Deficiency Of Plasma ADAMTS13 Activity Research Articles

Hereditary TTP/Upshaw-Schulman syndrome: the ductus arteriosus controls newborn survival.

Hereditary TTP (hTTP), termed Upshaw-Schulman syndrome, is an ultra-rare disorder caused by a severe deficiency of plasma ADAMTS13 activity that allows circulation of ultra-large von Willebrand factor (UL-VWF) multimers. The greatest risk for hTTP is in their first days after birth, when 35-50% of patients will have severe hemolysis, jaundice, and thrombocytopenia. It is often fatal without effective treatment. In utero, fetal blood flowing from the pulmonary artery through the ductus arteriosus (DA) to the aorta is under low-shear-force. At birth, blood flow through the DA reverses to a left-to-right shunt, and the diameter of the DA begins to decrease due to hyper-oxygenated blood and decreased plasma prostaglandin E2. This causes turbulent circulation that unfolds UL-VWF, allowing platelet aggregation. If the DA closes promptly, hTTP newborns survive, but if it remains patent, turbulent circulation persists, triggering microvascular thrombosis. hTTP is commonly diagnosed as hemolytic disease of the fetus and newborn (HDFN) caused by anti-red cell antibodies and treated with exchange blood transfusion, which prevents kernicterus even when the diagnosis of hTTP is missed. The diagnosis of newborn-onset hTTP should be considered because HDFN does not cause severe thrombocytopenia, which might be effectively treated with recombinant ADAMTS13.

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

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

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.

The Potential Role of ANKRD36 in Immune Thrombotic Thrombocytopenic Purpura

A.B. and L.Z. Contribute equally to this work. Background: Immune thrombotic thrombocytopenic purpura (iTTP), caused by autoantibody against ADAMTS13, is characterized by the formation of microvascular thrombosis, resulting in thrombocytopenia, microangiopathic hemolytic anemia, and organ damage. The mechanism underlying autoantibody formation and pathogenesis of iTTP are not fully understood. Our lab previously reported a strong association between mutations in the Ankyrin Repeat Domain 36 (ANKRD36) family genes and iTTP. To further understand the role of ANKRD36 in iTTP, we established a partial deletion mutant of Ankrd36 in mice, which were bred with Adamts13-/- mice to generate double homozygous mice. The role of Ankrd36 in a murine model of TTP and the inflammatory or immunological responses to potential viral infection were assessed. Methods: Mice with various genotypes including wild-type (WT), Adamts13-/- (A13-/-), Ankrd36-/-(AK36-/-), and Ankrd36-/-Adamts13-/- (AK36-/-A13-/-) were treated with 10 mg/kg polyinosinic-polycytidylic acid [Poly (I:C)] via intraperitoneal injection. Complete blood count in anti-coagulated whole blood was performed at baseline, 4, 24, and 72 h post-challenge. Major organ tissues were collected for histology, immunohistochemistry, and flow cytometric analyses from unchallenged animals or 72 h following Poly (I:C) injection. Results: All mice at baseline did not exhibit thrombocytopenia (Fig. 1A, left). Seventy-two hours following the challenge with Poly (I:C), platelet counts were significantly reduced in A13-/- with or without AK36-/- but not in AK36-/- alone mice compared with those in WT controls. Interestingly, the median platelet counts appeared to be the lowest in AK36+/-A13-/- among all groups (Fig. 1A, right). Histology and immunohistochemical analyses revealed occlusive thrombi in terminal arterioles as exemplified by the positive staining with anti-VWF or anti-fibrinogen IgG 72 hours following Poly (I:C) challenge (Fig. 1B). Moreover, our histological analysis demonstrated a consistent drop in the percentage of splenic white pulp following Poly (I:C) treatment in all genotypes with the most dramatic drop in the WT, followed by the AK36-/-A13-/-, then the A13-/-,and AK36-/-mice. Immunohistochemical staining of spleen tissue revealed a significant increase in both B and T cells in the red pulp, which led us to conduct the flow cytometrical experiments. Our flow results demonstrated that following Poly (I:C) treatment the percentage of CD3+ T cells, CD138+ plasma cells, and CD11b+F4/80+ macrophages decreased, while the percentage of B cells increased (Fig. 2). Specifically, within the T cell population, CD3+CD4+ helper T cells increase while CD3+CD8+ natural killer T cells decreased. In all cell types except for macrophages, the AK36-/- group shows no change from the wild type. Interestingly, our AK36-/-A13-/- mice showed similar or more profound changes compared to the WT mice in all cell types. Conclusions: Our results demonstrate for the first time a synergistic role of partial deletion of ANKRD36 protein in pathogenesis of TTP resulting from severe deficiency of plasma ADAMTS13 activity and potential viral infection. Additionally, our results indicate that the ANKRD36 family proteins may play a regulatory role in the immune and inflammatory responses. Further investigation into the interaction between ANKRD36 family protein and ADAMTS13 may help understand the pathogenic mechanism of iTTP and other immune inflammatory disorders. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ADAMTS13 protease or lack of von Willebrand factor protects irradiation and melanoma‐induced thrombotic microangiopathy in zebrafish

BackgroundSevere deficiency of plasma ADAMTS13 activity may result in potentially fatal thrombotic thrombocytopenic purpura and relative deficiency of plasma ADAMTS13 activity may be associated with adverse outcomes of certain malignancies. Here, we report the role of ADAMTS13 or lack of von Willebrand factor (VWF) in reducing irradiation and melanoma‐induced thrombotic microangiopathy (TMA) and mortality in zebrafish. MethodsZebrafish melanoma cell line (ZMEL) was injected subcutaneously into wild‐type (wt), adamts13−/− (a13−/−), von Willebrand factor (vwf−/−), and a13−/−vwf−/− zebrafish following total body irradiation; the tumor growth, its gene expression pattern, the resulting thrombocytopenia, and the mortality were determined. ResultsTotal body irradiation at 30 Gy alone resulted in a transient thrombocytopenia in both wt and a13−/− zebrafish. However, thrombocytopenia occurred earlier and more profound in a13−/− than in wt zebrafish, which was resolved 2 weeks following irradiation alone. An inoculation of ZMEL following the irradiation resulted in more severe and persistent thrombocytopenia, as well as earlier death in a13−/− than in wt zebrafish. The vwf−/− or a13−/−vwf−/− zebrafish were protected from developing severe thrombocytopenia following the same maneuvers. RNA‐sequencing revealed significant differentially expressed genes associated with oxidation–reduction, metabolism, lipid, fatty acid and cholesterol metabolic processes, steroid synthesis, and phospholipid efflux in the melanoma explanted from a13−/− zebrafish compared with that from the wt controls. ConclusionsOur results indicated that plasma ADAMTS13 or lack of VWF may offer a significant protection against the development of irradiation‐ and/or melanoma‐induced TMA. Such a microenvironment may directly affect melanoma cell phenotypes via alternation in the oxidation–reduction and lipid metabolic pathways.

Neutrophil Extracellular Traps (NETs) Contribute to the Formation of Microvascular Thrombosis in Immune Thrombotic Thrombocytopenic Purpura

Introduction. Immune thrombotic thrombocytopenic purpura (iTTP), a potentially fatal blood disorder, is primarily caused by severe deficiency of plasma ADAMTS13 activity resulting from immunoglobulin (Ig) G-mediated inhibition of plasma ADAMTS13 activity. However, severe ADAMTS13 deficiency is necessary but not sufficient to cause acute iTTP. An environmental factor such as infection or acute inflammation may be necessary to trigger the acute onset of the disease. We and others have previously reported that plasma markers of neutrophil activation and neutrophil extracellular traps (NETs) formation are significantly elevated in patients with acute iTTP, which returns to normal during remission. However, the pathogenetic role of NETs in acute iTTP is not fully understood.Methods and results. Using flow cytometry, microfluidic shear-based assay, and confocal imaging analysis, we determined the in vivo NETosis in blood samples obtained from patients with acute episode of iTTP and ex vivo NETs formation, as well as the therapeutic efficacy of DNase I on thrombus formation under flow. We showed that by flow cytometry that only very few CitH3+/MPO+ positive neutrophils were present in the healthy donor blood. This population of cells dramatically increased after being stimulated with a bacterial toxin (i.e., Shigatoxin-2) at ~100 ng/mL for 15 min. Importantly, the number of CitH3+/MPO+ positive neutrophils in the sample obtained from a patient with acute iTTP was ~1,000 times higher than that in the healthy controls (Fig. 1), suggesting a massive NETosis in patients with acute iTTP. Microfluidic shear-based assay and confocal imaging analysis further confirmed a dramatic increase in adhesion and aggregation of murine platelets (stained with Alexa647 anti-CD41) and neutrophil (stained with Hoechst), as well as formation of NETs (stained with Syto green) following a perfusion of an Adamts13 -/- murine whole blood (anti-coagulated with thrombin inhibitor, PPACK) under arterial shear (15 dyne/cm 2) over a stimulated murine endothelial surface. Interestingly, an addition of DNase I (100 U/mL) significantly reduced the overall surface coverage of platelets and neutrophils on the murine endothelial surface under the same conditions (Fig. 2).Conclusions. These results demonstrate for the first time NETosis and NETs formation are common in patients with acute iTTP and in Adamts13 -/- mice after being stimulated with shigatoxin; DNase I appears to be highly efficacious eliminating the NETs and platelet/neutrophil-dominant thrombosis under arterial flow. Our findings support the pathogenetic role of NETs in the onset and progression of iTTP, and the therapeutic potential of DNase I in such a fatal disease. [Display omitted] DisclosuresZheng: Alexion: Speakers Bureau; Sanofi-Genzyme: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria; Clotsolution: Other: Co-founder; AJMC: Honoraria.

Lysine-Histone Triggers Thrombotic Thrombocytopenic Purpura in Zebrafish and Mice

Background: Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal syndrome, primarily resulting from a severe deficiency of plasma ADAMTS13 activity due to mutations in ADAMTS13 gene or autoantibodies against ADAMTS13. However, severe deficiency of plasma ADAMTS13 activity alone is often not sufficient to cause an acute episode; additional environmental and/or genetic factors may be required to trigger acute TTP.Objectives: To address the potential triggers and identify novel therapeutic targets for TTP, we established and characterized a novel Adamts13-/- zebrafish line and determined the pathogenic role of lysine-histone in triggering TTP in both zebrafish and mice.Methods and Results:CRISPR/Cas9 was employed to create an 8-nt deletion mutation in the signal peptide region of zebrafish Adamts13, resulting in complete deficiency of Adamts13 protein. Adamts13-/- zebrafish on the double transgenic (fli1-eGFP/gata1-dsRed) background showed normal size, morphology, and fertility except for the consistent appearance of a branching interseptal vessel. Adamts13-/- zebrafish larvae exhibited a significantly increased response to oxidative stress with an increased mortality rate and an accelerated rate of thrombus formation in microvasculature and caudal vessels after oxidative injury. Additionally, Adamts13-/- zebrafish demonstrated a significantly reduced number of circulating mature thrombocytes (thrombocytopenia) and an increased number of fragmented red blood cells (schistocytosis), consistent with the clinical phenotype of a spontaneous TTP. Moreover, plasma levels of histone/DNA complexes were found to be highly elevated in patients with acute TTP, but their pathogenic role is not known. Wild-Type and Adamts13-/- zebrafish and mice were challenged with a lysine rich-histone (H5505) intraperitoneally (200 μg/g body weight) and intravenously (20, 60, and 120 μg/g body weight), respectively. Complete blood counts were determined over a period of 14 and 3 days, respectively. We showed a more severe and persistent thrombocytopenia, increased fragmentation of red blood cells, as well as increased mortality rate in Adamts13-/- compared to Wild-Type zebrafish (Figure). Similar dose-dependent and profound thrombocytopenia was observed in Adamts13-/- but not in Wild-Type mice. Histological analysis of the organ tissues from Adamts13-/- zebrafish that died after histone challenge revealed the presence of microvascular thrombosis, supporting the diagnosis of TTP in these zebrafish.Conclusions: These results demonstrate for the first time the success in establishing a zebrafish model of Adamts13-/- and TTP. Such a novel animal model enables us to rapidly evaluate the role of lysine-histone and many other potential environmental or genetic factors in the onset, progression, and outcome of TTP. This model may also help to identify novel therapeutic targets for both hereditary and acquired TTP in at an unprecedented pace. [Display omitted] DisclosuresZheng:Alexion: Speakers Bureau; Ablynx: Consultancy.

Hydrogen-Deuterium Exchange Plus Mass Spectrometry (HX-MS) Reveals a Novel Mechanism Underlying the pH-Dependent Regulation of ADAMTS13 Activity

Background: ADAMTS13 is a metalloprotease that is essential for the cleavage of von Willebrand factor (VWF). Deficiency of plasma ADAMTS13 activity causes a potentially fatal blood disorder, thrombotic thrombocytopenic purpura. Previous studies demonstrated a two-fold increase in the proteolytic activity of ADAMTS13 towards the VWF73 substrate by deleting the distal C-terminal domains, decreasing the pH, or binding a monoclonal antibody to the C-terminal domains. This suggests an allosteric interaction between the distal and proximal domains of ADAMTS13 modulated by pH.Objectives: To identify peptide regions within a recombinant full-length ADAMTS13 (rADAMTS13) responsible for the pH-modulated allosteric mechanism.Methods: To identify regions in the rADAMTS13 that undergo conformational changes in response to a change in pH, we performed hydrogen deuterium exchange plus mass spectrometry (HX-MS) at three different pH conditions, pH 6, pH 7, and pH 8. Because the rate of hydrogen and deuterium exchange is dependent upon the pH in a logarithmic fashion, a single time point from each of these pH conditions were compared. For instance, 6000 s was used for pH 6, 600 s for pH 7, and 60 s for pH 8. Any differences in the deuterium incorporation in these conditions in a given peptide should be contributed to conformational changes within the protein effecting accessibility and not simply the rate of hydrogen and deuterium exchange.Results: Purified rADAMTS13 and a variant truncated after the spacer domain (rMDTCS) were incubated with deuterium for various times (0- 6000 s). After quenching the reaction by lowering pH to 2.5 and temperature to 0 °C, the rADAMTS13 and rMDTCS were digested by pepsin and fungal protease-13 and then partially resolved by liquid chromatography before being injected into a LTQ ObitrapXL mass spectrometer for peptide identification. Upon comparing the mass spectra of these three conditions, the peptides with mass spectra shift greater than 1 m/z unit as a result of conformational changes from the pH change were identified in the metalloprotease, cysteine-rich, spacer, and CUB-2 domains. In the metalloprotease, three peptide regions were identified (i.e. residues 110 -131, 181-197, and 217-230) with 19, 6, and 1 overlapping peptides, respectively. Within the cysteine rich domain, there were two identified peptide regions, 468 - 480 and 502 - 503. Within the spacer domain, there was only one identified peptide region 629-630, with 5 overlapping peptides. Finally, In CUB-2, there were two peptide regions, 1327-1340 and 1366-1377, with 2 overlapping peptides each (see Figure). All of these regions, with only the exception of residues 217-230, were more accessible to deuterium at pH 6 than at pH 8.To determine if these results are contingent upon the whole protein being present, and possible intramolecular protein interactions, the HX experiment was repeated using two ADAMTS13 variants (i.e. rMDTCS and rT5C). The rT5C contains the C-terminal TSP1 5-8 repeats and CUB domains. Because of smaller size, more overlapping peptides were recovered, which allows for narrowing down the residues central to the conformational change. Two small areas were narrowed down to several peptides, residues 120-123, with 6 overlapping peptides, and residue 503 within the cysteine rich with 3 overlapping peptides. Curiously, both regions within the CUB-2 domain showed little change with pH, despite 6 overlapping peptides in the residues 1327 -1340 and residues 1366-1377 (see Figure), suggesting the necessity of the proximal domains of the protein to modulate the conformation as a function of pH.Finally, the regions within the rADAMTS13 that exhibit a pH dependent conformation change are similar to the regions we reported showing bimodal behavior previously. Regions within the metalloprotease, the cysteine rich, and CUB-2 are all shared in residues 217-230, 468-480, and 1327-1328, respectively (see Figure).Conclusions: Our results, along with the direct binding experiments using a surface plasmon resonance assay, suggest that the N- and C-terminus of ADAMTS13 interact with each other at multiple discrete sites, which mediates the pH-dependent regulation of ADAMTS13 conformation and function. [Display omitted] DisclosuresZheng:Alexion: Speakers Bureau; Ablynx: Consultancy.

Elevated plasma levels of syndecan-1 and soluble thrombomodulin predict adverse outcomes in thrombotic thrombocytopenic purpura

AbstractImmune-mediated thrombotic thrombocytopenic purpura (iTTP) is a potentially fatal blood disorder resulting from acquired deficiency of plasma ADAMTS13 activity. Despite recent advances in early diagnosis and novel therapeutics, the mortality rate of acute iTTP remains as high as 10% to 20%. Moreover, a reliable clinical and laboratory parameter that predicts disease severity and outcomes is lacking. We show in the present study that plasma levels of syndecan-1 (Sdc-1) and soluble thrombomodulin (sTM) on admission were dramatically increased in patients with acute iTTP and remained substantially elevated in a subset of patients compared with healthy controls. The elevated admission plasma levels of Sdc-1 and sTM were associated with abnormal Glasgow coma scale scores, low estimated glomerular filtration rates, the need for intensive care, and in-hospital mortality rates. Moreover, a further simultaneous increase in plasma Sdc-1 and sTM levels at the time of clinical response/remission (eg, when normalization of platelet counts and substantial reduction of serum lactate dehydrogenase activity were achieved) was highly predictive of iTTP recurrence. These results demonstrate that endothelial injury, resulting from disseminated microvascular thromboses, is severe and persistent in patients with acute iTTP. Plasma levels of Sdc-1 and sTM on admission and in remission are predictive of in-hospital mortality and recurrence of acute iTTP, respectively. Thus, an incorporation of such novel plasma biomarkers into the risk assessment in acute iTTP may help implement a more vigorous and intensive therapeutic strategy for these patients.

Factors Associated with Mortality in Patients Experiencing First Episodes of Acquired Thrombotic Thrombocytopenic Purpura (aTTP). Results of the Spanish TTP Registry

Introduction: Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare, but life-threatening, hematological disorder characterized by severe thrombocytopenia, hemolytic microangiopathic anemia, and frequent organ damage. The underlying pathophysiology of aTTP is a functional deficiency of plasma ADAMTS13 activity caused by antibodies directed against the ADAMTS13 protease. Despite plasma exchange (PEX) and immunosuppression with corticosteroids, and, more recently, rituximab, which achieve remission in most patients with aTTP, 10-20% of patients are refractory to treatment and die as a result of disease progression. Most of such deaths occur during first episodes of aTTP, as subsequent relapses tend to be milder. These patients would probably benefit from new therapies aimed at temporarily halting the microvascular thrombosis. This study was aimed at identifying predictive factors of mortality during a first episode of aTTP. Methods: We searched the Spanish TTP Registry (REPTT, Registro Español de la Purpura Trombocitopénica Trombótica) for patients with a clinical diagnosis of TTP (n = 345) with ADAMTS13 activity &lt;10%, and anti-ADAMTS13 inhibitors in plasma (n = 143). Among these, we selected 103 patients with complete information on their first episode of aTTP. The patients were diagnosed between 2004 and 2018, and all were treated with daily PEX and corticosteroids. Additional treatments (rituximab, vincristine, etc.) were used at the discretion of the attending hematologist. Clinical and laboratory data of the episodes were analyzed at diagnosis and during the course of the treatment. The impact of refractoriness, exacerbations (as defined by the international consensus; DOI: 10.1111/jth.13571) and platelet transfusions on mortality was also studied. Results: The 103 patients examined suffered a total of 111 episodes of aTTP (103 with a first episode and 8 with a relapse). Eight deaths (7.7%) took place during an initial episode of aTTP and none during relapses. The time elapsed from the diagnosis to the events of death ranged from a few hours to 36 days (Figure). One patient died before starting the treatment. In the multivariate analysis, stupor or coma at diagnosis was the only clinical and biological factor associated with mortality, as 6 out of 18 patients with stupor or coma died vs. 2 out of 85 without these symptoms (OR: 21.95% CI: 4-114; p = 0.001). Persistently low platelet counts during PEX (i.e. refractoriness) were also strong predictors of subsequent mortality, with a platelet count &lt;20 x109/L after 6 PEX procedures yielding the highest positive and negative values (Table). Exacerbation of aTTP while on treatment occurred in 44 of the 95 patients, all of whom eventually achieved remission with no events of death. Patients with exacerbations required a higher number of PEX procedures to achieve clinical remission (23, IQR: 19-31 vs. 12, IQR: 8-16; p &lt;0.001) and were more likely to receive rituximab (39% vs. 9%; p = 0.001). Platelets were transfused into 16 patients, including 2 patients who died a few days later and 14 survivors. Conclusions: Stupor or coma at diagnosis and lack of response to PEX by the 6th-7th day in patients experiencing first episodes of aTTP are strong predictors of mortality. These patients are candidates for new treatments aimed at controlling the microvascular thrombotic phenomena until effective immunosuppression is achieved. Disease exacerbation does not seem to increase mortality but requires a more intensive treatment. Disclosures De La Rubia: Celgene Corporation: Consultancy; AbbVie: Consultancy; AMGEN: Consultancy; Janssen: Consultancy; Takeda: Consultancy. Pascual Izquierdo:Novartis: Consultancy; Sanofi: Consultancy.

Longitudinal Assessment of Plasma ADAMTS13 Biomarkers Helps Predict Recurrence of Immune Thrombotic Thrombocytopenic Purpura

Background: Severe deficiency of plasma ADAMTS13 activity resulting from autoantibodies against ADAMTS13 is the primary cause of immune thrombotic thrombocytopenic purpura (iTTP). The anti-ADAMTS13 IgGs may bind and inhibit plasma ADAMTS13 activity and/or accelerate its clearance from circulation. Approximately 40% of iTTP patients who survived the initial episode may experience disease recurrence (e.g. exacerbation and/or relapse). However, a reliable biomarker that predicts disease recurrence is still lacking. The present study sought to determine the roles of plasma ADAMTS13 activity, antigen, inhibitors, and anti-ADAMTS13 IgG at various stages of the disease process in predicting recurrence (e.g. exacerbation and/or relapse) in adult patients with iTTP. Methods: 93 episodes from 83 unique iTTP patients who underwent therapeutic plasma exchange (TPE) therapy at UAB Medical center from April 2006 to December 2018 were enrolled into the study. Clinical and laboratory parameters were collected at diagnosis, and ADAMTS13 biomarkers (e.g. activity, inhibitors, antigen, and anti-ADAMTS13 IgG) were determined in samples collected on admission, 3-7 days following the first TPE, and at clinical response (prior to discharge). Various statistical analyses including Kaplan-Meier survival and Cox analyses were performed to assess the clinical relevance of each biomarker at various stages of the disease course. Results: We found that plasma ADAMTS13 activity and antigen on admission had no significant value in predicting iTTP exacerbation (disease recurrence within 30 days of discontinuation of TPE) or relapse (the disease recurrence after 30 days of discontinuation of TPE). However, high levels of anti-ADAMTS13 IgG on admission were associated with an increased risk of exacerbation (HR=2.1, 95% CI, 1.0-4.1, p=0.34) or recurrence within one year (HR=2.0, 95% CI 1.0-3.9, p=0.041). More interestingly, low plasma ADAMTS13 activity (&lt;10 U/dL) 3 to 7 days after initiation of TPE (HR=3.8, 95% CI 1.3-11.0, p=0.015) or at clinical remission (median 7 days, IQC 6-10 days) when platelet counts and lactate dehydrogenase (LDH) levels had normalized (HR=4.3, 95% CI 1.6-11.8, p=0.004) and low levels of plasma ADAMTS13 antigen at clinical remission (HR=3.2, 95% CI 1.1-7.5), even when body mass index, race, and partial thromboplastin time (PTT) were all included as covariates for the analysis, were associated with the increased risks of iTTP recurrence. Conclusions: Longitudinal assessment of plasma ADAMTS13 biomarkers (e.g., activity, antigen and anti-ADAMTS13 IgG) is of a critical value for predicting iTTP recurrence. These results suggest the need for additional therapeutic coverage (such as the use of rituximab, corticosteroids, and caplacizumab) to prevent iTTP recurrence after discharge from the hospital and supports the concept for achieving the biochemical remission (e.g. the normalization of ADAMTS13 activity, antigen, and anti-ADAMTS13 IgG) for the long-term management of patients with iTTP. Disclosures Zheng: Shire/Takeda: Research Funding; Clotsolution: Other: Co-Founder; Ablynx/Sanofi: Consultancy, Speakers Bureau; Alexion: Speakers Bureau.

Exome Sequencing Identifies Glycosylation Defects As a Probable Cause of Immune Thrombotic Thrombocytopenic Purpura

Background: Immune thrombotic thrombocytopenic purpura (iTTP) is a potentially fatal syndrome, resulting primarily from autoantibodies against ADAMTS13. However, the mechanism underlying the autoantibody formation and the contribution of other genomic alterations to the pathogenesis of iTTP are largely unknown. Methods: Whole exome sequencing (WES) and bioinformatic analyses were performed to determine the genetic variations in 40 patients with iTTP who had ADAMTS13 activity &amp;lt;10 IU/dL and a positive inhibitor or an elevated anti-ADAMTS13 IgG in concordance with clinical presentations of severe thrombocytopenia and microangiopathic hemolytic anemia with various degrees of organ injury. WES was also performed at the same time in fifteen age-, gender-, and ethnicity- matched individuals who did not have a history of iTTP or other hematological disorders as controls. Results: WES identified variants or mutations in the genes involving in glycosylation, including O-linked glycosylation, to be the major pathway affected in patients with iTTP. We propose that the altered glycosylation may be responsible for the development of autoantibodies against ADAMTS13 which impair the proteolytic cleavage of von Willebrand factor, accelerate the clearance of ADAMTS13 from circulation, and result in severe thrombocytopenia platelets in patients with iTTP. We also identified defects in ankyrin repeat containing protein ANKRD36C, a protein with hitherto unknown function, as the most statistically significant genomic alterations associated with iTTP (p &amp;lt; 10-5). Moreover, candidate gene analysis revealed that various genes involving in hemostasis, complement activation, platelet function and signaling pathway, and inflammation were all affected in patients with iTTP, which may contribute to the onset, progress, severity, and long-term outcome of iTTP. Finally, we also identified two patient subgroups where the disease mechanism might be different. Conclusion: Our findings provide novel insight into the pathogenic mechanism underlying ADAMTS13 autoantibody production and the potential contribution of other genetic abnormalities in modifying the iTTP clinical presentations in the individuals with severe deficiency of plasma ADAMTS13 activity. Disclosures Zheng: Alexion: Speakers Bureau; Ablynx/Sanofi: Consultancy, Speakers Bureau; Shire/Takeda: Research Funding; Clotsolution: Other: Co-Founder.

Plasma Levels of Histone-DNA Complex and Cell-Free DNA Predict Mortality in Patients with Immune Thrombotic Thrombocytopenic Purpura

Background: Deficiency of plasma ADAMTS13 activity resulting from acquired autoantibody against ADAMTS13 is the primary cause of immune thrombotic thrombocytopenic purpura (iTTP). Therapeutic plasma exchange plus corticosteroids and immunosuppression is the standard of care for patients with iTTP. However, mortality rate remains high and clinical factors or laboratory biomarkers that predict mortality are not fully established. Previous studies have demonstrated that plasma levels of histone-DNA complexes and cell-free DNA are dramatically elevated in patients with acute disease but reduced during remission. We hypothesize that elevated histone-DNA complexes or cell-free DNA may have a predictive role for mortality in patients with acute iTTP. Methods: 102 unique patients with a confirmed diagnosis of iTTP who underwent therapeutic plasma exchange (TPE) at UAB-Medical Center from April 2006 to December 2018 were enrolled into the study. Demographic information and laboratory parameters were collected on admission and during the follow-up. Plasma levels of histone-DNA complexes and cell-free DNA were determined by an enzyme-linked immunosorbent assay and PicoGreen dsDNA assay, respectively. Mann-Whitney, Fisher's exact, t test, log-rank test, and Cox proportional hazard regression analysis were performed to determine the significance of each marker in predicting death. Results: In this cohort, age was 44.7 ± 1.3 (mean ± SD) years old, 56.9% were female, 82.4% were African Americans, 61.8% had an initial episode, the median (IQR) body mass index (BMI) was 33.6 (29.3-41.5). 52.9% of patients had hypertension, 21.6% diabetes, 18.6% systemic lupus erythematous and 8.7% HIV. The overall mortality rate was 10.8% (11/102). Plasma levels, median (IQR), of histone-DNA complexes and cell-free DNA on admission were 56.3 (35.8-136.4) AU/mL and 952.2 (799.7-1431.4) ng/mL, respectively. Mann-Whitney test revealed that high plasma levels of histone-DNA complex (≥134.9 U/mL) (p=0.007) and cell-free DNA (≥952.2 ng/mL) (p=0.009) were associated with the increased risks of myocardial injury (e.g. elevated troponin). Cox proportional hazard regression analysis demonstrated that the increased levels of histone-DNA complexes and cell-free DNA were also predictive for the increased mortality rates in patients with iTTP with hazard ratios (HRs) of 4.1 (95% CI, 1.2-13.4) (p=0.02) and 8.5 (95% CI, 2.2-33.3 (p=0.002), respectively. Kaplan-Meier survival analysis revealed significant differences in the disease-free survival rates of patients with high levels (≥75 percentile) vs. low levels (&lt;75 percentile) of plasma histone-DNA complexes and cell-free DNA, respectively (see Fig. 1). Conclusion: Our results demonstrate that the elevated plasma levels of histone-DNA complexes and cell-free DNA predict the worse outcome (e.g. the increased mortality rates) in patients with iTTP. *SD, standard deviation; IQR, interquartile range; 95% CI, 95% confidential interval. Disclosures Zheng: Ablynx/Sanofi: Consultancy, Speakers Bureau; Alexion: Speakers Bureau; Shire/Takeda: Research Funding; Clotsolution: Other: Co-Founder.

Synergistic effects of ADAMTS13 deficiency and complement activation in pathogenesis of thrombotic microangiopathy

AbstractSevere deficiency of plasma ADAMTS13 activity is the primary cause of thrombotic thrombocytopenic purpura (TTP) whereas overwhelming activation of complement via an alternative pathway results in atypical hemolytic uremic syndrome (aHUS), the prototypes of thrombotic microangiopathy (TMA). However, clinical and pathogenic distinctions between TTP and aHUS are often quite challenging. Clinical reports have suggested that complement activation may play a role in the development of TTP, which is caused by severe deficiency of plasma ADAMTS13 activity. However, the experimental evidence to support this hypothesis is still lacking. Here, we show that mice with either Adamts13−/− or a heterozygous mutation of complement factor H (cfh) at amino acid residue of 1206 (ie, cfhW/R) alone remain asymptomatic despite the presence of occasional microvascular thrombi in various organ tissues. However, mice carrying both Adamts13−/− and cfhW/R exhibit thrombocytopenia, low haptoglobin, increased fragmentation of erythrocytes in peripheral blood smear, increased plasma levels of lactate dehydrogenase activity, blood urea nitrogen, and creatinine, as well as an increased mortality rate, consistent with the development of TMA. Moreover, mice with a homozygous mutation of cfh (ie, cfhR/R) with or without Adamts13−/− developed severe TMA. The mortality rate in mice with Adamts13−/−cfhR/R was significantly higher than that in mice with cfhR/R alone. Histological and immunohistochemical analyses demonstrated the presence of disseminated platelet-rich thrombi in terminal arterioles and capillaries of major organ tissues in these mice that were either euthanized or died. Together, our results support a synergistic effect of severe ADAMTS13 deficiency and complement activation in pathogenesis of TMA in mice.

Exome Sequencing Identifies Glycosylation Defects as a Probable Cause of Immune-Mediated Thrombotic Thrombocytopenic Purpura

Abstract Background Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a potentially fatal syndrome, resulting from autoantibodies against ADAMTS13. However, the mechanism underlying autoantibody formation is not known. Neither is known about the other genetic abnormality in the setting of severe deficiency of plasma ADAMTS13 activity. Methods Whole-exome sequencing (WES) was performed in 40 patients with iTTP who had plasma ADAMTS13 activity &lt;10% and a positive inhibitor or elevated anti-ADAMTS13 IgG. Fifteen age- and ethnicity-matched subjects who never had iTTP were recruited as healthy controls. Results WES identified mutations in the genes involved in glycosylation, including O-linked glycosylation to be the major pathway affected in patients with iTTP. Mass spectrometry confirmed the changes in plasma levels of various glycoproteins in patients with acute iTTP when compared with those in the healthy controls. The altered glycosylation in glycoproteins may be responsible for the development of autoantibodies, susceptibility of von Willebrand factor to proteolysis by ADAMTS13, and the clearance of platelets in iTTP patients. Moreover, candidate gene analysis revealed that various genes involving in hemostasis, complement activation, platelet number and function, and inflammation were all affected in patients with iTTP, which may contribute to the onset, progress, severity, and long-term outcome of iTTP. Conclusions Our findings provide novel insight into a pathogenic mechanism underlying autoantibody production and the potential contribution of other genetic abnormalities in pathogenesis of iTTP in the individuals with severe deficiency of plasma ADAMTS13 activity. Future Direction Further studies are warranted to determine the specific glycosylation patterns of various plasma and cellular proteins in patients with iTTP and to determine the synergistic role of various gene mutations and severe ADAMTS13 deficiency in the pathogenesis of iTTP.

Lysine Histone-Induced Thrombocytopenia in Zebrafish Depends on Endothelial Von Willebrand Factor

Thrombotic thrombocytopenic purpura (TTP), a potentially fatal blood disorder, is caused by severe deficiency of plasma ADAMTS13 activity, primarily resulting from acquired autoantibodies that inhibit ADAMTS13's ability to cleave von Willebrand factor (VWF). However, severe deficiency of plasma ADAMTS13 activity alone is often not sufficient to cause an acute episode of TTP; additional environmental or genetic factors may be required for the development of acute TTP. Here, we report a creation and characterization of a novel zebrafish model of TTP and use the model to identify a potential mechanistic link between an innate immunity and onset of TTP. Using CRISPR/cas9, we generated adamts13-/-,vwf-/-, and adamts13-/-vwf-/-zebrafish on a double transgenic (Fli1-eGFP/Gata1-dsRed) background in which erythrocytes, thrombocytes, and endothelial cells were uniquely labeled with an endogenously expressed fluorescent protein. Our results demonstrated that adamts13-/-zebrafish larvae (5-dpf) had an increased rate of developing occlusive thrombi in the caudal venues after FeCl3 injury; also, adamts13-/- adult zebrafish (3-4 months) exhibited ~27% reduction of their total or mature thrombocyte counts with a significantly increased number of fragmented erythrocytes; moreover, an intraperitoneal administration of a lysine-rich histone resulted in severe and more persistent thrombocytopenia with an increased mortality in adamts13-/-zebrafish than their wild-type littermate; finally, both spontaneous and histone-induced thrombocytopenia and thrombotic microangiopathy in adamts13-/-was eliminated when endothelial vwf was genetically deleted from zebrafish. Together, these results provide experimental evidences to support a mechanistic link of acute infection or inflammation to acute onset of TTP in patients lacking plasma ADAMTS13 activity. DisclosuresZheng:Alexion: Research Funding, Speakers Bureau.

Relative Deficiency of Plasma A Disintegrin and Metalloprotease with Thrombospondin Type 1 Repeats 13 Activity and Elevation of Human Neutrophil Peptides in Patients with Traumatic Brain Injury.

Traumatic microvascular injury (tMVI) is a universal endophenotype of traumatic brain injury (TBI) that is responsible for significant neurological morbidity and mortality. The mechanism underlying tMVI is not fully understood. The present study aims to determine plasma levels of von Willebrand factor (VWF), a disintegrin and metalloprotease with thrombospondin type 1 repeats (ADAMTS) 13 activity, and human neutrophil peptides (HNP) 1-3 and to correlate these biomarkers with functional outcomes after moderate-severe TBI. Thirty-one consecutive TBI patients (Glasgow Coma Scale [GCS] range, 3-12) were enrolled into the study between February 2010 and November 2014. Blood samples were collected on 0, 1, 2, 3, and 5 days after admission and analyzed for plasma levels of VWF antigen (VWFAg), collagen-binding activity (VWFAc), ADAMTS13 activity, and HNP1-3 proteins. Mean values of plasma VWFAg, VWFAc, and HNP1-3 were significantly increased in TBI patients compared to those in healthy controls (n = 30). Conversely, mean plasma values of ADAMTS13 activity in TBI patients were significantly decreased during the first 2 days after admission. This resulted in a dramatic reduction in the ratio of ADAMTS13 activity to VWFAg or ADAMTS13 to VWFAc in all 5 post-TBI days. Cluster analysis demonstrated that high median plasma levels of VWFAg and HNP1-3 were observed in the cluster with a high mortality rate. These results demonstrate that a relative deficiency of plasma ADAMTS13 activity, resulting from activation of neutrophils and endothelium, may contribute to the formation of microvascular thrombosis and mortality after moderate-severe TBI.

Plasma ADAMTS13 activity and von Willebrand factor antigen and activity in patients with subarachnoid haemorrhage.

Increased von Willebrand factor (VWF) and reduced ADAMTS13 activity are associated with arterial thrombosis. This may also be the culprit mechanism implicated in delayed cerebral ischaemia after aneurysmal subarachnoid haemorrhage (SAH). It was our objective to determine plasma VWF and ADAMTS13 in patients with SAH and healthy subjects; and to explore the levels of those markers and outcome after SAH. Forty consecutive patients were enrolled between September 2007 and April 2014 in a pilot study. Plasma samples were collected from SAH patients on post-bleed day (PBD) 0, 1, 3, 5, 7 and 10 and healthy controls. VWF antigen (VWFAg) and VWF activity (VWFAc) were determined by enzyme-linked immunoassay and collagen binding assay, respectively. ADAMTS13 activity was determined by the cleavage of a fluorescent substrate. Univariate descriptive statistics and cluster analyses were performed based on outcomes in the group with SAH only. Mean age of SAH patients was 52.4 years (26-84 years) and 30 (75 %) were women. 12/40 (30 %) had a high Hunt and Hess grade (IV-V) and 25 (62.5 %) were treated with coil embolisation. Plasma VWFAg and VWFAc were significantly higher in SAH patients than those in healthy subjects on each PBD (p<0.0001). Concurrently, plasma ADAMTS13 activity in SAH patients was significantly lower than that in healthy subjects (p<0.0001). Among those with SAH, cluster analysis demonstrated that patients with higher VWFAg and VWFAc and/or lower ADAMTS13 activity might be at risk of increased mortality. In conclusion, the relative deficiency of plasma ADAMTS13 activity in SAH patients may associate with worse outcome.

Therapeutic efficacy of the platelet glycoprotein Ib antagonist anfibatide in murine models of thrombotic thrombocytopenic purpura

AbstractThrombotic thrombocytopenic purpura (TTP), a potentially fatal blood clot disorder, is primarily caused by severe deficiency of plasma ADAMTS13 activity resulting from acquired autoantibodies. Plasma exchange is the only effective initial therapy. However, the high mortality rate and the complications associated with plasma exchange therapy remain a major concern. To address unmet clinical needs, therapeutic efficacies of anfibatide, a snake venom-derived platelet glycoprotein Ib antagonist, in murine models of spontaneous thrombocytopenia and shigatoxin-induced TTP were determined. A light scattering platelet aggregometry, microfluidic shear-based assay, and murine models of TTP were used in the study. We showed that purified anfibatide inhibits ristocetin- or botrocetin-induced human or murine platelet agglutination in the presence of von Willebrand factor in a concentration-dependent manner. Anfibatide could also dramatically inhibit the adhesion and aggregation of murine and human platelets on a collagen surface under arterial shear stress, in the presence or absence of plasma ADAMTS13 activity. Most importantly, we demonstrated that an intraperitoneal administration of anfibatide at the dose of 60 ng/g body weight twice daily mitigated spontaneous thrombocytopenia and prevented shigatoxin-induced TTP in Adamts13−/− and disease-susceptible mice (CAST/Ei strain). Thus, we conclude that anfibatide, when administered at the optimal dosage, route, and interval, is efficacious in treating spontaneous and bacterial shigatoxin-induced TTP in the murine models. Our findings may provide the basis for further development of anfibatide for the treatment of acute TTP in humans.

Human neutrophil peptides and complement factor Bb in pathogenesis of acquired thrombotic thrombocytopenic purpura.

Acquired thrombotic thrombocytopenic purpura is primarily caused by the deficiency of plasma ADAMTS13 activity resulting from autoantibodies against ADAMTS13. However, ADAMTS13 deficiency alone is often not sufficient to cause acute thrombotic thrombocytopenic purpura. Infections or systemic inflammation may precede acute bursts of the disease, but the underlying mechanisms are not fully understood. Herein, 52 patients with acquired autoimmune thrombotic thrombocytopenic purpura and 30 blood donor controls were recruited for the study. The plasma levels of human neutrophil peptides 1-3 and complement activation fragments (i.e. Bb, iC3b, C4d, and sC5b-9) were determined by enzyme-linked immunosorbent assays. Univariate analyses were performed to determine the correlation between each biomarker and clinical outcomes. We found that the plasma levels of human neutrophil peptides 1-3 and Bb in patients with acute thrombotic thrombocytopenic purpura were significantly higher than those in the control (P<0.0001). The plasma levels of HNP1-3 correlated with the levels of plasma complement fragment Bb (rho=0.48, P=0.0004) and serum lactate dehydrogenase (rho=0.28, P=0.04); in addition, the plasma levels of Bb correlated with iC3b (rho=0.55, P<0.0001), sC5b-9 (rho=0.63, P<0.0001), serum creatinine (rho=0.42, p=0.0011), and lactate dehydrogenase (rho=0.40, P=0.0034), respectively. Moreover, the plasma levels of iC3b and sC5b-9 were correlated (rho=0.72, P<0.0001), despite no statistically significant difference of the two markers between thrombotic thrombocytopenic purpura patients and the control. We conclude that innate immunity, i.e. neutrophil and complement activation via the alternative pathway, may play a role in the pathogenesis of acute autoimmune thrombotic thrombocytopenic purpura, and a therapy targeted at these pathways may be considered in a subset of these patients.