Recombinant Human Von Willebrand Factor Research Articles

High-Density Lipoprotein Inhibits Microvascular and Arterial Thrombosis in Mice By Attenuating VWF Self-Association

Background: Von Willebrand factor (VWF) mediates platelet adhesion and aggregation in primary hemostasis and thrombosis. The thrombogenicity of VWF depends on its self-association into fibers and its ability to efficiently capture platelets under high shear stress. We have shown that high shear promotes self-association of VWF into hyperadhesive strands and fibers, and that this process can be enhanced by low density lipoprotein (LDL) and attenuated by high density lipoprotein (HDL). In a model of thrombotic microangiopathy induced by VWF infusion into ADAMTS13-deficient mice, HDL attenuated the thrombocytopenia induced by the VWF challenge, suggesting that HDL may attenuate other types of thrombosis. Aims: To study the effect of HDL on microvascular and arterial thrombosis in mice by intravital microscopy. Methods: Microvascular thrombosis was induced in ADAMTS13-deficient mice by (1) local stimulation of mesenteric venules with calcium ionophore, and (2) infusion of high doses of recombinant human VWF (rVWF). The effect of HDL on VWF-platelet thrombus formation and deposition in the microvasculature was monitored microscopically. Arterial thrombosis was induced in wild-type (WT) mice by (1) laser-induced cremaster injury, and (2) FeCl 3-induced carotid artery injury. The effect of HDL on arterial thrombosis was also monitored microscopically. Results: When we induced microvascular thrombosis with ionophore, ADAMTS13-deficient mice evinced robust local platelet adhesion, platelet string formation, and thrombus embolization. Time to thrombus resolution was significantly prolonged (about 15 min) compared to that in WT mice (< 5 min). In mice pretreated with HDL (100 mg/kg), platelet deposition was largely prevented, with only occasional single platelets adhering. The differences in the extent of thrombi deposition and time to thrombus resolution between HDL-treated and untreated mice were both statistically significant. In the VWF challenge microvascular thrombosis model, rVWF infusion into ADAMTS13-deficient mice induced platelet adhesion to endothelium-associated VWF fibers in the cremaster microcirculation, forming long strings on the walls of arterioles and venules. Many microvessels became completely occluded. The prothrombotic effect persisted over 90 minutes. In contrast, pretreatment with HDL (100 mg/kg) led to significantly reduced platelet adhesion and string formation. Vessel occlusion was almost completely prevented. When thrombosis was induced in the cremaster arterioles of WT mice, HDL pretreatment resulted in notably decreased platelet accumulation and fibrin deposition in growing thrombi. HDL pretreatment also destabilized the thrombi and led to frequent embolization. In the FeCl 3-induced carotid artery injury model, HDL pretreatment slowed thrombus growth, enhanced thrombus embolization, and delayed vessel occlusion. Conclusion(s): The results demonstrate that VWF self-association is an important mechanism promoting thrombosis in large and small vessels. Our results show that HDL possesses a novel antithrombotic effect by inhibiting VWF self-association, preventing thrombosis in both small and large vessels. These results suggest that increasing the plasma HDL level can be exploited to prevent and treat thrombotic microangiopathies and arterial thrombosis.

Nanobody Activators of Von Willebrand Factor Via Targeting Its Autoinhibitory Module

Introduction: Von Willebrand factor (VWF) is a multimeric plasma glycoprotein responsible for platelet arrest during injury, especially at high shear. After immobilization to the vessel wall, a VWF multimer is unfurled and elongated. This leads to exposure of the A1 domain therein that in turn binds to platelet receptor GPIbα and starts the aggregation process. Recently, it was suggested that VWF activation involves force-dependent disruption of the autoinhibitory module (AIM) that flanks the A1 domain on both sides. In this scenario, the AIM could be targeted for both VWF inhibition (Caplacizumab) and activation (ristocetin), although the exact mechanism and binding site of ristocetin still remains murky. If the quasi-stable structure of the AIM is important to VWF autoinhibition, specific disruption of its confirmation may be able to activate VWF. To this end, we sought to identify AIM-targeting activators using yeast surface display of a llama nanobody library.Methods: One adult Lama glama was immunized with recombinant human VWF AIM-A1 protein produced from transfected Expi293F cells. VHH specific genes were amplified from cDNAs prepared from PBMCs of the animal and electroporated into EBY100 cells. The resulting yeast display library was screened for AIM-specific binders via selection against binding to recombinant A1 protein without an intact AIM, and then for binding to the complex of AIM-A1 with GPIbα. Positive hits were produced as His-tagged monomeric nanobodies in E. coli and purified with nickel-affinity and gel filtration chromatography. The affinity of nanobodies to AIM-A1 was determined using bio-layer interferometry. Platelet-rich plasma from healthy donors was used to assess the effect of nanobodies on platelet aggregation in a light transmission aggregometer with comparison to that of ristocetin.Results: An AIM-A1-specific nanobody yeast display library was established. Several rounds of flow cytometry-based cell sorting of yeast cells with aforementioned binding properties produced AIM-binding nanobodies. Nanobodies encoded in three single clones have been expressed from E. coli and they exhibited differential binding affinities towards AIM-A1. Clone 6C4 showed the lowest affinity (K D 120 ± 3 nM), 6D12 showed intermediate affinity (K D 31 ± 0.8 nM), and 6C11 showed the highest affinity (K D 13.5 ± 0.2 nM) as shown in Figure 1. These nanobodies showed no detectable affinity towards recombinant A1-CAIM protein (residues 1268-1493), indicating that their epitopes are located in the N-terminal portion of the AIM (residues 1238-1267).When added to human platelet-rich plasma, each nanobody dose-dependently activated platelets and rapidly induced full platelet aggregation at concentrations exceeding the affinity of the nanobody for VWF (Figure 2). The aggregation could be inhibited by the addition of antibodies that block the interaction between VWF and GPIbα. Plots of extents of aggregation as a function of nanobody concentration produced EC 50 values of ~100 nM for 6C11 and 6D12.Conclusion: By isolating nanobodies that can bind specifically to the AIM and activate plasma VWF, we add supporting evidence that the AIM protects the A1 domain from binding to platelets. Interestingly, these nanobodies bind to the NAIM, on the opposite side of the module compared to ristocetin, the only known AIM-activating agent until now. With higher VWF-binding affinities than ristocetin and a robust profile as stable monomers, these nanobodies may prove useful in VWF-related research and diagnostics. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Primary Myeloma Cell Induced Von Willebrand Factor Release from the Endothelium Is Mediated By VEGF and Attenuated By Heparin

IntroductionMultiple Myeloma (MM) remains an incurable disease and is associated with high rates of venous thromboembolism (VTE), the biological basis for which is not fully understood. Critically, VTE is associated with increased mortality in MM. This highlights the clinical importance of understanding cancer-coagulation crosstalk in MM. Accumulating evidence demonstrates that many solid tumours trigger endothelial cell (EC) activation with resultant von Willebrand Factor (VWF) secretion proposed to contribute to both risk of VTE and cancer metastasis. However, the interplay between VWF and MM disease biology remains poorly defined.Methods/Results100 patients with plasma cell disorders were recruited to this study. Significantly elevated plasma VWF antigen (VWF:Ag) levels were observed in patients with newly diagnosed MM (NDMM) compared to those with monoclonal gammopathy of undetermined significance (MGUS) or smouldering MM (SMM) (median 292.7 IU/dL vs 133.1 IU/dL; P<0.0001). Moreover, VWF:Ag levels in those with relapsed/refractory MM (RRMM) were further elevated (median 339 IU/dL; P<0.0001]). VWF:Ag levels also appear to correlate with disease response to therapy, with lower levels seen in patients achieving a complete response/very good partial response compared with those gaining only a partial response or worse (median 154.45 IU/dL vs 338.5 IU/dL; P<0.05).We also observed increased VWF collagen binding activity in NDMM/RRMM compared with MGUS/SMM (median 455.7 IU/dL vs 189.7 IU/dL; P<0.05). This qualitative measurement is sensitive to alterations in haemostatically active high molecular weight multimers of VWF secreted upon EC activation. Supporting this, raised plasma VWF propeptide (VWFpp) levels, a marker of acute EC activation, were seen in those with NDMM/RRMM compared with MGUS/SMM (median 182 IU/dL vs 133.01 IU/dL; P<0.05). Reduced VWFpp/VWF:Ag ratios were seen in those with NDMM/RRMM, indicating decreased circulatory clearance of VWF. We hypothesise that MM may exhibit a ‘double-hit’ in regulation of plasma VWF:Ag with increased secretion of VWF but also simultaneously reduced VWF clearance leading to sustained and markedly elevated systemic plasma VWF levels.Next we measured VWF:Ag levels in bone marrow (BM) samples from 10 patients and found that VWF is released locally within the BM niche (median 190.2 IU/dL). Co-culture of primary human ECs with supernatant from freshly isolated primary MM cells or several Human Myeloma Cell Lines (HMCLs) demonstrated that MM cells stimulate release of VWF from ECs in a rapid manner, suggestive of Weibel-Palade body exocytosis (median 34.9ng/ml vs 9.2ng/ml in untreated EC; P<0.05). Elevated levels of Vascular Endothelial Growth Factor-A (VEGF-A), a potent EC activator, were observed in MM cell supernatants. Pre-treatment of primary MM cells or HMCLs with anti-VEGF antibody bevacizumab abolished EC VWF secretion in a dose dependant manner (32.3ng/ml vs anti-VEGF treated 10.1ng/ml P<0.0001). Furthermore, clinically relevant doses of low molecular weight heparin (LMWH) also attenuated VWF secretion (29.6ng/ml vs LMWH treated 15ng/ml; P<0.05). Measurement of VEGF-A in primary MM cell and HMCL supernatant treated with LMWH or bevacizumab confirmed significant reductions in VEGF-A levels. Together these data indicate that stimulation of VWF release from the endothelium by MM cells is mediated by VEGF-A and directly inhibited by LMWH treatment.Using flow cytometry, we assessed the direct interaction of MM cells and VWF in vitro. Both primary MM cells and HMCLs bound significantly to human recombinant VWF in a dose-dependent manner. Binding was reduced by 50% following treatment with LMWH (P<0.05). Given that the VWF A1 domain contains a heparin binding motif, we speculate that MM cell adhesion to VWF may be at least in part mediated by this domain and future work focuses on elucidating the receptors involved.ConclusionThese data suggest that MM is not only associated with a marked quantitative increase in plasma VWF:Ag, but also impacts VWF functional activity. Our novel data help define the biological mechanisms underpinning elevated VWF levels in MM, with a key contribution of VEGF-A secreted directly from MM cells in the BM microenvironment. Collectively, our findings provide insights into cancer-coagulation crosstalk in MM and may help identify novel therapeutic targets to reduce VTE risk and disease progression. DisclosuresGlavey: Celgene and BMS company: Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding. Quinn: Takeda: Honoraria. O'Sullivan: Leo Pharma: Research Funding.

In Vitro Assessment of von Willebrand Factor in Cryoprecipitate, Antihemophilic Factor/VWF Complex (Human), and Recombinant von Willebrand Factor.

Patients with von Willebrand disease (VWD) often require treatment with supplemental von Willebrand factor (VWF) prior to procedures or to treat bleeding. Commercial VWF concentrates and more recently recombinant human VWF (rVWF) have replaced cryoprecipitate as the mainstay of therapy. In comparison with cryoprecipitate, the VWF content and multimer distribution under current manufacturing processes of these commercial products has not been reported. We measured the factor VIII (FVIII:C), VWF antigen (VWF:Ag), VWF collagen-binding activity (VWF:CB), VWF platelet-binding activity by GPIbM enzyme-linked immunosorbent assay (VWF:GPIbM), and percentage of high-molecular-weight (HMWM) VWF in 3 pools of group A and O cryoprecipitate, 3 vials of VWF concentrate (Humate-P), and 1 lot of rVWF (Vonvendi). We found that both group O and group A cryoprecipitate have significantly higher ratios of VWF:GPIbM activity and FVIII:C activity relative to VWF:Ag and have better preservation of HMWM than Humate-P. Although not compared statistically, rVWF appears to have more HMWM VWF and a higher ratio of VWF:GPIbM to VWF:Ag than Humate-P and cryoprecipitate. The estimated acquisition cost for our hospital for treating one major bleeding episode was more than 4-fold higher with Humate-P and 7- to 10-fold higher with rVWF than with cryoprecipitate.

Successful Management of Pregnancy with Severe Von Willebrand Disease in a Jehovah's Witness with Recombinant Von Willebrand Factor

Background: Von Willebrand disease (vWD) is the most common inherited bleeding disorder with a reported prevalence of 1% in epidemiological studies and symptomatic prevalence of 1 in 10,000.Pregnancy in vWD is associated with increased bleeding risk particularly postpartum hemorrhage. Treatment options include desmopressin acetate (DDAVP), plasma derived factor concentrates and antifibrinolytic agents. Human Recombinant von Willebrand factor (vWF) (Vonvendi®) has been approved in the United States for on demand treatment and perioperative management of adults with vWD. It has been shown to maintain sustained levels of VWF activity but requires co-administration with FVIII to achieve adequate FVIII levels. Recombinant VWF is an option for patients who refuse blood for religious reasons.Case: Here we describe a 39 year old patient in her third pregnancy who is a Jehovah's Witness. Consent was obtained from the patient for this report. She was initially diagnosed with von Willebrand's disease at the age of six, when she had hematuria. At the age of 11, with menarche, she had significant menorrhagia resulting in symptomatic anemia, a reduction in her hemoglobin concentration to 44 g/L, requiring uterine artery embolization. She was placed on an oral contraceptive pill for menorrhagia. She used DDAVP for trauma induced injury. Her first pregnancy resulted in spontaneous abortion. She required a D&C and DDAVP was used. Prior to delivery of the second pregnancy DDAVP was used but she had postpartum hemorrhage, requiring additional dosing of DDAVP and uterine artery embolization.In the current pregnancy, aPTT was 36.4 seconds with normal PT and platelet count and blood group O. At 15 weeks' gestational age, VWF antigen (ACL TOP 700 -IL HemosIL) was 0.11 units/ml (normal range for blood group O 0.45-1.5 unit/ml), ristocetin cofactor (ACL TOP 700 -IL HemosIL), 0.09 units/ml (normal range 0.48-2.0 units/ml for blood group O), and FVIII level (Sysmex CS5100 -Dade Actin FS) 0.09 units/ml (normal range 0.58-1.9 units/ml). Factor levels at 23 weeks' gestation 1 hour following DDAVP were FVIII 1.05 units/ml, VWF antigen 0.5 units/ mL, and ristocetin cofactor 0.52 units/ml. She was willing to accept recombinant factor concentrates only.Results: She had an elective admission for induction of labor at 37 weeks but proceeded to Cesarean Section due to non-progression of labor. Her PTT on admission was 40 seconds with VWF antigen of 0.11 units/ml, VWF activity < 0.07 units/ml and FVIII 0.13 units/ml. The Table describes her levels following the administration of recombinant vWF. She was administered Vonvendi® on a planned dose of 40 IU/kg Xynta® and rFVIII 30 IU/kg to increase factor levels to more than 50%; 60-min following infusions vWF : RCo was 0.64 U/ml with FVIII 0.79 U/ml. The Cesarean section was performed under spinal anesthesia without complications. Tranexamic acid was used intravenously before the delivery of the neonate and continued for first two days and then changed to oral dose.Target levels were achieved over the 5 days after delivery with the current regimen. She experienced a transient hypersensitivity reaction with urticaria and dyspepsia after the third dose of Vonvendi®. She did not have significant bleeding. Her hemoglobin concentration remained stable at 120 g/L throughout her inpatient stay.The neonate did not have bleeding with delivery but was found to have VWF antigen of 0.11 U /ml, VWF activity of 0.07 U/ml and FVIII of 0.16 U/ml. Genetic analysis of the mutation associated with her vWD is in progress.Conclusion: The use of rvWF and rVIII resulted in adequate hemostasis peripartum. Further prospective data are required to reaffirm the safety and dosing of rvWF for peripartum management of patients with vWD who require intervention. DisclosuresKazi:Shire: Other: Vonvendi was provided by Shire.

Platelet rescue by macrophage depletion in obese ADAMTS‐13‐deficient mice at risk of thrombotic thrombocytopenic purpura

Background Thrombotic thrombocytopenic purpura (TTP) is caused by the absence of ADAMTS-13 activity. Thrombocytopenia is presumably related to the formation of microthrombi rich in von Willebrand factor (VWF) and platelets. Obesity may be a risk factor for TTP; it is associated with abundance of macrophages that may phagocytose platelets. Objectives To evaluate the role of obesity and ADAMTS-13 deficiency in TTP, and to establish whether macrophages contribute to thrombocytopenia. Methods Lean or obese ADAMTS-13-deficient (Adamts-13-/- ) and wild-type (WT) mice were injected with 250 U kg-1 of recombinant human VWF (rVWF), and TTP characteristics were evaluated 24 h later. In separate experiments, macrophages were depleted in the liver and spleen of lean and obese WT or Adamts-13-/- mice by injection of clodronate-liposomes, 48 h before injection of rVWF. Results Obese Adamts-13-/- mice had a lower platelet count than their lean counterparts, suggesting that they might be more susceptible to TTP development. Lean Adamts-13-/- mice triggered with a threshold dose of rVWF did not develop TTP, whereas typical TTP symptoms developed in obese Adamts-13-/- mice, including severe thrombocytopenia and higher lactate dehydrogenase (LDH) levels. Removal of hepatic and splenic macrophages by clodronate injection in obese Adamts-13-/- mice before treatment with rVWF preserved the platelet counts measured 24 h after the trigger. In vitro experiments with cultured macrophages confirmed a VWF dose-dependent increase of platelet phagocytosis. Conclusions Obese Adamts-13-/- mice are more susceptible to the induction of TTP-related thrombocytopenia than lean mice. Phagocytosis of platelets by macrophages contributes to thrombocytopenia after rVWF injection in this model.

The role of ultralarge multimers in recombinant human von Willebrand factor - a review of physico-and biochemical studies and findings in in vivo models and in humans with von Willebrand disease.

Ultralarge multimers (ULM) of VWF are considered to be the most active with respect to binding to platelets and to subendothelial structures and therefore are of critical importance for the function of VWF in stabilizing the primary hemostatic plug. In contrast to plasma-derived FVIII-VWF concentrates, human rVWF obtained from mammalian cell culture retains the full-spectrum of intact multimers, including ULM, as physiologically formed in the Golgi apparatus and stored in platelet α-granules and endothelial cell Weibel-Palade bodies. In the course of physico and biochemical, functional and animal studies, rVWF exhibited superiority in structure and function compared to pdVWF. These effects seemed to correlate with the multimer size and therefore might be attributed to the presence of ULM in rVWF preparations. The pharmacokinetic (PK), safety and efficacy characteristics seen in preclinical studies were further demonstrated in clinical trials.

Phylogenetic Analysis Identifies a Subset of ADAMTS13 Domains That Are Highly Conserved and Essential for Allosteric Regulation

Introduction: Human ADAMTS13 has metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C) and spacer (S) domains (proximal domains), followed by 7 T and 2 CUB domains (distal domains).ADAMTS13 regulates von Willebrand factor (VWF) by cleaving a Tyr-Met bond in the VWF A2 domain that is exposed under shear force. Acquired or inherited ADAMTS13 deficiency impairs this regulatory mechanism and cause thrombotic thrombocytopenic purpura (TTP), a life threatening disorder. We recently showed that ADAMTS13 is autoinhibited by its distal T8-CUB domains and this inhibition is relieved by binding to VWF. Thus, VWF promotes its own destruction by allosterically activating ADAMTS13. We investigated the evolution and conservation of this remarkable regulatory mechanism.Methods:ADAMTS13 cDNA sequences for >300 species were assembled from genome assemblies and SRA transcriptomic and genomic data. Recombinant ADAMTS13 variants were expressed in HEK293 cells and purified chromatographically. Plasma was obtained from dog, armadillo, rabbit, primates (human, baboon, marmoset, macaque), rodents (rat, mouse), ungulates (cattle, sheep, pig, horse), birds (chicken, pigeon), and Xenopus laevis. Plasma ADAMTS13 activity of at least three members of each species was assayed with human-FRETS71, a fluorogenic ADAMTS13 substrate based on human VWF. Similar fluorogenic substrates were prepared with armadillo, rat, pigeon and cattle VWF sequences. Plasma ADAMTS13 also was assayed with allosteric activators of human ADAMTS13: monoclonal antibodies (MAbs) against distal T and CUB domains, low pH (pH 6), and recombinant human VWF D4 domain. ADAMTS13-VWF interactions were assessed with biolayer interferometry (BLI) and binding data fitted to 1:1 binding model.Results: The ancestral ADAMTS13 had a long propeptide that is characteristic of most metalloproteases but notably missing from human ADAMTS13, and 8 distal T domains compared to 7 distal T domains in human ADAMTS13. The ancestral structure today is found in most reptiles, amphibians, and marsupials. The propeptide, T3, T4, T6 and T6a have been deleted independently several times in mammals, birds, amphibians and fish. Most animal plasmas cleaved human-FRETS71 less rapidly than human plasma, although armadillo, rabbit, and dog were 2.4-, 3.0-, 4.0-fold more active, respectively. Human and cattle VWF have Tyr-Met cleavage sites, but armadillo VWF has a Tyr-Leu bond. Armadillo plasma cleaved armadillo-FRETS71 and human-FRETS71 at a similar rate; other species cleaved armadillo-FRETS71 15% to 88% as rapidly as human-FRETS71. Surprisingly, cattle-FRETS71 was cleaved ~2- to 15-fold faster than human-FRETS71 by most plasmas. Except for C57Bl/6 mice (which have ADAMTS13 truncated after T6), armadillo, and dog, all plasmas screened were activated by MAbs or VWF D4. Frog and pigeon ADAMTS13 were activated ~6-fold by D4, whereas human ADAMTS13 was activated only 2-fold. Absence of CUB domains in armadillo ADAMTS13 abolished activation by D4. In contrast, absence of all but 3 distal T domains in pigeon ADAMTS13 did not impair activation by VWF D4. Recombinant human ADAMTS13 constructed with deletions like those in armadillo and pigeon ADAMTS13 had similar properties: delT3-6 was activated by VWF D4 but M-T8 was not. All enzymes activated by VWF D4 showed enhanced cleavage of multimeric VWF under fluid shear stress compared to enzymes that are not activated by VWF D4 (Figure 1 and Table 1). When normalized for activity toward FRETS71, pigeon ADAMTS13 was ~2-fold more specific for VWF multimers than is human ADAMTS13. In agreement with the cleavage data, recombinant human and pigeon ADAMTS13, and MT-8 bound VWF D4-CK with Kd values in the 350-840 nM range, whereas MDTCS, MT-7 and delT2-7 did not bind.Conclusion: Allosteric regulation of ADAMTS13 by VWF is broadly conserved, with some apparent exceptions. The distal T3-T6 domains are dispensable for allosteric regulation, whereas T7, T8 and CUB domains are required. Furthermore, ADAMTS13 from different species exhibits striking variation in substrate specificity. For most species, altering the sequence of the VWF substrate can markedly increase the rate of cleavage, indicating that ADAMTS13 and VWF have not evolved to be optimal enzyme-substrate pairs. These properties are likely to reflect evolutionary pressure to balance the risk of VWF-dependent bleeding and thrombosis. [Display omitted] [Display omitted] DisclosuresSadler:Ablynx: Consultancy; 23andMe: Consultancy; BioMarin: Consultancy.

The co-influence of VWD type 2B/2M mutations in the A1 domain and platelet GPIbα on the rate of cleavage to VWF by ADAMTS13

IntroductionIn plasma, the size of the von Willebrand factor (VWF) multimer is down-regulated by ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13). The binding of platelets or glycoprotein (GP) Ibα recombinant fragment to VWF domain A1 may increase the cleavage by ADAMTS13 to VWF. Both type 2B and type 2M von Willebrand disease (VWD) result in bleeding disorders with the diathesis of increased and decreased binding affinity between GPIbα and VWF, respectively. However, the influence of 2B/2M VWD mutations in the A1 domain and GPIbα on cleavage by ADAMTS13 to VWF needs further study. Materials and MethodsDifferent types of full-length human recombinant VWF (rVWF) were expressed, including three type 2B mutations (P1337L, H1268D, and R1308C), one type 2M mutation (D1302G), and wild type (WT). The three characterized types of rVWF were digested by ADAMTS13 under static conditions or high-shear stress. The interaction of rVWF and ADAMTS13 was also tested by plate-binding assays. ResultsUnder static (natured) conditions or high-shear stress, type 2B mutants exhibited a higher susceptibility to ADAMTS13 than rVWF-WT, whereas type 2M mutant was normal. While under static (denatured) conditions or high-shear stress (with GPIbα fragment) rVWF-WT showed an even higher susceptibility to ADAMTS13 than the two type 2B mutants studied. ConclusionType 2B mutations localized in the A1 domain could enhance the sensitivity to ADAMTS13-mediated proteolysis. When GPIbα participated, there was a dramatically increased proteolytic cleavage of VWF by ADAMTS13 to rVWF-WT, excluding some type 2B mutants.

The novel ADAMTS13‐p.D187H mutation impairs ADAMTS13 activity and secretion and contributes to thrombotic thrombocytopenic purpura in mice

Congenital thrombotic thrombocytopenic purpura (TTP) is characterized by mutations in the ADAMTS13 gene, which either impair protein secretion or influence ADAMTS13 (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type-1 motif, member 13) activity. Phenotypic consequences of these mutations have not yet been evaluated in animal models for TTP. To identify the in vitro effect of a novel ADAMTS13 mutation and to investigate whether this mutation induces TTP in vivo. All 29 ADAMTS13 exons with exon-intron boundaries of a patient with pregnancy-onset TTP were sequenced. Wild-type and mutant ADAMTS13 proteins were both transiently and stably expressed in human embryonic kidney cells, and their activity was evaluated in vitro using fluorescence resonance energy transfer and flow assays. Molecular dynamics simulations were performed to study Ca(2+) stability. Adamts13(-/-) mice were hydrodynamically injected with wild-type and mutant expression plasmids and triggered with recombinant human von Willebrand factor. We identified a novel heterozygous c.559G>C mutation in exon 6 of the proposita's ADAMTS13 gene. This mutation resulted in a p.Asp187His substitution (p.D187H), which was located in the high affinity Ca(2+) -binding site in the metalloprotease domain of ADAMTS13. The homozygous p.D187H mutation down-regulated ADAMTS13 activity in vitro. Impaired proteolytic activity was linked to unstable Ca(2+) binding as visualized using a molecular dynamics simulation. In addition, the p.D187H mutation affects protein secretion in vitro. In Adamts13(-/-) mice, the homozygous p.D187H mutation reduced ADAMTS13 secretion and activity and contributed to TTP when these mice were triggered with recombinant human von Willebrand factor. Our data indicate that the p.D187H mutation impairs ADAMTS13 activity and secretion and is responsible for TTP onset in mice.

Assessment Of Endothelial Damage and Cardiac Injury In a Mouse Model Mimicking Thrombotic Thrombocytopenic Purpura

Background Thrombotic thrombocytopenic purpura (TTP) is a life-threatening illness caused by deficiency of the Von Willebrand factor (VWF) cleaving protease ADAMTS13. Endothelial injury is believed to be a key initiating event in the pathogenesis leading to platelet activation and formation of platelet-rich thrombi in microvasculature. However, the nature of endothelial injury in TTP is poorly defined and seems to differ among the microvascular territory. Especially, a cardiac injury has recently been identified and hypothesized as a major cause of death in TTP. Methods We used an animal model in which some TTP-like symptoms have been triggered in ADAMTS13 knockout mice (B6.129-ADAMTS13tm1Dgi ) by challenge with 2000 units/kg body weight of recombinant human VWF containing ultra large VWF (rhVWF, Baxter, Vienna Austria). rhVWF was also injected in 6 wild type mice (C57/bl6-B129 WT+VWF) while seven other wild type mice received placebo (WT). Cardiac involvement was assessed by transthoracic echocardiography using validated parameters i.e left ventricular ejection fraction (LVEF), fractional shortening (FS) and cardiac index (CI). Cardiac perfusion was determined by arterial spin labeling MRI (Biospec 47/40 advanced II, Brucker, Ettlingen, Germany). Ex vivo endothelial function analysis in isolated coronary, mesenteric and renal segments were performed using a myograph. Arteries were precontracted by phenylephrine or serotonin and the relaxations induced by increasing concentrations of acetylcholine (receptor-mediated, endothelium-dependent relaxation) or the nitric oxide (NO) donor sodium nitroprusside (SNP; endothelium-independent- relaxation) were assessed. Arteries were also incubated with the NO synthase-inhibitor N-Nitro L-Arginine (LNNA) to assess the contribution of NO to the relaxing responses to acetylcholine (endothelium-dependent NO-independent-relaxation). Results On day 1, VWF-challenged ADAMTS13 KO mice rapidly developed symptoms and severe thrombocytopenia (41.8G/L ± 6.9G/L vs 480.7G/L ± 86.5G/L (WT+VWF) and 1118.6G/L ± 160.8G/L (WT); p&lt;0.001), schistocytosis and a decrease in hematocrit levels (40.9% ± 0.8% vs WT+VWF: 51.3% ± 3,8%; p&lt; 0.05 and WT: 56.1% ± 3%; p&lt; 0.001). On day 2, necropsy revealed that macroscopic lesions were restricted to the heart and consisted of acute myocardial hemorrhage and necrosis. Myocardial necrosis was accompanied by infiltration of lymphocytes and interstitial edema. Minimal, acute tubular necrosis in the kidneys was seen in a few animals. Injection of rhVWF in ADAMTS13 KO mice resulted in a decrease in LVEF (73.8% ± 2.7% vs WT+VWF: 86.6% ± 1.3% and WT: 86.8% ± 1.3%; p&lt; 0.01), in FS (37.3% ± 2.1% vs WT+VWF: 50.1% ± 1.6%; p&lt;0.05 and WT: 50.9% ± 12.8%; p&lt; 0.01) and in CI (1.21 ml/min/g ± 0.09 vs WT+VWF: 1.70 ml/min/g ± 0.14 and WT: 1.66 ml/min/g ± 0.1; p&lt; 0.05) suggesting an early systolic dysfunction. However, cardiac perfusion was not significantly affected on day 2. Administration of rhVWF in ADAMTS13KO mice decreased the NO-mediated relaxing responses to acetylcholine in mesenteric (51.1% ± 14.8% vs WT+VWF: 94.4% ± 1.6%; p&lt; 0.05 and WT: 96.5% ± 2.3%; p&lt; 0.001) and coronary arteries (61.7% ± 12.4% vs WT+VWF: 89.9% ± 6.3% and WT: 96.8% ± 0.9%; p&lt; 0.05) but not in renal arteries. Interestingly, under LNNA, endothelium-dependent relaxations were also markedly affected in coronary arteries (19.3% ± 3.7% vs WT+VWF: 70.2% ± 4.7%; p&lt;0.001) in ADAMTS13KO mice suggesting the role of other endothelium-derived vasoactive factors. In parallel, the relaxing responses to SNP were slightly affected (47.8% ± 13.8% vs WT+VWF: 79.1% ± 13.3% and WT: 80.2% ± 7.2%; p&lt; 0.05), suggesting a strong coronary vascular dysfunction on day 2. Conclusions This experimental study reproduced an animal model in which TTP like symptoms are triggered in ADAMTS13 KO mice by administration of VWF and demonstrated that the heart is the most sensitive target organ with myocardial necrosis and systolic dysfunction. We showed for the first time a strong mesenteric and coronary endothelial dysfunction in an induced-TTP model. It may represent an important trigger of the systemic organs failure occurring in this disease, strengthening the need to define the mechanisms involved in this early vascular injury. Disclosures: Schiviz: Baxter: Employment.

Pharmacokinetics and safety of a novel recombinant human von Willebrand factor manufactured with a plasma-free method: a prospective clinical trial

AbstractSafety and pharmacokinetics (PK) of recombinant von Willebrand factor (rVWF) combined at a fixed ratio with recombinant factor VIII (rFVIII) were investigated in 32 subjects with type 3 or severe type 1 von Willebrand disease (VWD) in a prospective phase 1, multicenter, randomized clinical trial. rVWF was well tolerated and no thrombotic events, inhibitors, or serious adverse events were observed. The PK of rVWF ristocetin cofactor activity, VWF antigen, and collagen-binding activity were similar to those of the comparator plasma-derived (pd) VWF-pdFVIII. In vivo cleavage of ultra-large molecular-weight rVWF multimers by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; the endogenous VWF protease) and generation of characteristic satellite bands were demonstrated. In 2 subjects with specific nonneutralizing anti-VWF–binding antibodies already detectable before rVWF infusion, a reduction in VWF multimers and VWF activity was observed. Stabilization of endogenous FVIII was enhanced following post–rVWF-rFVIII infusion as shown by the difference in area under the plasma concentration curve compared with pdVWF-pdFVIII (AUC0-∞) (P < .01). These data support the concept of administering rVWF alone once a therapeutic level of endogenous FVIII is achieved. This trial was registered at www.clinicaltrials.gov as #NCT00816660.

Preclinical Efficacy Testing of Baxter's Recombinant ADAMTS13 in a Mouse Model of TTP.

Abstract 2216Baxter is currently developing a recombinant ADAMTS13 (rADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) product for potential prophylaxis and treatment of thrombotic thrombocytopenic purpura (TTP). We established a disease model in ADAMTS13 ko mice (B6.129-ADAMTS13tm1Dgi), in which the animals simultaneously and consistently develop TTP-like symptoms by challenge with a high dose of human recombinant von Willebrand factor (rVWF) containing ultra-large VWF multimers.To test the prophylactic efficacy of Baxter’s rADAMTS13, groups of 10 ADAMTS13 ko mice received single doses of rADAMTS13 (1, 5, 40, 80 or 200U/kg) before challenge with rVWF. Doses of 1 and 5mL/kg fresh frozen plasma corresponding to 1 and 5U/kg ADAMTS13 served as positive controls. In a second study, the prophylactic and therapeutic efficacy of Baxter’s rADAMTS13 was tested over time. Groups of 10 ADAMTS13 ko mice received a single dose of 200 FRETS-U/kg rADAMTS13 5min-120h before, or 15–180min after challenge with rVWF. In both studies, buffer was used as a negative control. Efficacy was defined as the degree of prevention of platelet drop and LDH increase. Schistocytosis and organ damage were also assessed.Morbidity in negative controls was 100%: all animals receiving rVWF containing ultra-large VWF multimers were severely thrombocytopenic, showed increased LDH levels, schistocytosis and organ damage.Prophylactic treatment with rADAMTS13 prevented LDH increase (p<0.0001), and dose-dependently prevented thrombocytopenia (p≤0.0003), schistocytosis and alleviated organ damage compared to treatment with buffer.Efficacy of rADAMTS13 was treatment interval-dependent in both studies. Platelet count at termination of all rADAMTS13-treated animals was statistically higher than that of buffer-treated controls (p≤0.0001). However, animals receiving prophylactic treatment 120h before administration of rVWF showed severe thrombocytopenia; clinically relevant protection was only seen with treatment intervals ≤72h. Therapeutic treatment with rADAMTS13 stabilized platelet count and prevented further thrombocytopenia. LDH, schistocytosis and organ damage confirmed the treatment interval-dependent improvement in platelet count.In summary, Baxter's rADAMTS13 was effective in an rVWF-induced animal model closely mimicking the situation in patients with hereditary TTP. Comparison with FFP, the current standard of treatment of TTP, showed Baxter's rADAMTS13 to have similar or slightly improved efficacy. Disclosures:Schiviz:Baxter Innovations GmbH: Employment. Resch:Baxter Innovations GmbH: Employment. Farnleitner:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.

Genetic Variability of the CLEC4M Endothelial Lectin Receptor Modulates Binding and Internalization of Von Willebrand Factor and Contributes to Variance in Plasma VWF Levels

Abstract 16Type 1 von Willebrand’s Disease (VWD) can result from decreased synthesis or accelerated clearance of von Willebrand Factor (VWF), resulting in partial quantitative deficiency. Approximately 35% of individuals with Type 1 VWD do not have a putative mutation in their VWF gene, suggesting that genes other than VWF may contribute to the pathophysiology of this disease. Recently, the CHARGE GWAS meta-analysis identified single nucleotide polymorphisms in the gene encoding the C-type lectin domain family 4 member M (CLEC4M) as being significantly associated with plasma VWF levels in normal individuals. CLEC4M is a pathogen recognition receptor with a polymorphic extracellular neck region consisting of a variable number of tandem repeats (VNTR) (3 – 9 repeats). We hypothesize that CLEC4M binds to and clears VWF from the circulation, and that different CLEC4M VNTR alleles may contribute to differences in plasma levels of VWF in normal subjects and patients with Type 1 VWD.Previously, genotyping of 555 subjects (196 cases with type 1 VWD, and 362 family members) for the CLEC4M VNTR number showed that the most frequently documented alleles were VNTR 5 (29%), 6 (15%), and 7 (53%). Family-based association analysis on kindreds with type I VWD has demonstrated a significant excess transmission of VNTR 6 to the type I VWD phenotype (p=0.005) and an association of this VNTR allele with VWF:RCo (p=0.037). In the present studies, we have complemented this genetic association data with experiments to directly evaluate the ability of CLEC4M to bind and internalize VWF. Further, we characterized the ability of different CLEC4M VNTR alleles to facilitate VWF clearance.Binding of VWF to CLEC4M was assessed with a modified ELISA using a recombinant CLEC4M-Fc chimera. CLEC4M-Fc bound to Humate P (plasma-derived VWF-FVIII) in a dose-dependent manner. CLEC4M-Fc also bound to recombinant human VWF, and factor VIII-free plasma-derived VWF. CLEC4M-Fc demonstrated a 70% increase in binding to de-O-glycosylated Humate P (p=0.041), and a 75% decrease in binding to de-N-glycosylated Humate P (p=0.046) relative to controls. Additionally, pre-incubation of CLEC4M-Fc with the polysaccharide mannan attenuated binding to all VWF preparations by approximately 50%.Binding and internalization of VWF by HEK 293 cells stably expressing CLEC4M (VNTR allele 7) was assessed with immunofluorescence and ELISA. Binding of VWF co-localized with CLEC4M expression on HEK 293 cells. CLEC4M and VWF co-localized with early endosomal antigen-1, suggesting that CLEC4M participates in receptor-mediated endocytosis of VWF. CLEC4M-expressing cells bound and internalized VWF in a dose- and time-dependent manner relative to controls. Preincubation of CLEC4M expressing cells with mannan inhibited VWF binding and internalization by 50% (p=0.0088).The contribution of CLEC4M genetic variability to VWF binding and internalization was measured using HEK 293 cells expressing CLEC4M with 4, 6, 7, and 9 tandem repeats. Decreased binding and internalization of VWF was observed with cells expressing CLEC4M 4 and 9 tandem repeat constructs as compared to CLEC4M with 7 tandem repeats (CLEC4M 4 – 60% reduction, p < 0.001; CLEC4M 9 – 45% reduction, p=0.006). Cells expressing the CLEC4M VNTR combination 4 and 9, had a 55% decrease in binding and internalization of VWF relative to cells expressing CLEC4M with 7 VNTRs (p < 0.001). These VNTR associated differences in VWF binding/internalization were not accounted for by variances in the CLEC4M expression levels in the transfected HEK 293 cells.These studies demonstrate that the C-type lectin CLEC4M binds to and internalizes VWF through an N-glycan-dependent mechanism. Additionally, it provides further evidence that polymorphisms in the CLEC4M gene contribute to quantitative VWF deficiency. Disclosures:Montgomery:Gen-Probe/GTI Diagnostics: Consultancy; CSL Behring: Consultancy; Octapharma: Consultancy. James:CSL-Behring, Baxter, Bayer: Honoraria, Research Funding.

Detection of Non Inhibitory Binding Antibodies to Von Willebrand Factor Affecting the Clearance of VWF:Ag in Von Willebrand Disease

Abstract▪2275▪This icon denotes a clinically relevant abstractVon Willebrand Disease (VWD) is an inherited rare bleeding disorder caused by a deficiency of von Willebrand factor (VWF). VWF, the largest multimeric plasma glycoprotein, facilitates platelet aggregation and stabilizes FVIII in the circulation. Inhibitory antibodies to VWF have been reported in 10% – 15% of type 3 VWD patients repetitively treated with plasma-derived VWF/FVIII concentrates. The clinical impact of non-inhibitory antibodies to VWF has not been previously reported. To investigate the immunogenicity of a novel recombinant human VWF (rhVWF), both total binding anti-VWF antibodies and inhibitory anti-VWF antibodies (VWF:Ristocetin Cofactor Activity [RCo], VWF:Collagen Binding Activity [CB], VWF:FVIII Binding [FVIIIB]) were assessed in a Phase 1 multicenter clinical study. Total binding anti-VWF antibodies were determined by an enzyme-linked immunosorbent assay (ELISA) employing polyclonal anti-human IgG antibodies. Plasma samples were analyzed in two steps employing a screening assay determining the titer of the binding antibodies and in a second step, confirming the specificity of the antibody in a competition assay. Samples were considered positive, when a sample was at least two titer steps lower than the antibody titer detected in the screening assay necessitating a screening titer of at least 1:80. Neutralizing antibodies to the key functional activities of VWF, such as VWF:RCo, VWF:CB and VWF:FVIIIB, were measured by assays based on the Bethesda assay established for quantitative analysis of FVIII inhibitors (Nijmegen modification of the Bethesda assay). To exclude false positive results, the detection limit for anti-VWF inhibitors was set to 1 BU/mL for all 3 assays. Three of 39 subjects screened had a pre-existing high titer non-neutralizing binding antibody (all 1/1280) to VWF. One of these 3 subjects was excluded from the study due to the presence of an inhibitory antibody to VWF:CB (1.3 BU/mL) at screening. Two of the 3 subjects were enrolled and treated with rVWF and/or pdVWF concentrate as part of the safety, tolerability and PK assessments. The high titer binding anti-VWF antibodies were associated with a significant decreased VWF:Ag activity post infusion of either pdVWF or rVWF and consequential decreased activity of VWF:RCo, VWF:CB and FVIII:C. For example, one of the subject had a reduced VWF:Ag incremental recovery of 1.1 IU/dL/(U VWF:RCo/kg) (mean of cohort 1.6 IU/dL/[U VWF:RCo/kg]) and a reduced VWF:Ag t1/2 2.4 hours (mean of cohort t1/2 25.3 hours) post infusion of rhVWF dosed at 50 IU VWF:RCo/kg. Relevant data of all PK assessments and antibody titers will be presented. None of the study subjects developed an inhibitory antibody to either VWF or FVIII and there was no impact on the subsequent treatment of bleeding episodes with commercial pdFVIII/VWF concentrates noted by the treating physician. The clinical relevance of non-neutralizing antibodies to VWF requires additional investigation. Disclosures:Suiter:Baxter Innovations GmbH: Employment. Horling:Baxter Innovations GmbH: Employment. Reipert:Baxter Innovations GmbH: Employment. Turecek:Baxter BioScience: Employment. Varadi:Baxter Innovations GmbH: Employment. Chapman:Baxter Innovations GmbH: Employment. Engl:Baxter Innovations GmbH: Employment. Wong:Baxter Healthcare Corp: Employment. Ewenstein:Baxter Healthcare Corp: Employment.

Recombinant Human Von Willebrand Factor (rhVWF): First-In-Human Study Evaluating Pharmacokinetics, Demonstrating Safety and Tolerability In Type 3 Von Willebrand Disease

AbstractAbstract 237Von Willebrand Disease (VWD) is an inherited rare bleeding disorder caused by a deficiency of von Willebrand factor (VWF). VWF is the largest soluble multimeric plasma glycoprotein, which facilitates platelet aggregation and stabilizes FVIII in the circulation. Patients with type 3 disease display severe hemorrhagic symptoms, mainly in mucosal tissues, muscle and joints. Replacement of VWF stabilizes endogenous FVIII to hemostatic levels within hours. Commercially available VWF/FVIII concentrates are plasma-derived (pd) and subject to limitations such as donor dependency, risk of blood-borne pathogen transmission, lack of high molecular weight VWF multimers, and variation in multimer composition. A novel recombinant human VWF (rhVWF) has been developed using a plasma-free method, which represents the largest protein ever produced using recombinant technology. Safety, tolerability and pharmacokinetics of the rhVWF combined at a fixed ratio with rFVIII were investigated in a Phase 1 multicenter, international clinical study in 31 patients with type 3 VWD and severe type 1 VWD. Four concentrations of rhVWF (2, 7.5, 20 and 50 IU VWF:RCo/kg) were administered in a dose-escalating manner in separate cohorts. rhVWF was well tolerated, and no thrombotic events, VWF inhibitors or other serious adverse reactions were observed. Pharmacokinetics of rhVWF/rhFVIII (50 IU VWF:RCo/kg and 38.5 IU FVIII/kg) compared with pdVWF/pdFVIII (50 IU VWF:RCo/kg and 21 IU/kg FVIII/kg) were evaluated in a sub-group of 8/31 patients using a randomized, crossover design (8-day minimum washout period). Interim data in 8 subjects show a higher degree of secondary FVIII activity with rhVWF/rhFVIII compared to pdVWF/pdFVIII (see Figure 1) that is not solely due the difference in the rhVVF:FVIII infusion ratio (1.3:1 rhVWF/rhFVIII vs. approximately 2:1 pdVWF/pdFVIII). The pharmacokinetics of the rhVWF:RCo and pdVWF:RCo were comparable and were also reflected in the VWF:Ag and collagen binding activity. Evidence is also provided for the in vivo cleavage of the ultra-high molecular weight multimers of rhVWF by endogenous ADAMTS13. In summary, interim data from the ongoing Phase 1 study, demonstrate that rhVWF is safe and well tolerated, has VWF:RCo pharmacokinetics that are comparable to pdVWF and enhances stabilization of endogenous FVIII. Multiple doses of rhVWF/rhFVIII would be expected to have beneficial effects in major surgery and severe mucosal bleeding events. These data would also support the treatment concept to administer rhVWF alone once a therapeutic baseline level of endogenous FVIII is achieved (after 1–2 doses). [Display omitted] Disclosures:Suiter:Baxter BioScience: Employment. Laffan:Baxter BioScience: Consultancy. Mannucci:Baxter BioScience: Consultancy. Kempton:Baxter BioScience: Consultancy. Romond:Baxter BioScience: Consultancy. Shapiro: Baxter BioSci- ence: Consultancy. Birschmann:Baxter BioScience: Consultancy. Gill:Baxter BioScience: Consultancy. Ragni:Baxter BioScience: Consultancy. Turecek:Baxter BioScience: Employment. Ewenstein:Baxter Bioscience: Employment. Baxter BioScience:Baxter BioScience: Employment.

Factor VIII Inhibitors: Von Willebrand Factor Makes A Difference In Vitroand In Vivo

AbstractAbstract 709The important association between von Willebrand factor (VWF) and factor VIII (FVIII) has been investigated for decades, but the effect of VWF on FVIII inhibitors is still controversial. Studies have demonstrated that some anti-FVIII inhibitory antibodies inhibit VWF-FVIII interaction, while others rely on the presence of VWF to inhibit FVIII activities. The influence of VWF on the Bethesda assay, which is routinely used in the clinic to determine the titer of FVIII-neutralizing inhibitors, is still uncertain because the plasma from hemophilia A patients with inhibitors contains normal levels of VWF. To explore the effect of VWF on the reactivity of FVIII inhibitors, we immunized VWF and FVIII double knockout (VWFnullFVIIInull) mice with recombinant human B-domain deleted FVIII (rhFVIII) to induce anti-FVIII inhibitory antibody development. Inhibitory plasma was collected and the titer of inhibitors was determined by Bethesda assay. Murine plasma-derived VWF (from FVIIInull mice) or recombinant human VWF (rhVWF) was used to study the influence of VWF on inhibitor inactivation of FVIII activity (FVIII:C). The remaining FVIII:C after inactivation was determined by chromogenic assay. When inhibitory plasma was incubated with rhFVIII in the presence of 1 U/ml VWF, the residual FVIII activity recovered was higher than in the absence of VWF, resulting in 6.82 ± 1.12 (n = 27) fold lower apparent inhibitor titers. This protective effect is VWF dose dependent. The source of VWF (plasma-derived murine VWF vs. rhVWF) did not affect its protection of FVIII from inhibitor inactivation and VWF does not affect FVIII:C measured in the chromogenic assay in the absence of inhibitors. Interestingly, we found that inhibitor inactivation of FVIII:C in the absence of VWF occurred much faster than in its presence. When the usual 2 hr. incubation at 37°C was omitted from the Bethesda assay, adding rhVWF to rFVIII before mixing with inhibitory plasma resulted in 67.29 ± 20.18 (n = 5) fold lower apparent inhibitor titers than without added VWF. In contrast, if VWF was added to inhibitory plasma first and then mixed with rhFVIII, the inhibitor titers were only 11.04 ± 3.56 (n = 5) fold lower than without added VWF. These results indicate that rhFVIII present in a preformed VWF-FVIII complex is protected from inhibitory antibody inactivation. Conversely, when VWF and inhibitory plasma are added to rhFVIII at the same time, the VWF and inhibitors appear to compete to bind to rhFVIII. Inhibitor titers were lower than in the absence of VWF, but the protective effect is not as efficient as when VWF and rhFVIII were already associated with one another before encountering inhibitors. To confirm the protective effect of VWF on FVIII from inhibitor inactivation, we infused FVIIInull or VWFnullFVIIInull mice with inhibitory plasma and rhFVIII followed by a tail clip survival test. When rhFVIII was infused into FVIIInull mice to 2% followed by inhibitory plasma infusion, all mice with inhibitor titer of 2.5 BU/ml (n = 4) survived tail clipping, and 2 of 4 survived with either 25 BU/ml or 250 BU/ml. If inhibitory plasma was infused first followed by rhFVIII infusion, then only 2 of 6 mice with inhibitor titers of 2.5 BU/ml survived tail clip challenge and none survived with 25 BU/ml and 250 BU/ml. In the first set of mice the infused FVIII was able to form a protective complex with endogenous VWF before encountering inhibitors, while in the second set FVIII is exposed to VWF and pre-infused inhibitory antibodies at the same time, a competitive binding that appears to reduce VWF's protective effect. In contrast, when rhFVIII was infused into VWFnullFVIIInull mice followed by inhibitory plasma infusion, no animals (n = 4 for each group) survived tail clipping with inhibitor titers of 2.5 BU/ml or higher. In summary, our studies demonstrate that VWF exerts a protective effect, reducing inhibitor inactivation of FVIII, both in vitro and in vivo. While the role of VWF in stabilizing plasma FVIII in a milieu rich in proteases has been appreciated for decades, our results indicate that treatment utilizing products containing a complex of FVIII with VWF may be especially beneficial in hemophilia A patients with inhibitors. Disclosures:No relevant conflicts of interest to declare.

Preclinical Testing of Human Recombinant von Willebrand Factor: ADAMTS13 Cleavage Capacity in Animals as Criterion for Species Suitability

Von Willebrand factor (VWF) is cleaved by the plasma metalloprotease ADAMTS13 ( A Disintegrin and Metalloproteinase with Thrombo Spondin repeats, number 13) that regulates the hemostatic activity of VWF by limiting its multimeric size in the human system. In vitro and ex vivo studies have shown that human recombinant VWF (rVWF) is virtually resistant to the proteolytic activity of murine ADAMTS13. In contrast, rabbit and cynomolgus ADAMTS13 is able to cleave human rVWF. These findings were consistent with in vivo results showing distinct pharmacological behavior of human rVWF depending on the cleaving capacity of ADAMTS13 present in the species tested. Studies were performed using three mouse strains (ADAMTS13 deficient, C57BL/6J [wild type], VWF deficient), rats, rabbits, and cynomolgus monkeys. All animals were infused once with different doses of human rVWF and, in addition, 14 daily doses were given to rats and cynomolgus monkeys. Exaggerated pharmacological effects were observed in mice, with the ADAMTS13 knockout mouse being the most sensitive strain. Similar findings with decreased incidence and severity were seen in normal C57BL/6J mice and also in VWF-deficient mice, where they were least pronounced. In rats, exaggerated pharmacological effects were observed only after 14 doses. Rabbits and cynomolgus monkeys showed no exaggerated pharmacological effects. These differences between species and between mouse strains suggest that the efficiency of ADAMTS13 to cleave rVWF determines the severity of clinical, laboratory and pathohistological findings. These observations highlight the importance of evaluating species' suitability for the generation of meaningful preclinical data for determining the therapeutic safety margins for human patients. Only animals with a sufficient rVWF cleavage capacity by endogenous ADAMTS13 (rabbits and cynomolgus monkeys) are considered appropriate animal models for preclinical evaluation of the rVWF product.

Species-dependent variability of ADAMTS13-mediated proteolysis of human recombinant von Willebrand factor.

von Willebrand factor (VWF) is composed of a series of multimers, the sizes of which are regulated by the plasma metalloprotease ADAMTS13. Proteolysis of human recombinant VWF (rVWF) by ADAMTS13 present in the plasma of different species typically used as preclinical animal models was investigated to evaluate the efficacy and safety of rVWF. Degradation of rVWF was studied in vitro under moderate denaturing conditions and was monitored by multimer analysis, residual collagen binding, and immunoblot analysis. In vivo cleavage was determined by administration of rVWF to cynomolgus monkeys, rabbits and VWF-deficient mice and subsequent analysis of plasma samples by immunoblot. Plasma ADAMTS13 levels were determined with a synthetic human VWF peptide (FRETS-VWF73). From the animals tested, only rabbit plasma was as efficient as human plasma in proteolysing rVWF in vitro. Mouse plasma virtually failed to cleave rVWF. Administration of human rVWF resulted in ADAMTS13-specific cleavage products in rabbits and, to a lesser extent, in cynomolgus monkeys at various doses of rVWF. Virtually no cleavage occurred in mice. ADAMTS13 activity levels in rabbit and monkey plasma were similar to those in human plasma and were not significantly altered upon infusion of rVWF up to very high doses, indicating that rVWF did not lead to an exhaustion of endogenous ADAMTS13 in both species. The differences in susceptibility to cleavage of rVWF by different species need to be considered when interpreting the physiology of human rVWF from results of tests in animal models.

Production of Recombinant Human Von Willebrand Factor in the Milk of Transgenic Pigs

Von Willebrand factor (vWF), a large multimeric glycoprotein present in blood plasma, is a blood protein of the coagulation system. It is defective in von Willebrand disease and is involved in a large number of other diseases, including thrombotic thrombocytopenic purpura-hemolytic uremic syndrome and heyde's syndrome. We have developed a line of transgenic swine harboring recombinant human von Willebrand factor (rhvWF) cDNA through microinjection of fertilized one-cell pig zygotes. Expression of rhvWF in the mammary gland and secretion of rhvWF into the milk of the transgenic swine were confirmed by immunohistochemical and western blot analyses, respectively, and rhvWF proteins were detected in milk from all lactating founder females at concentrations that were 28- to 56-folds greater than that in circulating human plasma. The amino acid sequence of rhvWF protein in the transgenic pig milk matched that of vWF produced from human blood plasma. This study provides evidence that production of rhvWF from transgenic pig milk is a potentially valuable technology and can be used as a cost-effective alternative in clinical applications.