Application Value of Plasma vWF and ADAMTS-13 in Evaluating Postoprative Vascular Endothelial Injury and Prognosis in Children with VSD / 中国实验血液学杂志

OBJECTIVE@#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.@*METHODS@#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.@*RESULTS@#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.@*CONCLUSION@#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.

Clinical diagnosis and treatment of hereditary thrombocytopenia and purpura: a report of five cases and literature review / 中华血液学杂志

Objective: To report the clinical manifestations and laboratory features of five patients with congenital thrombotic thrombocytopenic purpura (cTTP) and explore its standardized clinical diagnosis and treatment along with a review of literature. Methods: Clinical data of patients, such as age of onset, disease manifestation, personal history, family history, and misdiagnosed disease, were collected. Treatment outcomes, therapeutic effects of plasma infusion, and organ function evaluation were observed. The relationship among the clinical manifestations, treatment outcomes, and ADAMTS13 gene mutation of patients with cTTP was analyzed. Additionally, detection of ADAMTS13 activity and analysis of ADAMTS13 gene mutation were explored. Results: The age of onset of cTTP was either in childhood or adulthood except in one case, which was at the age of 1. The primary manifestations were obvious thrombocytopenia, anemia, and different degrees of nervous system involvement. Most of the patients were initially suspected of having immune thrombocytopenia. Acute cTTP was induced by pregnancy and infection in two and one case, respectively. ADAMTS13 gene mutation was detected in all cases, and there was an inherent relationship between the mutation site, clinical manifestations, and degree of organ injury. Therapeutic or prophylactic plasma transfusion was effective for treating cTTP. Conclusions: The clinical manifestations of cTTP vary among individuals, resulting in frequent misdiagnosis that delays treatment. ADAMTS13 activity detection in plasma and ADAMTS13 gene mutation analysis are important bases to diagnose cTTP. Prophylactic plasma transfusion is vital to prevent the onset of the disease.

A combined regimen based on bortezomib and glucocorticoids for 6 patients with recurrent/refractory immune thrombotic thrombocytopenic purpura / 中华血液学杂志

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

Research Advance in Treatment of Thrombotic Thrombocytopenic Purpura --Review / 中国实验血液学杂志

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy, in which a severe deficiency of von Willebrand factor lyase results in thrombocytopenic clots that block blood vessels and eventually lead to terminal organ failure. Therapeutic plasma exchange is the cornerstone of TTP treatment which can greatly improves the survival rate of the patients. With the further exploration to the pathophysiological mechanism of TTP, other alternative therapies, new immunosuppressive agents, targeted antagonists, gene therapy and other emerging means gradually emerge, which are expected to further reduce the mortality and recurrence rate of the patients. In this review, the new developments in TTP treatment were summarized briefly.

Advance in the diagnosis and treatment of hereditary thrombotic thrombocytopenic purpura / 中华医学遗传学杂志

Congenital thrombotic thrombocytopenic purpura, also known as Upshaw-Schulman syndrome, is a rare autosomal recessive genetic disorder. The main pathogenesis is homozygous or compound heterozygous variants of von Willebrand factor lyase (ADAMTS13) gene mapped to chromosome 9q34, which may result in severe lack of ADAMTS13 which cleaves von Willebrand factor (vWF) multimers in the plasma and increase the risk of microvascular thrombosis, leading to various complications. The advance of research on the pathogenesis of cTTP, recombinant human ADAMTS13 and gene therapy have made breakthroughs which may lead to cure of cTTP. This article has provided a review for the latest progress made in the diagnosis and treatment of cTTP.

Reference range for ADAMTS13 antigen, activity and anti-ADAMTS13 antibody in the healthy adult Singapore population

INTRODUCTION@#ADAMTS13 (a disintegrin-like and metalloproteinase with a thrombospondin Type 1 motif, member 13) plays a fundamental role in the regulation of haemostasis and thrombosis. Its deficiency leads to an accumulation of ultra-large von Willebrand multimers, inducing spontaneous platelet aggregation, thrombosis in the microvasculature, and thrombotic thrombocytopenic purpura (TTP), a condition with 90% mortality when left untreated. Prompt quantification of ADAMTS13 antigen, activity and autoantibody plays a crucial role in the diagnosis and management of TTP and can help differentiate it from other thrombotic microangiopathies (TMAs). Reference ranges for ADAMTS13 are generally derived from Caucasian patients. Given that polymorphism in the ADAMTS13 gene can be associated with variable ADAMTS13 levels, we aimed to establish the first reference range in Singapore and provide a crucial laboratory test for institutions here and elsewhere.@*METHODS@#150 healthy voluntary donors (75 men, 75 women) aged 21-60 years, with an ethnic mix mirroring Singapore's population profile, were recruited. ADAMTS13 antigen, activity and autoantibody levels were measured using the fluorescence resonance energy transfer-vWF73 and enzyme-linked immunosorbent assay methodologies.@*RESULTS@#Levels (activity 0.65-1.79 IU/mL, antigen 0.36-1.17 IU/mL, autoantibody 1.4-12.5 U/mL) were not statistically different between the genders and various age groups.@*CONCLUSION@#TTP and TMAs are encountered in a wide range of specialties. The availability of new assays in Singapore will aid clinicians in the timely management of these conditions. Standardising reference ranges established for Singapore against World Health Organization standards allows harmonisation of measurements between laboratories and for future research collaborations.

Expression of ADAMTS13 and PCNA in the placentas of gestational diabetic mothers / Expresión de ADAMTS13 y PCNA en las placentas de madres diabéticas gestacionales

SUMMARY: GDM is linked with overexpression of inflammatory cytokines and increased oxidative stress, leading to endothelial dysfunction and vascular disorder. Weaimed to examine the expression of ADAMTS13 and PCNA in the placentas of gestational diabetes mellitus (GDM) patients to investigate the effects of hypoxia, induced by GDM, on proliferation and extracellular matrix formation in the maternal and fetal placenta cells. A total of 60 placentas were collected from pregnant women admitted to the obstetrics clinic. Thirty of them were diagnosed with GDM, and 30 of them were diagnosed with non-GDM patients. Samples were fixed in 10 % formaldehyde, after routine follow-up, embedded in paraffin wax. Sections of 5 µm were cut stained with Mayer Hematoxylin-Eosin, examined under a light microscope. Sections for immunohistochemical analysis were cut and processed for antigen retrievalin citrate solution. Sections were incubated with ADAMTS13 and PCNA primary antibodies, counterstained with hematoxylin, and evaluate under a light microscope. In histopathological examination, the non-diabetic placentas showed that decidua cells in the maternal region were polygonal with oval nuclei and organized in groups. In the GDM group, there were pyknosis and apoptotic changes in decidua cell nuclei. Vacuolar areas were observed in large cavities in maternal connective tissue. Inflammation and dilatation with congestion were observed in the blood vessels of the villus. In the GDM group, positive ADAMTS13 expression was observed in the decidua cells vascular endothelial cells, and surrounding connective tissue fibroblast cells. In the GDM group, a significant increase in PCNA expression was observed in decidua cells, connective tissue cells and endothelial cells. Functional changes in ADAMTS13 proteases and PCNA were thought to induce maternal and fetal complications by stimulating extracellular matrix development.

RESUMEN: La diabetes gestacional está relacionada con la sobreexpresión de citocinas inflamatorias y aumento del estrés oxidativo, lo que lleva a una disfunción endotelial y un trastorno vascular. Nuestro objetivo fue examinar la expresión de ADAMTS13 y PCNA en las placentas con diabetes mellitus gestacional (DMG) para investigar los efectos de la hipoxia inducida por DMG sobre la proliferación y formación de matriz extracelular en células placentarias maternas y fetales. Se recolectaron un total de 60 placentas de mujeres embarazadas ingresadas a la consulta de obstetricia. Treinta de ellas fueron diagnosticadas con DMG y 30 diagnosticadas sin DMG. Las muestras se fijaron en formaldehído al 10 %, y luego de un seguimiento de rutina, fueron embebidas en parafina. Se cortaron secciones de 5 µm teñidas con hematoxilina-eosina de Mayer, las que fueron examinadas bajo un microscopio óptico. Se cortaron y procesaron las secciones para el análisis inmunohistoquímico para la recuperación de antígeno en solución de citrato. Las secciones se incubaron con anticuerpos primarios ADAMTS13 y PCNA, se contratiñeron con hematoxilina y se evalua- ron con un microscopio óptico. En el examen histopatológico, las placentas no diabéticas mostraron que las células de la decidua en la región materna eran poligonales con núcleos ovalados y organizadas en grupos. En el grupo de DMG, se observó picnosis y cambios apoptóticos en los núcleos de las células de la decidua. Se observaron áreas vacuolares en el tejido conectivo materno. En los vasos sanguíneos de las vellosidades se observó inflamación y dilatación con congestión. En el grupo de DMG, se observó expresión positiva de ADAMTS13 en las células de la decidua, en las células endoteliales vasculares y en los fibroblastos del tejido conectivo circundante. En el grupo de DMG se observó un aumento significativo de la expresión de PCNA en células de la decidua, células de tejido conectivo y en las células endoteliales. Se considera que los cambios funcionales en las proteasas ADAMTS13 y PCNA inducen a complicaciones maternas y fetales al estimular el desarrollo de la matriz extracelular.

Biological Function of CysR Domain of ADAMTS13 / 中国实验血液学杂志

OBJECTIVE@#To investigate the biological function of Cysteine rich (CysR) domain of a disintegrin and metalloprotease with thrombospondin type 1 repeats-13 (ADAMTS13) on cleavage of von Willebrand factor (vWF) and provide experimental evidence for exploring the pathogenesis of thrombotic thrombocytopenic purpura (TTP).@*METHODS@#The six amino acids (EDGTLS) in ADAMTS13 CysR domain were point mutated one by one, and the mutant ADAMTS13 proteins were expressed and purified. The cleavage products of vWF polymer by wild-type or mutant ADAMTS13 under denaturing condition or shear stress were separated by 1% SeaKem HGT agarose gel and detected by Western blot.@*RESULTS@#The mutant ADAMTS13 plasmids (M1: Glu515Ala; M2: Asp516Ala; M3: Gly517Ala; M4: Thr518Ala; M5: Leu519Ala; M6: Ser520Ala) were successfully constructed and the proteins of wild-type and mutant ADAMTS13 were purified. Wild-type ADAMTS13 almost completely cleaved the vWF polymer under denaturing condition, while the cleavage activity of M1 mutant was significantly reduced in the same condition (P<0.01). The cleavage activity of M1 mutant of ADAMTS13 was also significantly reduced compared with that of the wild-type under shear stress (P<0.01). The activity of M1 mutant to cleave the FRETS-vWF73 was dramatically reduced compared with that of wild-type ADAMTS13. However, the binding ability of M1 mutant to vWF was similar with that of wild-type ADAMTS13.@*CONCLUSION@#The CysR domain of ADAMTS13 plays an important role in the digestion of vWF under denaturing condition and shear stress. The Glu515 amino acid residue might be an important site for substrate recognition.

Recurrent thrombocytopenia with hemolytic anemia in a boy aged 3 years / 中国当代儿科杂志

A boy, aged 3 years and 8 months, had recurrent thrombocytopenia with hemolytic anemia for more than 3 years. The physical examination showed no enlargement of the liver, spleen, and lymph nodes or finger deformities. Laboratory results showed a negative result of the direct antiglobulin test, normal coagulation function, and increases in bilirubin, lactate dehydrogenase and reticulocytes. The results of von Willebrand factor-cleaving protease ADAMTS13 activity assay showed extreme deficiency, and antibody assay showed negative ADAMTS13 inhibitory autoantibodies. Next-generation sequence showed compound heterozygous mutation in the

Grupos sanguíneos y su relación con los niveles plasmáticos del Factor de von Willebrand / Blood groups and their relationship with plasma levels of von Willebrand Factor

Resumen Introducción: El tipo de grupo sanguíneo entre otros factores, influye en los niveles plasmáticos del Factor de von Willebrand (FvW), su actividad biológica podría incidir en el desarrollo de eventos trombóticos y hemorrágicos. Objetivo: Describir las características y los mecanismos de reacciones postrasduccionales del grupo sanguíneo que permiten la variación en la concentración plasmática del FvW. Materiales y métodos: Revisión teórico descriptiva de tipo documental. Las bases de datos consultadas fueron Medline, Lilacs, ScienceDirect, Scopus, SciELO, Proquest, Ovid y Pubmed. Como criterio de selección se incluyeron artículos en idioma inglés y español a partir del año 2010 y algunos anteriores como referente histórico. Resultados: Se describieron los principales mecanismos e investigaciones que evidencian la influencia del tipo de grupo sanguíneo ABO en los niveles plasmáticos del FvW, así como la estructura y función de dicha proteína. Conclusiones: Las concentraciones plasmáticas del FvW pueden depender del tipo de grupo sanguíneo, la edad, sexo, embarazo, ciclo menstrual, variación de proteínas y factores bioquímicos e inmunológicos. Se podría tener en cuenta el tipo de grupo sanguíneo de los pacientes como un posible factor predictor a futuro de complicaciones clínicas tanto trombóticas como hemorrágicas.

Abstract Introduction: The type of blood group among other factors influences the plasma levels of von Willebrand Factor (FvW) and its biological activity could influence the development of thrombotic and hemorrhagic events. Objective: To describe the characteristics and mechanisms of post-translational reactions of the blood group that generate variation in the plasma concentration of FvW. Materials and methods: A descriptive theoretical review of documentary type. The databases consulted were Medline, Lilacs, ScienceDirect, Scopus, SciELO, Proquest, Ovid and Pubmed. As a selection criterion, articles in English and Spanish were included beginning in 2010 and some previous ones as historical reference. Results: The main mechanisms and investigations that show the influence of the ABO blood group type on the plasma levels of FvW, as well as the structure and function of this protein were described. Conclusions: FvW plasma concentrations may depend on the type of blood group, age, sex, pregnancy, menstrual cycle, protein variation and biochemical and immunological factors. The type of blood group of patients could be considered as a possible future predictor of both thrombotic and hemorrhagic clinical complications.

Plasmic score applicability for the diagnosis of thrombotic microangiopathy associated with ADAMTS13-acquired deficiency in a developing country

ABSTRACT Background: Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease that requires early diagnosis and treatment that can be made possible by applying the PLASMIC score. This study aims to evaluate this score applicability for patients with suspected TTP in a developing country. Methods: This was a retrospective study performed at a tertiary hospital in the northeastern region of Brazil. Patients were analyzed in two groups: ADAMTS13 activity <10% and activity >10%. Patients were stratified according to the PLASMIC score, and the level of agreement between the PLASMIC score and the ADAMTS13 activity was evaluated. Results: Eight patients with thrombotic microangiopathy were included. Four patients had ADAMTS13 activity <10%, all with a PLASMIC score =6. The other four had ADAMTS13 activity >10%, all with a score <6. Based on a score =6 for presumptive diagnosis of TTP, we attained a 100% diagnostic accuracy in our sample. The PLASMIC score was also able to accurately predict response to plasma exchange and the risk of long-term unfavorable outcomes. Conclusions: The reproducibility of the PLASMIC score was quite satisfactory in our sample. It accurately discriminates between patients who had ADAMTS13 deficiency and those with normal enzyme activity, precluding the need for specific laboratory evaluation, which is not always available. This score can be useful for an early diagnosis and indicates which patients will benefit from the treatment in developing countries.

A 2-day-old neonate with hyperbilirubinemia and thrombocytopenia / 中国当代儿科杂志

A girl was admitted to the hospital on day 2 after birth due to jaundice of the skin and sclera for half a day. The main clinical manifestations were persistent severe jaundice and thrombocytopenia, and she was finally diagnosed with congenital thrombotic thrombocytopenic purpura (TTP). Her conditions were improved after phototherapy, exchange transfusion, and infusion of fresh frozen plasma, red blood cells, and platelets. Gene detection showed a homozygous mutation of c.3650T>C(p.I1217T) in the ADAMTS13 gene, while her parents had a heterozygous mutation at this locus. Congenital TTP is a rare autosomal recessive disease, and timely infusion of fresh frozen plasma can achieve a good clinical outcome. This is the first case of congenital TTP caused by homozygous mutation at this locus reported in China and overseas.

Effect of Recombinant Protein in the Spacer Domain on ADAMTS13 Activity / 中国实验血液学杂志

OBJECTIVE@#To obtain the recombinant protein of spacer domain in von Willebrand factor cleaving protease (ADAMTS13), and further study its biological function in ADAMTS13.@*METHODS@#The prokaryotic expression vector was constructed by using the template of plasmid with full-length ADAMTS13, and then transfected into E coli., following the induction of IPTG with the low temperature (30 ℃). The recombinant protein was purified with Ni-NTA agarose column by gradient imidazole. The purity and immune activity of purified products were identified with SDS-PAGE and Western blot respectively. By Adding the recombinant protein to the plasma of immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients, the activity of ADAMTS13 was tested.@*RESULTS@#The prokaryotic expression vector was successfully constructed and the protein of spacer domain with the high purity was obtained. Western blot showed that the recombinant fragment could both react with monoclonal antibody against 6×His and polyclonal sheep IgG against ADAMTS13 (Gln34-Trp688). The protein formed a main lane at the position of 15 kDa with SDS-PAGE. It was demonstrated that the recombinant protein could efficiently elevate the ADAMTS13 activity in plasma of iTTP patients to reach normal level by functional experiment.@*CONCLUSION@#The recombinant protein has high purity and immune activity, which provides the experimental basis for further research on mechanism of iTTP involved in spacer domain.

Research Advances of Acquired Thrombotic Thrombocytopenic Purpura--Review / 中国实验血液学杂志

Thrombotic thrombocytopenic purpura (TTP) is an acute, potentially life-threatening thrombotic microangiopathies (TMA). TTP has mainly been diagnosed by clinical findings, such as thrombocytopenia and microangiopathic haemolytic anemia. In addition, the reduced activity of von Willebrand factor-cleaving metalloproteinase ADAMTS13 below 10% has been accepted internationally as a diagnostic criterion for TTP. In clinic, the accurate diagnosis and early initiation of therapy can significantly improve the survival rate of patients. Therapy should be focused on increasing ADAMTS13 activity and eliminating or inhibiting ADAMTS13 antibody by the infusion or exchange of therapeutic plasma and corticosteroids. Both the administration of recombinant ADAMTS13 and reducing the release of human neutrophil peptides (HNPs) would be novel promising strategies for the prevention of platelet-vWFinteraction. This review briefly summarizes the recent advances in terms of pathogenesis, diagnosis and treatment of TTP.

Relapse of congenital thrombotic thrombocytopenic purpura, after spontaneous remission, in a second-trimester primigravida: case report and review of the literature / Recidiva, após remissão espontânea, de púrpura trombocitopênica trombótica congênita em primigesta no segundo trimestre de gestação: relato de caso e revisão da literatura

ABSTRACT CONTEXT: Thrombotic microangiopathy syndrome or thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS) describes distinct diseases sharing common pathological features: microangiopathic hemolytic anemia and thrombocytopenia, without any other apparent cause. CASE REPORT: An 18-year-old second-trimester primigravida presented with a history of fifteen days of intense weakness, followed by diarrhea over the past six days. She reported having had low platelets since childhood, but said that she had never had bleeding or menstrual abnormalities. Laboratory investigation showed anemia with schistocytes, thrombocytopenia and hypohaptoglobulinemia. Red blood cell concentrate and platelet transfusions were performed. The hypothesis of TTP or HUS was put forward and ADAMTS13 enzyme activity was investigated. The patient evolved with increasing platelet counts, even without specific treatment, and she was discharged. One month afterwards, she returned presenting weakness and swollen face and legs, which had developed one day earlier. The ADAMTS13 activity was less than 5%, without presence of autoantibodies. Regarding the two previous admissions (at 9 and 16 years of age), with similar clinical features, there was spontaneous remission on the first occasion and, on the second, the diagnosis of TTP was suspected and plasmapheresis was performed, but ADAMTS13 activity was not investigated. CONCLUSION: To date, this is the only report of congenital TTP with two spontaneous remissions in the literature This report reveals the importance of suspicion of this condition in the presence of microangiopathic hemolytic anemia and thrombocytopenia without any other apparent cause.

RESUMO CONTEXTO: A síndrome de microangiopatia trombótica, ou púrpura trombocitopênica trombótica-síndrome hemolítico urêmica (PTT-SHU), descreve doenças diversas com clínica e achados patológicos comuns: anemia hemolítica microangiopática e trombocitopenia, na ausência de outra causa aparente. RELATO DO CASO: Primigesta de 18 anos no segundo trimestre apresenta-se com quadro de 15 dias de fraqueza intensa seguida por diarreia há seis dias. Relata ter plaquetas baixas desde a infância e nega sangramentos e anormalidades menstruais. Investigação laboratorial identificou anemia com esquizócitos, plaquetopenia e hipo-haptoglobulinemia. Foi realizada transfusão de plaquetas e concentrado de hemácias. A hipótese de PTT ou SHU foi aventada e realizou-se pesquisa da atividade da enzima ADAMTS13. A paciente evoluiu com elevação das plaquetas, mesmo sem tratamento específico, tendo alta. Retornou após um mês da alta com queixa de fraqueza há um dia e inchaço de face e pernas. A atividade da ADAMTS13 foi menor que 5%, sem autoanticorpos. Nas duas internações anteriores (aos 9 e 16 anos), com quadros similares, houve remissão espontânea na primeira internação e, na segunda, o diagnóstico de PTT foi suspeitado e foi realizada plasmaférese, porém sem a pesquisa da atividade da ADAMTS13. CONCLUSÃO: Até esta data, este é único relato de TTP congênita com duas remissões espontâneas na literatura. Este relato revela a importância da suspeição desta patologia na presença de anemia hemolítica microangiopática e trombocitopenia sem outra causa aparente.

Effect of Vitamin D Status on Von Willebrand Factor and ADAMTS13 in Diabetic Patients on Chronic Hemodialysis

Von Willebrand factor (vWF) is a glycoprotein with a crucial role in the formation of platelet thrombi, and ADAMTS13 is the main enzyme responsible for vWF cleavage. Both are important in the relationship between diabetic nephropathy, hypercoagulability, and cardiovascular disease. This study evaluated a potential relationship between vitamin D (vitD) levels, vWF, ADAMTS13 activity, and inflammation in diabetic patients on chronic hemodialysis (HD). Blood samples from 52 diabetic patients on chronic HD were obtained to determine vitD levels, vWF, and ADAMTS13 activity, and inflammatory markers. HD patients were grouped according to 25-hydroxyvitamin D [25(OH) VitD]25 nmol/L (n=36). vWF antigen and vWF activity were elevated in both groups, with an average of 214.3±82.6% and 175.8±72.6%, respectively. Average ADAMTS13 activity was within the normal range in both groups. Blood samples from the vitD <25 nmol/L group showed a positive correlation between c-reactive protein (CRP) and vWF levels (P=0.023; r=0.564; 95% confidence interval=0.095-0.828), with a negative correlation between HbA1c and 25(OH) VitD (P=0.015; r=-0.337; 95% confidence interval=-0.337-0.19). Diabetic patients on chronic HD had elevated vWF levels and activity with no significant change in ADAMTS13 activity. The correlation between CRP and vWF levels in the 25(OH) VitD<25 nmol/L group suggests inflammatory-related endothelial dysfunction in these patients.

Influence of HSCT Preconditioning on ADAMTS-13 Activity and vWF level and Its Clinical Significance / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the influence of precondintioning on ADAMTS-13 activity and vWF level, and its clinical significance by measuring the alterations of ADAMTS-13 activity and vWF antigen levels before and after preconditioning of hematopoietic stem cell transplantation (HSCT) in the patients.</p><p><b>METHOD</b>A total of 113 patients received HSCT in the First Hospital affiliated to Soochow University were investigated, 20 healthy volunteers were used as the control. The ADAMTS-13 activity and vWF antigen level were measured by FRETS-vWF73 and ELISA respectively. Modified BU/CY, TBI/CY or BEAM were used as preconditioning regimen.</p><p><b>RESULTS</b>(1) out of all the patients enrolled in this study, 8 patients were diagnosed as thrombotic disorders, and 49 patients were with occurence of acute graft-versus-host disease (aGVHD); (2) comparing with the control group, the ADAMTS-13 activity were lower and vWF antigen was higher in the patients after preconditioning (P < 0.01). Among all the patients, 69 (59.3%) cases showed the decrease of ADAMTS-13 activity after preconditioning, including 9 patients with more than 60% (9/113, 8.0%) decrease, in the meantime, the average plasma vWF antigen level of these 69 patients was significantly increased after preconditioning (P < 0.05); (3) the plasma ADAMTS-13 activity in 8 patients with thrombotic complications decreased after preconditioning, and there was significant difference in comparision with that of patients without thrombotic complication (P < 0.01). The patients with decrease of ADAMTS-13 activity more than 60% accounted for 37.5% (3/8), at the same time, the the level of vWF antigen increased (P < 0.01); (4) The medium level of ADAMTS-13 activity was dropped in 49 patients with aGVHD after preconditoning, but there was no abvious difference in comparision with that of patients without aGVHD. Among them 25 patients were with significant drop actvity of ADAMTS-13 at that time when aGVHD occurred (P < 0.01). Out of them, the patients with drop more than 60% before preconditioning accounted for 60% (2/35). The logistic regression analyse showed that the drop of ADAMTS-13 activity more than 60% before preconditioning was the risk factor for occurence of thrombosis at the later stage (P < 0.01), but the drop of ADAMTS-13 activity after preconditioning did not was the risk factor for occurence of aGVHD.</p><p><b>CONCLUSION</b>The decrease of ADAMTS-13 activity after preconditioning of HSCT confirmed to be lower than that befor preconditioning of HSCT, and the level of vWF antigen after preconditioning of HSCT could be higher than that before precondifioning, especially in patients with thrombosis. The decrease of ADAMTS-13 activity after preconditioning has been found to be mone than 60%, then this decrease can be considered as an independent risk factor for later thrombogenesis, but the decrease of ADAMTS-13 after precoditioning activity did not associate with occurence of aGVHD, so the decrease of ADAMTS-13 activity can be used as an important predictor of thrombosis after HSCT.</p>

Identification of Monoclonal Antibodies against von Willebrand Factor Cleaving Protease (ADAMTS13) and Their Function / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To construct and identify the monoclonal antibodies against von willebrand factor cleaving protease(ADAMTS13), and to study their biological function.</p><p><b>METHODS</b>BALB/c mice were immunized by purified recombinant ADAMTS13 truncated eukaryotic protein (ADAMTS13-T7). Murine anti-human ADAMTS13 monoclonal antibodies (McAbs) were constructed by standard hybridoma technology and identified by ELISA. The recognition of McAbs with full-length recombinant ADAMTS13 protein was identified by Western blot. In function assay, the influence of McAbs on the proteolysis of vWF by ADAMTS13 was observed.</p><p><b>RESULTS</b>A group of 6 murine anti-ADAMTS13 McAbs was obtained with the clone number 1G11, 2F11, 6G3, 9E1, 10A8 and 10B4. In ELISA, the highest titers of 1G11 and 2F11 were observed, both of which showed a higher affinity to ADAMTS13-T7 than full-length ADAMTS13. The Western blot demonstrated that the 6 McAbs all could recognize ADAMTS13, among which 1G11 and 2F11 showed stronger reaction with ADAMTS13. In addition, under the denatured conditions, 1G11 and 2F11 could inhibit hydrolysis of vWF by ADAMTS13, and that was stronger with the increasing of McAbs concentration.</p><p><b>CONCLUSION</b>McAbs against ADAMTS13 have been gained, two of which are inhibitory antibodies.</p>

ADAMTS13 Level in Prothrombotic Status and Its Related Factor Analysis / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To detect the plasma activity of von Willebrand factor-cleaving protease (ADAMTS13) in the patients with prothrombotic status, and explore the effect and significance of ADAMTS13 in the prothrombotic status. The correlation of ADAMTS13 with von Willebrand factor (vWF), thrombospondin 1 (TSP1), C-reactive protein etc, and blood pressure was simultaneously analyzed．</p><p><b>METHODS</b>The activity of ADAMTS13 in patient groups (atherosclerosis, diabetes, acute promyelocytic leukemia, cancer and sepsis, a total of 260 cases) and in control group 50 cases were evaluated by residue collagen binding assay（R-CBA）, the protein levels of TSP1 and vWF were measured by ELISA kits； the correlation of ADAMTS13 activity with CRP, creatinine, and blood pressure was analyzed with statistical soft ware.</p><p><b>RESULTS</b>The activity of plasma ADAMTS13 in patient group was significantly lower than that in normal control group(P<0.05). And the protein levels of TSP1 and vWF in the patients with prothrombotic status were higher than those in the normal controls(P<0.05). Analysis of the correlation showed that the ADAMTS13 activity correlated negatively with the levels of TSP1 protein, blood sugar, blood pressure, D-dimer, creatinine,and CRP levels (P<0.05), however, the ADAMTS13 activity did not significantly correlate with the levels of serum lipids, blood type, platelet number and hemoglobin level(P>0.05).</p><p><b>CONCLUSION</b>The plasma ADAMTS13 activity is decreased in the patients with prothrombotic status, suggesting that the decreased ADAMTS13 activity may participate in the occurrence of prothrombotic status, and the dectection of plasma ADAMTS13 activity may help the diagnosis of pro-thrombotic disease.</p>

Púrpura trombocitopénica trombótica, presentación de tres casos en el Hospital Carlos Andrade Marín / Thrombotic thrombocytopenic purpura: presentation of three cases in the Carlos Andrade Marin Hospital

La púrpura trombocitopénica trombótica es un tipo raro de anemia hemolítica microangiopática ocasionada por un déficit de proteína ADAMTS 13 cuya mortalidad sin tratamiento es del 90%. En nuestro hospital no tenemos disponible la cuantificación de esta proteína. Debe tenerse una alta sospecha diagnóstica ante la presencia de anemia hemolítica, trombocitopenia y esquistocitos en sangre periférica. Se presentan 3 casos clínicos, el primero de una paciente de 52 años con una PTT idiopática adquirida que requirió plasmaféresis y corticoterapia sin buena respuesta. Dada la refractariedad fue necesario administración de Rituximab, Ciclofosfamida y Vincristina. El segundo caso una paciente femenino de 42 años con positividad de anticuerpos Anti DNA, desarrolló una PTT con deterioro del estado de conciencia, hemiparesia y crisis convulsivas que requirió manejo en terapia intensiva, luego de 5 sesiones de plasmaféresis y corticoterapia tuvo una evolución favorable. El tercer caso, paciente masculino de 45 años con insuficiencia respiratoria, signos de falla derecha, se documentó PTT por la presencia de abundantes esquistocitos en sangre periférica; el paciente falleció observándose en la autopsia infarto masivo de ventrículo derecho. Consideramos como un aporte a la bibliografía esta descripción de casos especialmente por la heterogeneidad de la presentación y respuesta al tratamiento.

Thrombotic thrombocytopenic purpura is a rare type of microangiopathic hemolytic anemia caused by a defciency of ADAMTS 13 protein whose mortality without treatment is 90%. In our hospital we cannot quantify this protein. Diagnostic should be strongly suspected in the presence of hemolytic anemia, thrombocytopenia and peripheral blood schistocytes. We describe 3 cases, the frst of a 52 years with idiopathic acquired TTP requiring plasma exchange and steroids without good response. Given the refractoriness was necessary administration of Rituximab, Cyclophosphamide and Vincristine. The second case, a 42 year old female patient with Anti DNA antibody positivity develops a PTT with impairment of consciousness, hemiparesis and seizures that required treatment in intensive care, after 5 sessions of plasmapheresis and steroids had a favorable outcome. The third case, 45 years old male patient who presents with dyspnea, diagnosis of TTP is documented by abundant Schistocytes in peripheral blood, develops secondary acute respiratory failure and died. At autopsy is was found a massive right ventricular infarction. We consider a contribution to the literature this description of cases especially by heterogeneity of presentation and response to treatment.