Novel interactions in platelet biology: CLEC-2/podoplanin and laminin/GPVI

Summary.  We have identified a novel platelet membrane protein, CLEC-2 as a receptor for rhodocytin, a platelet-activating snake venom. CLEC-2 is specifically expressed in platelets and megakaryocytes, and has an atypical ITAM, which undergoes tyrosine phosphorylation by Src kinases, resulting in downstream signaling including Syk, SLP-76 and PLCγ2. We found that CLEC-2 is the receptor for podoplanin, a sialoglycoprotein implicated in tumor-induced platelet aggregation and tumor metastasis. VWF bridges exposed collagen, at damaged vessels, to GPIb. Subsequently, GPVI binds to collagen, leading to integrin α2β1 activation. We found that platelets adhere to laminin, another major ECM component, through integrin α6β1, and are activated through GPVI. This is the first report on GPVI having a ligand, laminin, other than collagen. Laminin also interacts with VWF, leading to platelet adhesion via GPIb under sheer stress. The redundancy of platelet interactions with laminin and with collagen may serve to promote hemostasis at sites of damaged vessels.     

Synchronised therapy and high-dose cyclophosphamide in proliferative lupus nephritis

The aim of this open study was to compare the outcomes and side effects of plasmapheresis (PP) in patients with proliferative lupus nephritis treated with cyclophosphamide (Cyc) boluses. The study involved 28 consecutive patients. All of the patients met the ACR modified criteria for SLE and underwent a qualifying renal biopsy. In group I, patients were treated with synchronised therapy (PP, 50 ml/kg, followed by pulse Cyc, 750 mg/m(2), repeated monthly for 6 months), whereas in group II, they were given only intermittent Cyc boluses (at the same dosage). The data were collected in the patients' records according to a standardised protocol. Patients were followed-up for a mean of 4 years. The disease-free survival was analysed using Kaplan-Meier estimated survival curves ([S(t)]). At the end of the 6-month treatment period, a statistically significant number of patients in group I (75%) was in complete remission in comparison to group II (31%) (P < 0.02), whereas at long-term follow-up, these percentages were similar (41% vs. 50%, P = n.s.). The main functional and immunological parameters showed a normalisation in both groups. The risk of a poor renal outcome significantly correlated with high serum creatinine levels at the onset of nephritis (P < 0.05). We documented a higher rate of infectious complications in group I. This study reports that synchronised therapy is useful in inducing a faster remission in patients with proliferative lupus nephritis. However, it is not superior to conventional therapy at long term follow-up analysis. Positive results should be reinforced by a long-term maintenance therapy.     

免疫抑制剂不同冲击疗法治疗狼疮性肾炎的临床研究

目的：探讨免疫抑制剂不同疗法治疗狼疮性肾炎的疗效。方法：46例患者随机分为3组。甲基氢化泼尼松（MP）冲击组：15例患者采用MP1．0g 5％葡萄糖250ml静滴,每日1次,连用3日,以后根据病情可连续应用2－3个疗程。环磷酰胺（CTX）冲击组：15例患者采用CTX0．8－1．0g/1.73m^2静滴,每月1次,连续6－8个月。其他16例患者采用MP联合CTX冲击治疗（MP＋CTX冲击组）：方法同上。每组患者均常规应用泼尼松治疗。在治疗前后检测24小时尿蛋白定量、血肌酐、C3、C4、抗核抗体（ANA）的变化。结果：治疗6个月后,MP组、CTX组及MP＋CTX组对狼疮性肾炎的疗效分别为80．0％、81．3％、和93．3％。各组尿蛋白24小时定量、血肌酐、C3、C4升高治疗前后均有显著性差异（P均＜0．05）;而MP＋CTX组较MP组和CTX组差异更明显（P均＜0．05）。结论：MP与CTX冲击疗法治疗狼疮性肾炎的疗效肯定,而两者联合冲击的疗效更好。     

Plasmapheresis in lupus nephritis with nephrotic syndrome: A long-term followup

Lupus nephritis with nephrotic syndrome is one of the most serious complications of systemic lupus erythematosus. Six female patients with systemic lupus and nephrotic syndrome, refractory to immunosuppressive drug therapy, received 15–20 exchange plasmaphereses. One patient, treated concurrently with high-dose steroids, showed temporary improvement, and five patients, treated concurrently with steroids and either cyclophosphamide or azathioprine, had long-term remissions. Plasmapheresis is a promising therapeutic modality in cases of refractory lupus nephritis with nephrotic syndrome.     

狼疮性肾炎患者心理状态分析与对策

目的 探讨狼疮性肾炎患者心理状态并提出相应的护理措施。方法 采用症状自评量表（SCL-90）进行测试。结果 患者在躯体化、强迫症状、人际敏感、抑郁、焦虑、敌意、恐怖、偏执、精神病性9个因子评分与国内常模比较，差异具有非常显著性（P〈0．01）。结论 狼疮性肾炎患者心理状态差，应加强心理护理。     

应用酮酸疗法改善狼疮性肾炎患者营养状态的研究

目的研究酮酸疗法对狼疮性肾炎(LN)患者营养状态的治疗作用.方法选择我院门诊和住院的LN及非狼疮性慢性肾功能不全(NLN)患者共45例,所选患者血清尿素氮(BUN)均大于9 mmol/L,血清肌酐(Cr)大于178 μmol/L,且无肝脏酶学异常;将LN与NLN患者随机分为两组:实验组为极低蛋白饮食 α-酮酸治疗组,对照组为单纯低蛋白饮食治疗组,所有LN或NLN患者其他治疗均相同,患者治疗前及治疗后2周及2个月收集晨起空腹静脉血清,置于-30 ℃冻存,待批量检测,用酶标法测定血清前白蛋白(PA)、白蛋白(ALB)水平,血清BUN、Cr由我院检验科当日检测.结果酮酸疗法治疗2个月后,实验组较对照组血清BUN明显下降,LN与NLN两个实验组BUN分别为(9.29±4.70) mmol/L、(14.02±7.50) mmol/L,而LN与NLN两个对照组分别为(13.87±6.97) mmol/L、(19.48±7.32) mmol/L(P<0.05);实验组两组血清PA较对照组两组明显增高,分别为(347.99±112.61) mg/L、(381.27±86.13) mg/L和(282.13±66.27)、(352.98±94.21) mg/L(P<0.05).结论酮酸疗法能够降低LN及NLN患者的血清BUN水平,血清PA是较ALB敏感的反映营养状态的指标,酮酸疗法能够改善LN患者的营养状态.     

青蒿琥酯通过抑制ICAM-1治疗鼠狼疮性肾炎的研究

目的探索青蒿琥酯对狼疮模型鼠-MRL/lpr鼠狼疮性肾炎（LN）的疗效及作用机制。方法分别以青蒿琥酯50mg/（kg.d）和相同体积生理盐水灌胃治疗MRL/lpr鼠,治疗16周后检测小鼠24 h蛋白尿、血肌酐水平,检测小鼠外周血血清中ICAM-1浓度及淋巴细胞表面CD54阳性率,检测小鼠肾组织中ICAM-1表达。结果青蒿琥酯能够显著减少狼疮鼠的蛋白尿（P〈0.01）及血肌酐水平（P〈0.05）,显著降低狼疮鼠的血清中可溶性ICAM-1浓度（P〈0.05）及淋巴细胞表面CD54的阳性率（P〈0.05）,导致肾组织中ICAM-1的表达明显下降。结论青蒿琥酯显著缓解MRL/lpr鼠LN病情,此作用可能与抑制ICAM-1的表达有关。     

狼疮肾炎外周血单个核细胞LFA—1表达

目的检测淋巴细胞功能相关抗原-1(LFA-1)在狼疮肾炎外周血单个核细胞中的表达并对其临床价值进行探讨.方法通过分离外周血单个核细胞,利用流式细胞术检测了狼疮肾炎外周血单个核细胞LFA-1表达.结果狼疮肾炎活动组外周血单个核细胞LFA-1表达高于非活动组和健康对照组(7.49±1.17vs2.67±0.65、2.21±0.32,P值均小于0.01),而非活动组与健康对照组则无明显差异(P＞0.05).LFA-1表达与狼疮活动指数呈正相关(r=0.746,P＜0.01).与狼疮活动指标抗核抗体、抗双链DNA抗体呈正相关(r值分别为0.627、0.705,P＜0.01),与补体(C3)呈负相关(r=-0.538,P＜0.01).结论LFA-1分子与狼疮活动密切相关,临床上可以作为狼疮活动的参考指标.     

Glycoprotein (GP) VI dimer as a major collagen-binding site of native platelets: direct evidence obtained with dimeric GPVI-specific Fabs

Summary.  Background: The platelet collagen receptor glycoprotein (GP) VI is suggested to exist as a dimer on the platelet surface, but no direct proof of the functional importance of dimer formation has been provided. Objectives: To obtain direct evidence for GPVI dimers on the platelet membrane and their functional importance, Fab antibodies were developed that bind to GPVI dimer (GPVI-Fc₂) but not to GPVI monomer (GPVIex) through a phage display method. Results: Ssix Fabs were found: B–F, only reactive with GPVI-Fc₂, and A, mainly reactive with GPVI-Fc₂, with some reactivity towards GPVIex; each Fab (Fab-dHLX-MH) forms a bivalent dimer (b-Fab) by dimerizing the dHLX domains from two Fab molecules. Fab F was subcloned to a monovalent format by deleting its dHLX domain. All b-Fabs induced platelet aggregation, but the monomeric form of Fab F (m-Fab-F) specifically inhibited collagen-induced aggregation. All b-Fabs and m-Fab-F inhibited GPVI-Fc₂ binding to fibrous collagen. Immunoblotting showed that b-Fab-F and m-Fab-F bound weakly to GPVI-Fc₂. Adding the anti-GPVI monoclonal antibody 204-11 increased the B _max of m-Fab-F binding to GPVI-Fc₂, suggesting that 204-11 binds to GPVI-Fc₂ molecules not already in the appropriate conformation to recognize the Fab, converting them to a conformation reactive to the Fab. Conclusions: GPVI forms a specific structure by dimerization that is necessary for the binding of this receptor to collagen fibrils. The binding of m-Fab-F to platelets directly demonstrates that GPVI is present as a functionally relevant dimer on the platelet surface.     

Claviform aspergillus‐related vegetation in the left ventricle of a patient with systemic lupus erythematosus

A 38‐year‐old woman was diagnosed with systemic lupus erythematosus and received immunosuppressive therapy. After 6 months of treatment, workup for low‐grade fever yielded elevated enzyme‐linked immunosorbent assay titers for Aspergillus antigen in serum and ascites, leading to the diagnosis of disseminated aspergillosis. Transthoracic echocardiography revealed a claviform vegetation attached to the left ventricular anterior septum. Two days after the start of antifungal Amphotericin‐B therapy, the patient suffered from several neurologic disorders. A second transthoracic echocardiography revealed that the vegetation decreased in size. Two weeks later, the vegetation increased again. Combination therapy of Amphotericin‐B and Voriconazole was initiated, and the vegetation eventually disappeared completely. © 2017 Wiley Periodicals, Inc. J Clin Ultrasound 46 :231–232, 2018     

70例狼疮性肾炎患者肾脏纤维化及临床指标分析

目的总结狼疮性肾炎患者的临床表现,肾脏纤维化、血清学及免疫学指标变化。方法回顾性分析经肾穿刺活检组织病理证实70例狼疮性肾炎患者的临床资料。结果70例首发症状主要为皮疹、溃疡、水肿;病理类型中Ⅱ型13例,Ⅲ型16例,Ⅳ型24例,Ⅴ型12例,Ⅵ型1例,Ⅲ+Ⅴ型1例,Ⅳ+Ⅴ型3例;24例处于活动期,其中Ⅳ型12例;免疫荧光示免疫球蛋白(immunoglobulin,Ig)A、IgG、IgM、C3阳性沉积者分别有59、59、58、64例,IgA、IgG、IgM、C3均阳性沉积者54例,沉积部位位于系膜区、基底膜、上皮下等多处,肾小管上皮区有Nox4表达,肾小球系膜区、肾小管上皮及间质内见大量平滑肌肌动蛋白、纤维连接蛋白表达;70例抗核抗体均为阳性,活动期者抗干燥综合征抗原A抗体阳性率最高;活动期者血红蛋白、白蛋白、IgA、补体C3水平明显低于非活动期者,血尿素氮、血肌酐、24 h尿蛋白定量、C反应蛋白较非活动期者高,红细胞沉降率较非活动期者快(P<0.05)。结论狼疮性肾炎患者首发症状以皮疹、溃疡、水肿等肾外症状多见,病理类型以Ⅳ型为主,肾脏纤维化标志物在肾小球系膜区、肾小管上皮及间质内大量表达;活动期者Ⅳ型最多,氧化应激及炎症水平增高,营养状况较差。     

B细胞淋巴瘤/白血病-2蛋白在狼疮性肾炎肾组织表达及意义

目的 研究细胞凋亡调节蛋白B细胞淋巴瘤／白血病-2(Bel-2)在狼疮性肾炎(LN)患者肾组织的表达情况，探讨其与LN肾组织病理改变的关系及意义。方法 以免疫组化方法检测44例活动期LN患者肾组织中Bel-2蛋白、增殖细胞核抗原(PCNA)的表达，观察其与LN肾组织病理改变的关系。结果 ①增殖型LN肾小球内Bel-2及PCNA阳性细胞数均增高，且与球内细胞增殖程度呈正相关；②LN肾间质细胞Bel-2表达增高，与间质炎症细胞浸润程度正相关。结论 增殖型LN肾组织中Bel-2蛋白异常表达与病理改变有关，肾组织中Bel-2的过多表达对LN肾小球细胞增殖及间质炎症细胞浸润可能起重要作用。     

Compromised ITAM-based platelet receptor function in a patient with immune thrombocytopenic purpura

Summary.  Background:  Receptors on platelets that contain immunoreceptor tyrosine-based activation motifs (ITAMs) include collagen receptor glycoprotein (GP) VI, and FcγRIIa, a low affinity receptor for immunoglobulin (Ig) G. Objectives:  We examined the function of GPVI and FcγRIIa in a patient diagnosed with immune thrombocytopenic purpura (ITP) who had unexplained pathological bruising despite normalization of the platelet count with treatment. Methods and Results:  Patient platelets aggregated normally in response to ADP, arachadonic acid and epinephrine, but not to GPVI agonists, collagen or collagen-related peptide, or to FcγRII-activating monoclonal antibody (mAb) 8.26, suggesting ITAM receptor dysfunction. Plasma contained an anti-GPVI antibody by MAIPA and aggregated normal platelets. Aggregating activity was partially (∼60%) blocked by FcγRIIa-blocking antibody, IV.3, and completely blocked by soluble GPVI ectodomain. Full-length GPVI on the patient platelet surface was reduced to ∼10% of normal levels, and a ∼10-kDa GPVI cytoplasmic tail remnant and cleaved FcγRIIa were detectable by western blot, indicating platelet receptor proteolysis. Plasma from the patient contained ∼150 ng mL⁻¹ soluble GPVI by ELISA (normal plasma, ∼15 ng mL⁻¹) and IgG purified from patient plasma caused FcγRIIa-mediated, EDTA-sensitive cleavage of both GPVI and FcγRIIa on normal platelets. C onclusions:  In ITP patients, platelet autoantibodies can curtail platelet receptor function. Platelet ITAM receptor dysfunction may contribute to the increased bleeding phenotype observed in some patients with ITP.     

系统性红斑狼疮患者甲状腺疾病的发生率以及亚临床甲状腺功能减退症与自身抗体、狼疮肾炎的关系

目的研究系统性红斑狼疮(SLE)患者甲状腺疾病的发生率以及亚临床甲状腺功能减退症与自身抗体、狼疮肾炎(LN)的关系。方法选取我院2010年1月至2019年12月收治的66例SLE患者作为研究对象,根据是否发生甲状腺疾病及亚临床甲状腺功能减退症将患者分为SLE组(未发生甲状腺疾病与亚临床甲状腺功能减退症,45例)与合并组(发生甲状腺疾病或亚临床甲状腺功能减退症,21例)。比较两组患者的甲状腺功能水平、自身抗体阳性率及LN发生率。结果 66例患者中,18例(27.27%)发生甲状腺疾病,3例(4.55%)发生亚临床甲状腺功能减退症。合并组的T_3、T_4、FT_3、FT_4、TSH水平均高于SLE组,差异具有统计学意义(P<0.05)。两组的nRNP、Sm、SSA、Scl-70、Jo-1、RIB抗体阳性率比较,差异无统计学意义(P>0.05);SLE组的SSB抗体阳性率与LN发病率均低于合并组,差异具有统计学意义(P<0.05)。结论在SLE患者中,甲状腺疾病存在一定的发生率,也有部分患者存在亚临床甲状腺功能减退症,而此部分患者的自身SSB抗体阳性率及LN发生率均较高。因此对SLE患者进行自身抗体SSB与甲状腺功能的检测,可以辅助临床对患者疾病发生的预测。     

护理干预对狼疮性肾炎维持治疗期病人的影响

[目的]探讨护理干预对狼疮性肾炎（LN）维持治疗期病人的病情影响。[方法]将2003年—2007年出院的40例LN病人随机分为干预组和对照组,每组20例,干预组出院后进行护理干预,所有研究对象均随访1年,出院后1个月、3个月、6个月、9个月、12个月各到门诊随访1次,复查补体C3、C4降低,将C3、C4降低作为LN病人病情复发的实验室指标,比较两组复发率。[结果]随着时间延长,干预组复发率明显低于对照组,差异均有统计学意义（P〈0.05）。[结论]护理干预能增强病人的治疗依从性,提高病人的治疗效果和生活质量。     

狼疮肾炎患者血中抗磷脂抗体与血栓素变化的观察

目的：观察血抗磷脂抗体（ａＰＬ）与前列环素（ＰＧＩ２）和血栓素Ａ２（ＴＸＡ２）在狼疮肾炎（ＬＮ）中的变化,并探讨其临床意义。方法：用酶联免疫吸附法（ＥＬＩＳＡ）和放射免疫法分别检测ＬＮ患者血ａＰＬ、６酮前列腺素Ｆ１α（６ｋｅｔｏＰＧＦ１α）和血栓素Ｂ２（ＴＸＢ２）水平。结果：５１例ＬＮ中３５例ａＰＬ阳性（６８．６％）,ａＰＬ与血栓形成、系统性红斑狼疮（ＳＬＥ）活性及皮肤血管炎显著相关。ａＰＬ阳性组ＴＸＢ２〔（２５６５．８７±５９３．４７）ｎｇ／Ｌ〕较ａＰＬ阴性组〔（７１８．７４±１３７．６３）ｎｇ／Ｌ〕和正常对照组〔（４２１．３７±１０８．５９）ｎｇ／Ｌ〕均显著升高（Ｐ均＜０．０１）;６ｋｅｔｏＰＧＦ１α和ＴＸＢ２比值均显著降低（ａＰＬ阳性组：０．５７０±０．０６９;ａＰＬ阴性组：１．０３０±０．０７７;对照组：１．９１０±０．１１７;Ｐ均＜０．０１）。结论：ａＰＬ与ＬＮ患者的血栓形成有明显关系,其原因可能与体内ＰＧＩ２ＴＸＡ２平衡破坏及ＴＸＡ２升高有关。     

Differential roles for the adapters Gads and LAT in platelet activation by GPVI and CLEC‐2

Summary. Background: The adapter proteins SLP‐76 and LAT have been shown to play critical roles in the activation of PLCγ2 in platelets downstream of GPVI/FcRγ and the C‐type lectin receptor CLEC‐2. SLP‐76 is constitutively associated with the adapter Gads in platelets, which also binds to tyrosine phosphorylated LAT, thereby providing a potential pathway of regulation of SLP‐76. Objective: In the present study, we have compared the role of Gads alongside that of LAT following activation of the major platelet glycoprotein receptors using mice deficient in the two adapter proteins. Results: Gads was found to be required for the efficient onset of aggregation and secretion in response to submaximal stimulation of GPVI and CLEC‐2, but to be dispensable for activation following stronger stimulation of the two receptors. Gads was also dispensable for spreading induced through integrin α_IIbβ₃ or the GPIb–IX–V complex. Further, Gads plays a negligible role in aggregate formation on collagen at an arteriolar rate of shear. In stark contrast, platelets deficient in the adapter LAT exhibit a marked decrease in aggregation and secretion following activation of GPVI and CLEC‐2, and are unable to form stable aggregates on collagen at arteriolar shear. Conclusions: The results demonstrate that Gads plays a key role in linking the adapter LAT to SLP‐76 in response to weak activation of GPVI and CLEC‐2 whereas LAT is required for full activation over a wider range of agonist concentrations. These results reveal the presence of a Gads‐independent pathway of platelet activation downstream of LAT.     

99mTc—DTPA肾动态显像评估早期狼疮性肾炎患者肾小球功能

目的通过99mTc-DTPA肾动态显像评估早期狼疮性。肾炎患者肾小球功能损害情况。方法对55例肌酐正常的狼疮性肾炎患者进行99mTc-DTPA肾动态显像测定GFR,同时行血清肌酐、血尿素氮、血清补体C3、C4、ds—DNA测定和24h尿蛋白定量等常规检查。结果55例患者中,GFR在正常范围的仅有15例,占27．3％;GFR下降患者40例,占72．7％。40例肾小球损害的患者中,23例为轻度肾小球损害,17例为中度肾小球损害。肾小球损害组血清肌酐和尿素氮水平均高于正常组,差异具有统计学意义（P〈0．05）;同时肾小球损害组24h尿蛋白定量明显高于正常组（P=0．006）,补体C3、C4水平低于正常组（P〈0．05）。患者年龄、血清肌酐和24h尿蛋白定量均与GFR值负相关（P〈0．01）。结论99mTc-DTPA肾动态显像测定GFR,可判断SLE早期肾小球功能损害,为早期发现、早期治疗LN患者及对其进行随访提供依据。