Peter J. Lenting

Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant

Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAV-expression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non-codon-optimized variants. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 ± 162% of normal) in HA knock-out mice following administration of 2 × 10(12) vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 ± 4% of normal was observed at this dose in a nonhuman primate. hFVIII expression above 100% was observed in 3 macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy.

von Willebrand factor biosynthesis, secretion, and clearance: connecting the far ends

To understand the placement of a certain protein in a physiological system and the pathogenesis of related disorders, it is not only of interest to determine its function but also important to describe the sequential steps in its life cycle, from synthesis to secretion and ultimately its clearance. von Willebrand factor (VWF) is a particularly intriguing case in this regard because of its important auxiliary roles (both intra- and extracellular) that implicate a wide range of other proteins: its presence is required for the formation and regulated release of endothelial storage organelles, the Weibel-Palade bodies (WPBs), whereas VWF is also a key determinant in the clearance of coagulation factor VIII. Thus, understanding the molecular and cellular basis of the VWF life cycle will help us gain insight into the pathogenesis of von Willebrand disease, design alternative treatment options to prolong the factor VIII half-life, and delineate the role of VWF and coresidents of the WPBs in the prothrombotic and proinflammatory response of endothelial cells. In this review, an update on our current knowledge on VWF biosynthesis, secretion, and clearance is provided and we will discuss how they can be affected by the presence of protein defects.

The rate of hemolysis in sickle cell disease correlates with the quantity of active von Willebrand factor in the plasma

Vaso-occlusion, hemolysis, and oxidative stress are hallmarks of sickle cell disease (SCD). This pathology is accompanied by systemic endothelial activation, rendering the endothelium more adhesive for blood cells, including sickle erythrocytes. Activated endothelial cells display or secrete several adhesive molecules, including von Willebrand factor (VWF). We assessed several VWF parameters in SCD patients at baseline: multimer pattern, antigen concentration (VWF:Ag), activation factor (VWF:AF), and total active VWF (VWF:TA). VWF:AF was determined using a llama nanobody (AU/VWFa-11) that detects a platelet-binding conformation of the A1 domain; VWF:TA was calculated by multiplying VWF:Ag by VWF:AF. SCD plasma contained elevated VWF:Ag and ultralarge VWF multimers. VWF:TA, a measure of total VWF reactivity, correlated closely with hemolysis, as determined by serum lactate dehydrogenase. ADAMTS13 activity and antigen were normal in all patients. These findings suggest an important role for hyperreactive VWF in SCD pathology and connect SCD to other microangiopathies, particularly thrombotic thrombocytopenic purpura.

Of von Willebrand factor and platelets

Hemostasis and pathological thrombus formation are dynamic processes that require multiple adhesive receptor-ligand interactions, with blood platelets at the heart of such events. Many studies have contributed to shed light on the importance of von Willebrand factor (VWF) interaction with its platelet receptors, glycoprotein (GP) Ib-IX-V and αIIbβ3 integrin, in promoting primary platelet adhesion and aggregation following vessel injury. This review will recapitulate our current knowledge on the subject from the rheological aspect to the spatio-temporal development of thrombus formation. We will also discuss the signaling events generated by VWF/GPIb-IX-V interaction, leading to platelet activation. Additionally, we will review the growing body of evidence gathered from the recent development of pathological mouse models suggesting that VWF binding to GPIb-IX-V is a promising target in arterial and venous pathological thrombosis. Finally, the pathological aspects of VWF and its impact on platelets will be addressed.

Accelerated uptake of VWF/platelet complexes in macrophages contributes to VWD type 2B-associated thrombocytopenia

Von Willebrand disease (VWD) type 2B is characterized by mutations causing enhanced binding of von Willebrand factor (VWF) to platelets. Bleeding tendency is associated with heterogeneous clinical manifestations, including moderate to severe thrombocytopenia. The underlying mechanism of the thrombocytopenia has remained unclear. Here, a mouse model of VWD type 2B was used to investigate pathways contributing to thrombocytopenia. Immunohistochemical analysis of blood smears revealed that mutant VWF was exclusively detected on platelets of thrombocytopenic VWD type 2B mice, suggesting that thrombocytopenic VWD type 2B mice were elevated two- to threefold upon chemical macrophage depletion. Colocalization of platelets with CD68-positive Kupffer cells and CD168-positive marginal macrophages in liver and spleen, respectively, confirmed the involvement of macrophages in the removal of VWF/platelet complexes. Significantly more platelets were found in liver and spleen of VWD type 2B mice compared with control mice. Finally, platelet survival was significantly shorter in VWD type 2B mice compared with control mice, providing a rationale for lower platelet counts in VWD type 2B mice. In conclusion, our data indicate that VWF type 2B binds to platelets and that this is a signal for clearance by macrophages, which could contribute to the thrombocytopenia in patients with VWD type 2B.

Structure and function of a recombinant von Willebrand factor drug candidate.

The complex structure, large size, and multiple posttranslational modifications of von Willebrand factor (VWF) presented a technological challenge for the production of recombinant VWF (rVWF). Nonetheless, we developed an rVWF product for treating von Willebrand disease, whereupon rVWF is coexpressed with recombinant factor VIII (rFVIII) in Chinese hamster ovary cells used to produce rFVIII for the treatment of hemophilia A. Here we describe the characterization of the structure and function of the rVWF drug product, with a focus on its in vitro platelet aggregation and matrix protein binding functions. Electron microscopy and multimer analysis revealed a highly organized structure for the rVWF protein, with a homogeneous multimer distribution including ultrahigh molecular weight multimers. The specific activity for binding to collagen and platelets mediated by ristocetin is higher in rVWF than in commercial plasma-derived VWF-FVIII complex products. The affinity and binding capacity of rVWF to FVIII is comparable to VWF in plasma. rVWF effectively binds to platelets and promotes platelet adhesion under shear stress similar to VWF in human plasma.

Regulation of von Willebrand factor-platelet interactions

The formation of thrombi is a multistep process involving several components, including von Willebrand factor (VWF). VWF is an adhesive multimeric protein, which acts as a molecular bridge between the subendothelial matrix and the glycoprotein Ib/IX/V receptor complex. Furthermore, VWF promotes the expansion of the platelet plug by cross-linking platelets via binding to integrin alphaIIbbeta3. In terms of thrombus formation, it is essential that VWF-platelet interactions occur timely, that is: it should happen not too early or too late. Given the co-existence of VWF and platelets in the circulation, this implies that there must be regulatory mechanisms that prevent premature formation of VWF-rich platelet aggregates that could occlude the vasculature. Indeed, several mechanisms have been identified at the level of VWF, which are dedicated to the prevention of excessive VWF-platelet interactions following endothelial release of VWF (which may include limited exposure to shear stress, the presence of Mg2+ ions, inhibition of VWF-platelet interactions by endothelial proteins, ADAMTS13-mediated proteolysis) and of circulating VWF-platelet aggregates during normal circulation (shielding of the platelet-binding A1 domain by other regions of the VWF molecule, inhibition of VWF-platelet interactions by beta2-glycoprotein I). In the present review an overview of these mechanisms will be discussed.

von Willebrand Factor as a Biological Sensor of Blood Flow to Monitor Percutaneous Aortic Valve Interventions

RATIONALE Percutaneous aortic valve procedures are a major breakthrough in the management of patients with aortic stenosis. Residual gradient and residual aortic regurgitation are major predictors of midterm and long-term outcome after percutaneous aortic valve procedures. We hypothesized that (1) induction/recovery of high molecular weight (HMW) multimers of von Willebrand factor defect could be instantaneous after acute changes in blood flow, (2) a bedside point-of-care assay (platelet function analyzer-closure time adenine DI-phosphate [PFA-CADP]), reflecting HMW multimers changes, could be used to monitor in real-time percutaneous aortic valve procedures. OBJECTIVE To investigate the time course of HMW multimers changes in models and patients with instantaneous induction/reversal of pathological high shear and its related bedside assessment. METHODS AND RESULTS We investigated the time course of the induction/recovery of HMW multimers defects under instantaneous changes in shear stress in an aortic stenosis rabbit model and in patients undergoing implantation of a continuous flow left ventricular assist device. We further investigated the recovery of HMW multimers and monitored these changes with PFA-CADP in aortic stenosis patients undergoing transcatheter aortic valve implantation or balloon valvuloplasty. Experiments in the aortic stenosis rabbit model and in left ventricular assist device patients demonstrated that induction/recovery of HMW multimers occurs within 5 minutes. Transcatheter aortic valve implantation patients experienced an acute decrease in shear stress and a recovery of HMW multimers within minutes of implantation which was sustained overtime. In patients with residual high shear or with residual aortic regurgitation, no recovery of HMW multimers was observed. PFA-CADP profiles mimicked HMW multimers recovery both in transcatheter aortic valve implantation patients without aortic regurgitation (correction) and transcatheter aortic valve implantation patients with aortic regurgitation or balloon valvuloplasty patients (no correction). CONCLUSIONS These results demonstrate that variations in von Willebrand factor multimeric pattern are highly dynamic, occurring within minutes after changes in blood flow. It also demonstrates that PFA-CADP can evaluate in real time the results of transcatheter aortic valve procedures.

Models for Prediction of Factor VIII Half-Life in Severe Haemophiliacs: Distinct Approaches for Blood Group O and Non-O Patients

Background Von Willebrand factor (VWF) is critical for the in vivo survival of factor VIII (FVIII). Since FVIII half-life correlates with VWF-antigen pre-infusion levels, we hypothesized that VWF levels are useful to predict FVIII half-life. Methodology Standardized half-life studies and analysis of pre-infusion VWF and VWF-propeptide levels were performed in a cohort of 38 patients with severe haemophilia A (FVIII <1 IU/ml), aged 15–44 years. Nineteen patients had blood-group O. Using multivariate linear regression-analysis (MVLR-analysis), the association of VWF-antigen, VWF-propeptide, age and body-weight with FVIII half-life was evaluated. Principal Findings FVIII half-life was shorter in blood-group O-patients compared to non-O-patients (11.5±2.6 h versus 14.3±3.0 h; p = 0.004). VWF-antigen levels correlated with FVIII half-life considerably better in patients with blood-group non-O than O (Pearson-rank = 0.70 and 0.47, respectively). Separate prediction models evolved from MVLR-analysis for blood-group O and non-O patients, based on VWF-antigen and VWF/propeptide ratio. Predicted half-lives deviated less than 3 h of observed half-life in 34/38 patients (89%) or less than 20% in 31/38 patients (82%). Conclusion Our approach may identify patients with shorter FVIII half-lives, and adapt treatment protocols when half-life studies are unavailable. In addition, our data indicate that survival of FVIII is determined by survival of endogenous VWF rather than VWF levels per se.

Life in the shadow of a dominant partner: the FVIII-VWF association and its clinical implications for hemophilia A

A normal hemostatic response to vascular injury requires both factor VIII (FVIII) and von Willebrand factor (VWF). In plasma, VWF and FVIII normally circulate as a noncovalent complex, and each has a critical function in the maintenance of hemostasis. Furthermore, the interaction between VWF and FVIII plays a crucial role in FVIII function, immunogenicity, and clearance, with VWF essentially serving as a chaperone for FVIII. Several novel recombinant FVIII (rFVIII) therapies for hemophilia A have been in clinical development, which aim to increase the half-life of FVIII (∼12 hours) and reduce dosing frequency by utilizing bioengineering techniques including PEGylation, Fc fusion, and single-chain design. However, these approaches have achieved only moderate increases in half-life of 1.5- to 2-fold compared with marketed FVIII products. Clearance of PEGylated rFVIII, rFVIIIFc, and rVIII-SingleChain is still regulated to a large extent by interaction with VWF. Therefore, the half-life of VWF (∼15 hours) appears to be the limiting factor that has confounded attempts to extend the half-life of rFVIII. A greater understanding of the interaction between FVIII and VWF is required to drive novel bioengineering strategies for products that either prolong the survival of VWF or limit VWF-mediated clearance of FVIII.