ADAMTS13 Antigen Levels Research Articles

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.

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.

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.

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.

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.

Determination of the level of von willebrand factor and ADAMTS13 in sickle cell anaemia patients in steady state.

Sickle cell anaemia (SCA) is a hypercoagulable state with alteration in the haemostatic parameters and may contribute to thrombosis in a steady state. The levels of von Willebrand factor (VWF) and ADAMTS13 antigen in the steady state as markers of thrombotic risk have not been fully investigated in our environment. Evaluation of the level of VWF and ADAMTS13 as a marker of thrombotic risk in SCA subjects in the steady state at UCTH, Calabar. This is a comparative study carried out at UCTH, Calabar. Sixty SCA patients were evaluated in the steady state with apparently healthy controls matched for age and sex VWF.Ag, and ADAMTS13.Ag was evaluated using Assay Pro enzyme-linked immunosorbent assay kit. Data was analysed with IBM Statistical Package for Social Sciences Chicago Software version 26. The median age of SCA and controls were 23 years and 20 years, respectively (P = 0.962). There were no significant differences in their sex distribution (P = 0.063). The mean ± standard deviation (SD) of VWF in the steady state and control were 1.34 ± 0.23 IU/mL and 1.41 ± 0.23 IU/mL with no significant difference in their mean (P = 0.864). The mean ± SD of ADAMTS13 in the steady state and control were 0.44 ± 0.06 μg/L and 0.62 ± 0.10 μg/L, respectively, with no significant difference in their mean (P = 0.171). There was no significant difference between VWF.Ag, ADAMTS13, and VWF.Ag: ADAMTS13 antigen ratio in SCA in the steady state and control. There is a need for further research to determine their functionality.

Changes in the activity of metalloprotease ADAMTS13 antigen von Willebrand factor in patients with acute coronary syndrome

Introduction. Currently, considerable attention is being given to the study of the interaction of the von Willebrand factor (vWF) and its specific regulator of metalloprotease ADAMTS13 in various clinical conditions accompanied by the development of thrombosis.Aim — to assess the change in the activity of ADAMTS13 metalloprotease and vWF factor antigen in patients with acute coronary syndrome during the first five days after the onset of an anginal attack.Patients and methods: The study included 90 patients aged 42 to 86 years (Me — 61.5 y.o.) hospitalized with a diagnosis of acute coronary syndrome. Of these, 69 were men (Me — 59 y.o.) and 21 women (Me — 63 y.o.). All patients were tested for ADAMTS13 activity, ADAMTS13 antigen, and vWF antigen. Blood samples for the study were obtained upon admission to the hospital (arterial and venous blood), as well as 24, 72 and 120 hours after hospitalization (venous blood).Results. It was found that at all points of the study, in patients without coronary artery thrombosis, the average value of the ADAMTS13 activity indicator was significantly higher compared to the group of patients with coronary artery thrombosis. At the same time, there were no statistically significant differences in the average values of the ADAMTS13 antigen between the compared groups of patients. The mean value of vWF antigen in patients with coronary artery thrombosis was significantly higher compared to that of patients without coronary artery thrombosis.Conclusion. An increase in ADAMTS13 activity was noted more often in patients with acute coronary syndrome without coronary artery thrombosis than in patients with coronary artery thrombosis, which may indicate the antithrombotic effect of ADAMTS13 metalloprotease. No differences were found in ADAMTS13 antigen levels when comparing the study groups of patients, which indicates a greater significance for the anticoagulant activity of the functional characteristics of ADAMTS13. Significantly higher levels of the vWF antigen in patients with coronary artery thrombosis were due to the response to ischemic myocardial injury and stress but were not the primary cause of the thrombotic event.

Alterations in B- and circulating T-follicular helper cell subsets in immune thrombotic thrombocytopenic purpura

AbstractT follicular helper (Tfh) cells regulate development of antigen-specific B-cell immunity. We prospectively investigated B-cell and circulating Tfh (cTfh) cell subsets in 45 patients with immune thrombotic thrombocytopenic purpura (iTTP) at presentation and longitudinally after rituximab (RTX). B-cell phenotype was altered at acute iTTP presentation with decreased transitional cells and post–germinal center (post-GC) memory B cells and increased plasmablasts compared with healthy controls. A higher percentage of plasmablasts was associated with higher anti-ADAMTS13 IgG and lower ADAMTS13 antigen levels. In asymptomatic patients with ADAMTS13 relapse, there were increased naïve B cells and a global decrease in memory subsets, with a trend to increased plasmablasts. Total circulating Tfh (CD4+CXCR5+) and PD1+ Tfh cells were decreased at iTTP presentation. CD80 expression was decreased on IgD+ memory cells and double-negative memory cells in acute iTTP. At repopulation after B-cell depletion in de novo iTTP, post-GC and double-negative memory B cells were reduced compared with pre-RTX. RTX did not cause alteration in cTfh cell frequency. The subsequent kinetics of naïve, transitional, memory B cells and plasmablasts did not differ significantly between patients who went on to relapse vs those who remained in remission. In summary, acute iTTP is characterized by dysregulation of B- and cTfh cell homeostasis with depletion of post-GC memory cells and cTfh cells and increased plasmablasts. Changes in CD80 expression on B cells further suggest altered interactions with T cells.

Reference range for ADAMTS13 antigen, activity and anti-ADAMTS13 antibody in the healthy adult Singapore population

ADAMTS13 (a disintegrin-like and metalloproteinase with a thrombospondin Type 1 motif, member 13) plays a fundamental role in the regulation of haemostasis and thrombosis. Its deficiency leads to an accumulation of ultra-large von Willebrand multimers, inducing spontaneous platelet aggregation, thrombosis in the microvasculature, and thrombotic thrombocytopenic purpura (TTP), a condition with 90% mortality when left untreated. Prompt quantification of ADAMTS13 antigen, activity and autoantibody plays a crucial role in the diagnosis and management of TTP and can help differentiate it from other thrombotic microangiopathies (TMAs). Reference ranges for ADAMTS13 are generally derived from Caucasian patients. Given that polymorphism in the ADAMTS13 gene can be associated with variable ADAMTS13 levels, we aimed to establish the first reference range in Singapore and provide a crucial laboratory test for institutions here and elsewhere. 150 healthy voluntary donors (75 men, 75 women) aged 21-60 years, with an ethnic mix mirroring Singapore's population profile, were recruited. ADAMTS13 antigen, activity and autoantibody levels were measured using the fluorescence resonance energy transfer-vWF73 and enzyme-linked immunosorbent assay methodologies. Levels (activity 0.65-1.79 IU/mL, antigen 0.36-1.17 IU/mL, autoantibody 1.4-12.5 U/mL) were not statistically different between the genders and various age groups. TTP and TMAs are encountered in a wide range of specialties. The availability of new assays in Singapore will aid clinicians in the timely management of these conditions. Standardising reference ranges established for Singapore against World Health Organization standards allows harmonisation of measurements between laboratories and for future research collaborations.

Role of High VWF and Low ADAMTS-13 in Ischemic Stroke

Aim: To measure levels of VWF and ADAMTS-13 in patients of ischemic stroke and healthy controls. Method: Blood samples of forty five patients and forty five controls of age 30-65 years were taken from medical or neurology wards of Lahore General Hospital. VWF antigen assay was determined by Immuno-Turbidimetric method and quantitative ADAMTS-13 antigen assay was measured by ELISA. Results: The mean level of plasma VWF antigen of controls was 114.0±49.7% and mean plasma level of VWF antigen in patients was 137.0±54.3%. The mean plasma VWF antigen level was significantly elevated among patients as compared to controls (p=0.011). The mean ADAMTS-13 antigen level of controls was 151.7± 87.9 U/L and mean ADAMTS-13 antigen level of patients was 107.9±106.7 U/L. Our study also found that mean ADAMTS-13 was significantly lower in patients as compared to controls (p=0.018). Conclusion: In conclusion, Vwf and ADAMTS-13 antigen assays have an important role in pathogenesis of clot formation in patients of ischemic stroke. Keywords: Low ADAMTS, ischemic stroke, high WWF

Plasma and rhADAMTS13 reduce trauma-induced organ failure by restoring the ADAMTS13-VWF axis

AbstractTrauma-induced organ failure is characterized by endothelial dysfunction. The aim of this study was to investigate the role of von Willebrand factor (VWF) and its cleaving enzyme, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs, member 13) in the occurrence of endothelial permeability and organ failure in trauma. In an observational study in a level-1 trauma center, 169 adult trauma patients with clinical signs of shock and/or severe injuries were included. Trauma was associated with low ADAMTS13 and high VWF antigen levels, thus generating an imbalance of ADAMTS13 to VWF. Patients who developed organ failure (23%) had greater ADAMTS13-to-VWF imbalances, persistently lower platelet counts, and elevated levels of high-molecular-weight VWF multimers compared with those without organ failure, suggesting microthrombi formation. To investigate the effect of replenishing low ADAMTS13 levels on endothelial permeability and organ failure using either recombinant human ADAMTS13 (rhADAMTS13) or plasma transfusion, a rat model of trauma-induced shock and transfusion was used. Rats in traumatic hemorrhagic shock were randomized to receive crystalloids, crystalloids supplemented with rhADAMTS13, or plasma transfusion. A 70-kDa fluorescein isothiocyanate–labeled dextran was injected to determine endothelial leakage. Additionally, organs were histologically assessed. Both plasma transfusion and rhADAMTS13 were associated with a reduction in pulmonary endothelial permeability and organ injury when compared with resuscitation with crystalloids, but only rhADAMTS13 resulted in significant improvement of a trauma-induced decline in ADAMTS13 levels. We conclude that rhADAMTS13 and plasma transfusion can reduce organ failure following trauma. These findings implicate the ADAMTS13-VWF axis in the pathogenesis of organ failure.

Thrombosis and Von Willebrand Factor in Chuvash Polycythemia

Background: In Chuvash polycythemia (CP), homozygous germline VHLR200W results in augmented hypoxia-sensing, elevated erythropoietin and hemoglobin, and increased morbidity and mortality from venous and arterial thromboses.1 In CP patients and controls from Russia's Chuvash Republic, the probability of new thrombosis in CP increased with age, smoking, and higher expression of THBS1, the gene encoding thrombospondin-1.2 In mice, hypoxia leads to upregulation of THBS1 through HIF-23 and to upregulation of VWF .4 THBS1 reportedly regulates the size of circulating von Willebrand Factor (VWF) multimers5 through inhibiting proteolytic cleavage of VWF by ADAMTS13.6 We hypothesized that upregulation of THBS1 in CP may be a risk for thrombosis through an effect on ADAMTS13 and VWF.Methods: We enrolled into an observational study 155 patients with CP and 154 controls matched by age, sex and place of residence. Written informed consent was obtained from all subjects according to the Declaration of Helsinki. We genotyped for VHLR200W, measured baseline plasma THBS-1 concentrations2 and followed the subjects for a median of 9.4 years. Here, we report results for determining baseline circulating levels of ADAMTS13 antigen, activity and specific activity and of VWF antigen and differential multimers in up to 57 CP patients and 30 controls.Results: CP subjects had higher hemoglobin and erythropoietin concentrations and lower white blood cell and platelet counts (Table 1). They were also more likely to be current smokers. There was a trend to higher THBS-1 concentrations in the CP patients (p=0.084), which was significant after adjusting for platelet count (P=0.016). ADAMTS13 and VWF antigen levels did not differ between CP patients and controls but ADAMTS13 activity and especially specific activity were lower in CP. Plasma THBS-1 concentration correlated positively with ADAMTS13 antigen concentration (r=0.358, P=0.001) but negatively with specific activity (r=-0.363, P=0.001) (Figure 1). Eight (14.0%) CP patients (aged 27-76) years had a history of thrombosis (Table 1). The mean serum VWF antigen concentration (P=0.001) and differential VWF multimer size (P=0.090) were higher in CP patients with a history of thrombosis (Figure 2). During the prospective follow-up period, 10 CP patients (17.5%) experienced a new thrombosis at a median age of 46 (range 26-70) years. Three patients also experienced an additional thrombosis for a total of 13 new thromboses: myocardial infarction (n=5), stroke (n=3), splanchnic thrombosis (n=2), deep vein thrombosis (n=2), pulmonary embolism (n=1). Baseline serum VWF antigen levels (P=0.019) and VWF multimer size (P=0.099) were higher in subjects who developed a new thrombosis during follow-up (Figure 3), and these relationships persisted after adjusting for smoking history.Discussion: THBS1 is an adhesive glycoprotein that mediates cell-to-cell and cell-to-matrix interactions and facilitates platelet aggregation.7 In mice, platelet-derived THBS1 modulated arterial thrombosis only in the presence of VWF.8 Our present findings suggest that CP subjects have lower ADAMTS13 specific activity than controls and that this lower activity may be related to higher plasma THBS1 concentrations. Lower ADAMTS13 specific activity suggests the presence of an inhibitor or partial inactivation of the protease. Furthermore, higher VWF antigen and multimer size in CP patients is associated with a past history of thrombosis and a greater risk for new thrombosis in the future. More broadly, our findings suggest a possible implication of VWF in thrombosis of conditions associated with an up-regulated hypoxic response. Further studies may identify new pathways of prevention and treatment with broad applicability.

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

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

DEFICIENCY OF ADAMTS-13 IN SEPSIS PATIENTS AND ITS CORRELATION WITH MORBIDITY/ MORTALITY

Objective: To detect ADAMTS-13 deficiency in sepsis patients and its effect on in-hospital morbidity and mortality in adult patients of sepsis.&#x0D; Study Design: Cross sectional study.&#x0D; Place and Duration of Study: Armed Forces Institute of Pathology (AFIP) and Combined Military Hospital (CMH) Rawalpindi, from Apr 2017 to Apr 2018.&#x0D; Methodology: All indoor patients admitted at Combined Military Hospital Rawalpindi and diagnosed as suffering from sepsis at Armed Forces Institute of Pathology according to the guidelines of the Critical Care Society of Medicine Consensus Conference Committee. Forty sepsis patients were recruited for the study. Two groups were formed on the basis of ADAMTS-13 antigen levels, non-deficient and deficient. Basic haematological and clinical parameters were recorded. Levels of ADAMTS-13 antigen were analyzed by using Technozyme kit byenzyme-linked immunosorbent assay (ELISA) and level less than 0.40 IU/ml was taken deficient as defined by manufacturer. Comparison was done between ADAMTS-13 deficient and non-deficient group with regard to clinical and haematological characteristics, ICU stay, hospital admission days and in-hospital mortality. Healthy controls of same age and gender were also observed for ADAMTS-13 antigen level.&#x0D; Results: Among total 40 sepsis patients, thirty two (80%) were males and eight (20%) were females with a mean age of 61 ± 15 years (range: 30-85 years). ADAMTS-13 deficiency (&lt;0.40 IU/ml) was found in 28 (70%) of patients while 12 (30%) had normal antigen levels. In patients with ADAMTS-13 deficiency, twenty four (85.7%) had low platelet levels &lt;150 x 109/l, mean hospital stay 19.2 ± 7.6 days and in-hospital mortality was seen in eight (28.6%) patients, in comparison of non-deficient group in which platelet levels were equal or &gt;150 x 109/l, mean hospital stay was 10.2 ± 3.6 days and in-hospital mortality was in one (8.3%) patient respectively. ADAMTS-13 antigen level was found sufficient in healthy controls.&#x0D; Conclusion: Majority of the sepsis patients were found ADAMTS-13 deficient. Deficiency exhibit long term hospital admission, thrombocytopenia and increase in hospital mortality. ADAMTS-13 deficiency might play a role in sepsis induced thrombocytopenia and in hospital mortality. More studies are recommended on the subject with larger sample size to evaluate its role in sepsis.

Altered von Willebrand Factor and ADAMTS13 Levels in Children With Cirrhosis and Extrahepatic Portal Hypertension.

This study was concerned with whether vWF (von Willebrand factor) and a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13 (ADAMTS13) has altered in patients with cirrhosis and extrahepatic portal hypertension (EPH). We aimed to investigate changes to vWF and ADAMTS13 in children with cirrhosis and EPH. This study was conducted between January and October 2019 with both cirrhosis and EPH patients and with healthy volunteers. The von Willebrand factor antigen (vWF:Ag), von Willebrand Ristocetin cofactor (vWF:RCo), and ADAMTS13 antigen and activity were studied. Twenty-eight children with cirrhosis, 16 children with EPH, and 20 healthy controls were included in the study. vWF:Ag and vWF:RCo levels were higher in patients with cirrhosis than in healthy controls (171.65±101.67 vs. 85.86±30.58, P<0.01 and 121.62±55.83 vs. 61.52±27.03, P<0.01, respectively). vWF:Ag and vWF:RCo levels were higher in patients with EPH than in healthy controls (133.93±80.13 vs. 85.86±30.58, P<0.01 and 103.18±58.55 vs. 61.52±27.03, P=0.02, respectively). The ADAMTS13 antigen and activity levels were lower in patients with cirrhosis than in healthy controls (0.58±0.23 vs. 0.97±0.15, P<0.01 and 49.91±22.43 vs. 86.51±22.07, P=0.02, respectively). The ADAMTS13 antigen and activity levels were lower in patients with EPH than in healthy controls (0.69±0.11 vs. 0.97±0.15, P=0.03; and 68.50±13.29 vs. 86.51±22.07, P=0.02, respectively). The increase in vWF and the decrease in ADAMTS13 were more pronounced in cirrhotic patients with autoimmune hepatitis (AIH) than in non-AIH patients. While levels of vWF:Ag and vWF:RCo increased in children with cirrhosis and EPH, levels of the ADAMTS13 antigen and ADAMTS13 activity decreased. These alterations were more pronounced in patients with AIH-derived cirrhosis.

Congenital Thrombotic Thrombocytopenic Purpura With a Novel ADAMTS13 Gene Mutation

Congenital thrombotic thrombocytopenic purpura (TTP) is an autosomal recessive disorder characterized by thrombocytopenia, microangiopathic hemolytic anemia (MAHA), and thrombosis. Congenital TTP should also be considered while investigating neonatal hyperbilirubinemia, hemolytic anemia, or isolated thrombocytopenia. This case is of an 8-year-old male child who presented with prolonged and recurrent history of thrombocytopenia and MAHA, first identified when he was seven weeks of age preceding neonatal hyperbilirubinemia. Peripheral blood smear examination showed thrombocytopenia and schistocytes. He then went through a series of laboratory investigations until at the age of seven years, when the ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) antigen level was performed and found to be low: 40 ng/ml (630-850). Subsequently, he received a trial of steroids and rituximab which were found to be ineffective and associated with complications. In this case, a definitive diagnosis was delayed until the age of eight years when a novel homozygous pathogenic frameshift variant ADAMTS13 c.3033delC, p.Cys1012AlafsX109 in exon 23 was identified. After receiving regular plasma infusions, thrombocytopenia and hemolysis improved. Congenital TTP should be considered in every neonatal hyperbilirubinemia, thrombocytopenia or hemolytic anemia to avoid delay in diagnosis. Early diagnosis through analysis of the ADAMTS13 gene is crucial for optimal management as well as for genetic counselling.

Reduced Cleavage of Von Willebrand Factor By ADAMTS13 Exacerbates Acute Kidney Injury Secondary to Traumatic Injury

Introduction: Increases in plasma von Willebrand Factor (vWF) antigen levels, accompanied by decreases in its respective metalloprotease, ADAMTS13, have been demonstrated following severe injury. We hypothesized that following traumatic injury, endothelial damage and activation would result in the release of high molecular weight multimeric (HMWM) vWF forms into circulation with a concominant decrease in ADAMTS13. We further hypothesized that the alterations in circulating levels and functions of these factors, following traumatic injury, promote an increased thrombogenic and inflammatory response, which exacerbates the risk of developing post traumatic acute kidney injury (AKI). Methods: Plasma samples from a cohort of patients with trauma induced AKI, collected at 0-, 24-, and 72-hours after admission, were evaluated against plasma samples from trauma patients that were matched for injury characteristics, but were without AKI. VWF and ADAMTS13 activity and antigen levels were evaluated. Circulating vWF multimeric composition was determined by vertical agarose gel electrophoresis. We further evaluated the effects of traumatic injury on ADAMTS13 and vWF by means of a pre-clinical model of trauma and hemorrhage, which is comprised of a liver laceration, cardiac puncture, and soft tissue injury. Following this polytrauma model, hepatic ADAMTS13 RNA expression was evaluated by qRT-PCR of total liver RNA; analyzed liver samples were taken immediately before, 24-, and 72-hours after injury. The impact of the polytrauma model on renal function was evaluated via changes in plasma cystatin C, while the effect on the renal microvasculature was evaluated by means of immunofluorescent staining of vWF and fibrinogen in kidney sections. Results: VWF antigen was increased in trauma patients when compared to healthy controls upon admission (11.0 ± 3.72 ug/mL vs. 4.64 ± 0.91 ug/mL), persisting through 72-hours (13.11 ± 3.16 ug/mL). The percentage of HMWM vWF forms in trauma patient samples was elevated at all time points compared to pooled normal plasma (PNP) (27.9 ± 4.5% and 15.9 ± 0.8). Trauma patient ADAMTS13 activity was decreased at all time points (0.48 ± 0.14 IU/mL vs. 1.27 ± 0.26 IU/mL). AKI patients showed a 1.54 ± 0.48-fold increase in vWF antigen levels over the 72-hour period (p=0.016), while there was no change in the control trauma group. Those who developed AKI demonstrated a larger proportion of HMWM vWF at 72-hours than their non-AKI counterparts (32.9 ± 2.4% vs 28.8 ± 3.2%). qRT-PCR of murine total liver RNA, following the polytrauma model, exhibited ADAMTS13 RNA expression levels that were 35% of normal at 24-hours and 60% of normal at 72 hours post-injury. A matched analysis of murine plasma cystatin C levels demonstrated a marked elevation 24-hours post polytrauma model (sham: 510ng/mL (435-561) vs trauma: 550 (479-701)). H&amp;E staining of kidney sections, which were harvested following the polytrauma model, demonstrated generalized renal tubule dilation and denudation, suggestive of post-traumatic acute tubular necrosis. Immunofluorescent studies of kidney sections from the same mice exhibited 8-15µm microvascular plugs that were positive for fibrinogen with a vWF core; renal immunofluorescent studies of control mice demonstrated no microvascular depositions. Conclusions: These data indicate that traumatic injury alters the circulating composition of ADAMTS13 and its target, vWF, shifting their equilibrium to one that promotes inflammation and thrombosis. In the immediate moments following injury, these mechanisms support life-saving hemostasis; however, as these changes extend into the following days, the early hemostatic benefit quickly shifts to a burden that may produce microvascular clot deposition, exacerbating pathologic states such as acute kidney injury. Disclosures No relevant conflicts of interest to declare.