Ultra-large Von Willebrand Factor Research Articles

Improving our understanding on the clinical role of plasmin-mediated von Willebrand factor degradation.

Von Willebrand factor (VWF) plays a pivotal role in primary hemostasis. A Disintegrin And Metalloproteinase with a ThromboSpondin type 1 motif, member 13 (ADAMTS13) is primarily responsible for cleaving ultra-large VWF multimers into smaller, less adhesive forms. However, plasmin has also been shown to cleave VWF multimers. This proteolytic cleavage of VWF results in a decreased multimer size and, hence, a lower VWF activity. This review aims to present a comprehensive overview of the involvement of plasmin-mediated VWF proteolysis in (micro)thrombosis. Plasmin-mediated VWF proteolysis has been suggested to play a role in various pathologies involving microthrombosis in combination with an imbalance in VWF antigen levels and ADAMTS13 activity, as well as activation of the fibrinolytic system, but quantitative assays to demonstrate this were lacking. Recently, a VHH-based bioassay was developed designed specifically to quantify plasmin-cleaved VWF (cVWF). The novel ELISA assay holds significant promise for gaining further insights into the clinical relevance of plasmin-mediated VWF proteolysis in several pathologies. Furthermore, local plasmin activation at the site of microthrombosis has been shown to be a promising treatment strategy by degrading VWF-rich microthrombi. Plasmin-mediated proteolysis of VWF is observed during microthrombosis; however, it remains unclear whether it impacts disease severity. A novel ELISA method to detect cVWF will improve our understanding of the clinical role of plasmin-mediated VWF degradation.

Hereditary TTP/Upshaw-Schulman syndrome: the ductus arteriosus controls newborn survival.

Hereditary TTP (hTTP), termed Upshaw-Schulman syndrome, is an ultra-rare disorder caused by a severe deficiency of plasma ADAMTS13 activity that allows circulation of ultra-large von Willebrand factor (UL-VWF) multimers. The greatest risk for hTTP is in their first days after birth, when 35-50% of patients will have severe hemolysis, jaundice, and thrombocytopenia. It is often fatal without effective treatment. In utero, fetal blood flowing from the pulmonary artery through the ductus arteriosus (DA) to the aorta is under low-shear-force. At birth, blood flow through the DA reverses to a left-to-right shunt, and the diameter of the DA begins to decrease due to hyper-oxygenated blood and decreased plasma prostaglandin E2. This causes turbulent circulation that unfolds UL-VWF, allowing platelet aggregation. If the DA closes promptly, hTTP newborns survive, but if it remains patent, turbulent circulation persists, triggering microvascular thrombosis. hTTP is commonly diagnosed as hemolytic disease of the fetus and newborn (HDFN) caused by anti-red cell antibodies and treated with exchange blood transfusion, which prevents kernicterus even when the diagnosis of hTTP is missed. The diagnosis of newborn-onset hTTP should be considered because HDFN does not cause severe thrombocytopenia, which might be effectively treated with recombinant ADAMTS13.

Numerical study of ultra-large von Willebrand factor multimers in coagulopathy.

An excessive von Willebrand factor (VWF) secretion, coupled with a moderate to severe deficiency of ADAMTS13 activity, serves as a linking mechanism between inflammation to thrombosis. The former facilitates platelet adhesion to the vessel wall and the latter is required to cleave VWF multimers. As a result, the ultra-large VWF (UL-VWF) multimers released by Weibel-Palade bodies remain uncleaved. In this study, using a computational model based on first principles, we quantitatively show how the uncleaved UL-VWF multimers interact with the blood cells to initiate microthrombosis. We observed that platelets first adhere to unfolded and stretched uncleaved UL-VWF multimers anchored to the microvessel wall. By the end of this initial adhesion phase, the UL-VWF multimers and platelets make a mesh-like trap in which the red blood cells increasingly accumulate to initiate a gradually growing microthrombosis. Although high-shear rate and blood flow velocity are required to activate platelets and unfold the UL-VWFs, during the initial adhesion phase, the blood velocity drastically drops after thrombosis, and as a result, the wall shear stress is elevated near UL-VWF roots, and the pressure drops up to 6 times of the healthy condition. As the time passes, these trends progressively continue until the microthrombosis fully develops and the effective size of the microthrombosis and these flow quantities remain almost constant. Our findings quantitatively demonstrate the potential role of UL-VWF in coagulopathy.

To Gain Insights into the Pathophysiological Mechanisms of the Thrombo-Inflammatory Process in the Atherosclerotic Plaque.

Thromboinflammation, the interplay between thrombosis and inflammation, is a significant pathway that drives cardiovascular and autoimmune diseases, as well as COVID-19. SARS-CoV-2 causes inflammation and blood clotting issues. Innate immune cells have emerged as key modulators of this process. Neutrophils, the most predominant white blood cells in humans, are strategically positioned to promote thromboinflammation. By releasing decondensed chromatin structures called neutrophil extracellular traps (NETs), neutrophils can initiate an organised cell death pathway. These structures are adorned with histones, cytoplasmic and granular proteins, and have cytotoxic, immunogenic, and prothrombotic effects that can hasten disease progression. Protein arginine deiminase 4 (PAD4) catalyses the citrullination of histones and is involved in the release of extracellular DNA (NETosis). The neutrophil inflammasome is also required for this process. Understanding the link between the immunological function of neutrophils and the procoagulant and proinflammatory activities of monocytes and platelets is important in understanding thromboinflammation. This text discusses how vascular blockages occur in thromboinflammation due to the interaction between neutrophil extracellular traps and ultra-large VWF (von Willebrand Factor). The activity of PAD4 is important for understanding the processes that drive thromboinflammation by linking the immunological function of neutrophils with the procoagulant and proinflammatory activities of monocytes and platelets. This article reviews how vaso-occlusive events in thrombo-inflammation occur through the interaction of neutrophil extracellular traps with von Willebrand factor. It highlights the relevance of PAD4 in neutrophil inflammasome assembly and neutrophil extracellular traps in thrombo-inflammatory diseases such as atherosclerosis and cardiovascular disease. Interaction between platelets, VWF, NETs and inflammasomes is critical for the progression of thromboinflammation in several diseases and was recently shown to be active in COVID-19.

Sars-CoV2 ORF7a Protein Stimulates Synthesis and Secretion of Endothelial Von Willebrand Factor

Introduction: Patients with SARS-CoV-2 show significantly elevated plasma levels of von Willebrand factor (VWF) and increased thrombosis. However, the molecular mechanism underlying increased plasma levels of VWF in COVID-19 patients is not fully understood. Methods. Drosophila genetic screen, recombinant expression, endothelial culture, confocal microscopy, flow cytometry, and a microfluidic assay were employed for the study. Results. Drosophila genetic screen for SARS-CoV-2 proteins allowed us to identify Orf7a to be the key factor that induces Hml (drosophila homolog of human VWF)-dependent hypercoagulability. Recombinant Orf7a tagged with mCherry (mCh) and FLAG was expressed transiently and stably in a murine endothelial cell line SVEC4-10 and a HEK293 cell line. The ORF7a protein is localized predominantly to the Golgi and trans-Golgi network (TGN) in HEK293 cells ( Fig. 1A) but shows its expression in perinuclear area and cell surface in SVEC4-10 cells ( Fig. 1B). Significantly higher numbers of VWF-strings were formed in the ORF7a transfected SVEC4-10 cell line under static state than in the non-transfected control cells. These cell surface or extracellular VWF strings were readily removed by a human recombinant ADAMTS13 ( Fig. 1C & 1D). Flow cytometry analysis revealed that significant higher levels of intracellular VWF signal in the cells following transient expression of Orf7a than in those non-expressed controls ( Fig. 2A & 2B). Microfluidic shear-based assay and confocal imaging analysis further demonstrated that dramatically increased rate and surface coverage of fluorescent labeled murine platelets over a histone-stimulated SVEC surface expressing Orf7 comparing with over the same cells without expression of Orf7, following perfusion of Adamts13 -/- murine whole blood (anticoagulated with PPACK) under arterial shear ( Fig. 2C & 2D). Using proteomics, we identified conserved host proteins ARF4, GBF1 and ERGIC-53 (a COPII pathway component) as direct interacting factors for Orf7a and demonstrated that these proteins have conserved roles in VWF secretion. Conclusions:Our results demonstrate that ORF7a may upregulate VWF synthesis and secretion in endothelial cells, which enhances thrombus formation. Recombinant ADAMTS13 may rapidly remove endothelial surface ultra large VWF strings and exhibit therapeutic potential for COVID-19 associated thrombosis.

Caplacizumab Frontline Added to Therapeutic Plasma Exchange and Immunosuppression Prevents Unfavorable Outcomes in Immune-Mediated TTP: An International Real-World Study of the TTP-IWG (The Capla 500 Project)

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) results from autoantibody-mediated severe deficiency of ADAMTS13, the Von Willebrand factor (VWF)-cleaving protease. In this context, ultra-large VWF multimers accumulate in the circulation, leading to increased platelet clumping, with subsequent severe thrombocytopenia, microangiopathic hemolytic anemia and multiorgan failure. The anti-VWF nanobody caplacizumab is licensed for adults with iTTP. Prospective controlled trials and national real-world studies have provided evidence that caplacizumab improved outcome of the acute phase of the disease. However, whether caplacizumab decreases mortality, and the optimal timing of caplacizumab initiation, remain to be determined. To address these questions, an international survey, the Capla 500+ project, has been conducted. An academic call for observations involving experienced teams from 10 worldwide countries recruited 942 adult patients treated with a regimen of daily therapeutic plasma exchange (TPE), caplacizumab and immunosuppression with corticosteroids ± rituximab (caplacizumab group). Cases were compared to historic controls (control group) randomly selected from 2015 to 2018 by 4 teams in a 2:1 ratio (N=495), treated with TPE and immunosuppression. Primary outcome was 3-month survival post-first TPE. Key secondary outcomes were refractoriness and exacerbations, time to clinical response, number of TPE to achieve clinical response, time to ADAMTS13 activity recovery ≥20%, and caplacizumab-related adverse events. Clinical presentation and severity were comparable between groups ( Table). All patients received TPE in both caplacizumab and control groups, in association with corticosteroids (99% and 93%, respectively), and rituximab (92% and 71%, respectively) frontline or as salvage therapy. Caplacizumab was started within 3 days (715 patients), or delayed by≥4 days from first TPE (218 patients); for those with delayed initiation, caplacizumab was added for management of exacerbation or refractoriness (63 cases) or a slow improvement to TPE/immunosuppression (155 cases). The 3-month survival post-first TPE in the caplacizumab group was 98.6% vs 93.3% in the control group (p<0.0001). Failure to achieve clinical response was 5-times less likely in the caplacizumab group as compared to the control group (95% confidence interval, 0.1-0.37, p<0.0001). Deaths in the caplacizumab group occurred after a median of 4.5 days (interquartile range [IQR], 3-5), mostly due to uncontrolled iTTP. Patients receiving caplacizumab experienced less refractoriness and exacerbations, and required less TPE to achieve clinical remission than controls (p<0.0001 for all), regardless of the use of rituximab. Forty-six patients experienced an exacerbation following caplacizumab interruption while ADAMTS13 activity was still undetectable. Time to ADAMTS13 activity ≥20% was shorter in the caplacizumab group (p=0.01) ( Table), where rituximab was more systematically used. In patients with delayed caplacizumab initiation, clinical response was obtained after a median of 4 days (IQR, 2-8) following caplacizumab introduction. This result was comparable to the time to clinical response obtained in patients who initiated caplacizumab within 3 days of the first TPE (median, 4 days [IQR, 3-6], p=0.8). In patients with early caplacizumab initiation, unfavorable outcomes were particularly infrequent (death, 1%; refractoriness, 1.1%; exacerbations, 5.3%),andtime to clinical response in survivors was half the time compared to control group survivors (p<0.0001) ( Figure). Caplacizumab-related adverse events occurred in 220 patients (23%). They consisted of major bleeding (N=19, 2.2%), including: digestive tract bleeding (N=8), intracranial hemorrhage, severe central venous catheter insertion bleeding, metrorrhagia (N=2 cases each). Clinically relevant non-major bleeding (N=34, 3.7%), non-clinically relevant non-major bleeding (N=114, 14%), and inflammatory reaction at the injection site (N=35, 4.5%) were also reported. This unprecedented, international academic effort on such a rare disease provides convincing evidence that caplacizumab added to TPE and immunosuppression prevents unfavorable outcomes during the acute phase of iTTP, including mortality, and alleviates the burden of care at the potential expense of rare, major bleeding events.

Safety and Pharmacokinetics of Recombinant ADAMTS13 in Patients with Sickle Cell Disease: A Phase 1 Randomized, Double-Blind, Placebo-Controlled Study

Background Sickle cell disease (SCD) is an autosomal recessive hemoglobinopathy associated with chronic hemolysis and vaso-occlusive crises (VOCs). Despite the use of treatments such as hydroxyurea, many patients continue to experience VOCs. There is emerging evidence implicating the dysregulation of the von Willebrand factor (VWF)-ADAMTS13 axis in the pathophysiology of VOCs. The size and platelet-binding activity of ultra-large VWF multimers is regulated by the VWF-cleaving metalloprotease ADAMTS13; therefore, the administration of recombinant ADAMTS13 (rADAMTS13), particularly during VOCs, may be therapeutically beneficial in patients with SCD. Here, we present findings from a phase 1 study (NCT03997760) investigating the safety and pharmacokinetics (PK) of three doses of rADAMTS13 (TAK-755; Takeda Development Center Americas, Inc., Lexington, MA, USA) in patients with SCD. Aims To assess the safety, tolerability, and PK of 40 IU/kg, 80 IU/kg, and 160 IU/kg rADAMTS13 in patients with SCD at baseline health. Methods This phase 1, randomized, double-blind, placebo-controlled, multicenter, ascending single dose study (NCT03997760) enrolled patients aged between 18 and 65 years with a documented history of HbSS or HbSβ o thalassemia. Concurrent treatment with a stable dose of hydroxyurea was permitted. Patients were at baseline health: those diagnosed with acute VOC in the 21 days prior to dose administration were excluded. Patients who had received a blood transfusion within 30 days of screening were also excluded. Patients were randomized 3:1 to receive a single intravenous infusion of either rADAMTS13 or placebo in three sequential dose cohorts: cohort 1, 40 IU/kg; cohort 2, 80 IU/kg; cohort 3, 160 IU/kg. Patients were followed for 28 days post-infusion. Each dose cohort was opened sequentially following a review of safety data by a dose escalation committee. Primary safety endpoints included treatment-emergent adverse events (TEAEs), serious TEAEs, and the incidence of neutralizing antibodies against ADAMTS13. Secondary endpoints included an assessment of PK, comprising the maximum concentration (C max) and the area under the concentration-time curve (AUC) from zero (pre-dose) to 72 hours post-dose (AUC 0-72) estimated from ADAMTS13 antigen and activity levels, among other parameters. Results Five patients received placebo and 14 patients received a single dose of rADAMTS13 (cohort 1, n=4; cohort 2, n=6; cohort 3, n=4). The median age (range) of patients who received placebo was 36.0 (20-43) years and 2 (40.0%) were male. Median age (range) of patients who received rADAMTS13 was 38.5 (23-50) years and 9 (64.3%) were male. TEAEs were reported in 2 (40.0%) patients who received placebo and 8 (57.1%) patients who received rADAMTS13. TEAEs considered related to rADAMTS13 were reported in 4 patients (28.6%; Table). One serious TEAE (sickle cell anemia with crisis) not considered related to rADAMTS13 was reported in a patient who received 40 IU/kg rADAMTS13. No bleeding-related TEAEs were reported during the study and no TEAEs led to study discontinuation or death. No anti-ADAMTS13 neutralizing antibodies were detected within 28 days following rADAMTS13 administration. For the PK assessment, mean (standard deviation [SD]) baseline ADAMTS13 antigen was 0.68 (0.16) μg/mL, and rADAMTS13 demonstrated a dose-proportional increase in ADAMTS13 antigen level ( Figure). For cohorts 1, 2, and 3, respectively, baseline-adjusted ADAMTS13 antigen mean C max was 0.51 μg/mL (n=4), 1.07 μg/mL (n=5), and 2.08 μg/mL (n=4), and mean AUC 0-72 was 18.21 h*μg/mL (n=3), 43.01 h*μg/mL (n=5), and 74.51 h*μg/mL (n=4). Mean (SD) baseline ADAMTS13 activity was 1.06 (0.31) IU/mL. For cohorts 1, 2 and 3, respectively, mean C max for baseline-adjusted ADAMTS13 activity was 0.94 IU/mL (n=4), 2.09 IU/mL (n=5), and 3.08 IU/mL (n=4). Conclusions No safety signals or dose-limiting toxicities, such as bleeding events, were observed for the three evaluated doses of rADAMTS13 up to 160 IU/kg, and no rADAMTS13-related serious TEAEs were reported. No anti-ADAMTS13 neutralizing antibodies were detected during the study. rADAMTS13 demonstrated a dose-proportional increase in ADAMTS13 antigen levels, which is consistent with linear PK. Findings from this study support the further investigation of rADAMTS13 as a potential therapeutic option for patients with SCD.

Quantitative System Pharmacology Modeling of the Effect of Recombinant ADAMTS13 on Platelets in Patients with Congenital Thrombotic Thrombocytopenic Purpura

Background: Congenital thrombotic thrombocytopenic purpura (cTTP) is an ultra-rare, thrombotic microangiopathy due to an inherited deficiency of the von Willebrand factor (VWF)-cleaving protease, ADAMTS13.Accumulation of ultra-large VWF multimers increases blood clot formation in small blood vessels, leading to a low platelet count (thrombocytopenia). Treatment of cTTP consists of ADAMTS13 replacement with infusions of plasma-based therapies (PBT). Recombinant ADAMTS13 (rADAMTS13; Takeda Development Center Americas, Inc., Lexington, MA, USA) is in development for patients with cTTP.The ultra-rare nature of cTTP limits the sample size for a formally powered study to assess the impact of ADAMTS13 supplementation on efficacy outcome. This study was a proof-of-principle that model-based clinical trial simulations can be used to supplement clinical trial data in ultra-rare indications. Aims: To calibrate and validate a quantitative system pharmacology (QSP) model describing a mechanistic relationship between ADAMTS13, VWF and platelet numbers in patients with cTTP who received long-term prophylaxis with rADAMTS13 or PBT (fresh frozen or solvent/detergent treated plasma). In addition, to evaluate the treatment benefits of rADAMTS13 versus PBT using clinical trial simulations in virtual patients with cTTP as a supplement to the cTTP phase 3 study (NCT03393975) data. Methods: A QSP model was built using i) in vitro enzyme kinetic data and preclinical experimental data following administration of recombinant VWF and rADAMTS13 in ADAMTS13 knockout (KO) mice, and ii) data from the literature including platelet count and VWF activity from healthy human and untreated patients with cTTP. These data were used to calibrate the parameters corresponding to interactions between ADAMTS13 activity, VWF activity, and platelets. The model was validated against literature data from patients with cTTP treated with PBT and with data from a phase 2 study of patients with immune-mediated TTP treated with rADAMTS13 (NCT03922308). The model was then calibrated to longitudinal platelet data from the cTTP phase 3 study, in which patients with cTTP received prophylaxis for 6 months with PBT (exposure equivalent to ~10 IU/kg ADAMTS13 in the form of 10-20 mL/kg doses) and rADAMTS13 (40 IU/kg) either once every week (Q1W) or once every 2 weeks (Q2W) dosing intervals . The model was used to provide 1-year clinical trial simulations of isolated thrombocytopenia occurrences in a population of 1,000 virtual patients, phenotype-matched to patients in the cTTP phase 3 study. Results:Virtual clinical trial simulations using the QSP model suggested that rADAMTS13 administered over 1 year with a Q1W or Q2W schedule results in significantly fewer patients experiencing occurrences of thrombocytopenia (platelet count threshold of <150 x 10 9/L) compared to patients treated with PBT (hazard ratio [HR] relative to Q2W PBT: Q1W PBT, HR = 0.62; Q2W rADAMTS13, HR = 0.47; Q1W rADAMTS13, HR = 0.18). The treatment difference between rADAMTS13 and PBT was even more pronounced when a thrombocytopenia threshold of <100 x 10 9/L was used (Figure and Table). Simulations also predicted a prophylactic carry-over effect on platelets, with approximately 20 days taken to reach 90% of the long-term average platelet count when switching from rADAMTS13 to PBT or from PBT to rADAMTS13. A correlation analysis for a Q2W dosing of rADAMTS13 demonstrated that ADAMTS13 trough activity (C trough, Spearman ρ = 0.96) and time with ADAMTS13 activity above 10% (Spearman ρ = 0.93) showed stronger correlations with increased platelet count than the maximum plasma ADAMTS13 activity level (C max, Spearman ρ = 0.68). A correlation analysis for a Q1W dosing frequency produced similar results.Mean time above 10% ADAMTS13 activity was higher following rADAMTS13 versus PBT administration (5.2 vs 1.7 days). Conclusions: This model-based virtual clinical trial simulation predicts that dosing with rADAMTS13 would provide >50% reduction in the HR of thrombocytopenia occurrences over PBT. Patients may also have a higher level of protection for 2-3 weeks when switching from rADAMTS13 to PBT. These findings supplement the assessment of clinical benefit of rADAMTS13 in the cTTP phase 3 study and show that increased ADAMTS13 activity following rADAMTS13 administration is likely to reduce occurrences of thrombocytopenia in cTTP.

Thermodynamic Effects of Antibodies on VWF73 Cleavage By ADAMTS13

Background and objective: Immune thrombotic thrombocytopenic purpura (iTTP) is an antibody-mediated life-threatening disease, caused by severe deficiency a plasma metalloprotease ADAMTS13 that impairs the cleavage of ultra-large von Willebrand factor (VWF). This leads to catastrophic microvascular thrombosis and angiopathic hemolysis. Exploring the mechanism of antibodies that inhibit ADAMTS13 under physiologic conditions and standard assay conditions for diagnosis of iTTP (pH 6.0) may help better understand the pathophysiology of iTTP. Our previous study suggests that an inhibitory anti-ADAMTS13 antibody may affect catalytic turnover of the enzyme more than its apparent substrate binding affinity. Here, we describe a rigorous method to quantitatively determine thermodynamic linkage between antibody binding and substrate binding affinity under various conditions. We used well-characterized single chain fragments of the variable loop (scFv's) identified from a phage display library derived from several iTTP patients, and compare the apparent Michaelis constant ( K M,app,) and the maximal enzyme velocity ( V max) of ADAMTS13-mediated VWF cleavage in conditions that more closely represent plasma and in standard conditions. We also describe the stimulatory effect of a newly characterized scFv that targets the C-terminus of ADAMTS13. Methods: Single chain fragments of the variable region (scFv4-20, scFv3-3 and scFv4-41), isolated by phage display from a human anti-ADAMTS13 antibody library derived from iTTP patients, were expressed and purified. ADAMTS13 in normal human plasma was titrated with increasing concentrations of the fluorescently labeled surrogate substrate FRETS-VWF73 to derive V maxand K M,appin the standard assay conditions (pH 6.0) and the conditions closer to physiologic pH (pH 7.0). The same parameters were derived in the presence of saturating concentrations of scFv's. Reaction quotient (Q), which defines thermodynamic linkage between an allosteric effector and substrate, is derived from the ratio of K M,app when saturating scFv is present and K M,app without antibodies. Results: The V max of VWF73 cleavage by ADAMTS13 was lower at pH 7.0 than at pH 6.0 (5.23 ± 0.15 ΔFU/sec vs 7.48 ± 0.42 ΔFU/sec, respectively), and K M,app at pH 6.0 (2.54 ± 0.26 µM) was higher than at pH 7.0 (1.48 ± 0.11 µM) in the absence of scFv. Saturating inhibitory scFv4-20 decreased V max at both pH 6.0 and pH 7.0 (4.45 ± 0.16 ΔFU/sec and 1.00 ± 0.14 ΔFU/sec, respectively); K M,app was relatively unaffected at pH 6.0 (2.70 ± 0.07 µM, Q 1.06) and decreased at pH 7.0 (1.08 ± 0.27 µM, Q 0.73). Saturating scFv3-3 and scFv4-41 increased V max at both pH 6.0 (9.86 ± 0.46 ΔFU/sec and 10.05 ± 0.67 ΔFU/sec, respectively) and 7.0 (9.09 ± 0.25 ΔFU/sec and 10.26 ± 0.34 ΔFU/sec, respectively); while in the presence of scFv3-3 and scFv4-41, K M,app decreased slightly at pH 6.0 (2.20 ± 0.08 µM, Q 0.87; and 2.36 ± 0.26 µM, Q 0.93, respectively) and more dramatically at pH 7.0 (1.08 ± 0.05 µM, Q 0.73; and 1.16 ± 0.07 µM, Q 0.78, respectively). (Figure 1). Conclusions: These results indicate that anti-ADAMTS13 IgG-mediated effects on VWF cleavage may differ when comparing the standard and more physiologic pH conditions. At pH 7.0, both inhibitory and stimulatory scFvs exert a stronger effect on V max of VWF73 cleavage by ADAMTS13 than at pH 6.0. The scFv4-41, which was not previously characterized, had a stimulatory effect on ADAMTS13 very similar to the previously characterized scFv3-3. The K M,app decreases with the increasing of pH from 6.0 to 7.0, while the presence of saturated scFvs investigated in the study appears to increase apparent substrate binding affinity in conditions close to physiologic. The change of enzyme kinetics of VWF73 cleavage by ADAMTS13 in presence of saturated antibodies adds the understanding of iTTP pathophysiology and guide future investigative directions. Key words: Immune thrombotic thrombocytopenic purpura; ADAMTS13, inhibitory and stimulatory antibody

Estimating the Population-Based Prevalence of Congenital Thrombotic Thrombocytopenic Purpura Using Large-Scale Sequencing Data

Introduction Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder caused by severe ADAMTS13 deficiency. This severe deficiency (<10%) is classified into two forms of acquired or hereditary TTP. Acquired TTP is either due to circulating autoantibodies inhibiting ADAMTS13 activity or increasing its clearance, whereas congenital TTP results from homozygous or compound heterozygous ADAMTS13 mutations (cTTP, Upshaw-Schulman syndrome). Because of ADAMTS13 defects, an abnormal amount of ultra-large von Willebrand factor multimers is concentrated, resulting in the formation of platelet-rich microthrombi in the circulation. Given that the true prevalence of cTTP is not established, we sought to leverage the availability of exome or genome sequences of very large numbers of individuals (genome Aggregation Database, gnomAD) to estimate the worldwide and within-population prevalence of cTTP. We also aimed to examine the global mutational landscape of ADAMTS13. Methods Exome and genome sequencing data of 141,456 subjects of gnomAD were extracted and analyzed for ADAMTS13. We comprehensively reviewed literature to identify all ADAMTS13 variants that have been associated with cTTP so far. In addition, all variants reported to be associated with cTTP in HGMD and/or LOVD datasets were included. Finally, we searched for ADAMTS13 variants in ClinVar that have been classified as “pathogenic” or “likely pathogenic”. The following gnomAD variants were considered pathogenic: variants associated with cTTP in HGMD/LOVD; nonsense, frameshift, inframe deletion/insertion variants; splicing variants affecting the first/last two intronic nucleotides; splicing variants affecting the first/last eight intronic nucleotides predicted deleterious by 4 different in silico tools; missense variants predicted deleterious by 7 different in silico tools. Using the frequencies of known or predicted pathogenic variants in ADAMTS13 of the gnomAD population (n= 141,456), we estimated the prevalence of individuals homozygous or compound heterozygous for cTTP (assuming Hardy-Weinberg equilibrium). Results Following quality controls and removing variants with no clear association with cTTP, we found that 302 different ADAMTS13 variants have been so far reported to cause cTTP in the literature, HGMD and LOVD as well as ClinVar. We then collected high-quality data from gnomAD population including 141,456 subjects with different ethnicities: 12,487 Africans/African Americans, 17,720 Latinos/Admixed Americans, 5,185 Ashkenazi Jewish, 9,977 East Asians, 12,562 Finnish Europeans, 64,603 non-Finnish Europeans, 15,308 South Asians, and 3,614 persons without an assigned ethnicity. We identified a total number of 2212 variants within ADAMTS13 among 282,912 alleles analyzed in the gnomAD population. Of these, following a conservative approach, 250 different variants were pathogenic; 83 reported to be associated with cTTP and 167 predicted to be pathogenic. Among a maximum number of 282912 alleles analyzed, 2166 alleles were affected by novel or reported variants. Of which, 53% of the affected alleles were due to known pathogenic variants and the remaining 47% were due to novel predicted variants. Considering all pathogenic variants (n=250) and only those reported (n=83), we estimated an overall cTTP prevalence of 17 and 59 individuals per million people, respectively (Table 1). Considering reported variants only, the highest prevalence of cTTP was estimated in Europeans and Finnish population (30 and 26 per 10 6), followed by Latinos/Admixed Americans (14 per 10 6) and Africans/African Americans (6 per 10 6). East and South Asian ethnicities had a lower estimated prevalence of 4 and 3 per 10 6, respectively. Ashkenazi Jewish population had the lowest estimated prevalence of cTTP (0.6 per 10 6) among all populations (Table 1). Conclusion We have attempted to estimate the worldwide and within-population prevalence of cTTP using available genome/exome sequencing data of 141,456 individuals. Our results reveal that putative disease alleles and cTTP-associated variants are more prevalent than expected and the true cTTP prevalence is more than ten times higher than was reported. This finding suggests that a large number of patients might be undiagnosed. We also established the mutational burden of ADAMTS13 by identifying 167 novel variants in addition to the 302 previously reported.

A-154 Cost-effectiveness of On-site Vs Send-out Measurement of ADAMTS13 Activity in the Management of Acquired Thrombotic Thrombocytopenic Purpura

Abstract Background Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy (TMA) caused by congenital or acquired severe deficiency of disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13), an enzyme cleaving von Willebrand factor (VWF). ADAMTS-13 deficiency leads to the accumulation of ultra-large VWF multimers, and subsequent platelet aggregation and microthrombi formation. Aside clinical symptoms, the diagnosis of TTP is based thrombocytopenia, hemolytic anemia with the presence of schistocytes, and severely reduced activity of ADAMTS-13 (<10%). As TTP is a life-threatening condition, it is of great importance to shorten the time to the treatment, and according to the French recommendations, the triplet therapy including plasma exchange (PEX), rituximab, caplacizumab, plus corticosteroids, must be administered to patients with suspected TTP until the results of ADAMTS-13 was obtained, and be pursued only in the case of deficiency. Therefore, a rapid ADAMTS-13 testing is critical for early diagnosis and optimal management of acute TTP. Methods We compared the cost-effectiveness of a strategy based on on-site measurement of ADAMTS-13 activity using a rapid fully automated chemiluminescent assay (HemosIL AcuStar ADAMTS-13) vs its centralized measurement in a reference laboratory, as it is currently the case at our institution. For that purpose, we created two scenarios based on either its local measurement with a maximum time-to-result of 16 h (assay performed during daytime, 7/7) or its centralized measurement with a median time-to-result of 4 days (range: 2–8, as demonstrated from the 2018–2021 estimates). The triplet therapy was hypothesized to be started on admission and stopped if ADAMTS-13 activity was >10%. In case of ADAMTS-13 activity <10%, an inhibitor screening would be performed at the reference laboratory, as this assay was not expected to be locally implemented. For the calculation, we considered the costs of reagent, shipping/handling of samples to the reference laboratory, laboratory staff workload, reimbursement rates, and treatments administered in patients with suspected TTP until availability of the ADAMTS-13 result. Results Based on 2018 to 2021 estimates, there was a mean of 60 prescriptions of ADAMTS-13 activity per year. Most were for the follow-up of TTP patients and for patients with non-TTP TMA. Only seven were for the diagnosis of acute TTP, as suggested by the French score, which was confirmed in four cases (mean values). The local measurement of ADAMTS-13 activity was found to be cost-effective with a 16%-reduction of the costs compared to its centralized measurement in those seven patients with suspected TTP during the first 4 days. Conclusion Together with its short turnaround time (33 min) and its full automation, our results suggest that the HemosIL AcuStar ADAMTS-13 activity assay could be an assay of choice to rapidly measure ADAMTS-13 activity in the plasma and thus to establish the diagnosis of acute TTP in emergency settings. Considering the local costs, using the strategy based on the local evaluation of ADAMTS-13 activity led to a 16%-reduction of the overall costs during the first 4 days after admission of the seven patients with suspected acute TTP.

Effect of far-infrared radiation therapy on von Willebrand factor in patients with chronic kidney disease.

Hemostatic abnormality has contributed to vascular access thrombosis in patients with chronic kidney disease (CKD). Previous studies have demonstrated that far-infrared radiation (FIR) therapy can maintain the patency and maturity of arteriovenous fistulas of patients undergoing hemodialysis (HD). However, prolonged access bleeding is observed once FIR is conducted at the end of dialysis. FIR can block the binding of platelet and von Willebrand factor (vWF), a predictor of hemostatic abnormality and vascular access thrombosis. However, clinical studies exploring FIR and vWF are sparse. We recruited 20 HD patients, 21 CKD patients, and 20 controls to examine the alteration of vWF and a disintegrin and metalloproteinase with thrombospondin type 1 repeats 13 (ADAMTS13) following a single 40-min session of FIR therapy. In addition, the alteration of these factors in the HD group was examined following a 40-min FIR session thrice a week for 3 months. A decreasing trend in the vWF activity-antigen ratio of participants in all groups following a single FIR session was observed. In addition, the ratio in the HD group was significantly lower following 3 months of FIR therapy. The subgroup analysis revealed a consistent trend and multiple regression analysis showed that participants not taking hydroxymethylglutaryl-coenzyme A reductase inhibitor, diabetes mellitus, and higher hemoglobin levels were the significant factors. The alteration of the vWF activity-antigen ratio correlated moderately to that of ADAMTS13 antigen and activity. FIR may alter the ratio of ultra-large vWF multimers through ADAMTS13, contributing to inhibiting platelet-endothelium interactions of CKD patients.

Recombinant ADAMTS-13 Improves Survival of Mice Subjected to Endotoxemia.

When stimulated by proinflammatory mediators, endothelial cells release ultra-large von Willebrand factor (ULVWF) multimers that are hyperactive in activating and aggregating platelets. These ULVWF multimers can accumulate in the circulation and on the inflamed endothelium because they are insufficiently cleaved by the metalloprotease ADAMTS-13, which becomes moderately deficient under conditions of systemic inflammation. This moderate ADAMTS-13 deficiency may lead to thrombotic complications that contribute to ischemic tissue injury and organ failure that are associated with severe infections. To test this hypothesis, we investigated whether recombinant ADAMTS-13 improves the pathological course of endotoxemia in lipopolysaccharide (LPS)-treated mice. C57BL/J6 mice received a bolus infusion of either 5 µg/mouse of ADAMTS-13 or vehicle control 30 min after LPS challenge and were monitored for seven-day survival. During the monitoring period, platelet counts, VWF antigen, and ADAMTS-13 activity were measured. Thrombosis was also examined by the immunohistochemistry in the liver. We found that ADAMTS-13 reduced mortality from 66% to 34.9%. The improved survival was associated with a greater recovery from thrombocytopenia, higher plasma ADAMTS-13 activity, and less thrombotic vascular occlusion. These results suggest that systemic inflammation could result in deficient ULVWF proteolysis by ADAMTS-13 and that ADAMTS-13 improves the outcomes of endotoxemia-induced inflammation.

N-Acetylcistein for thrombotic thrombocytopenic purpura: an observational case series study.

Acquired thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder. N-Acetylcysteine (NAC) rapidly degrades ultra-large von Willebrand factor multimers by disrupting the disulfide bonds. We report a series of twelve consecutive patients diagnosed with acquired TTP successfully treated with high-dose NAC (150 mg/kg/day) in combination with plasma exchange and steroids. Eight patients also received rituximab. Two patients presented refractory TTP. All patients achieved a quick clinical response in a median time of 5.5 days after starting NAC and are alive after a median follow-up of 29 months. The treatment was feasible and well tolerated. These data provide further evidence of the potential benefit and safety of adding NAC to the standard of care.

Heat-inactivated Factor B inhibits alternative pathway fluid-phase activation and convertase formation on endothelial cell-secreted ultra-large von Willebrand factor strings

Defective regulation of the alternative complement pathway (AP) causes excessive activation and promotes the inflammation and renal injury observed in atypical hemolytic-uremic syndrome (aHUS). The usefulness of heat-inactivated Factor B (HFB) in reducing AP activation was evaluated in: fluid-phase reactions, using purified complement proteins and Factor H (FH)-depleted serum; and in surface-activated reactions using human endothelial cells (ECs). C3a and Ba levels, measured by quantitative Western blots, determined the extent of fluid-phase activation. In reactions using C3, FB, and Factor D proteins, HFB addition (2.5-fold FB levels), reduced C3a levels by 60% and Ba levels by 45%. In reactions using FH-depleted serum (supplemented with FH at 12.5% normal levels), Ba levels were reduced by 40% with HFB added at 3.5-fold FB levels. The effectiveness of HFB in limiting AP convertase formation on activated surfaces was evaluated using stimulated ECs. Fluorescent microscopy was used to quantify endogenously released C3, FB, and C5 attached to EC-secreted ultra-large VWF strings. HFB addition reduced attachment of C3b by 2.7-fold, FB by 1.5-fold and C5 by fourfold. Our data indicate that HFB may be of therapeutic value in preventing AP-mediated generation of C3a and C5a, and the associated inflammation caused by an overactive AP.

Functioning of the ADAMTS-13/vWF axis and its clinical significance

Advances in biology have allowed us to substantially deepen our knowledge about hemostasis functioning both in health and disease. ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) and von Willebrand factor (vWF) are components of the hemostasis system, which physiological interaction holds an important place in maintaining homeostasis. ADAMTS-13 is a metalloproteinase mainly acting to release vWF fragments into the blood plasma, as well as regulating its activity by cleaving ultra-large vWF multimers (UL-vWF) into smaller and less active forms. The study of such factors is of great clinical importance, since a decrease in ADAMTS-13 activity and an increase in vWF level can be predictors of microcirculatory disorders that play an important role in developing multiple organ failure. However, very few and fully contradictory studies devoted to the physiological aspects of the ADAMTS-13/vWF axis functioning in the mother–fetus system are available, therefore requiring to be further investigated.

An evaluation of von Willebrand factor (recombinant) therapy for adult patients living with severe type 3 von Willebrand disease

ABSTRACT Introduction Von Willebrand Factor (VWF) containing concentrates have been used for the treatment of von Willebrand Disease (VWD) for many years. Recently, however, a novel recombinant VWF (rVWF or vonicog alpha, VONVENDI [US], VEYVONDI [Europe]) has arrived to the market for the treatment of VWD. Initially, rVWF was approved by the U.S. Food and Drug Administration (FDA) for the on-demand treatment and control of bleeding episodes and for the perioperative management of bleeding for patients with VWD. More recently, however, the FDA has approved rVWF for routine prophylaxis to prevent bleeding episodes for those patients with severe type 3 VWD receiving on-demand therapy. Areas Covered This review will focus on recent phase III trial results from NCT02973087 regarding the use of long-term routine twice weekly prophylaxis with rVWF for the prevention of bleed events in patients with severe type 3 VWD. Expert Opinion A novel rVWF concentrate may have greater hemostatic potential over prior plasma-derived VWF concentrates and is now FDA approved for use in routine prophylaxis for patients with severe type 3 VWD in the United States. This greater hemostatic potential may be due to the presence of ultra-large VWF multimers and a more favorable high-molecular-weight multimer pattern compared to prior pdVWF concentrates.

The 'scintilla' starting vaso-occlusion in sickle cell disease.

RBCEV SS RBC SS Reduced adhesion to endothelial cells# RBCEV AA RBC AA ADAMTS-13 is a metalloproteinase that cleaves von vWF and ultra large vWF. The importance of ADAMTS-13 is well known for conditions where vaso-occlusion occurs in microvessels, such as in thrombotic thrombocytopenic purpura (TTP). Similarly to hemopexin, the demonstration of a direct involvement of ADAMTS-13 in the adhesion process of SCD is relevant for potential therapeutical development. In fact, An Ran et al. showed that in the presence of hemopexin and ADAMTS-13, adhesion of sickle erythrocytes is returned to the level seen when normal erythrocytes are tested. The finding of an involvement of vWF in sickle cell adhesion suggests a role for anticoagulant proteins in SCD. This is also in keeping with similar findings of reduced naturally occurring anticoagulant (Protein C and Protein S) and vaso-occlusion in SCD.2, 10 In our opinion, this study has a strong scientific value, since it has been conducted in both in vitro and in vivo studies, in a mouse model with SCD. It will now be important to validate these findings in human trials, possibly in a large cohort of patients with SCD. If these findings were to be further corroborated, RBCEVs could potentially be used as a new parameter for monitoring SCD patients in a steady state and during SCD crises. RBCEV trials may possibly even predict SCD crises. Finally, the link between RBCEV, hemopexin and ADAMTS-13 offer an excellent target for the development of future treatment strategies for SCD.

Proximity proteomics identifies septins and PAK2 as decisive regulators of actomyosin-mediated expulsion of von Willebrand factor

AbstractIn response to tissue injury, within seconds the ultra-large glycoprotein von Willebrand factor (VWF) is released from endothelial storage organelles (Weibel−Palade bodies) into the lumen of the blood vasculature, where it leads to the recruitment of platelets. The marked size of VWF multimers represents an unprecedented burden on the secretory machinery of endothelial cells (ECs). ECs have evolved mechanisms to overcome this, most notably an actomyosin ring that forms, contracts, and squeezes out its unwieldy cargo. Inhibiting the formation or function of these structures represents a novel therapeutic target for thrombotic pathologies, although characterizing proteins associated with such a dynamic process has been challenging. We have combined APEX2 proximity labeling with an innovative dual loss-of-function screen to identify proteins associated with actomyosin ring function. We show that p21 activated kinase 2 (PAK2) recruits septin hetero-oligomers, a molecular interaction that forms a ring around exocytic sites. This cascade of events controls actomyosin ring function, aiding efficient exocytic release. Genetic or pharmacological inhibition of PAK2 or septins led to inefficient release of VWF and a failure to form platelet-catching strings. This new molecular mechanism offers additional therapeutic targets for the control of thrombotic disease and is highly relevant to other secretory systems that employ exocytic actomyosin machinery.

Immune-mediated thrombotic thrombocytopenic purpura plasma induces calcium- and IgG-dependent endothelial activation: correlations with disease severity.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by a severe ADAMTS13 deficiency due to the presence of anti-ADAMTS13 auto-antibodies, with subsequent accumulation of circulating ultra-large von Willebrand factor (VWF) multimers. The role of endothelial cell activation as a trigger of the disease has been suggested in animal models but remains to be demonstrated in humans. We prospectively obtained plasma from the first plasma exchange of 25 patients during iTTP acute phase. iTTP but not control plasma, induced a rapid VWF release and P-selectin exposure on the surface of dermal human micro-vascular endothelial cell (HMVEC-d), associated with angiopoietin-2 and endothelin-1 secretion, consistent with Weibel-Palade bodies exocytosis. Calcium (Ca2+) blockade significantly decreased VWF release, whereas iTTP plasma induced a rapid and sustained Ca2+ flux in HMVEC-d which correlated in retrospect, with disease severity and survival in 62 iTTP patients. F(ab)'2 fragments purified from the immunoglobulin G fraction of iTTP plasma mainly induced endothelial cell activation with additional minor roles for circulating free heme and nucleosomes, but not for complement. Furthermore, two anti-ADAMTS13 monoclonal antibodies purified from iTTP patients' B cells, but not serum from hereditary TTP, induced endothelial Ca2+ flux associated with Weibel-Palade bodies exocytosis in vitro, whereas inhibition of endothelial ADAMTS13 expression using small intering RNA, significantly decreased the stimulating effects of iTTP immunoglobulin G. In conclusion, Ca2+-mediated endothelial cell activation constitutes a "second hit" of iTTP, is correlated with the severity of the disease and may constitute a possible therapeutic target.