An Overview of Laboratory Testing for ADAMTS13.

ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) is also called von Willebrand factor (VWF) cleaving protease (VWFCP). ADAMTS13 acts to cleave VWF multimers and thus reduce plasma VWF activity. In the absence of ADAMTS13 (i.e., in thrombotic thrombocytopenia purpura, TTP), plasma VWF can accumulate, in particular as "ultra-large" VWF multimers, and this can lead to thrombosis. Relative deficiencies in ADAMTS13 can also occur in a variety of other conditions, including secondary thrombotic microangiopathies (TMA). Of contemporary interest, COVID-19 (coronavirus disease 2019) may also be associated with relative reduction of ADAMTS13 and also pathological accumulation of VWF, with this likely contributing to the thrombosis risk seen in affected patients. Laboratory testing for ADAMTS13 can assist in the diagnosis of these disorders (i.e., TTP, TMA), as well as in their management, and can be achieved using a variety of assays. This chapter therefore provides an overview of laboratory testing for ADAMTS13 and the value of such testing to assist the diagnosis and management of associated disorders.

Automated and Rapid ADAMTS13 Testing Using Chemiluminescence: Utility for Identification or Exclusion of TTP and Beyond.

Thrombotic thrombocytopenic purpura (TTP) is a prothrombotic condition caused by a significant deficiency of the enzyme, ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). In the absence of adequate levels of ADAMTS13 (i.e., in TTP), plasma VWF accumulates, in particular as "ultra-large" VWF multimers, and this leads to pathological platelet aggregation and thrombosis. In addition to TTP, ADAMTS13 may be mildly to moderately reduced in a range of other conditions, including secondary thrombotic microangiopathies (TMA) such as those caused by infections (e.g., hemolytic uremic syndrome (HUS)), liver disease, disseminated intravascular coagulation (DIC), and sepsis, during acute/chronic inflammatory conditions, and sometimes also in COVID-19 (coronavirus disease 2019)). ADAMTS13 can be detected by a variety of techniques, including ELISA (enzyme-linked immunosorbent assay), FRET (fluorescence resonance energy transfer) and by chemiluminescence immunoassay (CLIA). The current report describes a protocol for assessment of ADAMTS13 by CLIA. This protocol reflects a rapid test able to be performed within 35 min on the AcuStar instrument (Werfen/Instrumentation Laboratory), although certain regional approvals may also permit this testing to be performed on a BioFlash instrument from the same manufacturer.

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

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

Analysis of thrombogenicity under flow reveals new insights into the prothrombotic state of patients with post-COVID syndrome.

BACKGROUND: Post-COVID syndrome (PCS) affects millions of people worldwide, causing a multitude of symptoms and impairing quality of life months or even years after acute COVID-19. A prothrombotic state has been suggested; however, underlying mechanisms remain to be elucidated. OBJECTIVES: To investigate thrombogenicity in PCS using a microfluidic assay, linking microthrombi, thrombin generation, and the von Willebrand factor (VWF):a Disintegrin and Metalloproteinase with a Thrombospondin Type 1 motif, member 13 (ADAMTS13) axis. METHODS: Citrated blood was perfused through microfluidic channels coated with collagen or an antibody against the VWF A3 domain, and thrombogenicity was monitored in real time. Thrombin generation assays were performed and α(2)-antiplasmin, VWF, and ADAMTS13 activity levels were also measured. RESULTS: We investigated thrombogenicity in a cohort of 21 patients with PCS with a median time following symptoms onset of 23 months using a dynamic microfluidic assay. Our data show a significant increase in platelet binding on both collagen and anti-VWF A3 in patients with PCS compared with that in controls, which positively correlated with VWF antigen (Ag) levels, the VWF(Ag):ADAMTS13 ratio (on anti-VWF A3), and inversely correlated with ADAMTS13 activity (on collagen). Thrombi forming on collagen presented different geometries in patients with PCS vs controls, with significantly increased thrombi area mainly attributable to thrombi length in the patient group. Thrombi length positively correlated with VWF(Ag):ADAMTS13 ratio and thrombin generation assay results, which were increased in 55.5% of patients. α(2)-Antiplasmin levels were normal in 89.5% of patients. CONCLUSION: Together, these data present a dynamic assay to investigate the prothrombotic state in PCS, which may help unravel the mechanisms involved and/or establish new therapeutic strategies for this condition.

Prognostic value of von Willebrand factor and ADAMTS13 in patients with COVID-19: A systematic review and meta-analysis.

BACKGROUND: Endotheliopathy and coagulopathy appear to be the main causes for critical illness and death in patients with coronavirus disease 2019 (COVID-19). The adhesive ligand von Willebrand factor (VWF) has been involved in immunothrombosis responding to endothelial injury. Here, we reviewed the current literature and performed meta-analyses on the relationship between both VWF and its cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) with the prognosis of COVID-19. METHODS: We searched MEDLINE, Cochrane Library, Web of Science, and EMBASE databases from inception to 4 March 2022 for studies analyzing the relationship between VWF-related variables and composite clinical outcomes of patients with COVID-19. The VWF-related variables analyzed included VWF antigen (VWF:Ag), VWF ristocetin cofactor (VWF:Rco), ADAMTS13 activity (ADAMTS13:Ac), the ratio of VWF:Ag to ADAMTS13:Ac, and coagulation factor VIII (FVIII). The unfavorable outcomes were defined as mortality, intensive care unit (ICU) admission, and severe disease course. We used random or fixed effects models to create summary estimates of risk. Risk of bias was assessed based on the principle of the Newcastle-Ottawa Scale. RESULTS: A total of 3764 patients from 40 studies were included. The estimated pooled means indicated increased plasma levels of VWF:Ag, VWF:Rco, and VWF:Ag/ADAMTS13:Ac ratio, and decreased plasma levels of ADAMTS13:Ac in COVID-19 patients with unfavorable outcomes when compared to those with favorable outcomes (composite outcomes or subgroup analyses of non-survivor versus survivor, ICU versus non-ICU, and severe versus non-severe). In addition, FVIII were higher in COVID-19 patients with unfavorable outcomes. Subgroup analyses indicated that FVIII was higher in patients admitting to ICU, while there was no significant difference between non-survivors and survivors. CONCLUSIONS: The imbalance of the VWF-ADAMTS13 axis (massive quantitative and qualitative increases of VWF with relative deficiency of ADAMTS13) is associated with poor prognosis of patients with COVID-19.

The use of obinutuzumab and ofatumumab in the treatment of immune thrombotic thrombocytopenic purpura.

Rituximab, an anti-CD20 monoclonal antibody, can be used to treat immune thrombotic thrombocytopenic purpura (iTTP) during acute presentation or disease relapse. Undesirable side-effects include severe hypersensitivity reactions, particularly anaphylaxis and rituximab-induced serum sickness, with a minority not maintaining a response to treatment. Alternative humanised anti-CD20 treatments, obinutuzumab and ofatumumab, have been used. A review of the UK TTP Registry showed 15 patients received these drugs over 26 treatment episodes (eight obinutuzumab and 18 ofatumumab). Indications for alternative anti-CD20 treatment were severe infusion-related reactions, acute rituximab-induced serum sickness and a short duration of disease remission. All patients achieved disease remission (ADAMTS13 [A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13] activity ≥30 iu/dl) after a median 15 days and 92% of episodes achieved complete remission (≥60 iu/dl). Seven patients required further treatment for disease relapse with a median relapse-free survival of 17.4 months. All patients continued to respond to re-treatment with the preceding drug when relapse occurred. There were four adverse events in 26 treatment episodes (15%) - two infections and two infusion reactions. These results suggest that obinutuzumab and ofatumumab may be considered as an alternative option to rituximab in the treatment of iTTP with a comparable safety profile, absence of significant hypersensitivity reactions and sustained normalisation of ADAMTS13.

ADAMTS13 factor deficiency in severe COVID-19 may not be immune mediated - report from a pilot study.

BACKGROUND: The assessment of ADAMTS13 factor activity and inhibitor levels was conducted in severe COVID-19 patients as an observational study. RESULTS: A total of 14 patients were included and the average ADAMTS13 activity level at the time of admission was 28.54±30.74% (range 1.83-86.67%) which was reduced compared to controls (88.09±14.77). Nine patients had reduced ADAMTS13 factor activity (<40%) and 77.7% among them had severe deficiency (<10% activity). ADAMTS13 inhibitor was positive (>15 IU/mL) only in two patients and an overall mean value was 8.15±5.8. Elevated D-Dimer and length of hospital stay had significant correlation with ADAMTS13 activity (-0.247 and 0.306 respectively). No features of thrombotic microangiopathy were observed and hence no plasma exchange was performed. CONCLUSION: Reduced ADAMTS13 factor activity without inhibitor development may give a clue to the disease progress in COVID-19.

Carriers of ADAMTS13 Rare Variants Are at High Risk of Life-Threatening COVID-19.

Thrombosis of small and large vessels is reported as a key player in COVID-19 severity. However, host genetic determinants of this susceptibility are still unclear. Congenital Thrombotic Thrombocytopenic Purpura is a severe autosomal recessive disorder characterized by uncleaved ultra-large vWF and thrombotic microangiopathy, frequently triggered by infections. Carriers are reported to be asymptomatic. Exome analysis of about 3000 SARS-CoV-2 infected subjects of different severities, belonging to the GEN-COVID cohort, revealed the specific role of vWF cleaving enzyme ADAMTS13 (A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 13). We report here that ultra-rare variants in a heterozygous state lead to a rare form of COVID-19 characterized by hyper-inflammation signs, which segregates in families as an autosomal dominant disorder conditioned by SARS-CoV-2 infection, sex, and age. This has clinical relevance due to the availability of drugs such as Caplacizumab, which inhibits vWF-platelet interaction, and Crizanlizumab, which, by inhibiting P-selectin binding to its ligands, prevents leukocyte recruitment and platelet aggregation at the site of vascular damage.

Impaired exercise capacity in post-COVID-19 syndrome: the role of VWF-ADAMTS13 axis.

Post-COVID syndrome (PCS), or long COVID, is an increasingly recognized complication of acute SARS-CoV-2 infection, characterized by persistent fatigue, reduced exercise tolerance, chest pain, shortness of breath, and cognitive slowing. Acute COVID-19 is strongly linked with an increased risk of thrombosis, which is a prothrombotic state quantified by an elevated von Willebrand factor (VWF) antigen (Ag)/ADAMTS13 ratio that is associated with severity of acute COVID-19 infection. We investigated whether patients with PCS also had evidence of a prothrombotic state associated with symptom severity. In a large cohort of patients referred to a dedicated post-COVID-19 clinic, thrombotic risk, including VWF(Ag)/ADAMTS13 ratio, was investigated. An elevated VWF(Ag)/ADAMTS13 ratio (≥1.5) was present in nearly one-third of the cohort and was 4 times more likely to be present in patients with impaired exercise capacity, as evidenced by desaturation ≥3% and/or an increase in lactate level >1 from baseline on a 1-minute sit-to-stand test and/or a 6-minute walk test (P < .0001). Of 276 patients, 56 (20%) had impaired exercise capacity, of which 55% (31/56) had a VWF(Ag)/ADAMTS13 ratio ≥1.5 (P < .0001). Factor VIII and VWF(Ag) were elevated in 26% and 18%, respectively, and support a hypercoagulable state in some patients with PCS. These findings suggest possible ongoing microvascular/endothelial dysfunction in the pathogenesis of PCS and suggest a role for antithrombotic therapy in the treatment of these patients.

Relapse of immune-mediated thrombotic thrombocytopenic purpura following mRNA COVID-19 vaccination: a prospective cohort study.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare and life-threatening disease. Vaccination has been reported to be a trigger of onset and relapse of autoimmune diseases. We evaluated after mRNA COVID-19 vaccination 32 adult patients previously diagnosed with iTTP by means of weekly monitoring of complete blood count and ADAMTS13 testing. Thirty of 32 patients received at least one dose of Pfizer-BioNTech, the remaining two received Moderna. A total of five patients, all vaccinated with Pfizer-BioNTech, had a biochemical relapse at a median post-vaccination time of 15 days following the second or third vaccine dose, presenting without measurable ADAMTS13 activity and a median anti- ADAMTS13 autoantibody value of 34 U/mL. Four of five cases had concomitant clinical relapse and were treated with corticosteroids alone or daily sessions of plasma exchange and caplacizumab, while one patient was closely monitored with ADAMTS13 with no onset of anemia and thrombocytopenia. Although the benefits of vaccination exceed its potential risks, clinicians should be aware that iTTP relapse might follow COVID-19 vaccination. Therefore, laboratory and clinical monitoring of iTTP patients should be done in the first post-vaccination month, in order to promptly diagnose and treat any relapse.

Refractory immune TTP following Pfizer-BioNTech COVID-19 vaccine successfully salvaged with caplacizumab.

Immune thrombotic thrombocytopenic purpura (iTTP) is a life-threatening thrombotic microangiopathy caused by antibodies against ADAMTS13. We report a young, healthy female who developed hematuria, vomiting, and hematemesis 3 weeks after her first dose of Pfizer Bio-NTech COVID-19 vaccine. Investigations confirmed iTTP with undetectable ADAMTS13 activity and a positive antibody assay. Despite initial response to standard treatment with plasma exchange and corticosteroids, she had an acute deterioration of her TTP with neurological and cardiac involvement. Fortunately, she then had prompt response to rituximab and emergently obtained caplacizumab and is now in remission. Although most cases of iTTP are of unknown etiology, we cannot exclude that her almost fatal iTTP episode was triggered by the Pfizer-BioNTech COVID-19 vaccine. This case also highlights the ability of caplacizumab to quickly halt disseminated thrombus formation in refractory TTP.

Acquired Thrombotic Thrombocytopenic Purpura After BNT162b2 COVID-19 Vaccine: Case Report and Literature Review.

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy that is deadly if not treated promptly. The treatment of choice in patients presenting with TTP is plasma exchanges. However, immunosuppressive therapy and caplacizumab have significantly improved outcomes in TTP. This microangiopathy is classically divided into 2 entities: hereditary and acquired TTP (aTTP), caused by an autoantibody against ADAMTS 13. We present a case study of a patient wth TTP occurring after a second dose of the BNT162b2 (Pfizer-BioNTech) COVID-19 vaccine along with a review of the literature. A 55-year-old patient presented with gastrointestinal symptoms, anemia, and severe thrombocytopenia. The blood film revealed the presence of schistocytes. A diagnosis of aTTP was established because the patient had severe ADAMTS 13 deficiency and autoantibodies against ADAMTS 13 were positive. This episode occurred 10 days after the patient received the COVID-19 vaccine. The patient received plasma exchanges, prednisone, rituximab, and caplacizumab and achieved complete remission. Ten patients with aTTP induced by the COVID-19 vaccine have been reported in the literature. Most of these situations occurred after the second dose of COVID-19 vaccine, and 7 patients were noted to have received the BNT162b2 vaccine. Caplacizumab was used in 6 patients, and complete remission was achieved in 8 patients.

COVID-19 microthrombosis: unusually large VWF multimers are a platform for activation of the alternative complement pathway under cytokine storm.

ADAMTS13, a metalloproteinase, specifically cleaves unusually large multimers of von Willebrand factor (VWF), newly released from vascular endothelial cells. The ratio of ADAMTS13 activity to VWF antigen (ADAMTS13/VWF) and indicators of the alternative complement pathway (C3a and sC5b-9) are both related to the severity of COVID-19. The ADAMTS13/VWF ratio is generally moderately decreased (0.18-0.35) in patients with severe COVID-19. When these patients experience cytokine storms, both interleukin-8 and TNFα stimulate VWF release from vascular endothelial cells, while interleukin-6 inhibits both production of ADAMTS13 and its interaction with VWF, resulting in localized severe deficiency of ADAMTS13 activity. Platelet factor 4 and thrombospondin-1, both released upon platelet activation, bind to the VWF-A2 domain and enhance the blockade of ADAMTS13 function. Thus, the released unusually-large VWF multimers remain associated with the vascular endothelial cell surface, via anchoring with syndecan-1 in the glycocalyx. Unfolding of the VWF-A2 domain, which has high sequence homology with complement factor B, allows the domain to bind to activated complement C3b, providing a platform for complement activation of the alternative pathway. The resultant C3a and C5a generate tissue factor-rich neutrophil extracellular traps (NETs), which induce the mixed immunothrombosis, fibrin clots and platelet aggregates typically seen in patients with severe COVID-19.