Significant gynecological bleeding in women with low von Willebrand factor levels.

Gynecological bleeding is frequently reported in women with von Willebrand disease (VWD). Low von Willebrand factor (VWF) may be associated with significant bleeding phenotype despite only mild plasma VWF reductions. The contribution of gynecological bleeding to this phenotype has yet to be described. The optimal clinical bleeding assessment tool (BAT) to evaluate bleeding remains unclear. Using a standardized approach to phenotypic assessment, we evaluated gynecological bleeding and directly compared the Condensed Molecular and Clinical Markers for the Diagnosis and Management of type 1 VWD (Condensed MCMDM-1 VWD) and International Society on Thrombosis and Haemostasis (ISTH) BAT scores in 120 women enrolled in the Low von Willebrand in Ireland Cohort study. Heavy menstrual bleeding (HMB) was reported in 89% of female participants; 45.8% developed iron deficiency. Using identical data, Condensed MCMDM-1 VWD menorrhagia domain scores were significantly lower than ISTH BAT scores (2 vs 3;</p

Manhattan plot GWAS with antithrombin phenotype.

<p>The thresholdof significance to select candidate SNPs for validation is also shown.</p

Consequences of <i>LARGE</i> gene silencing in HepG2 and HEK-EBNA cell lines.

<p>A) Secreted proteins to the conditioned medium evaluated by immunoblotting. B) Effect on intracellular antithrombin from HepG2 cells analyzed by immunofluorescence and immunoblotting. C) Effect on the levels of <i>SERPINC1</i> expression in HEK-EBNA and HepG2 cell lines. Immunoblots and immunofluorescence figures are representative of at least 3 independent experiments. Control represents cells transfected with scramble siRNA, although similar results were observed in cells transfected without siRNA.</p

<i>LARGE</i> haplotypes identified in the validation study and their correlation with anti-FXa activity.

<p><i>LARGE</i> haplotypes identified in the validation study and their correlation with anti-FXa activity.</p

Genotype-phenotype analysis in the validation study.

<p>Genotype-phenotype analysis in the validation study.</p

Glycomic and proteomic analysis of α-antithrombin purified from plasma of healthy subjects with the highest (blue) and lowest (red) <i>LARGE</i> expression.

<p>As controls we also used antithrombin glycoforms α (black), and β (green) purified from a pool of 100 healthy blood donors. The β glycoform has 3 <i>N</i>-glycans since it lacks N-glycosylation at N-135. A) MALDI TOF mass spectrometric analysis of: 1) Intact glycoproteins; 2) 2AB-labeled N-glycans. B) HPLC data. 1) Distribution of the glycan structures of antithrombin specimens. Values are represented as % of total glycan pool. Between brackets are the absolute fluorescence units. 2) HILIC HPLC profiles of antithrombin specimens.</p

Aptamer BT200 blocks interaction of K1405-1408 in the VWF-A1 domain with macrophage LRP1

Rondaptivan pegol (previously BT200) is a PEGylated RNA aptamer that binds to the A1 domain of VWF. Recent clinical trials demonstrated that BT200 significantly increased plasma VWF-FVIII levels by attenuating VWF clearance. The biological mechanism(s) through which BT200 attenuates in vivo clearance of VWF have not been defined. We hypothesized that BT200 interaction with the VWF-A1 domain may increase plasma VWF levels by attenuating macrophage-mediated clearance. We observed that full length- and VWF-A1A2A3 binding to macrophages, and VWF-A1 domain binding to LRP1 cluster II and cluster IV, were concentration-dependently inhibited by BT200. Additionally, full length VWF binding to LRP1 expressed on HEK293T (HEK-LRP1) cells was also inhibited by BT200. Importantly, BT200 interacts with the VWF-A1 domain in proximity to a conserved cluster of four lysine residues (K1405, K1406, K1407 and K1408). Alanine mutagenesis of this K1405-K1408 cluster (VWF-4A) significantly (p<0.001) attenuated binding of VWF to both LRP1 clusters II and IV. Furthermore, in vivo clearance of VWF-4A was significantly (p<0.001) reduced compared to wild type VWF. BT200 did not significantly inhibit binding of VWF-4A to LRP1 cluster IV or HEK-LRP1 cells. Finally, BT200 interaction with the VWF-A1 domain also inhibited binding to macrophage galactose lectin (MGL) and the SR-AI scavenger receptor. Collectively, our findings demonstrate that BT200 prolongs VWF half-life by attenuating macrophage-mediated clearance and specifically the interaction of K1405-1408 in the VWF-A1 domain with macrophage LRP1. These data support the concept that targeted inhibition of VWF clearance pathways represent a novel therapeutic approach for VWD and hemophilia A