Plasma Of Thrombotic Thrombocytopenic Purpura Patients Research Articles

Monitoring ADAMTS13 Autoantibody Titer Using a Novel Quantitative Radioimmunoprecipitation Assay In Patients With Acquired Thrombotic Thrombocytopenic Purpura

BackgroundADAMTS13-binding immunoglobulin G (IgG)-type autoantibodies are present in patients with acquired thrombotic thrombocytopenic purpura (TTP), thereby causing severe deficiency of plasma ADAMTS13 activity. Some specific autoantibodies directly inhibit the enzymatic activity by interfering with the access of its substrate, von Willebrand factor (VWF), particularly in the spacer domain, although others bind to almost all of the domains in the molecule, possibly leading to the acceleration of the clearance from blood stream. Not only the inhibitor titer, but the importance of ADAMTS13 autoantibody titer was highlighted by many previous clinical studies, associating with the prognosis such as recurrence rate. ObjectiveWe targeted to establish a novel high-sensitive assay to measure ADAMTS13-binding IgG autoantibody titer using three kinds of radioisotope-labeled antigens, ADAMTS13 whole molecule, MDTCS and T2-8/CUB, and aimed to analyze the association between the autoantibody titer and the clinical characteristics of TTP patients. Materials and MethodsHuman Cell-Free Protein Expression System (Takara #3281, Shiga, Japan) was used to synthesize radioisotope-labeled antigens. ADAMTS13 cDNA corresponding to whole molecule (A13), metalloprotease to spacer domains (MDTCS) and TSP1-2 to CUB2 domains (T2-8/CUB) were cloned into an expression vector pT7-IRES and mixed with Cell Lysate containing T7 RNA polymerase, methionine-free amino acids, ATP, translation enhancement factor and 35S-methionine. The correct synthesis and molecular size of the radiolabeled antigens were checked with SDS-PAGE. To assess the utility as a quantitative assay, each of the antigens was mixed with mouse anti-ADAMTS13 monoclonal antibodies, whose epitopes were determined in our previous study (Thromb Res. 2012; 130(3):e79-83), and the immune complex was precipitated with protein G beads, washed and measured in a liquid scintillation counter. Plasma samples from acquired TTP patients were tested to quantify the autoantibody titers using radiolabeled A13, MDTCS and T2-8/CUB antigens, respectively. As a control, plasma samples from healthy subjects with no histories of autoimmune disease were also tested. ResultsEach of the radiolabeled antigens was detected as a single band at the correct molecular weight size and successfully immnoprecipitated with several mouse anti-ADAMTS13 monoclonal antibodies, indicating the intact molecular conformation of the synthesized proteins using the cell-free protein synthesis system. Moreover, the appropriate dose-dependent escalation curves in accordance with the addition of the monoclonal antibodies were observed, thereby confirming the utility of the assay as a quantitative analysis. We tested TTP patient plasma at onset (n=5) and were able to detect ADAMTS13 autoantibody titers with each of the radiolabeled A13, MDTCS and T2-8/CUB antigens. We next applied this assay for monitoring ADAMTS13 autoantibody titer in a clinical course of TTP patient. The patient developed the first episode of TTP at the age of 2 month and treated with steroid pulse and plasma exchange therapy for six consecutive days. Remission was once achieved but 6 months later from the onset, the second episode of TTP occurred and the patient was treated with 11 plasma exchange and rituximab at the dose of 375 mg/m2 once a week for 4 weeks. Plasma samples at onset, after the first 6 consecutive plasma exchange and after rituximab administration were examined about autoantibody titer using this assay. Interestingly, the titers remained high even after the plasma exchange but declined clearly after the rituximab treatment, whereas no reduction of total IgG level was observed. These findings suggest that the autoantibody titration using this assay might be useful to assess the effect of treatment and associate with the prognosis related to the recurrence. ConclusionWe developed a novel quantitative radioimmunoprecipitation assay to measure ADAMTS13 autoantibody titer related to three antigens, ADAMTS13 whole molecule, MDTCS and TSP2-8/CUB. This assay may serve not only as a diagnostic test but as a monitoring index to evaluate the prognosis of TTP. Disclosures:No relevant conflicts of interest to declare.

Complement Activation Associated with ADAMTS13 Deficiency in Human and Murine Thrombotic Microangiopathy

This study addressed the contribution of ADAMTS13 deficiency to complement activation in thrombotic thrombocytopenic purpura (TTP). Renal tissue and blood samples were available from 12 TTP patients. C3 and C5b-9 deposition were demonstrated in the renal cortex of two TTP patients, by immunofluorescence and immunohistochemistry, respectively. C3 was also demonstrated in the glomeruli of Shiga toxin-2-treated Adamts13(-/-) mice (n = 6 of 7), but less in mice that were not Shiga toxin-2 treated (n = 1 of 8, p < 0.05) or wild-type mice (n = 0 of 7). TTP patient plasma (n = 9) contained significantly higher levels of complement-coated endothelial microparticles than control plasma (n = 13), as detected by flow cytometry. Exposure of histamine-stimulated primary glomerular endothelial cells to platelet-rich plasma from patients, or patient platelet-poor plasma combined with normal platelets, in a perfusion system, under shear, induced C3 deposition on von Willebrand factor-platelet strings (on both von Willebrand factor and platelets) and on endothelial cells. Complement activation occurred via the alternative pathway. No C3 was detected when cells were exposed to TTP plasma that was preincubated with EDTA or heat-inactivated, or to control plasma. In the perfusion system, patient plasma induced more release of C3- and C9-coated endothelial microparticles compared with control plasma. The results indicate that the microvascular process induced by ADAMTS13 deficiency triggers complement activation on platelets and the endothelium, which may contribute to formation of thrombotic microangiopathy.

Importance of Measuring Circulating ADAMTS13 Immune Complexes in Addition to Free Antibodies in Patients with Acquired TTP

Abstract 102A severe functional deficiency of the von Willebrand factor (VWF) cleaving protease ADAMTS13 results in accumulation of uncleaved, highly adhesive VWF multimers in plasma. This VWF multimer accumulation leads to increased platelet aggregation and thrombus formation in the microcirculation. Anti-ADAMTS13 autoantibodies that either neutralize ADAMT13's activity or enhance protein clearance are the main cause of severe ADAMTS13 deficiency in acquired thrombotic thrombocytopenic purpura (TTP). Circulating anti-ADAMTS13 immune complexes (CIC) that may be a factor in disease pathogenesis have been described in plasma of TTP patients. We have developed an ELISA assay to specifically determine the levels of CIC in which the antigen portion is immobilized by a polyclonal anti-ADAMTS13 IgG and the immunoglobulin component detected by a class-specific antibody. Samples (individual or longitudinal) from a total of 100 patients with acute idiopathic TTP were used to compare IC levels with those of the free anti-ADAMTS13 antibodies, ADAMTS13 antigen (ADAMTS13:Ag), ADAMTS13 activity (ADAMTS13:Ac) and inhibitor. This analysis was to provide for the first time a complete picture of the autoantibody response in this population.IgG-containing CIC were present in most of the patient samples, with IgG4-IC being the predominant type of CIC. A correlation between the subclasses of the free and complexed IgG anti-ADAMTS13 antibodies was observed, but the levels of CIC appeared to follow inverse kinetics to the free antibody and the inhibitory titer. We also found samples devoid of free ADAMTS13:Ag, but containing CIC, indicating that ADAMTS13, either newly synthesized or exogenously administered by plasma infusion during plasma exchange, had been quantitatively sequestered through formation of IC. ADAMTS13:Ag was also undetectable in some samples with high levels of free anti-ADAMTS13 antibodies but lacking CIC, suggesting that IC that had formed were already cleared at these time points.Our study suggests that in addition to characterization of autoantibodies against ADAMTS13, ADAMTS13-specific CIC also need to be determined at time of diagnosis and during treatment to obtain a full picture of disease status. The formation of ICs capturing all freely available ADAMTS13 may have a high impact on treatment response, especially in patients who are refractory to treatment. The gathered data may thus help to timely implement alternative treatment strategies in patients unresponsive to standard treatments, thereby reducing unexpected disease relapses with fatal outcome. Disclosures:Ferrari:Baxter Innovations GmbH: Employment. Plaimauer:Baxter Innovations GmbH: Employment. Gruber:Baxter Innovations GmbH: Employment. Palavra:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Detection of Circulating ADAMTS13 Immune Complexes by Antibody Class-Specific ELISA

Abstract 4361A severe functional deficiency of the von Willebrand factor (VWF) cleaving protease ADAMTS13 results in the accumulation of uncleaved, highly adhesive VWF multimers in plasma leading to increased platelet aggregation and thrombus formation in the microcirculation. Anti-ADAMTS13 autoantibodies that either neutralize ADAMT13's activity or enhance protein clearance are the major cause of severe ADAMTS13 deficiency in acquired thrombotic thrombocytopenic purpura (TTP). Circulating anti-ADAMTS13 immune complexes that may play a role in disease pathogenesis have been described in plasma of TTP patients. We have developed a new ELISA assay to specifically determine the levels of ADAMTS13-specific circulating immune complexes (CIC) in which the antigen portion is immobilized by a polyclonal anti-ADAMTS13 IgG and the immunoglobulin component detected by a class-specific antibody. An inverse correlation between levels of circulating anti-ADAMTS13 autoantibodies and ADAMTS13-specific CIC has been observed in plasma of acquired TTP patients. The correlation between free anti-ADAMTS13 titer and the level of CIC and insight into the immunoglobulin (sub) classes involved may provide valuable prognostic information about disease severity and progression, and aid patient treatment monitoring. Disclosures:Plaimauer:Baxter Innovations GmbH: Employment. Ferrari:Baxter Innovations GmbH: Employment. Gruber:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Epitope analysis of autoantibodies to ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura

Introduction Autoantibodies to ADAMTS13 have a pivotal role in the pathogenesis of acquired thrombotic thrombocytopenic purpura (TTP). By decreasing the function of ADAMTS13, autoantibodies impair the cleavage of ultra-large von Willebrand factor (UL-VWF) multimers into smaller sizes, leading to lethal platelet-VWF thrombi in the microcirculation. We therefore aimed to determine the sites of autoantibody recognition on ADAMTS13. Materials and Methods In this study, IgG purified from 13 acquired TTP patients were examined to determine their binding sites on ADAMTS13. Immobilized IgG on microtiter plate or proteinG beads was screened by phage library expressing various peptides of ADAMTS13. Results In screening, diverse peptide sequences were obtained from almost all of the ADAMTS13 domains, including the spacer domain, which is considered a major binding site. In particular, we detected an identical amino-acid sequence in the C-terminus of the spacer domain from Gly662 to Val687 that was recognized by autoantibodies from 5 TTP patients. The specific autoantibody was expected to be associated with the plasma levels of the ADAMTS13 antigen or activity, and with the quantity of ADAMTS13 autoantibodies or the inhibitory autoantibody titer in TTP patient plasma. These measurements, however, did not seem to be related to the presence or absence of the specific autoantibody. Conclusions These findings indicate that the specific autoantibody might be a feature of acquired TTP, although its clinical significance remains to be elucidated.

Identification of ADAMTS13 Peptide Sequences Recognized by Autoantibodies in Patients with Acquired Thrombotic Thrombocytopenic Purpura.

Anti-ADAMTS13 autoantibodies are considered to play pivotal roles in the pathophysiology of acquired thrombotic thrombocytopenic purpura (TTP). They inhibit the ADAMTS13 function resulting in the appearance of ultra-large von Willebrand factor (VWF) multimers. Major binding sites of the autoantibodies were reported to be in the cysteine-rich/spacer domains. To clarify the precise peptide sequences recognized by anti-ADAMTS13 IgG autoantibodies, we constructed a random cDNA fragment library expressing various peptides of ADAMTS13 on the surface of lambda phage and screened the library using purified IgG from 13 TTP patients. Diverse peptide sequences were obtained from almost entire ADAMTS13 domains such as metalloprotease, disintegrin, TSP1-1, cysteine-rich, spacer, TSP1- 2, 3, 4, 5, 7, 8 and CUB1. In particular, we detected an identical 26 amino-acid epitope sequence in the C-terminus of spacer domain from Gly662 to Val687 (sp662–687) shared by 5 TTP patients. Moreover, the peptide sequence was exactly included in one of the VWF binding epitope sites that we previously determined (Blood 110 (11), 795a, 2007). We then assessed the impact of specific autoantibody to ADAMTS13 activity measured by FRETS-VWF73 or EIA and ADAMTS13 inhibitor titer in each of TTP patient plasma. However, both of the ADAMTS13 activity and inhibitor titer seemed not correlated with the existence of specific sp662–687 IgG autoantibody. These observations suggest that the autoantibody to sp662–687 may be one specific feature of TTP, although other epitopes are also involved in the pathogenesis of the disorder.

In Vitro Efficacy of Recombinant ADAMTS13 in Acquired TTP Patients with Anti-ADAMTS13 Inhibitory Antibody.

Thrombotic thrombocytopenic purpura (TTP) is associated with a severe functional deficiency of the ADAMTS13 metalloprotease resulting in accumulation of highly adhesive ultra-large von Willebrand factor (ULVWF) multimers. ULVWF promote spontaneous platelet clumping eventually leading to life-threatening widespread microvascular thrombosis in multiple organs. Inhibitory and non-inhibitory anti-ADAMTS13 autoantibodies that result in functional deficiency and/or accelerated in vivo clearance of ADAMTS13 have been detected in patients suffering from acquired idiopathic TTP. Current treatment with plasma exchange therapy is considered to remove the autoantibodies and to replenish plasma with the deficient protease. While researching therapeutic recombinant ADAMST13 (rADAMTS13) as a possible treatment modality for TTP patients, we investigated the in vitro potency of rADAMTS13 to overwhelm and neutralize circulating anti-ADAMTS13 antibodies and concurrently to reinstate plasma ADAMTS13 to normal. Mixing and incubating patient plasma with normal reagent plasma and subsequent FRETSVWF73 activity analysis was used to calculate the individual anti-ADAMTS13 inhibitor titers in 36 untreated idiopathic TTP patient plasma samples with severe ADAMTS13 deficiency (<10% of normal). The inhibitor titers (IU/ml) ranged from 1.5 to 85IU/ml (median of 3.75 IU/ml). To compare the performance of the different patient samples at equal inhibitor titers, we diluted each sample to predefined inhibitor titers (e.g. in patient samples with inhibitor titers <10IU/ml we diluted to 1, 3, 6 and 9IU/ml). Then, incrementally increasing concentrations of purified rADAMTS13 derived from stably transfected CHO cells were added to each pre-set inhibitor titer done for each of the 36 patient samples. Complex formation of rADAMTS13 and anti-ADAMTS13 patient antibody was allowed and the remaining ADAMTS13 activity was analyzed. The input of rADAMTS13 required to saturate circulating inhibitors and to elevate the activity level up to 1U/ml in the plasma was calculated by regression analysis. Our data indicate a linear correlation between supplemented rADAMTS13 and anti-ADAMTS13 antibody titer (for titers <10IU/ml) suggesting that rADAMTS13 should be further investigated as a possible treatment of acquired TTP.

Beta2-Glycoprotein I: Implications for a Regulatory Role in Thrombotic Thrombocytopenic Purpura.

Introduction: Thrombotic thrombocytopenic purpura (TTP) is serologically characterized by a severe deficiency of ADAMTS-13 (von Willebrand factor (vWF)-cleaving protease) activity and a pentad of clinical symptoms; thrombocytopenia, microangiopathic hemolytic anemia, neurological symptoms, renal dysfunction and fever. Due to the absence of ADAMTS-13 activity, ultra-large vWF multimers can be detected in plasma of TTP patients. It is thought that these ultra-large vWF-multimers have a more active (=platelet-binding) conformation than normal vWF-multimers. One of the poorly understood observations in TTP is that some patients have no ADAMTS-13 activity for years without suffering from TTP-related clinical symptoms. Apparently, there are additional factors that regulate the activity of vWF and thereby possibly influences disease activity in TTP patients. It was recently published that plasma protein beta2-glycoprotein I (b2GPI) binds activated vWF and thereby impairs its platelet binding capacity. Given the presence of large, active vWF multimers in TTP, we speculated on a role for b2GPI in acute TTP.Methods: In a population of 38 patients suffering from acute acquired TTP blood samples were drawn. ADAMTS-13 activity, ADAMTS-13 inhibitor levels, anti-ADAMTS-13 IgG titre (Technoclone, Vienna Austria) and b2GPI plasma concentration were measured. Furthermore, we tested the influence of b2GPI on platelet adhesion onto vWF under flow in a flow based-assay (as described by Dong et al., Blood . 2002; 100:4033–9); Endothelial cells were stimulated with histamine and subsequently perfused with platelets in the presence/absence of b2GPI.Results: The median b2GPI plasma concentration in all 38 patients was significantly lower than in controls (87.6ug/ml +/− 7.7 vs 169.0ug/ml +/− 16.7, p=0.001). Furthermore, we found a significant difference in b2GPI plasma levels between patients with a high ADAMTS-13 inhibitor levels (bethesda titre > 3, n=19) and patients having low inhibitor levels (bethesda titre < 3, n=19) of 81.3ug/ml +/− 8.4 compared to 107.5ug/ml +/− 16.1 (p=0.03). In addition, a negative correlation with a trend towards significance was observed between the anti ADAMTS-13-IgG titre and the b2GPI plasma concentration (R=−0.28, p=0.09). In the flow-based assay, we found a concentration dependent inhibition of b2GPI on platelet adhesion. B2GPI coated beads bound directly to the freshly released vWF in a regular (string-like) pattern indicating specific binding of b2GPI to vWF.Conclusion: In this study, we show that b2GPI was able to inhibit platelet adhesion on freshly released VWF on endothelial cells. TTP patients seem to have lower b2GPI levels than healthy controls and the lowest levels were observed in the patient group with high functional ADAMTS-13 inhibitor titres. Together, our findings suggest that the inhibitory effect of b2GPI on vWF-platelet adhesion is attenuated in TTP possibly resulting in increased disease activity.

Development and Performance of the GTI Diagnostics ADAMTS-13 Activity Assay (ATS-13™) and ADAMTS-13 Inhibitor Assay (ATS-13™ INH).

Thrombotic Thrombocytopenic Purpura (TTP) is a disease characterized by the sudden onset of a classical pentad of symptoms: thrombocytopenia, fever, renal insufficiency, neurologic deficit, and microangiopathic hemolytic anemia. ADAMTS-13 is the protease responsible for cleaving von Willebrand Factor (VWF); decreased levels or deficiency of ADAMTS-13 activity has been demonstrated in the plasma of TTP patients. The lack of ADAMTS-13 activity results in the accumulation of multimers of VWF in the plasma and ultimately intravascular platelet aggregation resulting in the clinical symptoms associated with TTP. The presence or absence of inhibitory antibodies directed against ADAMTS-13 influences the treatment protocol for these patients. GTI has developed a rapid, quantitative assay for ADAMTS-13 activity that is based on the cleavage of a von Willebrand Factor substrate and subsequent fluorescent detection. A method comparison was done of GTI's ADAMTS-13 Activity Assay (n=397) using normal plasma, plasma from suspected TTP patients, and plasma from patients suspected to have other coagulation diseases (ITP, HUS, HIT, APS). All samples were also run on the collagen-binding (n=197) or the Furlan method (n=200) for comparison. This data demonstrated good correlation. Assay precision (with-in run, n=20 and assay-to-assay, n=40) was evaluated and there was less than 20% cv total imprecision for samples with levels of activity across the assay range. In addition, GTI has developed an assay for the semi-quantitative detection of inhibitory antibodies directed against the ADAMTS-13 protease. This assay is similar in format and principle to our ATS-13™ kit; however inhibition of the ADAMTS-13 protease activity is determined in the patient's heat-inactivated sample following mixing with a provided control at a single dilution. Assay precision and preliminary method comparison data will be presented.

4A.3 Thrombotic thrombocytopenic purpura

Historical background More than 80 years after the first description of thrombotic thrombocytopenic purpura (TTP) by Eli Moschcowitz [1,2], the pathogenesis of this syndrome is still a mystery and in spite of improved modalities of therapy mortality rate of around 15% presents a great challenge for the medical team. The originally described pentad, including fever, thrombocytopenia, microangiopathic hemolytic anemia, renal failure and neurological abnormalities, is no longer required for establishing the diagnosis of TTP. Thus, the association of thrombocytopenia and microangiopathic hemolytic anemia is sufficient for a TTP alert [3]. It was only in the early 1980s when ultra-large VWF (ULVWF) multimers were found in plasma of TTP patients, suggesting a defect in VWF multimers processing, yielding an increased platelet clumping as a basis for the various clinical manifestations of TTP [4]. In 1997 8 two different groups have described for the first time the lack of a cleaving protease activity among inherited as well as acquired TTP patients, and a serum IgG fraction was identified to induce this effect in the latter group, establishing the autoimmune nature of acquired TTP [5,6]. At that time it was established that a level of <5% of the cleaving protease is required for induction of increased platelet adhesion and TTP symptoms. In 2001 2 the VWF cleaving protease has been characterized as the 13th member of the ADAMTS (A Disintegrin and Metalloprotease with Thrombospondin type motifs) metalloprotease family; the gene was sequenced and mutations responsible for inherited TTP were identified [7 9]. Later studies have clarified in greater detail the mechanism of action of ADAMTS 13, its binding site on the A3 domain of the VWF molecule, and the cleavage site on the A2 domain (Tyr 842 843 Met) [10]. It was also discovered that optimal cleavage of ULVWF is executed under high shear flow conditions, when ULVWF is attached to the surface and stretched due to the flow effect, thus exposing the different sites of interaction with ADAMTS 13 [11 13]. These later discoveries opened the road to the development of new diagnostic as well as therapeutic approaches. Thus different new tests have been developed for diagnosis and monitoring of ADAMTS-13 activity, including the testing of the cleavage products of VWF by collagen binding assays, by electrophoretic as well as by ELISA methods. The latter method was based on the identification of the minimally 73 AA peptide required for cleavage by ADAMTS 13 [14]. In addition, physiological testing of ADAMTS 13 has been developed, based on platelet adhesion under flow as a function of ADAMTS-13 activity in the tested plasma [15]. Treatment of TTP is still a great challenge due to the stormy nature of the disease and the relatively high mortality rate of around 15%. Since 1991 it was established that plasma exchange (PE) is indeed the treatment of choice, yielding reduction in both the ULVWF multimers and the inhibitor of ADAMTS 13, and providing an active ADAMTS 13 and normal-size VWF multimers [16]. The role of other treatment modalities, including steroids, IVIG, aspirin and rituximab, is still a matter for future investigations. Treatment of inherited cases of TTP is based on replacement of the missing ADAMTS 13, routinely by plasma transfusion, and potentially by a recombinant product which is now available. In spite of these advancements there are still many open questions regarding the pathogenesis of TTP and its family of Thrombotic MicroAngiopathies (TMAs). Thus, although a <5% activity level of ADAMTS 13 was found to be necessary for the development of TTP, not all patients with such a low level will develop clinical symptoms. Pathogenesis of other TMAs, including HUS and those associated with bone marrow transplantation, with HIV and those which are drug induced is not yet established. The potential effect of PE in this group of disorders is also not established [17].

A study on the significance of plasma thrombospondin1 in thrombotic thrombocytopenic purpura and the relationship between thrombospondin1 and von Willebrand factor cleaving protease (ADAMTS13)

To measure plasma thrombospondin1 (TSP1) in thrombotic thrombocytopenic purpura (TTP) and other diseases such as idiopathic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE), myocardial infarction, brain infarction, and malignant tumor et al. and 8 patients after bone marrow transplantation (BMT) were also investigated. Then to study on the relationship between TSP1 and von Willebrand factor cleaving protease (ADAMTS13); and to identify the significance of plasma TSP1 in TTP. TSP1 was measured by a commercial kit and the activity of ADAMTS13 was evaluated by residue collagen binding assay. TSP1 in TTP plasma before plasma exchange or plasma infusion was 6.49 mg/L, and stepping up to 13.02 mg/L after therapy, but still significantly lower than 18.34 mg/L in normal control. While the decrease in different degree of ADAMTS13 activity was observed from 0%-52%, and there were 8 samples whose activity of ADAMTS13 were no more than 10%; the different extent of increase in those patients after therapy was demonstrated to be 2.9%-93.4%, only one patient's ADAMTS13 activity was below 10%. The activity of ADAMTS13 in some ITP and SLE patients were mildly decreased (63% +/- 16% and 70% +/- 14% respectively), TSP1 were also decreased (16 mg/L +/- 8 mg/L). TSP1 in patients of myocardial infarction and brain infarction were increased (24.0 mg/L +/- 2.9 mg/L), while ADAMTS13 activity had no significant change (72% +/- 16%). Same things happened in BMT patients. There was some concordance between the decrease of ADAMTS13 activity and TSP1 in plasma of TTP patients. And the change of TSP1 was restricted to TTP. TSP1 may contribute to the episode of TTP in a still unclear fashions.

Development and Performance of the GTI ADAMTS-13 Activity Assay.

Thrombotic Thrombocytopenic Purpura (TTP) is a disease characterized by the sudden onset of a classical pentad of symptoms: thrombocytopenia, fever, renal insufficiency, neurologic deficit, and microangiopathic hemolytic anemia. It was recently discovered that ADAMTS-13 is the protease responsible for cleaving von Willebrand Factor; deficiency of ADAMTS-13 activity has been demonstrated in the plasma of TTP patients. The lack of ADAMTS-13 activity results in the accumulation of multimers of vWF in the plasma and ultimately intravascular platelet aggregation resulting in the clinical symptoms associated with TTP. Currently there are no commercially available diagnostic assays for TTP. Several laboratories have developed “in house” assays that measure the activity of the ADAMTS-13 protease. However, these assays are cumbersome, complex, difficult to perform, and there is a long turn around time, as they require long incubations. The results are difficult to interpret and are not easily quantitated. In addition, there is no standardization of results from laboratory to laboratory, mainly because the assays use various technologies. Determining the extent to which the ADAMTS-13 enzyme activity is decreased is believed to be important in distinguishing a patient with TTP from those presenting with similar clinical symptoms. Until ADAMTS-13 assays are more quantitative, there is not a good method for studying patients with severe decreases in ADAMTS-13 levels versus those who have some intermediate but less than normal level of enzyme activity. Furthermore, the lack of standardized results makes the assays less useful in monitoring the effectiveness of treatment of the TTP patients. GTI has developed a rapid, quantitative assay for ADAMTS-13 activity that is based on the cleavage of a substrate which consists of a peptide fragment of von Willebrand Factor and subsequent fluorescent detection. An initial study using plasma (obtained from the Blood Center of Wisconsin) from 5 TTP patients demonstrated good correlation between the GTI assay and the collagen binding assay, thus demonstrating feasibility of the assay. A more extensive evaluation using 40 normal samples and 55 samples from suspected TTP patients was initiated, the results of which will be reported. Assay sensitivity/limit of detection (based on dilution studies) was 3% normal ADAMTS-13 activity. Assay precision using 3 samples (mean values of 96%, 55%, and 9% normal ADAMTS-13 activity) run in duplicate over 8 days was evaluated. Assay imprecision was less than 10% cv for all samples tested.

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Minimal evidence of platelet and endothelial cell reactive antibodies in thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a syndrome characterized by microvascular thrombosis with thrombocytopenia and end-organ injury. Evidence suggests that platelet or endothelial cell injury may be initial pathological events in TTP. A number of factors in patient plasma, including immunoglobulins, have been proposed to mediate cellular injury in TTP. However, systematic analyses of TTP patient plasma for the presence of platelet or endothelial cell antibodies are lacking. We, therefore, analyzed 48 TTP patient plasma samples for the presence of platelet and endothelial cell antibodies by using enzyme-linked immunosorbent assay, flow cytometry, and microlymphocytotoxicity. Twelve of 48 TTP patient samples (25%) reacted against purified platelet glycoproteins. Nine (19%) also contained antibodies that bound to allogeneic target platelets in flow-cytometric assays. Nine of 48 samples (19%) contained antibodies to human umbilical vein endothelial cells in flow-cytometric assays, and seven of 48 patient samples (15%) bound to human dermal microvascular endothelial cells. Six of 48 (13%) patient plasma samples contained antibodies that bound to human umbilical vein endothelial cells activated with gamma-interferon and tumor necrosis factor-alpha. Of twenty samples that were reactive in one or more platelet or endothelial cell assay, eight contained human leukocyte antigen antibodies reactive in microlymphocytotoxicity. These studies demonstrate that antibodies reactive against platelet or endothelial cell antigens are not prevalent in TTP, and that more than a third of antibodies detected are human leukocyte antigen alloantibodies. Our findings suggest that autoantibodies against platelets or endothelial cells are not important in the pathogenesis of this syndrome.

Thrombotic thrombocytopenic Purpura: Understanding a Disease No Longer Rare

Serial studies of plasma samples from patients during episodes of thrombotic thrombocytopenic purpura (TTP) have often shown either the presence of unusually large (UL) von Willebrand factor (vWf) multimers or, alternatively, absence of the largest plasma vWf forms. The presence of ULvWf multimers in TTP patient plasma may reflect impaired processing of the ULvWf forms released from endothelial cells. The disappearance of ULvWf and large vWf multimers in some TTP patient plasma samples during acute TTP episodes may be predominantly because these ULvWf forms, along with the largest vWf multimers, bind to platelets and cause aggregation. Serial flow cytometry studies of EDTA-whole blood samples from patients with initial episode, intermittent, and chronic relapsing types of TTP confirm that vWf is the likely aggregating agent, perhaps in association with fluid shear stress. The amount of vWf bound to single platelets has been found to be significantly increased during TTP relapses relative to remission periods in patients with all types of TTP. A substance in normal platelet-poor plasma and the cryoprecipitate-depleted fraction of normal plasma (cryosupernatant) is capable in vitro of reversibly reducing the size of ULvWf multimeric forms released by endothelial cells into the somewhat smaller vWf multimers ordinarily in circulation. This activity has characteristics of a limited disulfide bond reductase. The process of ULvWf breakdown may be made irreversible by the tandem proteolysis, catalyzed by a vWf metalloproteinase, of partially reduced vWf multimers. Several patients with chronic relapsing TTP have decreased or absent plasma vWf metalloproteinase activity, apparently on a congenital basis. Adult initial episode and intermittent TTP patients have been found to have vWf metalloproteinase activity inhibited by an autoantibody during, but not after, TTP epidsodes.

Thrombotic thrombocytopenic purpura: understanding a disease no longer rare.

Serial studies of plasma samples from patients during episodes of thrombotic thrombocytopenic purpura (TTP) have often shown either the presence of unusually large (UL) von Willebrand factor (vWf) multimers or, alternatively, absence of the largest plasma vWf forms. The presence of ULvWf multimers in TTP patient plasma may reflect impaired processing of the ULvWf forms released from endothelial cells. The disappearance of ULvWf and large vWf multimers in some TTP patient plasma samples during acute TTP episodes may be predominantly because these ULvWf forms, along with the largest vWf multimers, bind to platelets and cause aggregation. Serial flow cytometry studies of EDTA-whole blood samples from patients with initial episode, intermittent, and chronic relapsing types of TTP confirm that vWf is the likely aggregating agent, perhaps in association with fluid shear stress. The amount of vWf bound to single platelets has been found to be significantly increased during TTP relapses relative to remission periods in patients with all types of TTP. A substance in normal platelet-poor plasma and the cryoprecipitate-depleted fraction of normal plasma (cryosupernatant) is capable in vitro of reversibly reducing the size of ULvWf multimeric forms released by endothelial cells into the somewhat smaller vWf multimers ordinarily in circulation. This activity has characteristics of a limited disulfide bond reductase. The process of ULvWf breakdown may be made irreversible by the tandem proteolysis, catalyzed by a vWf metalloproteinase, of partially reduced vWf multimers. Several patients with chronic relapsing TTP have decreased or absent plasma vWf metalloproteinase activity, apparently on a congenital basis. Adult initial episode and intermittent TTP patients have been found to have vWf metalloproteinase activity inhibited by an autoantibody during, but not after, TTP epidsodes.

Increased von Willebrand factor binding to platelets in single episode and recurrent types of thrombotic thrombocytopenic purpura.

Extensive microvascular platelet aggregation is characteristic of thrombotic thrombocytopenic purpura (TTP). Previous studies have indicated that abnormalities of von Willebrand factor (vWf) are often present in TTP patient plasma. There has not been previously any direct evidence linking these abnormalities to the process of intravascular platelet aggregation in TTP. We used flow cytometry to analyze the binding of vWf to single platelets, and the presence of platelet aggregates, in the blood of 4 children with chronic relapsing (CR) TTP and 5 adults with single episode or recurrent TTP. vWf on the single platelets of CRTTP patients at all time points studied was significantly increased compared to controls, and was increased further as platelet counts decreased to levels below 40,000/microl. The single episode and recurrent adult TTP patients had platelet aggregates in the blood, as well as increased vWf on single platelets, before therapy commenced and thereafter until recovery was in process. In the one unresponsive single episode TTP patient, vWf on single platelets remained elevated, and platelet aggregates persisted, until her death. The platelet alpha-granular protein, P-selectin, was not increased on the single platelets of most TTP blood samples, suggesting that it is vWf from plasma (rather than from alpha-granules) that attaches to platelet surfaces in association with platelet aggregation. These results suggest that vWf-platelet interactions are involved in the platelet clumping process that characterizes TTP.