Conformational plasticity of ADAMTS13 in hemostasis and autoimmunity

A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) is a multidomain metalloprotease for which until now only a single substrate has been identified. ADAMTS13 cleaves the polymeric force-sensor von Willebrand factor (VWF) that unfolds under shear stress and recruits platelets to sites of vascular injury. Shear force–dependent cleavage at a single Tyr–Met peptide bond in the unfolded VWF A2 domain serves to reduce the size of VWF polymers in circulation. In patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP), a rare life-threatening disease, ADAMTS13 is targeted by autoantibodies that inhibit its activity or promote its clearance. In the absence of ADAMTS13, VWF polymers are not adequately processed, resulting in spontaneous adhesion of blood platelets, which presents as severe, life-threatening microvascular thrombosis. In healthy individuals, ADAMTS13–VWF interactions are guided by controlled conversion of ADAMTS13 from a closed, inactive to an open, active conformation through a series of interdomain contacts that are now beginning to be defined. Recently, it has been shown that ADAMTS13 adopts an open conformation in the acute phase and during subclinical disease in iTTP patients, making open ADAMTS13 a novel biomarker for iTTP. In this review, we summarize our current knowledge on ADAMTS13 conformation and speculate on potential triggers inducing conformational changes of ADAMTS13 and how these relate to the pathogenesis of iTTP.     

过敏性紫癜合并肺炎支原体感染患儿呼吸道菌群多样性与补体C3、C4及免疫指标的相关性

目的研究儿童过敏性紫癜(HSP)合并肺炎支原体(MP)感染患儿呼吸道菌群多样性与补体C3、C4及免疫指标的相关性。方法选择2017年3月至2020年3月于我院接受治疗的HSP患儿96例,其中48例合并MP感染的患儿设为合并MP组,48例未合并MP感染的患儿设为HSP组。另外选择同期我院健康体检儿童45例为对照组。比较3组对象呼吸道菌群多样性,血清免疫球蛋白IgG、IgA、IgM、IgE及补体C3、C4水平,并分析呼吸道菌群多样性与免疫指标间的相关性。结果合并MP组患儿呼吸道菌群Simpson指数显著高于对照组,Shannon指数显著小于对照组(均P<0.05)。HSP组患儿呼吸道菌群Simpson指数及Shannon指数与对照比较差异无统计学意义(均P>0.05)。合并MP组患儿血清IgA水平较对照组显著上升,IgM水平显著下降;HSP组患儿血清IgA、IgE水平较对照组显著上升;合并MP组患儿血清IgE、IgM水平显著低于HSP组(均P<0.05);而3组对象血清IgG水平比较差异无统计学意义(均P>0.05)。合并MP组和HSP组患儿血清补体C3水平显著低于对照组;合并MP组患儿血清补体C4水平显著低于对照组;合并MP组患儿血清补体C3、C4水平均显著低于HSP组(均P<0.05)。IgM、IgE及补体、C3、C4水平与Simpson指数呈负相关,与Shannon指数呈正相关(均P<0.05)。结论HSP合并MP感染患儿呼吸道菌群多样性与补体C3、C4及IgM、IgE水平具有显著相关性。     

腹型过敏性紫癜患儿急性期与恢复期肠道菌群变化的分析

目的探究腹型过敏性紫癜患儿急性期与恢复期肠道菌群的变化,为制定更针对性的过敏性紫癜治疗措施提供依据。方法选取28例腹型过敏性紫癜患儿粪便标本(急性期19例和恢复期9例),9例非腹型过敏性紫癜患儿粪便标本,一共37例粪便标本。采用Illumina MiSeq平台提取样本DNA进行高通量测序,对测序结果进行生物信息学分析。结果腹型过敏性紫癜患儿急性期肠道菌群α多样性与恢复期、非腹型过敏性紫癜存在显著差异,而β多样性没有显著差异,腹型过敏性紫癜患儿急性期粪便标本中韦荣球菌属的相对丰度显著高于无腹痛腹型过敏性紫癜和非腹型过敏性紫癜患儿,而瘤胃球菌属的相对丰度在腹型过敏性紫癜患儿恢复期粪便标本中最高。结论肠道菌群的变化可能参与腹型过敏性紫癜的发病,其中主要表现在急性期韦荣球菌属丰富度的增加和恢复期瘤胃球菌属丰富度的升高,为微生物制剂的应用提供依据。     

Relation between 3435C>T multidrug resistance 1 gene polymorphism with high dose methylprednisolone treatment of childhood acute idiopathic thrombocytopenic purpura

The current study was conducted to assess 3435C>T multidrug resistance 1 gene polymorphism and the efficacy of high dose methylprednisolone (HDMP) in childhood acute idiopathic thrombocytopenic purpura patients.

Methods

A total of 31 childhood acute Idiopathic thrombocytopenic purpura patients (17 females, 14 males) between the ages of 2 and 16 years of age were included in the study. High-dose methylprednisolone was given at a dose of 30 mg/kg/day for 3 days and 20 mg/kg/day for 4 days, consecutively and intravenously. Polymerase chain reaction-restriction fragment length polymorphism was used for the detection of C3435T single nucleotide polymorphism. Fragments obtained were 238 bp to T/T genotype, 172 bp and 60 bp fragments to the C/C genotype, and 238 bp, 172 bp and 60 bp to the C/T genotype.

Results

The distribution of CC, CT, and TT genotypes were 19.0%, 61.3%, and 19.4%, respectively. Both allele frequencies of C and T were the same — 50%. There was no significant difference in genotype and allele distribution between the patients with ITP and the control group (χ² = 0.84 p = 0.65, χ² = 0.2 p = 0.63, respectively). There were no significant differences in age, gender, and pre- and post-treatment platelet counts between CC, CT, and TT genotypes of the MDR gene. Response to treatment shows no significant difference between genotype and allele groups.

Conclusion

In our study, there was no difference in the HDMP treatment response between MDR1 gene genotypes. However, it should be noted that this study includes a small group of patients. Our data should therefore be considered preliminary, awaiting further confirmatory studies on an expanded patient base.     

Escherichia coli-derived von Willebrand factor-A2 domain fluorescence/Förster resonance energy transfer proteins that quantify ADAMTS13 activity

The cleavage of the A2 domain of von Willebrand factor (VWF) by the metalloprotease ADAMTS13 regulates VWF size and platelet thrombosis rates. Reduction or inhibition of this enzyme activity leads to thrombotic thrombocytopenic purpura (TTP). We generated a set of novel molecules called VWF-A2 FRET (fluorescence/Förster resonance energy transfer) proteins, where variants of yellow fluorescent protein (Venus) and cyan fluorescent protein (Cerulean) flank either the entire VWF-A2 domain (175 amino acids) or truncated fragments (141, 113, and 77 amino acids) of this domain. These proteins were expressed in Escherichia coli in soluble form, and they exhibited FRET properties. Results show that the introduction of Venus/Cerulean itself did not alter the ability of VWF-A2 to undergo ADAMTS13-mediated cleavage. The smallest FRET protein, XS-VWF, detected plasma ADAMTS13 activity down to 10% of normal levels. Tests of acquired and inherited TTP could be completed within 30 min. VWF-A2 conformation changed progressively, and not abruptly, on increasing urea concentrations. Although proteins with 77 and 113 VWF-A2 residues were cleaved in the absence of denaturant, 4 M urea was required for the efficient cleavage of larger constructs. Overall, VWF-A2 FRET proteins can be applied both for the rapid diagnosis of plasma ADAMTS13 activity and as a tool to study VWF-A2 conformation dynamics.     

Association of interleukin-(IL)10 haplotypes and serum IL-10 levels in the progression of childhood immune thrombocytopenic purpura

Derangement of genetic and immunological factors seems to have a pivotal role in the pathophysiology of immune thrombocytopenic purpura (ITP). We investigated interleukin(IL)-10 genetically determined expression in children with an acute progression of ITP (n=41) compared to young patients with chronic ITP (n=44) and healthy controls (n=60), and attempted to correlate IL-10 production with the course of the disease. We genotyped our study population for three single nucleotide polymorphisms at positions -1082 (A/G), -819 (C/T) and -592 (C/A) in the promoter region of the IL-10 gene. IL-10 levels were measured by enzyme-linked immunoassay. The IL-10 production in our study population was significantly higher in patients carrying the GCC haplotype than those bearing ACC and ATA haplotypes (6.9 ± 1.5 vs 3.6 ± 0.8 vs 3.3 ± 0.3, p=0.03). The serum concentration of IL-10 was significantly higher in patients with an acute course of their disease, who mainly carried the GCC haplotype (92%), compared to chronic subjects, bearing the non-GCC haplotypes, and controls [17 pg/mL (1.7-18) vs 3.5 pg/mL (0.6-11) vs 3 pg/mL (1-7), p<0.01)]. Our findings show that patients carrying the GCC-high producer IL-10 haplotype have an acute development of ITP and that IL-10 levels might represent a useful predictive biomarker of the disease course.     

DNA methyltransferase 3B (DNMT3B − 579 G > T) promotor polymorphism and the susceptibility to pediatric immune thrombocytopenic purpura in Egypt

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease characterized by increased platelet destruction. Although the etiology of ITP remains unclear, it is accepted that both environmental and genetic factors play an important role in the development of the disease. The present study aimed at exploring a novel molecular determinant that may influence the susceptibility and course of ITP in Egyptian children. To achieve our aim, genotyping of DNMT3B − 579 G > T promotor polymorphism by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay. The current study was conducted on 140 ITP patients and 150 age and gender matched healthy controls. The results obtained revealed that DNMT3B − 579 TT homotype was significantly higher in ITP patients and conferred almost three fold increased risk of ITP (OR = 3.16, 95%CI = 1.73–5.79). There was no statistically significant difference between ITP patients with wild or mutant genotypes as regards their clinical or laboratory data. Furthermore, there was no statistical difference in the distribution of DNMT3B − 579 G > T genotypes between acute and chronic ITP patients. In conclusion, DNMT3B − 579 G > T promotor polymorphism represents a novel genetic risk factor for ITP but not a predictor for tendency to chronicity in pediatric ITP in Egypt.     

儿童特发性血小板减少性紫癜与人巨细胞病毒感染的关系

目的:探讨特发性血小板减少性紫癜的治疗转归与巨细胞病毒感染的关系。为临床合理有效地治疗ITP提供部分参考。方法:用ELISA法和流式细胞仪分别检测患儿血清HCMV抗体及外周血淋巴细胞亚群。选取2005.1-2006.12在本院儿科就诊的68例新发ITP患儿,将其按有无HCMV感染分为HCMV感染组和非感染组,比较二者的免疫状态,近期(两周)疗效,远期(六月)疾病转归之间的关系。结果:68例ITP患儿中HCMV感染30例,非HCMV感染ITP存在T淋巴细胞亚群的异常,即CD4+细胞减低,CD8+细胞升高。HCMV感染患儿T淋巴细胞总数亦降低,CD4+细胞下降明显。近期疗效无明显差异,远期转归相比较有统计学意义。结论:HCMV感染诱发的细胞免疫异常是儿童ITP发病及病程迁延的重要因素。应同时进行抗病毒和免疫治疗。     

不同剂量丙种球蛋白联合地塞米松治疗儿童特发性血小板减少性紫癜的疗效观察

目的:探讨不同剂量丙种球蛋白联合地塞米松治疗儿童特发性血小板减少性紫癜(ITP)的临床疗效。方法:选择2010年7月至2015年7月间我院收治的特发性血小板减少性紫癜患儿78例,按照随机数字表法将其分为观察组38例及对照组40例。观察组采用200 mg/(kg·d)丙种球蛋白联合地塞米松进行治疗,对照组采用400 mg/(kg·d)丙种球蛋白联合地塞米松进行治疗。对两组患者的临床疗效、治疗前后血小板计数、血小板上升时间、血小板恢复正常时间、出血停止时间、住院时间、不良反应及医疗费用进行观察。结果:两组患儿血小板计数治疗前无显著差异(P0.05),治疗后两组血小板计数均呈逐渐升高趋势(P0.05),但组间比较无统计学差异(P0.05)。两组患儿血小板上升时间、血小板恢复正常时间、出血停止时间及住院时间均接近,无统计学差异(P0.05)。观察组治疗总有效率为94.74%,对照组为95.00%,两组无显著差异(P0.05)。观察组不良反应发生率为8.17%,对照组为12.50%,两者相比无统计学差异(P0.05)。观察组患儿平均医疗费用为(1.25±0.34)万元,对照组患儿平均医疗费用为(2.31±0.40)万元,观察组明显低于对照组,两者差异有统计学意义(P0.05)。结论:不同剂量丙种球蛋白联合地塞米松治疗ITP具有类似的临床疗效及安全性,但小剂量丙种球蛋白组治疗费用明显降低,因此值得临床推广应用。