Novel monoclonal antibody-based enzyme immunoassay for determining plasma levels of ADAMTS13 activity

BACKGROUND: ADAMTS13 specifically cleaves unusually large von Willebrand factor (VWF) multimers, which induce platelet thrombi formation under high shear stress. ADAMTS13 activity is deficient in patients with thrombotic thrombocytopenic purpura (TTP). The determination of plasma levels of ADAMTS13 activity is a prerequisite for a differential diagnosis of thrombotic microangiopathies. Here, a unique and highly sensitive enzyme immunoassay (EIA) of ADAMTS13 activity is described. STUDY DESIGN AND METHODS: ADAMTS13 hydrolyzes the peptide bond between Y1605 and M1606 of VWF. In this assay, a recombinant fusion protein (GST-VWF73-His) is used as a substrate. A panel of mouse monoclonal antibodies (MoAbs) that specifically recognizes Y1605, which is the C-terminal edge residue of the VWF-A2 domain and is generated by the enzymatic cleavage, has been produced. These antibodies were prepared with a synthetic decapeptide, termed N-10 (1596-DREQAPNLVY-1605), as the immunogen. Twenty-six clones specific to N10 were obtained, and one anti-N10 MoAb was used in this study. RESULTS: With horseradish peroxidase-conjugated anti-N10 MoAb, a standard enzyme assay was established. This assay was highly sensitive, and the detection limit was 0.5 percent of the normal. Further, an inhibitor of ADAMTS13 was measured to a level of 0.1 Bethesda units per mL. ADAMTS13 activity was measured in 20 patients with Upshaw-Schulman syndrome, a congenital TTP, and 61 acquired TTP patients. The activity measured by this assay and by the classic VWF multimer assay showed high correlation. CONCLUSION: A convenient and highly sensitive EIA for ADAMTS13 activity has been established. This assay can be introduced for routine laboratory work in transfusion medicine.     

A shear-based assay for assessing plasma ADAMTS13 activity and inhibitors in patients with thrombotic thrombocytopenic purpura

BACKGROUND: Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with hereditary and acquired thrombotic thrombocytopenic purpura (TTP). To date, plasma ADAMTS13 activity is determined by cleavage of either predenatured von Willebrand factor (VWF) or small peptides derived from the VWF‐A2 domain. The physiologic relevance of the assay results is uncertain. STUDY DESIGN AND METHODS: We sought to develop a novel shear‐based assay to assess plasma ADAMTS13 activity and inhibitors. We also compared this assay with a fluorogenic peptide assay. RESULTS: We found that an incubation of purified plasma VWF with 0.5 to 1.0 µL of citrated plasma under constant vortexing at 2500 rpm for 60 minutes in the presence of 5 mmol/L CaCl₂ and 1.7 µmol/L ZnCl₂ and low concentration of NaCl resulted in the maximal cleavage of VWF. The cleavage product could be separated by a 2.5% agarose gel and detected by Western blotting. The assay revealed that plasma and recombinant ADAMTS13 are highly sensitive to inhibition by zinc and chloride ions. Under the optimal conditions, the shear‐based assay appeared to be more sensitive than the guanidine‐denaturization assay for determining plasma ADAMTS13 activity. CONCLUSIONS: Our fluid shear‐based assay may be useful for investigating basic biologic function and regulation of ADAMTS13 metalloprotease. It may also be applicable for assessing plasma ADAMTS13 activity and inhibitors in TTP patients.     

ADAMTS13 and microvascular thrombosis

Interaction between platelet and von Willebrand factor, a circulating adhesive glycoprotein, is essential for hemostasis under the high shear environments of arterioles and capillaries. If unregulated, this interaction may lead to unwarranted platelet thrombosis. ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, number 13), a plasma zinc metalloprotease synthesized primarily in the stellate cells of the liver, cleaves shear stress-activated von Willebrand factor, thereby preventing the occurrence of von Willebrand factor-platelet interaction in the circulation. A profound deficiency of ADAMTS13, due to genetic mutations or autoimmune inhibition, results in intravascular von Willebrand factor platelet aggregation and widespread microvascular thrombosis characteristic of thrombotic thrombocytopenic purpura. Cloning of ADAMTS13 and structure-function analyses of the enzyme are leading to exciting advances in the diagnosis and therapy of this hitherto mysterious disease.     

房颤患者vWF与ADAMTS-13水平的变化及意义

目的 研究房颤患者vWF、ADAMTS-13的变化及意义.方法 分别测定27例持续性房颤、20例阵发性房颤患者和25例正常对照组血浆血管性血友病因子（vWF）、血管性血友病因子裂解酶（ADAMTS-13）水平、D-二聚体（D-D）水平,测vWF/ADAMTS-13比值.结果 与对照组比较,阵发性房颤组、持续性房颤组的vWF、D-D均显著增高、ADAMTS-13明显下降,vWF/ADAMTS-13比值明显增高;vWF/ADAMTS-13比值在阵发性房颤组和持续性房颤组间差异有统计学意义,且持续性房颤组比值与D-D水平呈正相关.结论 房颤患者存在有明显vWF、ADAMTS-13水平变化,且与其可能出现的血栓栓塞并发症有密切关系.     

ADAMTS13 and microvascular thrombosis

Interaction between platelet and von Willebrand factor, a circulating adhesive glycoprotein, is essential for hemostasis under the high shear environments of arterioles and capillaries. If unregulated, this interaction may lead to unwarranted platelet thrombosis. ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, number 13), a plasma zinc metalloprotease synthesized primarily in the stellate cells of the liver, cleaves shear stress-activated von Willebrand factor, thereby preventing the occurrence of von Willebrand factor–platelet interaction in the circulation. A profound deficiency of ADAMTS13, due to genetic mutations or autoimmune inhibition, results in intravascular von Willebrand factor platelet aggregation and widespread microvascular thrombosis characteristic of thrombotic thrombocytopenic purpura. Cloning of ADAMTS13 and structure–function analyses of the enzyme are leading to exciting advances in the diagnosis and therapy of this hitherto mysterious disease.     

Comparison of ADAMTS13 and Von Willebrand factor levels and activities,and plasminogen levels,in plasma products currently available for the treatment of thrombotic thrombocytopenic purpura in South Africa

Objective

Thrombotic thrombocytopenic purpura (TTP) results from a deficiency in the Von Willebrand factor (VWF) cleaving protease, ADAMTS13. Treatment involves plasma exchange (PEX) therapy with either fresh frozen plasma (FFP), cryosupernatant (CSP) or solvent/detergent-treated plasma (SDP), available in South Africa as Bioplasma FDP. The aim of the study was to generate in vitro data on these products, and to explore possible differences between the products that may offer treatment advantages.

血栓性血小板减少性紫癜患者vWF裂解蛋白酶抗原和活性的检测及意义

目的研究血管性血友病因子裂解蛋白酶(ADAMTS13)抗原含量和活性在血栓性血小板减少性紫癜(TTP)患者及遗传性 TTP 家族突变携带者中变化的情况。方法用残余胶原结合实验(RCBA)检测13例 TTP 患者共28份血浆标本[含血浆置换(PE)前后]及10例携带者的 ADAMTS13活性;用新近建立的三抗体夹心酶联免疫反应法检测标本的 ADAMTS13抗原含量。结果正常对照组 ADAMTS13含量为(600.93±145.36)mU/ml(设白种人混合血浆的 ADAMTS13抗原含量为1000mU/ml),活性为(74.79±11.81)%。遗传性 TTP 患者 ADAMTS13抗原含量和活性治疗前和发病间期均明显减低,PE 后恢复;其家族中携带者 ADAMTS13抗原含量为(331.40±109.85)mU/ml,活性为(66.79±12.82)%(与对照组比较,P 值分别<0.01和>0.05);原发性 TTP 患者 PE 前 ADAMTS13抗原含量为(98.7±82.08)mU/ml,活性为(22.23±19.07)%(与对照组比较,P 值均<0.01);PE 后ADAMTS13 抗原含量为(449.4±232.33)mU/ml,活性为(60.92±22.33)%(与对照组比较,P 值分别<0.01和>0.05);1例继发性 TTP 患者 PE 后 ADAMTS13抗原含量远高于正常,活性仅为6.00%结论治疗前的 TTP 患者 ADAMTS13抗原含量和活性均明显减低。大多数患者两指标变化趋势一致,也有个别患者两指标变化趋势相反,前者可能因为遗传因素或体内免疫系统的廓清作用,后者可能因为抗 ADAMTS13抗体仅抑制了 ADAMTS13的活性而未影响其抗原的含量或其他未知原因所致。     

ADAMTS13 and Thrombotic Thmmbocytopenic Purpura

Von Willebrand factor (vWF) is a large,multimeric blood protein that is made in endothelial cells,which store vWF multimers in unique organelles called Wcibel-Palade bodies.     

ADAMTS13蛋白C-末端结构调节其酶活性的研究

目的 初步研究并探讨ADAMTS13蛋白C-末端结构域变化在调节其酶活性方面的作用.方法 将人全长野生型及缺失C-末端TSP8和CUB结构域的截短型重组ADAMTS13基因转染Hela细胞并稳定表达.分别在静态尿素变性条件及蜗旋装置提供的高剪切力条件下观察这两种ADAMTS13蛋白酶切血管性血友病因子(vWF)的差异,同时利用Western blot技术及残余胶原结合实验对结果进行检测,另外利用ELISA法直接检测这两种ADAMTS13蛋白静态条件下结合vWF的能力.结果 在Hela细胞真核表达载体表达并纯化了全长野生型及缺失C-末端TSP8和CUB结构域的截短型ADAMTS13蛋白,分别利用鼠抗His-Tag单抗(抗载短型ADAMS113单)及兔抗人ADAMTS13多抗Western blot法鉴定了其特异性.静态尿素变性条件下,截短型ADAMTS13蛋白酶活性显著高于野生型,且静态条件下在结合vWF的能力方面,截短型也显著高于野生型;但在高剪切力作用下,只有全长野生型ADAMTS13可以对vWF进行剪切.结论 ADAMTS13蛋白C-末端TSP8和CUB结构域在不同条件下可以调节ADAMTS13蛋白结合vWF的能力,从而调节其酶活性的发挥,ADAMTS13蛋白C-末端在高剪切力作用条件下对ADAMTS13酶活性的发挥至关重要.     

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.     

The utility of patient characteristics in predicting severe ADAMTS13 deficiency and response to plasma exchange

BACKGROUND: Idiopathic thrombotic thrombocytopenic purpura (TTP) is a rare form of thrombotic microangiopathy (TMA) characterized by extreme deficiency of ADAMTS13, an enzyme responsible for cleavage of von Willebrand factor. Plasma exchange therapy is the cornerstone of current treatment and is ineffective for most other forms of TMA. The availability of ADAMTS13 testing has improved diagnostic accuracy and appropriate selection of patients who are most likely to respond to plasma exchange. STUDY DESIGN AND METHODS: We performed a retrospective chart review of 110 cases of clinically suspected TTP with ADAMTS13 test results from 2005 to the present. The primary goal was to identify presenting clinical and/or laboratory features of patients with ADAMTS13 deficiency that would prove useful in increasing the likelihood of, or excluding the possibility of, TTP. In addition, patient outcomes including alternative diagnoses and response to plasma exchange were reviewed. RESULTS: Significant correlations for severe ADAMTS13 deficiency were seen for four of the observed variables: indirect bilirubin, reticulocyte percentage, creatinine, and platelet count; a fifth variable, D‐dimer, just missed significance but performed well in subsequent analysis. Receiver operator characteristics curves for individual variables had area under the curve (AUC) values ranging from 0.75 to 0.85; a combined model had an AUC of 0.98. In addition, we constructed tree models both for clinical use as a diagnostic algorithm and for recursive partitioning to help establish cutoff points for categorical variables in developing an easy‐to‐use clinical prediction score. CONCLUSION: These results may enable the rapid exclusion and accurate diagnosis of TTP using readily available laboratory data.