Von Willebrand Factor Inhibitor Research Articles

102 BB-031 Recanalizes MCAO and Reduces Inflammation in Canine Model of LVO Stroke

INTRODUCTION: Acute ischemic stroke (AIS) is the leading cause of long term disability. rtPA treatment of AIS results in intracranial hemorrhage (ICH) in 7% of patients with 40% mortality. Increased matrix metalloprotease-9 (MMP-9) correlates with ICH, while reduced levels maintain integrity of the blood brain barrier (BBB), preventing ICH. Von Willebrand Factor (VWF) induces both thrombosis and inflammation in large vessel occlusion (LVO) stroke. BB-031 targets VWF, and lyses thrombotic occlusion. BB-025 reverses BB-031. METHODS: After 6 hours of autologous canine MCAO, animals were treated with vehicle, BB-031, and BB-031+BB-025. VWF inhibition, TICI scores, and plasma inflammatory markers [VWF, interleukin-1 (IL-1b), interleukin-6 (IL-6), tumor necrosis factor-a (TNF- a), and matrix metalloproteinase-9 (MMP-9)] were analyzed over the course of the study. RESULTS: Within 15 minutes of vehicle treatment, VWF increased by 3.102 ± .382 ng/ml (n = 6) whereas BB-031 reduced VWF by 0.223 ± .154 ng/ml (p < 0.05) (n = 8). BB-031-treated animals had TICI > 2B in 62.5% of the animals. Although IL-1b and IL-6 were unchanged, and TNF-a was not detectable, MMP-9 levels were lower in BB-031-treated animals at sacrifice compared to both vehicle and BB-031+BB-025 (p < 0.0001). CONCLUSIONS: BB-031 treatment inhibited VWF, increased reperfusion, and reduced MMP-9 after canine MCAO, suggesting that in addition to recanalization, inhibiting VWF mitigates inflammation and iatrogenic ICH.

Conformational activation and inhibition of von Willebrand factor by targeting its autoinhibitory module

AbstractActivation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify 2 groups of nanobodies. One group, represented by clone 6D12, is conformation insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1; 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.

Immunogenicity of a Plasma-Derived Von Willebrand Factor/Factor VIII Concentrate during the Wil-31 Prophylaxis Study in Patients with Von Willebrand Disease

Background: Long-term prophylaxis with a von Willebrand factor (VWF) concentrate is recommended in people with von Willebrand disease (VWD) with a history of frequent and severe bleeds. The WIL-31 study demonstrated the efficacy of prophylaxis with a plasma-derived VWF/factor VIII (pdVWF/FVIII) concentrate containing VWF and FVIII in a 1:1 activity ratio (wilate ®) in adults and children with VWD of all types. The primary endpoint was met, showing a decrease in mean total annualized bleeding rate (ABR) of 84% compared with prior on-demand treatment. Aim: To assess the immunogenicity of pdVWF/FVIII when used during the WIL-31 study in children and adults with VWD of all types. Methods: WIL-31 (NCT04052698) was a prospective, non-controlled, international, multicenter phase 3 trial that enrolled male/female patients aged ≥6 years with VWD type 1 (VWF: RCo <30 IU/dL), type 2 (except 2N) or type 3. Prior to entering the WIL-31 study, all patients had received on-demand treatment with a VWF-containing product during a 6-month, prospective, observational, run-in study (WIL-29); patients who experienced at least 6 bleeding episodes (BEs), excluding menstrual bleeds, of which ≥2 were treated with a VWF-containing product, were eligible to enter WIL-31. During WIL-31, patients received regular pdVWF/FVIII (containing VWF and FVIII in a 1:1 activity ratio) prophylaxis 2-3 times per week at a dose of 20-40 IU/kg for 12 months. FVIII (chromogenic and one-stage assays) and VWF (VWF:RCo assay) activities were assessed at baseline and throughout the study. Retention samples were collected at screening and baseline visits for possible later inhibitor testing (to be performed at a central laboratory using the Nijmegen-Bethesda assay for FVIII inhibitors and the mixing study based on VWF:RCo for VWF inhibitors). Inhibitor testing was also performed if inhibitor development was suspected. Results: Inhibitor testing was performed in 2 of 33 patients because of unusual pharmacokinetic results at the 6-month visits, even though neither patient had any clinical signs of an inhibitor. The VWF inhibitors were detected in samples from two brothers taken at their 6-month visits. However, when retained blood samples from the screening visits of both boys were tested, both samples tested inhibitor-positive, demonstrating that both boys had developed the inhibitors prior to starting prophylaxis during WIL-31. During and before the WIL-29 run-in study, both boys had been treated on demand with another pdVWF/FVIII concentrate. The boys had both been diagnosed with VWD shortly after birth, and it was suspected that they had type 2A based on family history and were 15 (patient A) and 11 years old (patient B) at the start of WIL-31. They had a family history of VWD (both parents were diagnosed with VWD), but no family history of inhibitors. After the completion of WIL-31, both patients were reclassified to type 3 VWD following further investigation including next-generation sequencing. Baseline VWF activity indicated low VWF:RCo levels (Patient A: 0.092 IU/mL and Patient B: 0.65 IU/mL; Figure 1). The boys received 38 (patient A) and 34 IU/kg (patient B) pdVWF/FVIII respectively, twice weekly during WIL-31, and their mean total ABRs decreased by 85% and 91%, respectively, during WIL-31 compared with WIL-29. All bleeding episodes during WIL-31 were spontaneous nose bleeds. Both boys continued the same prophylactic regimen until the end of WIL-31. In one patient the inhibitor titer was negative by the end of the study. Conclusion: VWF inhibitors were detected in 2 of the 33 patients on prophylaxis with pdVWF/FVIII in the WIL-31 study and subsequent investigation revealed that both patients were already positive for VWF inhibitors prior to the study start. In one of the patients, the inhibitor resolved during continued prophylaxis. Despite the inhibitors, prophylaxis was effective in both patients with an ≥85% reduction in ABR compared to on demand treatment.

Phase II Study of Danazol with Plasma Exchange and Corticosteroids for the Treatment of Thrombotic Thrombocytopenic Purpura

Background: Ahn and colleagues reported a decrease in plasma exchange (PE)/plasma infusion requirement with danazol in seven cases of thrombotic thrombocytopenic purpura (TTP) (Ahn Blood 2006;108(11):1057a). We reported an increase in platelet count (PLT) within 24 hours with danazol in two of three cases of human immunodeficiency virus-associated TTP resistant to PE (Torri Transfusion 2010;50:86a). In 2009, we initiated a phase II study of danazol with PE and corticosteroids (CS) for the treatment of TTP. This trial was approved by the institutional review board and was registered at ClinicalTrials.gov (ID# NCT00953771). We report the final results. Aims: To determine if danazol reduces number of PE's, time to remission (TR), and length of stay (LOS) when administered with standard therapy for the initial treatment of TTP. Methods: Experimental Group (EG): Patients with TTP (PLT < 100,000/µL and microangiopathy) receive daily PE, prednisone 1 mg/kg, and danazol 600 mg. Following remission (PLT > 150,000/µL for three consecutive days), PE is tapered to every other day for one week then stopped. Then prednisone is tapered weekly over four weeks then stopped. After one week danazol is tapered by 200 mg weekly then stopped. Historical controls (HC): Patients treated for TTP at St. Luke's-Roosevelt Hospital Center with PE with or without CS from 2000 to 2007 (n=20). Relapse definition: PLT < 100,000/µL with microangiopathy > 30 days after remission. Total number of PE's, TR, LOS, and continuous response rates are compared between the 2 groups. Accrual target: 16 patients to detect a 40% decrease in the number of PE's with 80% power and a level of significance of 0.05.The study was terminated in 2018 due to low accrual. Results: Nine patients were enrolled. Mean number of PE's (days) was 14.8 (EG) and 16.1 (HC), mean TR (days) was 11.4 (EG) and 11 (HC), and mean LOS (days) was 27.3 (EG) and 24.9 (HC), p > 0.05. Four patients in HC had a total of 6 relapses at our institution at a median of 28.25 months, range 2.75 to 97 months after the initial event. One patient in EG relapsed 43 months after study enrollment. One patient in EG died from an unrelated cause. None of the additional seven patients in EG have relapsed during a median follow-up of 49 months, range 12 to 125 months. One episode of pulmonary edema and one episode of hypotension occurred in EG, for which treatment was not discontinued. Discussion: Our study shows that danazol does not reduce PE requirement, TR, or LOS during treatment of TTP, but may prevent relapse. This pattern of response is similar to that which has been observed with rituximab. In a phase 2 study of rituximab in conjunction with PE and CS for the treatment of TTP (Scully Blood 2011), in which results were compared with historical controls who did not receive rituximab, median number of PE's (days) and median LOS (days) were 16.5 and 16.5 in the rituximab group versus 18.0 and 20 in the historical control group, p=ns. Ten percent of patients relapsed at a median of 27 months in the rituximab group whereas 57% of patients relapsed at a median of 18 months in the historical control group. Of interest, the cost of rituximab 375 mg/m 2 weekly for 4 weeks as given would have been approximately $ 25,366.50. The cost of danazol in our protocol would have been approximately $ 2196.00. Caplacizumab is a humanized monoclonal antibody fragment that inhibits von Willebrand factor binding to platelet glycoprotein lb-IX-V. In a phase 3 study (Scully N Engl J Med 2019), caplacizumab in conjunction with PE and CS for the treatment of TTP led to reduced PE's, time to normalization of platelet counts, and LOS, but increased relapses in comparison with placebo. In 2020, the International Society on Thrombosis and Haemostasis strongly recommended adding CS to PE for the treatment of TTP and conditionally recommended adding rituximab and caplacizumab to PE and CS (Zheng J Thromb Haemost 2020). Conclusion: Danazol in conjunction with PE and CS may be a low-cost option to prevent relapses of TTP.

PB1218 Aptamer Inhibitor of Von Willebrand Factor Induces Thrombolysis and Improves Vascular Patency Upon Immediate and Delayed Treatment in a Microfluidic Model of Stroke

PB1218 Aptamer Inhibitor of Von Willebrand Factor Induces Thrombolysis and Improves Vascular Patency Upon Immediate and Delayed Treatment in a Microfluidic Model of Stroke

Abstract WP210: Aptamer Inhibitor Of Von Willebrand Factor Induces Thrombolysis And Improves Vascular Patency Upon Immediate And Delayed Treatment Of Stroke In An In Vitro Microfluidic Model

Von Willebrand factor (VWF) is critical in arterial thrombosis. Previous studies have shown that aptamer BB-031, a VWF inhibitor, induces thrombolysis in vivo in ischemic stroke models, making it a promising stroke therapeutic. We hypothesized that BB-031 will induce thrombolysis in an ex vivo ischemic stroke model and that advanced imaging techniques will provide mechanistic information. A microfluidic model was utilized to simulate ischemic stroke and drug delivery without disturbing the occluding thrombus. Heparinized whole blood was obtained from healthy volunteers (N=10). A gravity-based constant pressure microfluidic system mimicking arterial perfusion induced occlusive thrombosis at a collagen-coated region of stenosis. After resting for 0, 3, or 6 hours to allow retraction in situ thrombi were reperfused with blood containing 1692nM (~1mg/kg) BB-031, 0.7nM alteplase (ALT), 0.7nM tenecteplase (TEN), or vehicle 500um upstream of the thrombus using a separate inlet. Platelets were labelled prior to thrombus formation and reperfusion using fluorescent anti-CD41. Thrombolysis and patency were then monitored for 2hrs via outlet mass recording and z-stacked immunofluorescent imaging. Upon immediate reperfusion, 1692nM (~1mg/kg) BB-031 increased mean mass output by 0.250±0.629g compared to 0.031±0.065g with vehicle, 0.074±0.061g with ALT, and 0.101±0.150g with 0.7nM TEN. After a 3-hour retraction period, BB-031 reperfusion mean mass output was 0.951±1.279g compared to 0.301±0.380g with vehicle, 0.412±0.681g with ALT, and 0.569±0.714g with TEN. After 6-hours resting, reperfusion mass output increased by 1.354±1.433g with BB-031, 0.368±0.538g with vehicle, 0.158±0.195g with ALT, and 0.520±0.539g with TEN. Representative z-stacked renderings of the thrombus pre and post reperfusion from a 0-hour resting period show a volumetric decrease in thrombus size of 39% with BB-031 compared to 25% and 18% reduction with vehicle and ALT respectively in a donor-matched dataset. BB-031 improves patency in a microfluidic model of biofidelic thrombolysis compared to current standards of care, which is enhanced at 3 and 6 hours post-occlusion. This may be due to VWF-dependent lysis resulting in reduced thrombus volume.

Abstract TMP4: BB-031 Reduces Platelet Reactivity And Attenuates Von Willebrand Factor Levels In A Large Vessel Occlusion Stroke Model

Introduction: Acute ischemic strokes (AIS) are a leading cause of death and long-term disability worldwide. Von Willebrand Factor (VWF) is an attractive target for anti-thrombotic therapeutics as it plays a pivotal role in platelet adhesion, activation, and aggregation with large vessel occlusion (LVO) stroke. VWF is stored in an ultra large form in platelet α-granules and Weibel-Palade bodies of endothelial cells from where it is released during injury or inflammation. BB-031 is an RNA aptamer that binds and inhibits VWF, and both prevents and lyses an occlusive thrombus. Hypothesis: We hypothesize BB-031 treatment will inhibit VWF thereby reducing platelet reactivity and attenuating the increase in VWF levels in a canine model of LVO stroke. Methods: A canine embolic MCAO model was used to assess BB-031 efficacy of VWF inhibition and platelet activity compared to placebo. After 6 hours of LVO with an autologous clot, intravenous treatment was initiated with placebo (n=7), or 0.5 (n=8), 1.0 (n=4), and 5.0 (n=4) mg/kg BB-031. Whole blood was collected at baseline, 6 hours after occlusion then at 10, 15, 20, 25, 30, 60 min after treatment and lastly, at time of sacrifice 9 hours after MCAO induction. Enzyme-linked immunosorbent assay (ELISA) was used to measure circulating VWF and platelet function analysis (PFA-100 Siemens) was utilized to determine platelet reactivity. Results: VWF levels immediately after LVO in vehicle control were significantly attenuated within 10 minutes of administration with all BB-031 concentrations (p<0.05). Within 15 minutes after vehicle treatment, VWF levels increased by 3.102 ± .382 ng/ml whereas BB-031 administration of 0.5, 1.0, and 5.0 mg/kg reduced VWF levels by 0.223 ± .154 ng/ml, 0.269 ± .304 ng/ml and 1.053 ± .267 ng/ml respectively. Platelet reactivity to ADP/Collagen (PFA-100) revealed significantly increased platelet closing time in all BB-031 treated canines from immediately after administration through 60 minutes. (p<0.05). Conclusion: BB-031 treatment demonstrated significant VWF inhibition and reduced platelet reactivity in a canine AIS LVO model compared to placebo. These findings indicate that BB-031 may offer a superior therapeutic treatment paradigm for ischemic stroke.

Abstract 94: Randomized, Double-blind, Placebo-controlled Phase 1 Study Of Vwf-binding Aptamer, Bb-031: Safety, Tolerability, Pharmacokinetic And Pharmacodynamic Activity In Healthy Volunteers

Introduction: Von Willebrand Factor (VWF) is a ubiquitous component of thrombi extracted by endovascular thrombectomy from patients with large vessel occlusion (LVO) stroke. BB-031 is a modified RNA aptamer that targets von Willebrand Factor (VWF) that lyses occlusive thrombi and improves outcomes in pre-clinical models of acute ischemic stroke (AIS). Hypothesis: BB-031 is safe, tolerable, and inhibits both VWF binding and activity in a dose-dependent manner. Methods: Healthy participants were randomized to receive BB-031 or placebo (6:2) by intravenous bolus injection at single ascending doses (SAD) of 0.1, 0.3, 1.0, 2.0 and 4.0 mg/kg (total n=40). Serial Clinical assessment, and blood sample collection for pharmacokinetic (PK) and pharmacodynamic (PD) analysis were performed. Non-compartmental PK analysis was conducted using Phoenix WinNonlin. PD evaluation included measurement of VWF inhibition and whole blood thrombosis (Platelet Function Analyzer [PFA-200]). Results: BB-031 was safe and well-tolerated for 28 days following single IV doses up to 4.0 mg/kg. There were no significant adverse events (SAEs), or treatment-emergent adverse events (TEAEs) leading to dose discontinuation. Minor events included bleeding at an IV site in 1 participant, and minor bleeding of an ulcer on the tongue in 1 participant. None of the minor TEAEs were dose-dependent. BB-031 demonstrated nonlinear, dose-dependent plasma PK across the dose range tested, with an apparent mean terminal half-life (t 1/2 ) of 18 min at 0.1 mg/kg to 67 min at 4.0 mg/kg. Dose-dependent changes in VWF binding were observed; >95% maximal mean change from baseline was reached following a single IV dose of 4.0 mg/kg. Finally, PFA-200 results showed complete inhibition of clot formation (closing time ≥300 seconds) at all doses tested with a dose-dependent duration of inhibition and return to normal range. Conclusion: This first-in-human SAD study of BB-031 demonstrated safety following a single IV dose of 0.1 to 4 mg/kg, and dose-dependent patterns of VWF binding and platelet function changes. These results lay the foundation for future studies in patients suffering from thrombotic conditions including AIS.

Von Willebrand factor neutralizing and non-neutralizing alloantibodies in 213 subjects with type 3 von Willebrand disease enrolled in 3WINTERS-IPS

BackgroundType 3 von Willebrand disease (VWD) is the most severe form of this disease owing to the almost complete deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived products containing VWF or recombinant VWF rarely cause the development of alloantibodies against VWF that may be accompanied by anaphylactic reactions. ObjectiveThe objective of this study was to assess the prevalence of anti-VWF alloantibodies in subjects with type 3 VWD enrolled in the 3WINTERS-IPS. MethodsAn indirect in-house enzyme-linked immunosorbent assay has been used to test all the alloantibodies against VWF. Neutralizing antibodies (inhibitors) have been tested with a Bethesda-based method by using a VWF collagen binding (VWF:CB) assay. Samples positive for anti-VWF antibodies were further tested with Bethesda-based methods by using the semiautomated gain-of-function glycoprotein-Ib binding (VWF:GPIbM) and a VWF antigen (VWF:Ag) enzyme-linked immunosorbent assay. ResultsIn total, 18 of the 213 (8.4%) subjects tested positive for anti-VWF antibodies and 13 of 213 (6%) had VWF:CB inhibitors. These 13 were among the 18 with anti-VWF antibodies. Of the 5 without VWF:CB inhibitors, 3 had non-neutralizing antibodies, 1 only inhibitor against VWF:GPIbM, and one could not be tested further. Ten of the 13 subjects with VWF:CB inhibitors also had VWF:GPIbM inhibitors, 6 of whom also had VWF:Ag inhibitors. Subjects with inhibitors were homozygous for VWF null alleles (11/14), homozygous for a missense variant (1/14), or partially characterized (2/14). ConclusionsAnti-VWF antibodies were found in 8.4% of subjects with type 3 VWD, whereas neutralizing VWF inhibitors were found in 6%, mainly in subjects homozygous for VWF null alleles. Because inhibitors may be directed toward different VWF epitopes, their detection is dependent on the assay used.

Dose-Dependent Von Willebrand Factor Inhibition by Aptamer BB-031 Correlates with Thrombolysis in a Microfluidic Model of Arterial Occlusion.

Von Willebrand Factor (VWF) plays a critical role in thrombus formation, stabilization, and propagation. Previous studies have demonstrated that targeted inhibition of VWF induces thrombolysis when administered in vivo in animal models of ischemic stroke. The study objective was to quantify dose-dependent inhibition of VWF-platelet function and its relationship with thrombolysis using BB-031, an aptamer that binds VWF and inhibits its function. VWF:Ac, VWF:RCo, T-TAS, and ristocetin-induced impedance aggregometry were used to assess BB-031-mediated inhibition of VWF. Reductions in original thrombus surface area and new deposition during administration of treatment were measured in a microfluidic model of arterial thrombolysis. Rotational thromboelastometry was used to assess changes in hemostasis. BB-031 induced maximal inhibition at the highest dose (3384 nM) in VWF:Ac, and demonstrated dose-dependent responses in all other assays. BB-031, but not vehicle, induced recanalization in the microfluidic model. Maximal lytic efficacy in the microfluidic model was seen at 1692 nM and not 3384 nM BB-031 when assessed by surface area. Minor changes in ROTEM parameters were seen at 3384 nM BB-031. Targeted VWF inhibition by BB-031 results in clinically measurable impairment of VWF function, and specifically VWF-GPIb function as measured by VWF:Ac. BB-031 also induced thrombolysis as measured in a microfluidic model of occlusion and reperfusion. Moderate correlation between inhibition and lysis was observed. Additional studies are required to further examine off-target effects of BB-031 at high doses, however, these are expected to be above the range of clinical targeted dosing.

The Empower Study - Design of a Randomized Clinical Trial to Assess the Efficacy and Safety of a Plasma-Derived Von Willebrand Factor / Factor VIII Concentrate for Heavy Menstrual Bleeding in Women with Von Willebrand Disease

Background: Although von Willebrand disease (VWD) is inherited by men and women equally, women are more likely to experience bleeding symptoms due to the challenges of menstruation and childbirth. Heavy menstrual bleeding (HMB) is the most common symptom observed in women and girls with VWD, and increases risk of iron deficiency anemia, blood transfusion, hospitalization and decreased health-related quality of life. While replacement therapy with von Willebrand factor (VWF) is recommended for prophylaxis in patients with VWD with severe and frequent bleeding, recommendations on its use for prophylaxis of HMB are limited due the lack of clinical trial data. Aims: The EMPOWER pilot trial will determine the feasibility of the trial design, as well as explore assay sensitivity to inform the determination of the primary outcome for the definitive randomized trial which will evaluate the effect of prophylaxis with plasma-derived VWF/FVIII (1:1; pdVWF:FVIII) concentrate compared with placebo on HMB in women with VWD. Methods: EMPOWER is a pilot, randomized, placebo-controlled, crossover, double-blind (patient and outcome assessor blinded), 2-year trial in female outpatients with VWD and HMB. We will evaluate the effect of 2-3 doses of prophylaxis with 40-60 IU VWF:RCo/kg pdVWF/FVIII concentrate when provided on the 2 heaviest days within the first 4 days of menstruation compared with placebo (normal saline). For the first treatment period, participants will be randomized to receive either pdVWF/FVIII concentrate or placebo for 4 cycles, crossing over to the comparator treatment during the second treatment period (see Figure 1). Non-pregnant female patients, ≥18 years of age with a diagnosis of inherited VWD (any type) with HMB defined as a modified pictorial blood assessment chart (mPBAC) score >100, on stable treatment for HMB and iron-deficiency anemia will be eligible. Patients with diagnosis of another bleeding disorder or the presence of >2 risk factors for venous thromboembolism will be excluded. The pilot trial viability outcomes include blinding index scores and participant drop-out rates. Feasibility outcomes include data completeness and rate of enrolment. The proposed primary clinical efficacy outcome is the mPBAC score and the key secondary efficacy outcome is the need for rescue therapy (i.e. >2 days of oral tranexamic acid use, additional treatment with pdVWF:FVIII concentrate, additional hormonal therapy for HMB, urgent/emergent gynecological surgery for HMB, treatment with intravenous iron, red blood cell transfusion, or hospital admission for HMB). Other secondary outcomes include major bleeding, clinically relevant non-major bleeding, red blood cell transfusion, fatigue and quality of life scores. Given the uncertainty in this area of research and the relative comparable clinical importance of proposed clinical efficacy outcomes, we will evaluate assay sensitivity to determine the outcome that has the greatest chance of detecting a difference between treatment periods. Assay sensitivity is defined as the ability of a scale to distinguish an effective from a less effective intervention or placebo. Assay sensitivity along with the proportion of missing data per clinical efficacy outcome and sample size considerations will influence the choice of primary outcome for the definitive trial. Safety will be evaluated by monitoring for hypersensitivity reactions, thromboembolic events and VWF inhibitors. The pilot trial will recruit 20 participants from Hemophilia Treatment Centres in Canada over 2 years. Results: The pilot trial is aiming to launch by the fall 2022. If the pilot trial is deemed feasible and viable, we will subsequently conduct a definitive international randomized controlled trial. Conclusion: Data from the EMPOWER trial will fill an important knowledge gap to facilitate evidence-based management of HMB in women with VWD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Ancient Herbal Formula Mahuang Lianqiao Chixiaodou Decoction Protects Acute and Acute-on-Chronic Liver Failure via Inhibiting von Willebrand Factor Signaling.

Acute liver failure (ALF) and acute-on-chronic liver failure (ACLF) are characterized by systemic inflammation and high mortality, but there is no effective clinical treatment. As a classic traditional Chinese medicine (TCM) formula, MaHuang-LianQiao-ChiXiaoDou decoction (MHLQD) has been used clinically for centuries to treat liver diseases. The LPS/D-GalN-induced ALF mice model and the CCl4+LPS/D-GalN-induced ACLF mice model were used to observe the therapeutic effects of MHLQD on mice mortality, hepatocytes death, liver injury, and immune responses. MHLQD treatment significantly improved mice mortality. Liver injury and systemic and hepatic immune responses were also ameliorated after MHLQD treatment. Mechanistically, proteomic changes in MHLQD-treated liver tissues were analyzed and the result showed that the thrombogenic von Willebrand factor (VWF) was significantly inhibited in MHLQD-treated ALF and ACLF models. Histological staining and western blotting confirmed that VWF/RAP1B/ITGB3 signaling was suppressed in MHLQD-treated ALF and ACLF models. Furthermore, mice treated with the VWF inhibitor ADAMTS13 showed a reduced therapeutic effect from MHLQD treatment. Our study indicated that MHLQD is an effective herbal formula for the treatment of ALF and ACLF, which might be attributed to the protection of hepatocytes from death via VWF/RAP1B/ITGB3 signaling.

Von Willebrand factor and cancer: Another piece of the puzzle

Von Willebrand factor and cancer: Another piece of the puzzle

Efficacy and safety of von Willebrand factor concentrate almost devoid of factor VIII (Wilfactin®) in paediatric patients under 6 years of age with severe von Willebrand disease.

Plasma-derived von Willebrand factor (VWF) (Wilfactin®, LFB, France) was developed for prophylaxis and treatment of haemorrhages in both adults and adolescents with von Willebrand disease (VWD). Replacement therapy in paediatric patients is a key element of the clinical trial programme. Patients aged <6 years with severe VWD were enrolled in a multinational, open-label study to evaluate the in vivo recovery for Wilfactin®, and its efficacy in preventing and treating bleeding episodes and during surgery. Overall haemostatic efficacy based on a 4-point scale was assessed by investigators. The treatment period ≥18 months investigated the long-term safety. Nine patients, including 7 with type 3 VWD were exposed to treatment with Wilfactin® for up to 4.2 years. Recovery of VWF in 7 patients (n=5 type 3, n=1 type 2, n=1 type 1) was 1.8±0.4 IU/dL per IU/kg. Of the 62 bleeds, 89% were controlled with one (73%) or two (16%) infusions of Wilfactin®. The median dose per infusion was 54 IU/kg. A factor VIII dose was co-administered in 1.6% of bleeds. "Excellent"/"Good" haemostatic efficacy was achieved in 90.3% of episodes. Six patients underwent 11 minor surgical interventions. Treatment duration was 1 day (range: 1-6 days) with a dose administered 30-60 minutes before procedure of 56 IU/kg (range: 41-106 IU/kg). Haemostasis was rated as "Excellent" in all surgeries. During 4-year prophylactic treatment in one patient, breakthrough bleeds were reported in 2.2% of infusions. No VWF inhibitors, thromboembolic events or allergic/anaphylactic-type reactions were observed following a total exposure of 770 days. The results show that Wilfactin® provides a safe and effective treatment in patients <6 years of age with severe VWD.

Abstract TP243: Treatment With Vwf Inhibitor Dtri-031 In Combination With Rtpa Increases Thrombolytic Efficacy Compared To Rtpa Or Aspirin In Mouse, Canine, And Human Blood

Introduction: We demonstrated that VWF inhibition thrombolyses stabilized clots in and decreases infarct size after middle cerebral artery occlusion (MCAO) in pre-clinical models of stroke. Objective: To compare the thrombolytic efficacy of von Willebrand Factor (VWF) inhibition by DTRI-031 versus rTPA and aspirin utilizing an ex vivo Halo assay. Hypothesis: We hypothesized that DTRI-031 treatment, would result in greater combinatorial efficacy with aspirin or rTPA than any one treatment alone. Methods: Ex vivo Halo assay was performed with whole blood from male and female, 15-month-old wild-type C57BL/6J adult mice, 1-year-old hounds, and healthy control patients 40-60 years of age). Results: Healthy patients The mean CLR max of the negative control group (n=6) was 0.024 ± 0.17 min -1 . Addition of 250 nM DTRI-031 (n=8) or 0.7 nM rtPA (n=8) increased the CLR max to 1.67 ± 1.56 min -1 and 1.80 ± 1.41 min -1 respectively. Addition of 500 nM DTRI-031 and 0.7 rtPA (n=4) resulted in a synergistic effect of both compounds with a CLR max of 10.15 ± 2.4 min -1 (p=.0003). Mice The CLR max of the negative control (n=6) was 0.013 ± 0.011 min -1 . Addition of 250 nM DTRI-031 (n=6) increased the CLR max to 1.19 ± 1.27 min -1 . Addition of 7 nM rtPA (n=6) increased the CLR max to 0.13 ± 0.13 min -1 , which was considerably muted compared to the assay with human blood. The synergistic effect of DTRI-031+rtPA was seen at doses of 250 nM DTRI + 7 nM rtPA (n=4) with a CLR max of 2.08 ± 1.45 min -1 and 500 nM + 7 nM rtPA (n=6) with a CLR max of 4.3 ± 2.5 min -1 compared to monotherapy (p&lt;0.0001). Canine The CLR max of the negative control (n=33) was 2.611 ± 2.659 min -1 . The addition of 250 nM DTRI-031 or 500 nM DTRI-031 and 0.7 rtPA (n=18) resulted in a synergistic effect of both compounds which were significantly greater than all monotherapies with a CLR max of 13.52 ± 8.985 min -1 and 9.152± 6.943 min -1 , respectively. Conclusion: VWF inhibition by DTRI-031 aptamer + rTPA significantly improves clot lysis rate compared to monotherapy with rTPA, DTRI-031, and aspirin in mouse, canine, and human blood.

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.

Phage display broadly identifies inhibitor‐reactive regions in von Willebrand factor

BackgroundCorrection of von Willebrand factor (VWF) deficiency with replacement products containing VWF can lead to the development of anti‐VWF alloantibodies (i.e., VWF inhibitors) in patients with severe von Willebrand disease (VWD). ObjectiveLocate inhibitor‐reactive regions within VWF using phage display. MethodsWe screened a phage library displaying random, overlapping fragments covering the full‐length VWF protein sequence for binding to a commercial anti‐VWF antibody or to immunoglobulins from three type 3 VWD patients who developed VWF inhibitors in response to treatment with plasma‐derived VWF. Immunoreactive phage clones were identified and quantified by next‐generation DNA sequencing (NGS). ResultsNext‐generation DNA sequencing markedly increased the number of phages analyzed for locating immunoreactive regions within VWF following a single round of selection and identified regions not recognized in previous reports using standard phage display methods. Extending this approach to characterize VWF inhibitors from three type 3 VWD patients (including two siblings homozygous for the same VWF gene deletion) revealed patterns of immunoreactivity distinct from the commercial antibody and between unrelated patients, though with notable areas of overlap. Alloantibody reactivity against the VWF propeptide is consistent with incomplete removal of the propeptide from plasma‐derived VWF replacement products. ConclusionThese results demonstrate the utility of phage display and NGS to characterize diverse anti‐VWF antibody reactivities.

Critical Extracellular Matrix Proteins Besides Collagen Bind Von Willebrand Factor

Introduction: Vascular endothelial injury exposes blood to many extracellular matrix proteins including collagen. Von Willebrand Factor (VWF) is a coagulation factor that binds platelets to collagen in the process of normal hemostasis. Collagen IV has been shown to bind VWF through the A1 domain. Many adhesive glycoproteins found in the extracellular matrix of vascular endothelium have been shown to induce hemostasis by binding coagulation factors. We hypothesized that in addition to collagen, other extracellular matrix proteins can bind VWF as well. We investigated VWF's interactions with four other extracellular matrix (ECM) proteins: laminin, fibronectin, thrombospondin, and vitronectin.Methods: Enzyme linked immunoassay (ELISA) was performed to measure the ability of laminin, fibronectin, thrombospondin, and vitronectin to bind to VWF. Each ECM protein was coated individually on a maleic anhydride plate. Detection of VWF:ECM complexes was performed using a combination of two biotinylated monoclonal anti-VWF antibodies. Inhibition of VWF:ECM interactions was tested using monoclonal and polyclonal antibodies against VWF and the individual ECM proteins. We also tested inhibition of VWF:ECM interactions by competing with collagen IV, laminin, fibronectin, thrombospondin, and vitronectin. ELISA was also used to test laminin, fibronectin, thrombospondin, and vitronectin binding to recombinant VWF (rVWF) constructs to determine a specific binding region and to determine the requirement for high molecular weight multimers with each ECM protein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on all ECM proteins to investigate purity.Results: Results for the four ECM proteins generally paralleled each other and will be referred to together unless otherwise specified. SDS-PAGE, however, showed independent bands for each ECM protein preparation, confirming that these interactions were with the individual proteins and not with a single protein contaminating all preparations. ELISA showed binding of laminin, fibronectin, thrombospondin, and vitronectin to wild type (WT) rVWF and plasma from heathy individuals with normal VWF. No binding was detected for individuals with type 3 VWD. Polyclonal anti-VWF (DAKO) inhibited all VWF:ECM interactions (average 99% inhibition), indicating that binding is VWF specific. Monoclonal anti-VWF A1 domain (AVW-3) also inhibited all VWF:ECM interactions (average 98% inhibition), confirming that the A1 domain of VWF is crucial for this interaction. Several rVWF variants were used to test the importance of VWF functional domains. Compared to WT rVWF, VWF:ECM binding was reduced or undetectable with all ECM proteins for 1395A (undetectable) and 1399H (average 4%) but normal for 1786D, 1392A and 1402A, which were shown to abrogate VWF binding to collagen III and collagen IV, respectively. An 11 amino acid deletion from 1392 to 1402 also showed undetectable binding. Reduced binding with these constructs indicate the critical nature of the 1395 to 1399 amino acid region of the A1 domain to the VWF:ECM interactions. Compared to WT rVWF, binding was decreased or undetectable on all ECM proteins for 1597W (average 26%), 2773R (undetectable) and Y87S (undetectable) rVWF constructs. The 1597W, 2773R, and Y87S rVWF constructs contain defects in domains of VWF crucial to multimerization. Reduced binding with these constructs indicate that the VWF:ECM interactions are multimer dependent. All VWF:ECM interactions were significantly inhibited by collagen IV (90% inhibition for thrombospondin, complete inhibition for all other ECMs). The VWF:ECM interactions were also partially inhibited by thrombospondin (average 46% inhibition) and vitronectin (average 62% inhibition). Laminin and fibronectin did not noticeably inhibit any VWF:ECM interactions.Conclusions: In addition to collagen, VWF binds other ECM proteins found in vascular endothelium including laminin, fibronectin, thrombospondin, and vitronectin. These four ECM proteins appear to bind to VWF in a restricted region of the A1 domain as competitive binding suggests differences between collagen IV and other ECM proteins. The role and importance of VWF's interaction with these ECM proteins offer alternative binding partners that may represent a unique contribution to hemostasis. DisclosuresFlood:CSL Behring: Consultancy; Shire: Consultancy.

Inhibition of the Shear-Induced Platelet Microparticle Formation by a mAb Against GPIb alpha Is Associated with Protein Tyrosine Dephosphorylation and the Blockade of Filamin Cleavage.

Platelet microparticle (MP) formation during shear-induced platelet aggregation (SIPA) contributes to a relevant part of thrombin generation. A monoclonal antibody (mAb) Ib-23 against platelet GPIb alpha (provided by B.S.) inhibits von Willebrand factor binding, ristocetin-induced platelet aggregation (RIPA), shear induced MP formation and thrombin generation. Platelet calpain affects several functions including MP-formation. One of several target substrates of calpain, filamin, binds to the cytoplasmic tail of GPIbalpha and mediates the assembly of the contractile elements during platelet activation. We therefore hypothesized that the inhibitory mechanism of our mAb Ib-23 on MP formation is a “calpeptin-like effect”, i.e. an inhibition of calpain affecting the protein tyrosine phosphorylation.PPACK-anticoagulated platelet-rich plasma or washed platelets (250′000/μl) were exposed to a shear rate of 5000 sec−1 for 5 min in a cone-plate viscometer. The mAb Ib-23 was added at the RIPA-inhibiting IC80–90. The samples were fluorescence-labelled with an anti-GPIIb-PE mAb to monitor SIPA and MP formation and analyzed by flow cytometry. Resting and sheared platelets were lysed in SDS-PAGE loading buffer for the analysis of filamin degradation and protein tyrosine phosphorylation.Results: Epitope mapping showed that mAb Ib-23 binds between residues 234 and 456 on GP Ib alpha. Exposure of platelets to high shear rates induced the activation of calpain as demonstrated by the degradation of filamin, a phenomenon blocked by the mAb Ib-23. Ib-23 did not inhibit the SIPA induced tyrosine phosphorylation of high molecular weight proteins like FAK, Syk and alpha-actinin, it even weakly enhanced the tyrosine phosphorylation of these proteins. Moreover, Ib-23 prevented the dephosphorylation of low molecular weight proteins (appr. 40 and 33kDa). Annexin V-binding was unaffected by Ib-23. In contrast, abciximab, a GP IIb/IIIa antagonist, partially inhibited SIPA and annexin binding, but not MP generation. Abciximab weakly inhibited the degradation of filamin, inhibited SIPA-induced tyrosine phosphorylation of FAK and Syk, but did not prevent the tyrosine dephosphorylation of the low molecular weight proteins. Control-experiments with calpeptin demonstrated a similar inhibition of filamin degradation as with mAb Ib-23. An isotype control antibody was without effects. The intact antibody Ib-23 was more effective than the Fab2 fragment. A mAb against the FcgammaRII receptor (IV.3, to exclude a bridging of the FcgammaRII and GpIbalpha via the antibody's Fc part) did not affect MP-formation.We conclude that binding of mAb Ib-23 on the outside of GP Ib alpha inhibits SIPA-induced MP formation and thrombin generation possibly via a “calpeptin-like effect” which inhibits the activation of calpain and of protein tyrosine phosphatases.

Monitoring of von Willebrand factor inhibitors in patients with type 3 von Willebrand disease using a quantitative assay.

Antibodies inhibiting von Willebrand factor (VWF) develop in a subset of patients with type 3 von Willebrand disease (VWD3) and may be detected by their inhibition of ristocetin cofactor activity (VWF:RCo). Some also inhibit factor VIII activity (VIII:C). To describe monitoring of ten VWD3 patients for VWF inhibitors using a quantitative assay. VWF inhibitor was measured by comparing VWF:RCo activity of a mix of patient and pooled normal plasma (PNP) with a mix of buffer and PNP, using agglutination of fixed normal platelets in microtiter plates or lyophilized platelets in an aggregometer. VIII:C inhibitor was measured by Bethesda assay. Preanalytical heat treatment of patient plasma was used during treatment episodes. Four of 10 patients monitored developed VWF inhibitors, two detected during bleeding episodes refractory to treatment and two on routine screening. Data from the first five patients were used to establish an arbitrary unit, VWU, defined as the amount of inhibitor per millilitre of patient plasma inactivating 25% of the activity of 1mL of PNP. In three of four patients, both VWF:RCo and VIIII:C were inhibited at some time points, although VIII:C inhibition sometimes disappeared. In one patient, no VIII:C inhibition was seen. Two patients remained inhibitor positive more than 15 years after inhibitor detection, one became negative following immune tolerance induction, and one was deceased. VWF inhibitors can be quantitatively monitored in VWD3 patients. Preanalytical heat treatment may be required for their detection post infusion.