scholarly journals Adamts-13 Inhibitor Testing Is Often Negative on Initial Testing

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5051-5051
Author(s):  
Hina Chaudhry ◽  
Michelle Sholzberg ◽  
Katerina Pavenski
Keyword(s):  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Genetic Defect ◽  
Reference Laboratory ◽  
Adamts13 Activity ◽  
Repeated Testing ◽  
Inhibitor Titre ◽  
Adamts13 Deficiency ◽  
Platelet Thrombus ◽  
Von Willebrand

Abstract Background: Thrombotic thrombocytopenic purpura (TTP) presents with microangiopathic hemolytic anemia and thrombocytopenia and is caused by severe ADAMTS13 deficiency. TTP can be the result of autoantibodies to ADAMTS13 or genetic defect in the ADAMTS13 gene. ADAMTS-13 is an enzyme that specifically cleaves unusually large von Willebrand Factor (VWF) multimers which mediate platelet thrombus formation under high shear. When ADAMTS13 is deficient, unusually large VWF multimers accumulate causing excessive platelet aggregation and thrombosis in the microvasculature. Methods: St. Michael's Hospital, Toronto is home to a large reference laboratory for special coagulation. We use a commercial ELISA Technoclone Technozym ADAMTS-13 activity assay for the determination of ADAMTS-13 activity and Technoclone Technozym ADAMTS-13 inhibitor assay to identify anti-ADAMTS-13 antibodies. We send samples to another Canadian laboratory for validation of our results as they use an in-house ELISA assay for ADAMTS13 activity and anti-ADAMTS13 antibody. Results: We performed a retrospective review of all ADAMTS13 activity tests performed by our laboratory between January 1, 2013 and June 30, 2018. The total number of tests was 466 from 203 unique patients. 24% had an ADAMTS-13 activity under 10% (N = 144) which is consistent with the diagnosis of TTP. When specimens with severe ADAMTS-13 deficiency were tested for presence of anti-ADAMTS13 antibody, 46% were negative. Four of these specimens were sent to the other laboratory and all had detectable, albeit very low titre, inhibitors. Furthermore, on repeated testing over the study period, the vast majority of patients who presented with low ADAMTS13 activity and no detectable antibody subsequently became antibody positive. Fifty-two patients remained antibody negative by our internal and send-out testing. Five of them were known to have or were subsequently diagnosed with hereditary TTP (hTTP). Only one patient continues to have negative antibody but whose clinical course is not consistent with hTTP. Conclusions: We found that a commercial ADAMTS13 (Technoclone Technozym) antibody assay is falsely negative in a substantial proportion of patients with autoimmune TTP, the majority of which likely had a low titer inhibitor, below the threshold of test detection. More sensitive assays and/or repeated testing, presumably as inhibitor titre increases during the course of the disease, may detect antibody presence in the majority of samples of patients with autoimmune TTP. This is an important finding as this could impact the types of therapies offered to patients with negative antibody screens and may also avoid unnecessary, expensive genetic testing . Disclosures No relevant conflicts of interest to declare.

Determinants of von Willebrand Factor Function

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-17-SCI-17
Author(s):  
Cécile V. Denis ◽  
Olivier D. Christophe ◽  
Peter J. Lenting
Keyword(s):  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Active Form ◽  
Regulatory Pathways ◽  
Spontaneous Interaction ◽  
Platelet Binding ◽  
Platelet Thrombus ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract SCI-17 Platelet thrombus formation is a multistep process involving a number of molecular players, including von Willebrand factor (vWF). vWF is an adhesive multimeric protein that acts as a molecular bridge between the subendothelium and the glycoprotein Ib/IX/V receptor complex on platelets. Furthermore, vWF promotes the expansion of the platelet plug by cross-linking platelets via binding to integrin αIIbβ3. It is important to keep in mind that before participating in the formation of platelet-rich thrombi, vWF and platelets coexist in the circulation without interacting with each other. For optimal function, it is essential that vWF-platelet interactions occur in a timely way, that is, not too early and not too late. In the former case, spontaneous interaction may lead to intravascular thrombosis, while in the latter, hemorrhagic complications may arise. In order to reach this fine balance of regulation, a number of mechanisms are in place that contribute to control vWF function. In the last few years, considerable progress has been made in either revealing or better understanding such determinants. Physiologically, most of these mechanisms are dedicated to the prevention of excessive vWF-platelet interactions. These include shear-stress-mediated vWF conformational changes that lead to exposure or nonexposure of the platelet-binding site and cleavage sites on the vWF molecule. Intramolecular shielding of the vWF-platelet binding domain by adjacent domains also contributes to vWF reactivity. A major determinant of vWF function is related to its multimeric size, which can be controlled by proteolysis by ADAMTS13 and by other proteases, such as granzyme B or neutrophil elastase. The thiol reductase activity of ADAMTS13 toward vWF also contributes to multimer regulation. Finally, interaction of vWF with plasma proteins such as β2-glycoprotein I, or with endothelial proteins such as osteoprotegerin and galectins, can also participate in keeping vWF from binding excessively to platelets. Pathologically, dysregulations of the above-mentioned mechanisms may lead to either an overly active form of vWF or, in contrast, to an inactive protein. Additional determinants can also become prominent, such as the presence of mutations in the vWF sequence, leading to the genetic bleeding disorder known as von Willebrand disease. Determinants affecting vWF-platelet function have been studied extensively, as vWF participation in platelet thrombus formation is its best known and most important role. However, rather fascinating mechanisms have been identified that can modulate other functions of vWF. An example thereof is the recent identification of vWF cleavage by ADAM28 expressed by carcinoma cells in order to escape the proapoptotic action of vWF on such cells. Another example is the regulation of the Factor VIII binding capacity of vWF that can be controlled by cleavage by granzyme M. Identification of these various regulatory pathways now opens new avenues to act upon in order to better control the fine balance between the prohemostatic and the prothrombotic roles of vWF. Disclosures: No relevant conflicts of interest to declare.

Elevated Threshold Shear Rate for the Dependence of Glycoprotein Ibα-Mediated Platelet Thrombus Formation onto Immobilized von Willebrand Factor in Mouse Blood.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3662-3662
Author(s):  
Patrizia Marchese ◽  
Taisuke Kanaji ◽  
Denisa D. Wagner ◽  
Jerry Ware ◽  
Zaverio M. Ruggeri
Keyword(s):  
Shear Rate ◽  
Collagen Type ◽  
Thrombus Formation ◽  
Collagen Type I ◽  
Type I ◽  
Shear Rates ◽  
Normal Platelet ◽  
Platelet Thrombus ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The interaction between platelet glycoprotein (GP) Ibα and von Willebrand Factor (VWF) is essential to initiate platelet deposition at sites of vascular injury and sustain platelet thrombus formation when the shear rate exceeds a threshold value. With human blood, the dependence of normal platelet adhesion and aggregation on VWF-GP Ibα function becomes evident at shear rates above 1,000 s−1. In the last several years, mouse models have been increasingly used to study the mechanisms of thrombus formation in circulating blood, and mice deficient in both VWF and GP Ibα have been generated. These animals offer the opportunity to evaluate whether the pathways of platelet adhesion and aggregation mediated by VWF and GP Ibα are equally important in mouse and human blood as well as to define the threshold shear rate at which the function of these pathways may become essential in the mouse circulation. To address this issue, we used an ex vivo perfusion system using fibrillar collagen type I as the thrombogenic surface and a flow chamber in which the shear rate varied according to a predictable function from the inlet to the outlet in relation to the x,y position in the flow path. Thus, wall shear rates between 5,000 at the inlet and 0 s−1 at the outlet could be evaluated in a single experiment, allowing a precise definition of the threshold at which platelet deposition on the surface could initiate. In these studies we used wild type control animals (WT), mice deficient in VWF (VWF-KO) and mice in which most of the extracellular domain of GP Ibα was replaced by a domain of the human interleukin 4 receptor (GPIb-KO/IL-4R). In the latter case, the ligand binding function of GP Ibα was obliterated, but unlike in GP Ib-KO mice platelet morphology and count were essentially normal. Blood was obtained from the retroorbital vein plexus and contained 100 u/ml heparin as an anticoagulant. Experiments were recorded in real time for the visualization of platelet-surface contacts and confocal videomicroscopy was used for the direct measurement of platelet thrombus volume. With normal mouse blood, platelet formed large thrombi throughout the tested range of shear rates. In contrast, with VWF-KO and GPIb-KO/IL-4R blood, thrombus volume was less than 5% of normal at 5,000 s−1, approximately 50% of normal at 3,000 s−1, but entirely normal at 1,500 s−1. Essentially the same results were observed when the extracellular matrix of mouse fibroblasts, which may better represent the complex thrombogenic properties of the vascular wall, was used as a reactive substrate instead of isolated collagen type I. The different threshold shear rate at which VWF and GP Ibα function are essential for thrombus formation with human and mouse platelets may be explained by the smaller size of the latter, which consequently are subjected to a lower drag at equivalent shear rate levels. Moreover, the similar behavior of VWF-KO and GPIb-KO/IL-4R platelets suggests that, under the conditions of these studies, VWF binding is the predominant GP Ibα function required for normal platelet thrombus formation at high shear rates. The present results should allow a more critical evaluation of the findings derived from mouse models of hemostasis and thrombosis.

Identification of a Binding Site for Integrin αIIbβ3 in the von Willebrand factor (VWF) A1 Domain: Dual Roles for the A1 Domain in Platelet Thrombus Formation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3658-3658
Author(s):  
Junmei Chen ◽  
Miguel A. Cruz ◽  
José A. López
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Shear Stresses ◽  
Platelet Surface ◽  
Rgd Sequence ◽  
Platelet Thrombus ◽  
A1 Domain ◽  
Von Willebrand

Abstract In 1999, Wu et al found that blood from patients with type 3 von Willebrand disease (lacking VWF in both plasma and platelets) could not form thrombi on a collagen surface (Arterioscler. Thromb. Vasc Biol2000, 201661–1667). This suggested that VWF was absolutely required for the accumulation of platelets in thrombi under flow, even in the presence of fibrinogen. Platelets have two VWF receptors, the GP Ib-IX-V complexes and αIIbβ3 , the former mediating the initial tethering and attachment of platelets onto VWF and the latter being involved in platelet-platelet contacts. GP Ib-IX-V binds VWF within the A1 domain and αIIbβ3 is known to bind an Arg-Gly-Asp (RGD) sequence in the C1 domain. In the study of Wu et al, reconstitution of the VWF-deficient plasma with recombinant VWF missing the A1 domain failed to restore thrombus formation, even when the collagen surface was first coated with wild-type VWF to allow platelet attachment. The A1 domain is thus important not only for initial platelet adhesion but also for thrombus accumulation, possibly by binding another platelet receptor. Consistent with this, the number of binding sites for the isolated A1 domain on the platelet surface is more than twice the number of GP Ibα polypeptides. The receptor responsible for these binding sites is unknown but αIIbβ3 is a good candidate given its high copy number and the marked defect seen in platelet thrombus formation in its absence or blockade. Of interest, while deletion of A1 prevented thrombus formation in the studies of Wu et al, mutation of the VWF RGD sequence did not. We therefore examined whether αIIbβ3 also binds within the VWF A1 domain. We found the following. 1) Purified, unactivated αIIbβ3 binds to immobilized A1 domain, binding blocked by antibodies to either αIIbβ3 or A1. 2) Unactivated αIIbβ3 does not interact with immobilized full-length VWF, but binds VWF in the presence of ristocetin. The binding of αIIbβ3 to both VWF and isolated A1 is blocked by the αIIbβ3 antibody c7E3 but not by RGD peptides, and by the A1 antibody 6G1. This suggests that the αIIbβ3 binding site in the A1 domain may overlap the 6G1 epitope (residues 700-709), which is distinct from the GPIbα binding site. 3) 6G1 inhibits shear-induced platelet aggregation—a process that requires both GP Ibα and αIIbβ3—without blocking GP Ibα binding. 4) Platelets firmly adhere on the surface containing A1 and cross-linked collagen-related peptide (CRP), a potent GP VI agonist, at high shear stresses. The CRP-GP VI interaction is not strong enough to arrest platelets under flow, suggesting that GP VI signals could activate αIIbβ3, and αIIbβ3 could mediate firm adhesion. Consistent with this, the αIIbβ3 antibody c7E3 prevented firm platelet adhesion. In summary, we find that αIIbβ3 binds to the A1 domain, in or near the sequence of Glu700-Asp709. In addition to its apparent role in platelet-platelet interactions during thrombus growth, the binding of αIIbβ3 to the VWF A1 domain may also facilitate the binding of GP Ibα to a distinct region of A1, as the site of αIIbβ3 overlaps the binding site of ristocetin and 6G1, both which induce VWF to bind GP Ibα. Therefore, by binding to the same site as 6G1 and ristocetin in the C-terminal peptide of A1, αIIbβ3 may regulate the affinity of A1 for GP Ibα in flowing blood.

Thienopyridine-Associated Thrombotic Thrombocytopenia Purpura: Updated Antibody Results From the SERF-TTP Study.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 892-892
Author(s):  
Anaadriana Zakarija ◽  
Thanh Ha Luu ◽  
Hau C. Kwaan ◽  
June McKoy ◽  
Ivy Weiss ◽  
...  
Keyword(s):  
Plasma Exchange ◽  
Neutralizing Antibodies ◽  
Conflicts Of Interest ◽  
Therapeutic Plasma Exchange ◽  
Severe Thrombocytopenia ◽  
Adamts13 Activity ◽  
Laboratory Findings ◽  
History Of ◽  
Adamts13 Deficiency ◽  
Drug Dependent

Abstract Abstract 892 Background: The thienopyridines, ticlopidine and clopidogrel, have been associated with thrombotic thrombocytopenia purpura (TTP). However, few studies have reported information on antibodies to ADAMTS13 among patients with thienopyridine-associated TTP. We previously reported on two mechanistic pathways of thienopyridine-associated TTP with some overlapping features. Evaluation of ADAMTS13 autoantibodies was undertaken to improve understanding of these syndromes. Methods: Clinical and laboratory findings were evaluated for 30 ticlopidine-, 10 clopidogrel-associated TTP cases, and 54 cases of idiopathic TTP. Results: Among patients with thienopyridine-induced TTP, those with a history of ticlopidine versus clopidogrel use were more likely to present with severe thrombocytopenia (platelet < 20,000) (90% versus 13%), severe ADAMTS13-deficiency (80% versus 0%), and neutralizing antibodies to ADAMTS13 (100% versus 0%), and were less likely to have less than a two week history of thienopyridine exposure (0% versus 50%) (p<0.05 for each comparison). They were also more likely to survive following therapeutic plasma exchange (TPE) (85% versus 50%). 2 patients exposed to clopidogrel later relapsed and had similar characteristics to idiopathic TTP patients with non-deficient ADAMTS13 activity. Conclusion: Ticlopidine causes TTP by a pathway involving a neutralizing autoantibody to ADAMTS13 while clopidogrel causes TTP by an ADAMTS13-independent pathway. Although ADAMTS13 autoantibodies are present in both idiopathic and ticlopidine-associated TTP, spontaneous relapses are not seen in ticlopidine-associaated TTP, suggesting that drug-dependent antibodies are present. Clopidogrel associated TTP is distinct from idiopathic TTP in that ADAMTS13 autoantibodies are absent and response to TPE is poor. Disclosures: No relevant conflicts of interest to declare.

Are Patients Who Have Recovered From ADAMTS13-Deficient Thrombotic Thrombocytopenia Purpura (TTP) at Risk for Developing Systemic Lupus Erythematosus (SLE)?

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2519-2519
Author(s):  
Deirdra R. Terrell ◽  
Zayd Al-Nouri ◽  
Judith A. James ◽  
Johanna A. Kremer Hovinga Strebel ◽  
Bernhard Lämmle ◽  
...  
Keyword(s):  
At Risk ◽  
Clinical Features ◽  
Lupus Erythematosus ◽  
Conflicts Of Interest ◽  
Population Data ◽  
First Episode ◽  
Adamts13 Activity ◽  
Acr Criteria ◽  
Adamts13 Deficiency

Abstract Abstract 2519 TTP associated with acquired, ADAMTS13-deficiency and SLE are both autoimmune disorders that occur preferentially in young, black women and they have many similar clinical features. TTP may occur in patients previously diagnosed with SLE, or patients may develop SLE following recovery from TTP. In addition, TTP may be quite difficult to distinguish from SLE patients with severe hematologic manifestations. We compared the prevalence of SLE-associated autoantibodies in TTP patients to published population data using 95% confidence intervals (CI). The Oklahoma TTP Registry enrolled 292 consecutive patients with their first episode of clinically diagnosed TTP from 11–13-1995 (date of our initial ADAMTS13 measurement) to 7–31-2009; ADAMTS13 activity was measured in 271 (93%) patients; 64 (24%) patients had ADAMTS13 activity <10%, 63 were evaluated for SLE-associated autoantibodies, including 2 patients with a previous diagnosis of SLE. Serum from the patient's acute initial episode was used for analysis. The prevalence of ANA, anti-dsDNA, anti-Ro, and aPL in TTP patients was significantly higher than published population data; prevalences of anti-nRNP, anti-Sm, and anti-La were not different. Autoantibody TTP (95% CI) Population % ANA  ≥1:40 89% (78%–95%) 0–27%  ≥1:120 56% (42%–68%) 0% Anti-dsDNA  ≥1:30 43% (30%–56%) 3% Anti-Ro  OD>0.350 17% (8%–29%) 3% aPL IgM  ≥20 PL units 15% (7%–26%) 2% Because of the increased prevalence of SLE-associated autoantibodies, we evaluated our TTP patients for the America College of Rheumatology (ACR) criteria for SLE (presence of ≥4 of 11 criteria suggests the diagnosis of lupus); abnormalities associated with any TTP episode were not counted in this evaluation of clinical criteria for SLE. By definition ACR criteria can be fulfilled serially or simultaneously over a lifetime. Evaluations were completed between 6-1-2007 and 5-1-2009 on 38/42 (90%) eligible patients (alive, non-institutionalized, no previous SLE diagnosis) consisting of physical examination, review of available lifetime medical records, and serial laboratory evaluations. Patients have been followed for a median of 8.3 years (range, 1–14 years). During this time, 3 (8%) developed clinically evident SLE requiring treatment 1, 5, and 70 months after their initial TTP episodes. Among the other 35 patients, 3 (8%) have ≥4 SLE classification criteria by medical record review (1 had pre-existing Sjögren's syndrome and receives treatment; 2 have minimal clinical features and are not actively treated for SLE); 9 (24%) have 3 criteria; 16 (42%) have 2 criteria; 6 (16%) have 1 criterion; and 1 (2%) patient has no ACR criteria for SLE. All patients continue to be followed and clinically evaluated for potential intervention. SLE diagnosis is a clinical designation and because of the lack of disease modifying drugs, routine follow-up is standard of care unless the patient is symptomatic. Conclusions: [1] A high prevalence of SLE-associated autoantibodies was present in a cohort of consecutive patients with TTP associated with acquired severe ADAMTS13 deficiency. [2] The presence of anti-dsDNA, anti-Ro, aPL and high titers of ANA suggest that patients with ADAMTS13-deficient TTP may be at risk for developing SLE. [3] During long-term follow-up, 6 (16%) of 38 patients have developed overt SLE or ACR criteria without an established diagnosis of SLE. Careful continuing evaluation following recovery from TTP is important. Disclosures: No relevant conflicts of interest to declare.

ADAMTS13 In 4 Different VWF/VIII Concentrates and Its Impact on Therapy.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3677-3677
Author(s):  
Mirjeta Qorraj ◽  
Tanja Falter ◽  
Sarah Steinemann ◽  
Thomas Vigh ◽  
Inge Scharrer
Keyword(s):  
Von Willebrand Factor ◽  
Gel Electrophoresis ◽  
Conflicts Of Interest ◽  
Von Willebrand Disease ◽  
Relative Reduction ◽  
Hemorrhagic Diathesis ◽  
Adamts13 Activity ◽  
Kinetic Measurements ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 3677 Introduction: The hemostatic activity of von Willebrand Factor (VWF) is mainly controlled by the plasma metalloprotease ADAMTS13, which cleaves ultralarge VWF multimers. A qualitative or quantitative deficiency of VWF induces the most common hemorrhagic diathesis, the von Willebrand Disease (VWD). The current classification graduates the VWD in three major types. Depending on severity and the type of VWD the treatment with VWF/FVIII concentrates may by necessary. The commercially available VWF/FVIII concentrates differ in their multimer structure and furthermore also in their pharmacokinetics. We investigated commercial VWF concentrates with respect to their ADAMTS 13 activity and antigen levels with the newest available methods. Moreover, to detect a possible correlation, we analysed the VWF multimer structure of the concentrates. Methods: We analysed 4 human derived VWF/VIII-concentrates (over all 7charges) after reconstitution according to the manufacturer's instructions in different dilutions. Following methods were used: BCS Method according to Böhm detects the capacity of the concentrates for autoproteolysis. The VWF solutions were diluted with 5mol/l urea and then incubated for 14–16h at 37°C in low ionic TRIS buffer containing BaCl2 and different plasma samples: pool plasma; plasma from patients with TTP with neutralizing ADAMTS13 auto-antibodies; plasma from patients with TTP without auto-antibodies. The residual VWF:Ristocetin Cofactor (VWF:RCo) activity was subsequently measured using the BC von Willebrand Reagent from Dade Behring. ELISA Technozym®ADAMTS13 and Actifluor TM ADAMTS13 are based on the kinetic measurements of the activity with fluorescence resonance energy transfer (FRET). ADAMTS13 antigen was measured by use of the Technozym ELISA kit. SDS-Gel electrophoresis in 1% Agarose Gel was used to investigate the structure of VWF multimers. Results: The BCS Method according to Böhm is an indirect measurement for endogenous ADAMTS13 activity in the investigated concentrate. Important is the loss of the residual VWF:RCo in the concentrates in presence of TTP-plasma without antibodies and pool plasma compared to the residual VWF:RCo in presence of TTP-plasma with antibodies. All concentrates show some ADAMTS13 activity, however product 1 contains more ADAMTS13 than the other concentrates. The results of the two FRETS-assays correspond very well to the BCS-method results; in addition the assays detect directly the ADAMTS13 activity also in very low measurement range. In a dilution of 16U VWF per ml concentrate the ADAMTS13 activity in product 1 with 4.3% was the highest compared to product 2: 3.2%, product 3: 2.6% and product 4: 2%. The great variability of the test results in higher concentrations may be caused by interferences between some constituents of the concentrates and the analysis. In the same sample set and dilution the ADAMTS13 antigen values correlate very well with ADAMTS13 activity values. The SDS gel electrophoresis reveals the different VWF structure of product1; it has less large and ultralarge multimers. There could be a correlation to the relatively higher ADAMTS13 activity and antigen level. Conclusion: All the investigated VWF/VIII concentrates contain some ADAMTS13 activity and antigen. This was found especially by FRETs assay due to the high sensitivity. Because of the correlation between ADAMTS13 activity and modified VWF multimer structure we like to conclude that ADAMTS13 has influence on stability and therefore also on quality of the concentrates. This might have a therapeutic consequence especially for VWD type 2A. Type 2A is characterized by a relative reduction of intermediate and large VWF multimer. The multimeric abnormalities are commonly the result of in vivo proteolytic degradation of the von Willebrand factor caused by ADAMTS13. Disclosures: No relevant conflicts of interest to declare.

Investigation of Side Effects of Plasmaexchange In the Treatment of Thrombotic Thrombocytopenic Purpura

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4661-4661
Author(s):  
Sarah Steinemann ◽  
Tanja Falter ◽  
Mirjeta Qorraj ◽  
Thomas Vigh ◽  
Inge Scharrer
Keyword(s):  
Side Effects ◽  
Thrombotic Thrombocytopenic Purpura ◽  
Side Effect ◽  
Conflicts Of Interest ◽  
Allergic Reactions ◽  
Adamts13 Activity ◽  
Plasma Therapy ◽  
Thrombocytopenic Purpura ◽  
Wide Range ◽  
Von Willebrand

Abstract Abstract 4661 Introduction: Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia, hemolytic anemia and microthrombi. A deficiency of the metalloprotease ADAMTS 13, which cleaves a Tys1605-Met1606 bond in the A2 subunit of von Willebrand factor (VWF), leads to formation of ultra large von Willebrand multimers (UL-VWF) and can cause platelet aggregation and mircovascular thrombosis. Treatment of choice is the substitution of plasma with plasmaexchange. There are two different plasma types available: Fresh Frozen Plasma (FFP) and solvent/detergent (s/d) treated plasma. This treatment may carry significant risks and side effects for the patients. Therefore we investigated the side effects of the therapy and furthermore the ADAMTS13 activity of the two plasma types. Methods: A questionnaire was send to 66 TTP patients of the Department of Hematology to evaluate different side effects of the therapy. 20 batches of FFP and 4 batches of s/d plasma of all blood groups were investigated on ADAMTS13 activity. The ADAMTS13 activity was detected with BCS-Method according to Böhm and two commercial FRET assays. Results: So far 34 patients were inquired about age, weight and suspected trigger situations that might have caused their TTP manifestation. The mean age of the patients was 34 years with a mean weight of 70kg. A previous infection caused TTP manifestation in 42% of the patients; drug therapy (22%) and pregnancy (17%) were other mentioned triggers. 94% of the patients suffered from an acquired TTP and only 6% had a hereditary TTP. The patients had 2.88 relapses and were treated with 16.27 plasmaexchanges. 56% had an additional therapy with Rituximab to achieve a faster remission of the disease. These patients needed less plasmaexchanges for recovery, which proofed to be significant at 2% level in a one sided t-test. Tingling (64.7%) and shivering (51%) were the most often mentioned side effects and simultaneously described as the strongest. Shivering was significantly correlated to tachycardia (p<0.01). Headaches were significantly correlated to hot flushes, tingling and collapse (p< 0.05). Side effects and allergic reactions occurred in the therapy with FFP as well as with s/d plasma. Another side effect was the complication that came along with infection of the venous access. Most patients had a central venous catheter (72%) and described infections and pruritus (60%), 50% of them mentioned this complication more than once. We found in usual FFP slightly higher ADAMTS13 activity levels (696.97 ng/ml) than in s/d virus inactivated plasma (643.86 ng/ml). The ADAMTS13 activity varied between the different assays (normal range: 666 ± 135ng/ml). Conclusion: Our investigation demonstrated that plasmaexchange therapy is still associated with a wide range of side effects. Side effects of plasmaexchange that were most frequently described by patients were tingling and shivering. Headaches also occurred in various cases. Patients suffered generally from more than one side effect at the same time during the treatment. Allergic reactions to the plasma therapy were mentioned by 65% of the patients. Disclosures: No relevant conflicts of interest to declare.

A Novel Mutation of ADAMTS13 Gene in Congenital Thrombotic Thrombocytopenic Purpura

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4674-4674
Author(s):  
Xia Bai ◽  
Jian Su ◽  
Minghua Jiang ◽  
Zhaoyue Wang ◽  
Changgeng Ruan
Keyword(s):  
Conflicts Of Interest ◽  
Novel Mutation ◽  
Gene Mutations ◽  
Type I ◽  
Adamts13 Activity ◽  
Intron Boundary ◽  
Congenital Thrombotic Thrombocytopenic Purpura ◽  
Peripheral Leukocytes ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 4674 Congenital thrombotic thrombocytopenic purura (TTP) is caused by gene mutations of von Willebrand factor-cleaving protease (a disintegrin and metalloprotease with thrombospondin type I domains 13, ADAMTS13). In this study, one novel mutation in the ADAMTS13 gene was found in a woman whose presents are first cousins. Thrombocytopenia occurred during the second trimesters in her first pregnancy, and she died of recurrent attacks after diagnosis of TTP. The ADAMTS13 activity measured using the recombinant FRET-VWF73 during her acute episode was less than 5%. ADAMTS13 inhibitor was negative measured by 9:1 mixture of patient and pooled normal plasma followed by ADAMTS13 activity assay using the VWF multimer electrophoresis. The 29 exons and exon-intron boundary sites of ADAMTS13 gene was analyzed using the human genomic DNA extracted from peripheral leukocytes of the patient. The results demonstrated she was homozygous for R498C. This novel mutant was constructed using the expression plasmid pSectag containing ADAMTS13 cDNA, and the vector was introduced by linpofectamine 2000 to Hela cells. Western Blot revealed that rADAMTS13-wide type (WT) was synthesized as a single band with molecular mass close to 190 Kda in the conditioned media, however, no detectable ADAMTS13 of this mutant existed. The lysates of cells expressing the mutant showed the same protein amounts compared to the rADAMTS13-WT. The immunofluorescence study demonstrated that mutant had the same localization pattern at Endoplasmic Reticulum(ER)and Golgi-compartments compared to the rADAMTS13-WT. The results imply that this mutant may be retained in the cellular ER and Golgi-comparments, but rapidly degraded or insufficiently secreted. Disclosures: No relevant conflicts of interest to declare.

FRETS-VWF73 rather than CBA assay reflects ADAMTS13 proteolytic activity in acquired thrombotic thrombocytopenic purpura patients

2014 ◽  
Vol 112 (08) ◽  
pp. 297-303 ◽  
Author(s):  
Ilaria Mancini ◽  
Carla Valsecchi ◽  
Luca Lotta ◽  
Louis Deforche ◽  
Silvia Pontiggia ◽  
...  
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Proteolytic Activity ◽  
Binding Activity ◽  
Adamts13 Activity ◽  
Flow Conditions ◽  
Thrombocytopenic Purpura ◽  
Severe Deficiency ◽  
Adamts13 Deficiency ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryCollagen-binding activity (CBA) and FRETS-VWF73 assays are widely adopted methods for the measurement of the plasmatic activity of ADAMTS13, the von Willebrand factor (VWF) cleaving-protease. Accurately assessing the severe deficiency of ADAMTS13 is important in the management of thrombotic thrombocytopenic purpura (TTP). However, non-concordant results between the two assays have been reported in a small but relevant percentage of TTP cases. We investigated whether CBA or FRETS-VWF73 assay reflects ADAMTS13 proteolytic activity in acquired TTP patients with non-concordant measurements. Twenty plasma samples with non-concordant ADAMTS13 activity results, <10% using FRETS-VWF73 and ≥20% using CBA, and 11 samples with concordant results, <10% using either FRETS-VWF73 and CBA assays, were analysed. FRETS-VWF73 was performed in the presence of 1.5 M urea. ADAMTS13 activities were also measured under flow conditions and the VWF multimer pattern was defined in order to verify the presence of ultra-large VWF due to ADAMTS13 deficiency. In FRETS-VWF73 assay with 1.5 M urea, ADAMTS13 activity significantly increased in roughly 50% of the samples with non-concordant results, whereas it remained undetectable in all samples with concordant measurements. Under flow conditions, all tested samples showed reduced ADAMTS13 activity. Finally, samples with non-concordant results showed a ratio of high molecular weight VWF multimers higher than normal. Our results support the use of FRETS-VWF73 over CBA assay for the assessment of ADAMTS13 severe deficiency and indicate urea as one cause of the observed differences.

scholarly journals Systemic antithrombotic effects of ADAMTS13

2006 ◽  
Vol 203 (3) ◽  
pp. 767-776 ◽  
Author(s):  
Anil K. Chauhan ◽  
David G. Motto ◽  
Colin B. Lamb ◽  
Wolfgang Bergmeier ◽  
Michael Dockal ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Type I ◽  
Inhibitory Antibody ◽  
Antithrombotic Agent ◽  
Antithrombotic Effects ◽  
Life Threatening ◽  
Adamts13 Deficiency ◽  
Von Willebrand ◽  
Willebrand Factor

The metalloprotease ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type I repeats 13) cleaves highly adhesive large von Willebrand factor (VWF) multimers after their release from the endothelium. ADAMTS13 deficiency is linked to a life-threatening disorder, thrombotic thrombocytopenic purpura (TTP), characterized by platelet-rich thrombi in the microvasculature. Here, we show spontaneous thrombus formation in activated microvenules of Adamts13−/− mice by intravital microscopy. Strikingly, we found that ADAMTS13 down-regulates both platelet adhesion to exposed subendothelium and thrombus formation in injured arterioles. An inhibitory antibody to ADAMTS13 infused in wild-type mice prolonged adhesion of platelets to endothelium and induced thrombi formation with embolization in the activated microvenules. Absence of ADAMTS13 did not promote thrombi formation in αIIbβ3 integrin-inhibited blood. Recombinant ADAMTS13 reduced platelet adhesion and aggregation in histamine-activated venules and promoted thrombus dissolution in injured arterioles. Our findings reveal that ADAMTS13 has a powerful natural antithrombotic activity and recombinant ADAMTS13 could be used as an antithrombotic agent.

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