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.     

von Willebrand因子裂解酶活性水平的检测及其临床应用

血管性血友病因子裂解酶 (vWF cp)是新近发现的一个金属蛋白酶 ,其活性在多种生理病理状态下发生改变。为了研究vonWillebrand因子裂解酶活性检测及其临床应用 ,用ELISA法检测了vWF cp酶解前及裂解后血清 (浆 )中血管性血友病因子 (vWF)的胶原结合能力 ,两者之比作为该待测血清 (浆 )的相对vWF cp活性水平。同时观察血栓性血小板减少性紫癜 (TTP)患者与肿瘤患者体内vWF cp活性的改变。结果表明 ,该方法准确测出了87名健康成人和 79例患者血样的残余胶原结合力 (R CBA) ,批内变异和批间变异分别为 3.6 0 % (n =9)和 8.35 %(n =5 )。 5 3名健康成人血清vWF cp活性水平为 (79.4 7± 10 .78) % ,30名健康成人血浆vWF -cp活性水平为(78.79± 9.17) %。 6例TTP患者中 5例血浆vWF cp活性水平显著降低 ,2 5例良性肿瘤患者血清vWF cp水平轻度降低 ,4 9例恶性肿瘤患者则明显下降 (P值分别小于 0 .0 0 1,0 .0 3和 0 .0 0 1)。结论 :以R CBA检测vWF cp活性水平简单易行 ,可应用于临床检验。恶性肿瘤患者 ,尤其是TTP患者血浆 (清 )vWF cp活性水平显著降低。     

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.     

Cloning, expression and functional characterization of the full-length murine ADAMTS13.

Functional deficiency or absence of the human von Willebrand factor (VWF)-cleaving protease (VWF-cp), recently termed ADAMTS13, has been shown to cause acquired and congenital thrombotic thrombocytopenic purpura (TTP), respectively. As a first step towards developing a small animal model of TTP, we have cloned the complete (non-truncated) murine Adamts13 gene from BALB/c mice liver poly A+ mRNA. Murine ADAMTS13 is a 1426-amino-acid protein with a high homology and similar structural organization to the human ortholog. Transient expression of the murine Adamts13 cDNA in HEK 293 cells yielded a protein with a molecular weight of approximately 180 kDa which degraded recombinant murine VWF (rVWF) in a dose-dependent manner. The cleavage products of murine rVWF had the expected size of 140 and 170 kDa. Murine ADAMTS13 was inhibited by EDTA and the plasma from a TTP patient.     

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.     

Recombinant ADAMTS13 normalizes von Willebrand factor‐cleaving activity in plasma of acquired TTP patients by overriding inhibitory antibodies

Summary. Background: Severe deficiency of the von Willebrand factor (VWF)‐cleaving protease ADAMTS13 as observed in acquired thrombotic thrombocytopenic purpura (TTP) is caused by inhibitory and non‐inhibitory autoantibodies directed against the protease. Current treatment with plasma exchange is considered to remove circulating antibodies and to concurrently replenish the deficient enzyme. Objectives: To explore the use of recombinant ADAMTS13 (rADAMTS13) as a potential therapeutic agent in acquired TTP, we investigated its efficacy in normalizing VWF‐cleaving activity in the presence of ADAMTS13 inhibitors. Methods: Thirty‐six plasma samples from TTP patients were adjusted to predefined inhibitor titers, and recovery of ADAMTS13 activity was analyzed following supplementation with rADAMTS13. Results: We showed a linear relation between the inhibitor titer measured and effective rADAMTS13 concentration necessary for reconstitution of VWF‐cleaving activity in the presence of neutralizing autoantibodies. Conclusions: Our results support the further investigation of the potential therapeutic applicability of rADAMTS13 as an adjunctive therapy in acquired TTP.     

Rituximab and intermediate‐purity plasma‐derived factor VIII concentrate (Koate®) as adjuncts to therapeutic plasma exchange for thrombotic thrombocytopenic purpura in patients with an ADAMTS13 inhibitor

Thrombotic thrombocytopenic purpura (TTP) results from a congenital or acquired deficiency of the von Willebrand factor (vWF)‐cleaving protease ADAMTS13. The disease can be fatal and hence treatment should be initiated promptly. Therapeutic plasma exchange (TPE) remains the standard treatment along with adjunct therapies including steroids and immunosuppressive drugs. Addition of rituximab to TPE has been shown to be beneficial in refractory/relapsing TTP; however, TPE results in removal of rituximab from the circulation requiring more frequent dosing of rituximab to achieve a favorable outcome. The intermediate‐purity plasma‐derived Factor VIII concentrate (FVIII) Koate® contains the highest amount of ADAMTS13 activity yet reported and has been used successfully in treating congenital TTP. Here we report our experience with addition of this FVIII concentrate to rituximab, corticosteroids and TPE in three TTP patients with an ADAMTS13 inhibitor to permit withholding TPE for 48 h after rituximab infusion. J. Clin. Apheresis 30:50–54, 2015. © 2014 Wiley Periodicals, Inc.     

Relapsed thrombotic thrombocytopenic purpura presenting as an acute cerebrovascular accident

Thrombotic thrombocytopenic purpura (TTP) is an uncommon but severe disorder that classically presents with microangiopathic hemolytic anemia (MAHA), thrombocytopenia, and fluctuating neurological changes. Previously, it was impossible to make a diagnosis of TTP in the absence of thrombocytopenia or microangiopathic hemolysis (MAHA). We describe two cases of relapsing TTP that presented with acute cerebrovascular accident (CVA) without concurrent thrombocytopenia or MAHA after initial classical presentation of TTP. In both cases, the diagnosis of TTP as the cause of the CVA was attributed to severe deficiency of the von Willebrand factor cleaving protease, ADAMTS13 in plasma (11 and 12%, normal 79-127%). Each patient had a dramatic clinical improvement in response to therapeutic plasma exchange. The experience in these two cases suggests that TTP should be considered as a potential cause among patients presenting with a CVA, particularly if the patients have a history of TTP.     

The role of ADAMTS13 in the pathogenesis of thrombotic thrombocytopenic purpura-hemolytic uremic syndrome

The identification, characterization, and clinical observation of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin-1-like domains) have provided important insights into the pathogenesis of thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is a plasma enzyme essential for postsecretion proteolytic processing of von Willebrand factor (VWF). Absence of ADAMTS13 is associated with the occurrence of abnormally large multimers of VWF and is also associated with the occurrence of TTP. Initial assumptions that absent ADAMTS13 was itself the etiology of TTP have been tempered by subsequent observations that ADAMTS13 activity can be severely deficient without clinical abnormalities and that patients can have characteristic clinical features of TTP without severe ADAMTS13 deficiency. A current interpretation of these observations is that ADAMTS13 deficiency is a major risk factor for the development of TTP, but it is neither always necessary nor sufficient to cause TTP. This interpretation is consistent with other vascular and thrombotic disorders in which multiple risk factors and associated conditions contribute to the etiology of acute events.     

Comparison and stability of ADAMTS13 activity in therapeutic plasma products

BACKGROUND: The von Willebrand factor (VWF)-cleaving protease, ADAMTS13, is often deficient in cases of thrombotic thrombocytopenic purpura (TTP). The primary treatment of TTP is therapeutic plasma exchange (TPE) utilizing a variety of plasma products that help restore ADAMTS13 activity. However, multiple replacement products are available to choose from. Thawed plasma products have a variable refrigerated shelf life depending on the product type; stability of ADAMTS13 in thawed products stored at 1 to 6 degrees C has not been determined. STUDY DESIGN AND METHODS: ADAMTS13 activity was measured in three types of plasma products and cryoprecipitate. Fresh-frozen plasma (FFP) aliquots and cryoprecipitate-poor plasma (CPP) products were produced from 10 whole-blood (WB) donations. Twenty-four-hour plasma products were manufactured from 10 additional WB donations. ADAMTS13 activity in these products at time of thaw and after 5 days of storage at 1 to 6 degrees C was measured with a modified version of the FRETS-VWF73 fluorogenic assay. ADAMTS13 activity at time of thaw was measured in 10 units of cryoprecipitate and five related CPP products. RESULTS: ADAMTS13 is present in similar amounts in FFP, CPP, and 24-hour plasma products. Storage at 1 to 6 degrees C for up to 5 days did not significantly diminish ADAMTS13 activity. The concentration of ADAMTS13 in cryoprecipitate was significantly higher than that observed in plasma products. CONCLUSION: FFP, CPP, and 24-hour plasma products should be equally effective for ADAMTS13 restoration through TPE and should remain so for the duration of the shelf life of the thawed products.     

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。     

The use of 50% albumin/plasma replacement fluid in therapeutic plasma exchange for thrombotic thrombocytopenic purpura

Background: Acquired thrombotic thrombocytopenic purpura (TTP) is caused by a deficiency of von Willebrand factor‐cleaving protease (ADAMTS13) and is often associated with the presence of an antibody inhibiting the activity of the protease. Typically, 1–1.5 plasma volume exchanges are performed daily until symptoms have resolved and the platelet count exceeds 150,000/µl. Plasma is the usual replacement fluid as it provides a source of functional ADAMTS13, thus exposing patients to large volumes of plasma. Historically, Puget Sound Blood Center (PSBC) has performed therapeutic plasma exchange (TPEs) for TTP using 5% albumin for the first half of the procedure followed by plasma for the remainder. We sought to assess the efficacy of this approach. Study Design and Methods: All TPEs performed for the diagnosis of TTP by the PSBC apheresis service from January 1, 2004 through December 31, 2011 were reviewed. Response time, remission rates, relapses, and adverse events were evaluated for those patients with documented ADAMTS13 levels ≤10%. Comparisons were made with published data on TTP patients treated using 100% plasma replacement. Results: Twenty‐one patients required a median of 11 TPEs. Median time to response was 14 days. Ninety percent of patients responded to TPE. Among patients achieving remission, 53% relapsed. Out of 283 total procedures, there were 74 procedures with a documented adverse event (26%), mostly mild allergic reactions. Conclusions: TPE with an albumin/plasma replacement is safe and well‐tolerated. Remission and relapse rates were comparable to those reported using 100% plasma replacement. J. Clin. Apheresis, 28:416–421, 2013. © 2013 Wiley Periodicals, Inc.     

Inactivation of ADAMTS13 by plasmin as a potential cause of thrombotic thrombocytopenic purpura

Summary. Background: ADAMTS13 deficiency causes accumulation of unusually large von Willebrand factor molecules, which cross‐link platelets in the circulation or on the endothelial surface. This process of intravascular agglutination leads to the microangiopathy thrombotic thrombocytopenic purpura (TTP). Most TTP patients have acquired anti‐ADAMTS13 autoantibodies that inhibit enzyme function and/or clear it from the circulation. However, the reason for ADAMTS13 deficiency is not always easily identified in a subset of patients. Objectives: To determine the origin of ADAMTS13 deficiency in a case of acquired TTP. Methods: Western blotting of ADAMTS13 in plasmas from acute and remission phases was used. Results: The ADAMTS13 deficiency was not caused by mutations or (detectable) autoantibodies; however, an abnormal ADAMTS13 truncated fragment (100 kDa) was found in acute‐phase but not remission‐phase plasma. This fragment resulted from enzymatic proteolysis, as recombinant ADAMTS13 was also cleaved when in the presence of acute‐phase but not remission‐phase plasma. Inhibitor screening showed that ADAMTS13 was cleaved by a serine protease that could be dose‐dependently inhibited by addition of exogenous α₂‐antiplasmin. Examination of the endogenous α₂‐antiplasmin antigen and activity confirmed deficiency of α₂‐antiplasmin function in acute‐phase but not remission‐phase plasma. To investigate the possibility of ADAMTS13 cleavage by plasmin in plasma, urokinase‐type plasminogen activator was added to an (unrelated) congenital α₂‐antiplasmin‐deficient plasma sample to activate plasminogen. This experiment confirmed cleavage of endogenous ADAMTS13 similar to that observed in our TTP patient. Conclusion: We report the first acquired TTP patient with cleaved ADAMTS13 and show that plasmin is involved.     

Bench-to-bedside review: thrombocytopenia-associated multiple organ failure--a newly appreciated syndrome in the critically ill

New onset thrombocytopenia and multiple organ failure (TAMOF) presages poor outcome in critical illness. Patients who resolve thrombocytopenia by day 14 are more likely to survive than those who do not. Patients with TAMOF have a spectrum of microangiopathic disorders that includes thrombotic thrombocytopenic purpura (TTP), disseminated intravascular coagulation (DIC) and secondary thrombotic microanigiopathy (TMA). Activated protein C is effective in resolving fibrin-mediated thrombosis (DIC); however, daily plasma exchange is the therapy of choice for removing ADAMTS 13 inhibitors and replenishing ADAMTS 13 activity which in turn resolves platelet: von Willebrand Factor mediated thrombosis (TTP/secondary TMA).     

Bench-to-bedside review: Thrombocytopenia-associated multiple organ failure – a newly appreciated syndrome in the critically ill

New onset thrombocytopenia and multiple organ failure (TAMOF) presages poor outcome in critical illness. Patients who resolve thrombocytopenia by day 14 are more likely to survive than those who do not. Patients with TAMOF have a spectrum of microangiopathic disorders that includes thrombotic thrombocytopenic purpura (TTP), disseminated intravascular coagulation (DIC) and secondary thrombotic microanigiopathy (TMA). Activated protein C is effective in resolving fibrin-mediated thrombosis (DIC); however, daily plasma exchange is the therapy of choice for removing ADAMTS 13 inhibitors and replenishing ADAMTS 13 activity which in turn resolves platelet: von Willebrand Factor mediated thrombosis (TTP/secondary TMA).     

Bench-to-bedside review: Thrombocytopenia-associated multiple organ failure – a newly appreciated syndrome in the critically ill

New onset thrombocytopenia and multiple organ failure (TAMOF) presages poor outcome in critical illness. Patients who resolve thrombocytopenia by day 14 are more likely to survive than those who do not. Patients with TAMOF have a spectrum of microangiopathic disorders that includes thrombotic thrombocytopenic purpura (TTP), disseminated intravascular coagulation (DIC) and secondary thrombotic microanigiopathy (TMA). Activated protein C is effective in resolving fibrin-mediated thrombosis (DIC); however, daily plasma exchange is the therapy of choice for removing ADAMTS 13 inhibitors and replenishing ADAMTS 13 activity which in turn resolves platelet: von Willebrand Factor mediated thrombosis (TTP/secondary TMA).     

Update on thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a thrombotic microangiopathy, which is classically associated with signs and symptoms of fever, thrombocytopenia, neurologic deficits, hemolytic anemia, and renal failure. It is caused by a deficiency of A Disintegrin-like And Metalloprotease with a ThromboSpondin type1 motif 13 (ADAMTS13), which may be an inherited disorder, but more commonly is an acquired disease due to autoantibodies directed against ADAMTS13. Low ADAMTS13 levels result in increased ultra-large von Willebrand factor multimers, which induce platelet adhesion and thrombosis. Plasma exchange therapy is the standard of care, and has greatly reduced morbidity and mortality. A recent TTP case is reviewed, and treatments for recurrent or refractory TTP are summarized. A scoring system using clinical and laboratory parameters to evaluate which suspected TTP patients will benefit from plasma exchange therapy is also discussed.     

Complement activation in thrombotic thrombocytopenic purpura

Summary. Background: Ultra‐large von Willebrand factor and deficiency of its cleaving protease are important factors in the events leading to thrombotic microangiopathy; however, the mechanisms involved are only partly understood. Whereas pathological activation of the alternative complement pathway is linked to atypical hemolytic uremic syndrome, the role of complement activation in thrombotic thrombocytopenic purpura (TTP) is unknown. The aim of this study was to investigate whether signs of complement activation are characteristic of TTP. Patients and methods: Twenty‐three patients with TTP (18 women, median age 38 years) and 17 healthy controls (13 women, median age 38 years) were included. Complement parameters (C3, Factors H, I, B and total alternative pathway activity) together with complement activation fragments (C3a) or complexes (C1rs‐INH, C3bBbP, sC5b9) were measured by ELISA or RID. ADAMTS13 activity and anti‐ADAMTS13 inhibitory antibodies were measured by the VWF‐FRET73 assay. Results: Increased levels of C3a, and SC5b9 were observed in TTP during acute episodes, as compared with healthy controls. Decreased complement C3 levels indicative of complement consumption occurred in 15% of acute TTP patients. Significant decrease of complement activation products C3a and SC5b9 was observed during plasma exchange (PEX). The sustained presence of anti‐ADAMTS13 inhibitory antibodies in complete remission was associated with increased complement activation. Conclusion: These data document in an observational study the presence of complement activation in TTP. Further investigation is needed to determine its potential pathogenetic significance.     

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.     

Emerging roles of adjunct therapies in acquired thrombotic thrombocytopenia purpura

• Acquired thrombotic thrombocytopenia purpura (aTTP) is caused by autoantibody‐mediated severe deficiency of the von Willebrand factor (vWF) cleaving protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13), with subsequent accumulation of ultra‐large vWF‐multimers that spontaneously form platelet‐VWF complexes and microthrombi within the microcirculation.
• Therapeutic plasma exchange (TPE), by removing autoantibodies and excess ultra‐large vWF multimers and replenishing ADAMTS13 activity, remains the urgent primary initial treatment. Although heterogeneity in treatment exists, most centers add upfront immunosuppression with steroids, and many also add upfront rituximab. Refractoriness, exacerbation and relapse are commonly treated with adjunct rituximab.
• Despite adjunct steroids and rituximab, TTP refractoriness, exacerbation, relapse, morbidity, and mortality remain problematic. Newer adjunct therapies include suppression of ADAMTS13 autoantibody production via plasma cell depletion, inhibition of vWF‐platelet interaction, and replenishment of ADAMTS13 function with recombinant ADAMTS13 protein.