Acquired platelet GPVI receptor dysfunction in critically-ill patients with sepsis

Blood ◽  
2021 ◽  
Author(s):  
Lukas Johannes Weiss ◽  
Georgi Manukjan ◽  
Annerose Pflug ◽  
Nadine Winter ◽  
Mathis Leonard Weigel ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Pilot Study ◽  
Animal Models ◽  
Disease Onset ◽  
Vascular Integrity ◽  
Sepsis Diagnosis ◽  
Syk Kinase ◽  
Healthy Donors ◽  
Icu Discharge

GPVI, the platelet immunoreceptor tyrosine activating motif (ITAM) receptor for collagen plays a striking role on vascular integrity in animal models of inflammation and sepsis. Understanding ITAM-receptor signaling defects in humans suffering from sepsis may improve our understanding of the pathophysiology, especially during disease onset. In a pilot study, platelets from fifteen septic patients were assessed consecutively at day of admission, day 5-7 and the day of ICU-discharge, and subjected to comprehensive analyses by flow cytometry, aggregometry and immunoblotting. Platelet function was markedly reduced in all patients. The defect was most prominent after GPVI stimulation with collagen-related peptide. In 14/15 patients GPVI-dysfunction was already present at time of ICU admission, considerably before the critical drop in platelet counts. Sepsis platelets failed to transduce the GPVI-mediated signal to trigger tyrosine phosphorylation of Syk kinase or LAT. GPVI deficiency was in part inducible in platelets of healthy donors through co-incubation in whole blood, but not in plasma from sepsis patients. Platelet aggregation upon GPVI stimulation increased only in those patients whose condition ameliorated. As blunted GPVI signaling occurred early at sepsis onset this defect could be exploited as an indicator for early sepsis diagnosis, which needs to be confirmed in prospective studies.

A novel flow cytometry–based platelet aggregation assay

Blood ◽  
2013 ◽  
Vol 121 (10) ◽  
pp. e70-e80 ◽  
Author(s):  
Iris M. De Cuyper ◽  
Marjolein Meinders ◽  
Edith van de Vijver ◽  
Dirk de Korte ◽  
Leendert Porcelijn ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Animal Models ◽  
Small Volume ◽  
Aggregation Assay ◽  
Kinetic Measurements ◽  
Experimental Animal Models ◽  
Single Receptor ◽  
Key Points ◽  
Platelet Aggregation Assay

Key Points FCA is a novel flow cytometry–based platelet aggregation assay that allows single receptor analysis in small volume/thrombocytopenic samples FCA facilitates platelet studies in experimental animal models even during gestation and allows kinetic measurements in individual animals

scholarly journals Acquired Platelet GPVI Dysfunction As Possible Predictor for Early Sepsis Diagnosis and Poor Outcome

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3609-3609
Author(s):  
Lukas J Weiss ◽  
Georgi Manukjan ◽  
Nils Nagler ◽  
Markus Kredel ◽  
Thien-Tri Lam ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Whole Blood ◽  
Early Time ◽  
Bacterial Isolates ◽  
Vascular Integrity ◽  
Platelet Counts ◽  
Sepsis Diagnosis ◽  
Bona Fide ◽  
Early Sepsis

Introduction: Sepsis is a major contributor to the global disease burden with estimated 20 million cases per year. With every hour prior to begin of therapy mortality increases by over 7%. Hence, early diagnosis is indispensable for patient survival, though it is hampered by the lack of known pathognomonic symptoms. Sepsis diagnosis is set according to an increase of the sequential organ failure assessment (SOFA) score, reflecting organ dysfunction. Severe symptoms comprising dysfunctional hemostasis are disseminated intravascular coagulation and formation of massive edema due to loss of vascular integrity. Functional hemostasis and maintenance of vascular integrity depends particularly on the platelet surface receptor glycoprotein (GP)VI that dimerizes upon activation and transduces its signals via the associated immunoreceptor tyrosine activation motif (ITAM) of the Fc-gamma chain. Sepsis-associated thrombocytopenia has been extensively investigated and low platelet counts are implemented in the SOFA score.Platelet function during sepsis, however, remains ill-defined with few reports using ADP or thrombin receptor activating peptide (TRAP) and overall conflicting results. Objectives: We assessed platelet function in patients with sepsis III criteria in a single center study at three times during disease: (I) intensive care unit (ICU) admission day; (II) day five to seven at ICU; (III) day of ICU discharge. Methods: Fifteen patients were recruited from the ICU of the University Hospital Würzburg, Germany (m:f ratio 9:6; median age 70; mortality: 40%). Platelets in whole blood were stimulated with ADP, TRAP, or the GPVI agonist collagen-related peptide (CRP). Activation was detected flow cytometrically by P-selectin (CD62P) exposure and integrin GPIIb/IIIa activation (PAC-1 binding). Results were correlated with light transmission aggregometry (LTA) and immunoblotting of phospho-Syk and -LAT. Co-incubation experiments were performed with plasma, whole blood or patient-borne bacterial isolates comprising Gram-negative (E. coli, K. pneumoniae) or Gram-positive (E. faecalis) strains. Results: Platelet function was markedly reduced in all patients assessed by flow cytometry and LTA despite overall unaltered receptor expression. The defect was most prominent after GPVI stimulation with CRP(Mean Geo-MFI ± SD; Control: 9356 ± 3460; Sepsis: 1438 ± 2090; p<.0001). In 14/15 patients GPVI-dysfunction was already present at time (I). In contrast, only 7/15 patients were thrombocytopenic at this early time. We did not consistently find elevated GPVI ectodomain plasma levels, low platelet counts, increased mean platelet volume, or elevated reticulated platelet levels in our cohort at early time points and minor alterations only late during disease progression. We conclude that none of these is a bona fide biomarker, neither for early sepsis diagnosis nor as a prognostic marker. Sepsis platelets failed to transduce the GPVI signal to induce tyrosine phosphorylation of Syk kinase or LAT. ITIM receptor-based tyrosine phosphatases SHP1 and SHP2 were neither preactivated in resting platelets nor responsive to GPVI stimulation. Of note, platelet aggregation upon GPVI stimulation increased in those patients whose condition ameliorated. We found a strong correlation of restored platelet aggregation by LTA with survival and could distinguish survivors from non-survivors using this marker for stratification (%max aggregation ± SD; survivors: 25 ± 8%; non-survivors: 4 ± 3%; p<0.01). Responsiveness to CRP was unaltered when platelets of healthy donors were co-incubated with patient blood borne bacterial isolates. Platelet reactivity was also unaltered when they were pre-incubated in plasma of sepsis patients. However, pre-incubation in whole blood clearly diminished the response toward CRP,suggesting that the abrogated GPVI signaling is in part mediated by cellular components. Conclusion: Our results strongly imply that GPVI hypo-responsiveness could serve as a bona fide marker for early sepsis diagnosis and also bears potential to act as a prognostic indicator for therapy monitoring, independent from the source of infection. Acknowledgement: Financial support was granted by the Deutsche Forschungsgemeinschaft (374031971 - TRR 240). Disclosures No relevant conflicts of interest to declare.

Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

1991 ◽  
Vol 65 (04) ◽  
pp. 432-437 ◽  
Author(s):  
A W J Stuttle ◽  
M J Powling ◽  
J M Ritter ◽  
R M Hardisty
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Calcium Ions ◽  
Human Platelet ◽  
Human Platelets ◽  
In Vivo Detection ◽  
Fibrinogen Binding ◽  
Specific Antagonist

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

Quantitative Sensory Testing in Animal Models of Chronic Pain: A Pilot Study

2018 ◽  
Author(s):  
B. Monteiro ◽  
M. Moreau ◽  
C. Otis ◽  
L. De Lorimier ◽  
J. Pelletier ◽  
...  
Keyword(s):  
Chronic Pain ◽  
Pilot Study ◽  
Animal Models ◽  
Quantitative Sensory Testing ◽  
Sensory Testing

scholarly journals Emerging hiPSC Models for Drug Discovery in Neurodegenerative Diseases

2021 ◽  
Vol 22 (15) ◽  
pp. 8196
Author(s):  
Dorit Trudler ◽  
Swagata Ghatak ◽  
Stuart A. Lipton
Keyword(s):  
Drug Discovery ◽  
Animal Models ◽  
Neurodegenerative Diseases ◽  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Neural Function ◽  
Neural Cells ◽  
Human Samples ◽  
Healthy Donors ◽  
Induced Pluripotent

Neurodegenerative diseases affect millions of people worldwide and are characterized by the chronic and progressive deterioration of neural function. Neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD), represent a huge social and economic burden due to increasing prevalence in our aging society, severity of symptoms, and lack of effective disease-modifying therapies. This lack of effective treatments is partly due to a lack of reliable models. Modeling neurodegenerative diseases is difficult because of poor access to human samples (restricted in general to postmortem tissue) and limited knowledge of disease mechanisms in a human context. Animal models play an instrumental role in understanding these diseases but fail to comprehensively represent the full extent of disease due to critical differences between humans and other mammals. The advent of human-induced pluripotent stem cell (hiPSC) technology presents an advantageous system that complements animal models of neurodegenerative diseases. Coupled with advances in gene-editing technologies, hiPSC-derived neural cells from patients and healthy donors now allow disease modeling using human samples that can be used for drug discovery.

scholarly journals OP0045 NLRP12 INVOLVES IN THE LUPUS WITH POSITIVE INTERFERON SIGNATURE

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 23.2-24
Author(s):  
Y. P. Tsao ◽  
F. Y. Tseng ◽  
C. W. Chao ◽  
M. H. Chen ◽  
S. T. Chen
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Animal Models ◽  
Nucleic Acids ◽  
Disease Activity ◽  
Interferon Signature ◽  
Healthy Donors ◽  
Healthy Control ◽  
Lupus Disease Activity

Background:Systemic lupus erythematous (SLE) is a systemic autoimmune disease with diverse etiological factors. It was recognized that interferon (IFN) signature involved in the progress of SLE. NLRP12 (NOD-like receptor family (NLR) pyrin domain containing 12) is a pyrin containing NLR protein that we had linked its new biological function to the cross-regulation of Toll like receptor (TLRs) and Rig-I like receptor (RIG-I) pathways. NLPR12 acts as an innate immune check-point in regulating type I IFNs expression during TLRs and RIG-I activation. The importance of NLRP12 in lupus disease activity remained to be elucidated.Objectives:To clarify the role of NLRP12 in regulating the interferon signature.Methods:Peripheral blood mononuclear cells (PBMCs) were collected from SLE patients and healthy donors for analysis of NLRP12 and IFN-α gene expression by RT-QPCR. PBMCs were applied for Chromatin immuneprecipitation (ChIP) assay and electrical mobility shift assay (EMSA) to determine the putative transcription factor that regulates NLRP12 expression. An involvement of epigenetic regulation of NLRP12 expression in SLE patients was also analyzed. Bone marrow derived dendritic cells (BMDCs) were collected from wild type mouse and Nlrp12 knocked-out mice. Another CD14+ monocytes were isolated from 10 cases of lupus patients and 8 cases of healthy control, following by stimulating different type of nucleic acids, and IFN-α and IL-6 were measured with ELISA assay. CD14+ monocytes in lupus patients were also pre-treated with IFNAR2 antibody for further nucleic acid stimulation. Two mice models were applied for evaluation the role of Nlrp12: intraperitoneal injection of TMPD (2,6,10,14-tetramethylpentadecane, or pristane) in C57BL/6 mice and Faslpr mice. Both models were conducted with and without Nlrp12 knockout.Results:NLRP12 expression was significantly lower in PBMC isolated from SLE patients compared to healthy donors. The inverse correlation was observed in NLRP12 and IFNA gene expression as well as NLRP12 expression and amount of double-stranded DNA autoantibody in SLE patients. NLRP12 expression showed negative correlations with IFN-α treatment, as well as herpes simplex virus-1 (HSV-1) infection. Results from ChIP and EMSA analysis indicated a potential transcription factor 1 (TF-1) regulating NLRP12 promoter activity. TF-1 lead to transcriptional suppression of NLRP12 in SLE PBMC, and it was gradually induced after IFN treatment. Recruitment of TF-1 to NLRP12 promoter in SLE PBMC compared to the healthy PBMC was detected, and increased when treating with IFN. Human CD14+ monocytes collected from lupus and healthy control stimulating with different type of nucleic acids revealing significant increasing level of IFN-α and IL-6 in lupus patients. Among animal models, both pristine induced mice and Faslpr mice revealed increasing autoantibodies production and severity of glomerulonephritis in Nlrp12-/- group in comparison with Nlrp12+/+ ones, indicating the role of NLRP12 in maintaining positive interferon signature as well as disease activity.Conclusion:Expression level of NLRP1.2 has been demonstrated to be a biomarker of disease activity in SLE patients. The NLRP12 was involved in the interferon signature, which was also negatively regulated by TF-1. Both clinical samples and animal models revealed NLRP12 in maintaining the positive interferon signature, indicating the possible role of exacerbating factor for lupus disease activity.Disclosure of Interests:None declared

scholarly journals A Multiplex, Droplet Digital PCR Assay for the Detection of T-Cell Receptor Excision Circles and Kappa-Deleting Recombination Excision Circles

2019 ◽  
Vol 66 (1) ◽  
pp. 229-238 ◽  
Author(s):  
Tracie Profaizer ◽  
Patricia Slev
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
T Cell Receptor ◽  
Reference Gene ◽  
Cell Receptor ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Blood Samples ◽  
Healthy Donors ◽  
Excision Circles

Abstract BACKGROUND T-cell receptor excision circles (TREC) and κ-deleting recombination receptor excision circles (KREC) concentrations can be used to assess and diagnose immune deficiencies, monitor thymic and bone marrow immune reconstitution, or follow responses to drug therapy. We developed an assay to quantify TREC, KREC, and a reference gene in a single reaction using droplet digital PCR (ddPCR). METHODS PCR was optimized for 3 targets: TREC, KREC, and ribonuclease P/MRP subunit p30 (RPP30) as the reference gene. Multiplexing was accomplished by varying the target's fluorophore and concentration. Correlation with clinical results was evaluated using 47 samples from healthy donors, 59 samples with T-cell and B-cell markers within the reference interval from the flow cytometry laboratory, 20 cord blood samples, and 34 samples submitted for exome sequencing for severe combined immunodeficiency disease (SCID). RESULTS The limit of the blank was 4 positive droplets, limit of detection 9 positive droplets, and limit of quantification 25 positive droplets, or 2.0 copies/μL. TREC and KREC copies/μL were as expected in the healthy donors and cord blood samples and concordant with the healthy flow cytometry results. Of the samples from the SCID Panel, 56.5% had a TREC count <20 copies/μL and 17.7% had a KREC count <20 copies/μL, suggestive of low T- and B-cell numbers, respectively. CONCLUSIONS Our multiplex ddPCR assay is an analytically sensitive and specific method for the absolute quantification of TREC and KREC. To the best of our knowledge, this paper is the first to describe the simultaneous quantification of TREC, KREC, and a reference gene by use of ddPCR.

scholarly journals ROS Promote Ox-LDL-Induced Platelet Activation by Up-Regulating Autophagy Through the Inhibition of the PI3K/AKT/mTOR Pathway

2018 ◽  
Vol 50 (5) ◽  
pp. 1779-1793 ◽  
Author(s):  
Xiang Wang ◽  
Yun-Feng Fu ◽  
Xiao Liu ◽  
Guo Feng ◽  
Dan Xiong ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Mtor Pathway ◽  
Enhanced Chemiluminescence ◽  
Transmission Electron ◽  
Related Proteins

Background/Aims: Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in patients with dyslipidemic disorders. Although oxLDL stimulates activating signaling, researchers have not clearly determined how these events drive accelerated thrombosis. Here, we describe the mechanism by which ROS regulate autophagy during ox-LDL-induced platelet activation by modulating the PI3K/AKT/mTOR signaling pathway. Methods: For in vitro experiments, ox-LDL, the ROS scavenger N-acetylcysteine (NAC), the mTOR inhibitor rapamycin and the autophagy inhibitor 3-MA were used alone or in combination with other compounds to treat platelets. Then, platelet aggregation was evaluated on an aggregometer and platelet adhesion was measured under shear stress. The levels of a platelet activation marker (CD62p) were measured by flow cytometry, reactive oxygen species (ROS) levels were then quantified by measuring DCFH-DA fluorescence intensity via flow cytometry. Nitric oxide (NO) and superoxide (O2·-) levels were determined by the nitric acid deoxidize enzyme method and lucigenin-enhanced chemiluminescence (CL), respectively. Transmission electron microscopy was used to observe the autophagosome formation, immunofluorescence staining was employed to detect LC3 expression and western blotting was used to measure the levels of PI3K/AKT/mTOR pathway- and autophagy-related proteins. Results: Ox-LDL-induced platelets showed a significant increase in platelet aggregation and adhesion, CD62p expression, ROS level and O2·- content, with an elevated LC3II/LC3I ratio and Beclin1 expression, but a dramatic reduction in the levels of p62 and pathway-related proteins (all P < 0.05). However, platelet activation and autophagy were aggravated by the Rapamycin treatment, and decreased following treatment with NAC, 3-MA, or NAC and 3-MA, together with increased activity of the PI3K/AKT/mTOR pathway. Additionally, decreased platelet activation and autophagy were observed in platelets treated with NAC and Rapamycin or Rapamycin and 3-MA compared with platelets treated with Rapamycin alone, suggesting that both NAC and 3-MA reversed the effects of Rapamycin. Conclusion: Inhibition of ROS production may reduce autophagy to suppress ox-LDL-induced platelet activation by activating PI3K/AKT/mTOR pathway.

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