Autoantibodies directed against ADAMTS13 prohibit the processing of von Willebrand factor multimers, initiating a rare and life-threatening disorder called acquired thrombotic thrombocytopenic ...purpura (TTP). Recently, HLA-DRB1*11 has been identified as a risk factor for the development of acquired TTP. Here, we identified ADAMTS13-derived peptides presented on MHC class II alleles from 17 healthy donors. Dendritic cells from a panel of both HLA-DRB1*11–positive and -negative donors were pulsed with ADAMTS13, and the HLA-DR–presented peptide repertoire was analyzed by mass spectrometry. Interestingly, at low antigen concentrations, HLA-DRB1*11- or DRB1*03-positive donors presented a limited number of CUB2-derived peptides. Pulsing of dendritic cells using higher concentrations of ADAMTS13 resulted in the presentation of larger numbers of ADAMTS13-derived peptides by both HLA-DRB1*11–positive and -negative donors. Although the presented peptides were derived from several ADAMTS13 domains, inspection of the peptide profiles revealed that CUB2 domain–derived peptides were presented with a higher efficiency when compared with other peptides. Remarkably, dendritic cells from DRB1*11 donors pulsed with higher concentrations of ADAMTS13-present derivatives of a single CUB2-derived peptide. We hypothesize that functional presentation of CUB2-derived peptides on HLA-DRB1*11 contributes to the onset of acquired TTP by stimulating low-affinity, self-reactive CD4+ T cells.
•ADAMTS13 derived peptides presented on HLA-DR; implications for acquired TTP.•CUB2 domain peptide binds to risk-allele HLA-DRB1*11.
BACKGROUND
Platelet (PLT) concentrates are prophylactically given to prevent major bleeding complications. The corrected count increment (CCI) is currently the only tool to monitor PLT transfusion ...efficacy. PLT function tests cannot be performed in patients with thrombocytopenia. Therefore, an optimized agonist‐induced assay was used to determine PLT function, in patients with severe thrombocytopenia before and after transfusion.
STUDY DESIGN AND METHODS
PLT reactivity toward adenosine diphosphate (ADP), thrombin receptor–activating peptide SFLLRN (TRAP), and convulxin (CVX) was assessed by flow cytometry. P‐selectin expression was measured on PLTs from 11 patients with thrombocytopenia before and 1 hour after transfusion, on stored PLTs, and on stored PLTs incubated for 1 hour in whole blood from patients ex vivo.
RESULTS
The mean (±SEM) CCI after 1 hour was 11.4 (±1.5). After transfusion, maximal agonist‐induced PLT P‐selectin expression was on average 29% higher for ADP (p = 0.02), 25% higher for TRAP (p = 0.007), and 24% higher for CVX (p = 0.0008). ADP‐induced reactivity of stored PLTs increased with 46% after ex vivo incubation (p = 0.007). These PLTs also showed an overall higher P‐selectin expression compared to PLTs 1 hour after transfusion (p = 0.005). After normalization for this background expression, a similar responsiveness was observed.
CONCLUSIONS
Our study shows recovery of PLT function after transfusion in patients with thrombocytopenia. The majority of functional PLTs measured after transfusion most likely represents stored transfused PLTs that regained functionality in vivo. The difference in baseline P‐selectin expression in vivo versus ex vivo suggests a rapid clearance from circulation of PLTs with increased P‐selectin expression.
Summary
Platelets and platelet-monocyte interaction play an important role in inflammation. Both pro- and anti-inflammatory effects of platelet inhibition have been reported in animal models. This ...study aimed to investigate the effect of platelets and platelet inhibition by the new P2Y
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receptor antagonist ticagrelor on monocyte function, as assessed by cytokine responses to Toll-like Receptor (TLR) ligands. In a set of
in vitro
experiments, peripheral blood mononuclear cells (PBMC) incubated with the TLR2 ligand Pam3CSK4 produced less cytokines in the presence of platelets, whereas platelets increased the production of cytokines when PBMC were exposed to TLR4 ligand lipopolysaccharide (LPS). These effects of platelets were dependent on direct platelet-leukocyte aggregation and for the Pam3CSK4-induced response, on phagocytosis of platelets by monocytes. In a double blind, placebo-controlled crossover trial in healthy volunteers, a single oral dosage of 180 mg ticagrelor reduced platelet-monocyte complex (PMC) formation. This was associated with an increase in pro-inflammatory cytokines in blood exposed to Pam3CSK4, but a decrease in these cytokines in blood exposed to LPS. These findings show that platelets differentially modulate TLR2- and TLR4-mediated cytokine responses of PBMC. Through inhibition of platelet-leukocyte interaction, P2Y
12
receptor antagonists may either exert a pro- or anti-inflammatory effect during infections depending on the TLR primarily involved.
Acquired thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder that results from the development of auto-antibodies against ADAMTS13. Recent research suggests that HLA-DRB1*11 ...provides a risk factor for the development of acquired TTP. Previous work in our department identified ADAMTS13 derived peptides that are presented on MHC class II (Sorvillo et al. 2013). Pulsing of dendritic cells from HLA typed healthy donors showed preferential presentation of the CUB-2 domain derived peptides "FINVAPHAR" and "ASYILIRD" on HLA-DRB1*11 and HLA DRB1*03 respectively. In this study we screened peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with acquired TTP for reactive CD4+ T cells against the previously identified peptides.
The presence of CD4+ T cells recognizing FINVAPHAR or the ASYILIRD peptide was addressed by flow cytometry. PBMCs were stimulated for 24 hours and up regulation of CD40L was visualized on CD4+ T cells. Stimulation with Staphylococcus aureus enterotoxin B (SEB) and overlapping peptides from cytomegalovirus (CMV) pp65 protein were used as controls. Several samples derived from patients in the acute phase of the disease were analyzed in this study.
A high frequency of FINVAPHAR responsive CD4+ T cells was identified by monitoring the up regulation of CD40L on CD4+ T cells in a sample from a HLA-DRB1*1104 patient. Four percent of the CD4+ T cells were activated in response to the FINVAPHAR peptide. A control peptide in which the anchor-residues of the FINVAPHAR peptide were modified did not induce activation of CD4+ T cells. Incubation with full length ADAMTS13 also induced activation of CD4+ T cells although to a lesser extent. No FINVAPHAR reactive CD4+ T cells were observed after stimulation in HLA-DRB1*1104 positive healthy individuals.
PBMC samples from HLA-DRB1*0301 positive patients were screened for CD4+ T cells reactive with the ASYILIRD peptide. ASYILIRD positive CD4+ T cells were detected in samples of 2 DRB1*0301 positive patients. In one patient 0.5% reactive CD4+ T cells were detected after stimulation with the ASYILIRD peptide. In samples derived from a second patient 1.5% of CD4+ T cells reactive against the ASYILIRD peptide were detected. Stimulation with a control peptide in which the anchor residues were modified did not result in activation of CD4+ T cells in both patients. Stimulation with SEB and CMV derived peptides resulted in activation of CD4+ T cells in the samples analyzed. No CD40L+ CD4+ T cells were observed after incubation with full length ADAMTS13, this may be due to insufficient processing of ADAMTS13 under our experimental conditions.
Taken together our data provide the first evidence for the presence of ADAMTS13 reactive CD4+ T cells in acquired TTP. Based on our results we propose that FINVAPHAR and ASYILIRD CD4+ T cells are involved in the onset and/or relapse of acquired TTP.
No relevant conflicts of interest to declare.
Introduction: Immune Thrombocytopenia (ITP) is still diagnosed by exclusion of many other causes for thrombocytopenia. In order to prevent misdiagnosis, an ITP-specific diagnostic test would be very ...helpful. In addition, characterization of glycoprotein specificity of platelet autoantibodies may explain (the severity of) bleeding symptoms and response to therapy. In this regard, we optimized the cut-off value of the direct monoclonal antibody immobilization of platelet antigens (MAIPA) assay for detection of platelet glycoprotein directed autoantibodies and re-evaluated its sensitivity and specificity for the diagnosis of ITP.
Materials and Methods: The MAIPA was performed as part of our routine protocol, described by Kiefel et al. (1985). For the determination of a new cut-off value, and to calculate the sensitivity and specificity, blood samples were tested from 462 healthy blood donors and 43 non-immune-mediated thrombocytopenic patients, suffering from either hematological malignancies or aplastic anemia (n=20), hepato-splenomegalic pooling (n=3), drug-induced thrombocytopenias (n=4), viral infections (n=6), pregnancy related thrombocytopenia (n=7), pseudothrombocytopenias (n=2) and microangiopathy (n=1) and from 60 known ITP patients. We then have tested 120 prospectively collected samples from thrombocytopenic patients, sent for diagnostic tests to our laboratory, and categorized these samples based on subsequently obtained clinical evaluation into 'most likely ITP' (n=64) or 'most likely non-ITP' (n=56).
Results: The calculated direct MAIPA sensitivity and specificity, using a cut-off value of E=0.130, in the ITP and non-ITP control groups (n=103) were 85% (95% CI, 73-93%) and 100% (95% CI, 92-100%), respectively (see Figure). The platelet auto-antibodies in the ITP control group (n=60) were directed against glycoprotein (GP)IIb/IIIa (66.7%), GPIb/IX (60%), GPV (51.7%), GPIa/IIa (40.6%) and/or GPIV (26.9%). The calculated sensitivity and specificity for detection of platelet auto-antibodies in the prospective diagnosed ITP and non-ITP patient control groups (n=120) were 75% (95% CI, 63-85%) and 96% (95% CI, 88-100%), respectively (see Figure). For this group of patients, the direct MAIPA showed, for diagnosis of ITP, a negative predictive value (NPV) of 77% (95% CI, 66-86%) and a positive predictive value (PPV) of 96% (95% CI, 86-100%).
Furthermore, in 23 ITP patients the sequential sampling in a rituximab-treatment protocol showed platelet counts that were significantly and inversely correlated with the direct-MAIPA extinctions (p=0.006). In this respect, we excluded that higher platelet counts impaired the detection of platelet autoantibodies - e.g. by diluting them over an higher platelet mass-since autoantibodies were successfully detected in samples from ITP patients (n=4) with, as a result of splenectomy, platelet counts above 100 x 109/L and in untreated ITP patients with platelet counts between 75 and 100 x 109/L. These findings may implicate that response to rituximab as reflected by a rise in platelet counts is dependent on antibody presence, but the mechanism of effective lowering of platelet autoantibody levels by rituximab is still unclear.
In conclusion, the revisited direct MAIPA showed to be a valuable technique for the detection of platelet autoantibodies both at diagnosis and during treatment and can possibly become a guide for optimizing therapy towards a more personalized treatment of ITP.
Direct MAIPA O.D. above 0.13 is considered positive. Control samples: historically well characterized ITP patients. Prospective study: requests for serological ITP diagnostics, after final clinical evaluation classified as ITP or non-ITP.
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Schipperus:Novartis: Consultancy.
Platelets are key cells in atherosclerosis and acute cardiovascular events. Platelet hyperreactivity and increased platelet-monocyte aggregation (PMA) are found in HIV-infected patients and may ...contribute to the excess cardiovascular risk. The integrase inhibitor raltegravir (RAL) has been associated with better residual viral suppression and reduction in inflammatory and coagulation biomarkers. The aim of our study was to investigate whether RAL-treated patients have reduced platelet reactivity and PMA.
We performed a cross-sectional study involving 80 virologically suppressed adult HIV1-infected patients on a RAL-based (n = 25), nonnucleoside reverse transcriptase inhibitor (NNRTI)-based (n = 30) or a protease inhibitor based (n = 25) regimen and 30 healthy controls. Platelet reactivity was determined by measuring platelet P-selectin expression and the binding of fibrinogen to platelets to stimulation with two concentrations of ADP. PMA was determined by measuring the expression of the platelet marker CD42b on CD14 positive cells.
HIV-infected individuals had higher platelet reactivity and PMA than controls. RAL-treated individuals showed significantly lower P-selectin expression to stimulation with low (P = 0.026 vs. NNRTI and P = 0.005 vs. protease inhibitor group) and high-dose ADP (P = 0.009 vs. NNRTI and P = 0.003 vs. protease inhibitor group). A similar trend for was found for fibrinogen binding, although only the difference in P-selectin expression between RAL and protease inhibitor treated patients reached statistical significance (P = 0.038). PMA was also lower in the RAL group than in the NNRTI (P = 0.037) and protease inhibitor (P = 0.034) groups.
Use of a RAL-based regimen was associated with a reduction in persistent HIV-induced platelet hyperreactivity and PMA compared with NNRTI and protease inhibitor based regimen.
Abstract
Von Willebrand factor (VWF) is one of the most important proteins of the hemostatic system. Its multimeric state is essential for its natural function to guide platelets to sites of injury. ...ADAMTS13 is the key protease that regulates the multimeric state of VWF. Without ADAMTS13, VWF multimers can grow to pathologically large sizes. This is a risk factor for the life-threatening condition thrombotic thrombocytopenic purpura (TTP). In this condition, VWF-rich thrombi occlude the microvasculature of various tissues. Intriguingly, a complete ADAMTS13 deficiency does not cause continuous TTP, either in patients or genetically targeted mice. Instead, TTP occurs in episodes of disease, separated by extended periods of remission. This indicates that regulating factors beyond ADAMTS13 are likely involved in this pathologic cascade of events. This raises the question of what really happens when ADAMTS13 is (temporarily) unavailable. In this review, we explore the possible role of complementary mechanisms that are capable of modifying the thrombogenic potential of VWF.
Abstract only
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Background: This randomized phase III trial assessed whether intensification of rituximab (R) during the first 4 cycles of R-CHOP can improve outcome of diffuse large B-cell ...lymphoma (DLBCL) patients compared with standard R-CHOP. Patients in complete remission (CR) after induction treatment were randomized between rituximab maintenance and observation. Intensification of rituximab was not more effective than standard R-CHOP, showing same CR-rates and progression free survival after induction (ASCO 2016 # 7504). Here, we report the results of the second randomization for rituximab maintenance therapy. Methods: Patients in CR after R-CHOP were randomized between 24 months of rituximab maintenance 375 mg/m
2
intravenous every 8 weeks (n = 199 ) or observation (n = 199). CT scans were performed at 6, 12, 18 and 24 months in both arms. The primary endpoint was disease free survival (DFS) from maintenance randomization. Secondary endpoints were overall survival (OS) and adverse events (AEs). ( www.trialregister.nl NTR1014). Results: Median age was 65 years (range 31-80), 48% were 66 years or older and 49% were male. The majority of patients (54%) had a high-intermediate or high aa-IPI score. After a median follow-up of 79.9 months (maximum 125.7 months), the 5-year DFS rate was 79% for rituximab maintenance versus 74% for observation. This difference was not statistically significant, with a hazard ratio of 0.83 (95% confidence interval 0.57-1.19, p= 0.31, adjusted for age and aa-IPI). The secondary endpoint OS was also not significantly different (85% versus 83% at 5 years). No clinical subgroup benefited from rituximab maintenance. Toxicity was mild. Among patients who received rituximab maintenance CTCAE grade 3 and 4 AEs were reported in 17% and 6% of patients, respectively. Infection was the most frequent AE, a grade 3 infection occurred in 6% of patients. Neutropenia was seen in 1% (grade 3) and 3% (grade 4) of patients. Conclusions: Rituximab maintenance therapy provides no additional benefit for DLBCL patients in first CR after R-CHOP. Clinical trial information: www.trialregister.nl NTR1014.
IntroductionThe risk of major bleeding complications in catheter directed thrombolysis (CDT) for acute limb ischemia (ALI) remains high, with reported major bleeding complication rates in up to 1 in ...every 10 treated patients. Fibrinogen was the only predictive marker used for bleeding complications in CDT, despite the lack of high quality evidence to support this. Therefore, recent international guidelines recommend against the use of fibrinogen during CDT. However, no alternative biomarkers exist to effectively predict CDT-related bleeding complications. The aim of the POCHET biobank is to prospectively assess the rate and etiology of bleeding complications during CDT and to provide a biobank of blood samples to investigate potential novel biomarkers to predict bleeding complications during CDT.MethodsThe POCHET biobank is a multicentre prospective biobank. After informed consent, all consecutive patients with lower extremity ALI eligible for CDT are included. All patients are treated according to a predefined standard operating procedure which is aligned in all participating centres. Baseline and follow-up data are collected. Prior to CDT and subsequently every six hours, venous blood samples are obtained and stored in the biobank for future analyses. The primary outcome is the occurrence of non-access related major bleeding complications, which is assessed by an independent adjudication committee. Secondary outcomes are non-major bleeding complications and other CDT related complications. Proposed biomarkers to be investigated include fibrinogen, to end the debate on its usefulness, anti-plasmin and D-Dimer.Discussion and conclusionThe POCHET biobank provides contemporary data and outcomes of patients during CDT for ALI, coupled with their blood samples taken prior and during CDT. Thereby, the POCHET biobank is a real world monitor on biomarkers during CDT, supporting a broad spectrum of future research for the identification of patients at high risk for bleeding complications during CDT and to identify new biomarkers to enhance safety in CDT treatment.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background. Deficient ADAMTS13 activity (TS13:act <10%) induced by anti-ADAMTS13 autoantibodies (autoAbs) causes immune-mediated thrombotic thrombocytopenic purpura (iTTP). Recently we showed that an ...open ADAMTS13 conformation is characteristic for acute iTTP patients, while folded ADAMTS13 was found in 78% of iTTP patients in remission with an TS13:act >50%. However, also iTTP patients in remission with a persistent (<10%) or moderately restored (10-50%) TS13:act have been described, but their ADAMTS13 conformation is unknown. Intriguingly, the factor responsible for inducing open ADAMTS13 in iTTP patients remains elusive. Identifying the cause of open ADAMTS13 in iTTP will help better understand the pathophysiology of iTTP and could help appreciate the prognosis and better manage the prevention of subsequent relapses.
Aim. Determine ADAMTS13 conformation in plasma of iTTP patients during acute TTP and remission when TS13:act is <10%, moderately restored (10-50%) or >50% and investigate if anti-ADAMTS13 autoAbs induce conformational changes in ADAMTS13.
Methods. TS13:act was determined in 120 iTTP plasma samples from 4 different centers (Marseille, Milan, Budapest, Utrecht). Samples were categorized according to the presence of clinical symptoms (acute versusremission) and their TS13:act in remission (>50%, 10-50%,<10%). Next, ADAMTS13 conformation was determined in all samples using our ADAMTS13 conformation ELISA. Additionally, presence of anti-ADAMTS13 autoAbs was also determined via ELISA. Finally, IgG's from 18 acute iTTP plasma samples were purified and added to folded ADAMTS13 from healthy donor (HD) plasma to test whether iTTP IgG's are able to induce the open HD ADAMTS13 conformation.
Results. Of the 120 iTTP plasma samples, 46 were obtained during the acute (clinical signs present) and 74 during the remission phase (clinical signs absent). Further subdividing remission samples showed that TS13:act was >50% in 41, 10-50% in 14 and <10% in 19 samples. ADAMTS13 was open in 98% (45/46) of the acute samples and folded in 71% (29/41) of the remission samples with TS13:act >50%, confirming our previous results. Interestingly, ADAMTS13 was open in 93% and 89% of remission samples with TS13:act 10-50% and <10%, respectively (chi square, P<0.0001). Since anti-ADAMTS13 autoAbs influence TS13:act in iTTP patients, we next could demonstrate that open ADAMTS13 conformation was linked with presence of anti-ADAMTS13 autoAbs (chi square, P<0.0001) suggesting that anti-ADAMTS13 autoAbs could be a factor able to induce an open ADAMTS13 conformation in iTTP patients. To further test this hypothesis, we purified IgG's from 18 acute iTTP plasma's with open ADAMTS13 and added them to HD plasma containing closed ADAMTS13, where 14 of the 18 patient IgG pools (78%) did induce the open conformation in HD ADAMTS13, indicating that patient anti-ADAMTS13 autoAbs indeed can induce conformational changes in ADAMTS13.
Conclusion. We show that ADAMTS13 is not only in the open conformation in iTTP patient plasma during the acute phase but also in remission when TS13:act is <10% or 10-50%. Hence, the presence of open ADAMTS13 in those remission patients indicates that the underlying pathophysiology is still ongoing, emphasizing the need for a close monitoring of those patients. In addition, anti-ADAMTS13 autoAbs were identified as a factor responsible for the change in conformation in ADAMTS13 in iTTP.
Peyvandi:Octapharma US: Honoraria; Kedrion: Consultancy; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Grifols: Speakers Bureau; Sobi: Speakers Bureau; Shire: Speakers Bureau; Novo Nordisk: Speakers Bureau; Octapharma US: Honoraria; Octapharma US: Honoraria; Novo Nordisk: Speakers Bureau; Kedrion: Consultancy; Roche: Speakers Bureau; Novo Nordisk: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Roche: Speakers Bureau; Roche: Speakers Bureau; Shire: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Octapharma US: Honoraria; Roche: Speakers Bureau; Roche: Speakers Bureau; Grifols: Speakers Bureau; Sobi: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Sobi: Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Novo Nordisk: Speakers Bureau; Shire: Speakers Bureau; Kedrion: Consultancy; Novo Nordisk: Speakers Bureau; Grifols: Speakers Bureau; Grifols: Speakers Bureau; Kedrion: Consultancy; Kedrion: Consultancy; Grifols: Speakers Bureau; Octapharma US: Honoraria. Coppo:Ablynx: Consultancy. Veyradier:LFB: Other: Investigator. Vanhoorelbeke:Shire: Consultancy; Ablynx: Consultancy.
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