Thrombospondin Type Research Articles

Mechanisms of inhibition of human monoclonal antibodies in immune thrombotic thrombocytopenic purpura

AbstractAntibody binding to a plasma metalloprotease, a disintegrin and metalloproteinase with thrombospondin type 1 repeats 13 (ADAMTS13), is necessary for the development of immune thrombotic thrombocytopenic purpura (iTTP). Inhibition of ADAMTS13-mediated von Willebrand factor (VWF) cleavage by such antibodies clearly plays a role in the pathophysiology of the disease, although the mechanisms by which they inhibit ADAMTS13 enzymatic function are not fully understood. At least some immunoglobulin G–type antibodies appear to affect the conformational accessibility of ADAMTS13 domains involved in both substrate recognition and inhibitory antibody binding. We used single-chain fragments of the variable region previously identified via phage display from patients with iTTP to explore the mechanisms of action of inhibitory human monoclonal antibodies. Using recombinant full-length ADAMTS13, truncated ADAMTS13 variants, and native ADAMTS13 in normal human plasma, we found that, regardless of the conditions tested, all 3 inhibitory monoclonal antibodies tested affected enzyme turnover rate much more than substrate recognition of VWF. Hydrogen-to-deuterium exchange plus mass spectrometry experiments with each of these inhibitory antibodies demonstrated that residues in the active site of the catalytic domain of ADAMTS13 are differentially exposed to solvent in the presence and absence of monoclonal antibody binding. These results support the hypothesis that inhibition of ADAMTS13 in iTTP may not necessarily occur because the antibodies directly prevent VWF binding, but instead because of allosteric effects that impair VWF cleavage, likely by affecting the conformation of the catalytic center in the protease domain of ADAMTS13. Our findings provide novel insight into the mechanism of autoantibody-mediated inhibition of ADAMTS13 and pathogenesis of iTTP.

Heterogeneity of Target Antigens in Sarcoidosis-Associated Membranous Nephropathy

Membranous nephropathy (MN) is the most common glomerular disease associated with sarcoidosis. The target antigen M-type phospholipase A2 receptor 1 (PLA2R) has been identified in a subset of sarcoidosis-associated MN. The target antigen is not known in the remaining sarcoidosis-associated MN. Data of patients with history of sarcoidosis and biopsy-proven MN were retrieved and analyzed. Mass spectrometry (MS/MS) was performed on all kidney biopsies of sarcoidosis-associated MN to detect the target antigens. Immunohistochemistry (IHC) studies were performed to confirm and localize the target antigens along the glomerular basement membrane (GBM). Eighteen patients with history of sarcoidosis and biopsy-proven MN were identified, of whom 3 were known to be PLA2R-negative, and in the remaining patients the target antigen was unknown. Thirteen (72%) patients were males; the median age at MN diagnosis was 54.5 years. The median proteinuria at presentation was proteinuria 9.8 g/24 h. Eight patients (44.4%) had concurrent sarcoidosis. Using MS/MS, we detected PLA2R and neural epidermal growth factor-like-1 protein (NELL1) in 7 (46.6%) and 4 (22.2%) patients, respectively. In addition, 1 case each (5.5%) was positive for thrombospondin type 1 domain-containing 7A (THSD7A), protocadherin-7 (PCDH7), and putative antigen Serpin B12. No known target antigen was detected in the remaining 4 patients (22.2%). Patients with sarcoidosis and MN exhibit heterogeneous target antigens. We identified, along with PLA2R, the presence of previously unreported antigens, including NELL1, PCDH7, and THSD7A. The incidence of the target antigens in sarcoidosis appears to mirror the overall incidence of target antigens in MN. MN in sarcoidosis may be the result of a heightened immune response and is not associated with a single target antigen.

Editor’s Choice Articles for April

Editor’s Choice Articles for April

Clinical Significance of Thrombospondin Type 1 Domain-Containing 7A and Neural Epidermal Growth Factor-Like 1 Protein in M-Type Phospholipase A2 Receptor-Negative Membranous Nephropathy

Objective To investigate the clinical significance of thrombospondin type 1 domain-containing 7A (THSD7A) and neural epidermal growth factor-like 1 protein (NELL1) in phospholipase A2 receptor (PLA2R)-negative membranous nephropathy (MN). Methods A total of 116 PLA2R-negative MN patients treated in Hangzhou TCM Hospital Affiliated to Zhejiang Chinese Medical University from 2014 to 2021 were enrolled in this study.Immunohistochemistry was employed to detect THSD7A and NELL1 in the renal tissue.The pathological characteristics,treatment,and prognosis were compared between positive and negative groups. Results The 116 PLA2R-negative MN patients included 23 THSD7A-positive patients and 9 NELL1-positive patients.One patient was tested positive for both proteins.The THSD7A-positive group showed higher positive rate of IgG4 (P=0.010),more obvious glomerular basement membrane (GBM) thickening (P=0.034),and higher proportion of stage Ⅱ MN and lower proportion of stage I MN (P=0.002) than the THSD7A-negative group.The NELL1-positive group had lower positive rates of C1q and IgG2 (P=0.029,P=0.001),less obvious GBM thickening (P<0.001),more extensive inflammatory cell infiltration (P=0.033),lower proportion of deposits on multi-locations (P=0.001),and lower proportion of atypical MN (P=0.010) than the NELL1-negative group.One patient with THSD7A-positive MN was diagnosed with colon cancer,while none of the NELL1-positive patients had malignancy.Survival analysis suggested that THSD7A-positive MN had worse composite remission (either complete remission or partial remission) of nephrotic syndrome than the negative group (P=0.016),whereas NELL1-positive MN exhibited better composite remission of nephrotic syndrome than the negative group (P=0.015).The MN patients only positive for NELL1 showed better composite remission of nephrotic syndrome than the MN patients only positive for THSD7A (P<0.001). Conclusions THSD7A- and NELL1-positive MN is more likely to be primary MN,and there is no significant malignancy indication.However,it might have a predictive value for the prognosis of MN.

Elevated Expression of ADAMTS-12 in Maternal and Umbilical Cord Serum of Placenta Accreta Spectrum

Objective We investigated a disintegrin and metalloproteinase with thrombospondin type 1 motif-12 (ADAMTS-12) levels in both fetal umbilical cord and maternal blood of pairs diagnosed with placenta accreta spectrum (PAS). Method We analyzed ADAMTS-12 level by ELISA in 160 samples that included 30 control pairs (30 maternal and 30 umbilical cord serums), 20 pairs in the placenta previa (PP), and 30 pairs in the PAS group (30 maternal and 30 umbilical cord serums). Results There were increased serum levels of ADAMTS-12 in maternal serum (p = 0.037) and umbilical cord serum level (p = 0.004) of PAS group compared with the PP and healthy control groups. There was a positive correlation between maternal ADAMTS-12 and fetal ADAMTS-12 serum levels (r = 0.53, p = 0.002). Conclusion Our findings indicate that ADAMTS-12 could be a ­diagnostic biomarker for PAS.

Functions of Protein &lt;i&gt;C&lt;/i&gt;-Mannosylation in Physiology and Pathology

Protein C-mannosylation is a unique type of protein glycosylation in which a single α-mannose is attached to the indole C2 of tryptophan (Trp) through a C–C bond. The Trp-x-x-Trp (WxxW) sequences, whose first Trp residue may be C-mannosylated, constitute the consensus motifs of this rare glycosylation modification. Dpy-19 was recognized as a gene encoding C-mannosyltransferase in Caenorhabditis elegans. DPY19L1 and DPY19L3 were later confirmed as mammalian C-mannosyltransferases. The consensus motif can be found in the thrombospondin type 1 repeat and cytokine receptor type I families as well as in many other proteins, and recent studies suggest critical roles of C-mannosylation in the folding, sorting, and/or secretion of the substrate proteins. We successfully synthesized C-mannosylated Trp-containing Trp-Ser-Pro-Trp (WSPW) peptides. As a result of using these peptides in our investigations, we proposed that C-mannosylation may have biological functions in addition to contributing to the folding and stability of the substrate proteins. In this mini-review, we discuss the biological roles of C-mannosylation in physiology and pathology as based on our recent findings.

Diagnostic utility of serum and urine biomarkers in idiopathic membranous nephropathy: a systematic review and meta-analysis.

Membranous nephropathy is an autoimmune nephropathy that is one of the most common pathological types of nephrotic syndrome. It is important to find and apply specific biomarkers for the noninvasive diagnosis of idiopathic membranous nephropathy (IMN). However, there are limited data about their diagnostic value. Therefore, an overall meta-analysis helps to identify effective biomarkers for the clinical diagnosis of IMN. A systematic literature search was carried out in PubMed, Embase, Cochrane and Web of Science from inception until December 31, 2020. Two researchers searched for studies that met the inclusion criteria. The results of the joint study were expressed in terms of sensitivity and specificity. The meta-analysis included 24 studies with biomarkers for the clinical diagnosis of IMN, including antibody against phospholipase A2 receptor (PLA2R-AB), antibody against thrombospondin type I domain-containing 7A (THSD7A-AB), lysosome membrane protein-2 (LIMP-2) and circular RNAs. The diagnostic efficiency of PLA2R-AB for IMN had a combined sensitivity of 60% and a combined specificity of 100%. The diagnostic efficiency of THSD7A-AB for IMN had a combined sensitivity of 3% and a combined specificity of 99%. The diagnostic efficiency of urinary LIMP-2 for IMN was 100%, and the specificity was 100%. The diagnostic efficiency of exosomal circRNAs for IMN was 100%, and the specificity was 100%. This meta-analysis shows that PLA2R-AB and THSD7A-AB are of important diagnostic value for IMN. More studies are needed in the future to reveal the diagnostic value of LIMP-2 and circRNAs for IMN.

A Genetic Risk Score Distinguishes Different Types of Autoantibody-Mediated Membranous Nephropathy

Introduction: Membranous nephropathy (MN) is the leading cause of nephrotic syndrome in adults and is characterized by detectable autoantibodies against glomerular antigens, most commonly phospholipase A2 receptor 1 (PLA2R1) and thrombospondin type-1 domain containing 7A (THSD7A). In Europeans, genetic variation in at least five loci, PLA2R1, HLA-DRB1, HLA-DQA1, IRF4, and NFKB1, affects the risk of disease. Here, we investigated the genetic risk differences between different autoantibody states. Methods: 1,409 MN individuals were genotyped genome-wide with a dense SNV array. The genetic risk score (GRS) was calculated utilizing the previously identified European MN loci, and results were compared with 4,929 healthy controls and 422 individuals with steroid-sensitive nephrotic syndrome. Results: GRS was calculated in the 759 MN individuals in whom antibody status was known. The GRS for MN was elevated in the anti-PLA2R1 antibody-positive (N = 372) compared with both the unaffected control (N = 4,929) and anti-THSD7A-positive (N = 31) groups (p < 0.0001 for both comparisons), suggesting that this GRS reflects anti-PLA2R1 MN. Among PLA2R1-positive patients, GRS was inversely correlated with age of disease onset (p = 0.009). Further, the GRS in the dual antibody-negative group (N = 355) was intermediate between controls and the PLA2R1-positive group (p < 0.0001). Conclusion: We demonstrate that the genetic risk factors for PLA2R1- and THSD7A-antibody-associated MN are different. A higher GRS is associated with younger age of onset of disease. Further, a proportion of antibody-negative MN cases have an elevated GRS similar to PLA2R1-positive disease. This suggests that in some individuals with negative serology the disease is driven by autoimmunity against PLA2R1.

Clinical significance of assessing ADAMTS-13 and von Willebrand factor level in COVID-19 convalescent pregnant women

Introduction. Coronavirus infection is associated with severe endotheliopathy, thromboinflammation and immunothrombosis leading to excessive release of von Willebrand factor (vWF) multimers from Weibel–Palade bodies, which can affect activity of ADAMTS-13 metalloproteinase (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) and the ADAMTS-13/vWF axis previously shown by us to be altered in non-pregnant women with severe COVID-19. Aim: to study a clinical role of hemostasis activation particularly ADAMTS-13/vWF axis in pregnant women after COVID-19. Materials and Methods. A prospective case–control study was conducted with pregnant women (n = 135) divided into 3 groups: group 1 included 45 women with prior COVID-19 during pregnancy, group 2 – 45 women in the acute phase of the infection during pregnancy, group 3 – 45 healthy pregnant women. The level of vWF and ADAMTS-13 was assessed in all patients. Results. The concentration of vWF antigen (vWF:Ag) in the acute period of the disease in pregnant women with COVID-19 was significantly higher compared to the control group (p &lt; 0.001). ADAMTS-13 level in pregnant women after COVID-19 did not differ from that of in control group, while vWF level was significantly higher in 66.7 % (30/45). The ADAMTS-13/vWF ratio was increased and significantly differed both in pregnant patients during the acute period of the disease (p &lt; 0.001) and pregnant women after infection (p = 0.0002) compared with the control group. Conclusion. Our results show that endotheliopathy was prominently manifested in pregnant women with COVID-19 and persisted for several months after disease. The ADAMTS-13/vWF ratio determines the pathway functioning, the risk of microcirculation disorders and clinical complications.

Functioning of the ADAMTS-13/vWF axis and its clinical significance

Advances in biology have allowed us to substantially deepen our knowledge about hemostasis functioning both in health and disease. ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) and von Willebrand factor (vWF) are components of the hemostasis system, which physiological interaction holds an important place in maintaining homeostasis. ADAMTS-13 is a metalloproteinase mainly acting to release vWF fragments into the blood plasma, as well as regulating its activity by cleaving ultra-large vWF multimers (UL-vWF) into smaller and less active forms. The study of such factors is of great clinical importance, since a decrease in ADAMTS-13 activity and an increase in vWF level can be predictors of microcirculatory disorders that play an important role in developing multiple organ failure. However, very few and fully contradictory studies devoted to the physiological aspects of the ADAMTS-13/vWF axis functioning in the mother–fetus system are available, therefore requiring to be further investigated.

Hemoperfusion and plasmapheresis in the intensive care unit

In addition to kidney replacement procedures, several other extracorporeal procedures are employed in the intensive care unit. Hemoperfusion with activated charcoal was the predominant treatment used for removal of toxins from the 1970s until the millennium. Nowadays, this treatment does no longer play aclinically meaningful role as even strongly protein-bound toxins can be removed by effective dialysis procedures in case poisoning. The concept of acytokine adsorber was introduced 10years ago, which is directed towards withstanding the cytokine storm. Despite negative data from prospective randomized controlled studies, its use is steadily increasing in Germany. Atotally different treatment concept is the biomimetic pathogen adsorber, which removes bacteria, viruses and fungi from the bloodstream by binding to immobilized heparin. Whether this rapid reduction of the pathogen load translates into an improvement of clinically relevant endpoints is unclear, as prospective randomized controlled studies are lacking. For the early hours of septic shock a very old procedure, plasmapheresis, has recently regained interest. The results of two large randomized controlled studies in this setting from Europe and Canada will become available in 2025/2026. The rationale to use plasma exchange in early sepsis is that this procedure not only removes cytokines but also replenishes reduced levels of protective factors, such as angiopoietin‑1, adisintegrin and metalloproteinase with a thrombospondin type1 motif, member13 (ADAMTS-13) and proteinC, if fresh plasma is used as exchange fluid. All afore mentioned procedures do not only have a different mode of action but are also used at seperate time points of bloodstream infections and/or sepsis.

Development of Selective ADAMTS-5 Peptide Substrates to Monitor Proteinase Activity.

The dysregulation of proteinase activity is a hallmark of osteoarthritis (OA), a disease characterized by progressive degradation of articular cartilage by catabolic proteinases such as a disintegrin and metalloproteinase with thrombospondin type I motifs-5 (ADAMTS-5). The ability to detect such activity sensitively would aid disease diagnosis and the evaluation of targeted therapies. Förster resonance energy transfer (FRET) peptide substrates can detect and monitor disease-related proteinase activity. To date, FRET probes for detecting ADAMTS-5 activity are nonselective and relatively insensitive. We describe the development of rapidly cleaved and highly selective ADAMTS-5 FRET peptide substrates through in silico docking and combinatorial chemistry. The lead substrates 3 and 26 showed higher overall cleavage rates (∼3-4-fold) and catalytic efficiencies (∼1.5-2-fold) compared to the best current ADAMTS-5 substrate ortho-aminobenzoyl(Abz)-TESE↓SRGAIY-N-3-[2,4-dinitrophenyl]-l-2,3-diaminopropionyl(Dpa)-KK-NH2. They exhibited high selectivity for ADAMTS-5 over ADAMTS-4 (∼13-16-fold), MMP-2 (∼8-10-fold), and MMP-9 (∼548-2561-fold) and detected low nanomolar concentrations of ADAMTS-5.

The C-terminal domains of ADAMTS1 contain exosites involved in its proteoglycanase activity

A disintegrin-like and metalloproteinase with thrombospondin type 1 motifs (ADAMTS1) is a protease involved in fertilization, cancer, cardiovascular development, and thoracic aneurysms. Proteoglycans such as versican and aggrecan have been identified as ADAMTS1 substrates, and Adamts1 ablation in mice typically results in versican accumulation; however, previous qualitative studies have suggested that ADAMTS1 proteoglycanase activity is weaker than that of other family members such as ADAMTS4 and ADAMTS5. Here, we investigated the functional determinants of ADAMTS1 proteoglycanase activity. We found that ADAMTS1 versicanase activity is approximately 1000-fold lower than ADAMTS5 and 50-fold lower than ADAMTS4 with a kinetic constant (kcat/Km) of 3.6× 103M-1s-1 against full-length versican. Studies on domain-deletion variants identified the spacer and cysteine-rich domains as major determinants of ADAMTS1 versicanase activity. Additionally, we confirmed that these C-terminal domains are involved in the proteolysis of aggrecan as well as biglycan, a small leucine-rich proteoglycan. Glutamine scanning mutagenesis of exposed positively charged residues on the spacer domain loops and loop substitution with ADAMTS4 identified clusters of substrate-binding residues (exosites) in β3-β4 (R756Q/R759Q/R762Q), β9-β10 (residues 828-835), and β6-β7 (K795Q) loops. This study provides a mechanistic foundation for understanding the interactions between ADAMTS1 and its proteoglycan substrates and paves the way for development of selective exosite modulators of ADAMTS1 proteoglycanase activity.

Frontiers in pathophysiology and management of thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a fatal disease in which platelet-rich microthrombi cause end-organ ischemia and damage. TTP is caused by markedly reduced ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity. Hereditary or congenital TTP (cTTP) is caused by ADAMTS13 gene mutations. In acquired or immune TTP (iTTP), ADAMTS13 activity is reduced by anti-ADAMTS13 autoantibodies. TTP is characterized by thrombocytopenia, hemolytic anemia, fever, renal dysfunction, and neuropsychiatric symptoms. Therapeutic plasma exchange (TPE) and immunosuppressive therapy are the mainstays of treatment. As untreated TTP has a high mortality rate, immediate initiation of TPE is recommended when TTP is suspected. Conventionally, corticosteroids have been used for immunosuppressive therapy. Current drug therapies include rituximab, an anti-CD20 antibody that is effective in newly diagnosed cases and refractory cases, as well as for relapse prevention, and caplacizumab, an anti- von Willebrand factor (VWF) nanobody that inhibits the binding of platelets to VWF and prevents microthrombi formation. Recombinant human ADAMTS13 is a promising treatment for cTTP. Although these therapeutic advances have improved the outcomes of TTP, early diagnosis and prompt initiation of appropriate therapy are necessary to achieve these outcomes.

A case of systemic lupus erythematosus having concurrent Evans syndrome and acquired thrombotic thrombocytopenic purpura.

An 18-year-old Japanese woman with systemic lupus erythematosus experienced dyspnoea, headache, tinnitus, and purpura for 2 weeks and was admitted to our hospital. The patient had been diagnosed with systemic lupus erythematosus and secondary immune thrombocytopenia 8 years before and treated with high-dose prednisolone and mycophenolate mofetil. Since the blood test on admission showed haemolytic anaemia with a positive direct Coombs test and anti-glycoprotein IIb/IIIa antibodies, the patient was initially diagnosed with Evans syndrome (ES). The patient was treated with pulse intravenous methylprednisolone followed by 45 mg/day prednisolone; however, the patient's platelet count did not normalise. Based on a low level of a disintegrin-like and metalloproteinase with thrombospondin type 1 motif 13 (ADAMTS-13) activity and a high level of ADAMTS-13 inhibitors, a diagnosis of acquired thrombotic thrombocytopenic purpura (TTP) was confirmed. After undergoing therapeutic plasma exchange for 6 consecutive days, the patient's platelet count recovered rapidly. Although concurrent acquired TTP and ES have not been reported previously, the findings from this case highlight the importance of measuring ADAMTS-13 activity and inhibitors to rule out acquired TTP, especially when ES is refractory to glucocorticoids.

The expression of SERPINE1 in colon cancer and its regulatory network and prognostic value

BackgroundSerpin Peptidase Inhibitor 1 (SERPINE1) promotes cancer progression by making it easier for cancer cells to spread to surrounding normal tissue. We expect to understand the prognostic value and regulatory network of SERPINE1 in colon cancer using bioinformatics methods.MethodsThe expression of target gene SERPINE1 in varying cancers was analyzed by the Tumor Immune Estimation Resource (TIMER) database. SERPINE1 expression in Colon Adenocarcinoma and normal tissue samples was assessed by starBase and UALCAN databases. SERPINE1 expression in clinical tissues was assayed using quantitative reverse transcription Polymerase Chain Reaction (qRT-PCR). SERPINE1 expression was detected in colon cancer patients with various clinical features (age, gender, nodal metastasis status, race, stages, and subtype) using analysis of variance. Survival curve was used to analyze the effect of high and low expression of SERPINE1 on the survival time of patients with different clinical phenotypes. Gene Set Enrichment Analysis (GSEA) was conducted on the results of LinkFinder calculation using LinkInterpreter module, which was combined with Pearson correlation analysis to obtain the kinase targets and miRNA targets, transcription factor targets, and corresponding signaling pathways associated with SERPINE1. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were performed on GSEA result. Finally, Gene Multiple Association Network Integration Algorithm (GeneMANIA) was utilized to establish a network of genes related to the kinases MAPK1, miR-18a, and SRF_Q, and biological functions were analyzed.ResultsBased on TIMER, starBase, and UALCAN databases, SERPINE1 was found to be remarkably highly expressed in colon cancer patients, which was further verified by clinical tissue. It was also associated with different clinical features (nodal metastasis status, stages, subtypes). Additionally, survival analysis showed that patients with low expression of SERPINE1 had a longer survival time, suggesting that SERPINE1 was a prognostic risk factor for colon cancer. Pearson correlation analysis revealed that the expression of Integrin Alpha 5 (ITGA5), Matrix Metallopeptidase 19 (MMP19), and ADAM Metallopeptidase with Thrombospondin Type 1 Motif, 4 (ADAMTS4) had the highest correlation with that of SERPINE1. The GSEA results indicated that these genes were mainly enriched in the pathways of RNA expression and kinases. Finally, GeneMANIA analysis was introduced to construct the molecular network of SERPINE1.ConclusionOverall, our bioinformatics analyses comprehensively described the networks involved SERPINE1 in colon cancer and the potentially associated molecular mechanisms.

Delayed normalization of ADAMTS13 activity in acute thrombotic thrombocytopenic purpura in the caplacizumab era

AbstractThe benefits of caplacizumab in acute immune-mediated thrombotic thrombocytopenic purpura (iTTP) are well established. We identified a delayed normalization of a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13 (ADAMTS13) activity (>30%) in a subgroup treated with caplacizumab, not evident in the precaplacizumab era. Patients treated with caplacizumab (n = 64) achieved ADAMTS13 activity >30% at median 31 days after plasma exchange (PEX), compared with 11.5 days in the noncaplacizumab group (n = 50, P = .0004). Eighteen of 64 (28%) patients treated with caplacizumab had ADAMTS13 activity <10% at stopping caplacizumab with a longer time to ADAMTS13 activity >30% (median, 139 days after completing PEX). Eighteen of 64 (28%) patients receiving extended caplacizumab (31-58 days) failed to achieve ADAMTS13 activity >30% at the time of caplacizumab cessation, compared with 4 of 47 (8.5%) historical controls at a similar timepoint (30 + 28 days, P < .0001). Failure to achieve ADAMTS13 activity >30% within 30 + 28 days was 6 times more likely with caplacizumab (odds ratio, 6.3; P = .0006). ADAMTS13 antigen <30% at caplacizumab cessation was associated with increased iTTP recurrence (4/10 vs 0/9 in patients with ADAMTS13 antigen ≥30%). Admission anti-ADAMTS13 immunoglobulin G (IgG) antibody level did not predict recurrence. Anti-ADAMTS13 IgG antibody levels, immunosuppression, and ethnicity did not account for differences in ADAMTS13 activity response. ADAMTS13 antigen levels ≥30% may be useful to guide stopping caplacizumab therapy after extended use with ADAMTS13 activity <10%. The reason for delayed ADAMTS13 normalization is unclear and requires further investigation.

Application Value of Plasma vWF and ADAMTS-13 in Evaluating Postoprative Vascular Endothelial Injury and Prognosis in Children with VSD

To dynamically observe the levels and activities of von Willebrand factor (vWF) and ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) in plasma of children with congenital ventricular septal defect (VSD) during perioperative period, and explore the value of plasma vWF antigen (vWF:Ag) and ADAMTS-13 activity (ADAMTS-13: AC) in evaluating vascular endothelial injury and prognosis in children with VSD. In this cross-sectional study, a total of 74 children with VSD who underwent surgical treatment in TEDA International Cardiovascular Hospital from September 2018 to March 2019 were enrolled in the observation group. Among them, there were 28 cases of pure VSD, 32 cases of VSD combined with pulmonary hypertension, and 14 cases of VSD combined with valvular heart disease. 31 healthy children who underwent physical examination in Tianjin Children's Hospital during the same period were collected as the control group. The biochemical indexes of the children at admission were recorded. Peripheral plasma was collected at admission, postsurgery day 0 and day 1, respectively, and the levels of vWF activity (vWF:AC), vWF:Ag, ADAMTS-13 antigen (ADAMTS-13:Ag) and ADAMTS-13:AC were detected. The level of plasma vWF:Ag and vWF:AC in the observation group before surgery were significantly lower than those in the control group (P<0.001), and increased continuously, on postsurgery day 0 and day 1 (P<0.001). The level of ADAMTS-13:Ag in the observation group before surgery was significantly higher than that in the control group (P<0.001), which decreased significantly on postsurgery day 0 (P<0.001), and increased significantly on postsurgery day 1 compared with postsurgery day 0 (P=0.033). The level of ADAMTS-13:AC in the observation group before surgery was significantly lower than that in the control group (P=0.015), which decreased significantly on postsurgery day 0 (P=0.037), and increased on postsurgery day 1, but the difference was not statistically significant (P=0.051). The changes of vWF and ADAMTS-13 in the three subgroups were basically similar to the observation group. vWF: Ag/ADAMTS-13: AC ratio on postsurgery day 0 and day 1 had high diagnostic value in vascular endothelial injury (AUC=0.80, P<0.001; AUC=0.93, P<0.001). Preoperative vWF and ADAMTS-13 levels, and related baseline indicators were not correlated with postoperative infection, bleeding, thrombosis,etc. Preoperative vWF: Ag, vWF: AC and ADAMTS-13: AC levels in children with VSD are low, while the level of ADAMTS-13: Ag is high. After surgery, the levels of vWF: Ag and vWF: AC are increased and the level of ADAMTS-13: Ag is decreased. The postoperative vWF: Ag/ADAMTS-13: AC ratio shows high diagnostic value in evaluating vascular endothelial injury. There is no correlation between preoperative vWF and ADAMTS-13 levels with perioperative clinical events.

Abstract WP212: Association Of Thrombin Generation With Leukocyte Inflammatory Profile In Patients With Acute Ischemic Stroke

Thrombosis is central to the pathogenesis of acute ischemic stroke, with higher thrombin generation being associated with increased stroke risk. The immune system may contribute to thrombin generation in stroke and thus may offer novel strategies for stroke prevention. This study addresses the research question regarding the relationship of thrombin generation to leukocyte gene expression in patients with acute ischemic stroke. We isolated RNA from whole blood and examined the relationship to thrombin generation capacity in patients with acute ischemic stroke. Due to its effects on thrombin generation, patients on anticoagulants were excluded from the study. The relationship of gene expression with peak thrombin was evaluated by analysis of covariance across peak thrombin quartiles adjusted for sex and age. In 97 patients with acute ischemic stroke, peak thrombin was variable, ranging from 252.0 to 752.4 nM. Increased thrombin generation was associated with differences in thromboinflammatory leukocyte gene expression, including a decrease in ADAM metallopeptidase with thrombospondin type 1 motif 13 and an increase in nuclear factor B (NF-kB)-activating protein, protein disulfde isomerase family A member 5, and tissue factor pathway inhibitor 2. Pathways associated with peak thrombin included interleukin 6 signaling, thrombin signaling, and NF-kB signaling. A linear discriminant analysis model summarizing the immune activation associated with peak thrombin in a first cohort of stroke could distinguish patients with low peak thrombin from high peak thrombin in a second cohort of 112 patients with acute ischemic stroke.The identified genes and pathways support a role of the immune system contributing to thrombus formation in patients with stroke. These may have relevance to antithrombotic strategies for stroke prevention.

ADAMTS4 is involved in the production of the Alzheimer disease amyloid biomarker APP669-711

Amyloid-β (Aβ) deposition in the brain parenchyma is one of the pathological hallmarks of Alzheimer disease (AD). We have previously identified amyloid precursor protein (APP)669-711 (a.k.a. Aβ(-3)-40) in human plasma using immunoprecipitation combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (IP-MALDI-MS). Furthermore, we found that the level of a composite biomarker, i.e., a combination of APP669-711/Aβ1-42 ratio and Aβ1-40/Aβ1-42 ratio in human plasma, correlates with the amyloid PET status of AD patients. However, the production mechanism of APP669-711 has remained unclear. Using in vitro and in vivo assays, we identified A Disintegrin and Metalloproteinase with a Thrombospondin type 1 motif, type 4 (ADAMTS4) as a responsible enzyme for APP669-711 production. ADAMTS4 cleaves APP directly to generate the C-terminal stub c102, which is subsequently proteolyzed by γ-secretase to release APP669-711. Genetic knockout of ADAMTS4 reduced the production of endogenous APP669-711 by 30% to 40% in cultured cells as well as mouse plasma, irrespectively of Aβ levels. Finally, we found that the endogenous murine APP669-711/Aβ1-42 ratio was increased in aged AD model mice, which shows Aβ deposition as observed in human patients. These data suggest that ADAMTS4 is involved in the production of APP669-711, and a plasma biomarker determined by IP-MALDI-MS can be used to estimate the level of Aβ deposition in the brain of mouse models.