ADAMTS13 Inhibitor Research Articles

Frontline use of rituximab may prevent ADAMTS13 inhibitor boosting during caplacizumab treatment in patients with iTTP: post hoc analysis of a phase 2/3 study in Japan

BackgroundA recent Phase 2/3 study in Japanese patients showed that caplacizumab was effective in treating immune-mediated thrombotic thrombocytopenic purpura (iTTP), with a low rate of iTTP recurrence. ADAMTS13 activity is monitored weekly during caplacizumab treatment to guide discontinuation of caplacizumab and consequently avoid exacerbations or relapse. The aim of this study was to assess changes in ADAMTS13 activity/inhibitor levels during caplacizumab treatment in this patient population.MethodsA post hoc analysis of the Phase 2/3 study in Japanese patients was conducted. Patients ≥ 18 years old with confirmed iTTP received 10 mg of caplacizumab daily in conjunction with therapeutic plasma exchange (TPE) and immunosuppression for 30 days post-TPE. Outcomes included time to recovery of ADAMTS13 activity, ADAMTS13 activity level at treatment end, incidence of ADAMTS13 inhibitor re-elevation (ie, inhibitor boosting) during treatment, time to platelet count recovery, number of days of TPE, and safety. Outcomes according to presence of inhibitor boosting were also assessed.ResultsNineteen patients had confirmed iTTP and were included in this analysis. Median (95% confidence interval) time to recovery of ADAMTS13 activity to ≥ 10%, ≥ 20%, and ≥ 60% was 14.6 (5.9–24.8), 18.5 (5.9–31.8), and 47.5 (18.5–60.9) days, respectively. Median (range) ADAMTS13 activity level at caplacizumab treatment end was 62.0% (29.0–101.0). Nine patients had ADAMTS13 inhibitor boosting. Delayed response of ADAMTS13 activity was observed in patients with inhibitor boosting. The median time to platelet count response and median number of TPE days were shorter in patients with inhibitor boosting compared with patients without inhibitor boosting. Rituximab was administered to almost all patients with inhibitor boosting (88.9%), after completion of TPE. Patients without inhibitor boosting who were treated with rituximab received it prior to completion of TPE. Only one patient experienced a recurrence, which occurred shortly after caplacizumab discontinuation due to an adverse event.ConclusionsIn patients with iTTP, caplacizumab with TPE and immunosuppression may reduce the risk of ADAMTS13 inhibitor boosting if rituximab is administered early in the iTTP treatment period. Early administration of rituximab in addition to caplacizumab may prevent iTTP recurrence with inhibitor boosting.Trial registrationNCT04074187.

COVID-19 impact on thrombotic complications in cancer patients

Aim: to study the features of thrombotic complications in cancer patients during COVID-19 infection, and identify the most significant diagnostic and prognostic criteria.Materials and Methods. Within the framework of cohort non-randomized study, there were analyzed the course of coronavirus infection in 72 hospitalized patients with uterine cancer (n = 22), cervical cancer (n = 19), ovarian cancer (n = 24) as well as vaginal and vulvar cancer (n = 7). All patients hospitalized for COVID-19 were examined and treated in accordance with the Interim guidelines “Prevention, diagnosis and treatment of novel coronavirus infection (COVID-19)” effective at the time of therapy. Additionally, on days 3–7 after hospitalization, a blood test was performed once to determine the level of metalloproteinase ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13), ADAMTS-13 activity, ADAMTS-13 inhibitor and von Willebrand factor (vWF) level.Results. It was shown that the average age of the patients was 56.96 ± 7.55 years, the length of hospitalization ranged from 7 to 19 (13.0 ± 3.79) days. The disease severity was assessed by the degree of lung tissue damage based on computed tomography (CT), respiratory failure and development of systemic inflammatory response syndrome (SIRS). During hospitalization, CT-2 was detected in 46 (63.9 %) patients, CT-3 – in 26 (36.1 %) patients; 37 (51.4 %) patients were transferred to the intensive care unit (ICU). Clinically significant deep vein thrombosis (DVT) was diagnosed in 9 (12.5 %) patients, and pulmonary embolism (PE) – in 4 (5.6 %) patients. Of these, 6 cases of DVT and 3 cases of PE were fatal. A total of 14 (19.44 %) patients deceased due to developing acute cardiopulmonary failure. A vWF/ADAMTS-13 ratio greater than 2.1 was found in all ICU patients. Despite anticoagulant therapy, patients with DVT and PE had this ratio higher than 3.3 (4.00 ± 0.48), whereas in all 14 deceased patients it exceeded 2.98.Conclusion. Venous thromboembolism, including PE and DVT, has been identified as а serious complication of COVID-19. An opportunity to predict them early is of special importance because they may lead to serious complications such as disseminated intravascular coagulation, SIRS, cardiopulmonary failure, and death. In patients suffering from cancer infected with COVID-19, not only a decline in ADAMTS-13 activity and level was detected, but also a parallel increase in vWF level. A vWF/ADAMTS-13 ratio may be an early indicator of COVID-19 severity in such patients: a vWF/ADAMTS-13 ratio exceeding 2.1 was common for all ICU patients. Hence, it evidences about a potential for using this parameter to early identify such risk patients who may require more intensive care and medical intervention.

Persistent ADAMTS13 inhibitor delays recovery of ADAMTS13 activity in caplacizumab-treated Japanese patients with iTTP

AbstractFor patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP), caplacizumab, a nanobody against von Willebrand factor A1 domain, has become crucial. Delayed normalization of ADAMTS13 activity during caplacizumab therapy has been identified. In a retrospective analysis, we compared platelet count, ADAMTS13 activity, its inhibitor, and anti-ADAMTS13 immunoglobulin G (IgG) levels in acute iTTP cases treated with caplacizumab (n = 14) or without it (n = 16). The median time from initial therapeutic plasma exchange (TPE) to the first rituximab administration was 12 days in the caplacizumab group (n = 11) and 10 days in the group without caplacizumab (n = 13). We evaluated ADAMTS13-related parameters at onset and once a week until day 28 after the first TPE. The number of days until the platelet counts reached ≥150 × 109/L was significantly shorter in the caplacizumab group than in the non-caplacizumab group. The median ADAMTS13 activity levels on days 14, 21, and 28 were significantly lower in the caplacizumab group. The median titers of the ADAMTS13 inhibitor and anti-ADAMTS13 IgG on the same days were significantly higher in the caplacizumab group. Furthermore, the median number of days from the first TPE until finally achieving an ADAMTS13 activity of ≥10% was significantly longer in the caplacizumab group than in the non-caplacizumab group (42 vs 23 days, P = .014). We observed delayed ADAMTS13 activity recovery and continued inhibitor and anti-ADAMTS13 IgG detection in patients with acute iTTP on caplacizumab, possibly because of the decreased number of TPEs and delayed frontline rituximab.

Acquired thrombotic thrombocytopenic purpura with rituximab-induced serum sickness

The patient was a 38-year-old man who was admitted to our hospital with fever, vomiting and unclear speech. Blood tests showed hemolytic anemia, thrombocytopenia, and elevated creatinine, and a Coombs test was negative. ADAMTS13 activity was <1% and ADAMTS13 inhibitor was 0.7 BU/ml, so we diagnosed acquired thrombotic thrombocytopenic purpura (aTTP). We started plasma exchange and prednisolone 1 mg/kg on the day of admission (day 1). The platelet count recovered to normal on day 5, and hemolytic findings improved. However, symptoms worsened again, and thus we started rituximab for refractory aTTP on day 24. After rituximab administration, the patient showed no signs of infusion reaction (IR) or recurrence. On day 31, he received the second dose of rituximab and was discharged. On day 38, he had knee pain, fever, erythema, and difficulty moving. CRP was elevated and CH50 slightly decreased. Imaging findings were normal. Anti-rituximab antibody was positive (197 ng/ml), leading to the diagnosis of rituximab-induced serum sickness. Rituximab administration was discontinued, and the serum sickness has not recurred. The presence of anti-rituximab antibodies may aid diagnosis in patients with suspected symptoms of serum sickness or recurring IR-like symptoms.

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 (&amp;lt;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 &amp;lt;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 (&amp;lt;0.5% vs. 8.7%, p=0.029), 21 (&amp;lt;0.5% vs. 25.5%, p=0.003), and 28 (&amp;lt;0.5% vs. 27.8%, p&amp;lt;0.001), whereas the median ADAMTS13 inhibitor level was significantly higher in the caplacizumab group on the same days: day 14 (5.6 vs. &amp;lt;0.5 BU/mL, p=0.005), day 21 (2.0 vs. &amp;lt;0.5 BU/mL, p=0.009), and day 28 (0.8 vs. &amp;lt;0.5 BU/mL, p&amp;lt;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.

Post Hoc Analysis of a Phase 2/3 Study in Japanese Patients with iTTP Treated with Caplacizumab

Introduction Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare, life-threatening disease caused by a severe deficiency of a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13). Caplacizumab is a von Willebrand factor-directed antibody fragment designed to treat iTTP in conjunction with therapeutic plasma exchange (TPE) and immunosuppressive therapy. Caplacizumab was approved in Europe in 2018, in the United States in 2019, and in Japan in 2022. Methods In the open-label, Phase 2/3 study (NCT04074187), Japanese patients ≥18 years old with a clinical diagnosis of iTTP and ≤1 prior TPE received caplacizumab in combination with TPE and immunosuppression for ≥30 days post-TPE and were followed for 4 weeks after the end of treatment. Patients with persistent ADAMTS13 deficiency (eg, &amp;lt;10%) were allowed treatment extension of up to 8 weeks. This post hoc analysis assessed outcomes including patient characteristics, time to platelet count recovery, time to normalization of all 3 organ damage markers (lactate dehydrogenase, cardiac troponin I, and serum creatinine), safety, time to recovery of ADAMTS13 activity, ADAMTS13 activity at end of treatment, and incidence of ADAMTS13 inhibitor elevation during treatment stratified by iTTP severity. Very severe iTTP at baseline was defined as follows: French severity score ≥3 or severe neurological involvement (eg, coma, seizures, focal deficit), or cardiac involvement (cardiac troponin level &amp;gt;2.5×ULN). Results Overall, 21 patients were enrolled and treated with ≥1 dose of caplacizumab (modified intent-to-treat population [mITT] and safety population). Of these,19 patients had confirmed iTTP (less severe iTTP, n=8; very severe iTTP, n=11; Table). Median (95% confidence interval [CI]) time to platelet count response and median (range) days of TPE was 3.28 (1.69-4.01) and 5.5 (3-15) days in patients with less severe iTTP and 3.49 (1.76-3.98) and 6.0 (4-20) days in patients with very severe iTTP, respectively. Median (95% CI) time to normalization of all 3 organ damage marker levels was 1.73 (0.79-2.94) days in patients with less severe iTTP and 8.66 (2.65-12.55) days in patients with very severe iTTP, which may reflect the fact that patients with very severe iTTP had worse organ damage at baseline. The number of patients experiencing recurrence, ≥1 major thromboembolic event, or TTP-related death in patients with less severe and very severe iTTP was 1 for each subgroup. Two patients in each disease severity subgroup had ≥1 treatment-emergent serious adverse event. Of the 19 patients with confirmed iTTP, median (95% CI) time to recovery of ADAMTS13 activity to ≥10, ≥20, and ≥60% was 14.57 (5.95-24.76),18.49 (5.95-31.75), and 47.51 (18.49-60.94) days, respectively. Patients with ADAMTS13 inhibitor levels ≥2 BU/mL at baseline (n=7) took approximately twice as long to recover ADAMTS13 activity to ≥10, ≥20, and ≥60% versus patients with baseline inhibitor levels &amp;lt;2 BU/mL. Median (range) ADAMTS13 activity level at the end of caplacizumab treatment was 62.0% (29.0%-101.0%). ADAMTS13 inhibitor elevation occurred in 9 patients (Figure). Among them, 7 patients extended caplacizumab treatment. Only 1 patient with inhibitor re-increase experienced a recurrence 2 days after discontinuing caplacizumab due to an adverse event. Conclusions These results suggest that caplacizumab in conjunction with TPE and immunosuppression is effective for patients with iTTP regardless of their baseline severity and may reduce the risk of recurrence due to inhibitor re-increase.

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

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

A combined regimen based on bortezomib and glucocorticoids for 6 patients with recurrent/refractory immune thrombotic thrombocytopenic purpura

Objective: To observe the efficacy and adverse reactions of a combination therapy regimen based on bortezomib and glucocorticoids in recurrent/refractory immune thrombocytopenic purpura (iTTP) . Methods: Six patients with recurrent/refractory TTP were included and treated with a glucocorticoid and two courses of bortezomib-based regimen. The clinical remission status of patients, changes in ADAMTS13 activity/ADAMTS13 inhibitor, and the occurrence of treatment-related adverse reactions were observed. Results: Of the 6 patients, 2 were males and 4 were females, with a median age of 21.5 (18-68) years. Refractory TTP was found in 1 case and recurrent TTP in 5 cases. Glucocorticoids were administered with reference to prednisone at 1 mg·kg(-1)·d(-1), and gradually reduced in dosage after achieving clinical remission. Bortezomib is subcutaneously administered at 1.3 mg/m(2) on days 1, 4, 8, and 11 with a 28-day treatment course consisting of 2 courses. Six patients achieved clinical remission after receiving bortezomib as the main treatment. ADMATS13 activity returned to normal in all patients with TTP after treatment, and the ADAMTS13 inhibitor turned negative. Thrombocytopenia is the most common adverse reaction after treatment, with other adverse reactions, including peripheral neuritis and abdominal pain, but ultimately all patients returned to normal. In a median follow-up of 26 (9-41) months, 5 patients maintained sustained remission, and 1 patient relapsed after 16 months of bortezomib treatment. Conclusion: Combination therapy of bortezomib and glucocorticoids has a satisfactory therapeutic effect and controllable adverse reactions for recurrent/refractory iTTP.

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.

Two ischemic stroke events within 48 h: a case report of an unusual presentation of thrombotic thrombocytopenic purpura

BackgroundThrombotic thrombocytopenic purpura (TTP) considers a rare cause of ischemic stroke (IS). We reported a case of a newly diagnosed patient with acquired immune-mediated TTP (iTTP), in whom two IS events developed during 48 h.Case presentationA 59-year-old diabetic male was presented to the hospital 24 h after symptoms onset, including left hemiparesis, dysarthria, and decreased consciousness. A brain CT scan was performed with the suspicion of acute IS, indicating infarct lesions in the right middle cerebral artery (MCA) territory. The patient was not eligible for thrombolytic therapy due to admission delay. Over the next 24 h, the patient’s neurological condition deteriorated, and the second brain CT scan showed new ischemic lesions in the left MCA territory. Initial laboratory evaluation indicated thrombocytopenia without evidence of anemia. However, in the following days, thrombocytopenia progressed, and microangiopathic hemolytic anemia (MAHA) developed. The ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity and inhibitors assay confirmed the diagnosis of iTTP. The patient underwent plasma exchange activity and inhibitors assay confirmed the diagnosis of iTTP. The patient underwent and pulse IV methylprednisolone. Rituximab was also added due to the refractory course of the disease. After a prolonged hospital course, he had considerable neurologic recovery and was discharged.ConclusionsClinicians should consider two points. First, TTP should be considered in any patient presenting with IS and having thrombocytopenia or anemia without other symptoms of TTP. Second, worsening the patient's condition during hospitalization may indicate a new stroke and should be investigated immediately.

Differential Diagnosis Distinguishing Immune TTP from Septic DIC Using Plasma Levels of Haptoglobin and Factor XIII Activity

Introduction Differential diagnosis of immune thrombotic thrombocytopenic purpura (iTTP) and septic disseminated intravascular coagulation (DIC) in daily clinical practice is difficult. iTTP is diagnosed by deficiency of ADAMTS13 activity and ADAMTS13 inhibitor positivity. However, it is difficult to rapidly measure ADAMTS13 activity in real-time in clinical practice. Prompt therapeutic interventions are directly dependent on iTTP prognosis. A method for differentiating between these two disorders using common laboratory tests is needed. Plasma haptoglobin levels can indicate hemolysis and are reported to be severely decreased in iTTP. Coagulation factor XIII (FXIII) activity is also severely decreased in septic DIC. To date, no reports have observed haptoglobin in septic DIC or FXIII activity in iTTP. We analyzed the plasma levels of haptoglobin and FXIII activity in patients with iTTP and septic DIC to investigate whether these markers can be used for differential diagnosis. Methods iTTP was defined as <10% normal ADAMTS13 activity, and >0.5 BU/mL ADAMTS13 inhibitor. Septic DIC was defined as meeting both Third International Consensus Definitions for Sepsis and Septic Shock, and ISTH DIC diagnostic criteria. Thirty-five patients with iTTP and 30 with septic DIC were enrolled in this study. Plasma haptoglobin levels were measured using Human Quantikine ELISA Kits (#DPHAGO, R&D Systems, Inc., Minneapolis, USA), which is a highly sensitive method compared to methods used in clinical practice. Values are expressed as medians (first quartile-third quartile). Results As shown in the table, iTTP patients were significantly younger than those with septic DIC. The iTTP group showed significantly lower ADAMTS13 activity, higher ADAMTS13 inhibitor titer, lower hemoglobin level, lower platelet counts, higher LDH level, and lower creatinine level than the septic DIC group. In a comparison of coagulation and fibrinolytic markers, the septic DIC group had significantly higher PT-INR, prolonged APTT, higher FDP and D-dimer levels, and lower AT activity than the iTTP group. The plasma haptoglobin level was 0.39 mg/dL (0.18-0.76 mg/dL) in the iTTP group, and 54.2 mg/dL (0.6-143.9 mg/dL) in the septic DIC group (P < 0.01). FXIII activity was 91.3% (80.4-115.0%) in the iTTP group, and 36.3% (14.7-61.9%) in the septic DIC group (p<0.01). The ROC curve of haptoglobin and FXIII activity in the differential diagnosis of TTP and septic DIC were investigated. The cut-off level of haptoglobin was 2.868 mg/dL, the area under the curve (AUC) was 0.832 (95% CI:0.727-0.938), specificity was 66.7%, and sensitivity was 100%. The cutoff level of FXIII activity was 76.0%, AUC was 0.931 (95%CI:0.873-0.989), specificity was 77.1%, and sensitivity was 93.3%. We developed a scoring system to differentiate iTTP from septic DIC using a combination of FXIII activity and haptoglobin; TTP/DIC index = plasma FXIII activity (%) - plasma haptoglobin (mg/dL). Laboratory TTP was defined when the index was ≥60, and laboratory DIC was defined when the index was <60. The AUC for the combination of FXIII activity and haptoglobin was 0.968 (95% CI: 0.9354-1). The accuracy of iTTP diagnosis in laboratory TTP was 89.2% (33/37), and the accuracy of septic DIC diagnosis in laboratory DIC was 92.9% (26/28). Conclusions A comparison of iTTP and septic DIC showed significant differences in haptoglobin and FXIII activity levels. A combination of haptoglobin and FXIII activity was considered helpful in differentiating iTTP from septic DIC. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Blood Transfusion Ameliorates Free Haem-Induced Loss of Thrombomodulin and Endothelial Cell Activation in Paediatric Sickle Cell Anaemia

Background Sickle cell anaemia (SCA) is responsible for major paediatric morbidity including risk of stroke in up to 10% of children. Mainstays of therapy include hydroxyurea (HU) and chronic blood transfusion (BT), with BT considered gold standard for stroke prevention. However, the biological mechanisms through which BT is significantly superior to HU in reducing stroke risk have not been clearly defined. SCA complications are due in part to free haem-mediated damage, leading to endothelial cell (EC) activation, tissue factor (TF) expression and release of von Willebrand Factor (VWF), which binds platelets and sickle RBCs. Recently loss of EC Thrombomodulin (TM) has been shown to promote EC activation and VWF release and soluble TM (sTM) plays a role in disseminated intravascular coagulation and COVID-19. Hypothesis We hypothesize free haem alters EC TM expression, promoting EC activation and VWF release. Intravascular hemolysis and free haem may be reduced in children on chronic BT, maintaining EC quiescence and enhancing protection against stroke compared with HU. Methods Following ethical approval and informed consent 180 SCA children (median age 12 years, 49% male) were recruited. Steady-state plasma samples were collected prior to BT (n=84) or at outpatient clinic for HU (n=96). Paired crisis samples were obtained in a subset of HU (n=30). EC activation markers and Weibel Palade Body (WPB) proteins were evaluated by ELISA, VWF multimers were assessed by 1.8% agarose gels and ADAMTS13 activity assessed by FRET. Free haem levels were quantified and thrombin generation (TG) assays performed. Factor VIII:C was measured in the National Coagulation Centre at St James's Hospital. Human umbilical vein endothelial cells (HUVEC) were exposed to cell-free haem (Hemin), hydroxyurea, cytokines (IL-1β and TNFα) or patient plasma; VWF was measured in the supernatant and HUVEC TM expression assessed by flow cytometry. Results VWF:Ag and VWF propeptide (VWFpp) levels were significantly increased in HU compared to BT children, suggesting acute EC activation (median plasma VWF:Ag levels 1.29 v 1.03 IU/ml; p=0.0005, median VWFpp levels 1.63 v 1.17 IU/ml p=0.006). Furthermore, plasma levels of other WPB stored proteins were also significantly elevated in the HU cohort, including Angiopoietin-2 (Ang-2) (4214 v 2337 pg/ml, p<0.0001 and Osteoprotegerin (OPG) (1825 v 1507 pg/ml p=0.006). Abnormalities of high-, intermediate- and low molecular weight VWF multimers were noted in HU but not BT. While putative inhibitors of ADAMTS13 were increased in HU, including Platelet Factor 4 and Interleukin-6, ADAMTS13 activity did not differ between groups. FVIII:C levels were significantly elevated in 65% of the total cohort and were higher in HU than BT (1.62 IU/ml v 1.30 IU/ml p<0.0001). Free haem levels were increased in HU versus BT (12.5 v 9.5μM p=0.0003), as well as levels of LDH and reticulocyte %, pointing to increased intravascular hemolysis. EC markers and FVIII:C correlated strongly with hemolytic indices. In addition to increased EC activation, increased endogenous thrombin potential (ETP), peak thrombin and velocity index were observed in HU versus BT, pointing to enhanced TG, possibly driven by elevated circulating TF. TG parameters correlated with plasma FVIII:C levels. Exposure of HUVEC to free haem in vitro resulted in a significant decrease in surface TM expression and increased VWF secretion (p<0.05). Conversely exposure to hydroxyurea or cytokines did not alter TM expression. Exposure of HUVEC to plasma from crisis but not paired steady-state plasma induced EC activation and VWF release compared with control plasma. Importantly, free haem levels ≥20 μM were observed in 25% HU children in steady state and 40% in crisis, compared with 0% of BT. Furthermore, significantly elevated sTM levels in SCA children with a history of cerebrovascular disease compared to those without (5.7 v 4.6 ng/ml p=0.02), suggesting that sTM may predict end-organ dysfunction. Conclusions We have shown in a large cohort of SCA children that EC activation, TG and intravascular haemolysis are increased in HU-treated children compared with BT. Free haem induces loss of EC TM and VWF release in vitro, leading to EC activation. Elevated sTM levels were a biomarker of end-organ damage. These data suggest that reduction of free haem in children undergoing regular BT is a key mechanism through which EC quiescence is maintained in paediatric SCA.

The Mechanism of Antibody-Mediated Inhibition of ADAMTS13 in Immune Thrombotic Thrombocytopenic Purpura

Background and objective: Immune thrombotic thrombocytopenic purpura (iTTP) is a potentially life-threatening microangiopathic disorder resulting from inhibitory immunoglobulin (Ig) G-type antibodies that target plasma metalloprotease ADAMTS13. This leads to the accumulation of ultra-large multimers of von Willebrand factor (VWF) that occludes small arterioles and capillaries and causes end-organ damage. The way inhibitory antibodies work to inhibit ADAMTS13 is an active area of investigation and may lead to a better tool for diagnosing and treating iTTP. To determine whether inhibitory antibodies prevent substrate binding to ADAMTS13, which would increase the substrate concentration at which velocity is the half maximal (K0.5); decrease the maximal enzyme velocity (Vmax) of ADAMTS13 by affecting catalytic turnover; or both, we developed an assay using a well-characterized inhibitor of ADAMTS13 to explore this important question. Method: A single chain fragment of the variable region (scFv4-20), derived from a human monoclonal antibody isolated from an iTTP patient by phage display with potent inhibitory effects on ADAMTS13 (Casina et al., PNAS. 2015; 112:9620-25), was expressed and purified. Recombinant full-length ADAMTS13 and native ADAMTS13 in normal human plasma (NHP) were titrated with a fluorescein-labeled surrogate substrate (FRETS-VWF73) in the presence of an increasing fixed concentration of scFv4-20. Titrations were analyzed both individually and applied to a global fit, which allowed for evaluation of the relative effects of the inhibitor on Vmax and K0.5. The relative effect of scFv4-20 on K0.5 was inferred from the global fit via the parameter α; values of α within an order of magnitude of unity suggest little to minimal effect of the inhibitor on K0.5. Results: When native ADAMTS13 in normal human plasma was titrated with FRETS-VWF73 in the presence of increasing concentrations of scFv4-20, the Vmax decreased with increasing concentration of scFv4-20, but the K0.5 did not show a significant change under the standard assay conditions (pH 6.0, 25 ˚C) (Fig. 1). Similar results were observed when acidic conditions closer to physiologically relevant pH and temperature were used (Table 1). Titration of recombinant ADAMTS13 with FRETS-VWF73 revealed similar results regarding the effect of scFv4-20 on the Vmax and K0.5 (Table 1) The parameter α was close to 1 when the titrations were analyzed by global fit, regardless of the reaction conditions or type of ADAMTS13 used, suggesting little to no effect of scFv4-20 on K0.5 (Table 1). Conclusions: The antibody-mediated inhibition of ADAMTS13 by the inhibitory scFv4-20 likely results primarily from reducing catalytic turnover of the enzyme rather than the binding of ADAMTS13 to VWF. Our ongoing study is to determine whether this is a general feature of inhibitory antibodies against ADAMTS13 or if other inhibitory mechanisms are also involved in the pathophysiology of iTTP. The assay developed and described here can be applicable to explore the way antibodies affect the activity of ADAMTS13 in this polyclonal autoimmune disease. Key words: Immune thrombotic thrombocytopenic purpura; ADAMTS13, inhibitory antibody 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.

ADAMTS13 Biomarkers in Management of Immune Thrombotic Thrombocytopenic Purpura.

Immune thrombotic thrombocytopenic purpura (iTTP) is a rare but potentially fatal blood disorder resulting from acquired deficiency of plasma ADAMTS13, a metalloprotease that cleaves endothelium-derived ultralarge von Willebrand factor. Standard of care for iTTP including therapeutic plasma exchange, caplacizumab, and immunosuppressives, known as triple therapy, has led to a significant reduction in the disease-related mortality rate. The first International Society of Thrombosis and Haemostasis TTP guideline stresses the importance of having plasma ADAMTS13 activity testing in the algorithm for diagnosis and management of iTTP. However, the predictive role of assessing plasma ADAMTS13 activity and inhibitors or other ADAMTS13-related parameters in patients with acute iTTP and during remission has not been systematically evaluated. To review and assess the predictive values of testing plasma ADAMTS13 activity, antigen, and inhibitors or anti-ADAMTS13 immunoglobulin G at various stages of disease in outcomes of iTTP. Peer-reviewed publications and personal experience. We conclude that assessing ADAMTS13 biomarkers is not only essential for establishing the initial diagnosis, but also crucial for risk stratification and the early detection of disease recurrence. This may guide therapeutic interventions during acute episodes and for long-term follow-up of iTTP patients.

Prognostic factors in thrombotic thrombocytopenic purpura

Abstract Objectives Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy with no standardized prognostic model to predict mortality. The aim of the study is to determine parameters associated with TTP-related mortality. Methods In this cross-sectional, retrospective study, 77 TTP patients, treated with therapeutic plasma exchange between 2001 and 2019 in Ankara University Faculty of Medicine were included. Results There was no significant relationship between ADAMTS13 inhibitor levels, activity and mortality. Median number of plasmapheresis was 10 (2–32), higher in patients with complete response. Anemia, kidney injury and LDH levels were associated with survival; there was no significant relationship between platelet counts at the time of diagnosis and mortality. Mortality was lower in patients with platelet counts above 100 × 109/L and normalized LDH after treatment. Hemoglobin, albumin, LDH and creatinine levels at the time platelet counts exceeded 50 × 109/L were associated with survival. Conclusions We determined several clinical and laboratory parameters associated with mortality. Fewer numbers of plasmapheresis was associated with mortality; thus other treatments as rituximab and caplacizumab should be considered early in non-responders. Including changes in laboratory parameters may be considered in prognostic scoring systems to be developed in the future.

How I treat immune-mediated thrombotic thrombocytopenic purpura after hospital discharge

AbstractImmune-mediated thrombocytopenic purpura (iTTP) is a thrombotic microangiopathy characterized by an acquired ADAMTS13 deficiency as a result of the presence of an antibody inhibitor of ADAMTS13 leading to the formation of ultralarge von Willebrand multimers. Treatment of iTTP includes plasma exchange, high-dose glucocorticoids, rituximab, and, more recently, caplacizumab, to prevent the development of exacerbations. There is the risk of both relapse and long-term complications that include neurocognitive deficits and cardiovascular events that occur in patients in remission after recovery from an acute iTTP episode. Data on the risk factors for the development of these complications, the appropriate screening, and treatment are limited due to the paucity of research. This article is a review of the current understanding on the risk factors for exacerbation, relapse, and long-term complications of iTTP and discusses an approach to observing patients with iTTP after hospital discharge and during the long-term follow-up in the outpatient setting.

POS-011 PRIMARY ATYPICAL HEMOLYTIC-UREMIC SYNDROME ASSOCIATED WITH COVID-19: A BRIEF REVIEW

POS-011 PRIMARY ATYPICAL HEMOLYTIC-UREMIC SYNDROME ASSOCIATED WITH COVID-19: A BRIEF REVIEW

Naturally Occurring Anti-Idiotypic Antibodies Portray a Largely Private Repertoire in Immune-Mediated Thrombotic Thrombocytopenic Purpura.

Rare immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a life-threatening disease resulting from a severe autoantibody-mediated ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motifs, member 13) deficiency. Acute iTTP episodes are medical emergencies, but when treated appropriately &amp;gt;95% of patients survive. However, at least half of survivors will eventually experience a relapse. How remission of an initial episode is achieved and factors contributing to reemergence of anti-ADAMTS13 Abs and a relapsing course are poorly understood. In acquired hemophilia and systemic lupus erythematosus, anti-idiotypic Abs counteracting and neutralizing pathogenic autoantibodies contribute to remission. We selected and amplified the splenic anti-idiotypic IgG&lt;sub&gt;1&lt;/sub&gt; Fab κ/λ repertoire of two relapsing iTTP patients on previously generated monoclonal inhibitory anti-ADAMTS13 Fabs by phage display to explore whether anti-idiotypic Abs have a role in iTTP. We obtained 27 single anti-idiotypic Fab clones, half of which had unique sequences, although both patients shared four H chain V region genes (V&lt;sub&gt;H&lt;/sub&gt;1-69*01, V&lt;sub&gt;H&lt;/sub&gt;3-15*01, V&lt;sub&gt;H&lt;/sub&gt;3-23*01, and V&lt;sub&gt;H&lt;/sub&gt;3-49*03). Anti-idiotypic Fab pools of both patients fully neutralized the inhibitor capacity of the monoclonal anti-ADAMTS13 Abs used for their selection. Preincubation of plasma samples of 22 unrelated iTTP patients stratified according to functional ADAMTS13 inhibitor titers (&amp;gt;2 Bethesda units/ml, or 1-2 Bethesda units/ml), with anti-idiotypic Fab pools neutralized functional ADAMTS13 inhibitors and restored ADAMTS13 activity in 18-45% of those cases. Taken together, we present evidence for the presence of an anti-idiotypic immune response in iTTP patients. The interindividual generalizability of this response is limited despite relatively uniform pathogenic anti-ADAMTS13 Abs recognizing a dominant epitope in the ADAMTS13 spacer domain.

Molecular Diagnosis Is Vital to the Accurate Classification and Management of Thrombotic Thrombocytopenic Purpura in Children.

Thrombotic thrombocytopenic purpura (TTP) is a rare but potentially life-threatening hematologic disease, presenting a myriad of diagnostic and management challenges in children. Here, we provide a review of this disorder and discuss 2 exemplary cases of TTP occurring in adolescents, emphasizing the need for consideration of late-onset congenital TTP (cTTP). We demonstrate the importance of early confirmation of ADAMTS13 enzyme deficiency and the presence or absence of ADAMTS13 inhibitor in order to rapidly initiate the appropriate life-saving therapies. Ultimately, molecular testing is paramount to distinguishing between congenital and acquired immune-mediated TTP.