Vector integration and fate in the hemophilia dog liver multiple years after AAV-FVIII gene transfer.

Gene therapy using adeno-associated virus (AAV) vectors is a promising approach for the treatment of monogenic disorders. Long-term multiyear transgene expression has been demonstrated in animal models and clinical studies. Nevertheless, uncertainties remain concerning the nature of AAV vector persistence and whether there is a potential for genotoxicity.

The contribution of the sinusoidal endothelial cell receptors CLEC4M, stabilin-2, and SCARA5 to VWF-FVIII clearance in thrombosis and hemostasis.

Quantitative abnormalities in factor VIII (FVIII) and its binding partner, von Willebrand factor (VWF), are associated with an increased risk of bleeding or thrombosis, and pathways that regulate the clearance of VWF-FVIII can strongly influence their plasma levels. In 2010, the Cohorts for Heart and Aging Research in Genome Epidemiology (CHARGE) on genome-wide association study meta-analysis identified variants in the genes for the sinusoidal endothelial receptors C-type lectin domain family 4 member M (CLEC4M), stabilin-2, and scavenger receptor class A member 5 (SCARA5) as being associated with plasma levels of VWF and/or FVIII in normal individuals. The ability of these receptors to bind, internalize, and clear the VWF-FVIII complex from the circulation has now been reported in a series of studies using in vitro and in vivo models.

Current Understanding of Inherited Modifiers of FVIII Pharmacokinetic Variation.

The inherited bleeding disorder hemophilia A involves the quantitative deficiency of the coagulation cofactor factor VIII (FVIII). Prophylactic treatment of severe hemophilia A patients with FVIII concentrates aims to reduce the frequency of spontaneous joint bleeding and requires personalized tailoring of dosing regimens to account for the substantial inter-individual variability of FVIII pharmacokinetics. The strong reproducibility of FVIII pharmacokinetic (PK) metrics between repeat analyses in the same individual suggests this trait is genetically regulated.

The mechanistic and structural role of von Willebrand factor in endotoxemia-enhanced deep vein thrombosis in mice.

Background: Although the concept of immunothrombosis has established a link between inflammation and thrombosis, the role of inflammation in the pathogenesis of deep vein thrombosis remains to be fully elucidated. Further, although various constituents of venous thrombi have been identified, their localizations and cellular and molecular interactions are yet to be combined in a single, multiplexed analysis.

Objectives: The objective of this study was to investigate the role of the von Willebrand factor (VWF) in inflammation-associated venous thrombosis.

Novel cysteine substitution p.(Cys1084Tyr) causes variable expressivity of qualitative and quantitative VWF defects.

von Willebrand factor (VWF) is an extremely cysteine-rich multimeric protein that is essential for maintaining normal hemostasis. The cysteine residues of VWF monomers form intra- and intermolecular disulfide bonds that regulate its structural conformation, multimer distribution, and ultimately its hemostatic activity. In this study, we investigated and characterized the molecular and pathogenic mechanisms through which a novel cysteine variant p.

Stabilin-2 deficiency increases thrombotic burden and alters the composition of venous thrombi in a mouse model.

Background: Stabilin-2 is an endocytic scavenger receptor that mediates the clearance of glycosaminoglycans, phosphatidylserine-expressing cells, and the von Willebrand factor-factor VIII (FVIII) complex. In a genome-wide screening study, pathogenic loss-of-function variants in the human STAB2 gene associated with an increased incidence of unprovoked venous thromboembolism (VTE). However, the specific mechanism(s) by which stabilin-2 deficiency influences the pathogenesis of VTE is unknown.

Factor VIII pharmacokinetics associates with genetic modifiers of VWF and FVIII clearance in an adult hemophilia A population.

Background: Factor VIII (FVIII) pharmacokinetics (PK) in adult hemophilia A populations are highly variable and have been previously determined to be influenced by von Willebrand factor:antigen (VWF:Ag), ABO blood group, and age. However, additional genetic determinants of FVIII PK are largely unknown.

Objectives: The contribution of VWF clearance, VWF-FVIII-binding activity, and genetic variants in VWF clearance receptors to FVIII PK in adult patients were assessed.

The challenge of genetically unresolved haemophilia A patients: Interest of the combination of whole F8 gene sequencing and functional assays.

Background: The causative variant remains unidentified in 2%-5% of haemophilia A (HA) patients despite an exhaustive sequencing of the full F8 coding sequence, splice consensus sequences, 5'/3' untranslated regions and copy number variant (CNV) analysis. Next-generation sequencing (NGS) has provided significant improvements for a complete F8 analysis.

Aim: The aim of this study was to identify and characterize pathogenic non-coding variants in F8 of 15 French and Canadian HA patients genetically unresolved, through the use of NGS, mRNA sequencing and functional confirmation of aberrant splicing.

von Willebrand Factor Is a Critical Mediator of Deep Vein Thrombosis in a Mouse Model of Diet-Induced Obesity.

Objective: Obesity is characterized by chronic low-grade inflammation and consequentially a hypercoagulable state, associating with an increased incidence of venous thromboembolism. Increased VWF (von Willebrand factor) plasma concentration and procoagulant function are independent risk factors for venous thromboembolism and are elevated in obese patients. Here, we explore the pathobiological role of VWF in obesity-associated venous thrombosis using murine models.

Genetic determinants of VWF clearance and FVIII binding modify FVIII pharmacokinetics in pediatric hemophilia A patients.

Factor VIII (FVIII) pharmacokinetic (PK) properties show high interpatient variability in hemophilia A patients. Although previous studies have determined that age, body mass index, von Willebrand factor antigen (VWF:Ag) levels, and ABO blood group status can influence FVIII PK, they do not account for all observed variability. In this study, we aim to describe the genetic determinants that modify the FVIII PK profile in a population of 43 pediatric hemophilia A patients.

The scavenger receptor SCARA5 is an endocytic receptor for von Willebrand factor expressed by littoral cells in the human spleen.

Background: Scavenger receptors play a significant role in clearing aged proteins from the plasma, including the large glycoprotein coagulation factors von Willebrand factor (VWF) and factor VIII (FVIII). A large genome-wide association study meta-analysis has identified genetic variants in the gene SCARA5, which encodes the class A scavenger receptor SCARA5, as being associated with plasma levels of VWF and FVIII.

Objectives: The ability of SCARA5 to regulate the clearance of VWF-FVIII was characterized.

Shear stress and platelet-induced tensile forces regulate ADAMTS13-localization within the platelet thrombus.

Background: The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion and aggregation at the site of vessel injury. The adhesive activity of VWF is influenced by its multimer length which is regulated by the metalloprotease ADAMTS13. The ability of ADAMTS13 to regulate platelet thrombus growth in a shear-dependent manner has been described, however, the mechanistic basis of this action has not been well characterized.

The endothelial lectin clearance receptor CLEC4M binds and internalizes factor VIII in a VWF-dependent and independent manner.

Essentials CLEC4M is an endocytic receptor for factor FVIII. CLEC4M interacts with FVIII in a VWF-dependent and independent manner. CLEC4M binds to mannose-containing glycans on FVIII.

The endothelial cell receptor stabilin-2 regulates VWF-FVIII complex half-life and immunogenicity.

Quantitative abnormalities of the von Willebrand factor-factor VIII (VWF-FVIII) complex associate with inherited bleeding or thrombotic disorders. Receptor-mediated interactions between plasma VWF-FVIII and phagocytic or immune cells can influence their hemostatic and immunogenic activities. Genetic association studies have demonstrated that variants in the STAB2 gene, which encodes the scavenger receptor stabilin-2, associate with plasma levels of VWF-FVIII.

N-linked glycosylation modulates the immunogenicity of recombinant human factor VIII in hemophilia A mice.

Immune responses to factor VIII remain the greatest complication in the treatment of severe hemophilia A. Recent epidemiological evidence has highlighted that recombinant factor VIII produced in baby hamster kidney cells is more immunogenic than factor VIII produced in Chinese hamster ovary cells. Glycosylation differences have been hypothesized to influence the immunogenicity of these synthetic concentrates.

The common single nucleotide variants c.2365A>G and c.2385T>C modify VWF biosynthesis and clearance.

Plasma levels of von Willebrand factor (VWF) vary considerably in the general population and this variation has been linked to several genetic and environmental factors. Genetic factors include 2 common single nucleotide variants (SNVs) located in , rs1063856 (c.2365A>G) and rs1063857 (c.

Investigating von Willebrand Factor Pathophysiology Using a Flow Chamber Model of von Willebrand Factor-platelet String Formation.

Von Willebrand factor (VWF) is a multimeric glycoprotein coagulation factor that mediates platelet adhesion and aggregation at sites of endothelial damage and that carries factor VIII in the circulation. VWF is synthesized by endothelial cells and is either released constitutively into the plasma or is stored in specialized organelles, called Weibel-Palade bodies (WPBs), for on-demand release in response to hemostatic challenge. Procoagulant and proinflammatory stimuli can rapidly induce WPB exocytosis and VWF release.

Genetic diagnosis in hemophilia and von Willebrand disease.

Phenotypic assays are first-line in terms of diagnostic testing for inherited bleeding disorders. However, since the characterization of the genes that encode coagulation factors in the 1980s, significant progress has been made in translating this knowledge for diagnostic and therapeutic purposes. For the hemophilias, molecular genetic testing can be used to determine carrier status, establish a prenatal diagnosis and predict the likelihood of inhibitor development or anaphylaxis in response to infused coagulation factor concentrates.

How much do we really know about von Willebrand disease?

Purpose Of Review: In the last nine decades, large advances have been made toward the characterization of the pathogenic basis and clinical management of von Willebrand disease (VWD), the most prevalent inherited bleeding disorder. Pathological variations at the von Willebrand factor (VWF) locus present as a range of both quantitative and qualitative abnormalities that make up the complex clinical spectrum of VWD. This review describes the current understanding of the pathobiological basis of VWD.

Extracellular Histones Increase Tissue Factor Activity and Enhance Thrombin Generation by Human Blood Monocytes.

Objectives: Sepsis is characterized by systemic activation of inflammatory and coagulation pathways in response to infection. Recently, it was demonstrated that histones released into the circulation by dying/activated cells may contribute to sepsis pathology. Although the ability of extracellular histones to modulate the procoagulant activities of several cell types has been investigated, the influence of histones on the hemostatic functions of circulating monocytes is unknown.

The role of leukocytes in thrombosis.

In recent years, the traditional view of the hemostatic system as being regulated by a coagulation factor cascade coupled with platelet activation has been increasingly challenged by new evidence that activation of the immune system strongly influences blood coagulation and pathological thrombus formation. Leukocytes can be induced to express tissue factor and release proinflammatory and procoagulant molecules such as granular enzymes, cytokines, and damage-associated molecular patterns. These mediators can influence all aspects of thrombus formation, including platelet activation and adhesion, and activation of the intrinsic and extrinsic coagulation pathways.

Gene Therapy for Coagulation Disorders.

Molecular genetic details of the human coagulation system were among the first successes of the genetic revolution in the 1980s. This information led to new molecular diagnostic strategies for inherited disorders of hemostasis and the development of recombinant clotting factors for the treatment of the common inherited bleeding disorders. A longer term goal of this knowledge has been the establishment of gene transfer to provide continuing access to missing or defective hemostatic proteins.

Using genetic diagnostics in hemophilia and von Willebrand disease.

Most bleeding disorders encountered in clinical practice will be diagnosed, at least initially, by phenotypic assays. However, since the characterization of the genes that encode coagulation factors in the 1980s, significant progress has been made in translating this knowledge for diagnostic and therapeutic purposes. For hemophilia A and B, molecular genetic testing to determine carrier status, prenatal diagnosis, and likelihood of inhibitor development or anaphylaxis to infused coagulation factor concentrates is an established component of comprehensive clinical management.

FVIII stabilization: VWF D'D3 will do.

In this issue of Blood, Yee et al1 have demonstrated that expression or infusion of a truncated von Willebrand factor (VWF) fragment containing the factor VIII (FVIII)-binding D′D3 region of VWF is sufficient to stabilize endogenous FVIII levels in VWF-deficient mice. In the absence of the carrier function of VWF, FVIII is susceptible to rapid proteolysis and clearance resulting in markedly reduced plasma levels of FVIII that contribute to a bleeding diathesis.

Neutrophil extracellular traps promote thrombin generation through platelet-dependent and platelet-independent mechanisms.

Objective: Activation of neutrophils by microbial or inflammatory stimuli results in the release of neutrophil extracellular traps (NETs) that are composed of DNA, histones, and antimicrobial proteins. In purified systems, cell-free DNA (CFDNA) activates the intrinsic pathway of coagulation, whereas histones promote thrombin generation through platelet-dependent mechanisms. However, the overall procoagulant effects of CFDNA/histone complexes as part of intact NETs are unknown.