ADAMTS-13 conformation influences autoimmune recognition in immune thrombotic thrombocytopenic purpura.

BACKGROUND: Patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) have anti-ADAMTS-13 immunoglobulin G (IgG) autoantibodies that enhance ADAMTS-13 clearance and/or inhibit its function. ADAMTS-13 normally circulates in a closed conformation, which is manifested by the interaction of the CUB domains with the central spacer domain. Disruption of the spacer-CUB interaction opens ADAMTS-13, which augments its proteolytic function but may also expose cryptic autoimmune epitopes that promote further autoantibody recognition. OBJECTIVES: To explore differences in autoantibody binding to ADAMTS-13 in its closed or open conformations in patients with iTTP and to correlate these differences with disease-related parameters. METHODS: We developed a novel assay to measure autoantibodies binding to closed and open ADAMTS-13. Autoantibody titer and IgG subclass binding to open or closed ADAMTS-13 were measured in 70 iTTP first presentation samples and correlated with clinical data, remission, and relapse. RESULTS: In 70 patients with iTTP, the mean autoantibody titer against open ADAMTS-13 was, on average, approximately 2-fold greater than that against closed ADAMTS-13, suggesting that ADAMTS-13 opening increases epitope exposure and immune complex formation. Autoantibody titer against closed/open ADAMTS-13 and IgG subclass did not correlate with ADAMTS-13 antigen at presentation. Two patients with iTTP and persistent autoantibodies lost specificity for closed ADAMTS-13 in remission. Recognition of closed/open ADAMTS-13 and autoantibody IgG subclass between the first and second iTTP episodes were very similar. CONCLUSION: ADAMTS-13 autoantibody binding is highly influenced by ADAMTS-13 conformation. Although this does not appear to modify the pathogenicity of autoantibodies, the autoantibody signature at relapse suggests that relapse represents re-emergence of the original autoimmune response rather than de novo presentation.

Autoantibodies enhance ADAMTS-13 clearance in patients with immune thrombotic thrombocytopenic purpura.

BACKGROUND: Severe deficiency in ADAMTS-13 (<10%) and the loss of von Willebrand factor-cleaving function can precipitate microvascular thrombosis associated with thrombotic thrombocytopenic purpura (TTP). Patients with immune-mediated TTP (iTTP) have anti-ADAMTS-13 immunoglobulin G antibodies that inhibit ADAMTS-13 function and/or increase ADAMTS-13 clearance. Patients with iTTP are treated primarily by plasma exchange (PEX), often in combination with adjunct therapies that target either the von Willebrand factor-dependent microvascular thrombotic processes (caplacizumab) or the autoimmune components (steroids or rituximab) of the disease. OBJECTIVES: To investigate the contributions of autoantibody-mediated ADAMTS-13 clearance and inhibition in patients with iTTP at presentation and through the course of the PEX therapy. PATIENTS/METHODS: Anti-ADAMTS-13 immunoglobulin G antibodies, ADAMTS-13 antigen, and activity were measured before and after each PEX in 17 patients with iTTP and 20 acute TTP episodes. RESULTS: At presentation, 14 out of 15 patients with iTTP had ADAMTS-13 antigen levels of <10%, suggesting a major contribution of ADAMTS-13 clearance to the deficiency state. After the first PEX, both ADAMTS-13 antigen and activity levels increased similarly, and the anti-ADAMTS-13 autoantibody titer decreased in all patients, revealing ADAMTS-13 inhibition to be a modest modifier of the ADAMTS-13 function in iTTP. Analysis of ADAMTS-13 antigen levels between consecutive PEX treatments revealed that the rate of ADAMTS-13 clearance in 9 out of 14 patients analyzed was 4- to 10-fold faster than the estimated normal rate of clearance. CONCLUSION: These data reveal, both at presentation and during PEX treatment, that antibody-mediated clearance of ADAMTS-13 is the major pathogenic mechanism that causes ADAMTS-13 deficiency in iTTP. Understanding the kinetics of ADAMTS-13 clearance in iTTP may now enable further optimization of treatment of patients with iTTP.

Identification of a novel genetic locus associated with immune-mediated thrombotic thrombocytopenic purpura.

Immune thrombotic thrombocytopenic purpura (iTTP) is an ultra-rare, life-threatening disorder, mediated through severe ADAMTS13 deficiency causing multi-system micro-thrombi formation, and has specific human leukocyte antigen associations. We undertook a large genome-wide association study to investigate additional genetically distinct associations in iTTP. We compared two iTTP patient cohorts with controls, following standardized genome-wide quality control procedures for single-nucleotide polymorphisms and imputed HLA types. Associations were functionally investigated using expression quantitative trait loci (eQTL), and motif binding prediction software. Independent associations consistent with previous findings in iTTP were detected at the HLA locus and in addition a novel association was detected on chromosome 3 (rs9884090, P=5.22x10-10, odds ratio 0.40) in the UK discovery cohort. Meta-analysis, including the French replication cohort, strengthened the associations. The haploblock containing rs9884090 is associated with reduced protein O-glycosyltransferase 1 (POGLUT1) expression (eQTL P<0.05), and functional annotation suggested a potential causative variant (rs71767581). This work implicates POGLUT1 in iTTP pathophysiology and suggests altered post-translational modification of its targets may influence disease susceptibility.

Shrinking Weibel-Palade bodies prevents high platelet recruitment in assays using thrombotic thrombocytopenic purpura plasma.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP), caused by a genetic or autoimmune-driven lack of ADAMTS-13 activity, leads to high levels of the ultra-large von Willebrand factor (VWF) multimers produced by endothelial cells, causing excess platelet recruitment into forming thrombi, often with mortal consequences. Treatments include plasma infusion or replacement to restore ADAMTS-13 activity, or prevention of platelet recruitment to VWF. OBJECTIVES: We tested a different approach, exploiting the unique cell biology of the endothelium. Upon activation, the VWF released by exocytosis of Weibel-Palade bodies (WPBs), transiently anchored to the cell surface, unfurls as strings into flowing plasma, recruiting platelets. Using plasma from patients with TTP increases platelet recruitment to the surface of cultured endothelial cells under flow. WPBs are uniquely plastic, and shortening WPBs dramatically reduces VWF string lengths and the recruitment of platelets. We wished to test whether the TTP plasma-driven increase in platelet recruitment would be countered by reducing formation of the longest WPBs that release longer strings. METHODS: Endothelial cells grown in flow chambers were treated with fluvastatin, one of 37 drugs shown to shorten WPBs, then activated under flow in the presence of platelets and plasma of either controls or patients with TTP. RESULT: We found that the dramatic increase in platelet recruitment caused by TTP plasma is entirely countered by treatment with fluvastatin, shortening the WPBs. CONCLUSIONS: This potential approach of ameliorating the endothelial contribution to thrombotic risk by intervening far upstream of hemostasis might prove a useful adjunct to more conventional and direct therapies.

Immune thrombocytopenia in adults: A single-centre review of demographics, clinical features and treatment outcomes.

OBJECTIVES: Primary immune thrombocytopenia (ITP) is a bleeding disorder characterised by an isolated thrombocytopenia in the absence of an alternative diagnosis. The condition is highly heterogeneous with some patients requiring multiple of therapy before achieving response. In this study, we collected data on a large cohort of primary ITP patients with the objective of identifying variables which may predict treatment requirements. METHODS: We collected data on 379 patients, 275 with a confirmed diagnosis of primary ITP included demographics, baseline laboratory results and treatments. These were compared against treatment responses and lines of therapy. RESULTS: Patients who presented with a platelet count of <30 × 109 /L or bleeding symptoms were observed to require more subsequent lines of therapy (P-value <0.001). 32% of patients (n = 87) received no treatment, and these patients had a significantly higher median count compared to those with required >2 lines of therapy (P-value <0.001). Superior response rates were demonstrated with thrombopoietin receptor agonists when compared with other agents irrespective of baseline characteristics. CONCLUSIONS: Platelet counts at diagnosis are a potentially strong predictive indicator of subsequent lines of therapy. Patients with bleeding symptoms at diagnosis were more likely to have lower median platelets counts.

Comparison of Rituximab originator (MabThera) to biosimilar (Truxima) in patients with immune-mediated thrombotic thrombocytopenic purpura.

Immune thrombotic thrombocytopenic purpura (iTTP) is an acute, multisystem thrombotic microangiopathy mediated by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) autoantibodies. Immunosuppression with anti-CD20 therapy is the mainstay of treatment. MabThera's patent has now expired and biosimilars have been approved. Eighty-four consecutive patient episodes over 2 years, prior to and following our switch to Truxima are presented. Day 1 (D1), Day 28 (D28) and 3-month platelet counts, ADAMTS13 activity, and CD19 levels, adverse reactions and infective complications were recorded. Platelet counts were not significantly different between acute MabThera and Truxima treatment (D1 P = 0.085, D28 P = 0.77, 3 months P = 0.71) and electively (D1 P = 0.79, D28 P = 0.68, 3 months P = 0.99). ADAMTS13 recovery also was not significantly different acutely (D1 P = 0.99, D28 P = 0.27, 3 months P = 0.26) and electively (D1 P = 0.59, D28 P = 0.61, 3 months P = 0.34). CD19% depletion at D1 and 3 months was not significantly different acutely (D1 P = 0.52, 3 months P = 0.56) and electively (D1 P = 0.22, 3 months P = 0.19). Infusion reactions and infective complications were comparable with both therapies. This is the first series of the Rituximab biosimilar Truxima to be reported in iTTP, demonstrating equivalence to MabThera in terms of ADAMTS13 recovery, CD19 depletion, and platelet count at D28 and 3 months post-administration, with comparable infusion and infective complications. The financial benefit of the biosimilar anti-CD20 is considerable.

How we manage thrombotic microangiopathies in pregnancy.

Differentiation between the thrombotic microangiopathies (TMAs) that present in pregnancy may be clinically challenging, but is critical to ensure correct management because of the impact on fetal and maternal outcomes. Thrombotic thrombocytopenic purpura (TTP) and atypical haemolytic uraemic syndrome (aHUS) are medical/obstetric emergencies that require specialist input, both at the time of acute diagnosis and follow-up in subsequent pregnancies. Features of preeclampsia and HELLP syndrome (haemolysis, elevated liver enzymes, low platelets) may precede or be present in evolving TTP or aHUS. Clinicians need to be mindful of how a presumed diagnosis of a specific TMA in pregnancy may evolve and be prepared to frequently reassess signs and symptoms and revise the diagnosis and management plan accordingly.

Pathogenicity of Anti-ADAMTS13 Autoantibodies in Acquired Thrombotic Thrombocytopenic Purpura.

BACKGROUND: Acquired thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease in which anti-ADAMTS13 autoantibodies cause severe enzyme deficiency. ADAMTS13 deficiency causes the loss of regulation of von Willebrand factor multimeric size and platelet-tethering function, which results in the formation of disseminated microvascular platelet microthrombi. Precisely how anti-ADAMTS13 autoantibodies, or antibody subsets, cause ADAMTS13 deficiency (ADAMTS13 activity generally < 10%) has not been formally investigated. METHODS: We analysed 92 acquired TTP episodes at presentation, through treatment and remission/relapse using epitope mapping and functional analyses to understand the pathogenic mechanisms of anti-ADAMTS13 IgG. RESULTS: 89/92 of TTP episodes had IgG recognising the ADAMTS13 N-terminal domains. The central spacer domain was the only N-terminal antigenic target detected. 38/92 TTP episodes had autoantibodies recognising the N-terminal domains alone; 54/92 TTP episodes also had antibodies against the ADAMTS13 C-terminal domains (TSP2-8 and/or CUB domains). Changes in autoantibody specificity were detected in 9/16 patients at relapse, suggesting a continued development of the disease. Functional analyses on IgG from 43 patients revealed inhibitory IgG were limited to anti-spacer domain antibodies. However, 15/43 patients had autoantibodies with no detectable inhibitory action and as many as 32/43 patients had autoantibodies with inhibitory function that was insufficient to account for the severe deficiency state, suggesting that in many patients there is an alternative pathogenic mechanism. We therefore analysed plasma ADAMTS13 antigen levels in 91 acquired TTP presentation samples. We demonstrated markedly reduced ADAMTS13 antigen levels in all presentation samples, median 6% normal (range 0-47%), with 84/91 patients having < 25% ADAMTS13 antigen. ADAMTS13 antigen in the lowest quartile at first presentation was associated with increased mortality (odds ratio 5.7). CONCLUSIONS: Anti-spacer domain autoantibodies are the major inhibitory antibodies in acquired TTP. However, depletion of ADAMTS13 antigen (rather than enzyme inhibition) is a dominant pathogenic mechanism. ADAMTS13 antigen levels at presentation have prognostic significance. Taken together, our results provide new insights into the pathophysiology of acquired TTP.

Thrombotic thrombocytopenic purpura: basic pathophysiology and therapeutic strategies.

VWF is a multimeric plasma glycoprotein that specifically recruits platelets to sites of vessel injury. VWF multimeric size is central to this function, with larger multimers being more hemostatically active. Regulation of VWF multimeric size is mediated by the plasma metalloprotease ADAMTS13 (A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motifs, member 13). This enzyme can only recognize and cleave VWF when it is unraveled by rheological shear forces of the flowing blood. After the exposure of cryptic exosites, VWF recognition by ADAMTS13 involves multiple interactions that enable the protease to cleave VWF. Loss of VWF multimer size regulation caused by severe ADAMTS13 deficiency (either inherited or acquired) is associated with the microvascular thrombotic disorder thrombotic thrombocytopenic purpura (TTP). The sequelae associated with TTP are widely thought to be linked to hyperreactive circulating VWF that cause unwanted platelet aggregation in the high shear environment of the microvasculature. Diagnosis of TTP is primarily made through a combination of symptoms, analysis of plasma ADAMTS13 activity, and detection of inhibitory anti-ADAMTS13 antibodies. Current frontline treatments for TTP include plasma exchange, which serves to remove inhibitory antibodies (in acquired TTP) and provide a source of functional ADAMTS13, and steroids to treat the autoimmune component of acquired TTP. The use of anti-CD20 therapy has also exhibited encouraging results in the treatment of acquired TTP. Newer therapeutic strategies that are currently being explored or are in development include recombinant ADAMTS13, a hyperreactive ADAMTS13 variant, and anti-VWF therapy. This review discusses the basic biochemistry of VWF and ADAMTS13, their dysfunction in TTP, and therapeutic approaches for the amelioration of TTP.

Residual plasmatic activity of ADAMTS13 is correlated with phenotype severity in congenital thrombotic thrombocytopenic purpura.

The quantification of residual plasmatic ADAMTS13 activity in congenital thrombotic thrombocytopenic purpura (TTP) patients is constrained by limitations in sensitivity and reproducibility of commonly used assays at low levels of ADAMTS13 activity, blunting efforts to establish genotype-phenotype correlations. In the present study, the residual plasmatic activity of ADAMTS13 was measured centrally by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (limit of detection = 0.5%) in 29 congenital TTP patients. The results were used to study correlations among ADAMTS13 genotype, residual plasmatic activity, and clinical phenotype severity. An ADAMTS13 activity above 0.5% was measured in 26 (90%) patients and lower levels of activity were associated with earlier age at first TTP episode requiring plasma infusion, more frequent recurrences, and prescription of fresh-frozen plasma prophylaxis. Receiver operating characteristic curve analysis showed that activity levels of less than 2.74% and 1.61% were discriminative of age at first TTP episode requiring plasma infusion < 18 years, annual rate of TTP episodes > 1, and use of prophylaxis. Mutations affecting the highly conserved N-terminal domains of the protein were associated with lower residual ADAMTS13 activity and a more severe phenotype in an allelic-dose dependent manner. The results of the present study show that residual ADAMTS13 activity is associated with the severity of clinical phenotype in congenital TTP and provide insights into genotype-phenotype correlations.

Evidence of persistent neurologic injury following thrombotic thrombocytopenic purpura.

Despite improvements in our understanding of the pathophysiology of thrombotic thrombocytopenic purpura (TTP), little data exist regarding the long-term sequelae following a diagnosis of TTP. We present the results of a comprehensive evaluation of neurologic injury that included a magnetic resonance imaging (MRI), a neurocognitive testing, and an evaluation of health-related quality of life. Twenty-seven patients with a history of idiopathic TTP functioning normally in their activities of daily living were recruited from existing patient cohorts at both the Ohio State University (n 5 12) (Columbus) and the University College London Hospitals (n 5 15) (London, UK). Nine of 23 (39%) of the MRI studies were abnormal; 17/27 (63%) patients demonstrated neurocognitive impairment, particularly in tests of visual learning and memory. Health-related quality of life scores were also significantly lower than age- and gender-matched US norms for both the composite mental component score and physical component score. These data suggest that the prevalence of neurologic findings in TTP patients in remission is quite high and is largely undetected by routine clinical evaluations. Further longitudinal study will be required to define the risk for neurologic injury and the long-term prognosis in patients previously diagnosed with TTP.

Isolated bone marrow involvement in diffuse large B cell lymphoma: a report of three cases with review of morphological, immunophenotypic and cytogenetic findings.

Diffuse large B cell lymphoma (DLBL) comprises a heterogenous entity characterized by the presence of large cells, exhibiting a mature B cell phenotype. The high proliferation rate and aggressive disease remain a therapeutic challenge, but the apparent biological diversity permits a risk-stratification model for prognostic grouping through the International Prognostic Index (IPI). Empirical to this approach is the consideration of cytogenetic data, offering an insight into the pathogenetic events which may underlie neoplastic clonal evolution and disease progression. We describe three cases of DLBL presenting with isolated marrow disease, a rare primary finding in this lymphoma. All three cases showed involvement of blood and bone marrow without evidence of splenic or lymph node involvement on imaging studies. Histological and immunophenotypic findings were similar in all three cases, outlining the phenotypic maturity of this disease. Cytogenetic analysis revealed complex karyotypes in the two cases examined. M-FISH (multicolour fluorescent in situ hybridization) performed on bone marrow from case 1 showed several cryptic translocations not evident on G-banding, including a novel translocation between 2p and 9p, and an unbalanced translocation between 14q and 11q. Cytogenetic analysis in case 2 showed abnormalities involving 7q, 9p at the site of the INK4a gene, and the bcl-2 locus, findings confirmed by M-FISH. These cases serve to highlight the biological and cytogenetic heterogenity of DLBL and emphasize the need for complementary investigations in the characterization of this entity.