Metalloprotease ADAMTS13 Research Articles

Recombinant ADAMTS13 for Patients with Severe Congenital Thrombotic Thrombocytopenic Purpura: Design of a Phase 3b Open-Label Continuation Study of Prophylactic and on-Demand Treatment

BackgroundCongenital thrombotic thrombocytopenic purpura (cTTP) is an ultra-rare and potentially life-threatening autosomal recessive inherited thrombotic microangiopathy with an estimated prevalence of <1 in 1 million people. cTTP is caused by a deficiency of the metalloprotease ADAMTS13. Although patients can receive replacement therapy with infusions of either fresh-frozen or solvent/detergent-treated plasma, the level of ADAMTS13 that can be achieved is restricted by infusion volume, and patients can also experience adverse reactions to plasma infusions. A recombinant ADAMTS13 (rADAMTS13; TAK-755; Takeda Development Center Americas, Inc., Lexington, MA, USA) is being developed for use as a replacement therapy for patients with cTTP. A phase 1 study (NCT02216084) in patients with severe cTTP has shown the pharmacokinetic (PK) parameters of rADAMTS13 to be comparable with those estimated in previous plasma infusion studies. A phase 3 study (NCT03393975) to assess the safety and efficacy of rADAMTS13 in patients with severe cTTP was initiated in October 2017 and is ongoing. Here, we report the design of a continuation study, which follows on from the phase 3 study.Study Design and MethodsThis is a phase 3b, prospective, open-label, multicenter, single-arm continuation study (NCT04683003) to evaluate the long-term safety and efficacy of rADAMTS13 for prophylactic and on-demand treatment in patients with severe cTTP. The study will enroll approximately 77 patients and include approximately 57 patients who completed the phase 3 study (48 patients from the prophylaxis cohort; 9 patients from the on-demand cohort) and at least 20 patients who are naïve to rADAMTS13. Patients from expanded access programs or who had an allergic reaction to prophylactic standard of care therapy in the phase 3 study are also eligible for enrollment. Patients who were 0 to 70 years of age at screening for either the phase 3 study or the continuation study and were diagnosed with severe congenital ADAMTS13 deficiency (ADAMTS13 activity <10%) are eligible for inclusion. Patients will be excluded if they have a known life-threatening hypersensitivity to any constituents of rADAMTS13 or have functional ADAMTS13 inhibitors at screening. Patients will be enrolled into either the prophylaxis or on-demand cohort depending on their current therapy and physician consultations (Figure). Patients who completed the phase 3 study and those naïve to rADAMTS13 treatment who are enrolled in the prophylaxis cohort will receive 40 IU/kg rADAMTS13 intravenous infusions either once a week (Q1W) or once every 2 weeks (Q2W). Patients in the prophylaxis cohort can opt to receive at-home rADAMTS13 infusions and will be required to attend interval study visits every 12 weeks. Patients experiencing an acute TTP event will be enrolled in the on-demand cohort and receive 40 IU/kg rADAMTS13 on day 1, 20 IU/kg on day 2, and 15-IU/kg infusions daily from day 3 until 2 days after resolution of the acute event; patients can then choose to move into the prophylaxis cohort or discontinue from the study. Patients in the prophylaxis cohort who experience an acute TTP event during the study will also receive rADAMTS13 in the same dosing regimen as the on-demand cohort (Figure). The primary objective is to evaluate the long-term safety and tolerability of rADAMTS13, measured by the incidence of related treatment-emergent adverse events and serious adverse events, as well as clinically significant changes in vital signs, clinical chemistry, and hematology. The efficacy of rADAMTS13 prophylaxis is a secondary objective of the study, as is the incidence of isolated TTP manifestations. Other secondary outcomes include immunogenicity, health-related quality of life, and healthcare resource utilization in both the prophylaxis and on-demand cohorts. PK and pharmacodynamic parameters of rADAMTS13 will also be assessed. Patient participation is estimated to be either up to 3 years in duration or dependent on the commercial availability of rADAMTS13 in the respective country. The study was initiated in April 2021; estimated completion is September 2026.ConclusionTreatment options for cTTP are limited. This study will evaluate the long-term safety and efficacy of rADAMTS13 for the treatment of patients with severe cTTP. [Display omitted] DisclosuresJain: Takeda: Current equity holder in publicly-traded company; Takeda Development Center Americas, Inc.,: Current Employment. Marquez: Takeda Development Center Americas, Inc.,: Current Employment; Takeda: Current equity holder in publicly-traded company. Martell: Takeda: Current equity holder in publicly-traded company; Takeda Development Center Americas, Inc.,: Current Employment.

Abstract 1122‐000037: LVO in the Setting of TTP

Introduction : Thrombotic Thrombocytopenic Purpura (TTP) is a disorder of coagulation caused by a deficiency of ADAMTS13 due to either hereditary mutations or acquired autoimmune inhibitors. Low levels of ADAMTS13 metalloprotease leads to Von Willebrand Factor (VWF)‐platelet aggregation and microvascular thrombosis when patients with TTP are exposed to high shear stress in the microcirculation. [1] Hematologic disorder or coagulopathies are the major cause of 1–4% of all ischemic strokes. [2] TTP patients usually present with small vessel strokes or sometimes very distal branches of large arteries. The gold standard treatment for acute stroke in these patients is plasma exchange, however in patients presenting with LVO strokes, recanalization therapies should be considered. [3] Here we present a case of large vessel occlusion stroke in the setting of TTP. Methods : 31‐year‐old African American female with TTP and Strokes admitted to with right sided hemiparesis, and expressive aphasia within 2h of symptom onset. National Institute of Health Stroke Scale (NIHSS) of 16. Laboratory work up significant for Platelets 114k, Hemoglobin 12, PTT 24 seconds, Serum glucose 107, ADAMTS‐13 &lt;1 IU/dL and negative COVID‐19 PCR. CTH showing ASPECTS of 9 (Picture A), CTA head and neck revealing a left MCA occlusion and intracranial vessel irregularities in the left Anterior Cerebral Artery (ACA) and bilateral Posterior Cerebral Arteries (PCA) (Picture B). CTP with 24cc core and 132 cc mismatch (Picture C). Patient was taken to the Angiogram Suite for urgent thrombectomy with a final TICI score of 3 achieved after one pass (Picture D/E). Results : Post procedure NIHSS was 1 for mild expressive aphasia. Brain MRI revealed Left MCA and right parietal territories infarct (Picture F). Transesophageal Echocardiogram was unremarkable. She underwent urgent Plasma Exchange (PLEX) for TTP flair in light of undetectable ADAMTS‐13, significant drop in platelet count and thrombotic event. After 5 PLEX sessions platelets remained stable with counts greater than 150.000. Immunotherapy with Prednisone, Caplacizumab, Atovaquone and Mycophenolate Mofetil was continued and she was discharged home on day 7 from admission. Conclusions : Our patient had severe and refractory disease presenting with LVO and underwent an unusual therapeutic approach considering the etiology of her disease. IV thrombolysis and arterial thrombectomy are well established therapies for acute strokes in the general population, but are rarely performed in patients affected by Thrombotic Microangiopathy (TMA) such as TTP. Safety and efficacy of these procedures have not been studied in this population and it is usually selected by extrapolating data from studies that used individuals without TMA. We aim to illustrate this uncommon case of LVO stroke in the setting of TTP and potentially stimulate the elaboration of studies that include these patients.

Characterization of the interactions of ADAMTS13 CUB1 domain to WT- and GOF-Spacer domain by molecular dynamics simulation

Metalloprotease ADAMTS13 specifically cleaves VWF (von Willebrand Factor) to prevent excessive platelet aggregation and thrombus formation at the sites of vascular injury. To avoid non-specific cleavage, ADAMTS13 has the auto-inhibition effect in which the Spacer domain in N-terminal interacts with the CUB1 domain in C-terminal, resulting in decreased proteolytic activity. Previous studies reported that exosite-3 in the Spacer domain was a key binding site in the Spacer-CUB1 interaction. When exosite-3 was mutated (R660K/F592Y/R568K/Y661F/Y665F, GOF), the auto-inhibition of ADAMTS13 was disrupted and the enzymatic activity was markedly increased. However, the characteristics of the Spacer-CUB1 interaction is not fully understood. Here, we constructed the model of Spacer-CUB1 complex by homologous modeling and molecular docking to characterize the Spacer-CUB1 binding and predict key amino acid residues via molecular dynamics simulation. Our data showed that G607-S610 was a non-reported potential binding site in the Spacer domain; GOF mutation attenuated the formation of hydrogen bond between exosite-3 and the CUB1 domain; Residues E1231, R1251, L1258, D1259 and T1261 in the CUB1 domain might play an important role in the Spacer-CUB1 interaction. Our study advances the understanding of the structural basis of the auto-inhibition of ADAMTS13 and provides information about the key residues in the binding interface.

Gold nanoparticle enhanced multiplexed biosensing on a fiber optic surface plasmon resonance probe

We present an innovative multiplexing concept on a fiber optic surface plasmon resonance (FO-SPR) platform and demonstrate for the first time the simultaneous detection of two targets using the same FO sensor probe. Co(III)-NTA chemistry was used for oriented and stable co-immobilization of two different His6-tagged bioreceptors. T2C2 and MDTCS (i.e. fragments of the ADAMTS13 metalloprotease linked to the thrombotic thrombocytopenic purpura disorder) served as model system bioreceptors together with their respective targets (4B9 and II-1 antibodies). Gold nanoparticles were used here in an original way for discriminating the two targets in the same sample, in addition to their traditional signal amplification-role. After verifying the specificity of the selected model system, we studied the bioreceptor surface density and immobilization order. Innovative approach to lower the bioreceptor concentration below surface saturation resulted in an optimal detection of both targets, whereas the order of immobilization of the two bioreceptors did not give any significant difference. By sequentially immobilizing the T2C2 and MDTC bioreceptors, we established calibration curves in buffer and 100-fold diluted human blood plasma. This resulted in calculated limits of detection of 3.38 and 2.31 ng/mL in diluted plasma for 4B9 and II-1, respectively, indicating almost the same sensitivity as in buffer. Importantly, we also proved the applicability of the established calibration curves for quantifying the targets at random and more realistic ratios, directed by the design of experiments. This multiplexing study further expands the repertoire of applications on the FO-SPR biosensing platform, which together with its intrinsic features opens up great opportunities for diagnostics and life sciences.

Von Willebrand Factor in Health and Disease

—Von Willebrand factor (vWF), the key component of hemostasis, is synthesized in endothelial cells and megakaryocytes and released into the blood as high molecular weight multimeric glycoproteins weighing up to 20 million Daltons. Blood plasma metalloprotease ADAMTS13 cleaves ultra-large vWF multimers to smaller multimeric and oligomeric molecules. The vWF molecules attach to the sites of damage at the surface of arterioles and capillaries and unfold under conditions of shear stress. On the unfolded vWF molecule, the regions interacting with receptors on the platelet membrane are exposed. After binding to the vWF filaments, platelets are activated; platelets circulating in the vessels are additionally attached to them, leading to thrombus formation, blocking of microvessels, and cessation of bleeding. This review describes the history of the discovery of vWF, presents data on the mechanisms of vWF secretion and its structure, and characterizes the processes of vWF metabolism in the body under normal and pathological conditions.

Covid Causing Thrombotic Thrombocytopenic Purpura

COVID-19 has been causing havoc for over a year now. As more cases of COVID have been diagnosed, we are beginning to see various sequelae emerge after patients contract the virus. Specifically, we have seen a patient develop Thrombotic Thrombocytopenic Purpura (TTP) after being diagnosed with COVID-19. Thrombotic thrombocytopenic purpura is a hematologic disorder in which an enzyme deficiency, specifically of metalloprotease ADAMTS-13, causes abnormal multimer formation with the consequences of fever, microangiopathic hemolytic anemia, thrombocytopenia, renal disease, and neurological abnormalities. The coronavirus (COVID-19) has been observed across the world since first emerging in 2019 in China. Although both diseases have been widely observed independently, the development of TTP shortly after contracting COVID has not frequently been reported [1, 2, 3]. Thus, we are bringing to light a case of a patient who developed TTP shortly after being diagnosed with COVID.

The Role of VWF, FVIII, ADAMTS13 and Inflammatory Response in the Outcome of Acute Ischemic Stroke Mechanical Thrombectomy Procedure

Background and Purpose: Mechanical thrombectomy is the method of choice for the intracranial arteries large vessel occlusions therapy in acute ischemic stroke after failed intravenous thrombolysis. Stent retrievers, aspiration catheters or combination are the most often techniques used for the intracranial thrombus extraction. Procedure length, TICI grade of recanalisation and the risk factors like diabetes, hypertension and arhytmia are the critical parameters influencing 3month-mRS clinical outcome after ischemic stroke treatment. Procedure time of the sticky emboli extraction and the endothelial cells damage are influenced with blood coagulation parameters, especially platelet adhesion molecules, D-dimers, fibrinogen, FVIII, von Willebrand factor multimers and by the inflammatory response of the body. Large vWF-multimers are cleaved into smaller, less coagulant forms by the metalloprotease ADAMTS13. In case of ADAMTS13 deficiency, large vWF-multimers are not cleaved from the circulation with the higher risk of thrombosis. Methods: 120 consecutive patients with acute ischemic stroke underwent mechanical thrombectomy. Clinical and interventional procedure parameters, as well as blood coagulation factors and cell counts, extracted emboli light microscopy and peripheral blood inflammatory respond were analysed. Results: Significantly higher vWF/ADAMTS13 ratio was found in patients with higher NIHSS in the time of the admission with a worse outcome in 3month-mRS. Extractedvthromboemboli contained significantly higher amount of the inflammatory cells granulocytes, monocytes, vWF/FVIII with fibrin and platelets. Also significantly higher inflammatory respond was observed in patients with NIHSS >15 which correlates with the thromboemboli histology and procedure length prolongation. Conclusion: Platelets, coagulation factors vWF/FVIII and metalloprotease ADAMTS13, in conjunction with inflammatory responsive cells are important factors influencing patient's NIHSS outcome at Day 7 and 3 month after mechanical extraction procedure. High level of the vWF/FVIII and low ADAMTS13 protease activity are responsible for the resistance of the large vessel occlusions to the rt-PA thrombolytic efficacy. The sticky emboli contains red blood cells, fibrin, vWF/FVIII complexes, platelets and inflammation accompanying cells - granulocytes, monocytes and eosinophils. DisclosuresHajek:Novartis: Consultancy, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Pharma MAR: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

LDL and HDL Have Opposing Effects on VWF Self-Association

Von Willebrand Factor (VWF) is a large, multimeric plasma protein with roles in primary hemostasis and thrombosis. VWF unfolds in response to shear stress and self-associates into adhesive fibers that can capture and hold platelets at sites of vascular injury, initiating platelet plug formation. VWF has also been implicated in pathologies associated with microvascular dysfunction and occlusion, and dysregulation of VWF can produce the catastrophic microvascular thrombotic disorder thrombotic thrombocytopenic purpura. VWF self-association is regulated by several factors: it is enhanced by shear stress, it is decreased by regulatory cleavage by ADAMTS13 metalloprotease[Dong et al. Blood, 2002, 100:4033], and our group recently showed that high density lipoprotein (HDL) attenuates VWF self-association [Chung et al. Blood, 2016, 127:637]. High levels of HDL are associated with decreased risk of thrombosis, whereas high levels of low density lipoprotein (LDL) is a strong risk factor. This led us to investigate whether LDL also influences VWF self-association and fiber formation.We studied the effects of lipoproteins on VWF self-association by applying shear stress to VWF solutions by vortexing, which caused VWF loss from solution through a process of adsorption and self-association [Chung et al. Blood, 2016, 127:637]. We inhibited the ADAMTS13 in citrated-plasma with EDTA, sheared the treated plasma in the presence of exogenous HDL or LDL, and measured the VWF remaining in solution by sandwich ELISA. HDL attenuated VWF self-association in plasma, whereas LDL had the opposite effect, enhancing self-association. Using purified components, we sheared recombinant VWF in the presence of a fixed concentration of HDL that stabilized the VWF, and added increasing amounts of LDL to attain LDL/HDL ratios found in physiological and pathophysiological conditions. As the molar ratio of LDL/HDL particles increased from 0.17 to 0.67, VWF self-association also increased. When we repeated these assays with double the concentrations of both lipoproteins, the effect on VWF self-association was comparable. We tested EDTA-treated platelet-poor-plasma with known LDL and HDL concentrations and found that the LDL/HDL ratio regulated VWF self-association in a similar manner, but the effect was less pronounced than with the purified components.To further assess the effect of LDL and HDL on VWF self-association under shear, we perfused purified plasma VWF in a microcolumn impingement device [Herbig & Diamond JTH, 2015, 13:1699] at a wall shear rate of 10,000/s, in the presence of PBS buffer, LDL, HDL, and LDL/HDL molar ratios of 0.17 and 0.67. VWF fiber formation was monitored by differential interference contrast and immunofluorescence microscopy. The width of the bundles of VWF fibers were measured and total fluorescence was quantified using image analysis software. The presence of LDL dramatically enhanced the size of VWF fibers and the rate of formation and deposition on the microcolumn, indicating that LDL interacts directly with VWF to enhance self-association. Perfusing HDL with VWF had the opposite effect and attenuated self-association. When VWF was perfused with LDL and HDL together, the LDL/HDL ratio regulated the thickness and length of the VWF fibers formed. Our findings reveal a novel role by which LDL, by enhancing VWF self-association, may contribute to VWF-mediated thrombosis. DisclosuresNo relevant conflicts of interest to declare.

Von Willebrand factor, ADAMTS13 and mortality in dialysis patients

BackgroundVon Willebrand Factor (VWF) multimers are cleaved into smaller and less coagulant forms by the metalloprotease ADAMTS13. The aim of this study was to investigate the association between VWF and ADAMTS13 and mortality in dialysis patients.MethodsWe prospectively followed 956 dialysis patients. VWF levels and ADAMTS13 activity were measured. Cox proportional hazard analyses were used to calculate hazard ratios (HRs) with 95 % confidence intervals (CIs) to investigate the association between quartiles of VWF levels and ADAMTS13 activity and all-cause mortality. HRs were adjusted for age, sex, body mass index, cardiovascular disease, dialysis modality, primary kidney disease, use of antithrombotic medication, systolic blood pressure, albumin, C-reactive protein and residual GFR.ResultsOf the 956 dialysis patients, 288 dialysis patients died within three years (mortality rate 151 per 1000 person-years). The highest quartile of VWF as compared with lower levels of VWF was associated with a 1.4-fold (95 %CI 1.1–1.8) increased mortality risk after adjustment. The lowest quartile of ADAMTS13 activity as compared with other quartiles was associated with a 1.3-fold (95 %CI 1.0-1.7) increased mortality risk after adjustment. The combination of the highest VWF quartile and lowest ADAMTS13 quartile was associated with a 2.0-fold (95 %CI 1.3-3.0) increased mortality risk as compared with the combination of the lowest VWF quartile and highest ADAMTS13 quartile.ConclusionsHigh VWF levels and low ADAMTS13 activity were associated with increased mortality risks in dialysis patients.

Quantification of von Willebrand factor and ADAMTS-13 after traumatic injury: a pilot study

BackgroundVon Willebrand factor (VWF) is an acute phase reactant synthesized in the megakaryocytes and endothelial cells. VWF forms ultra-large multimers (ULVWF) which are cleaved by the metalloprotease ADAMTS-13, preventing spontaneous...

Secretion of von Willebrand Factor and Suppression of ADAMTS-13 Activity by Markedly High Concentration of Ferritin.

Hyperferritinemia is associated with poor outcomes in critically ill patients with sepsis, hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndromes (MAS) and coronavirus disease 19 (COVID-19). Autopsies of hyperferritinemic patients that succumbed to either sepsis, HLH, MAS or COVID-19 have revealed disseminated microvascular thromboses with von Willebrand factor (VWF)-, platelets-, and/or fibrin-rich microthrombi. It is unknown whether high plasma ferritin concentration actively promotes microvascular thrombosis, or merely serves as a prognostic biomarker in these patients. Here, we show that secretion of VWF from human umbilical vein endothelial cells (HUVEC) is significantly enhanced by 100,000 ng/ml of recombinant ferritin heavy chain protein (FHC). Ferritin fraction that was isolated by size exclusion chromatography from the plasma of critically ill HLH patients promoted VWF secretion from HUVEC, compared to similar fraction from non-critically ill control plasma. Furthermore, recombinant FHC moderately suppressed the activity of VWF cleaving metalloprotease ADAMTS-13. These observations suggest that a state of marked hyperferritinemia could promote thrombosis and organ injury by inducing endothelial VWF secretion and reducing the ADAMTS-13 activity.

Insights Into Immunothrombosis: The Interplay Among Neutrophil Extracellular Trap, von Willebrand Factor, and ADAMTS13.

Both neutrophil extracellular traps (NETs) and von Willebrand factor (VWF) are essential for thrombosis and inflammation. During these processes, a complex series of events, including endothelial activation, NET formation, VWF secretion, and blood cell adhesion, aggregation and activation, occurs in an ordered manner in the vasculature. The adhesive activity of VWF multimers is regulated by a specific metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13). Increasing evidence indicates that the interaction between NETs and VWF contributes to arterial and venous thrombosis as well as inflammation. Furthermore, contents released from activated neutrophils or NETs induce the reduction of ADAMTS13 activity, which may occur in both thrombotic microangiopathies (TMAs) and acute ischemic stroke (AIS). Recently, NET is considered as a driver of endothelial damage and immunothrombosis in COVID-19. In addition, the levels of VWF and ADAMTS13 can predict the mortality of COVID-19. In this review, we summarize the biological characteristics and interactions of NETs, VWF, and ADAMTS13, and discuss their roles in TMAs, AIS, and COVID-19. Targeting the NET-VWF axis may be a novel therapeutic strategy for inflammation-associated TMAs, AIS, and COVID-19.

Therapeutic application of recombinant human ADAMTS-13 improves shock reversal and coagulation status in a trauma hemorrhage and transfusion rat model

IntroductionIn hemorrhaging trauma patients, the endothelium is activated, resulting in excessive endothelial synthesis of von Willebrand Factor (vWF), which may enhance micro-thrombi formation, resulting in obstruction of the microcirculation and endothelial injury, aggravating bleeding, as well as contributing to organ failure. Under normal conditions, vWF is cleaved by the metalloprotease ADAMTS-13. After trauma, ADAMTS-13 levels are reduced.ObjectivesTo assess whether recombinant human ADAMTS-13 inhibits endothelial injury and organ failure in a rat trauma-transfusion model.MethodsBlood products were prepared from syngeneic rat blood according to blood bank standards. Polytrauma was induced in rats by crush injury to the intestines and liver and by fracture of the femur. The rats were hemorrhaged until a mean arterial pressure (MAP) of 40 mmHg was reached. Rats were randomized to receive transfusion of RBCs, FFPs, and platelets in a 1:1:1 ratio to achieve a MAP of 70 mmHg, with or without the addition of ADAMTS-13 (50 μg/kg). Blood samples were assessed for biochemistry and rotational thromboelastometry (ROTEM). Syndecan-1 and VE-cadherin levels were measured as a reflection of endothelial integrity. The amount of leakage of dextran-FITC from the vascular system to the parenchyma in lungs was quantified. To assess inflammation, IL-6 and IL-8 levels were determined. Organ damage was assessed by histopathology.ResultsAll rats were severely shocked, with no significant differences in shock parameters between groups. Rats treated with ADAMTS-13 showed signs of a more effective shock reversal (higher blood pressure, lower lactate levels) compared to controls. Also, ROTEM parameters of clot formation in rats receiving ADAMTS-13 improved compared to controls, which was mainly platelet-dependent. Syndecan-1 levels relative to baseline trended to be lower in ADAMTS-13 treated rats compared to controls (107 vs 149%, p = 0.08). ADAMTS-13 reduced albuminuria (1.7 vs 4.4 g/L, p < 0.01) and organ-specific inflammation (pulmonary IL-6 243 vs 369 pg/mL, p = 0.08; splenic IL-6 253 vs 307, p = 0.03) compared to controls, but did not improve histopathological scores.ConclusionsThe use of ADAMTS-13 in a rat trauma-transfusion model improves parameters of shock, platelet-driven coagulation, endothelial damage, and organ inflammation. These results suggest that ADAMTS-13 is important in mediating outcome of trauma. Whether ADAMTS-13 can be used as a therapeutic adjunct to treat bleeding trauma patients remains to be determined.

Role of Von Willebrand Factor after Injury: It May Do More Than We Think.

Acute traumatic coagulopathy is a complex phenomenon following injury and a main contributor to hemorrhage. It remains a leading cause of preventable death in trauma patients. This phenomenon is initiated by systemic injury to the vascular endothelium that is exacerbated by hypoperfusion, acidosis, and hypothermia and leads to systemic activation of the coagulation cascades and resultant coagulopathy. Many previous studies have focused on endotheliopathy with targeted markers such as syndecan-1, soluble thrombomodulin, and plasma adrenaline as potential culprits for initiation and propagation of this state. However, in more recent studies, hyperadhesive von Willebrand factor (VWF), which is released following endothelial injury, and its cleaving metalloprotease ADAMTS13 have emerged as significant targets of the downstream effect of endothelial breakdown and coagulation dysregulation. Elucidation of the mechanism by which the dysregulated VWF-ADAMTS13 axis leads to endothelial dysfunction and coagulopathy after trauma can help identify new targets for therapy and sites for intervention. Much of what is known mechanistically regarding VWF stems from work done in traumatic brain injury. Following localized brain injury, brain-derived extracellular vesicles are released into circulation where they induce a hypercoagulable state that rapidly turns into consumptive coagulopathy. VWF released from injured endothelial cells binds to these extracellular vesicles to enhance their activity in promoting coagulopathy and increasing endothelial permeability. However, there are numerous gaps in our knowledge of VWF following injury, providing a platform for further investigation.

P0240ACQUIRED THROMBOTIC THROMBOCYTOPENIC PURPURA IN HIV POSITIVE PATIENTS SUCCESSFULLY TREATED WITH CAPLACIZUMAB: ABOUT TWO CASES

Abstract Background and Aims Acquired thrombotic thrombocytopenic purpura (aTTP) is a thrombotic microangiopathy still nowadays associated with a high risk of death. Standards of care included at first the association of plasmatic exchanges, Rituximab and steroids. The emergence of Caplacizumab, a nanobody targeting the A1 receptor of Von Willebrand Factor (VWF), significantly changed the prognosis of the disease. Thrombocytopenia is frequent in HIV positive patients, and aTTP is frequently considered. Prevalence of HIV positive in aTTP patients ranges from less than 1% in western countries, up to more than 50% in African countries. The pathophysiology still remains partially understood, leading to the development of autoantibodies targeting the ADAMTS13 metalloprotease. For now, no specific recommendations are available, and standards of care consist in treating HIV positive patient the same as other aTTP patients. We present the first description of two HIV positive patients, successfully treated with caplacizumab. Method We report there two cases of black women, respectively aged 31 (Patient 1) and 52 years old (Patient 2), both positive for HIV for several years. The first one had stopped any antiviral treatment few months ago, and had a positive blood viral load of 110764 copies/ml. Her CD4+ rate was 30/mm3. The second one was under antiviral medication, with a negative viral load and a CD4+ rate of 295/mm3. Both were admitted in our department because of low platelet levels and hemolysis, associated with inflammatory triggers. Bacteriema for Streptococcus mitis was discovered on admission in Patient 1 and Patient 2, who had a long-term follow-up for Still disease, presented with a flare-up. Results Both patients had positive anti-ADAMTS13 autoantibodies (Patient 1: titer 42 UI/ml, Patient 2: 45 UI/ml), with undetectable ADAMTS-13 activities. They were treated with plasmatic exchanges until platelets levels reached over 150 G/L, steroids 1.5 mg/kg/j, Rituximab 375 mg/m2 at day 1, 4 and 8 and finally caplacizumab 10 mg before the 2nd plasmatic exchange and every day until the ADAMTS13 activity reached over 20%. Antiviral therapy and antibiotics were initiated in Patient 1 and antiviral therapy was maintained in Patient 2. The evolution was promptly favorable with normalization of platelets levels at Day 4 in Patient 1, and Day 15 in Patient 2. Patient 1 stopped caplacizumab at Day 12 and Patient 2 at day 17, when ADAMTS 13 reached over 20%. None of them relapsed after treatment, with a respective follow-up of 4 and 2 months. Conclusion Caplacizumab seems to be a safe and efficient treatment for aTTP in HIV positive patients.

Traumatic injury results in prolonged circulation of ultralarge von Willebrand factor and a reduction in ADAMTS13 activity.

Increases in plasma von Willebrand Factor (VWF) levels, accompanied by decreases in the metalloprotease ADAMTS13, have been demonstrated soon after traumatic injury while downstream effects remain unclear. A cohort of 37 injured trauma patients from a randomized control trial investigating the use of prehospital plasma transfusion were analyzed for activity and antigen levels of ADAMTS13 and VWF at 0 and 24 hours after admission. Relevant clinical data were abstracted from the medical records. Trauma patient plasma was analyzed via agarose gel electrophoresis to evaluate the effects of injury on VWF multimer composition compared to healthy controls. von Willebrand factor levels were elevated at presentation (189% [110%-263%] vs. 95% [74%-120%]), persisting through 24 hours (213% [146%-257%] vs. 132% [57%-160%]), compared to healthy controls. Ultralarge VWF (UL-VWF) forms were elevated in trauma patients at both 0 and 24 hours, when compared to pooled normal plasma (10.0% [8.9%-14.3%] and 11.3% [9.1%-21.2%], respectively, vs. 0.6%). Circulating plasma ADAMTS13 activity was decreased at 0 hours (66% [47%-86%] vs. 100% [98%-100%]) and at 24 hours (72.5% [56%-87.3%] vs. 103% [103%-103%]) in trauma patients. ADAMTS13 activity independently predicted the development of coagulopathy and correlated with international normalized ratio, thromboelastography values, injury severity, and blood product transfusion. Traumatic injury is associated with acute coagulopathy that is characterized by increased UL-VWF multimers and reduction in ADAMTS13, which correlates with blood loss, transfusion requirement, and injury severity. These findings suggest the potential for future trials targeting ADAMTS13 repletion to enhance clearance of VWF multimers.

METALLOPROTEASE ADAMTS-13

Introduction. The signifi cance of ADAMTS-13 extends beyond its key role in the pathogenesis of thrombotic thrombocytopenic purpura (TTP); there is evidence of a relationship between a decrease in the ADAMTS-13 activity and thrombotic events in acute myocardial infarction and ischemic stroke.Aim. To generalise available information on the structure and function of the metalloprotease ADAMTS-13.General findings. The biological function of ADAMTS-13 consists in the cleavage of ultra-large von Willebrand factor (vWF) multimers. The fact that its defi ciency causes the development of TTP provides a basis for understanding the function of vWF–cleaving protease. ADAMTS-13 has a domain structure. The functional roles of most ADAMTS-13 domains, as well as the key role of the ADAMTS-13-vWF interaction in the regulation of haemostasis, are defi ned. The conformational activation of ADAMTS-13 by vWF constitutes an important aspect of its function. After getting into the bloodstream, ultra-large vWF multimers quickly adopt a closed conformation, which becomes very resistant to ADAMTS-13 proteolysis in the absence of shear stress. Ultra-large plasma vWF multimers regain their sensitivity to ADAMTS-13 after being exposed to high fl uid shear stress, which unfolds the central vWF-A2 domain. The unfolding of a vWF molecule under shear stress conditions reveals previously hidden exosites in domain A2, which gradually increase the binding affi nity between ADAMTS-13 and vWF. The mechanism underlying the production of autoantibodies against ADAMTS-13 is unknown and requires further study. The masking of cryptic epitopes in the closed conformation of ADAMTS-13 prevents the formation of autoantibodies. Early antigen recognition of ADAMTS-13 occurs through surface-exposed epitopes in the C-terminal domains. More detailed information on the mechanisms underlying the interaction between ADAMTS-13 and the vWF can improve the understanding of mechanisms involved in the regulation of the coagulation system.Conflict of interest: the authors declare no confl ict of interest.Financial disclosure: the study had no sponsorship.

ADAMTS13, an Anti-thrombotic Protein: Evidence Outside of Thrombotic Thrombocytopenic Purpura

von Willebrand factor (VWF) is a large multimeric plasma glycoprotein that plays a crucial role in hemostasis and thrombosis. VWF recruits platelets at sites of vascular injury by acting as a molecular bridge between circulating platelets and the site of injured or activated blood vessels. Biosynthesis of VWF is restricted to endothelial cells and megakaryocytes. Endothelial VWF is constitutively secreted into plasma and subendothelium, or is stored as "ultra-large" (UL)-VWF multimers in endothelial Weibel-Palade bodies. VWF produced in megakaryocytes is packaged as UL-VWF in the a-granules of platelets. VWF stored in endothelial and platelet storage organelles is secreted in a regulated process in response to stimulation by secretagogues. Absence or dysfunction of VWF results in bleeding symptoms, as observed in patients with von Willebrand disease. An abnormally high activity of VWF can lead to thrombotic events. Interestingly, the activity of VWF is determined by the size of its multimers and UL-VWF can spontaneously form platelet aggregates. An important regulator of VWF size is the metalloprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), which digests large thrombogenic VWF molecules into smaller, less reactive multimers via cleavage of the Y1605-M1606 bond in the VWF A2 domain. ADAMTS13 is mainly synthesized in the liver by hepatic stellate cells, but other sites of synthesis, including renal podocytes, tubular epithelial cell, platelets and endothelial cells, have also been described. ADAMTS13 is released as an active enzyme into the circulation with no physiological inhibitors. Reduced or absent ADAMTS13 activity causes the microangiopathic disorder thrombotic thrombocytopenic purpura (TTP), characterized by VWF and platelet-rich microthrombi that cause multiple organ failure and even death when left untreated. Besides its clear role in the pathophysiology of TTP, the anti-thrombotic and even anti-inflammatory properties of ADAMTS13 have also become apparent in various other thrombotic conditions. High VWF levels and low ADAMTS13 levels are associated with increased risk or even worse outcome of cardiovascular disease, including ischemic stroke and myocardial infarction. Preclinical studies in mouse models showed the beneficial effect of ADAMTS13 in both cerebral and myocardial ischemia/ reperfusion injury by decreasing both thrombosis and inflammation. In addition, ADAMTS13 was shown to also exert a direct thrombolytic effect on VWF-rich thrombi. In a mouse model of ischemic stroke, this thrombolytic activity resulted in efficient lysis of intracranial thrombi that were resistant to standard treatment with tissue plasminogen activator. Hence, ADAMTS13, as a therapeutic agent, could become an interesting avenue, not only to manage TTP, but also to treat other thrombotic complications. Disclosures De Meyer: Fonds voor Wetenschappelijk Onderzoek: Research Funding; KU Leuven: Employment, Research Funding; Ablynx: Consultancy, Research Funding; Cerenovus: Membership on an entity's Board of Directors or advisory committees; WhiteSwell: Consultancy.

Binding of Factor VIII to D'D3 and A2 Domains of Von Willebrand Factor Facilitates Mechanical Unraveling of the A2 Domain

Background: Proteolytic cleavage of von Willebrand factor (VWF) by a plasma metalloprotease ADAMTS13 is critical for normal hemostasis. Our previous studies demonstrate that binding of coagulation factor VIII (FVIII) through its light chain to VWF significantly accelerates the proteolytic cleavage of VWF by ADAMTS13 under fluidic shear. However, the mechanism underlying the FVIII-cofactor activity enhancing VWF proteolysis by ADAMTS13 under shear is not fully understood. Present study aims to test a hypothesis that FVIII binding to the D'D3 and/or other adjacent domains may facilitate a mechanic-induced conformational change in the central A2 domain of VWF, resulting in an increased accessibility and the cleavage of Tyr1605-Met1606 bond by ADAMTS13. Methods: Recombinant B-domainless FVIII and various VWF fragments (D'D3, D'D3A1, D'D3A1A2, and A2) with an Avi-His-tag on their N-termini and a Spy-tag on their C-termini were expressed and affinity-purified to homogeneity. Laser optical tweezers analysis determined the force-induced conformational changes of VWF fragments in the absence or presence of FVIII. Atomic force microscopy (AFM) and surface plasmon resonance (SPR) determined the binding events and kinetics between FVIII and various VWF domains. Results: Laser optical tweezers analysis demonstrated a Gaussian distribution of unfolding extension events upon pulling the single D'D3A1 fragment with the most probable extension distance of 22.8 ± 1.9 nm (mean ± SEM). Addition of 5 nM FVIII to the D'D3A1 or D'D3 fragment resulted in only a small decrease (~25%) or no change in the pulling extension profile. However, two peaks of Gaussian distribution were detected when pulling the D'D3A1A2 with one peak at ~18 nm and the other at ~40 nm. Addition of 5 nM FVIII to the D'D3A1A2 fragment, only one unfolding event was detected with the most probable unfolding extension distance of 24.5 ± 0.4 nm. Moreover, a single Gaussian distribution of unfolding extension profile was detected when pulling an isolated A2 fragment with the most probable unfolding extension of 31 ± 1 nm. Interestingly, addition of 5 nM FVIII resulted in a significant reduction of the most probable unfolding extension to 16.0 ± 0.4 nm, suggesting that FVIII may directly interact with A2 domain. AFM and SPR demonstrated the binding kinetics between FVIII and various VWF fragments. As shown, the intrinsic off-rate (k0) between FVIII and D'D3 was 0.19 ± 0.9 s-1 and the barrier width (Δx‡) between the naive state and the transition state was 1.5 ± 0.2 Å; the k0 between FVIII and D'D3A1A2 was 2.8 ± 1.0 s-1 and the barrier width (Δx‡) between the naive state and the transitional state was 1.7 ± 0.2 Å. Surprisingly, there was a detectable, but relatively weak binding between FVIII and A2 domain using AFM. SPR experiments confirmed the binding of FVIII to D'D3, D'D3A1A2, and A2 with the dissociation constant (KD) of ~2.9 nM, ~5.2 nM and ~1.6 nM, respectively. These results demonstrate that the specific interaction between FVIII and D'D3 or A2 domain. Conclusions: We have demonstrated for the first time using single-molecule force spectroscopy that binding of FVIII to the D'D3 and/or A2 domain of VWF may facilitate the force-induced conformational changes of the A2 domain, resulting in the enhanced proteolysis by ADAMTS13 under force. Our findings shed new light on how FVIII may regulate VWF proteolysis and function under (patho) physiological conditions. Disclosures Zheng: Shire/Takeda: Research Funding; Alexion: Speakers Bureau; Clotsolution: Other: Co-Founder; Ablynx/Sanofi: Consultancy, Speakers Bureau.

Crystal structure and substrate-induced activation of ADAMTS13

Platelet recruitment to sites of blood vessel damage is highly dependent upon von Willebrand factor (VWF). VWF platelet-tethering function is proteolytically regulated by the metalloprotease ADAMTS13. Proteolysis depends upon shear-induced conformational changes in VWF that reveal the A2 domain cleavage site. Multiple ADAMTS13 exosite interactions are involved in recognition of the unfolded A2 domain. Here we report through kinetic analyses that, in binding VWF, the ADAMTS13 cysteine-rich and spacer domain exosites bring enzyme and substrate into proximity. Thereafter, binding of the ADAMTS13 disintegrin-like domain exosite to VWF allosterically activates the adjacent metalloprotease domain to facilitate proteolysis. The crystal structure of the ADAMTS13 metalloprotease to spacer domains reveals that the metalloprotease domain exhibits a latent conformation in which the active-site cleft is occluded supporting the requirement for an allosteric change to enable accommodation of the substrate. Our data demonstrate that VWF functions as both the activating cofactor and substrate for ADAMTS13.