ADAMTS13 Activity In Patients Research Articles

Persistent ADAMTS13 Inhibitor May Lead to Delayed ADAMTS13 Recovery in Japanese Patients with Caplacizumab-Treated Immune-Mediated Thrombotic Thrombocytopenic Purpura

Introduction Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare condition that causes fatal ischemic end-organ damage due to autoantibodies against ADAMTS13. iTTP patients treated with caplacizumab in addition to standard treatments show a faster time to platelet recovery and fewer incidences of TTP-related organ damage and death. Caplacizumab got approval in Japan in December 2022, while it had been approved in Western countries as far back as 2018 and 2019. Recently, it has been reported that caplacizumab may delay the recovery of ADAMTS13 activity. Therefore, we analyzed the temporal changes of ADAMTS13 activity and its inhibitor in acute iTTP cases before and after the introduction of caplacizumab. Methods iTTP was diagnosed when ADAMTS13 activity was severely decreased (<10%) and ADAMTS13 inhibitor level was ≥0.5 BU/mL. Patients with caplacizumab-treated iTTP underwent once a week or more monitoring for ADAMTS13 activity and inhibitor during the acute phase (caplacizumab group). In addition, cases not treated with caplacizumab were selected that matched the inhibitor levels of the TTP registry since 2019 (non-caplacizumab group). The activity of the plasma ADAMTS13 and its inhibitor were measured using a chromogenic ADAMTS13 activity ELISA (Kinos Laboratories, Tokyo, Japan). Anti-ADAMTS13 IgG levels were analyzed using the TECHNOZYM ADAMTS13 INH ELISA (Tecnoclone, Vienna, Austria). The ADAMTS13 activity, ADAMTS13 inhibitor, and anti-ADAMTS13 IgG antibody levels in both groups were compared on day of onset, day 7, day 14, day 21, and day 28 from the initial therapeutic plasma exchange (TPE) (error margin of ±2 days for each time point). Continuous data were statistically compared using the Mann-Whitney U test. P values of <0.05 were judged as statistically significant. This study was approved by the ethics committee of our university. Results Of the 22 patients included in this study, 11 were treated with caplacizumab (caplacizumab group) and 11 were not (non-caplacizumab group). The median follow-up period was 55 and 74 days in the caplacizumab and non-caplacizumab groups, respectively. The demographic data for both groups are shown in Table. The median duration of caplacizumab administration in the caplacizumab group was 46 days. ADAMTS13 activity was less than 0.5% on admission in all cases. The ADAMTS13 inhibitor was detected in all cases, with no significant difference in the median titer between the caplacizumab group (7.2 BU/mL) and the non-caplacizumab group (5.0 BU/mL) (p=0.2). There was also no statistical significance in the median value of anti-ADAMTS13 IgG antibody between the caplacizumab group (133.6 U/mL) and non-caplacizumab group (73.9 U/mL) (p=0.439). All patients received TPE and corticosteroids. The median number of TPE in the caplacizumab group was significantly lower than that in the non-caplacizumab group (8 vs. 16 days, p=0.003). There was no significant difference in the median number of days from the first TPE to the start of rituximab administration, or in the proportion of rituximab administered between the two groups. After the final TPE, the caplacizumab group took a median of 43 days compared to the non-caplacizumab group (median, 3 days; p=0.003) to achieve ≥20% ADAMTS13 activity, which was significantly longer. The median ADAMTS13 activity level was significantly lower in the caplacizumab group than in the non-caplacizumab group on days 14 (<0.5% vs. 8.7%, p=0.029), 21 (<0.5% vs. 25.5%, p=0.003), and 28 (<0.5% vs. 27.8%, p<0.001), whereas the median ADAMTS13 inhibitor level was significantly higher in the caplacizumab group on the same days: day 14 (5.6 vs. <0.5 BU/mL, p=0.005), day 21 (2.0 vs. <0.5 BU/mL, p=0.009), and day 28 (0.8 vs. <0.5 BU/mL, p<0.001). Similarly, the median level of the anti-ADAMTS13 IgG antibody was significantly higher in the caplacizumab group compared to the non-caplacizumab group at identical time points on day 14 (55.4 vs. 8.3 U/mL, p=0.005) and 21 (median, 27.6 vs. 7.0 U/mL; p=0.016), and day 28 (median, 17.5 vs. 3.7 U/mL; p=0.008). Conclusion We found a delay in the recovery of ADAMTS13 activity in patients treated with caplacizumab, especially after the final TPE. This is potentially due to the persistent presence of ADAMTS13 inhibitor. When administering caplacizumab, closely monitoring for ADAMTS13 activity and its inhibitor is necessary in order to discontinue caplacizumab at the appropriate time.

Effects of convalescent plasma infusion on the ADAMTS13-von Willebrand factor axis and endothelial integrity in patients with severe and critical COVID-19

BackgroundConvalescent plasma infusion (CPI) was given to patients with COVID-19 during the early pandemic with mixed therapeutic efficacy. However, the impacts of CPI on the ADAMTS13-von Willebrand factor (VWF) axis and vascular endothelial functions are not known. ObjectivesTo determine the impacts of CPI on the ADAMTS13-VWF axis and vascular endothelial functions. MethodsSixty hospitalized patients with COVID-19 were enrolled in the study; 46 received CPI and 14 received no CPI. Plasma ADAMTS13 activity, VWF antigen, endothelial syndecan-1, and soluble thrombomodulin (sTM) were assessed before and 24 hours after treatment. ResultsPatients with severe and critical COVID-19 exhibited significantly lower plasma ADAMTS13 activity than the healthy controls. Conversely, these patients showed a significantly increased VWF antigen. This resulted in markedly reduced ratios of ADAMTS13 to VWF in these patients. The levels of plasma ADAMTS13 activity in each patient remained relatively constant throughout hospitalization. Twenty-four hours following CPI, plasma ADAMTS13 activity increased by ∼12% from the baseline in all patients and ∼21% in those who survived. In contrast, plasma levels of VWF antigen varied significantly over time. Patients who died exhibited a significant reduction of plasma VWF antigen from the baseline 24 hours following CPI, whereas those who survived did not. Furthermore, patients with severe and critical COVID-19 showed significantly elevated plasma levels of syndecan-1 and sTM, similar to those found in patients with immune thrombotic thrombocytopenic purpura. Both syndecan-1 and sTM levels were significantly reduced 24 hours following CPI. ConclusionOur results demonstrate the relative deficiency of plasma ADAMTS13 activity and endothelial damage in patients with severe and critical COVID-19, which could be modestly improved following CPI therapy.

Prediction of Relapse Based on Remission ADAMTS13 Activity in Patients with Immune-Mediated Thrombotic Thrombocytopenic Purpura

Background: Acquired, immune-mediated thrombotic thrombocytopenic purpura (iTTP) is defined by thrombocytopenia and microangiopathic hemolytic anemia due to ADAMTS13 deficiency. After the first episode of TTP, about 30-40% of the patients will experience one or more relapse. It has been hypothesized that relapses of iTTP may be associated with severely deficient ADAMTS13 activity during remission. However, some patients with iTTP will remain on remission despite persisting severely decreased ADAMTS13 activity. Moreover, spontaneous recovery of ADAMTS13 activity has also been reported. Whether severe ADAMTS13 deficiency during remission increases risk for relapse is not certain. Therefore we have evaluated the ability of ADAMTS13 activity levels measured during clinical remission to predict relapses of iTTP.Methods: Between March 2004 and June 2017, we collected clinical data and plasma samples from patients who presented to The Ohio State University with clinical diagnosis of iTTP (defined as thrombocytopenia, microangiopathic hemolytic anemia, without an alternative explanation). The diagnosis was confirmed by severely deficient ADAMTS13 activity (<10%). All ADAMTS13 measurements in this cohort were obtained from patients in remission with a platelet count >150 x109/uL and no clinical signs of active TTP. ADAMTS13 activity was determined using a SELDI-TOF mass-spectrometer-based method. For the present study, we only included samples obtained from patients that had never received any prophylactic treatment including rituximab or cyclosporine. To determine the ability of ADAMTS13 activity to predict a relapse in the following 30 and 90 days, we calculated the sensitivity, specificity and likelihood ratio of the test when the ADAMTS13 activity was less than 10% and less than 20%.Results: A total of 557 ADAMTS13 activity measurements were drawn in 39 patients while in remission, with a median interval of 91 days between samples. The demographic characteristics of the population are shown in Table 1. Fourteen patients had no history of relapses and 25 patients had at least one prior relapse. There were 23 relapses in 13 patients during the study period. Of the 557 samples, 145 (26%) were read as less than 10%, 52 (9.4%) were between 10% and 20%, and 360 (64.6%) were >20%. Only 4 (2.78%) and 11(7.59%) of the samples with ADAMTS13 activity <10%, were followed by a relapse in the next 30 and 90 days, respectively. The details of the ADAMTS13 activity during remission are listed in Table 2. The median number of samples from each patient was 10 (range 2 to 41). The sensitivity, specificity and likelihood ratio for relapse for ADAMTS13 activity <10% and 20%, over the following 30 and 90 days are shown in Table 3.Conclusions:- There is a high variability on the ADAMTS13 activity during remission from iTTP, with 25% of the samples showing severe deficiency (<10%) and 35% with activity less than 20%.- With only a small portion of severely deficient samples leading to relapses, both for the 30 and 90-day period, an ADAMTS13 activity of <10% measured during remission does not uniformly lead to relapse.- An ADAMTS13 activity < 20% is a better marker for relapse prediction than < 10%.- These findings call for prospective, randomized studies to evaluate the best biomarker or combination of biomarkers to predict a relapse. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Severely Deficient ADAMTS13 Activity Predicts Relapse of Immune-Mediated Thrombotic Thrombocytopenic Purpura in Pregnancy

Background: It is commonly accepted that severely deficient ADAMTS13 activity in remission increases the risk for relapse, but relapse in severely deficient ADAMTS13 activity in patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) is not uniform. Mouse models and published studies have suggested that a "second hit" is required in addition to severely deficient ADAMTS13 activity to lead to clinical relapse. Our initial published experience led us to preliminarily conclude that pregnancy could serve as the second hit in addition to severely deficient ADAMTS13 activity and lead to relapse. The aim of this study was to evaluate the risk of relapse and outcomes of pregnant patients with a diagnosis of iTTP. Methods: Since the initiation of the Ohio State University TTP Research Program in 2003, patients were consented at enrollment and followed longitudinally in our IRB approved iTTP registry, usually every three to six months. During pregnancy, patients were seen and CBC, LDH and ADAMTS13 activity were obtained monthly. Due to prior reports of iTTP relapses during pregnancy, it is our practice to offer prophylaxis with cyclosporine to pregnant patients with a diagnosis of iTTP and severely deficient ADAMTS13 activity. The clinical diagnosis of iTTP (defined as thrombocytopenia, microangiopathic hemolytic anemia, without an alternative explanation was confirmed by severely deficient ADAMTS13 activity (&lt;10%) in all cases. ADAMTS13 activity was determined using a SELDI-TOF mass-spectrometer-based method. Results: During the study time, we have followed 11 pregnancies from eight patients with iTTP. Two pregnancies occurred at the time of their initial diagnosis, and nine pregnancies occurred during follow up. Two patients were black, nine were white. Median age was 26 ( range 20-36). Of the nine pregnancies in patients with a previous diagnosis of iTTP, three resulted in relapses, two in the same patient; one at 23 weeks that resulted in fetal demise, and the next one occurred one week after delivery at 36 weeks. The other patient relapsed at 35 weeks and the baby was delivered early at 36 weeks. All relapses were preceded by a documented ADAMTS13 &lt;10%, with a normal platelet count, LDH and no associated symptoms, while none of the other pregnancies had a documented ADAMTS13 &lt;10% (Figure 1). Interestingly, one of the relapses occurred while the patient was taking cyclosporine during pregnancy as preemptive therapy to prevent relapses. She initially responded to therapy with an increase in her ADAMTS13 activity but subsequently lost the response which preceded her relapse post-partum. Conclusions: Pregnancy in patients with a diagnosis of iTTP is a risk factor for iTTP relapse. To our knowledge, this is the first report of the relationship between ADAMTS13 and the risk of relapse during pregnancy. Our results demonstrate that severely deficient ADATMS13 activity uniformly leads to relapse in pregnancy. It also highlights the clinical relevance of monitoring ADAMTS13 activity closely in patients with a history of iTTP that become pregnant. Figure 1 Disclosures Masias: Rigel Pharmaceuticals: Consultancy. Cataland:Alexion: Consultancy, Research Funding; Ablynx/Sanofi: Consultancy, Research Funding. OffLabel Disclosure: the use of cyclosporine for prevention of TTP relapses

ADAMTS-13 level in children with severe diarrhea-associated hemolytic uremic syndrome: Unmasking new association.

Severe deficiency of ADAMTS-13 leads to thrombotic thrombocytopenic purpura. Few studies have reported reduced activity of ADAMTS-13 in patients with atypical and typical hemolytic uremic syndrome (HUS). We hypothesized that ADAMTS-13 deficiency might play a role in the pathogenesis of severe HUS. This study aimed to evaluate the ADAMTS-13 level in severe typical HUS. This prospective case-control study was carried out in the Pediatric Nephrology Unit and Clinical Pathology Department, Faculty of Medicine, Zagazig University from February 2013 to February 2014. The study included 15 consecutive children with typical HUS as well as 15 healthy children as a control group. Routine laboratory investigations were performed. Assessment of serum ADAMTS-13 level was performed using the Quantikine human ADAMTS-13 ELISA kit. Data were analyzed using Statistical Package for Social Sciences version 16. Nonparametric values were expressed as median and range, and the median of two groups was tested by Mann-Whitney test. The serum ADAMTS-13 level was significantly lower in HUS patients when compared to the control group (P < 0.05). There were significant negative correlations between ADAMTS-13 level and duration on dialysis, as well as serum urea and creatinine. Furthermore, there were significant positive correlations between serum ADAMTS-13 level and both hemoglobin level and platelet count. Our study suggests that the ADAMTS-13 level was decreased in children with severe typical HUS and its deficiency correlated with disease severity.

Plasma ADAMTS-13 activity in proliferative lupus nephritis: a large cohort study from China.

Objectives The aim of this study was to investigate plasma ADAMTS-13 activity in patients with proliferative lupus nephritis and to evaluate the role of clinical, laboratory and pathological features, especially the vascular lesions in lupus nephritis. Methods Plasma samples from 163 class III and IV lupus nephritis patients confirmed by biopsy examinations and 98 normal controls were collected. ADAMTS-13 activity was evaluated by a residual collagen binding assay. IgG autoantibodies against ADAMTS-13 were detected by ELISA using recombinant ADAMTS-13 as a solid-phase ligand. Levels of vWF were measured by ELISA. Their associations with clinical, laboratory and pathological features were further assessed. Results Plasma ADAMTS-13 activity in lupus nephritis patients was significantly lower than that in normal controls (84 ± 21% vs. 90 ± 13%, p = 0.005). IgG ADAMTS-13 autoantibodies were detected in only three patients. The plasma level of vWF was significantly higher in the lupus nephritis group than in normal controls (1.00 ± 0.79 vs. 0.70 ± 0.30, p = 0.025). Plasma ADAMTS-13 activity was negatively correlated with the level of serum creatinine and proteinuria ( r = -0.354, p < 0.001; r = -0.200, p = 0.011, respectively). Patients with a higher level of ADAMTS-13 activity had significantly higher levels of factor H (401.51 ± 183.01 µg/ml vs. 239.02 ± 155.45 µg/ml, p = 0.005). Plasma ADAMTS-13 activity was negatively associated with total pathological AI scores ( r = -0.326, p < 0.001), endocapillary hypercellularity ( r = -0.419, p < 0.001), cellular crescents ( r = -0.274, p < 0.001), subendothelial hyaline deposits ( r = -0.266, p = 0.001), interstitial inflammatory cell infiltration ( r = -0.304, P < 0.001), tubular atrophy ( r = -0.199, p = 0.011), acute glomerular vascular lesions ( r = -0.344, p < 0.001) and acute renal vascular lesions ( r = -0.338, p < 0.001). No association was found between level of vWF and plasma ADAMTS-13 activity ( r = 0.033, p = 0.671). Low level of ADAMTS-13 activity was a risk factor for renal outcomes ( p = 0.039, HR = 0.047, 95% CI: 0.120-1.005). Conclusions Decreased ADAMTS-13 activity was found in patients with proliferative lupus nephritis, and plasma ADAMTS-13 activity was closely associated with renal injury indices, especially pathological vascular scores. The role of ADAMTS-13 in the disease remains to be further investigated.

Thrombotic risk of reduced ADAMTS13 activity in patients with antiphospholipid antibodies.

Antiphospholipid syndrome (APS) is an autoimmune disease in which antiphospholipid antibodies (aPLs) are generated. Previous studies show concurrence of APS and thrombotic thrombocytopenic purpura; therefore it is plausible to assume that anti-ADAMTS13 autoantibody is also involved in the pathophysiology of APS. We investigated the clinical significance of ADAMTS13 activity and anti-ADAMTS13 antibody in patients with aPLs. Two hundred and sixteen patients with positive lupus anticoagulant and/or anticardiolipin antibody were included. ADAMTS13 activity and anti-ADAMTS13 antibody were measured using fluorescence resonance energy-transfer technology and ELISA, respectively. Reduced ADAMTS13 activity was observed in 40.3% (87/216) of patients with aPLs. Although 33.8% (73/216) of patients were positive for anti-ADAMTS13 antibody, 41 of these 73 patients had normal levels of ADAMTS13 activity. Reduced ADAMTS13 activity was a significant risk factor for thrombotic events. Thrombotic events and age contributed to the reduced level of ADAMTS13 activity. Presence of anti-ADAMTS13 antibody did not show any association with the level of ADAMTS13 activity. Patients with autoimmune diseases tended to show higher levels of anti-ADAMTS13 antibody. Our findings suggest that reduced ADAMTS13 activity is a significant thrombotic risk factor in patients with aPLs irrespective of the presence of anti-ADAMTS13 antibody. Presence of anti-ADAMTS13 antibody is not seen with reduced activity and it tends to be increased in patients with autoimmune diseases.

Changes of ADAMTS13 Activity and TSP1 Level in Patients with Hematologic Malignancies

To investigate the changes of thrombospondin 1(TSP1) level and von Willebrand factor cleaving protease(ADAMTS13) activity in the patients with hematologic malignancies before and after treatment and to evaluate their clinical significance. Eighty-two patients with hematologic malignancies were enrolled in this study, among them 20 patients were with acute leukemia, 48 patients were with lymphoma and 14 patients were with multiple myeloma. The plasma samples of 82 patients with hematologic malignancies and 45 healthy controls were collected. The activities of ADAMTS13 were evaluated by residue collagen binding assay(R-CBA), the levels of TSP1 and vWF antigen were measured by enzyme-linked immunosorbent assay(ELISA). The activity of plasma ADAMTS13 in patients with hematologic malignancies was lower than that of normal controls(P<0.05). The levels of vWF antigen and TSP1 in the patients with hematologic malignancies were higher than those in normal controls(P<0.05). After standard induction chemotherapy, the ADAMTS13 activity of the patients with hematologic malignancies at the complete remission was higher than that before therapy(P<0.05); the vWF antigen level was significantly lower than that in the patients with hematologic malignancies before therapy(P<0.05), but still higher than that in controls(P<0.05). There were 25 infection patients in 82 cases of hematologic malignancies, and the ADAMTS13 activity in the patients with newly diagnosed hematologic malignancies complicated with infection before therapy was obviously lower than that in the patients with hematologic malignancies without infection(P<0.05), the levels of vWF antigen and TSP1 were significantly lower than that in patients without infection (P<0.05). In the process of treatment, 8 patients have been speculated to suffer from thrombus, and the ADAMTS13 activity in the patients with thrombus was obviously lower than that in the patients without thrombus(P<0.05). Low ADAMTS13 activity and high TSP1 level may participate in the progress of hematologic malignancies, the infection and thrombotic events may lead to further reduction of the ADAMTS13 activity. Assaying the level of ADAMTS13 activity in the patients with malignant tumor may be helpful to prevent the infection and thrombosis in the patients with hematologic malignancies.

Unconjugated bilirubin inhibits proteolytic cleavage of von Willebrand factor by ADAMTS13 protease

Bilirubin is a yellow breakdown product of heme catabolism. Increased serum levels of unconjugated bilirubin are conditions commonly seen in premature neonates and adults with acute hemolysis including thrombotic microangiopathy. Previous studies have shown that unconjugated bilirubin lowers plasma ADAMTS13 activity, but the mechanism is not fully understood. The study is to determine whether unconjugated bilirubin directly inhibits the cleavage of von Willebrand factor (VWF) and its analogs by ADAMTS13. Fluorogenic, surface-enhanced laser desorption/ionization time-of-flight mass spectrometric assay, and Western blotting analyses were used to address this question. Unconjugated bilirubin inhibits the cleavage of F485-rVWF73-H, D633-rVWF73-H, and GST-rVWF71-11K by ADAMTS13 in a concentration-dependent manner with a half-maximal inhibitory concentration of ~13, ~70, and ~17 μmol L(-1) , respectively. Unconjugated bilirubin also dose-dependently inhibits the cleavage of multimeric VWF by ADAMTS13 under denaturing conditions. The inhibitory activity of bilirubin on the cleavage of D633-rVWF73-H and multimeric VWF, but not F485-rVWF73-H, was eliminated after incubation with bilirubin oxidase that converts bilirubin to biliverdin. Furthermore, plasma ADAMTS13 activity in patients with hyperbilirubinemia increased after treatment with bilirubin oxidase. Unconjugated bilirubin directly inhibits ADAMTS13's ability to cleave both peptidyl and native VWF substrates in addition to its interference with certain fluorogenic assays. Our findings may help proper interpretation of ADAMTS13 results under pathological conditions. Whether elevated serum unconjugated bilirubin has prothrombotic effect in vivo remains to be determined in our future study.

Pregnancies Complicated By Severe Preeclampsia Exhibit Perturbed Von Willebrand Factor (VWF)-Associated Parameters

BackgroundElevated levels of the critical coagulation glycoproteins von Willebrand Factor (VWF) and Factor VIII (FVIII) are associated with thrombovascular disease. VWF and FVIII levels are elevated in healthy pregnancy compared with the nonpregnant state, and further increases have been reported in pregnancies complicated by preeclampsia, a potentially life-threatening vascular disorder of pregnancy. Aside from the quantitative increases in VWF and FVIII, data is inconsistent or little is known regarding other key VWF-associated parameters in pregnancies complicated by preeclampsia. HypothesisWe hypothesized that pregnancies complicated by severe preeclampsia would exhibit distinct changes in VWF-associated parameters compared to healthy pregnancies. MethodsWe assayed VWF-associated parameters in subjects newly diagnosed with severe preeclampsia, based on standard blood pressure (BP) and proteinuria criteria. Severe preeclampsia was defined on the basis of one or more of the following: severe hypertension (systolic BP >160 or diastolic BP >110) for at least 6 hours, seizures (eclampsia), hemolysis, elevated liver enzymes, thrombocytopenia, pulmonary edema, renal dysfunction, or fetal growth restriction. We compared results from preeclamptic pregnancies to gestational age-matched healthy pregnant controls. VWF antigen (VWF:Ag), VWF propeptide (VWF:pp), and FVIII antigen (FVIII:Ag) levels were determined by ELISA. Factor VIII activity (FVIII:C) was measured using a one-stage assay and ADAMTS13 activity was determined via cleavage of an enzyme-linked peptide substrate. ResultsForty-two patients with severe preeclampsia and a cohort of thirty-nine controls of similar gestational ages met study inclusion criteria (Table 1). The results of the coagulation assays are presented in Table 2. In summary, preeclamptic pregnancies were notable for statistically significant (p<0.001) increases in VWF:Ag (1.4-fold), VWF:pp (1.5-fold), and FVIII:Ag (1.2-fold) compared to controls. FVIII:Ag and FVIII:C were similar, and FVIII:Ag was used in the calculation of the VWF:FVIII ratio. The VWF:FVIII ratio trended higher in preeclamptic pregnancies, but this did not reach statistical significance (p=0.058). The mean ADAMTS13 activity in patients with preeclampsia was also significantly lower (0.76-fold, p<0.001) than healthy pregnancy controls. ConclusionsIn pregnancies complicated by severe preeclampsia, VWF:Ag and FVIII were significantly elevated, as expected. VWF:FVIII ratios also tended to be higher in preeclamptic pregnancies compared to controls. Interestingly, activity of the VWF-cleaving protease ADAMTS13 was modestly but significantly decreased in preeclampsia pregnancies, a finding for which there is conflicted literature. Three subjects with thrombocytopenia had ADAMTS13 activities >85%, indicating the thrombocytopenia was unrelated to a relative ADAMTS13 deficiency. These findings support the hypothesis that pregnancies complicated by severe preeclampsia exhibit VWF biology that is distinct from healthy pregnancy. We speculate that preeclampsia-associated VWF differences could play a direct role in the vascular pathogenesis and end-organ damage of preeclampsia. These data offer new clues towards better understanding of the mechanisms of severe preeclampsia and represent promising future targets for diagnosis and/or treatment. Disclosures:No relevant conflicts of interest to declare.

Increased ADAMTS13 activity in patients with venous thromboembolism

Increased levels of inflammatory markers and clotting factors have been related to the pathogenesis and prognosis of venous thromboembolism. In particular, the imbalance between VWF and ADAMTS13 has been described in patients with arterial thrombosis. In this study, 77 patients with previous VTE and 77 matched controls were selected for the evaluation of the inflammatory markers, FVW, ADAMTS 13 and D-dimer. The presences of post-thrombotic syndrome and residual vein obstruction were also assessed in patients. Serum levels of TNF-α and IL-6 were significantly increased in patients compared to controls (median=2.25 vs 1.59pg/mL, P≤0.001; 1.16 vs 0.98pg/ml, P=0.013, respectively). Plasma levels and activity of VWF (median=150.25 vs 95.39U/dL, P≤0.001; 145.26% vs 92.39%, P≤0.001) and ADAMTS 13 (median=1088.84 vs 950.80ng/mL, P≤0.001; 96.03 vs 83.64%, P≤0.001) were also higher in patients. We further analysed the subgroups of patients with higher risk for VTE recurrence or VTE sequelae, defined as the presence of high D-dimer levels, RVO or PTS. All inflammatory markers were significantly higher in patients with increased D-dimer. The presence of PTS or RVO was not associated with higher inflammatory or coagulation parameters. The increased levels of inflammatory markers and VWF may suggest that there is a persistence of inflammatory activity in patients even at long periods after the VTE episode. In this context, it may be postulated that increased levels of ADAMTS13 could represent a compensatory mechanism against persistently increased levels of VWF. Moreover, increased inflammatory activity was associated with increased D-dimer levels, thus it is possible that this inflammatory activity may also be related to the risk of VTE recurrence.

Role of a Tissue Factor Pathway Inhibitor, the Plasma Proteinase ADAMTS-13 and its Inhibitor in the Pathogenesis of Acute Respiratory Distress Syndrome in A/H1N1 Influenza

Objective: to study the role of a tissue factor pathway inhibitor (TFPI), the metalloproteinase ADAMTS-13, and its inhibitor in the pathogenesis of acute respiratory distress syndrome (ARDS) in A/H1N1 influenza. Subjects and methods. The study enrolled 52 patients of both sexes aged 19 to 40 years with different clinical forms of A/H1N1 influenza, of whom 14 patients developed Stages I-II ARDS in the presence of pneumonia. An enzyme immunoassay was used to estimate the concentrations of TFPI, ADAMTS-13, and its inhibitor, the antibodies of the IgG class. Results. The level of TFPI increased in parallel with the progression of the disease, its highest concentration was found in patients with ARDS-complicated influenza pneumonia. There was a higher relative activity of ADAMTS-13 in patients with moderate pneumonia while this enzyme was relatively deficient in those with ARDS-complicated pneumonia. Conclusion. Relative ADAMTS-13 deficiency is one of the triggers of ARDS in severe forms of pneumonia in the presence of A/H1N1 influenza. The high level of TFPI may predict the development of respiratory distress syndrome in pneumonia in the presence of influenza infection. Key words: A/H1N1 influenza, respiratory distress syndrome, tissue factor pathway inhibitor, ADAMTS-13.

Decreased Activity of ADAMTS13 In Patients with Deep Vein Thrombosis Associated with Inflammatory Markers

AbstractAbstract 3181 Introduction:Elevated levels of inflammatory markers and clotting factors have been related to the pathogenesis of DVT. Particularly, the balance between VWF and ADAMTS 13 activity has been previously described in patients with thrombosis. VWF levels are also described to be elevated during inflammatory processes and therefore could play a role linking the activation of inflammatory and coagulation systems in patients with DVT. Objective:To evaluate the activity of ADAMTS13 and VWF in patients with DVT and its association with inflammatory markers. Patients and Methods:Fifty-five patients with DVT, 6 months to five years after the acute episode, attended at the Hematology Center of UNIFESP were included in this study and 121 normal subjects were selected as controls. The activity of ADAMTS 13 was performed by binding of residual VWF to collagen; VWF antigen, IL-6, IL-8, F1+2 and MCP1 was determined by ELISA and D-dimer and CRP was performed by turbidimetry. Continuous variables were analyzed by Mann- Whitney test, categorical variables by the chi-square test and Spearman rank test was used for correlation analysis. Results:Thirty-tree patients had DVT caused by transient risk factor, especially the use of oral contraceptives, and 22 patients had spontaneous DVT. No patient had renal, hepatic or malignant disease. The median ADAMTS 13 activity was not statistically different between patients (median= 65.8%) and controls (median=78.5%, p= 0.3043). The plasma levels of VWF antigen, IL-6 and CRP were higher in patients than in controls (p= 0.008, p= 0.0003 and p= 0.008, respectively). The levels of IL-8 and MCP1 did not differ betwee n patients and controls (p= 0.65 and p= 0.07, respectively). The levels of VWF correlated significantly with CRP (r= 0.28, p= 0.005) and F1+2 (r= 0.3, p= 0,02) in patients. There was an inverse correlation between ADAMTS13 activity and F1+2 concentrations (r= -0,33, p= 0.01). Conclusion:This study suggests that, even after the acute episode of DVT, a chronic inflammatory state would contribute to increasing levels of VWF in patients' plasma and hypercoagulability. The downregulation of ADAMTS13 activity may also contribute to the activation of the coagulation. However the role of ADAMTS13 in the interaction between inflammatory and coagulation systems in patients with thrombosis is yet to be determined. These factors together may contribute to recurrent DVT, which affects over 25% of patients within 10 years from the first episode. Disclosures:No relevant conflicts of interest to declare.

DECREASED ACTIVITY of ADAMTS13 IN PATIENTS with DEEP Vein Thrombosis.

Abstract 3991Poster Board III-927 IntroductionSeveral risk factors, including increased levels of factor VIII (FVIII) and von Willebrand factor (VWF), have already been identified in patients with deep vein thrombosis (DVT). Recently we published a study showing that in a Brazilian population, plasma levels of FVIII over 180U/dl and VWF over 165U/dl were associated with the occurrence of venous thrombosis (odds ratio = 4.1 and 3.8 respectively) (Mello TB et al, 2009). The level of VWF in plasma and consequently FVIII is the result of genetic and acquired factors. ADAMTS13 (ADisintegrin And Metalloprotease with Thrombospondin type 1 repeats) is an enzyme responsible for cleavage of VWF, and its activity could contribute to VWF and FVIII plasmatic levels in patients with DVT. ObjectiveTo evaluate the activity of ADAMTS13 in patients with DVT associated with an increase of VWF and FVIII. Patients and methods: Fifty-six patients with FVIII > 180U/dl or FVW>165U/dl were selected from a cohort of 175 patients with DVT from the study mentioned above. Fifty-four normal subjects were selected as controls. The activity of ADAMTS 13 was performed by binding of residual VWF to collagen; VWF activity was measured by collagen binding, VWF antigen was determined by ELISA and FVIII was measured by a one-stage coagulation assay. Continuous variables were analyzed by Mann-whitney test and categorical variables by the Chi-square test. ResultsThe demographic distribution of patients and controls were similar. Among the 56 patients the median age was 37.5 years, 39 were women, 46 had blood typing “non-O”, 34 had DVT caused by transient risk factor, especially the use of oral contraceptives, 10 patients had a hereditary thrombophilia and 3 were carriers of antiphospholipid antibodies. No patient had renal, hepatic or malignant disease. The median ADAMTS 13 activity was significantly lower in patients (112.9%, 44.4 - 327.6%) when compared to controls (142.9%, 76.7 - 323.6%), P = 0.001. The VWF activity was also higher in patients (109.7%, 26.8 - 422.6%) when compared to controls, (79.1%, 45.5 - 203.8%), P=0.038. The median level of VWF antigen was significantly higher in the group of patients when compared to the control group (178.1U/dl versus 111.9 U/dl respectively, P <0.0001). There was an inverse correlation between ADAMTS13 activity and VWF activity. Conclusion: This study suggests that the increased VWF and FVIII activity in patients with DVT can be a result of decreased ADAMTS13 activity. The decreased activity of ADAMTS13 may be influenced by the action of cytokines in inflammatory processes, even after acute period. Future studies will be important to determine the correlation between activity of ADAMTS 13, VWF, and inflammatory markers in the pathogenesis of DVT. Disclosures:No relevant conflicts of interest to declare.

Decreased ADAMTS13 Levels in Patients after Living Donor Liver Transplantation

Introduction Thrombotic microangiopathy (TMA) is a complication occurring after liver transplantation (LT), and an unusually large multimer (ULM) of Von Willebrand factor (VWF) and ADAMTS13 may play an important role in the onset of TMA during LT. Material and Methods Eight-one patients underwent living donor LT (LDLT). Seventeen of those patients had both severe thrombocytopenia and hemolytic anemia with fragmented red cells and were diagnosed as TMA- like syndrome (TMALS). Results and Conclusions A significant reduction of ADAMTS13 and an increase of VWF were observed in the patients with TMALS. The ADAMTS13 activity in patients after LDLT was significantly reduced from day 1 to day 21, and it was significantly low in those with TMALS at day 14 and 28. The VWF levels in patients with LDLT were significantly high, and the VWF/ADAMTS13 ratio was significantly increased in patients at 7, 14 and 28 days after LDLT, especially in patients with TMALS at day 14 and 28 after LDLT. High molecular weight multimers of VWF were observed to have increased in patients with LDLT, and the high molecular weight multimers of VWF were further increased in those with mild TMALS but they decreased in those with severe TMA. These findings suggest that ULM- VWF and ADAMTS13 might be associated with the onset of TMA after LT.

ADAMTS13: what it does, how it works, and why it's important

ADAMTS13: what it does, how it works, and why it's important

Identification of Trigger Factors Initiating an Acute TTP Episode in 68 Patients.

Recent research efforts have provided significant insight into the pathogenesis of thrombotic thrombocytopenic purpura (TTP). However, the low incidence of the disease hampers the identification of trigger factors initiating an acute TTP episode. In the present work data from 68 TTP patients were analyzed. The patients were asked by a questionnaire to provide data concerning age, gender, medical history, underlying and concomitant diseases and potential triggers. Additional laboratory and clinical data from onset of disease and remission phase were obtained by a second questionnaire completed by the physicians. Our study showed that long term abuse of nicotine is a predisposing factor with an OR of 3.19. The OR for obesity and development of TTP was 2.01. The influence of nicotine abuse has not yet been described whereas the association between obesity and development of TTP is already known. The intake of oral contraceptives (13 of 46 women/28%) of Tamoxifen, of Fluvastatin, of Ticlopidine and of Quinine was identified to be potential triggers. 55% (34 patients) of the patients reported infections and inflammations as triggers (mostly influenza (n=16) and gastroenteritis (n=14)). The relation of female and male patients was 3:1. The mean age, in which the initial TTP episode occurred, was significantly lower in female patients compared with the male patients (33.8 versus 43.8 years). Severe ADAMTS-13 deficiency was found in 91% in all investigated episodes of the patients. Inhibitory activity was associated with severe ADAMTS-13 deficiency in 81% of the cases. The comparison of patients during remission with and without relapses of TTP revealed a significant lower ADAMTS-13 activity in patients with relapsing TTP compared to patients without a relapse of TTP. This result suggests that ADAMTS-13 activity has prognostic value with respect to the occurrence of relapse.

Increased Plasma VWF Antigen over Decreased ADAMTS13 Activity in Patients with Liver Cirrhosis: Relationship to Degree of Liver Disturbance and Clinical Features.

Background/aims: Deficiency of plasma ADAMTS13 activity (:act) accumulates unusually large von Willebrand factor multimer (UL-VWFM) in circulation that might induce platelet thrombi formation. We demonstrated that hepatic stellate cells are major ADAMTS13-producing cells in human liver using in situ hybridization and immunohistochemistry (Blood , 2005, 106:922), and a decreased plasma ADAMTS13:act in patients with cirrhotic biliary atresia can be fully restored after living-related liver transplantation (Blood 2000, 96:636a). Taken these findings together, ADAMTS13 may play a role on the regulation of sinusoidal microcirculation in the liver. We, therefore, investigated the relationship of ADAMTS13 and its substrate (UL-VWFM) to clinical features in patients with chronic liver diseases.Methods: Plasma levels of ADAMTS13:act, ADAMTS13 antigen (:ag) and VWF antigen (VWF:ag) were determined in 33 patients with chronic hepatitis (CH) and 109 liver cirrhosis (LC). ADAMTS13:act was measured by both the classic VWFM assay and the novel monoclonal antibody-based ELISA (Transfusion 2006, 46:1444). The ADAMTS13:ag was quantified by a sandwich ELISA using two anti-ADAMTS13 murine monoclonal Abs (A10 and C7), and the UL-VWFM was analyzed by a SDS−0.9% agarose gel electrophoresis.Results: ADAMTS13:act in LC progressively decreased from the highest in Child A (mean 79%), to Child B (63%), to the lowest in Child C (30%), compared with CH (87%) and normal healthy subjects (N, 102%). The activity measured by VWFM assay highly correlated with that assayed by ELISA (r=0.75, p<0.001), which was also correlated with ADAMTS13:ag (r=0.79, p<0.001). Markedly decreased ADAMTS13 :act was noted in cirrhotics with hepatic encephalopathy (28%), refractory ascites (25%), and hepatorenal syndrome (13%). The multivariate analysis showed Child-Pugh score and spleen volume as independent factors contributing to the activity (r=0.69, p<0.001). VWF:ag was remarkably high with the progression of liver disturbance (N 100%, CH 245%, Child A LC 320%, Child B LC 436%, Child C LC 486%, respectively), resulting in extremely increased ratio of VWF:ag to ADAMTS13 activity in advanced cirrhotics (N 1.0, CH 2.9, Child A LC 4.3, Child B LC 13.3, Child C LC 43.2, respectively). The cirrhotics with UL-VWFM showed higher Child-Pugh score (10.8 vs. 7.7, p<0.001), lower hemoglobin levels (9.6 g/dl vs. 11.3 g/dl, p<0.005), severe thrombocytopenia (6.2 x104/mm3 vs. 9.2 x104/mm3, p<0.05), higher C-reactive protein (3.8 mg/dl vs. 0.9 mg/dl, p<0.001), and higher serum creatinine (1.5 mg/dl vs. 0,9 mg/dl, p<0.005) than those without.Conclusions: Both the activity and antigen of plasma ADAMTS13 decrease with increasing severity of cirrhotics. The imbalance of a larger amount of UL-VWFM over the decreased plasma level of ADAMTS13 may reflect the predisposing state for the development of microcirculatory disturbance in advanced cirrhotics.

Microparticles Are Active Components of Therapeutic Plasma Exchange: Correlation with ADAMTS13 Activity in Patients with Thrombotic Thrombocytopenic Purpura.

INTRODUCTION: Therapeutic plasma exchange (TPE) is a widely accepted therapy for TTP. A leading hypothesis for its benefit is that TPE replenishes ADAMTS13 with fresh frozen plasma (FFP) and removes inhibitory antibodies. We have reported that endothelial microparticles (EMP) positive for ULvWF are elevated in acute TTP, decline with repeated TPE, and can inhibit ADAMTS13. Presence of platelet microparticles (PMP) in FFP has been described. This led us to the hypothesis that the benefit of TPE is partly due to its effect on microparticles (MP). We investigated the effect of TPE on MP and ADAMTS13 in TTP.METHODS: Nine patients were studied longitudinally during an episode of TTP. Blood samples were obtained upon admission and daily pre- and post-TPE. Additionally, the infused and removed plasma were studied. EMP defined by CD31+/CD42b− (EMP31), CD62E+ (EMP62) and CD62E+/vWF+ (EMPVWF); PMP were defined by CD31+/CD42b+ (PMP42) and CD41+ (PMP41), all measured in platelet-poor plasma by flow cytometry; concentrations are in millions / mL. ADAMTS13 activity was assayed by a FRETS-vWF73 method of Kokame et al [ Br J Haematol 2005; 129:93]. vWF multimers were analyzed following Raines et al [ Thromb Res 1990; 60:201].RESULTS: At admission, TTP patients exhibited very low ADAMTS13 levels, 0–10% of controls (ctl), and elevated EMP31 (2.5 ±0.7 vs. 0.35 ±0.09 ctl) and EMP62 (6.3 ±1.9 vs. 0.25 ±0.07 ctl), both p<0.001. After the first TPE, EMP fell significantly, to 1.76 ±0.85 for EMP31 and 3.4 ±1.3 for EMP62, both p<0.02. Similarly, EMPVWF dropped from 3.7 ±1.8 at admission (ctl = 0.28 ±0.20) to 2.6 ±1.0 after the first TPE, p=0.02. In parallel, ADAMTS13 activity slowly increased with each TPE, finally to near normal, 85 ±22% of ctl. These findings suggest a relationship between ADAMTS13 activity and MP counts. The MP fraction isolated from TTP patients had no detectable ADAMTS13 activity. When the MP fraction from TTP patients was incubated with normal particle free plasma (PFP) for 6hr, then centrifuged to remove MP, a significant fraction of activity was lost (25 ±8%, p=0.02), and no activity could be detected in the resuspended MP. Examination of the vWF multimer composition of the MP fraction of TTP patients exhibited enrichment of large vWF. When we assayed FFP for EMP, PMP and ADAMTS13 activity, we found EMP and LMP comparable to controls (EMP31 = 0.4 ±0.25; EMP62 = 0.31 ±0.2). Only PMP were significantly elevated in FFP (PMP42 = 4.4 ±1.8; PMP41 = 9.0 ±3.2). When we compared ADAMTS13 activity in the MP fraction and the PFP of FFP, the MP fraction accounted for 35 ±12% of the total activity. MP from FFP did not exhibit ULvWF. We consistently observed that immediately following each TPE, both EMP31 and EMP62 were reduced by 15–30%, with a parallel increase in both species of PMP of 10–15%. Concomitantly, EMP and PMP profiles in plasma removed through TPE from TTP patients remained comparable to samples obtained prior to the TPE.CONCLUSION: These results suggest that one action of TPE in TTP is to remove EMP which tend to bind and inactivate ADAMTS13. Thus, the basis of TPE is two-fold: infusion of ADAMTS13 by FFP infusion, and reduction of high levels of inhibitors including inhibitory antibodies as well as EMP.

In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a disease characterized by microvascular thrombosis, often associated with deficiency of the vonWillebrand factor (VWF) cleaving protease ADAMTS13. We investigated the spectrum of ADAMTS13 gene mutations in patients with TTP and congenital ADAMTS13 deficiency to establish the consequences on ADAMTS13 processing and activity. We describe five missense (V88M, G1239V, R1060W, R1123C and R1219W), 1 nonsense (W1016Stop) and 1 insertion (82_83insT) mutations. In two patients no mutation was identified despite undetectable protease activity. Expression in HEK293 mammalian cells (V88M, G1239V, R1123C and R1219W) documented that three missense mutants were not secreted, whereas theV88M was secreted at low levels and with reduced activity. We also provide evidence that impaired secretion of ADAMTS13 mutants observed in vitro translates into severely reduced ADAMTS13 antigen levels in patients in vivo. To evaluate whether the small amounts of mutant protease present in the circulation of patients had VWF cleaving activity, WT and mutant rADAMTS13 were stably expressed in Drosophila S2 cells under the influence of the Drosophila BiP protein signal sequence, which allows protein secretion. Drosophila expression system showed a 40-60% protease activity in the mutants. Several single nucleotide polymorphisms (SNPs) within exons and intron boundaries were found in patients, suggesting that the interplay of SNPs could at least in part account for ADAMTS13 functional abnormalities in patients without mutations. In conclusion, defective secretion and impaired activity of the mutants concur to determine an almost complete deficiency of ADAMTS13 activity in patients with a homozygous or two heterozygous ADAMTS13 mutations.