scholarly journals Activation of Neutrophil Granulocytes by Platelet-Activating Factor Is Impaired During Experimental Sepsis

2021 ◽  
Vol 12 ◽  
Author(s):  
Stefan Hug ◽  
Stefan Bernhard ◽  
Alexander Elias Paul Stratmann ◽  
Maike Erber ◽  
Lisa Wohlgemuth ◽  
...  
Keyword(s):  
Cell Size ◽  
Intracellular Ph ◽  
Whole Blood ◽  
Physiological Response ◽  
Ex Vivo ◽  
Platelet Activating Factor ◽  
Neutrophil Granulocytes ◽  
Dependent Manner ◽  
Activation Markers ◽  
Cellular Swelling

Platelet-activating factor (PAF) is an important mediator of the systemic inflammatory response. In the case of sepsis, proper activation and function of neutrophils as the first line of cellular defense are based on a well-balanced physiological response. However, little is known about the role of PAF in cellular changes of neutrophils during sepsis. Therefore, this study investigates the reaction patterns of neutrophils induced by PAF with a focus on membrane potential (MP), intracellular pH, and cellular swelling under physiological and pathophysiological conditions and hypothesizes that the PAF-mediated response of granulocytes is altered during sepsis. The cellular response of granulocytes including MP, intracellular pH, cellular swelling, and other activation markers were analyzed by multiparametric flow cytometry. In addition, the chemotactic activity and the formation of platelet–neutrophil complexes after exposure to PAF were investigated. The changes of the (electro-)physiological response features were translationally verified in a humanex vivowhole blood model of endotoxemia as well as during polymicrobial porcine sepsis. In neutrophils from healthy human donors, PAF elicited a rapid depolarization, an intracellular alkalization, and an increase in cell size in a time- and dose-dependent manner. Mechanistically, the alkalization was dependent on sodium-proton exchanger 1 (NHE1) activity, while the change in cellular shape was sodium flux- but only partially NHE1-dependent. In a pathophysiological altered environment, the PAF-induced response of neutrophils was modulated. Acidifying the extracellular pHin vitroenhanced PAF-mediated depolarization, whereas the increases in cell size and intracellular pH were largely unaffected.Ex vivoexposure of human whole blood to lipopolysaccharide diminished the PAF-induced intracellular alkalization and the change in neutrophil size. During experimental porcine sepsis, depolarization of the MP was significantly impaired. Additionally, there was a trend for increased cellular swelling, whereas intracellular alkalization remained stable. Overall, an impaired (electro-)physiological response of neutrophils to PAF stimulation represents a cellular hallmark of those cells challenged during systemic inflammation. Furthermore, this altered response may be indicative of and causative for the development of neutrophil dysfunction during sepsis.

EspP, An Extracellular Serine Protease From Enterohemorrhagic E. Coli, Induces Coagulopathy In Human Whole Blood and Plasma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4416-4416
Author(s):  
Kevin H.M. Kuo ◽  
Shekeb Khan ◽  
Elena Brnjac ◽  
Emil F. Pai ◽  
Alden E. Chesney
Keyword(s):  
Serine Protease ◽  
Whole Blood ◽  
Ex Vivo ◽  
Coagulation Factor ◽  
Time Dependent ◽  
Coagulation Cascade ◽  
Factor V ◽  
Dependent Manner ◽  
E Coli ◽  
Negative Controls

Abstract Abstract 4416 EspP (E. coli secreted serine protease, large plasmid encoded) is an extracellular serine protease produced by enterohemorrhagic E. coli (EHEC) O157:H7. Brunder et al. (Mol Microbiol 1997, 24:767–78) have shown that EspP cleaves, amongst other proteins, human coagulation factor V, and the authors hypothesized that it may contribute to the mucosal hemorrhage in patients with EHEC infection. We have since shown that EspP also cleaves factor VIII. Since the mechanism by which EHEC induces diarrhea-associated Hemolytic Uremic Syndrome (D+HUS) has not been fully elucidated, and EspP has been cited as a putative virulence factor in D+HUS, we investigated the role of EspP in primary and secondary hemostasis in the pathogenesis of D+HUS. Wild type EspP (EspPwt) and EspPS263A, where the serine at the active site was mutated to an alanine thereby abolishing its proteolytic activity, were expressed in the non-pathogenic E. coli host BL21(DE3) and purified by hydrophobic interaction and size-exclusion chromatography. EspPwt at 1.0 mg/mL was incubated for 0.5, 2.0 and 4.0 hours ex vivo with citrated plasma from 6 healthy adults. EspPS263A, bovine serum albumin (BSA) and phosphate buffer saline-glycerol (PBS-G) served as negative controls. PT, aPTT and TT were found to be significantly prolonged and activity of factors V, VII, VIII and XII were reduced in a time- and concentration-dependent manner (Figures 1 and Figure 2). When citrated plasma was incubated with 1 mg/mL EspPwt at 37°C for 4 hours, PT was prolonged by 23.2 +/− 3.8 s, aPTT by 41.6 +/− 8.3 s and TT by 6.1 +/− 0.6 s, relative to the negative controls. Factor V activity decreased by 0.82 +/− 0.14 U/mL, factor VII by 0.72 +/− 0.28 U/mL, factor VIII by 0.69 +/− 0.31 U/mL and factor XII by 0.36 +/− 0.09 U/mL, relative to the negative controls. Prothrombin activity was significantly reduced (0.16 +/− 0.08 U/mL) compared to all negative controls but remained above 0.75 U/mL. Factors IX, × and XI activity, and fibrinogen concentration were not significantly different from the controls. To determine whether any cellular components in whole blood contribute to EspP's effect on the coagulation cascade, the experiment was repeated using citrated whole blood in place of plasma during the incubation phase. Plasma was then recovered and analyzed. Similar results were observed. The results suggest that EspP has proteolytic activity against specific coagulation factors at least in an ex vivo setting. In patients with EHEC infection, EspP may contribute to the hemorrhagic diarrhea by impairing the coagulation cascade. Further studies are needed to determine whether EspP is able to induce coagulopathy in vivo and if so, whether induction of such a coagulopathic state may favour the entry of Shiga toxin into systemic circulation in patients with D+HUS. Figure 1 EspP prolongs PT, aPTT and TT in a time-dependent manner. Figure 1. EspP prolongs PT, aPTT and TT in a time-dependent manner. Figure 2 EspP reduces coagulation factor activity in a time-dependent manner. Figure 2. EspP reduces coagulation factor activity in a time-dependent manner. Disclosures: No relevant conflicts of interest to declare.

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.

Endothelin has potent ulcerogenic and vasoconstrictor actions in the stomach

1989 ◽  
Vol 256 (4) ◽  
pp. G661-G666 ◽  
Author(s):  
J. L. Wallace ◽  
G. Cirino ◽  
G. De Nucci ◽  
W. McKnight ◽  
W. K. MacNaughton
Keyword(s):  
Vascular Tone ◽  
Ex Vivo ◽  
Platelet Activating Factor ◽  
Dependent Manner ◽  
Leukotriene D4 ◽  
Chamber Model ◽  
Dose Dependent ◽  
Factor Antagonist

The role of endothelin, an endothelium-derived constricting factor, as a mediator of gastric ulceration was assessed using an ex vivo gastric chamber model in the rat. The rats were pretreated with indomethacin, and endothelin was infused intravenously or intra-arterially at various doses while the stomach was topically "challenged" with 20% ethanol. This concentration of ethanol did not, by itself, produce hemorrhagic damage in the stomach. However, infusion of endothelin rendered the mucosa vulnerable to damage induced by the ethanol. In a dose-dependent manner, endothelin increased the extent of hemorrhagic damage and the efflux of protein from the gastric mucosa. The effects of endothelin were less marked in rats not pretreated with indomethacin. The ulcerogenic actions of endothelin were not significantly affected by pretreatment with a platelet-activating factor antagonist or a leukotriene D4 antagonist. However, topical pretreatment with sodium nitroprusside produced a significant reduction in the damage induced by intravenous endothelin and topically applied ethanol. Endothelin also rendered the stomach vulnerable to damage induced by hydrochloric acid at a concentration (0.15 M) tolerated by control mucosa. Using an in vitro vascularly perfused rat stomach preparation, we found that endothelin produces marked increases in gastric vascular tone at nanomolar concentrations. These results suggest that the factors regulating the release of endothelin, and the balance between endothelial production of relaxing and contracting factors, may be important in the pathogenesis of ulcerative diseases of the stomach.

scholarly journals Burkholderia pseudomallei Triggers Altered Inflammatory Profiles in a Whole-Blood Model of Type 2 Diabetes-Melioidosis Comorbidity

2012 ◽  
Vol 80 (6) ◽  
pp. 2089-2099 ◽  
Author(s):  
Jodie Morris ◽  
Natasha Williams ◽  
Catherine Rush ◽  
Brenda Govan ◽  
Kunwarjit Sangla ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Oxidative Burst ◽  
Whole Blood ◽  
Burkholderia Pseudomallei ◽  
Inflammatory Responses ◽  
Ex Vivo ◽  
Bacterial Load ◽  
Activation Markers ◽  
Content Type

ABSTRACTMelioidosis is a potentially fatal disease caused by the bacteriumBurkholderia pseudomallei. Type 2 diabetes (T2D) is the most common comorbidity associated with melioidosis.B. pseudomalleiisolates from melioidosis patients with T2D are less virulent in animal models than those from patients with melioidosis and no identifiable risk factors. We developed anex vivowhole-blood assay as a tool for comparison of early inflammatory profiles generated by T2D and nondiabetic (ND) individuals in response to aB. pseudomalleistrain of low virulence. Peripheral blood from individuals with T2D, with either poorly controlled glycemia (PC-T2D [n= 6]) or well-controlled glycemia (WC-T2D [n= 8]), and healthy ND (n= 13) individuals was stimulated withB. pseudomallei. Oxidative burst, myeloperoxidase (MPO) release, expression of pathogen recognition receptors (TLR2, TLR4, and CD14), and activation markers (CD11b and HLA-DR) were measured on polymorphonuclear (PMN) leukocytes and monocytes. Concentrations of plasma inflammatory cytokine (interleukin-6 [IL-6], IL-12p70, tumor necrosis factor alpha [TNF-α], monocyte chemoattractant protein 1 [MCP-1], IL-8, IL-1β, and IL-10) were also determined. Following stimulation, oxidative burst and MPO levels were significantly elevated in blood from PC-T2D subjects compared to controls. Differences were also observed in expression of Toll-like receptor 2 (TLR2), CD14, and CD11b on phagocytes from T2D and ND individuals. Levels of IL-12p70, MCP-1, and IL-8 were significantly elevated in blood from PC-T2D subjects compared to ND individuals. Notably, differential inflammatory responses of PC-T2D, WC-T2D, and ND individuals toB. pseudomalleioccur independently of bacterial load and confirm the efficacy of this model of T2D-melioidosis comorbidity as a tool for investigation of dysregulated PMN and monocyte responses toB. pseudomalleiunderlying susceptibility of T2D individuals to melioidosis.

Parnaparin, a low-molecular-weight heparin, prevents P-selectindependent formation of platelet-leukocyte aggregates in human whole blood

2007 ◽  
Vol 97 (06) ◽  
pp. 965-973 ◽  
Author(s):  
Giovannina Di Fabio ◽  
Miriam Barbanti ◽  
Giovanni de Gaetano ◽  
Maria Benedetta Donati ◽  
Chiara Cerletti ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Platelet Activation ◽  
Whole Blood ◽  
Low Molecular Weight Heparin ◽  
Low Molecular Weight ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Activation Markers ◽  
Fibrinogen Binding ◽  
Vitro Effect

SummaryParnaparin, a low-molecular-weight heparin (LMWH), prevents platelet activation and interaction with polymorphonuclear leukocyte (PMN) in a washed cell system. The in-vitro effect of parnaparin was studied here on platelet-PMN aggregates formed with more physiologic approaches in whole blood, in parallel with unfractionated heparin and enoxaparin, another LMWH. Citrated blood from healthy subjects was stimulated: i) from passage through the “Platelet Function Analyzer” (PFA-100), a device that exposes blood to standardized high shear flow through collagen/ADP cartridges; ii) by collagen and ADP (2 and 50 µg/ml, respectively) added in combination under stirring in an aggregometer cuvette; iii) with recombinant Tissue Factor, to generate thrombin concentrations able to activate platelets without inducing blood clotting, or iv) the Thrombin Receptor Activating Peptide-6 (TRAP-6). Platelet P-selectin and platelet-PMN aggregates were measured by flow cytometry upon stimulation of blood. Fibrinogen binding to platelets and markers of PMN activation were also detected. Platelet P-selectin expression and platelet-PMN aggregate formation were induced in all four activation conditions tested. Parnaparin prevented in a concentration-dependent manner (0.3–0.8 IUaXa/ml) the expression of P-selectin and the formation of mixed aggregates, while the two reference heparin preparations had a much weaker effect. Platelet fibrinogen binding and PMN activation markers (fibrinogen binding, CD11b and CD40) were also prevented by parnaparin. These data extend in more physiological systems of platelet activation, the anti-inflammatory profile of parnaparin, previously reported in washed cells. The greater effect of parnaparin, as compared to the reference heparins, could be due to chemico-physical differences possibly unrelated to their anticoagulant effect.

scholarly journals Effects of Anticoagulants and Temperature on Expression of Activation Markers CD11b and HLA-DR on Human Leukocytes

1998 ◽  
Vol 5 (5) ◽  
pp. 695-702 ◽  
Author(s):  
Sharon Shalekoff ◽  
Liesl Page-Shipp ◽  
Caroline T. Tiemessen
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Venous Blood ◽  
Polymorphonuclear Neutrophils ◽  
Preliminary Evaluation ◽  
Activation Markers ◽  
Gm Csf ◽  
Hla Dr ◽  
Ifn Γ ◽  
Peripheral Leukocytes

ABSTRACT A whole-blood model was used to evaluate the effects of temperature and anticoagulant on the expression of activation markers HLA-DR and CD11b on peripheral leukocytes. Venous blood, anticoagulated with either EDTA or heparin, was obtained from six healthy blood donors and 13 hospitalized patients (8 human immunodeficiency virus type 1-seropositive individuals with concurrent pulmonary tuberculosis and 5 patients with pneumonia). A preliminary evaluation was carried out with whole blood from two of the normal donors, and cells were stained immediately for HLA-DR and CD11b markers or stained after incubation at room temperature or 37°C for 18 h with or without the addition of the cytokines gamma interferon (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ plus GM-CSF, tumor necrosis factor beta, or interleukin-6. Of the cytokines tested, the combination of IFN-γ and GM-CSF had the most pronounced modulation of marker expression on polymorphonuclear neutrophils (PMN), in particular, HLA-DR expression, which required induction for its detection. These cytokines were therefore used in further evaluations that considered the above-mentioned effects in the presence of disease. Results indicated that the expression of activation markers on PMN and lymphocytes in whole blood are influenced by the temperature of incubation and the choice of anticoagulant and the effects noted were dependent on (i) the particular cell surface marker, (ii) the cell type being studied, and (iii) the presence or absence of disease. It is therefore recommended that ex vivo whole-blood models for evaluating phenotype or immune function be carefully evaluated for the above-mentioned effects.

Thrombosomes Show Dose-Dependent Increase in Thrombus Formation in a Model of Deep Arterial Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2319-2319
Author(s):  
Nikhil Vilas Joshi ◽  
Jennifer Raftis ◽  
AJ Lucking ◽  
Narendra Tandon ◽  
M Fitzpatrick ◽  
...  
Keyword(s):  
Whole Blood ◽  
Immunohistochemical Staining ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Arterial Injury ◽  
Dependent Manner ◽  
Flow Conditions ◽  
Structural And Functional Properties ◽  
Dose Dependent

Abstract Abstract 2319 Introduction Thrombosomes are novel lyophilized human platelet derivatives that retain a number of key platelet structural and functional properties. Building on preliminary in vitro studies, we here sought to investigate whether Thrombosomes, suspended in platelet free plasma (PFP), would enhance and be incorporated in thrombus generated under flow conditions within a validated and well-characterised model of deep arterial injury. Methods PFP was obtained by centrifuging citrated whole blood from six healthy non-smoking volunteers and filtering with a 0.22μm filter. Thrombosomes were suspended in 60 mL of PFP to generate final concentrations of 20 and 200 × 106Thrombosomes /mL. Immediately prior to use, 1.2 mL of 1M CaCl2 was added to permit fibrin generation. Thrombus formation was assessed using the Badimon Chamber at low (212 s−1) and high (1690 s−1) shear rates with porcine aortic tunica media as the thrombogenic substrate. Total thrombus area and platelet-rich area were measured histomorphometrically using conventional and immunohistochemical staining respectively. Fluorescent labeled Thrombosomes were added to the extracorporeal circuit in the Badimon chamber to study the ex vivo thrombus generation in the whole blood. Electron microscopy of Thrombosomes and platelets was undertaken. Results Thrombosomes contributed towards thrombus formation in whole human blood as evidenced by incorporation of fluorescent-labeled Thrombosomes into the thrombus. Immunohistochemical staining of the glycoprotein IIb/IIIa receptor confirmed incorporation of Thrombosomes into the thrombus. In the high shear chamber, mean thrombus area increased in a dose-dependent manner following the addition of Thrombosomes (704 μm2 [95% CI, 224 – 1184 μm2], 1511 μm2 [95% CI, 687 – 2336 μm2] and 2378 μm2 [95% CI, 1567 – 3189 μm2] for PFP and Thrombosomes at concentrations of 20 and 200 × 106/mL respectively [P= 0.003]). In the low shear chamber, total thrombus area for the PFP was 4962 μm2 (95% CI, 2489 – 7434 μm2). The addition of Thrombosomes at concentrations of 20 and 200 × 106/mL led to a numerical increase in mean thrombus area to 6170 μm2 (95% CI, 3944 – 8397 μm2) and 7504 μm2 (95% CI, 3864 – 11144 μm2) respectively, although this was not statistically significant (P= 0.2969). Conclusions Thrombosomes suspended in PFP and exposed to injured arterial tissue under physiologically relevant flow conditions are incorporated into thrombus and enhance thrombus formation in a dose dependent manner. These findings act as impetus to undertake clinical studies of this rehydrated, lyophilized infusible hemostatic platelet product. Disclosures: Fitzpatrick: Cellphire Inc.: Employment, Equity Ownership. Feuerstein:Cellphire: Consultancy. Newby:Cellphire: Research Funding.

Spindle scaling mechanisms

2020 ◽  
Vol 64 (2) ◽  
pp. 383-396
Author(s):  
Lara K. Krüger ◽  
Phong T. Tran
Keyword(s):  
Cell Size ◽  
Spindle Assembly ◽  
Dependent Manner ◽  
Post Translational Modification ◽  
Size Dependent ◽  
A Cell ◽  
Chromosome Separation ◽  
Spindle Elongation ◽  
Protein Gradients ◽  
Spindle Structure

Abstract The mitotic spindle robustly scales with cell size in a plethora of different organisms. During development and throughout evolution, the spindle adjusts to cell size in metazoans and yeast in order to ensure faithful chromosome separation. Spindle adjustment to cell size occurs by the scaling of spindle length, spindle shape and the velocity of spindle assembly and elongation. Different mechanisms, depending on spindle structure and organism, account for these scaling relationships. The limited availability of critical spindle components, protein gradients, sequestration of spindle components, or post-translational modification and differential expression levels have been implicated in the regulation of spindle length and the spindle assembly/elongation velocity in a cell size-dependent manner. In this review, we will discuss the phenomenon and mechanisms of spindle length, spindle shape and spindle elongation velocity scaling with cell size.

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