血管性血友病因子裂解酶功能检测在血栓性血小板减少性紫癜诊断中的意义

目的 建立血栓性血小板减少性紫癜（TTP）的实验诊断方法,并用于TTP患者的诊断.方法 将微管透析前、后的正常人混合血浆、TTP患者血浆进行琼脂糖凝胶水平电泳与WesternBlot,分析TTP患者血浆中vWF多聚体的组成变化;以残余胶原结合力检测血管性血友病因子裂解酶（vWF-cp）的活性水平.结果 正常人混合血浆的vWF-cp活性为88.79%,4例TTP患者vWF-cp活性分别为8.90%,4.72%,28.73%,10.35%.正常混合血浆透析后只存在小分子量vWF;TTP患者血浆透析前、后的多聚体分布无变化或变化不明显.结论 残余胶原结合力测定结合裂解后的vWF多聚体电泳与Western Blot技术可准确分析vWF-cp活性与vWF多聚体结构组成的变化,为TTP诊断提供新的有用的实验诊断方法.     

von Willebrand 因子胶原结合试验的建立及其临床应用

目的　建立了一种新的检测vonWillebrand因子 (vWF)功能的方法。　方法　用ELISA法检测血浆中vWF与胶原的结合能力。　结果　该法敏感性为 0 0 0 1U/ml,批内和批间变异为3 34%和 6 70 %。 2 0名正常人血浆的胶原结合试验 (vWF :CBA)值为 (90 2 4± 2 2 87) %。 5 4例初步诊断为血管性血友病 (vWD)患者vWF :CBA值为 (31 94± 2 7 36 ) % ,10例 1型vWD为 (35 2 2± 2 0 0 2 ) % ,10例 2A型vWD为 (8 74± 6 38) % ,6例 3型vWD为 (0 70± 0 5 8) % ,以上各组值均低于正常值 (P <0 0 1) ;2A型vWD患者vWF :CBA值小于 1型vWD(P <0 0 1)。　结论　vWF胶原结合试验是一项简单易行、准确反映vWF功能的实验 ,为研究血管性血友病 (vWD)和血栓性疾病提供了一种新的方法 ,可向临床推广。     

ADAMTS-13活性水平检测在ITP与TTP鉴别中的意义

目的　探讨VWF裂解蛋白酶(ADAMTS -13,vWP cp)活性水平的检测在特发性血小板减少性紫癜(ITP)与血栓性血小板减少性紫癜(TTP)鉴别中的意义。方法　采用残余胶原结合实验(Residual CollagenBindingAssay)分别对35例ITP、6例TTP及32例健康人血浆ADAMTS 13活性水平进行检测。结果　ITP及TTP患者血浆ADAMTS 13活性水平[(47.6 2±2 4 . 2 2 ) %、(12 .5 4±11.6 9) % ]显著低于正常人[(78 .6 0±10 .0 3) % ](P <0 . 0 1) ,ITP患者血浆ADAMTS 13活性水平明显高于TTP患者(P<0. 0 1) ,ITP患者血浆ADAMTS 13活性水平阳性率[6 8 5 7% (2 4 /35 ) ]明显低于TTP患者[10 0 % (6 /6 ) ](P <0 . 0 1)。结论　ADAMTS 13活性水平检测在ITP与TTP鉴别诊断中具有一定的意义。     

ADAMTS13和TSP-1在糖尿病视网膜病变的意义

目的研究血管性血友病因子裂解蛋白酶(ADAMTS13)及凝血酶敏感蛋白-1(TSP-1)与糖尿病眼底视网膜病变(DR)的关系。方法选取114例糖尿病患者分为单纯糖尿病(DM)组38例和糖尿病视网膜病变(DR)组76例,同时选取30例健康体检者作为对照组。用残余胶原结合试验法检测ADAMTS13活性;同时用酶联免疫吸附试验法检测血浆中TSP-1及血管性血友病因子(vWF)抗原水平。结果 DM组患者血浆中ADAMTS13活性低于对照组,差异有统计学意义(P0.01),vWF抗原和TSP-1水平均高于对照组,差异有统计学意义(P0.01);DR组ADAMTS13活性明显低于DM组,差异有统计学意义(P0.05),vWF抗原和TSP-1水平均高于DM组,差异有统计学意义(P0.05)。结论 ADAMTS13活性降低及TSP-1水平升高可能导致糖尿病眼底视网膜病变的发生和发展。     

血管性血友病因子裂解蛋白酶活性的检测方法及评估

血管性血友病因子裂解酶（ADAMTS13，vWF—CP）裂解血浆中具有高黏附能力的超大分子量血管性血友病因子（UL—vWF），防止血小板因其引起聚集形成血栓。ADAMTS13活性异常是临床上血栓性血小板减少性紫癜（TTP），特别是遗传性TTP和特发性TTP发病的基础。其活性的检测在TTP临床诊断和治疗上具有日益重要的意义。目前报道的一些用于检测ADAMTS13活性的方法有十余种，本文对此进行综述。     

ADAMTS13的结构、功能及其与TTP诊疗的关系

血管性血友病因子裂解蛋白酶（ADAMTS 13）是一种能够特异性裂解血管性血友病因子（vWF）的血浆金属蛋白酶,严重的ADAMTS13活性缺失可导致特发性血栓性血小板减少性紫癜（TTP）的发生.本文简要综述了ADAMTS13的结构和功能及其与TTP的发病机制、诊断和治疗之间的关系.     

血管性假性血友病因子及其裂解酶在原发性肝癌患者中的变化及临床意义

血管性假性血友病因子裂解酶(von Willebrand factor cleaving protease,vWF-cp)是正常人血浆中存在的一种相对大分子质量的金属蛋白酶,它可专一性作用于血管性假性血友病因子(von Willebrand factor,vWF)亚单位,将vWF多聚体裂解为不具有黏附活性的片段,从而阻止血小板与血管内皮下胶原的连接,防止微血管内血小板血栓的过分形成.     

汉族正常人群与血栓性疾病患者vWF裂解酶C1423T基因多态性调查

目的　研究我国汉族人群中血管性血友病因子裂解酶 (vWF cp)基因C14 2 3T多态性的分布频率及其与血栓性疾病的相关性。方法　应用PCR技术结合酶切分析、聚丙烯酰胺凝胶电泳研究来自我国三个不同地区 4 0 0名汉族人vWF cp中C14 2 3T多态性的分布特点。同时 ,根据病例对照研究 ,比较了健康对照和血栓性疾病患者中该多态性的分布频率。结果　我国汉族人群中C14 2 3T的基因频率分别为 98.5 %和 1.5 % ,总理论杂合率为 2 .96 % ,不同地区人群杂合率不同 ,但未发现 14 2 3T T纯合子。该多态性的基因型与基因频率在健康对照和血栓性疾病患者中相似。结论　vWF cp基因C14 2 3T多态性在我国汉族人群中频率较低 ,尽管它影响vWF cp的酶活性。     

妊娠高血压综合征患者血浆血管性血友病因子及其裂解酶的测定及临床意义

目的检测正常孕妇及妊娠高血压综合征(PIH)患者血浆血管性血友病因子(vWF:Ag)水平及vWF裂解酶(vWF-CP)活性,探讨vWF及vWF-CP在PIH中的临床意义。方法采用残余胶原结合实验法及ELISA法分别对35例正常孕妇及52例PIH患者血浆vWF-CP活性及vWF:Ag水平进行检测。结果正常妊娠妇女血浆vWF:Ag(103．4±68．4)％显著高于正常非孕妇(78．4±51．6)％(P＜0．01),而血浆vWF-CP活性(76．3±1 8．4)％显著低于正常非孕妇(84．7±2．1)％(P＜0．05)。轻度妊高征妇女血浆vWF:Ag(98．8±48．5)％及vWF-CP活性(70．4±2 1．8)％与正常妊娠妇女无显著性差别(P＞0．05),而中、重度妊高征妇女血浆vWF:Ag(148．4±75．6)％显著高于正常妊娠妇女(P＜0．01),血浆vWF-CP活性(60．8±19．8)％显著低于正常妊娠妇女(P＜0．05)。结论妊高征患者血浆vWF升高,vWF-CP活性低下,参与妊高征的发生、发展,内皮损伤可能是致妊高征患者血浆vWF升高,vWF-CP活性低下的原因。     

慢性肾脏病患者血浆血管性血友病因子裂解酶活性测定

目的检测慢性肾脏病（CKD）患者血浆血管性血友病因子裂解酶（vWF-CP）活性及血浆血管性血友病因子（vWF）水平,探讨其在慢性肾脏病的意义。方法将139例CKD患者根据不同种类肾病及不同肾功能状态分组,其中狼疮肾炎（LN）31例,原发性肾病综合征（NS）25例,糖尿病肾病（DN）45例及慢性肾炎（CGN）38例;CKDⅠ期38例,CKDⅡ期30例,CKDⅢ期39例,CKDⅣ期19例,CKDⅤ期13例。采用残余胶原结合实验法及ELISA法对患者血浆vWF-CP活性及vWF水平进行检测,观察不同组患者血浆vWF-CP活性及vWF水平,并与正常对照组（40名）比较。结果LN、NS、DN及CGN患者血浆vWF-CP活性显著低于对照组,且以LN、NS组下降最明显,vWF水平均显著高于正常对照组,血浆vWF含量均与血浆vWF-CP活性呈负相关。CKDⅡ期、Ⅲ期血浆vWF-CP活性显著低于Ⅰ、Ⅳ、Ⅴ期．而Ⅰ、Ⅳ、Ⅴ期之间无显著区别,vWF含量各期之间无显著差别。结论CKD患者血浆vWF-CP活性明显降低,其低下程度与肾病的病种有关。内皮损伤、自身抗体可能是致CKD患者vWF-CP活性低下的原因之一。它参与了CKD的发生发展,可能是CKD血栓形成的机理之一。     

Deficient activity of von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a dramatic intravascular platelet-clumping disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, neurologic abnormalities, renal insufficiency and fever. TTP is a rare disease but is almost always fatal if untreated. More than 80% of patients survive with plasma therapy. In healthy individuals, the proteolytic cleavage of ultralarge von Willebrand factor (vWF) multimers prevents spontaneous clumping of platelets in the microcirculation. Patients with TTP have either severe congenital deficiency of von Willebrand factor-cleaving protease (vWF-cp), or have autoantibodies that inhibit the protease. Determination of vWF-cp levels in patient plasma helps to distinguish between TTP and other thrombotic microangiopathies with similar clinical signs and symptoms. vWF-cp is a member of the ADAMTS family of metalloproteases and has been designated ADAMTS13.     

Deficient activity of von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a dramatic intravascular platelet-clumping disorder characterized by microangiopathic hemolytic anemia, thrombocytopenia, neurologic abnormalities, renal insufficiency and fever. TTP is a rare disease but is almost always fatal if untreated. More than 80% of patients survive with plasma therapy. In healthy individuals, the proteolytic cleavage of ultralarge von Willebrand factor (vWF) multimers prevents spontaneous clumping of platelets in the microcirculation. Patients with TIP have either severe congenital deficiency of von Willebrand factor-cleaving protease (vWF-cp), or have autoantibodies that inhibit the protease. Determination of vWF-cp levels in patient plasma helps to distinguish between TTP and other thrombotic microangiopathies with similar clinical signs and symptoms. vWF-cp is a member of the ADAMTS family of metalloproteases and has been designated ADAMTS13.     

血小板内血管性血友病因子裂解酶表达的鉴定

目的　血管性血友病因子裂解酶(vWF cp)是一种新发现的血浆金属蛋白酶,他在人外周血细胞中的 表达尚待确定。本研究确定了该酶在血小板中的表达。方法　应用逆转录 聚合酶链反应(RT PCR)、流式细胞 术(FCM)和免疫共沉淀鉴定了血小板内vWF cpmRNA和蛋白的表达情况。同时应用FCM检测了白细胞、红细 胞内vWF cp蛋白的表达。结果　RT PCR显示,血小板中存在该蛋白酶mRNA的表达。同时,免疫沉淀及蛋白 印迹结果表明,血小板内存在相对分子质量约200000的vWF cp蛋白。FCM结果显示,经穿膜处理的血小板可 检测到vWF cp蛋白的明显表达,而不经穿膜处理以及经过穿膜处理的白细胞和红细胞中不存在该酶的表达。 结论　本研究不仅证实了外周血血小板中存在vWF cp,由于血小板亦为血管性血友病因子的一个主要合成/贮 存场所,这将有助于深入阐明血小板在血栓形成与止血中的作用。     

Comparison of von Willebrand factor antigen, von Willebrand factor-cleaving protease and protein S in blood components used for treatment of thrombotic thrombocytopenic purpura

Replacement of normal levels of von Willebrand factor-cleaving protease (VWF:CP, ADAMTS13) activity from infused plasma is important in plasma exchange (PEX) for the treatment of thrombotic thrombocytopenic purpura (TTP) patients. We have studied the VWF:CP activity, VWF multimer distribution, VWF:Ag, protein S (PS) activity and free PS antigen levels in fresh frozen plasma (FFP), cryosupernatant (CSP) and virally inactivated components treated with methylene blue/light (MB) or solvent detergent (SD) processes. VWF:CP activity was normal in all components tested and was retained following overnight storage at room temperature. CSP and SD plasma contained reduced levels of the highest molecular weight VWF multimers. Protein S activity was reduced below the normal range in SD plasma, but within the normal range for the other components tested. Virally inactivated SD- and MB-treated plasma may be an effective alternative to FFP and CSP in PEX for TTP. Reduced PS activity in SD plasma may predispose to venous thromboembolism, especially if infused in large volumes.     

血管性血友病因子裂解酶活性域的表达及其单克隆抗体的制备

血管性血友病因子裂解酶(vWF-cp)是一种新发现的金属蛋白酶,与血栓性微血管病的发生密切相关。本研究应用IPTG从pET28a( )-vWF-cp/MD质粒中诱导表达了vWF-cp的金属蛋白酶域,重组蛋白经Ni-NTA柱纯化,复性后免疫BALB/c小鼠,利用杂交瘤技术获得了稳定分泌抗vWF-cp金属蛋白酶域抗体的细胞株,进一步对其单克隆后,收集细胞接种于小鼠腹腔产生腹水,鉴定所获得的单克隆抗体,同时,利用单克隆抗体确定vWF-cp在组织细胞中的位置及其在各种正常组织中的表达情况。结果表明,重组蛋白主要以包涵体形式存在,占菌体总蛋白的28%。利用杂交瘤技术制备出3株抗vWF-cp单克隆抗体,对其中2株单克隆抗体作了进一步的鉴定。免疫双扩散和竞争ELISA结果显示,它们均属IgG1亚类,腹水中的含量约为4mg/ml,效价达1×10-5,而且识别vWF-cp金属蛋白酶域不同的表位。Westernblot和免疫沉淀结果表明,2株单克隆抗体能够识别重组蛋白和正常血小板中200kD的蛋白。在组织细胞中,2株单克隆抗体识别的蛋白位于细胞胞质内,而且在多种正常组织如肝、前列腺和卵巢等组织中表达,但脑组织中无该蛋白的表达。结论:本研究为了解vWF-cp在组织中的分布以及进一步研究该蛋白酶的结构和功能奠定了基础,并且可用所制备的单克隆抗体建立vWF-cp抗原的检测方法。     

The "cutting" edge: von Willebrand factor-cleaving protease activity in thrombotic microangiopathies.

The thrombotic microangiopathies (TMAs), including thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS), are classically defined by constellations of clinical findings. In the late 1990's, several groups reported that a single test might be able to diagnose TTP and distinguish it from HUS. This test was an assay to detect von Willebrand factor-cleaving protease activity. Although there has been debate in the literature as to the utility of this assay, review of the available data suggests that this test can separate the idiopathic TMAs into three distinct categories: (1) patients who are protease positive with severe renal failure (similar to classic HUS), (2) patients who are protease positive without severe renal failure and (3) patients who are protease negative. While the latter two categories may appear similar to TTP at presentation, the prognosis differs in each of these groups of patients. Through use of the protease assay clinicians will be better able to diagnose, treat, predict outcome and design clinical trials.     

Two generations with familial thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a rare multi-system disease characterised by the pentad of microangiopathic haemolytic anaemia, thrombocytopenia, renal dysfunction, fever and neurologic changes. A hereditary form of recurrent familial TTP has been described, which usually presents in adolescence or early adulthood and can lead to recurrent or chronic relapsing TTP. Genetic analyses of patients with familial TTP have linked the disease to chromosome 9q34, and an increased incidence is seen in people with HLA-B40 group antigens. We describe here an 11-year-old Egyptian girl with no significant past medical history who presented with new onset of bruising, petechial rash, fatigue and fevers and was diagnosed with familial TTP. Further testing revealed that both the patient and her father had the HLA-B40 group antigen and also had ADAMTS-13 von Willebrand factor-cleaving protease deficiency as well as factor-H deficiency.     

A randomized, controlled Phase III trial of therapeutic plasma exchange with fresh-frozen plasma (FFP) prepared with amotosalen and ultraviolet A light compared to untreated FFP in thrombotic thrombocytopenic purpura

BACKGROUND: Photochemical treatment of fresh-frozen plasma (FFP) with amotosalen and ultraviolet (UV) A light (PCT FFP) results in inactivation of a broad spectrum of pathogens while retaining coagulation factor activity, antithrombotic proteins, and von Willebrand factor-cleaving protease (VWF-CP) activity. STUDY DESIGN AND METHODS: A randomized, controlled, double-blind Phase III trial was conducted with PCT FFP or control FFP for therapeutic plasma exchange (TPE) in patients with thrombotic thrombocytopenic purpura (TTP). Owing to the rarity of this diagnosis, the trial was not powered to demonstrate small differences between treatment groups. Patients were treated with study FFP for a maximum of 35 days until remission was achieved (for a maximum of 30 daily study TPEs with no remission) plus an additional 5 days after remission. RESULTS: Among the 35 patients treated, the primary endpoint, remission within 30 days, was achieved by 14 of 17 (82%) PCT patients and 16 of 18 (89%) control patients (p = 0.658) The 90 percent confidence interval for treatment difference in remission rate for test - control was (-0.291 to 0.163). Time to remission, relapse rates, time to relapse, total volume and number of FFP units exchanged, and number of study TPEs were not significantly different between groups. Improvement in VWF-CP and inhibitors was similar for both groups. The overall safety profile of PCT FFP was similar to control FFP. No antibodies to amotosalen neoantigens were detected. CONCLUSION: The comparable results between treatment groups observed from this small trial suggest that TPE with PCT FFP was safe and effective for treatment of TTP.