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

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

Plasmin-cleaved von Willebrand factor as a biomarker for microvascular thrombosis.

ABSTRACT: von Willebrand factor (VWF) is an essential contributor to microvascular thrombosis. Physiological cleavage by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) limits its prothrombotic properties, explaining why ADAMTS13 deficiency leads to attacks of microthrombosis in patients with thrombotic thrombocytopenic purpura (TTP). We previously reported that plasminogen activation takes place during TTP attacks in these patients. Furthermore, stimulation of plasminogen activation attenuates pathogenesis in preclinical TTP models in vivo. This suggests that plasmin is an endogenous regulator of VWF thrombogenicity, in particular when ADAMTS13 falls short to prevent microvascular occlusions. VWF cleavage by plasmin is biochemically distinct from cleavage by ADAMTS13. We hypothesized that plasmin-cleaved VWF (cVWF) holds value as a biomarker of microvascular thrombosis. Here, we describe the development of a variable domain of heavy-chain-only antibody (VHH)-based bioassay that can distinguish cVWF from intact and ADAMTS13-cleaved VWF in plasma. We validate this assay by tracking cVWF release during degradation of microthombi in vitro. We demonstrate that endogenous cVWF formation takes place in patients with TTP during acute attacks of thrombotic microangiopathy but not in those in remission. Finally, we show that therapeutic plasminogen activation in a mouse model of TTP amplifies cVWF formation, which is accompanied by VWF clearance. Our combined findings indicate that cVWF is released from microthrombi in the context of microvascular occlusion.

Effectiveness of Caplacizumab Nanobody in Acquired Thrombotic Thrombocytopenic Purpura Refractory to Conventional Treatment.

Acquired thrombocytopenic thrombotic purpura (aTTP) is an autoantibody-mediated disease against the enzyme A Disintegrin and Metalloprotease domain with ThromboSpondin-1 type motif 13, which until now has been treated with plasma exchange (PEX) and corticosteroids. A 29-year-old female patient, who presented with aTTP in the context of pregnancy, has developed multiple relapses after treatment with PEX, corticosteroids, and rituximab. Recently, caplacizumab, a nanobody against von Willebrand factor, has been approved for the treatment of aTTP. In our patient, caplacizumab achieved better disease control, with a lower platelet count restoration time, days of PEX and hospitalization duration, as compared to standard therapy, reproducing the results of clinical trials. Caplacizumab represents a significant advance in the treatment of aTTP, especially in cases of recurrent relapses.

A Case Report of Congenital Thrombotic Thrombocytopenic Purpura: The Peripheral Blood Smear Lights the Diagnosis.

We report on a 12-year-old boy with congenital thrombotic thrombocytopenic purpura, on who had an erroneous diagnosis as chronic immune thrombocytopenia. The patient presented with complaints of jaundice and skin rash. Laboratory analysis showed nonimmune hemolytic anemia and severe thrombocytopenia. Peripheral blood smear showed 8% schistocytes, polychromasia, and anisocytosis. The ADAMTS13 antigen and activity were suspected to be lower than 5% with any antibodies against the enzyme. The DNA sequence analyses resulted in compound heterozygosity consisting of c.291_391del in exon 3 and c.4143dupA in exon 29. Schistocyte (fragmented erythrocytes) on the peripheral blood smear is a light that illuminates the diagnosis. Early recognition of the disease can prevent inappropriate treatments and morbidities due to organ damage.

Consumption of complement in a 26-year-old woman with severe thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination.

Extremely rare reactions characterized by thrombosis and thrombocytopenia have been described in subjects that received ChAdOx1 nCoV-19 vaccination 5-16 days earlier. Although patients with vaccine-induced thrombotic thrombocytopenia (VITT) have high levels of antibodies to platelet factor 4 (PF4)-polyanion complexes, the exact mechanism of the development of thrombosis is still unknown. Here we reported serum studies as well as proteomics and genomics analyses demonstrating a massive complement activation potentially linked to the presence of anti-PF4 antibodies in a patient with severe VITT. At admission, complement activity of the classical and lectin pathways were absent (0% for both) with normal levels of the alternative pathway (73%) in association with elevated levels of the complement activation marker sC5b-9 (630 ng/mL [n.v. 139-462 ng/mL]) and anti-PF4 IgG (1.918 OD [n.v. 0.136-0.300 OD]). The immunoblotting analysis of C2 showed the complete disappearance of its normal band at 110 kDa. Intravenous immunoglobulin treatment allowed to recover complement activity of the classical pathway (91%) and lectin pathway (115%), to reduce levels of sC5b-9 (135 ng/mL) and anti-PF4 IgG (0.681 OD) and to normalize the C2 pattern at immunoblotting. Proteomics and genomics analyses in addition to serum studies showed that the absence of complement activity during VITT was not linked to alterations of the C2 gene but rather to a strong complement activation leading to C2 consumption. Our data in a single patient suggest monitoring complement parameters in other VITT patients considering also the possibility to target complement activation with specific drugs.

Thrombotic thrombocytopenic purpura: a new menace after COVID bnt162b2 vaccine.

Thrombotic thrombocytopenic purpura (TTP) is a known menace in hematology and is quite rare in practice with known triggers. Lately, in the COVID-19 pandemic, hematology has seen a new pathology amongst which TTP associated with COVID-19 messenger RNA (mRNA) vaccine is unique. We report a case of a 69-year-old male with multiple comorbidities who presented to the hospital with severe fatigue and shortness of breath. Labs were significant for thrombocytopenia, anemia, and hemolysis with schistocytes consistent with TTP with a second dose of BNT162b2 mRNA vaccine as a likely culprit been documented.

Thrombotic Thrombocytopenic Purpura in Dengue Fever.

Thrombotic thrombocytopenic purpura is a rare but life threatening medical condition. Early recognition and treatment of thrombotic thrombocytopenic purpura is important especially in patients who do not present with the classic pentad to reduce the high mortality. Herein, we describe a case of a patient who does not fulfil the classic pentad features thrombotic thrombocytopenic purpura that was induced by dengue fever. The patients' initial full blood picture did not have all the typical features of microangiopathic haemolytic anaemia but there were fragmented red blood cells. However, even a small number of fragmented red blood cells in the peripheral blood should alert physicians of the possible diagnosis of thrombotic thrombocytopenic purpura together with other symptoms. Furthermore, signs and symptoms of thrombotic thrombocytopenic purpura and dengue fever can overlap such as fever, thrombocytopenia, neurological deficit mimicking dengue encephalopathy and dengue induced acute kidney injury.

Thrombotic microangiopathy during pregnancy.

Pregnancy is a high-risk time for the development of different kinds of thrombotic microangiopathy (TMA). Three major syndromes including TTP (thrombotic thrombocytopenic purpura), PE/HELLP (preeclampsia/hemolysis, elevated liver function tests, low platelets), and aHUS (atypical hemolytic- uremic syndrome) should be sought in pregnancy-TMA. These severe disorders share multiple clinical features and overlaps and even the coexistence of more than one pathologic mechanism. Each of these disorders finally ends in endothelial damage and fibrin thrombi formation within the microcirculation that fragments RBCs (schystocytes), aggregates platelets, and creates ischemic injury in the targeted organs i.e.; kidney and brain. Although the mechanisms of these severe disorders have been revealed, pregnancy-related TMA still interfaces with diagnostic and therapeutic challenges. Here, we highlight the current knowledge of diagnosis and management of these complications during pregnancy.

ADAMTS13 testing update: Focus on laboratory aspects of difficult thrombotic thrombocytopenic purpura diagnoses and effects of new therapies.

TTP is a life-threatening disorder diagnosed using a combination of clinical information and laboratory results. ADAMTS13 activity and antibody testing represent a major advance in the field, but results can sometimes be difficult to interpret due to technical aspects of the tests and characteristics of the causative antibodies in acquired TTP. Genetic testing for ADAMTS13 mutations is also now available to assist with the diagnosis of inherited TTP. This review will focus on ADAMTS13 testing and will highlight patient and laboratory aspects that can lead to diagnostic difficulty. The effects of TTP therapies on test results will also be discussed.

Acute Ischemic Stroke Revealing ChAdOx1 nCov-19 Vaccine-Induced Immune Thrombotic Thrombocytopenia: Impact on Recanalization Strategy.

Vaccine-induced immune thrombotic thrombocytopenia is a rare syndrome following the ChAdOx1 nCov-19 or Ad26.COV2.S vaccine. Reported patients developed mainly venous thrombosis. We describe a case of a young healthy women suffering from acute ischemic stroke due to large vessel occlusion without cerebral venous thrombosis 8 days after vaccination and its consequences on recanalization strategy. Considering the thrombocytopenia, intravenous thrombolysis was contraindicated. She underwent mechanical thrombectomy with complete recanalization and dramatically improved clinically. Positive detection of anti-PF4-heparin-antibodies confirmed vaccine-induced immune thrombotic thrombocytopenia diagnosis. In case of acute ischemic stroke after recent ChAdOx1 nCov-19 or Ad26.COV2.S vaccine, platelet count should be systematically checked before giving thrombolysis, and direct mechanical thrombectomy should be proposed in patients with large vessel occlusion.

Vaccine-Induced Immune Thrombotic Thrombocytopenia with Disseminated Intravascular Coagulation and Death following the ChAdOx1 nCoV-19 Vaccine.

Coronavirus is a novel human pathogen causing fulminant respiratory syndrome (COVID-19). Although COVID-19 is primarily a disease of the lungs with florid respiratory manifestations, there are increasing reports of cardiovascular, musculoskeletal, gastrointestinal, and thromboembolic complications. Developing an effective and reliable vaccine was emergently pursued to control the catastrophic spread of the global pandemic. We report a fatal case of vaccine-induced immune thrombotic thrombocytopenia (VITT) after receiving the first dose of the ChAdOx1 nCoV-19 vaccine. We attribute this fatal thrombotic condition to the vaccine due to the remarkable temporal relationship. The proposed mechanism of VITT is production of rogue antibodies against platelet factor-4 resulting in massive platelet aggregation. Healthcare providers should be aware of the possibility of such fatal complication, and the vaccine recipients should be warned about the symptoms of VITT.

Laboratory testing for ADAMTS13: Utility for TTP diagnosis/exclusion and beyond.

The metalloproteinase ADAMTS13 (a disintegrin with a thrombospondin type 1 motif, member 13), also known as VWF (von Willebrand factor) protease, may be assessed in a vast array of clinical conditions. Notably, a severe deficiency of ADAMTS13 characterizes TTP (thrombotic thrombocytopenic purpura), a rare but potentially fatal disorder associated with thrombosis due to accumulation of prothrombotic ultra-large VWF multimers. Although prompt identification/exclusion of TTP can be facilitated by rapid ADAMTS13 testing, the most commonly utilized assays are based on ELISA (enzyme linked immunosorbent assay) and require long turnaround time and have relatively limited throughput. Nevertheless, several rapid ADAMTS13 assays are now available, at least in select geographies. The current mini-review discusses these issues, as well as the potential utility of ADAMTS13 testing in a range of other conditions, including coronavirus disease 2019 (COVID-19).

COVID 19 infection associated with thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) which can cause significant mortality is a thrombotic microangiopathy due to deficiency of VWF cleaving protease ADAMTS13 and as per medical literature there are examples that TTP can be caused by COVID 19 infection. A 35 years old female after admission with right sided weakness and slurring of speech was found to be COVID positive and diagnosed as a case of TTP. Patient had absent ADAMTS13 level on day 1. Treatment was started with therapeutic plasma exchange (TPE) later injection Vincristine and Rituximab was given after 4th TPE as it was suspected as refractory case. Finally patient received 16 TPE procedures with cryo poor plasma as exchange fluid and gradually her platelet count started to maintain normal and she was discharged. Specific management and such association of this type of cases need to be studied more judiciously.

Unfolding the pathophysiology of congenital thrombotic thrombocytopenic purpura in pregnancy: lessons from a cluster of familial cases.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP), a rare, potentially life-threatening thrombotic microangiopathy, manifests either as congenital TTP or acquired forms. It is caused by the absence or severe depletion of a disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) protease, leading to the accumulation of ultra large von Willebrand factor multimers as well as extensive platelet adhesion and clumping, which can ultimately cause severe secondary end-organ damage. Pregnancy can provoke or exacerbate TTP, leading to maternal and fetal complications. OBJECTIVE: In this report, we focused on pregnancy outcomes in a recently recognized cohort of congenital TTP patients of Bedouin Arab descent in southern Israel who were all homozygous for a novel c.3772delA variant of the ADAMTS13 gene, leading to the clinical manifestations of TTP largely during pregnancy. STUDY DESIGN: All patients presented in this study belong to 2 closely related families of Arab Bedouin descent and were found to be homozygous for a novel ADAMTS13-c.3772delA variant. The cohort consisted of 19 females; 16 of them had congenital TTP and had been pregnant and were thus included. Patient data were collected from electronic medical records. RESULTS: Of note, 13 women from our cohort, who delivered 14 fetuses (owing to 1 twin pregnancy), were diagnosed with congenital TTP following complicated pregnancies, which included recurrent pregnancy loss, stillbirth, early onset preeclampsia (both mild and severe), hemolysis, elevated liver enzymes and low platelet count syndrome, intrauterine growth restriction with abnormal Doppler flow, preterm premature rupture of membranes, and a total perinatal mortality rate of 30.7% (4/13). An additional 3 women, who were diagnosed owing to complications outside of pregnancy and at older ages, experienced TTP during their pregnancies, which occurred before diagnosis. Subsequent pregnancies were treated with fresh frozen plasma leading to a 100% fetal survival rate in the pregnancies that reached fetal viability. All placentas had lesions consistent with maternal vascular underperfusion. However, the severity and frequency of these lesions were lower in the 8 placentas from pregnancies treated with fresh frozen plasma. CONCLUSION: This case series details a distinctive cohort of congenital TTP patients, all homozygous for the same, novel ADAMTS13 variant, who presented with clinical complications during pregnancy and maternal vascular lesions of underperfusion in the placenta. Our findings imply that the variant identified in the ADAMTS13 gene in our cohort may have a specific functional impact on the placenta, and that treatment with fresh frozen plasma during pregnancy ameliorates the course of the disease, leading to a milder phenotype or a normal pregnancy in the majority of cases.

Hematologic autoimmune disorders in the course of COVID-19: a systematic review of reported cases.

OBJECTIVE: As COVID-19 is a new emerging disease, the hematological/immunological changes that develop in the infected patients remain unknown. This study aims to systematically review the hematologic autoimmune complications in these patients. METHOD: Data from three online databases including Medline (via PubMed), Scopus and Web of Science were searched on 19 December 2020, and after excluding duplicate, irrelevant and inappropriate records, eligible documents were identified. Afterwards, information such as patients' history, presentations, paraclinical data, treatment course and outcome were extracted from the records. RESULTS: A total of 58 documents were considered to be eligible for data extraction which described 94 patients with COVID-19 who developed hematologic autoimmune disorder in their course of infection. Of these patients with COVID-19, the most common hematologic autoimmune disorder was immune thrombocytopenic purpura (55 cases) followed by autoimmune hemolytic anemia (22 cases). Other hematologic autoimmune disorders include antiphospholipid syndrome, thrombotic thrombocytopenic purpura, Evans syndrome and autoimmune neutropenia. CONCLUSION: The current study would help us to always consider an autoimmune etiology for cases with abnormal hematologic finding which further lead to an appropriate treatment of the patients, especially when the symptoms present in about 1-2 weeks after the first manifestation of the infection symptoms. Maybe, at least in this pandemic, it should be recommended to evaluate patients with unexpected and unexplained decrease in their hemoglobulin or platelet count for COVID-19. Another challenging issue is the treatment options. Given the multiorgan involvement and multifaceted nature of the infection, an individualized approach should be taken for each patient.

Redefining outcomes in immune TTP: an international working group consensus report.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a potentially fatal thrombotic microangiopathy caused by autoantibody-mediated severe deficiency of ADAMTS13. Standardized definitions of response, exacerbation, remission, and relapse were initially proposed in 2003 and modified by the International Working Group for TTP in 2017. These definitions, which have been widely used in clinical practice and research, are based primarily on the platelet count and are benchmarked against the timing of discontinuation of therapeutic plasma exchange (TPE). They do not incorporate ADAMTS13 activity or the temporizing effects on the platelet count of caplacizumab, a novel anti-von Willebrand factor (VWF) nanobody. In light of these limitations, the IWG aimed to develop revised consensus outcome definitions that incorporate ADAMTS13 activity and the effects of anti-VWF therapy, by using an estimate-talk-estimate approach. The updated definitions distinguish clinical remission and clinical relapse (defined primarily by platelet count) from ADAMTS13 remission and ADAMTS13 relapse (defined by ADAMTS13 activity). The revised definitions of exacerbation and remission are benchmarked against not only the timing of discontinuation of TPE but also that of anti-VWF therapy. Retrospective validation of the revised definitions is described, although they have yet to be prospectively validated. Clinical implications of the updated outcome definitions are also discussed and an example of their application to clinical practice is provided to highlight their clinical relevance.

COVID-19 associated thrombotic thrombocytopenic purpura (TTP) ; A case series and mini-review.

INTRODUCTION: Thrombotic microangiopathies are a group of disorders that are mainly related to endothelial dysfunction. This category of endothelial dysfunction results of several imbalances between platelets, endothelium and immune system, also cytokine production. AIM OF THIS STUDY: To report cases with thrombotic thrombocytopenic purpura (TTP) and COVID-19 and review COVID-19 endothelial dysfunction literature. METHODS: Primary laboratory data, peripheral blood smear, ADAMTS13 antigen activity level, and antibody ordered for each of these four patients. Treatments for COVID-19 administered for all patients. Traditional treatments for TTP also were administered. RESULTS: There were numerous schistocytes (more than 5%) in peripheral blood smears for each patient. ADAMTS13 antigen activity level was below 10%, and ADAMTS13 antibody was elevated for each patient. COVID-19 PCR was positive for all patients, and CT-Scans were indicative of the involvement of COVID-19. CONCLUSION: In this case series, we reported four COVID-19 patients who presented with signs and symptoms of anemia and thrombocytopenia, resulting in thrombotic thrombocytopenic purpura.