Prophylactic application of clotting factor concentrates is the basis of modern treatment of severe hemophilia A. In children, the early start of prophylaxis as primary or secondary prophylaxis has ...become the gold standard in most countries with adequate resources. In adults, prophylaxis is reasonably continued when started as primary or secondary prophylaxis in childhood to maintain healthy joint function. Initial data support that adult patients with already existing advanced joint arthropathy benefit from tertiary prophylaxis with significantly lowered number of bleeds, almost complete absence of target joints, and less time off from work. Current prophylactic regimens, although very effective, do not completely prevent joint disease in a long-term perspective. Joint arthropathy in primary prophylaxis develops over many years, sometimes over a decade or even longer time periods. The ankle joints are the first and most severely affected joints in those patients and thus may serve in outcome assessment as an indicator of early joint arthropathy when followed by ultrasound or magnetic resonance imaging. Optimized outcome and best use of available resources is expected from individualization of therapy regimens, which comprises the individual’s bleeding pattern, condition of the musculoskeletal system, level of physical activity and the pharmacokinetic profile of the substituted coagulation factor, and most recently includes novel products with extended half-lives.
Purpose
Recent findings demonstrated anti-PEG antibody formation in some healthy individuals and patients who have not received PEGylated biotherapeutics. Some of these findings evoked criticism ...because of shortcomings in the antibody assays used. To better understand this topic, we established robust antibody analytics and screened two cohorts of healthy individuals and one cohort of hemophilia patients for the expression of anti-PEG antibodies.
Methods
A flow cytometry approach and a fully validated ELISA platform were established to detect specific anti-PEG antibodies. Immunohistochemistry was used to test for potential binding of anti-PEG antibodies to human tissues.
Results
IgM and/or IgG anti-PEG antibodies are expressed by some healthy individuals and by some patients with hemophilia who have not received PEGylated biotherapeutics. These antibodies can be either transient or persistent and recognize PEGs of different sizes with or without terminal methoxy groups. Age and location of healthy individuals influence the prevalence of IgG but not of IgM antibodies. Anti-PEG antibodies do not cross-react with human tissues supporting the safety of the antibodies.
Conclusion
We confirm that some healthy individuals and some patients with hemophilia express specific antibodies against PEG which are not associated with any pathology and do not bind to human tissues.
Emicizumab mimics the hemostatic activity of activated factor VIII (FVIIIa) within the tenase complex. Despite functional similarities between FVIIIa and emicizumab, conventional laboratory methods ...designed for monitoring of FVIII activity are inappropriate for the measurement of emicizumab. At present, a modified one stage (FVIII) assay (mOSA) is mainly used for emicizumab monitoring. Two-stage chromogenic FVIII assays based on human factors can be used, although limited performance due to lack of corresponding optimization might be observed. Furthermore, the presence of FVIII or anticoagulants in the patient sample may falsify assay results. To address these issues, we optimized and evaluated a two-stage chromogenic assay (emi-tenase) for measurement of emicizumab in plasma samples. Heat inactivation of samples was established to abolish the influence of endogenous or substituted FVIII. The lower limit of quantification (LLoQ) was found to be 2 μg/ml in a manual assay format and 9.5 μg/ml on an automated coagulation analyzer. Intra- and inter-assay coefficients of variation (CV) did not exceed 20%. Analysis of 17 patient plasma samples with severe haemophilia A under emicizumab treatment showed good correlation of results between the emi-tenase assay and the mOSA (Cohens Kappa coefficient = 0.9). Taken together, the emi-tenase assay allows specific measurement of emicizumab plasma levels over a broad concentration range (10 μg/ml to 100 μg/ml). The assay can be applied on an automated coagulation analyzer, demonstrating its applicability within a routine laboratory setting.
This multinational, randomized, single-blind trial investigated the safety and efficacy of nonacog beta pegol, a recombinant glycoPEGylated factor IX (FIX) with extended half-life, in 74 previously ...treated patients with hemophilia B (FIX activity ≤2 IU/dL). Patients received prophylaxis for 52 weeks, randomized to either 10 IU/kg or 40 IU/kg once weekly or to on-demand treatment of 28 weeks. No patients developed inhibitors, and no safety concerns were identified. Three hundred forty-five bleeding episodes were treated, with an estimated success rate of 92.2%. The median annualized bleeding rates (ABRs) were 1.04 in the 40 IU/kg prophylaxis group, 2.93 in the 10 IU/kg prophylaxis group, and 15.58 in the on-demand treatment group. In the 40 IU/kg group, 10 (66.7%) of 15 patients experienced no bleeding episodes into target joints compared with 1 (7.7%) of 13 patients in the 10 IU/kg group. Health-related quality of life (HR-QoL) assessed with the EuroQoL-5 Dimensions visual analog scale score improved from a median of 75 to 90 in the 40 IU/kg prophylaxis group. Nonacog beta pegol was well tolerated and efficacious for the treatment of bleeding episodes and was associated with low ABRs in patients receiving prophylaxis. Once-weekly prophylaxis with 40 IU/kg resolved target joint bleeds in 66.7% of the affected patients and improved HR-QoL. This trial was registered at www.clinicaltrials.gov as #NCT01333111.
•Nonacog beta pegol, a recombinant glycoPEGylated FIX with extended half-life, was developed to improve care for patients with hemophilia B.•Weekly prophylaxis with nonacog beta pegol was well tolerated and was associated with low bleeding rates and an improved quality of life.
A type 3 von Willebrand disease (VWD) index patient (IP) remains mutation-negative after completion of the conventional diagnostic analysis, including multiplex ligation-dependent probe amplification ...and sequencing of the promoter, exons, and flanking intronic regions of the VWF gene (VWF). In this study, we intended to elucidate causative mutation through next-generation sequencing (NGS) of the whole VWF (including complete intronic region), mRNA analysis, and study of the patient-derived endothelial colony-forming cells (ECFCs). The NGS revealed a variant in the intronic region of VWF (997 + 118 T > G in intron 8), for the first time. The bioinformatics assessments (e.g., SpliceAl) predicted this variant creates a new donor splice site (ss), which could outcompete the consensus 5′ donor ss at exon/intron 8. This would lead to an aberrant mRNA that contains a premature stop codon, targeting it to nonsense-mediated mRNA decay. The subsequent quantitative real-time PCR confirmed the virtual absence of VWF mRNA in IP ECFCs. Additionally, the IP ECFCs demonstrated a considerable reduction in VWF secretion (~6% of healthy donors), and they were devoid of endothelial-specific secretory organelles, Weibel−Palade bodies. Our findings underline the potential of NGS in conjunction with RNA analysis and patient-derived cell studies for genetic diagnosis of mutation-negative type 3 VWD patients.
Congenital factor XIII (FXIII) deficiency is a rare, autosomal-recessive disorder, with most patients having an A-subunit (FXIII-A) deficiency. Patients experience life-threatening bleeds, impaired ...wound healing, and spontaneous abortions. In many countries, only plasma or cryoprecipitate treatments are available, but these carry a risk for allergic reactions and infection with blood-borne pathogens. The present study was a multinational, open-label, single-arm, phase 3 prophylaxis trial evaluating the efficacy and safety of a novel recombinant FXIII (rFXIII) in congenital FXIII-A subunit deficiency. Forty-one patients ≥ 6 years of age (mean, 26.4; range, 7-60) with congenital FXIII-A subunit deficiency were enrolled. Throughout the rFXIII prophylaxis, only 5 bleeding episodes (all trauma induced) in 4 patients were treated with FXIII-containing products. The crude mean bleeding rate was significantly lower than the historic bleeding rate (0.138 vs 2.91 bleeds/patient/year, respectively) for on-demand treatment. Transient, non-neutralizing, low-titer anti-rFXIII Abs developed in 4 patients, none of whom experienced allergic reactions, any bleeds requiring treatment, or changes in FXIII pharmacokinetics during the trial or follow-up. These non-neutralizing Abs declined below detection limits in all 4 patients despite further exposure to rFXIII or other FXIII-containing products. We conclude that rFXIII is safe and effective in preventing bleeding episodes in patients with congenital FXIII-A subunit deficiency. This study is registered at http://www..clinicaltrials.gov as number NCT00713648.
Coagulation factor XIII (FXIII) circulates in plasma as a pro-transglutaminase heterotetrameric complex (FXIIIA
B
), which upon activation by thrombin and calcium covalently crosslinks preformed ...fibrin polymers. The heterotetrameric complex is composed of a catalytic FXIIIA
subunit and a protective/regulatory FXIII-B
subunit coded by
and
genes, respectively. The catalytic FXIIIA
subunit is encoded by the
gene, expressed primarily in cells of mesenchymal origin, whereas the FXIIIB subunit encoded by the
gene is expressed and secreted from hepatocytes. The plasma FXIIIA
subunit, which earlier was believed to be secreted from cells of megakaryocytic lineage, is now understood to result primarily from resident macrophages. The regulation of the FXIII subunits at the genetic level is still poorly understood. The current study adopts a purely bioinformatic approach to analyze the temporal, time-specific expression array-data corresponding to both the subunits in specific cell lineages, with respect to the gene promoters. We analyze the differentially expressed genes correlated with
and
expression levels in an array of cell types, utilizing publicly available microarray data. We attempt to understand the regulatory mechanism underlying the variable expression of FXIIIA
subunit in macrophages (M0, M1, M2 and aortic resident macrophages). Similarly, the FXIIIB
subunit expression data from adult, fetal hepatocytes and embryonic stem cells derived hepatoblasts (hESC-hepatoblast) was analyzed. The results suggest regulatory dependence between the two FXIII subunits at the transcript level. Our analysis also predicts the involvement of the FXIIIA
subunit in macrophage polarization, plaque stability, and inflammation.
Vitamin K dependent coagulation factor deficiency type 1 (VKCFD1) is a rare hereditary bleeding disorder caused by mutations in γ-Glutamyl carboxylase (
) gene. The GGCX enzyme catalyzes the ...γ-carboxylation of 15 different vitamin K dependent (VKD) proteins, which have function in blood coagulation, calcification, and cell signaling. Therefore, in addition to bleedings, some VKCFD1 patients develop diverse non-hemorrhagic phenotypes such as skin hyper-laxity, skeletal dysmorphologies, and/or cardiac defects. Recent studies showed that
mutations differentially effect γ-carboxylation of VKD proteins, where clotting factors are sufficiently γ-carboxylated, but not certain non-hemostatic VKD proteins. This could be one reason for the development of diverse phenotypes. The major manifestation of non-hemorrhagic phenotypes in VKCFD1 patients are mineralization defects. Therefore, the mechanism of regulation of calcification by specific VKD proteins as matrix Gla protein (MGP) and Gla-rich protein (GRP) in physiological and pathological conditions is of high interest. This will also help to understand the patho-mechanism of VKCFD1 phenotypes and to deduce new treatment strategies. In the present review article, we have summarized the recent findings on the function of GRP and MGP and how these proteins influence the development of non-hemorrhagic phenotypes in VKCFD1 patients.
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