scholarly journals Autoantibody-mediated desialylation impairs human thrombopoiesis and platelet lifespan

Haematologica ◽  
2019 ◽  
Vol 106 (1) ◽  
pp. 196-207 ◽  
Author(s):  
Irene Marini ◽  
Jan Zlamal ◽  
Christoph Faul ◽  
Ursula Holzer ◽  
Stefanie Hammer ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Immune Thrombocytopenia ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Human Platelets ◽  
Matrix Proteins ◽  
Ex Vivo Model ◽  
Effector Functions ◽  
Clinical Cohort ◽  
Significant Impairment

Immune thrombocytopenia is a common bleeding disease caused by autoantibody-mediated accelerated platelet clearance and impaired thrombopoiesis. Accumulating evidence suggests that desialylation affects platelet life span in immune thrombocytopenia. Herein, we report on novel effector functions of autoantibodies from immune thrombocytopenic patients which might interfere with the clinical picture of the disease. Data from our study show that a subgroup of autoantibodies is able to induce cleave of sialic acid residues from the surface of human platelets and megakaryocytes. Moreover, autoantibody-mediated desialylation interferes with the interaction between cells and extracellular matrix proteins leading to impaired platelet adhesion and megakaryocyte differentiation. Using a combination of ex vivo model of thrombopoiesis, a humanized animal model, and a clinical cohort study, we demonstrate that cleavage of sialic acid induces significant impairment in production, survival as well as function of human platelets. These data may indicate that prevention of desialylation should be investigated in the future in clinical studies as a potential therapeutic approach to treat bleeding in immune thrombocytopenia.

Glycosylation of autoantibodies: Insights into the mechanisms of immune thrombocytopenia

2013 ◽  
Vol 110 (12) ◽  
pp. 1259-1266 ◽  
Author(s):  
Tamam Bakchoul ◽  
Kathrin Walek ◽  
Annika Krautwurst ◽  
Mathias Rummel ◽  
Gregor Bein ◽  
...  
Keyword(s):  
Immune Thrombocytopenia ◽  
Laser Scanning ◽  
Human Platelets ◽  
Laser Scanning Microscopy ◽  
Effector Functions ◽  
Scid Mouse Model ◽  
Rapid Clearance

SummaryImmune thrombocytopenia (ITP) is a bleeding disorder caused by IgG autoantibodies (AAbs) directed against platelets (PLTs). IgG effector functions depend on their Fc-constant region which undergoes post-translational glycosylation. We investigated the role of Asn279-linked N-glycan of AAbs in vitro and in vivo. AAbs were purified from ITP patients (n=15) and N-glycans were enzymatically cleaved by endoglycosidase F. The effects of native AAbs and deglycosylated AAbs were compared in vitro on enhancement of phagocytosis of platelets by monocytes and complement fixation and activation applying flow cytometry, laser scanning microscopy, and a complement consumption assay. AAb-induced platelet phagocytosis was inhibited by N-glycan cleavage (median phagocytic activity: 8% vs 0.8%, p=0.004). Seven out of 15 native AAbs bound C1q and activated complement. N-glycan cleavage significantly reduced both effects. In vivo survival of human PLTs was assessed after co-transfusion with native or N-glycan cleaved AAbs in a NOD/SCID mouse model. Injection of AAbs resulted in rapid clearance of human platelets compared to control (platelet clearance after 5h (CL5h) 75% vs 30%, p<0.001). AAbs that were able to activate complement induced more pronounced platelet clearance in the presence of complement compared to the clearance in the absence of complement (CL5h 82% vs 62%, p=0.003). AAbs lost their ability to destroy platelets in vivo after deglycosylation (CL5h 42%, p<0.001). N-glycosylation of human ITP AAbs appears to be required for platelet phagocytosis and complement activation, reducing platelet survival in vivo. Posttranslational modification of AAbs may constitute an important determinant for the clinical manifestation of ITP.

Regulation of tissue factor-induced coagulation and platelet aggregation in flowing whole blood

2005 ◽  
Vol 93 (01) ◽  
pp. 97-105 ◽  
Author(s):  
Marijke Kuijpers ◽  
Cécile Nieuwenhuys ◽  
Marion Feijge ◽  
Willem Kloots ◽  
Peter Giesen ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Aggregate Formation ◽  
Ex Vivo Model ◽  
Platelet Surface ◽  
Inhibiting Effect ◽  
Activated Platelets ◽  
Adp Receptor

SummaryPhotochemically induced thrombosis (a thrombin-dependent process) was measured in rats treated with moderate doses of anticoagulants, but which appeared to be unchanged. We considered the possibility that platelet-inhibiting agents, which also indirectly inhibit coagulation, would act as more potent antithrombotic agents.Inhibitors used as such were prostaglandin E1 (PGE1), which elevates cyclic AMP levels, and the P2Y12 ADP-receptor antagonist,AR-C69931MX. Effects of these agents were investigated in an ex vivo model system, in which whole blood under coagulant conditions was perfused over fibrinogen at defined wall shear rate. Perfusion of blood (rat or human) in the presence of tissue factor resulted in deposition of activated platelets and subsequent aggregate formation, along with exposure of procoagulant phosphatidylserine (PS) on the platelet surface and formation of fibrin fibers. In the presence of PGE1 aggregation was completely inhibited,but platelet adhesion and PS exposure were only party reduced, while fibrin formation was hardly affected. Treatment with AR-C69931MX caused similar, but less complete effects.These results indicate that in tissue factor- triggered blood under conditions of flow:(i) the platelet procoagulant response is independent of aggregate formation; (ii) the platelet-inhibiting effect of PGE1 and AR-C69931MX is sufficient to suppress aggregation, but not platelet adhesion and coagulation. These platelet inhibitors thus maintain their aggregation- inhibiting effect at sites of thrombin formation.

scholarly journals Glycomic Characterization of Platelets from Patients with Immune Thrombocytopenia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3158-3158
Author(s):  
Nora V. Butta ◽  
Stuart M Haslam ◽  
Anne Dell ◽  
Leow Ke Xuan ◽  
Sophie Ball ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Research Funding ◽  
Immune Thrombocytopenia ◽  
Human Platelets ◽  
T Antigen ◽  
Lewis X ◽  
Maldi Tof ◽  
Lewis Y ◽  
Complex Glycans ◽  
Tof Ms

Abstract Introduction: Platelet glycoproteins are key contributors to platelet function but their glycans structure is unclear. Alterations in glycan composition have been reported to impact platelet clearance under physiological conditions and in the disease mechanism of immune thrombocytopenia (ITP). Therefore, this study sought to characterize glycan structures in human platelets from healthy control individuals and ITP patients using mass spectrometry (MS)-based glycomics approach, andto compare their glycomic profiles to facilitate understanding of glycan alterations in ITP. Methods: Glycan residues on platelet surface were determined by flow cytometry. Platelet lysates (1×10 8 platelets) from 4 healthy controls and from 4 ITP patients with a clear anomalous glycosylation pattern were characterized by MALDI-MS based glycomic approaches. N-linked glycans were released from platelet glycoproteins by PNGase F digestion and subsequently purified with a Sep-Pak C18 reverse phase cartridge. O-linked glycans were released by reductive elimination. Both pools of glycans were permethylated prior to MALDI-TOF MS to obtain an initial carbohydrate profile. Selected glycan molecular ion species were analyzed by MALDI-TOF-TOF MS/MS before and after digesting with exoglycosidases. Results: Glycans present in platelets from healthy controls and from ITP patients were largely consistent. The MS spectra for N-glycans showed a mixture of high mannose glycans (m/z 1579.8, 1783.9, 1988.0, 2192.1 and 2396.2); complex glycans (m/z 2966.5, 3776.9 and 4587.4); and bisected or truncated glycans (m/z 3211.6 and 4022.1). The spectra showed the presence of bi-, tri- and tetra-antennary complex glycans, which varied in their level of sialylation.Figure 1 shows the relative abundance ratio within eight families of core glycan structures. Platelets from ITP patients showed a consistent increase in desialylated structures such as Hex 5HexNAc 4Fuc 1. In addition to different amounts of attached sialic acid, varying levels of fucosylation were observed; ranging from the addition of a single core Fuc (m/z 2244.1), to the addition of up to three Fuc residues (m/z 3402.7). Collision-activated decomposition (CAD) MALDI-TOF/TOF analysis was performed to generate fragment ions from molecular ions detected in MALDI-TOF profiling for detailed sequencing of platelet N-glycans. This analysis suggested the presence of three isoforms: (i) sialylated tetra-antennary structure with core fucosylation and one Lewis x/a antenna; (ii) sialylated tetra-antennary structure with one Lewis y/b antenna; (ii) sialylated, core-fucosylated tri-antennary structure with one LacNAc extension and one Lewis x/a antenna. Sialidase S digestion was used to highlight the extent of desialylation in the presence of sialidase. Noteworthy, the ratio of non-sialylated bi-and tri-antennary N-glycans in ITP patients were higher than that in controls. This enzymatic digestion confirms the presence of α2,3-linked Neu5Ac on platelet glycans. Remaining sialylated structures may possess α2,6-linked Neu5Ac. O-glycan profiles obtained showed the predominance of core 1 and core 2 structures (Figure 2). The two most dominant glycan structures in core 1 were sialyl T antigen (GalNAc 1Gal 1NeuAc 1; m/z 895.5) and disialyl T antigen (GalNAc 1Gal 1NeuAc 2; m/z 1256.7), being the latter less abundant in ITP patients. The core 2 structure was modified by the addition of fucose and/or sialic acid residues (m/z 1157.7, 1344.8, 1518.9 and 1706.0). Conclusion: N- and O-glycan structures in human platelets were characterized by MALDI-TOF MS profiling to reveal interesting structural features including the presence of sialylLewis x/a epitope, Lewis x/a epitope, Lewis y/b epitope and LacNAc extensions in complex type N-glycans. Presence of terminal sialic acid and sialylLewis x/a on platelet N-glycan antenna also suggest their potentialfunction as ligands for siglecs that are associated with cell signaling functions. Siglec-1 and -2 have been suggested to have potential roles in ethiopathogenesis of autoimmune diseases. Desialylation observed in glycans of platelets from ITP patients, might trigger immune system activation in these patients. This research was funded by ISCIII-Fondos FEDER PI19/00772 and Platelet Disorder Support Association Figure 1 Figure 1. Disclosures Butta: Roche: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; CSL-Behring: Research Funding; Novo-Nordisk: Speakers Bureau. Canales: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Speakers Bureau; Karyopharm: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Speakers Bureau; iQone: Honoraria; Sandoz: Honoraria, Speakers Bureau; Incyte: Consultancy; Janssen: Consultancy, Honoraria, Speakers Bureau; Gilead/Kite: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Consultancy; Eusa Pharma: Consultancy, Honoraria; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria. Jiménez-Yuste: Pfizer: Consultancy, Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; BioMarin: Consultancy; Sobi: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding. Alvarez Román: Octapharma: Consultancy, Honoraria, Research Funding; Biomarin: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; CSL-Behring: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Novo-Nordisk: Consultancy, Honoraria, Research Funding; Sobi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding.

Lyophilised reconstituted human platelets increase thrombus formation in a clinical ex vivo model of deep arterial injury

2012 ◽  
Vol 108 (07) ◽  
pp. 176-182 ◽  
Author(s):  
Jennifer Raftis ◽  
Andrew Lucking ◽  
Amanda Hunter ◽  
Mike Millar ◽  
Mike Fitzpatrick ◽  
...  
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Morphological Characteristics ◽  
Principal Component ◽  
Arterial Injury ◽  
Human Platelets ◽  
Dependent Manner ◽  
Ex Vivo Model ◽  
Electron Microscopic

SummaryPlatelets are the principal component of the innate haemostatic system that protect from traumatic bleeding. We investigated whether lyophilised human platelets (LHPs) could enhance clot formation within platelet-free and whole blood environments using an ex vivo model of deep arterial injury. Lyophilised human platelets were produced from stored human platelets and characterised using conventional, fluorescent and electron microscopic techniques. LHPs were resuspended in platelet-free plasma (PFP) obtained from citrated whole human blood to form final concentrations of 0,20 and 200 × 109 LHPs/L. LHPs with recalcified PFP or whole blood were perfused through the chamber at low (212 s-1) and high (1,690 s-1) shear rates with porcine aortic tunica media as thrombogenic substrate. LHPs shared morphological characteristics with native human platelets and were incorporated into clot generated from PFP or whole blood. Histomorphometrically measured mean thrombus area increased in a dose-dependent manner following the addition of LHPs to PFP under conditions of high shear [704 μm2 ± 186 μm2 (mean ± SEM), 1,511 μm2 ± 320 μm2 and 2,378 μm2 ± 315 μm2, for LHPs at 0, 20 and 200 × 109 /l, respectively (p= 0.012)]. Lyophil-ised human platelets retain haemostatic properties when reconstituted in both PFP and whole blood, and enhance thrombus formation in a model of deep arterial injury. These data suggest that LHPs have the potential to serve as a therapeutic intervention during haemorrhage under circumstances of trauma, and platelet depletion or dysfunction.

1849: Systematic Evaluation of 21μm Thulium Laser Device for Treatment of Benign Prostatic Enlargement in an Ex-VIVO Model

2007 ◽  
Vol 177 (4S) ◽  
pp. 614-614 ◽  
Author(s):  
Gunnar Wendt-Nordahl ◽  
Stefanie Huckele ◽  
Patrick Honeck ◽  
Peter Aiken ◽  
Thomas Knoll ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Systematic Evaluation ◽  
Thulium Laser ◽  
Laser Device ◽  
Benign Prostatic Enlargement ◽  
Ex Vivo Model ◽  
Prostatic Enlargement

Porcine small intestine, a good ex vivo model to investigate absorption and metabolism of natural products

2017 ◽  
Author(s):  
J Houriet ◽  
YE Arnold ◽  
C Petit ◽  
YN Kalia ◽  
JL Wolfender
Keyword(s):  
Natural Products ◽  
Small Intestine ◽  
Ex Vivo ◽  
Ex Vivo Model

Effects of Bupranolol, a New β-Blocker, on Platelet Functions of Rabbit and Human in Vitro

1976 ◽  
Vol 36 (02) ◽  
pp. 376-387 ◽  
Author(s):  
Teruhiko Umetsu ◽  
Kazuko Sanai ◽  
Tadakatsu Kato
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Human Platelets ◽  
Rabbit Platelet ◽  
Rabbit Platelets ◽  
Β Blocker ◽  
Platelet Aggregates ◽  
Induced Aggregation ◽  
Platelet Functions

SummaryThe effects of bupranolol, a new β-blocker, on platelet functions were investigated in vitro in rabbits and humans as compared with propranolol, a well-known β-blocker. At first, the effect of adrenaline on ADP-induced rabbit platelet aggregation was studied because adrenaline alone induces little or no aggregation of rabbit platelets. Enhancement of ADP-induced rabbit platelet aggregation by adrenaline was confirmed, as previously reported by Sinakos and Caen (1967). In addition the degree of the enhancement was proved to be markedly affected by the concentration of ADP and to increase with decreasing concentration of ADP, although the maximum aggregation (percent) was decreased.Bupranolol and propranolol inhibited the (adrenaline-ADP-)induced aggregation of rabbit platelets, bupranolol being approximately 2.4–3.2 times as effective as propranolol. Bupranolol stimulated the disaggregation of platelet aggregates induced by a combination of adrenaline and ADP, but propranolol did not. Platelet adhesion in rabbit was also inhibited by the β-blockers and bupranolol was more active than propranolol. With human platelets, aggregation induced by adrenaline was inhibited by bupranolol about 2.8–3.3 times as effectively as propranolol.From these findings. We would suggest that bupranolol might be useful for prevention or treatment of thrombosis.

Reaction of Acetaldehyde with Human Platelets

1992 ◽  
Vol 67 (01) ◽  
pp. 126-130 ◽  
Author(s):  
Olivier Spertini ◽  
Jacques Hauert ◽  
Fedor Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Action ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Reaction Medium ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

Effects of Two Different Doses of Hirudin on APTT, Determined with Eight Different Reagents

1995 ◽  
Vol 73 (02) ◽  
pp. 219-222 ◽  
Author(s):  
Manuel Monreal ◽  
Luis Monreal ◽  
Rafael Ruiz de Gopegui ◽  
Yvonne Espada ◽  
Ana Maria Angles ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Anticoagulant Activity ◽  
Subcutaneous Administration ◽  
In Vitro Study ◽  
Ex Vivo Model ◽  
Suitable Candidate ◽  
Significant Prolongation ◽  
High Doses ◽  
Different Doses

SummaryThe APTT has been considered the most suitable candidate to monitor the anticoagulant activity of hirudin. However, its use is hampered by problems of standardization, which make the results heavily dependent on the responsiveness of the reagent used. Our aim was to investigate if this different responsiveness of different reagents when added in vitro is to be confirmed in an ex vivo study.Two different doses of r-hirudin (CGP 39393), 0.3 mg/kg and 1 mg/kg, were administered subcutaneously to 20 New Zealand male rabbits, and the differences in prolongation of APTT 2 and 12 h later were compared, using 8 widely used commercial reagents. All groups exhibited a significant prolongation of APTT 2 h after sc administration of hirudin, both at low and high doses. But this prolongation persisted 12 h later only when the PTTa reagent (Boehringer Mannheim) was used. In general, hirudin prolonged the APTT most with the silica- based reagents.In a further study, we compared the same APTT reagents in an in vitro study in which normal pooled plasma was mixed with increasing amount of hirudin. We failed to confirm a higher sensitivity for silica- containing reagents. Thus, we conclude that subcutaneous administration of hirudin prolongs the APTT most with the silica-based reagents, but this effect is exclusive for the ex vivo model.

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