ADAMTS13 Antigen Research Articles

Clinical Utility of Recently FDA Approved IntelliSep test (Sepsis biomarker) for early Diagnosis of Sepsis: Comparison with other Biomarkers and von Willebrand Factor/ADAMTS13 Ration

Abstract Background Sepsis, a relatively common presentation in emergency departments, is a life-threatening disease. Recently, IntelliSep test by Cytovale (San Francisco, CA) received FDA approval as sepsis biomarker. We investigated clinical utility of this test using 44 patients (19 disease group and 25 non-disease group). Materials and Methods IntelliSep assesses cellular host response via deformability cytometry of leukocyte biophysical properties and intended for use in conjunction with clinical assessments and laboratory findings for early detection of sepsis. Test results are available within 10 minutes. Results consists of IntelliSep Band 1 (low risk, score: 0.1-4.9) to Band 3 (high risk, score: 6.3-10; total scale: 0.1-10). We measured IntelliSep score in 44 patients using EDTA blood. Left over plasma was used for measuring both plasma von Willebrand factor (vWF) antigen and ADAMTS13 antigen by ELISA assays. Monocyte distribution width (MDW) were obtained during routine CBC analysis using Beckman hematology analyzer. Lactate and high sensitivity troponin I were measured using Beckman analyzer. Results The median IntelliSep score was two-fold higher in the disease group (mean score 7.1, SD: 1.5, median: 7.4) compared to non-disease group (mean score 4.0, SD: 1.4, Median: 3.7) indicating that this new test can identify patients with high risk of developing sepsis and also rule out low risk patients (p<0.01 by both Mann-Whitney U test two tailed and t-test two tailed). The median MDW was also 34.8 % higher in the disease group (mean:25.6, SD: 5.0, Median: 24.4) compared to non-disease group (mean: 18.9, SD: 2.4, Median: 18.1) and difference was also statistically significant (p < 0.01 by both Mann-Whitney U test and t-test, two tailed). However, correlation between IntelliSep scores and MDW was only modest (overall, r= 0.66). Both lactate and procalcitonin concentrations were significantly higher in the disease group compared to the non-disease group. Interestingly, high-sensitivity troponin I concentrations were also significantly higher in the disease group. In addition, we observed significantly higher plasma vWF antigen in the disease group compared to the non-disease group (mean: 67.4 ug/mL in disease group vs 34.1 ug/mL in the non-disease group. However, in some patients in the disease group plasma vWF values were 6.8-fold higher than normal. In addition, vWF/ADAMTS13 ratio was also significantly elevated in the disease group compared to non-disease group. Discussion New IntelliSep test is useful in early prediction of sepsis in patients admitted to emergency department and may be superior to MDW and lactate for early diagnosis. Significant increase in plasma vWF in these patients indicate that vWF may also be a surrogate marker for endotheliopathy in sepsis. Lack of correlation between plasma VWF and Intellisep results indicates that these two markers probably measure different pathways for sepsis (endothelial activation vs neutrophil activation).

Clinical Utility of Recently Food and Drug Administration-Approved IntelliSep Test (Sepsis Biomarker) for Early Diagnosis of Sepsis: Comparison with Other Biomarkers.

Context: IntelliSep by Cytovale has received United States (U.S.) Food and Drug Administration (FDA) approval as a sepsis biomarker test. However, the clinical utility of this new test is not assessed in emergency departments. Objective: We investigated the clinical utility of this test using 44 patients visiting the emergency department at The University of Kansas Medical Center by comparing it with the monocyte distribution width (MDW) and other biomarkers including the von Willebrand factor (vWF) and ADAMTS13. Design and Methods: IntelliSep assesses the cellular host response via deformability cytometry of biophysical leukocyte properties and produces a score (IntelliSep Index; ISI: from 0.1 (lowest risk) to 10 (highest risk). We measured the ISI in 44 patients (19 high probability and 25 low probability of sepsis groups) using EDTA-anticoagulated blood. Left over plasma was used for measuring the plasma von Willebrand factor (vWF) and ADAMTS13 antigen by ELISA assays. The MDW was obtained during routine CBC analysis using a Beckman hematology analyzer. The lactate and high-sensitivity troponin I levels were measured using a Beckman analyzer. Procalcitonin was measured using a Cobas e801 analyzer. Results: The median ISI was twofold higher in the high-probability group than in the low-probability group (p < 0.01) while the median MDW was 34.5% higher in the high-probability group than in the low-probability group (p < 0.01). However, the correlation between the ISI and MDW was only modest (r = 0.66). In addition, significantly higher levels of plasma vWF antigen but lower levels of plasma ADAMTS13 antigen in the high-probability group were found, resulting in significantly higher vWF/ADAMTS13 ratios in the high-probability group than in the low-probability group. Conclusions: The new IntelliSep test along with vWF/ADAMTS13 ratios may be useful for the early diagnosis of sepsis in patients visiting the emergency department, which appears to be superior to the traditional marker, MDW.

Diagnosis of thrombotic thrombocytopenic purpura: easy-to-use fiber optic surface plasmon resonance immunoassays for automated ADAMTS-13 antigen and conformation evaluation

BackgroundLaboratory diagnosis of immune-mediated thrombotic thrombocytopenic purpura (iTTP) remains challenging when ADAMTS-13 activity ranges between 10% and 20%. To prevent misdiagnosis, open ADAMTS-13 conformation gained clinical attention as a novel biomarker, especially to diagnose acute iTTP in patients with diagnostic undecisive ADAMTS-13 activity. Plasma ADAMTS-13 conformation analysis corrects for ADAMTS-13 antigen, with both parameters being characterized in enzyme-linked immunosorbent assay (ELISA)-based reference assays requiring expert technicians. ObjectivesTo design ADAMTS-13 antigen and conformation assays on automated, easy-to-use fiber optic surface plasmon resonance (FO-SPR) technology to promote assay accessibility and diagnose challenging iTTP patients. MethodsADAMTS-13 antigen and conformation assays were designed on FO-SPR technology. Plasma of 20 healthy donors and 20 acute iTTP patients were quantified, and data from FO-SPR and ELISA reference assays were compared. ResultsFollowing assay design, both antigen and conformation FO-SPR assays were optimized and characterized, presenting strong analytical sensitivity (detection limit of 0.001 μg/mL) and repeatability (interassay variation of 14.4%). Comparative analysis suggested positive correlation (Spearman r of 0.92) and good agreement between FO-SPR and ELISA assays. As expected, FO-SPR assays showed a closed or open ADAMTS-13 conformation in healthy donors and acute iTTP patients, respectively. ConclusionBoth ADAMTS-13 antigen and conformation assays were transferred onto automated, easy-to-use FO-SPR technology, displaying potent analytical sensitivity and reproducibility. ADAMTS-13 antigen and conformation were determined for healthy donors and acute iTTP patients showing strong correlation with ELISA reference. Introducing FO-SPR technology in clinical context could support routine diagnosis of acute iTTP patients, notably when ADAMTS-13 activity fluctuates between 10% and 20%.

Mechanism underlying severe deficiency of plasma ADAMTS-13 activity in immune thrombotic thrombocytopenic purpura

BackgroundImmune-mediated thrombotic thrombocytopenic purpura is caused by autoantibodies against ADAMTS-13, a plasma enzyme that cleaves von Willebrand factor. However, the mechanism resulting in severe deficiency of plasma ADAMTS-13 activity remains controversial. ObjectivesTo determine the mechanism of autoantibody-mediated severe deficiency of plasma ADAMTS13 activity in immune-mediated thrombotic thrombocytopenic purpura. MethodsFluorescence resonance energy transfer-VWF73 was used to determine plasma ADAMTS-13 activity. Enzyme-linked immunosorbent assay (ELISA) was used to determine anti–ADAMTS-13 immunoglobulin G. ELISA and capillary electrophoresis–based Western blotting were employed to assess plasma ADAMTS-13 antigen. ResultsWe showed that plasma ADAMTS-13 antigen levels varied substantially in the samples collected on admission despite all showing plasma ADAMTS-13 activity of <10 IU/dL (or <10% of normal level) using either ELISA or Western blotting. More severe deficiency of plasma ADAMTS-13 antigen (<10%) was detected in admission samples by ELISA than by capillary Western blotting. There was a significant but moderate correlation between plasma ADAMTS-13 activity and ADAMTS-13 antigen by either assay method, suggesting that severe deficiency of plasma ADAMTS-13 activity is not entirely associated with low levels of ADAMTS-13 antigen. ConclusionWe conclude that severe deficiency of plasma ADAMTS-13 activity primarily resulted from antibody-mediated inhibition, but the accelerated clearance of plasma ADAMTS-13 antigen via immune complexes may also contribute significantly to severe deficiency of plasma ADAMTS-13 activity in a subset of patients with acute immune-mediated thrombotic thrombocytopenic purpura.

Determination of vWF, ADAMTS-13 and Thrombospondin-1 in Venous Thromboembolism and Relating Them to the Presence of Factor V Leiden Mutation.

Thrombophilia in venous thromboembolism (VTE) is multifactorial. Von Willebrand factor (vWF) plays a major role in primary hemostasis. While elevated vWF levels are well documented in VTE, findings related to its cleaving protease (ADAMTS-13) are contradicting. The aim of this study was to determine vWF, ADAMTS-13, and the multifactorial Thrombospondin-1 (TSP-1) protein levels in patients after 3-6 months following an unprovoked VTE episode. We also explored a possible association with factor V Leiden (FVL) mutation. vWF, ADAMTS-13 and TSP-1 were analyzed using ELISA kits in 60 VTE patients and 60 controls. Patients had higher levels of vWF antigen (P = .021), vWF collagen-binding activity (P = .008), and TSP-1 protein (P < .001) compared to controls. ADAMTS-13 antigen was lower in patients (P = .046) compared to controls but ADAMTS-13 activity was comparable between the two groups (P = .172). TSP-1 showed positive correlation with vWF antigen (rho = 0.303, P = .021) and negative correlation with ADAMTS-13 activity (rho = -0.244, P = .033) and ADAMTS-13 activity/vWF antigen ratio (rho = -0.348, P = .007). A significant association was found between the presence of FVL mutation and VTE (odds ratio (OR): 9.672 (95% confidence interval (CI) 2.074-45.091- P = .004), but no association was found between the mutation and the studied proteins (P > .05). There appears to be an imbalance between vWF and ADAMTS-13 in VTE patients even after 3-6 months following the onset of VTE. We report that the odds of developing VTE in carriers of FVL mutation are 9.672 times those without the mutation, but the presence of this mutation is not associated with the studied proteins.

ADAMTS-13 conformation influences autoimmune recognition in immune thrombotic thrombocytopenic purpura

BackgroundPatients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) have anti–ADAMTS-13 immunoglobulin G (IgG) autoantibodies that enhance ADAMTS-13 clearance and/or inhibit its function. ADAMTS-13 normally circulates in a closed conformation, which is manifested by the interaction of the CUB domains with the central spacer domain. Disruption of the spacer–CUB interaction opens ADAMTS-13, which augments its proteolytic function but may also expose cryptic autoimmune epitopes that promote further autoantibody recognition. ObjectivesTo explore differences in autoantibody binding to ADAMTS-13 in its closed or open conformations in patients with iTTP and to correlate these differences with disease-related parameters. MethodsWe developed a novel assay to measure autoantibodies binding to closed and open ADAMTS-13. Autoantibody titer and IgG subclass binding to open or closed ADAMTS-13 were measured in 70 iTTP first presentation samples and correlated with clinical data, remission, and relapse. ResultsIn 70 patients with iTTP, the mean autoantibody titer against open ADAMTS-13 was, on average, approximately 2-fold greater than that against closed ADAMTS-13, suggesting that ADAMTS-13 opening increases epitope exposure and immune complex formation. Autoantibody titer against closed/open ADAMTS-13 and IgG subclass did not correlate with ADAMTS-13 antigen at presentation. Two patients with iTTP and persistent autoantibodies lost specificity for closed ADAMTS-13 in remission. Recognition of closed/open ADAMTS-13 and autoantibody IgG subclass between the first and second iTTP episodes were very similar. ConclusionADAMTS-13 autoantibody binding is highly influenced by ADAMTS-13 conformation. Although this does not appear to modify the pathogenicity of autoantibodies, the autoantibody signature at relapse suggests that relapse represents re-emergence of the original autoimmune response rather than de novo presentation.

Comparing the Impact of Rituximab Dosing on Longitudinal ADAMTS13 Parameters in TTP

Introduction The use of rituximab to prevent clinical TTP relapse is widely accepted practice. However, the optimal dosing and regimen remains unclear with a variety used across treating centres. ‘Standard’ dosing is extrapolated from dosing for lymphoproliferative diseases: an equally efficacious lower dose would be time &amp; cost-saving. We are investigating the impact of low dose (4 x 200mg weekly) vs standard dose rituximab (4 x 375mg/m 2 weekly) on ADAMTS13 response utilising prospectively collected samples from 69 ADAMTS13 relapse episodes from 4 UK sites who received elective treatment between 2017-2022 (REC: 17/LO/1055). A subset analysis of the cohort was performed, investigating open/closed ADAMTS13 conformation from time of elective rituximab treatment to subsequent rituximab retreatment. Method ADAMTS13 activity was measured at fixed timepoints from first day of elective rituximab on days 1, 8, 15 &amp; 22, month 1, 3, 6 and then 6 monthly until retreatment. Open/closed ADAMTS13 conformation was analysed in a subset of 31 episodes with the 1C4 ELISA and ADAMTS13 antigen ELISA at 4 different timepoints (ADAMTS13 activity &amp;lt;15IU/dL or %; peak ADAMTS13 activity; interim follow-up sample after peak ADAMTS13 and at next ADAMTS13 relapse). Results Median time from one elective rituximab treatment to the next was 16 months (range: 2 - 64, IQR: 9 - 24) for the subset. Time to ADAMTS13 activity normalization was compared in 26 episodes: 5 episodes were excluded as they failed to achieve ADAMTS13 normalization. 17/31 had low dose and 14/31 standard dose. There was no significant difference in the rate of ADAMTS13 normalization in the low dose compared to standard dose rituximab regime, with median 34 days ( IQR: 18 - 99, range: 14 - 266) and 21 days (IQR: 15 - 65, range: 14 - 94) respectively. 13 patients remained in an open ADAMTS13 conformation at all four timepoints, of which 9 had received low dose rituximab. 5 of 13 patients did not achieve a normal ADAMTS13 activity (i.e. &amp;gt;60IU/dL) at any timepoint, with 3 having received low dose &amp; 2 standard dose rituximab. These 13 patients had a slightly shorter time period between elective rituximab treatment episodes with a median of 8 months (range: 2 - 25, IQR: 6 - 15). In 3 patients, ADAMTS13 remained closed on the follow up sample 3 months after peak ADAMTS13 activity (mean 100IU/dL), despite subsequently relapsing 2, 8 &amp; 12 months later. Two had received standard dose rituximab. 14 patients had a closed conformation at peak ADAMTS13 activity, but a subsequent open conformation on follow up sample testing, despite a normal ADAMTS13 activity (median: 82IU/dL, IQR: 72-86). The 7 patients receiving standard dose rituximab converted to an open conformation in 6 months (IQR: 3 - 12) compared to the 7 low dose patients who switched at 4 months (IQR: 3 - 12). Subsequent ADAMTS13 relapse after detection of an open conformation occurred at a median of 6 months later (range: 4 - 8) in the standard dose group and 8 months (range: 4 - 19) in the low dose group. Lastly, 1 patient had no follow up sample after peak ADAMTS13 activity (&amp; closed confirmation), presented with ADAMTS13 relapse (&amp;lt;15IU/dL) and open conformation 7 months later. Of the 31 episodes, ADAMTS13 activity &amp; antigen results were not significantly different between the two rituximab groups for both peak ADAMTS13 activity and follow up timepoints. At peak ADAMTS13 activity, approximately 31% of patients had an open conformation in the standard dose group compared to 69% in the low dose group. In the follow up samples, this increased to 85% open conformation in the standard dose group and 93% open in the low dose group. For two of the patients, with more than one episode of elective rituximab treatment during the study, a closed ADAMTS13 conformation was again achieved after subsequent rituximab treatment. Conclusion We studied the impact of low dose and standard dose rituximab on longitudinal parameters in ADAMTS13 relapse in TTP. Overall, there were no significant differences between open vs. closed conformation at peak ADAMTS13 activity and an interim follow up timepoint between low dose and standard dose rituximab. A higher proportion of patients on low dose rituximab failed to achieve closed conformation at any timepoint but numbers are small. Importantly, our data suggests that regardless of rituximab dose, the open conformation is not indicative of imminent ADAMTS13 relapse as patients required retreatment after a further 7 months on average.

Dynamic Assessment of Plasma von Willebrand Factor and ADAMTS13 Predicts Mortality in Hospitalized Patients with SARS-CoV-2 Infection.

Plasma levels of von Willebrand factor (VWF) are significantly elevated in patients with coronavirus disease 2019 (COVID-19). However, dynamic changes and prognostic value of this biomarker in hospitalized patients with COVID-19 have not been determined. A total of 124 patients infected with SARS-CoV-2 were prospectively recruited for the study. Serial blood samples were obtained at the time of admission (D1), 3-4 days following standard-care treatments (D2), and 1-2 days prior to discharge or any time collected prior to death (D3). Plasma VWF antigen, ADAMTS13 antigen, and ADAMTS13 proteolytic activity, as well as the ratio of VWF/ADAMTS13 were determined, followed by various statistical analyses. On admission, plasma levels of VWF in COVID-19 patients were significantly elevated compared with those in the healthy controls, but no statistical significance was detected among patients with different disease severity. Plasma ADAMTS13 activity but not its antigen levels were significantly lower in patients with severe or critical COVID-19 compared with that in other patient groups. Interestingly, the ratios of plasma VWF antigen to ADAMTS13 antigen were significantly higher in patients with severe or critical COVID-19 than in those with mild to moderate disease. More importantly, plasma levels of VWF and the ratios of VWF/ADAMTS13 were persistently elevated in patients with COVID-19 throughout hospitalization. Kaplan-Meier and Cox proportional hazard regression analyses demonstrated that an increased plasma level of VWF or ratio of VWF/ADAMTS13 at D2 and D3 was associated with an increased mortality rate. Persistent endotheliopathy, marked by the elevated levels of plasma VWF or VWF/ADAMTS13 ratio, is present in all hospitalized patients following SARS-CoV-2 infection, which is strongly associated with mortality.

Comparison of quantitative and qualitative characteristics of the system “P-selectin — platelets — von Willebrand factor — metalloprotease ADAMTS13” in patients with sepsis, idiopathic thrombocytopenic purpura and healthy individuals

Introduction. The blood coagulation system plays a significant role in the pathogenesis of sepsis. Aim: to evaluate changes in the quantitative and qualitative characteristics of von Willebrand factor (vWF), ADAMTS13 metalloprotease and P-selectin in patients with sepsis, comparing them with those in patients with idiopathic thrombocytopenic purpura and in healthy individuals. Materials and methods. The study included 56 patients who received inpatient treatment due to the development of sepsis of various etiologies, 12 patients with idiopathic thrombocytopenic purpura (ITP) and 50 healthy volunteers. In a venous blood sample, the following studies were performed: vWF antigen (vWF:Ag), GPIb-binding ability of vWF (vWF:GPIb), binding ability of vWF to type I collagen (vWF:CBAI), binding ability of vWF to type III collagen I (vWF:CBAIII), P-selectin, ADAMTS13 antigen (ADAMTS13:Ag), ADAMTS13 activity (ADAMTS13:AC) and antibodies to ADAMTS13 (ADAMTS13:AB). Results. The median values of vWF:Ag, vWF:GPIb, vWF:CBAI, and vWF:CBAIII were significantly higher in patients with sepsis compared to healthy individuals and patients with ITP. The median P-selectin in patients with sepsis with thrombocytopenia 1213 ng/mL [1104–1302] was statistically significantly higher than in healthy individuals 74.5 ng/mL [65.0–84.5] (p = 0.0001), and with patients with ITP 389.0 ng/ml [276.2–589.2] (p = 0.0001). Median ADAMTS13:Ag in patients with sepsis with thrombocytopenia &lt; 100×109/l 0.49 U/ml [0.44–0.71] and in patients with ITP 0.67 U/ml [0.62–0.76] were significantly lower compared with healthy individuals 0.97 U/ml [0.84–1.08] (p = 0.001 and p = 0.034, respectively), but the median ADAMTS13:Ag in patients with sepsis with thrombocytopenia was statistically significantly lower than in patients with ITP (p = 0.038). In 19.6 % of cases in patients with sepsis, the ADAMTS13:AB index was higher than 10.0 U/ml (19.6 U/ml [12.1–26.7]), while the median P-selectin indicator in this group of patients was significantly higher when compared with a group of patients with sepsis with an ADAMTS13:AB score of less than 10.00 U/mL (3.04 [2.11–3.94]): 1342 ng/mL[1271–1374] vs. 1130 ng/mL [1087–1271], p = 0.003, respectively. Conclusion. Activation of endothelial cells may underlie the increase in quantitative and qualitative indicators of vWF. Meanwhile, the effect of the adhesive characteristics of vWF (vWF:GPIb, vWF:CBAI and vWF:CBAIII) on the number of platelets in the blood of patients with sepsis was not revealed. Adhesion of vWF to the endothelium through interaction with P-selectin through the platelet GPIb receptor can promote the transition of vWF into a conformational-active state and lead to a decrease in the concentration of ADAMTS13 in the circulation.

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

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

ADAMTS13 and Von Willebrand Factor Levels in Patients with Chronic hepatitis C: Relations to Hemostatic Imbalance and Bacterial Infection

Abstract Background Liver cirrhosis is in close association with bacterial translocation as a result of alteration of intestinal barrier. In response to inflammation, the endothelial cells release the content of WPBs, VWF in the extracellular portion. The aim of the study was to assess the levels of ADAMTS13 and their impact on the prothrombotic function of VWF in patients with chronic hepatitis C with or without SBP. Patients and methods 75 individuals were the study population, 60 patients with CLD classified into: Child A (n = 15), Child B (n = 15) and Child C (n = 30) the latter is further classified into Child C (n = 15) and Child C patients with SBP (n = 15); healthy controls (n = 15). This study was performed at Theodor Bilharz Research Institute (TBRI), Imbaba, Giza. Determination of CRP by fixed time nephelometry. Determination of LBP, VWF, ADAMTS13 and D-dimer by magnetic multiplexed flowcytometry based immunoassay Luminex®. Results VWF levels were significantly increased (p &amp;lt; 0.01) in different groups of patients with CLD compared to controls. A similar significant increase (p &amp;lt; 0.01) was also noticed in Child C patients with SBP compared to other study groups. ADAMTS13 antigen levels were significantly decreased (p &amp;lt; 0.01) in different groups of patients with CLD compared to controls. D-dimer levels were significantly increased (p &amp;lt; 0.01) in different groups of patients with CLD compared to controls. Conclusion The levels of VWF correlated with the systemic inflammatory and bacterial translocation markers. ADAMTS13 levels were decreased in cases of systemic inflammatory process that accompanies SBP, leading to increased VWF levels.

Clinical significance of ADAMTS-13/vWF axis in pregnant women at different trimesters of gestation

Introduction. The pandemic of a novel coronavirus infection has demonstrated the importance of assessing the ADAMTS-13/vWF axis in patients with COVID-19, because a decline in this ratio mirrors disease severity. However very few data in the global literature on crosstalk and ADAMTS-13/vWF levels in pregnant women remaining very contradictory are available. Taking into consideration an impact of the ADAMTS-13/vWF axis on prevalence of thrombosis and disorders in the hemostasis system, investigation of this issue is highly demanded.Aim: to assess the functioning of the ADAMTS-13/vWF axis during physiological pregnancy.Materials and Methods. A controlled non-randomized study was conducted: main group included 44 women with physiologically occurring pregnancies at I, II and III trimesters; the control group consisted of 45 healthy non-pregnant women. The plasma level of ADAMTS-13 inhibitor (ADAMTS-13:i), ADAMTS-13 antigen (ADAMTS-13:Ag), vWF antigen (vWF:Ag), and ADAMTS-13 activity (ADAMTS-13:Ac) as well as relevant ratio (ADAMTS-13:Ac/vWF:Ag) were measured.Results. It was shown that in parallel with increasing gestational age, significant changes occurred in the ADAMTS-13:Ac/vWF:Ag ratio. In main group, patients at II trimester were found to have level of ADAMTS-13:Ac/vWF:Ag 0.359 ± 0.121, in III trimester –0.253 ± 0.741, which significantly differed (p &lt; 0.01) compared to control group with non-pregnant women (1,134 ± 0,308).Conclusion. Our study provides new insights into the functioning of the ADAMTS-13:/vWF axis in women with physiologically occurring pregnancy at I, II and III trimesters. Decline in ADAMTS-13:Ac was demonstrated along with increasing vWF:Ag level observed in parallel with increasing gestational age. Apparently, the progressive decrease of ADAMTS-13 concentration during pregnancy is associated with its increased consumption due to high vWF level. However, due to the small single-center patient cohort, further studies with larger-scale studies are needed.

Autoantibodies enhance ADAMTS-13 clearance in patients with immune thrombotic thrombocytopenic purpura

BackgroundSevere deficiency in ADAMTS-13 (<10%) and the loss of von Willebrand factor–cleaving function can precipitate microvascular thrombosis associated with thrombotic thrombocytopenic purpura (TTP). Patients with immune-mediated TTP (iTTP) have anti-ADAMTS-13 immunoglobulin G antibodies that inhibit ADAMTS-13 function and/or increase ADAMTS-13 clearance. Patients with iTTP are treated primarily by plasma exchange (PEX), often in combination with adjunct therapies that target either the von Willebrand factor-dependent microvascular thrombotic processes (caplacizumab) or the autoimmune components (steroids or rituximab) of the disease. ObjectivesTo investigate the contributions of autoantibody-mediated ADAMTS-13 clearance and inhibition in patients with iTTP at presentation and through the course of the PEX therapy. Patients/MethodsAnti-ADAMTS-13 immunoglobulin G antibodies, ADAMTS-13 antigen, and activity were measured before and after each PEX in 17 patients with iTTP and 20 acute TTP episodes. ResultsAt presentation, 14 out of 15 patients with iTTP had ADAMTS-13 antigen levels of <10%, suggesting a major contribution of ADAMTS-13 clearance to the deficiency state. After the first PEX, both ADAMTS-13 antigen and activity levels increased similarly, and the anti-ADAMTS-13 autoantibody titer decreased in all patients, revealing ADAMTS-13 inhibition to be a modest modifier of the ADAMTS-13 function in iTTP. Analysis of ADAMTS-13 antigen levels between consecutive PEX treatments revealed that the rate of ADAMTS-13 clearance in 9 out of 14 patients analyzed was 4- to 10-fold faster than the estimated normal rate of clearance. ConclusionThese data reveal, both at presentation and during PEX treatment, that antibody-mediated clearance of ADAMTS-13 is the major pathogenic mechanism that causes ADAMTS-13 deficiency in iTTP. Understanding the kinetics of ADAMTS-13 clearance in iTTP may now enable further optimization of treatment of patients with iTTP.

Delayed normalization of ADAMTS13 activity in acute thrombotic thrombocytopenic purpura in the caplacizumab era

AbstractThe benefits of caplacizumab in acute immune-mediated thrombotic thrombocytopenic purpura (iTTP) are well established. We identified a delayed normalization of a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13 (ADAMTS13) activity (>30%) in a subgroup treated with caplacizumab, not evident in the precaplacizumab era. Patients treated with caplacizumab (n = 64) achieved ADAMTS13 activity >30% at median 31 days after plasma exchange (PEX), compared with 11.5 days in the noncaplacizumab group (n = 50, P = .0004). Eighteen of 64 (28%) patients treated with caplacizumab had ADAMTS13 activity <10% at stopping caplacizumab with a longer time to ADAMTS13 activity >30% (median, 139 days after completing PEX). Eighteen of 64 (28%) patients receiving extended caplacizumab (31-58 days) failed to achieve ADAMTS13 activity >30% at the time of caplacizumab cessation, compared with 4 of 47 (8.5%) historical controls at a similar timepoint (30 + 28 days, P < .0001). Failure to achieve ADAMTS13 activity >30% within 30 + 28 days was 6 times more likely with caplacizumab (odds ratio, 6.3; P = .0006). ADAMTS13 antigen <30% at caplacizumab cessation was associated with increased iTTP recurrence (4/10 vs 0/9 in patients with ADAMTS13 antigen ≥30%). Admission anti-ADAMTS13 immunoglobulin G (IgG) antibody level did not predict recurrence. Anti-ADAMTS13 IgG antibody levels, immunosuppression, and ethnicity did not account for differences in ADAMTS13 activity response. ADAMTS13 antigen levels ≥30% may be useful to guide stopping caplacizumab therapy after extended use with ADAMTS13 activity <10%. The reason for delayed ADAMTS13 normalization is unclear and requires further investigation.

Application Value of Plasma vWF and ADAMTS-13 in Evaluating Postoprative Vascular Endothelial Injury and Prognosis in Children with VSD

To dynamically observe the levels and activities of von Willebrand factor (vWF) and ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) in plasma of children with congenital ventricular septal defect (VSD) during perioperative period, and explore the value of plasma vWF antigen (vWF:Ag) and ADAMTS-13 activity (ADAMTS-13: AC) in evaluating vascular endothelial injury and prognosis in children with VSD. In this cross-sectional study, a total of 74 children with VSD who underwent surgical treatment in TEDA International Cardiovascular Hospital from September 2018 to March 2019 were enrolled in the observation group. Among them, there were 28 cases of pure VSD, 32 cases of VSD combined with pulmonary hypertension, and 14 cases of VSD combined with valvular heart disease. 31 healthy children who underwent physical examination in Tianjin Children's Hospital during the same period were collected as the control group. The biochemical indexes of the children at admission were recorded. Peripheral plasma was collected at admission, postsurgery day 0 and day 1, respectively, and the levels of vWF activity (vWF:AC), vWF:Ag, ADAMTS-13 antigen (ADAMTS-13:Ag) and ADAMTS-13:AC were detected. The level of plasma vWF:Ag and vWF:AC in the observation group before surgery were significantly lower than those in the control group (P<0.001), and increased continuously, on postsurgery day 0 and day 1 (P<0.001). The level of ADAMTS-13:Ag in the observation group before surgery was significantly higher than that in the control group (P<0.001), which decreased significantly on postsurgery day 0 (P<0.001), and increased significantly on postsurgery day 1 compared with postsurgery day 0 (P=0.033). The level of ADAMTS-13:AC in the observation group before surgery was significantly lower than that in the control group (P=0.015), which decreased significantly on postsurgery day 0 (P=0.037), and increased on postsurgery day 1, but the difference was not statistically significant (P=0.051). The changes of vWF and ADAMTS-13 in the three subgroups were basically similar to the observation group. vWF: Ag/ADAMTS-13: AC ratio on postsurgery day 0 and day 1 had high diagnostic value in vascular endothelial injury (AUC=0.80, P<0.001; AUC=0.93, P<0.001). Preoperative vWF and ADAMTS-13 levels, and related baseline indicators were not correlated with postoperative infection, bleeding, thrombosis,etc. Preoperative vWF: Ag, vWF: AC and ADAMTS-13: AC levels in children with VSD are low, while the level of ADAMTS-13: Ag is high. After surgery, the levels of vWF: Ag and vWF: AC are increased and the level of ADAMTS-13: Ag is decreased. The postoperative vWF: Ag/ADAMTS-13: AC ratio shows high diagnostic value in evaluating vascular endothelial injury. There is no correlation between preoperative vWF and ADAMTS-13 levels with perioperative clinical events.

Decreased protein C activity, lower ADAMTS13 antigen and free protein S levels accompanied by unchanged thrombin generation potential in hospitalized COVID-19 patients

IntroductionCOVID-19 is associated with an increased thromboembolic risk. However, the mechanisms triggering clot formation in those patients remain unknown. Patients and methodsIn 118 adult Caucasian severe but non-critically ill COVID-19 patients (median age 58 years; 73 % men) and 46 controls, we analyzed in vitro plasma thrombin generation profile (calibrated automated thrombogram [CAT assay]) and investigated thrombophilia-related factors, such as protein C and antithrombin activity, free protein S level, presence of antiphospholipid antibodies and factor V Leiden R506Q and prothrombin G20210A mutations. We also measured circulating von Willebrand factor (vWF) antigen and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) antigen and activity. In patients, blood samples were collected on admission to the hospital before starting any therapy, including heparin. Finally, we examined the relationship between observed alterations and disease follow-up, such as thromboembolic complications. ResultsCOVID-19 patients showed 17 % lower protein C activity, 22 % decreased free protein S levels, and a higher prevalence of positive results for IgM anticardiolipin antibodies. They also had 151 % increased vWF, and 27 % decreased ADAMTS13 antigens compared with controls (p < 0.001, all). On the contrary, thrombin generation potential was similar to controls. In the follow-up, pulmonary embolism (PE) occurred in thirteen (11 %) patients. They were characterized by a 55 % elevated D-dimer (p = 0.04) and 2.7-fold higher troponin I (p = 0.002) during hospitalization and 29 % shorter time to thrombin peak in CAT assay (p = 0.009) compared to patients without PE. ConclusionsIn COVID-19, we documented prothrombotic abnormalities of peripheral blood. PE was characterized by more dynamic thrombin generation growth in CAT assay performed on admittance to the hospital.

Immunothrombosis in cancer patients: contribution of neutrophil extracellular traps, ADAMTS-13 and von Willebrand factor

Introduction. Neutrophil extracellular traps (NETs) and von Willebrand factor (vWF) are integral players in thrombosis and inflammation in cancer patients. It has been increasingly evident that an active interplay exists between NETs and vWF. Some studies suggest that NETs cause decrease in ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) activity, being an arm in the pathogenesis of both thrombotic microangiopathies (TMA) and other thrombotic complications during oncological process.Aim: to assess a crosstalk between NETs, vWF, and ADAMTS-13 in uterine, ovarian, breast malignant neoplasms as well as cervical canal adenocarcinoma.Materials and Methods. From September 2019 to July 2022, a prospective controlled interventional non-randomized study was carried out with 106 patients hospitalized for planned surgical treatment aged 30 to 72 years. The main group included 73 patients with malignant neoplasms of the female genital organs and mammary glands, stage I–III: uterine cancer (subgroup 1; n = 18), ovarian cancer (subgroup 2; n = 21), cervical cancer – adenocarcinoma of cervical canal (subgroup 3; n = 9) and breast cancer (subgroup 4; n = 25). The control group consisted of 33 women with female genital tract and breast benign neoplasms. In all patients, serum levels of vWF, citrullinated histone H3 (citH3), MPO (myeloperoxidase) antigen, ADAMTS-13 activity, ADAMTS-13 antigen, and D-dimer were evaluated.Results. The study revealed significant differences in the concentration of NETosis markers between the main and control groups. Patients with uterine cancer and adenocarcinoma of the cervical canal peaked at NETosis markers. At the same time, there were significant differences in citH3 concentration among patients with «early» (stage I) and «not early» (stage II–III) disease forms. While assessing level of von Willebrand factor (vWF:Ag), antigen (ADAMTS-13:Ag), and ADAMTS-13 activity (ADAMTS-13:Ac), significant differences were found between the main and control groups (p &lt; 0.0001). The vWF in the main groups was sharply increasedwhereas ADAMTS-13 antigen concentration and activity were decreased. A сorrelation analysis among oncological patients in main group showed that while citH3 level increased, it was also paralleled with rise in vWF:Ag (ρ = 0.80; p &lt; 0.01) and MPO:Ag (ρ = 0.87; p &lt; 0.01); increase in MPO:Ag level was coupled to rise in vWF:Ag (ρ = 0.70; p&lt; 0.01), but increase in vWF:Ag occurred along with decline in ADAMTS-13:Ac (ρ = –0.43; p &lt; 0.01) and ADAMTS-13:Ag (ρ= –0.42; p &lt; 0.01).Conclusion. The interplay between NET, vWF, and ADAMTS-13 leads to a vicious circle, reduces ADAMTS-13 activity by increasing serum vWF concentration, which positively correlates with severity and mortality in TMA, acute ischemic infarction, and COVID-19. Targeting the NETs-vWF axis may pave the way for therapeutic strategies for immunothrombosis in various diseases, including cancer.

In vitro characterization of a novel Arg102 mutation in the ADAMTS13 metalloprotease domain

BackgroundCongenital thrombotic thrombocytopenic purpura is caused by defects in the ADAMTS13 gene. ADAMTS13 is normally preactivated by conformational changes of the Metalloprotease (M) domain. Studying a novel congenital thrombotic thrombocytopenic purpura p.R102S mutation in the M domain, which results in undetectable ADAMTS13 activity in the patient, could help to explain the patients’ phenotype and to elucidate the currently unclear mechanism of allosteric preactivation. ObjectivesTo investigate the in vitro effect of p.R102S mutation on ADAMTS13 secretion, activity, and allosteric preactivation. MethodsMolecular modeling was used to study the effect of the mutation on the stability of ADAMTS13. Recombinant mutant ADAMTS13 was generated by transient and stable transfection of, respectively, CHO K1 and HEK293-T cells. ADAMTS13 antigen was measured in enzyme-linked immunosorbent assay. ADAMTS13 activity was measured in a FRETS-VWF73 assay. Allosteric preactivation was assessed in FRETS-VWF73 assay, using monoclonal antibody (mAb) 17G2 that normally induces a ∼2-fold increase in activity, and in enzyme-linked immunosorbent assay using mAb 6A6 recognizing a cryptic epitope in the M domain that becomes exposed after binding of 17G2. Resultsp.R102S mutation destabilizes the interactions between the M and Disintegrin-like (D) domain. p.R102S mutant secretion was impaired (35% of wild type) and activity was severely reduced (12% of wild type). p.R102S mutant could still be activated and the cryptic epitope of 6A6 was still fully exposed by 17G2 addition. Conclusionp.R102S mutation destabilizes the M–D domain interactions, causing impaired ADAMTS13 secretion and activity, which explains the patients’ phenotype. Allosteric preactivation of ADAMTS13 remains conserved in the presence of the p.R102S mutation.

Elevated Plasma Levels of Von Willebrand Factor and Syndecan-1 Predict 60-Day Mortality in Patients with Severe and Critical COVID-19

Introduction: Endotheliopathy is common pathologic findings in acute and long COVID-19, which is associated with the disease severity and predisposes to long-term complications. Previous studies have demonstrated that plasma levels of von Willebrand factor (VWF) and proteoglycan syndecan-1 are significantly elevated in patients with severe COVID-19. However, the role of these endothelial biomarkers for predicting a long-term outcome is still lacking. Patients and Methods:151 consecutive hospitalized patients between January and March 2022 at the University of Kansas Hospital with a confirmed SARS-CoV-2 infection by polymerase chain reaction were prospectively enrolled for the study. Whole blood anticoagulated with EDTA or sodium citrate was collected at least 3 times on admission (T1), 4-5 days following treatment (T2), and a few days prior to discharge or death (T3). Plasma levels of VWF antigen, ADAMTS13 antigen/activity, and syndecan-1 were determined using immunoassays or a functional assay. Kaplan-Meier survival analysis was performed to determine the predictive value of each biomarker for 60-day mortality. Results. Of 151 patients with a positive PCR test for SARS-CoV2, 124 patients were studied and analyzed. These patients were categorized into an asymptomatic control, a moderate, a severe, and a critical group according to an updated World Health Organization (WHO) guidance. The admission plasma levels (median, IQR) of VWF in patients of critical (334%,164.7-526.2%) (p=0.0049) and severe (255%, 143.1-410.9%) (p=0.0088) groups were significantly higher than in those of moderate (153%, 186.3-240.1%) (Fig. 1A). Interestingly, no statistically significant difference in the admission plasma levels of ADAMTS13 antigen among all groups (p>0.05) (Fig. 1B) despite a significantly lower plasma ADAMTS13 activity (median, IQR) in patients with severe (39.8%, 30.0-49.3%) (p=0.0067) and critical (38.6%, 26.9-51.5%) (p=0.047) COVID-19 than in those with a asymptomatic disease (49.2%, 37.5-103.4%) (Fig. 1C). Furthermore, plasma levels of syndecan-1 on admission were not different among all groups (p>0.05) (Fig. 1D). Longitudinal analysis demonstrated that plasma levels of VWF antigen and syndecan-1 were persistently elevated from baseline in patients who died but not in those who survived (Fig. 2A-D). Kaplan-Meier survival analysis revealed that an elevated level of plasma VWF (> 320%) on T2 (p<0.0001) (Fig. 2E) or an elevated level of plasma syndecan-1 on admission (T1) (> 260 ng/mL) (p=0.0002) (Fig. 2F), but not 4-5 days following therapy (T2) or before being discharged home or death (T3) predicted the 60-day mortality rate. Conclusions: Our results demonstrate significantly elevated plasma VWF antigen and reduced ADAMTS13 activity on admission in patients with severe and critical COVID-19. The dynamic change in plasma VWF or the initial plasma levels of syndecan-1 on admission predict the 60-day survival. The findings suggest that the additional therapeutic strategies may be needed to alleviate endothelial activation and damage to improve survival and long-term complications in patients with severe and critical COVID-19. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ADAMTS13 conformation and immunoprofiles in Japanese patients with immune-mediated thrombotic thrombocytopenic purpura

AbstractImmune-mediated thrombotic thrombocytopenic purpura (iTTP) is an ultrarare thrombotic disease caused by autoantibody-induced ADAMTS13 deficiency. Open ADAMST13 conformation, induced by autoantibodies, was identified as a novel biomarker for iTTP. Determining immunoprofiles in patients with iTTP has been shown to guide the development of novel targeted therapies. However, these studies were done in mainly Caucasian iTTP cohorts. To validate those findings across other ethnic cohorts, we investigated 195 acute TTP plasma samples from the Japanese iTTP registry. Seventy-six of the 195 samples had detectable ADAMTS13 antigen levels, of which 94.7% were shown to have an open ADAMTS13 conformation. A positive correlation was observed between ADAMTS13 inhibitor titers (a diagnostic parameter in Japan) and anti-ADAMTS13 immunoglobulin G autoantibody titers. Studying anti-M, anti-DT, anti-CS, anti-T2-T5, anti-T6-T8, anti–CUB1-2 autoantibodies and the corresponding immunoprofile showed that 73% of the patients had anti-CS autoantibodies and 25.8% had anti-M autoantibodies, with the latter being higher than in Caucasians. Stratifying patients according to their immunoprofiles revealed that the profile with only anti-CS autoantibodies was the most common immunoprofile similar to that in Caucasians (28.9%). Although this profile did not affect the 1-year TTP-related mortality rate, patients with autoantibodies against all 6 ADAMTS13 fragments had a higher risk for TTP-related death than other patients (P = .02). We here validated open ADAMTS13 as a novel biomarker for acute iTTP and determined the dominant immunoprofiling in the Japanese cohort, contributing to setting up the diagnosis and managing guidelines across different ethnic cohorts and developing ADAMTS13 variants that do not bind to the anti-CS autoantibodies.