding scores and laboratory results were analyzed. RESULTS: Fifty patients had a clinically relevant genetic variant in the A1 domain. Median bleeding score was 5. Compared with the nationwide Willebrand in the Netherlands study type 2 cohort, bleeding after surgery or delivery was reported more frequently and mucocutaneous bleedings less frequently. Median VWF activity/VWF antigen (VWF:Act/VWF:Ag) ratio was 0.32, whereas VWF collagen binding activity/VWF antigen (VWF:CB/VWF:Ag) ratio was 0.80. Variants in the A3 domain were only found in two patients with low to normal VWF:Act/VWF:Ag ratios (0.45, 1.03) and low VWF:CB/VWF:Ag ratios (0.45, 0.63). CONCLUSION: Genetically confirmed VWD type 2M patients have a relatively mild clinical phenotype, except for bleeding after surgery and delivery. Laboratory phenotype is variable and depends on the underlying genetic variant. Addition of genotyping to the current phenotypic characterization may improve diagnosis and classification of VWD.

Combining factor VIII levels and thrombin/plasmin generation: A population pharmacokinetic-pharmacodynamic model for patients with haemophilia A.

AIMS: Prophylactic treatment of haemophilia A patients with factor VIII (FVIII) concentrate focuses on maintaining a minimal trough FVIII activity level to prevent bleeding. However, due to differences in bleeding tendency, the pharmacokinetic (PK)-guided dosing approach may be suboptimal. An alternative approach could be the addition of haemostatic pharmacodynamic (PD) parameters, reflecting a patient's unique haemostatic balance. Our aim was to develop a population PK/PD model, based on FVIII activity levels and Nijmegen Haemostasis Assay (NHA) patterns, a global haemostatic assay that measures thrombin/plasmin generation simultaneously. METHODS: PK/PD measurements were collected from 30 patients treated with standard half-life FVIII concentrate. The relationship between FVIII activity levels and the thrombin/plasmin generation parameters (thrombin potential, thrombin peak height and plasmin peak height), were described by sigmoidal Emax functions. RESULTS: The obtained EC50 value was smallest for the normalized thrombin potential (11.6 IU/dL), followed by normalized thrombin peak height (56.6 IU/dL) and normalized plasmin peak height (593 IU/dL), demonstrating that normalized thrombin potential showed 50% of the maximal effect at lower FVIII activity levels. Substantial inter-individual variability in the PD parameters, such as EC50 of thrombin potential (86.9%) was observed, indicating that, despite similar FVIII activity levels, haemostatic capacity varies significantly between patients. CONCLUSION: These data suggest that dosing based on patients' individual PK/PD parameters may be beneficial over dosing solely on individual PK parameters. This model could be used as proof-of-principle to examine the application of PK/PD-guided dosing. However, the relation between the PD parameters and bleeding has to be better defined.