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BACKGROUND: Acquired von Willebrand syndrome (aVWS) is common in patients with mechanical circulatory support (MCS) devices. In these patients, the high shear stress in the device leads to increased shear-induced proteolysis of von Willebrand factor (VWF) by ADAMTS13 (A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, number 13). As a result, the high molecular weight (HMW) VWF multimers are lost, leading to a decreased VWF function and impaired haemostasis which could explain the bleeding complications that are frequently observed in these patients. To counteract this abnormal VWF degradation by ADAMTS13, we developed a novel targeted therapy, using an anti-ADAMTS13 monoclonal antibody (mAb) that inhibits the shear-induced proteolysis of VWF by ADAMTS13. METHODS: Human or bovine blood was circulated through in vitro MCS device systems with either inhibitory anti-ADAMTS13 mAb 3H9 or 17C7 (20 μg/mL) or control anti-ADAMTS13 mAb 5C11 or PBS. VWF multimers and function (collagen binding activity) were determined at different time points. Next, Impella® pumps were implanted in calves and the calves were injected with PBS and subsequently treated with mAb 17C7. VWF, ADAMTS13 and blood parameters were determined. RESULTS: We demonstrated that blocking ADAMTS13 could prevent the loss of HMW VWF multimers in in vitro MCS device systems. Importantly, our antibody could reverse aVWS in a preclinical Impella®-induced aVWS calf model. CONCLUSION: Hence, inhibition of ADAMTS13 could become a novel therapeutic strategy to manage aVWS in MCS device patients.

Recombinant ADAMTS13 is currently undergoing clinical trials as a treatment for hereditary thrombotic thrombocytopenic purpura, a lethal microvascular condition resulting from ADAMTS13 deficiency. Preclinical studies have also demonstrated its efficacy in treating arterial thrombosis and inflammation without causing bleeding, suggesting that recombinant ADAMTS13 may have broad applicability as an antithrombotic agent. Despite this progress, we currently do not understand the mechanisms that regulate ADAMTS13 activity in vivo. ADAMTS13 evades canonical means of protease regulation because it is secreted as an active enzyme and has a long half-life in circulation, suggesting that it is not inhibited by natural protease inhibitors. Although shear can spatially and temporally activate von Willebrand factor to capture circulating platelets, it is also required for cleavage by ADAMTS13. Therefore, spatial and temporal regulation of ADAMTS13 activity may be required to stabilize von Willebrand factor-platelet strings at sites of vascular injury. This review outlines potential mechanisms that regulate ADAMTS13 in vivo including shear-dependency, local inactivation, and biochemical and structural regulation of substrate binding. Recently published structural data of ADAMTS13 is discussed, which may help to generate novel hypotheses for future research.

BACKGROUND: Recombinant human ADAMTS13 (rADAMTS13) is a key protein in fundamental research for investigating its mode of action and the pathophysiology of thrombotic thrombocytopenic purpura (TTP). However, the expression of rADAMTS13 is quite low in mammalian cells, which makes the production of the protein time-consuming and labor-intensive. OBJECTIVES: We aimed at increasing the yield of rADAMTS13 by (1) using a more optimal signal peptide (SP) and (2) constructing an N-terminal fusion protein of ADAMTS13 with human serum albumin domain 1 (AD1-ADAMTS13). METHODS: Six SPs were investigated to select the most optimal SP. Expression plasmids containing the most optimal SP and ADAMTS13 cDNA or the fusion construct AD1-ADAMTS13 were generated and transiently transfected into CHOEBNALT85 cell-line. Expression levels of rADAMTS13 in expression medium were analyzed and compared with the expression level of rADAMTS13 with native SP (nat-SP). RESULTS: Expression of rADAMTS13 with coagulation factor VII (FVII) SP was 3-fold higher (16.00 μg/ml) compared with the expression with nat-SP (5.03 μg/ml). The highest yields were obtained with AD1-ADAMTS13 protein with a 15-fold higher concentration (78.22 μg/ml) compared with the expression with nat-SP. The rADAMTS13 expressed with FVII-SP retained its activity (104.0%) to cleave von Willebrand factor, whereas AD1-ADAMTS13 demonstrated even higher activity (144.3%). CONCLUSION: We succeeded in generating expression vectors that yield (1) rADAMTS13 at higher levels because of more optimal FVII-SP and (2) high levels of AD1-ADAMTS13 N-terminal fusion protein. The highest expression levels were obtained with AD1-ADAMTS13 N-terminal fusion protein, which is paving the way for highly efficient protein production. ispartof: JOURNAL OF THROMBOSIS AND HAEMOSTASIS vol:20 issue:10 pages:2379-2385 ispartof: location:England status: published

BACKGROUND: Patients with rheumatoid arthritis (RA) have frequent thrombotic events with endothelial dysfunction. Von Willebrand factor (VWF) has been shown to bind neutrophil extracellular traps (NETs) and NETs are part of RA etiology. OBJECTIVES: This study aims to elucidate whether this prothrombotic status exacerbates inflammation in arthritis. Here we focus on the involvement of A Disintegrin And Metalloprotease with ThromboSpondin type 1 motif, member 13 (ADAMTS-13), an enzyme cleaving VWF and its effect on NET deposition and RA development. METHODS: We evaluated the influence of the Adamts13 gene and recombinant human ADAMTS-13 (rhADAMTS-13) on arthritis in the mouse models of collagen-induced arthritis (CIA). We also assessed VWF and NETs in synovial tissue. RESULTS: Several Adamts13(−/−) mice developed arthritis, while Adamts13(+/+) siblings did not. Synovial tissue from Adamts13(−/−) showed accumulation of NETs. Treatment of DBA/1 J mice, an arthritis-susceptible strain, with well-tolerated doses of rhADAMT13 reduced arthritis incidence and alleviated the severity of arthritis. Mice treated with rhADAMT13 presented less serum interleukin 6 and less bone erosion determined by micro-computed tomography. The effects on arthritis severity were observed both when administering rhADAMTS-13 prophylactically and also when given after arthritis has developed. In both conditions, rhADAMTS-13 reduced VWF and NET deposition on proliferated synovial tissue evaluated by immunoblotting. CONCLUSIONS: Our results demonstrate the inhibitory role of Adamts13 in murine arthritis and the effectiveness of rhADAMTS-13 treatment. Additionally, this study suggests that deposition of VWF in the synovium and subsequent pathogenic NET retention promotes arthritis. Treatment with rhADAMTS-13 provides a potential therapeutic approach targeting inflammation and pro-thrombotic state in arthritis.

Prolonged recovery is common after acute SARS-CoV-2 infection; however, the pathophysiological mechanisms underpinning Long COVID syndrome remain unknown. VWF/ADAMTS-13 imbalance, dysregulated angiogenesis, and immunothrombosis are hallmarks of acute COVID-19. We hypothesized that VWF/ADAMTS-13 imbalance persists in convalescence together with endothelial cell (EC) activation and angiogenic disturbance. Additionally, we postulate that ongoing immune cell dysfunction may be linked to sustained EC and coagulation activation.Fifty patients were reviewed at a minimum of 6 weeks following acute COVID-19. ADAMTS-13, Weibel Palade Body (WPB) proteins, and angiogenesis-related proteins were assessed and clinical evaluation and immunophenotyping performed. Comparisons were made with healthy controls (n = 20) and acute COVID-19 patients (n = 36).ADAMTS-13 levels were reduced (p = 0.009) and the VWF-ADAMTS-13 ratio was increased in convalescence (p = 0.0004). Levels of platelet factor 4 (PF4), a putative protector of VWF, were also elevated (p = 0.0001). A non-significant increase in WPB proteins Angiopoietin-2 (Ang-2) and Osteoprotegerin (OPG) was observed in convalescent patients and WPB markers correlated with EC parameters. Enhanced expression of 21 angiogenesis-related proteins was observed in convalescent COVID-19. Finally, immunophenotyping revealed significantly elevated intermediate monocytes and activated CD4+ and CD8+ T cells in convalescence, which correlated with thrombin generation and endotheliopathy markers, respectively.Our data provide insights into sustained EC activation, dysregulated angiogenesis, and VWF/ADAMTS-13 axis imbalance in convalescent COVID-19. In keeping with the pivotal role of immunothrombosis in acute COVID-19, our findings support the hypothesis that abnormal T cell and monocyte populations may be important in the context of persistent EC activation and hemostatic dysfunction during convalescence.

Severe deficiency of plasma ADAMTS13 activity may result in potentially fatal thrombotic thrombocytopenic purpura and relative deficiency of plasma ADAMTS13 activity may be associated with adverse outcomes of certain malignancies. Here, we report the role of ADAMTS13 or lack of von Willebrand factor (VWF) in reducing irradiation and melanoma-induced thrombotic microangiopathy (TMA) and mortality in zebrafish.Zebrafish melanoma cell line (ZMEL) was injected subcutaneously into wild-type (wt), adamts13Total body irradiation at 30 Gy alone resulted in a transient thrombocytopenia in both wt and a13Our results indicated that plasma ADAMTS13 or lack of VWF may offer a significant protection against the development of irradiation- and/or melanoma-induced TMA. Such a microenvironment may directly affect melanoma cell phenotypes via alternation in the oxidation-reduction and lipid metabolic pathways.

Patients with essential thrombocythemia (ET) often experience bleeding associated with acquired von Willebrand syndrome (AVWS) when the platelet count is markedly increased.We investigated whether von Willebrand factor (VWF) degradation is enhanced in patients with ET.Seventy patients with ET underwent VWF multimer (VWFM) analysis and measurement of VWF-related parameters. We calculated the VWFM index, defined as the ratio of intensities of a patient's molecular weight-categorized VWFMs, and those of a healthy subject's, using densitometric analysis. VWF degradation product (DP) was measured via ELISA using a monoclonal antibody that specifically recognizes Y1605 at the C-terminal boundary, which is exposed following ADAMTS13-mediated cleavage of the Y1605-M1606 bond of the VWF A2 domain.Patients with higher platelet counts had a significantly reduced high molecular weight (HMW)-VWFM index and an increased VWF-DP:VWF antigen (Ag) ratio compared to those with lower platelet counts. On multivariate analysis, the VWF-DP/VWF:Ag ratio was an independent predictor of the HMW-VWFM index. Patients who underwent cytoreductive therapy had a significantly higher HMW-VWFM index and lower VWF-DP/VWF:Ag ratio than those who did not. Among individual patients, there was also a significant increase in the HMW-VWFM index and a decrease in the VWF-DP/VWF:Ag ratio after cytoreductive therapy compared to pre-therapy values.In patients with ET, an increased platelet count is associated with enhanced cleavage of VWF at the Y1605-M1606 bond, primarily by ADAMTS13, leading to AVWS. Cytoreductive therapy reduces the platelet count, prevents excessive VWF cleavage, and improves VWFM distributions.

The anti-von Willebrand factor (VWF) nanobody caplacizumab directly prevents the fatal microthrombi formation in immune-mediated thrombotic thrombocytopenic purpura (iTTP), thereby adding a new therapeutic principle to the treatment of this disorder. However, real-world treatment modalities beyond clinical trials remain heterogeneous.Here, we describe the risks and benefits of an alternate-day dosing regimen for caplacizumab by thoroughly analyzing the timing and outcome of this approach in a retrospective cohort of 25 iTTP patients treated with caplacizumab at seven different medical centers in Austria and Germany between 2018 and 2021.Alternate-day dosing of caplacizumab appeared feasible and led to persisting normal platelet counts in most patients. Five patients experienced iTTP exacerbations or relapses that led to the resumption of daily caplacizumab application. VWF activity was repeatedly measured in 16 of 25 patients and documented sufficient suppression by caplacizumab after 24 and 48 h in line with published pharmacodynamics.Extension of caplacizumab application intervals from daily to alternate-day dosing may be safely considered in selected patients after 3 to 4 weeks of daily treatment. Earlier modifications may be discussed in low-risk patients but require close monitoring for clinical and laboratory features of thrombotic microangiopathy.

Background: Endothelial activation has been proposed as a potential mechanism for the increased risk of venous thromboembolism (VTE) in human immunodeficiency virus (HIV)–infected pregnancy. Objectives: To assess the state of endothelial activation in HIV-infected pregnancy by measuring the von Willebrand factor (VWF) propeptide-to-antigen ratio, as an index of acute endothelial activation. Methods: VWF antigen and VWF propeptide were measured in HIV-negative participants (n = 85), HIV-infected virologically suppressed participants, (n = 89) and HIV-infected participants with HIV viral load (VL) of >50 copies/ml (n = 63) in each trimester. Results were correlated with multimer patterns and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13) antigen, activity, and antibody levels. Results: VWF propeptide-to-antigen ratio was increased, in the first, second, and third trimester, in the HIV-infected virologically suppressed group (1.7 ± 0.7, 1.7 ± 0.4, 1.6 ± 0.5) and the HIV-infected group with VL > 50 copies/ml (1.9 ± 0.9, 1.7 ± 0.9, 1.6 ± 1.1) compared to the HIV-negative group (1.4 ± 0.6, 1.3 ± 0.4, 1.2 ± 0.3, P

Background Consistent with fulminant endothelial cell activation, elevated plasma von Willebrand factor (VWF) antigen levels have been reported in patients with COVID‐19. The multimeric size and function of VWF are normally regulated through A Disintegrin And Metalloprotease with ThrombSpondin Motif type 1 motif, member 13 (ADAMTS‐13)‐‐mediated proteolysis. Objectives This study investigated the hypothesis that ADAMTS‐13 regulation of VWF multimer distribution may be impaired in severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2) infection contributing to the observed microvascular thrombosis. Patients and Methods Patients with COVID‐19 (n = 23) were recruited from the Beaumont Hospital Intensive Care Unit (ICU) in Dublin. Plasma VWF antigen, multimer distribution, ADAMTS‐13 activity, and known inhibitors thereof were assessed. Results We observed markedly increased VWF collagen‐binding activity in patients with severe COVID‐19 compared to controls (median 509.1 versus 94.3 IU/dl). Conversely, plasma ADAMTS‐13 activity was significantly reduced (median 68.2 IU/dl). In keeping with an increase in VWF:ADAMTS‐13 ratio, abnormalities in VWF multimer distribution were common in patients with COVID‐19, with reductions in high molecular weight VWF multimers. Terminal sialylation regulates VWF susceptibility to proteolysis by ADAMTS‐13 and other proteases. We observed that both N‐ and O‐linked sialylation were altered in severe COVID‐19. Furthermore, plasma levels of the ADAMTS‐13 inhibitors interleukin‐6, thrombospondin‐1, and platelet factor 4 were significantly elevated. Conclusions These findings support the hypothesis that SARS‐CoV‐2 is associated with profound quantitative and qualitative increases in plasma VWF levels, and a multifactorial down‐regulation in ADAMTS‐13 function. Further studies will be required to determine whether therapeutic interventions to correct ADAMTS‐13‐VWF multimer dysfunction may be useful in COVID‐microvascular thrombosis and angiopathy.

Background Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare, life‐threatening autoimmune thrombotic microangiopathy. Current standard of care is therapeutic plasma exchange, immunosuppression, and caplacizumab, an anti‐von Willebrand factor nanobody, which is effective in treating aTTP episodes. Patients/Methods Here we report on seven episodes of aTTP treated without plasma exchange in six female patients in Germany and Austria. Two episodes were initial presentations of aTTP; in five instances, patients experienced a relapse. In four episodes, moderate to severe organ dysfunction was observed; three cases presented with a mild course. All patients received caplacizumab immediately once aTTP was suspected or diagnosed, and plasma exchange was omitted based on shared decision making between patient and the treating physicians. Results We observed a rapid and robust increase of platelet counts already after the first dose of caplacizumab, leading to a doubling of platelet counts within 17 hours (median), platelet counts normalized (>150 G/L) after median 84 hours. Lactate dehydrogenase, as a surrogate parameter of organ damage, improved in parallel to the platelet counts, indicating resolving microangiopathy. Conclusions In conclusion, in selected cases of acute bouts of aTTP, it seems feasible to delay or omit plasma exchange if platelet counts increase and organ function is stable after start of caplacizumab therapy.

Background Despite an increase in our understandings of pathogenesis of thrombotic thrombocytopenic purpura (TTP), the approaches for initial diagnosis and management of TTP vary significantly. Objective The evidence-based guidelines of the International Society on Thrombosis and Haemostasis (ISTH) are intended to support patients, clinicians, and other health care professionals in their decisions about the initial diagnosis and management of acute TTP. Methods In June 2018, ISTH formed a multidisciplinary panel that included hematologists, an intensive care physician, nephrologist, clinical pathologist, biostatistician, and patient representatives, as well as a methodology team from McMaster University. The panel composition was designed to minimize the potential conflicts of interests. The panel used the Grading of Recommendations Assessment, Development, and Evaluation approach and the Population, Intervention, Comparison, Outcome framework to develop and grade their recommendations. Public comments were sought and incorporated in the final document. Results The panel agreed on three recommendations covering the initial diagnosis with emphasis on the importance of ADAMTS13 testing (eg, activity, anti-ADAMTS13 IgG or inhibitor) and assessment of the pretest probability of TTP by clinical assessment and/or the risk assessment models like the PLASMIC or French score. The panel noted how availability and turnaround time of ADAMTS13 test results might affect early diagnosis and management, in particular the use of caplacizumab. Conclusions There is a lack of high-quality evidence to support strong recommendations for the initial diagnosis and management of a suspected TTP. The panel emphasized the importance of obtaining ADAMTS13 testing in a proper clinical context. Future research should focus on how to monitor and act on ADAMTS13 levels during remission.

Background Despite advances in treatment options for thrombotic thrombocytopenic purpura (TTP), there are still limited high quality data to inform clinicians regarding its appropriate treatment. Methods In June 2018, the ISTH formed a multidisciplinary guideline panel to issue recommendations about treatment of TTP. The panel discussed 12 treatment questions related to immune-mediated TTP (iTTP) and hereditary or congenital TTP (cTTP). The panel used the Grading of Recommendations Assessment, Development, and Evaluation approach, including evidence-to-decision frameworks, to appraise evidence and formulate recommendations. Results The panel agreed on 11 recommendations based on evidence ranging from very low to moderate certainty. For first acute episode and relapses of iTTP, the panel made a strong recommendation for adding corticosteroids to therapeutic plasma exchange (TPE) and a conditional recommendation for adding rituximab and caplacizumab. For asymptomatic iTTP with low plasma ADAMTS13 activity, the panel made a conditional recommendation for the use of rituximab outside of pregnancy, but prophylactic TPE during pregnancy. For asymptomatic cTTP, the panel made a strong recommendation for prophylactic plasma infusion during pregnancy, and a conditional recommendation for plasma infusion or a wait and watch approach outside of pregnancy. Conclusions The panel's recommendations are based on all the available evidence for the effects of an individual component of various treatment approaches, including suppressing inflammation, blocking platelet clumping, replacing the missing and/or inhibited ADAMTS13, and suppressing the formation of ADAMTS13 autoantibody. There was insufficient evidence for further comparing different treatment approaches (eg, TPE, corticosteroids, rituximab, and caplacizumab, etc.), for which high quality studies are needed.

Background ADAMTS13, a plasma metalloprotease, cleaves von Willebrand factor (VWF) to regulate its function. Additionally, ADAMTS13 is thought to regulate lateral association of VWF multimers to form fibrillar structures through its free thiols. Objective The purpose of the present study is to obtain direct evidence for ADAMTS13 to engage in thiol/disulfide exchange reactions. Methods Covalent complexes between ADAMTS13 and VWF were determined by agarose gel electrophoresis under nonreducing conditions. Free thiols in ADAMST13 were identified by a reversed phase high-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry system. Results We demonstrate formation of covalent linkage between ADAMTS13 and VWF, which is time, concentration, temperature, and shear dependent. This interaction is independent of proteolytic activity of ADAMTS13 but depends on the C-terminal domains comprising the fifth through eighth thrombospondin type 1 repeats and C1r/C1s, Uegf, Bmp1 (CUB) domains. The interaction can be blocked by thiol-reactive agents, indicating that association is accomplished through disulfide bridge formation. Several partially reduced free thiols are identified in ADAMTS13, with cysteines 1254 and 1275 being the most prominent, although a point mutation (C1275S) in ADAMTS13 does not alter its ability to form covalent linkages with VWF. This suggests functionally relevant disulfide plasticity in ADAMTS13. Interestingly, ADAMTS13 also forms homo-oligomers under the same conditions as required for the generation of hetero-oligomeric complexes of ADAMTS13 and VWF. Conclusions Our results suggest that a dynamic network of free thiols in ADAMTS13 undergoing intra- and inter-molecular redox reactions may add another layer of regulation to VWF function under various conditions.

BACKGROUND: Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a lethal complication of severe malaria, characterized by marked pulmonary inflammation. Patient studies have suggested a link between von Willebrand factor (VWF) and malaria severity. OBJECTIVES: To investigate the role of VWF in the pathogenesis of experimental MA-ARDS. METHODS: Plasmodium berghei NK65-E (PbNK65) parasites were injected in Vwf+/+ and Vwf-/- mice. Pathological parameters were assessed following infection. RESULTS: In accordance with patients with severe malaria, plasma VWF levels were increased and ADAMTS13 activity levels were reduced in experimental MA-ARDS. ADAMTS13- and plasmin-independent reductions of high molecular weight VWF multimers were observed at the end stage of disease. Thrombocytopenia was VWF-independent because it was observed in both Vwf+/+ and Vwf-/- mice. Interestingly, Vwf-/- mice had a shorter survival time compared with Vwf+/+ controls following PbNK65 infection. Lung edema could not explain this shortened survival because alveolar protein levels in Vwf-/- mice were approximately two times lower than in Vwf+/+ controls. Parasite load, on the other hand, was significantly increased in Vwf-/- mice compared with Vwf+/+ mice in both peripheral blood and lung tissue. In addition, anemia was only observed in PbNK65-infected Vwf-/- mice. Of note, Vwf-/- mice presented with two times more reticulocytes, a preferential target of the parasites. CONCLUSIONS: This study suggests that parasite load together with malarial anemia, rather than alveolar leakage, might contribute to shortened survival in PbNK65-infected Vwf-/- mice. VWF deficiency is associated with early reticulocytosis following PbNK65 infection, which potentially explains the increase in parasite load. ispartof: JOURNAL OF THROMBOSIS AND HAEMOSTASIS vol:17 issue:8 pages:1372-1383 ispartof: location:England status: published

Background ADAMTS13 specifically cleaves the peptide bond between Y1605 and M1606 within the VWF-A2 domain. Objective The VWF contains ABO(H) blood group antigens, which may influence the susceptibility of VWF to ADAMTS13. Methods Using a unique monoclonal antibody recognizing the Y1605 residue, we have developed a sandwich ELISA to analyze the generation of a VWF-DP by ADAMTS13 quantitatively. Results Production of VWF-DP after exposure to four different degrees of high shear stress was validated in comparison to the reduction in high-molecular-weight multimers using VWF multimer analysis. In analysis of plasma from 259 healthy individuals, plasma levels of VWF antigen (VWF:Ag) were significantly lower in blood group O than in the other groups and were significantly correlated with plasma VWF-DP levels. The ratio between VWF-DP and VWF:Ag was significantly higher in blood group O than in blood groups A and AB. The ratio in blood group B was also significantly higher than those in A and AB, but did not differ from blood group O. Finally, to examine whether ABO(H) blood group antigens contributed to VWF cleavage, 82 plasma samples were exposed to high shear stress using a cone-plate shear stress inducer. The difference in the VWF-DP/VWF:Ag ratio before and after high shear stress in blood group O was significantly greater than those in groups A and AB. Conclusion These results indicate that blood group antigen A on VWF was more protective against ADAMTS13 cleavage than antigens B and H.

Background The catalytic domain of ADAMTS13 possesses one Zn2+ and up to three putative Ca2+ binding sites and can be inactivated by chelating agents. Although replenishment with an appropriate metallic cation is thought to restore the enzyme's proteolytic activity fully, ADAMTS13 stability in a metal ion-depleting environment has not been explored. Objectives To address the stability of ADAMTS13 in citrated human plasma. Methods ADAMTS13 activity was measured using the FRETS-VWF73 fluorogenic assay. The molar ratio of metals bound to ADAMTS13 was determined by size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS). Higher-order structural changes were analyzed using Fourier-transformed infrared spectroscopy and dynamic light scattering. Results ADAMTS13 was stable at room temperature for up to 24 hours irrespective of the presence of citrate (0.38%). However, at 37°C, citrate caused a time-dependent activity decrease. No ADAMTS13 activity decrease was seen in heparinized plasma, but the addition of citrate again caused ADAMTS13 instability at 37°C. Scavenging of citrate by the addition of Ca2+ or Zn2+ prior to but not postincubation prevented the activity decrease of the enzyme. The SEC-ICP-MS analyses showed that ADAMTS13 only bound Zn2+ and that its reduced activity correlated with a gradual loss of bound Zn2+ . Concomitant higher-order structural analyses demonstrated structural changes in ADAMTS13 that are typical of less-ordered protein structures. Conclusions Zn2+ is required to stabilize ADAMTS13 structure at physiologic temperature, thereby preventing irreversible loss of enzyme activity. This finding is particularly important to consider when using citrated human plasma as a source of ADAMTS13 in clinical settings.

Essentials Biological activity of human neutrophil peptide (HNP)-1 in hemostasis under physiological conditions is not fully understood. HNP-1 inhibits the adhesion/aggregation of murine platelets on a fibrillar collagen surface or an activated endothelial cell surface under flow. The anti-adhesion activity appears to depend on the terminal free thiols of HNP-1, which may inhibit VWF-VWF lateral associations. Our results suggest a protective role and potential novel therapeutic use of HNP-1 for arterial thrombosis. SUMMARY: Background Human neutrophil peptides (HNPs), also known as α-defensins, are released from degranulated neutrophils and play an important role in innate immunity. However, their biological roles in hemostasis under flow are not fully explored. Objective This study aims to determine the role of HNP-1 on platelet adhesion and aggregation on a collagen surface or ultra large von Willebrand factor (ULVWF) on endothelium under flow and elucidate the structural elements required for its activity. Methods Anticoagulated whole blood from wild-type or Adamts13-/- mice was incubated with a fluorescein-conjugated anti-human CD41 in the presence of increasing concentrations of a synthetic HNP-1 and perfused over a collagen surface or a tumor necrosis factor (TNF)-α activated murine endothelial cell surface under arterial flow. The rate of accumulation and the final surface coverage of fluoresceinated murine platelets or the rate of forming platelet-decorated ULVWF strings were determined using the BioFlux microfluidic system. Results HNP-1 inhibited the rate and final coverage of fluorescein-labeled murine platelets on a fibrillar collagen surface under flow (100 dyne/cm2 ) in a concentration-dependent manner and the anti-adhesive activity of HNP-1 depended on its terminal free cysteine thiols. HNP-1 (20 μM) also dramatically inhibited the formation of platelets-decorated ULVWF strings on TNF-α activated murine endothelial surface under arterial flow. Conclusions Our results demonstrate for the first time an antiplatelet adhesion or antithrombotic activity of HNP-1; this activity depends on its terminal free thiols, likely affecting VWF-VWF lateral associations. These findings may suggest a potential novel therapeutic strategy for arterial thrombosis.

Essentials Congenital thrombotic thrombocytopenic purpura (TTP) is primarily treated with plasma infusion. We present a pharmacokinetic analysis of ADAMTS-13 in six patients following plasma infusion. A median half-life of 130 h was demonstrated, ranging between 82.6 and 189.5 h. Investigation of interindividual clearance of ADAMTS-13 is necessary to optimize treatment. SUMMARY: Background Congenital thrombotic thrombocytopenic purpura (TTP) is defined by persistent severe deficiency of ADAMTS-13 in the absence of anti-ADAMTS-13 inhibitory antibodies, confirmed by mutational analysis. Replacement of the missing protease prevents disease relapse, primarily using plasma infusion (PI). Objectives, patients and methods There is scant evidence regarding optimal dose and frequency of treatment, which tends to be empirically guided. We present a pharmacokinetic analysis of ADAMTS-13 in six patients with congenital TTP on established regimes following PI. Results We found a median clearance of 25.41 mL h-1 and half-life of 130 h, ranging between 82.6 and 189.5 h (3.4-7.9 days, respectively). All patients reached baseline ADAMTS-13 level within 7-10 days post-plasma. Median ADAMTS-13 activity peak post-PI was 24.05 IU dL-1 . Variation was related to elimination rate, which, in turn, was affected by weight and metabolism, but not to von Willebrand factor antigen or activity levels. Using the pharmacokinetic parameters, we simulated individualized protocols based on PI dose or frequency to target hypothetical optimal plasma levels of ADAMTS-13 of 10 and 50 IU dL-1 , respectively. Results suggest a target trough ADAMTS-13 of 10 IU dL-1 is feasible but 50 IU dL-1 would not be achievable taking into account volume required. Conclusions Further work is needed to compare treatment of congenital TTP with PI vs. recombinant ADAMTS-13. PI may provide longer duration of ADAMTS-13 effect, but is limited by plasma volume required, whereas recombinant therapy can provide a higher ADAMTS-13 peak. We propose that investigation of interindividual clearance of ADAMTS-13 is necessary to optimize treatment and provide the rationale for dose and frequency of prophylaxis.

Essentials von Willebrand Factor (VWF) and ADAMTS13 may affect early injury after subarachnoid hemorrhage (SAH). Early brain injury was assessed in VWF−/−, ADAMTS13−/− and recombinant (r) ADAMTS13 treated mice. VWF−/− and rADAMTS13 treated mice had less brain injury than ADAMTS13−/− and wild-type mice. Early administration of rADAMTS13 may improve outcome after SAH by reducing early brain injury. Summary: Background Early brain injury is an important determinant of poor functional outcome and case fatality after aneurysmal subarachnoid hemorrhage (SAH), and is associated with early platelet aggregation. No treatment exists for early brain injury after SAH. We investigated whether von Willebrand factor (VWF) is involved in the pathogenesis of early brain injury, and whether ultra-early treatment with recombinant ADAMTS-13 (rADAMTS-13) reduces early brain injury after experimental SAH. Methods Experimental SAH in mice was induced by prechiasmatic injection of non-anticoagulated blood from a littermate. The following experimental SAH groups were investigated: C57BL/6J control (n = 21), VWF−/− (n = 25), ADAMTS-13−/− (n = 23), and C57BL/6J treated with rADAMTS-13 (n = 26). Mice were killed at 2 h after SAH. Primary outcome measures were microglial activation (IBA-1 surface area) and neuronal injury (number of cleaved caspase-3-positive neurons). Results As compared with controls, microglial activation was decreased in VWF−/− mice (mean difference of − 20.0%, 95% confidence interval [CI] − 4.0% to − 38.6%), increased in ADAMTS-13−/− mice (mean difference of + 34.0%, 95% CI 16.2–51.7%), and decreased in rADAMTS-13-treated mice (mean difference of − 22.1%, 95% CI − 3.4% to − 39.1%). As compared with controls (185 neurons, interquartile range [IQR] 133–353), neuronal injury in the cerebral cortex was decreased in VWF−/− mice (63 neurons, IQR 25–78), not changed in ADAMTS-13−/− mice (53 neurons, IQR 26–221), and reduced in rADAMTS-13-treated mice (45 neurons, IQR 9–115). Conclusions Our findings suggest that VWF is involved in the pathogenesis of early brain injury, and support the further study of rADAMTS-13 as a treatment option for early brain injury after SAH.

The interaction between platelets and the vessel wall is mediated by various receptors and adhesive proteins, of which von Willebrand factor (VWF) is the most prominent. The multimeric size of VWF is an important determinant of a more intense platelet-vessel wall interaction, and is regulated by the VWF-cleaving protease ADAMTS-13. A deficiency in ADAMTS-13 leads to higher concentrations of ultralarge VWF multimers and pathological platelet-vessel wall interactions, in its most typical and extreme form leading to thrombocytopenic thrombotic purpura, a thrombotic microangiopathy characterized by thrombocytopenia, non-immune hemolysis, and organ dysfunction. Thrombotic microangiopathy associated with low levels of ADAMTS-13 may be a component of the coagulopathy observed in patients with sepsis. Here, we review the potential role of ADAMTS-13 deficiency and ultralarge VWF multimers in sepsis, and their relationship with sepsis severity and prognosis. In addition, we discuss the possible benefit of restoring ADAMTS-13 levels or reducing the effect of ultralarge VWF as an adjunctive treatment in patients with sepsis.

SummaryBackground Thrombotic thrombocytopenic purpura (TTP) is caused by the absence of ADAMTS13 activity. Thrombocytopenia is presumably related to the formation of microthrombi rich in von Willebrand Factor (VWF) and platelets. Obesity may be a risk factor for TTP; it is associated with abundance of macrophages that may phagocytose platelets. Objectives To evaluate the role of obesity and ADAMTS13 deficiency in TTP, and to establish whether macrophages contribute to thrombocytopenia. Methods Lean or obese ADAMTS13 deficient (Adamts13-/-) and wild-type (WT) mice were injected with 250 U/kg of recombinant human VWF (rVWF), and TTP characteristics were evaluated 24h later. In separate experiments, macrophages were depleted in the liver and spleen of lean and obese WT or Adamts13-/- mice by injection of clodronate-liposomes, 48h before injection of rVWF. Results Obese Adamts13-/- mice had a lower platelet count than their lean counterparts suggesting that they might be more susceptible to TTP development. Lean Adamts13-/- mice triggered with a threshold dose of rVWF did not develop TTP, while typical TTP symptoms developed in obese Adamts13-/- mice, including severe thrombocytopenia and higher LDH levels. Removal of hepatic and splenic macrophages by clodronate injection in obese Adamts13-/- mice, before treatment with rVWF preserved the platelet counts, measured 24h after the trigger. In vitro experiments with cultured macrophages confirmed a VWF dose-dependent increase of platelet phagocytosis. Conclusions Obese Adamts13-/- mice are more susceptible to the induction of TTP-related thrombocytopenia than lean mice. Phagocytosis of platelets by macrophages contributes to thrombocytopenia after rVWF injection in this model. This article is protected by copyright. All rights reserved.

SummaryBackground The surgical repair of congenital heart lesions is frequently complicated by perioperative thrombosis of unclear etiology. An imbalance between von Willebrand Factor (VWF) and ADAMTS-13 is an emerging variable in thrombosis. Objectives To describe perioperative changes to VWF, ADAMTS-13 and NETosis and evaluate clinical and biochemical associations with postoperative thrombosis. Methods Neonates and infants undergoing palliation or definitive repair of congenital heart disease (CHD) were recruited (n =133). Pre- and postoperative plasma levels of VWF, ADAMTS-13 and markers of NETosis were determined. Patients were followed for up to 30 days for the occurrence of thrombosis. Univariate and multivariate logistic regression analyses were conducted to identify variables associated with thrombosis. Results We identified significant postoperative increases in VWF activity, VWF concentration, DNA-histone complexes and cell-free DNA with an overall decrease in ADAMTS-13 activity. Patients experiencing postoperative thrombotic events (9%) were characterized by surgery performed at lower intraoperative temperature, higher preoperative lactic acid levels and higher preoperative VWF activity and concentration. A multivariate logistic regression model identified preoperative VWF activity (odds ratio: 8.39 per IU/mL, 95% CI: 1.73-40.55) and transfusion of cryoprecipitate (odds ratio: 1.10 per mL/kg, 95% CI: 1.03-1.17) to be associated with thrombosis. Conclusions Pediatric patients undergoing surgical repair of congenital heart disease are exposed to high levels of VWF with diminished or minimal change to ADAMTS-13 in the immediate postoperative period. Elevated preoperative VWF activity is associated with postoperative thrombosis in pediatric CHD. This article is protected by copyright. All rights reserved.

Thrombotic thrombocytopenic purpura (TTP), a potentially fatal clinical syndrome, is primarily caused by autoantibodies against the von Willebrand factor (VWF)-cleaving metalloprotease ADAMTS13. In general, severe deficiency of plasma ADAMTS13 activity (20 IU/dL) in a similar clinical context supports an alternative diagnosis such as atypical hemolytic uremic syndrome (aHUS) or other types of TMA. Prompt differentiation of TTP from other causes of TMA is crucial for the initiation of an appropriate therapy to reduce morbidity and mortality. While plasma infusion is often sufficient for prophylaxis or treatment of hereditary TTP due to ADAMTS13 mutations, daily therapeutic plasma exchange remains the initial treatment of choice for acquired TTP with autoantibodies. Immunomodulatory therapies, including corticosteroids, rituximab, vincristine, cyclosporine, cyclophosphamide, and splenectomy, etc. should be considered to eliminate autoantibodies for sustained remission. Other emerging therapeutic modalities, including recombinant ADAMTS13, adeno-associated virus (AAV) 8-mediated gene therapy, platelet-delivered ADAMTS13, and antagonists targeting the interaction between platelet glycoprotein 1b and VWF are under investigation. This review highlights the recent progress in our understanding of the pathogenesis, diagnosis, and current and potential novel therapies for hereditary and acquired TTP.

Essentials The impact of N-linked glycosylation on ADAMTS-13 function has not been fully explored. The activity of glycan modified ADAMTS-13 was investigated under static and shear stress conditions. Terminal sialic acid on the metalloprotease domain glycans are important for ADAMTS-13 activity. The CUB domain glycans modulate ADAMTS-13 activity. SummaryBackground ADAMTS-13 activity can be regulated by its conformation, whereby interactions between the C-terminal CUB domains and the spacer domain maintain ADAMTS-13 in a closed conformation. ADAMTS-13 contains 10 N-linked glycans, with four sites present in theTSP2 through to CUB domains that may contribute to its conformation. Objectives/Methods We hypothesized that glycosylation contributes to ADAMTS-13 conformation and function. The proteolytic activity of glycan-modified ADAMTS-13 was assessed under static and shear stress conditions. Results Enzymatic removal of terminal silaic acid or entire N-linked glycan chains decreased activity against FRETS-VWF73 at pH 7.4 and against full-length von Willebrand factor (VWF) under shear stress. Using truncated ADAMTS-13, we demonstrated that this was attributable to loss of sialic acid from the glycans in the metalloprotease domain and an effect of N-linked glycosylation in the TSP2 through to CUB domains. Mutation of the N-linked glycan sites in the MDTCS domains reduced or abolished protein expression. However, the N707Q, N828Q, N1235Q and N1354Q (TSP2, TSP4, CUB1, and CUB2 domains, respectively) variants were expressed normally. Interestingly, the N707Q and N828Q variants showed reduced activity against FRETS-VWF73, but normal activity under flow conditions. In contrast, the N1235Q and N1354Q variants had enhanced activity against FRETS-VWF73 and VWF under shear stress. Immunoprecipitation experiments confirmed that loss of N-linked glycans in the CUB domains significantly reduced the interaction with the spacer domain and enhanced binding to the 6A6 anti-ADAMTS-13 antibody, which recognizes a cryptic epitope in the metalloprotease domain. Conclusions Together, these data demonstrate that the N-linked glycans of ADAMTS-13 play a crucial role in regulating ADAMTS-13 activity.

Essentials An association between ADAMTS-13 and coronary heart disease (CHD) has been suggested. 5688 participants ≥ 55 years from the Rotterdam Study without a history of CHD were included. Over a median follow-up time of 9.7 years, 456 individuals suffered from CHD. Low ADAMTS-13 activity was associated with an increased CHD risk.Background The metalloprotease ADAMTS-13 cleaves high-molecular-weight von Willebrand factor multimers into smaller, less procoagulant forms. Low ADAMTS-13 activity is associated with an increased risk of ischemic stroke but its pathogenic role in coronary heart disease (CHD) is unclear. Objectives We aimed to determine the association between ADAMTS-13 activity and the risk of CHD in a large prospective population-based cohort study. Methods A total of 5688 participants of the Rotterdam Study, a population-based cohort study involving individuals aged ≥ 55 years without a history of CHD, were included. ADAMTS-13 activity was measured by the FRETS-VWF73 assay and VWF:Ag levels by ELISA. We assessed the association between ADAMTS-13 activity, VWF:Ag levels and CHD using Cox proportional hazard regression analysis, adjusting for cardiovascular risk factors. Results Over a median follow-up time of 9.7 years, 456 individuals suffered from CHD. A low ADAMTS-13 activity (quartile 1) was associated with an increased CHD risk (HR 1.42, 95% CI 1.07-1.89) compared with the reference highest quartile. Conclusions Low ADAMTS-13 activity is associated with an increased risk of CHD in the elderly, independently of VWF and established cardiovascular risk factors.

Essentials The population prevalence of hereditary thrombotic thrombocytopenic purpura (TTP) is unknown. We studied the prevalence of hereditary TTP and population frequencies of two ADAMTS-13 mutations. A high frequency of hereditary TTP related to ADAMTS-13 mutation c.4143_4144dupA was found. Vicinity of ABO blood group and ADAMTS-13 loci may facilitate screening of ADAMTS-13 mutations.Background Hereditary thrombotic thrombocytopenic purpura (TTP) caused by ADAMTS-13 mutations is a rare, but serious condition. The prevalence is unknown, but it seems to be high in Norway. Objectives To identify all patients with hereditary TTP in central Norway and to investigate the prevalence of hereditary TTP and the population frequencies of two common ADAMTS-13 mutations. Patients/Methods Patients were identified in a cross-sectional study within the Central Norway Health Region by means of three different search strategies. Frequencies of ADAMTS-13 mutations, c.4143_4144dupA and c.3178 CT (p.R1060W), were investigated in a population-based cohort (500 alleles) and in healthy blood donors (2104 alleles) by taking advantage of the close neighborhood of the ADAMTS-13 and ABO blood group gene loci. The observed prevalence of hereditary TTP was compared with the rates of ADAMTS-13 mutation carriers in different geographical regions. Results We identified 11 families with hereditary TTP in central Norway during the 10-year study period. The prevalence of hereditary TTP in central Norway was 16.7 × 10(-6) persons. The most prevalent mutation was c.4143_4144dupA, accounting for two-thirds of disease causing alleles among patients and having an allelic frequency of 0.33% in the central, 0.10% in the western, and 0.04% in the southeastern Norwegian population. The allelic frequency of c.3178 CT (p.R1060W) in the population was even higher (0.3-1%), but this mutation was infrequent among patients, with no homozygous cases. Conclusions We found a high prevalence of hereditary TTP in central Norway and an apparently different penetrance of ADAMTS-13 mutations.

SummaryBackground Acquired thrombotic thrombocytopenic purpura (TTP) is caused by an autoantibody-mediated deficiency of the von Willebrand factor-cleaving protease ADAMTS-13. Acute episodes of the disease are treated with a combination of immunosuppression and repeated cycles of plasma exchange to remove anti-ADAMTS-13 autoantibodies and, at the same time, replenish functional ADAMTS-13. Although this is often effective, the mortality rate has remained between 10% and 20%, highlighting the need for safer treatment options. Objectives We previously showed that, in vitro, human recombinant ADAMTS-13 (rADAMTS-13) is able to override neutralizing antibodies and restore ADAMTS-13 activity in plasma from patients with acquired TTP. In the present study, we assessed the in vivo feasibility of this strategy by using a rat model. Methods Wild-type rats were adjusted to an ADAMTS-13 inhibitor (inhibitor) titer of ~ 10 BU mL−1 with goat anti-ADAMTS-13 IgG, and treated with increasing doses of rADAMTS-13. Blood samples were drawn and analyzed for ADAMTS-13-specific parameters, including FRETS-VWF73 activity, inhibitor, and ADAMTS-13-specific immune complexes (ICs). The pharmacokinetics of ADAMTS-13 activity and inhibitors were evaluated. Results Administration of inhibitor titer-adjusted doses of rADAMTS-13 to inhibitor-treated rats predictably restored activity. Inhibitors were readily neutralized through formation of ADAMTS-13-specific ICs, which were cleared at a higher rate than the free inhibitor. Surplus protease was enzymatically active in plasma, and showed similar pharmacokinetics to ADAMTS-13 in not inhibitor-treated rats. Conclusions Defined doses of rADAMTS-13 neutralized circulating anti-ADAMTS-13 antibodies and enabled reconstitution of ADAMTS-13 activity in plasma in our model, indicating that the protease may be a promising candidate for further exploration in treating acute episodes of acquired TTP.

Summary Background Recently, conformational activation of ADAMTS-13 was identified. This mechanism showed the evolution from a condensed conformation, in which the proximal MDTCS and distal T2-CUB2 domains are in close contact with each other, to an activated, open structure due to binding with von Willebrand factor (VWF). Objectives Identification of cryptic epitope/exosite exposure after conformational activation and of sites of flexibility in ADAMTS-13. Methods The activating effect of 25 anti-T2-CUB2 antibodies was studied in the FRETS-VWF73 and the vortex assay. Cryptic epitope/exosite exposure was determined with ELISA and VWF binding assay. The molecular basis for flexibility was hypothesized through rapid automatic detection and alignment of repeats (RADAR) analysis, tested with ELISA using deletion variants and visualized using electron microscopy. Results Eleven activating anti-ADAMTS-13 antibodies, directed against the T5-CUB2 domains, were identified in the FRETS-VWF73 assay. RADAR analysis identified three linker regions in the distal domains. Interestingly, identification of an antibody recognizing a cryptic epitope in the metalloprotease domain confirmed the contribution of these linker regions to conformational activation of the enzyme. The proof of flexibility around both the T2 and metalloprotease domains, as shown by by electron microscopy, further supported this contribution. In addition, cryptic epitope exposure was identified in the distal domains, because activating anti-T2-CUB2 antibodies increased the binding to folded VWF up to ~3-fold. Conclusion Conformational activation of ADAMTS-13 leads to cryptic epitope/exosite exposure in both proximal and distal domains, subsequently inducing increased activity. Furthermore, three linker regions in the distal domains are responsible for flexibility and enable the interaction between the proximal and the T8-CUB2 domains.

Summary Background Essential thrombocythemia (ET) is characterized by increased platelets and prevalent thrombosis. An acquired von Willebrand factor (VWF) disease has been hypothesized and inconsistently associated with extreme thrombocytosis or rare bleeding in ET. Whether VWF is modified in ET patients with controlled platelet count remains unclear. Objectives We studied different VWF- and platelet-associated parameters in ET patients treated according to current recommendations. Patients/Methods Sixty-nine ET patients (M = 29; median age, 62 [48–70] years; platelets, 432 [337–620] × 103 μL−1), 69 matched controls and 10 subjects with reactive thrombocytosis (RT) were studied. VWF:antigen (Ag), activity (act), electrophoretic patterns, VWF:propeptide, plasma glycocalycin (GC), glycoproteinV (GpV), ADAMTS-13, elastase, C-reactive protein and serum thromboxane (TX)B2 were measured. Results In ET patients, VWF:Ag was increased by 31 ± 13% vs. controls (P

Summary Background A common complication after aneurysmal subarachnoid hemorrhage (SAH) is delayed cerebral ischemia (DCI), which is associated with vasospasm and other mechanisms such as microthrombosis. ADAMTS-13 activity plays a role in the prevention of thrombus formation in the cerebral microvasculature. Previously, we observed that patients with DCI have lower levels of ADAMTS-13. Objectives To examine whether recombinant human ADAMTS-13 (rADAMTS-13) reduces cerebral microthrombus formation and brain injury in an experimental mouse model of SAH including wild-type and ADAMTS-13−/− mice. Methods Experimental SAH was induced with the prechiasmatic blood injection model. The following experimental groups were investigated: (i) C57BL/6J mice (n = 10); (ii) C57BL/6J mice (n = 10) treated with rADAMTS-13 20 min after SAH; (iii) ADAMTS-13−/− mice (n = 10); and (iv) ADAMTS-13−/− mice (n = 10) treated with rADAMTS-13 20 min after SAH. Mice were killed at 48 h. Results are presented as means with standard errors of the mean. Results Infusion with rADAMTS-13 reduced the extent of microthrombosis by ~ 50% in both wild-type mice (mean fibrinogen area: 0.28% ± 0.09% vs. 0.15% ± 0.04%; P = 0.20) and ADAMTS-13−/− mice (mean fibrinogen area: 0.32% ± 0.05% vs. 0.16% ± 0.03%; P = 0.016). In addition, rADAMTS-13 reduced brain injury by > 60% in both wild-type mice (mean microglia area: 0.65% ± 0.18% vs. 0.18% ± 0.04%; P = 0.013) and ADAMTS-13−/− mice (mean microglia area: 1.24% ± 0.36% vs. 0.42% ± 0.13%; P = 0.077). Conclusions Our results support the further study of rADAMTS-13 as a treatment option for the prevention of microthrombosis and brain injury after SAH.

Summary Background Microthrombosis and reactive inflammation contribute to neuronal injury after subarachnoid hemorrhage (SAH). ADAMTS-13 cleaves von Willebrand factor multimers, and inhibits thrombus formation and, seemingly, inflammatory reactions. Objective To investigate the effect of ADAMTS-13 in experimental SAH. Methods A total of 100 male C57/BL6 mice were randomly assigned to four groups: sham (n = 15), SAH (n = 27), vehicle (n = 25), and ADAMTS-13 (n = 23; 100 μL per 10 g of body weight of 100 μg of ADAMTS-13 per 1 mL of 0.9% NaCl; 20 min after SAH). Neurologic performance was assessed on days 1 and 2 after SAH. Animals were killed on day 2. The amounts of subarachnoid blood, microthrombi, apoptosis and degenerative neurons were compared. The degree of neuronal inflammation and vasospasm was also compared. In five mice each (SAH and ADAMTS-13 groups), bleeding time was assessed 2 h after SAH. Results Systemic administration of ADAMTS-13 achieved significant amelioration of microthrombosis and improvement in neurologic performance. ADAMTS-13 reduced the amount of apoptotic and degenerative neurons. A tendency for decreased neuronal inflammation was observed. ADAMTS-13 did not show any significant effect on vasospasm. The degree of systemic inflammation was not changed by ADAMTS-13 administration. ADAMTS-13 neither increased the amount of subarachnoid blood nor prolonged the bleeding time. Conclusions ADAMTS-13 may reduce neuronal injury after SAH by reducing microthrombosis formation and neuronal inflammation, thereby providing a new option for mitigating the severity of neuronal injury after SAH.

Summary Background An East Asian-specific P475S polymorphism in the gene encoding ADAMTS-13 causes an approximately 16% reduction in plasma ADAMTS-13 activity. Objectives To demonstrate the impact of this dysfunctional polymorphism by characterizing the structure and activity of the P475S mutant protein. Methods We determined the crystal structure of the P475S mutant of ADAMTS-13-DTCS (DTCS-P475S, residues 287–685) and compared it with the wild-type structure. We determined the enzymatic parameters of ADAMTS-13-MDTCS (residues 75–685) and MDTCS-P475S, and further examined the effects of denaturants and reaction temperature on their activity. We also examined the cleavage of shear-treated von Willebrand factor (VWF) by MDTCS-P475S. Results MDTCS-P475S showed a reaction rate similar to that of wild-type MDTCS, but showed two-fold lower affinity for the peptidyl substrate, indicating that the Pro475-containing V-loop (residues 474–481) in the CA domain is a substrate-binding exosite. Structural analysis showed that the conformation of the V-loop was significantly different in DTCS-P475S and the wild type, where no obvious interactions of Ser475 with other residues were observed. This explains the higher susceptibility of the enzymatic activity of MDTCS-P475S to reaction environments such as denaturants and high temperature. MDTCS-P475S can moderately cleave shear-treated VWF. Conclusions We have provided structural evidence that the P475S polymorphism in ADAMTS-13 leads to increased local structural instability, resulting in lowered affinity for the substrate without changing the reaction rate. The moderate activity of ADAMTS-13-P475S for shear-treated VWF is sufficient to prevent thrombotic thrombocytopenic purpura (TTP) onset.

ADAMTS-13, a plasma reprolysin-like metalloprotease, cleaves von Willebrand factor (VWF). Severe deficiency of plasma ADAMTS-13 activity results in thrombotic thrombocytopenic purpura (TTP), while mild to moderate deficiencies of plasma ADAMTS-13 activity are emerging risk factors for developing myocardial and cerebral infarction, pre-eclampsia, and malignant malaria. Moreover, Adamts13(-/-) mice develop more severe inflammatory responses, leading to increased ischemia/perfusion injury and formation of atherosclerosis. Structure-function studies demonstrate that the N-terminal portion of ADAMTS-13 (MDTCS) is necessary and sufficient for proteolytic cleavage of VWF under various conditions and attenuation of arterial/venous thrombosis after oxidative injury. The more distal portion of ADAMTS-13 (TSP1 2-8 repeats and CUB domains) may function as a disulfide bond reductase to prevent an elongation of ultra-large VWF strings on activated endothelial cells and inhibit platelet adhesion/aggregation on collagen surface under flow. Remarkably, the proteolytic cleavage of VWF by ADAMTS-13 is accelerated by FVIII and platelets under fluid shear stress. A disruption of the interactions between FVIII (or platelet glycoprotein 1bα) and VWF dramatically impairs ADAMTS-13-dependent proteolysis of VWF in vitro and in vivo. These results suggest that FVIII and platelets may be physiological cofactors regulating VWF proteolysis. Finally, the structure-function and autoantibody mapping studies allow us to identify an ADAMTS-13 variant with increased specific activity but reduced inhibition by autoantibodies in patients with acquired TTP. Together, these findings provide novel insight into the mechanism of VWF proteolysis and tools for the therapy of acquired TTP and perhaps other arterial thrombotic disorders.

SummaryBackground Rituximab has been documented in the treatment of acute (≤ 3 days from admission), relapsed/refractory thrombotic thrombocytopenic purpura (TTP) and given as prophylaxis in selected cases to prevent acute relapse. The precise timing of rituximab in acute TTP has not been determined. Objective To perform a retrospective analysis of rituximab use in a large TTP referral center over an 8-year period. Patients/Methods We assessed response to treatment and outcome for all patients treated with rituximab, including 91 patients presenting with 104 episodes of acute TTP and 15 patients given rituximab as prophylaxis to prevent relapse. In the acute TTP group we assessed the benefit of giving early (≤ 3 days from admission) vs. later (> 3 days) rituximab. Results In acute de novo TTP, previously untreated with rituximab, rituximab was given ≤ 3 days from admission to 54 patients and > 3 days from admission to 32 patients. Earlier administration (≤ 3 days) was associated with faster attainment of remission (12 vs. 20 days, P

Background: ADAMTS13 mutations play a role in thrombotic thrombocytopenic purpura (TTP) pathogenesis. Objectives: To establish a phenotype-genotype correlation in a cohort of congenital TTP patients. Patients/Methods: Clinical history and ADAMTS13 activity, antigen and anti-ADAMTS13 antibody assays were used to diagnose congenital TTP, and DNA sequencing and in vitro expression were performed to identify the functional effects of the ADAMTS13 mutations responsible. Results: Seventeen (11 novel) ADAMTS13 mutations were identified in 17 congenital TTP patients. All had severely reduced ADAMTS13 activity and antigen levels at presentation. Six patients with pregnancy-associated TTP and six patients with childhood TTP were homozygous or compound heterozygous for ADAMTS13 mutations located in the metalloprotease (MP), cysteine-rich, spacer and/or distal thrombospondin type 1 domains. The adults had TTP precipitated by pregnancy, and had overall higher antigen levels (median, 30ngmL-1; range

Summary. Background: ADAMTS13 cleaves hyperactive ultra-large von Willebrand factor (ULVWF) multimers into smaller and less active forms. It remains unknown whether VWF-mediated inflammatory processes play a role in the enhanced brain injury due to ADAMTS13 deficiency. Objective: We tested the hypothesis that the deleterious effect of ADAMTS13 deficiency on ischemic brain injury is mediated through VWF-dependent enhanced vascular inflammation. Methods: Transient focal cerebral ischemia was induced by 60 min of occlusion of the right middle cerebral artery. Myeloperoxidase (MPO) activity and inflammatory cytokines in the infarcted region were evaluated 23 h after reperfusion injury. Neutrophil infiltration within the infarct and surrounding areas was quantitated by immunohistochemistry. Results: We report that ADAMTS13-deficient mice exhibited significantly enlarged infarct size, concordant with increased myeloperoxidase (MPO) activity, neutrophil infiltration and expression of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In contrast, VWF-deficient mice exhibited significantly reduced MPO activity, neutrophil infiltration and inflammatory cytokine induction, demonstrating a role of VWF in these inflammatory processes. Mice deficient for both ADAMTS13 and VWF exhibited an identical reduction of the same inflammatory parameters, demonstrating that the increased inflammation observed in ADAMTS13-deficient mice is VWF dependent. Finally, the increased infarct size observed in ADAMTS13-deficient mice was completely abrogated by prior immunodepletion of neutrophils, demonstrating a causal role for acute inflammation in the enhanced brain injury that occurs in the setting of ADAMTS13 deficiency. Conclusion: These findings provide new evidence for ADAMTS13 in reducing VWF-mediated acute cerebral inflammation following ischemic stroke.

Summary. Background: Ultra-large von Willebrand factor and deficiency of its cleaving protease are important factors in the events leading to thrombotic microangiopathy; however, the mechanisms involved are only partly understood. Whereas pathological activation of the alternative complement pathway is linked to atypical hemolytic uremic syndrome, the role of complement activation in thrombotic thrombocytopenic purpura (TTP) is unknown. The aim of this study was to investigate whether signs of complement activation are characteristic of TTP. Patients and methods: Twenty-three patients with TTP (18 women, median age 38 years) and 17 healthy controls (13 women, median age 38 years) were included. Complement parameters (C3, Factors H, I, B and total alternative pathway activity) together with complement activation fragments (C3a) or complexes (C1rs-INH, C3bBbP, sC5b9) were measured by ELISA or RID. ADAMTS13 activity and anti-ADAMTS13 inhibitory antibodies were measured by the VWF-FRET73 assay. Results: Increased levels of C3a, and SC5b9 were observed in TTP during acute episodes, as compared with healthy controls. Decreased complement C3 levels indicative of complement consumption occurred in 15% of acute TTP patients. Significant decrease of complement activation products C3a and SC5b9 was observed during plasma exchange (PEX). The sustained presence of anti-ADAMTS13 inhibitory antibodies in complete remission was associated with increased complement activation. Conclusion: These data document in an observational study the presence of complement activation in TTP. Further investigation is needed to determine its potential pathogenetic significance.

Moreira et al describe a fascinating case of a five-year-old who developed microangiopathic hemolytic anemia (MAHA) a week after presentation with idiopathic systemic capillary leak syndrome (SCLS) and propose ADAMTS-13 loss in the third-space fluid as the cause of MAHA [1]. It would however be useful if the authors could provide the values of vascular endothelial growth factor (VEGF) and von willebrand factor (VWF) levels in this patient during the illness episode before this conclusion is made. Endothelial dysfunction is well-known to cause very high levels of both VEGF and VWF, to the extent in many experimental studies; these markers are used as a marker for endothelial activation and altered function [2, 3]. This article is protected by copyright. All rights reserved.

Summary. Background: The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentified. Methods: Seven C-terminally truncated murine ADAMTS13 (mADAMTS13) mutants were constructed and characterized in vitro. Their ability to cleave VWF strings in vivo was studied in the ADAMTS13 )/) mouse. Results: Murine MDTCS (devoid of T2-8 and CUB domains) retained full enzyme activity in vitro towards FRETS-VWF73 and the C-terminal T6-8 (del(T6CUB)) and CUB domains (delCUB) are dispensable under these assay conditions. In addition, mADAMTS13 fragments without the spacer domain (MDT and M) had reduced catalytic efficiencies. Our results hence indicate that similar domains in murine and human ADAMTS13 are required for activity in vitro, supporting the use of mouse models to study ADAMTS13 function in vivo. Interestingly, using intravital microscopy we show that removal of the CUB domains abolishes proteolysis of platelet-decorated VWF strings in vivo. In addition, whereas MDTCS is fully active in vivo, partial (del(T6-CUB)) or complete (delCUB) addition of the T2-8 domains gradually attenuates its activity. Conclusions: Our data demonstrate that the ADAMTS13 CUB and T2-8 domains influence proteolysis of platelet-decorated VWF strings in vivo.

Summary. Background: ADAMTS-13 proteolytic activity is controlled by the conformation of its substrate, von Willebrand factor (VWF), and changes in the secondary structure of VWF are essential for efficient cleavage. Substrate recognition is mediated through several non-catalytic domains in ADAMTS-13 distant from the active site. Objectives: We hypothesized that not all binding sites for ADAMTS-13 in VWF are cryptic and analyzed binding of native VWF to ADAMTS-13. Methods: Immunoprecipiation of VWF–ADAMTS-13 complexes using anti-VWF antibodies and magnetic beads was used. Binding was assessed by Western blotting and immunosorbent assays. Results: Co-immunoprecipitation demonstrated that ADAMTS-13 binds to native multimeric VWF (Kd of 79 ± 11 nmol L−1) with no measurable proteolysis. Upon shear-induced unfolding of VWF, binding increased 3-fold and VWF was cleaved. Binding to native VWF was saturable, time dependent, reversible and did not vary with ionic strength (I of 50–200). Moreover, results with ADAMTS-13 deletion mutants indicated that binding to native VWF is mediated through domains distal to the ADAMTS-13 spacer, probably thrombospondin-1 repeats. Interestingly, this interaction occurs in normal human plasma with an ADAMTS-13 to VWF stoichiometry of 0.0040 ± 0.0004 (mean ± SEM, n = 10). Conclusions: ADAMTS-13 binds to circulating VWF and may therefore be incorporated into a platelet-rich thrombus, where it can immediately cleave VWF that is unfolded by fluid shear stress.

BACKGROUND: ADAMTS13-neutralizing IgG autoantibodies are the major cause of acquired thrombotic thrombocytopenic purpura (TTP). OBJECTIVE: To analyze the IgG subclass distribution of anti-ADAMTS13 antibodies and a potential relationship between subclass distribution and disease prognosis. METHODOLOGY: An enzyme-linked immunosorbent assay-based method was used to quantify the relative amounts of IgG subclasses of anti-ADAMTS13 antibodies in acquired TTP plasma. RESULTS: IgG(4) (52/58, 90%) was the most prevalent IgG subclass in patients with acquired TTP, followed by IgG(1) (52%), IgG(2) (50%), and IgG(3) (33%). IgG(4) was found either alone (17/52) or with other IgG subclasses (35/52). IgG(4) was not detected in 10% of the patients. There was an inverse correlation between the frequency and abundance of IgG(4) and IgG(1) antibodies (P < 0.01). Patients with high IgG(4) levels and undetectable IgG(1) are more prone to relapse than patients with low IgG(4) levels and detectable IgG(1). CONCLUSIONS: All IgG subclasses of anti-ADAMTS13 antibodies were detected in patients with acquired TTP, with IgG(4), followed by IgG(1), antibodies dominating the anti-ADAMTS13 immune response. Levels of IgG(4) could be useful for the identification of patients at risk of disease recurrence.

Summary. Background: Functional deficiency of ADAMTS13 in thrombotic thrombocytopenic purpura (TTP) patients is associated with circulating ultralarge von Willebrand factor (VWF) molecules that display spontaneous platelet-binding capacities. Upon remission, however, ADAMTS13 activity does not always return to baseline. Objective: To study ADAMTS13 and VWF-related features in TTP patients in remission. Methods: ADAMTS13 activity, anti-ADAMTS13 antibodies, VWF antigen, ultralarge VWF and levels of VWF that circulate in a glycoprotein Ibα-binding conformation were determined in plasma samples of 22 acquired TTP patients in remission between 1 month and 6 years after achieving remission. The composition of active multimers was investigated with a novel immunoprecipitation assay based on monoclonal antibody AU/VWF-a12, which specifically recognizes the active conformation of VWF. Results: ADAMTS13 activity was undetectable in 23% of the patients, even years after they had achieved remission, and lack of ADAMTS13 activity was associated with increased active VWF levels and the presence of ultralarge VWF multimers. Active VWF levels and ultralarge VWF were also associated with blood groups. Results from immunoprecipitation experiments revealed the full range of multimers to be present. Conclusion: ADAMTS13 deficiency and the concurrent presence of ultralarge VWF and increased active VWF levels can be detected in TTP patients for years after they have achieved remission. Immunoprecipitation results suggest that the active conformation of VWF may be present in the lower molecular weight multimers, but future studies are necessary to confirm our findings.

International audience; Summary. Background: During sepsis, von Willebrand factor (VWF) is abundantly secreted; the main mechanism regulating its size involves specific proteolysis by the metalloprotease ADAMTS-13. Objectives: To determine whether ADAMTS-13 consumption due to its binding to, and/or cleavage, of VWF contributes to its decrease during sepsis and whether abrogating or enhancing ADAMTS-13 activity influences sepsis outcome. Methods: ADAMTS-13 activity was evaluated in a model of sepsis induced by cecum ligature and puncture (CLP) in wild-type and Vwf−/− mice. Sepsis outcome was studied in those mice and in Adamts-13−/− mice. Finally, survival was studied in wild-type mice injected hydrodynamically with the human ADAMTS-13 gene. Results: In wild-type mice, CLP-induced sepsis elicited a significant ADAMTS-13 decrease, and a strong negative correlation existed between VWF and ADAMTS-13. In Vwf−/− mice, CLP also induced severe sepsis, but ADAMTS-13 was not significantly diminished. Notably, Vwf−/− mice lived significantly longer than wild-type mice. In contrast, Adamts-13−/− mice and wild-type mice were comparable with regard to thrombocytopenia, VWF concentrations, absence of thrombi, and survival. Hydrodynamic hADAMTS-13 gene transfer with the pLIVE expression vector resulted in high and stable ADAMTS13 activity in CLP mice; however, no impact on survival was observed. Conclusions: VWF secretion is a major determinant of ADAMTS-13 decrease in the CLP model, and plays an important role in sepsis-induced mortality, but the complete absence of its regulating protease, ADAMTS-13, had no detectable impact in this sepsis model. Furthermore, increasing ADAMTS-13 activity had no impact on survival.

Evidence for cardiac involvement in thrombotic thrombocytopenic purpura (TTP) is uncommonly described.We retrospectively reviewed 41 patients assessing troponin T as a marker for cardiac involvement in acute TTP with clinical symptoms, electrocardiograms (ECG) and echocardiograms. A histopathological review of five patients who died of acute TTP was also undertaken.In 54% (22/41) of patients, troponin T wasor=0.05microg L(-1) (normal range 0-0.01 microg L(-1)). Half (12/22) had cardiac symptoms and 8/22 with a raised troponin T reported chest pain. ECG changes were present in 62% of patients with a raised troponin T. Median anti-ADAMTS 13 IgG antibody was significantly higher (P=0.018) in patients with troponin Tor=0.05 microg L(-1) (58.5% (range 17-162%), compared with patients with troponin T0.05 microg L(-1) (35%, range 9-134%). Patients who died had higher troponin T levels (median 0.305 microg L(-1)) and raised anti-ADAMTS 13 IgG (median 66.5%). On admission, there were no deaths in those with troponin Tor=0.04microg L(-1). Histology confirmed widespread myocardial microvascular thrombi.Clinical symptoms, ECG changes and echocardiograms are poor predictors of cardiac disease in acute TTP. Troponin T is specific for cardiac muscle and a sensitive marker of myocardial damage. In TTP patients, raised levels (or=0.05 microg L(-1)) signify myocardial necrosis associated with microvascular thrombi. Mortality and acute morbidity was associated with higher admission troponin T and raised IgG antibody (67%) to ADAMTS 13.

Summary. Background: Autoantibodies directed towards ADAMTS13 are present in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). Analysis of a set of antibodies derived from two patients with acquired TTP revealed frequent use of the VH1-69 heavy chain gene segment for the assembly of anti-ADAMTS13 antibodies. Objective: We explored the ability of two VH1-69 germline gene-encoded antibodies to inhibit the von Willebrand factor (VWF)-processing activity of ADAMTS13 under different experimental conditions. Furthermore, the presence of VH1-69 encoded anti-ADAMTS13 antibodies in 40 patients with acquired TTP was monitored using monoclonal antibody G8, which specifically reacts with an idiotype expressed on VH1-69 encoded antibodies. Methods and Results: Binding of the two VH1-69 encoded monoclonal antibodies was dependent on the presence of the spacer domain. Both antibodies inhibited ADAMTS13 activity under static conditions, as measured by cleavage of FRETS-VWF73 substrate and cleavage of VWF multimers. The recombinant antibodies were also capable of inhibiting the processing of UL-VWF strings on the surface of endothelial cells. G8-reactive antibodies directed towards ADAMTS13 were present in plasma of all patients containing anti ADAMTS13 antibodies. Conclusions: These results suggest that VH1-69 derived antibodies directed towards ADAMTS13 develop in the majority of patients with acquired TTP.