In Vitro Methods to Characterize the Effects of Tobacco and Nontobacco Products on Human Platelet Function

Loelius, Shannon G.; Spinelli, Sherry L.; Lannan, Katie L.; Phipps, Richard P.

doi:10.1002/cptx.46

Cited by 3 publications

(3 citation statements)

References 14 publications

(11 reference statements)

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“…Following thrombin stimulation, normal platelets attach rapidly to immobilized fibrinogen and follow sequential phases of spreading, creating filopodia followed by lamellipodia extensions, until achieving the fully spread state . Using IRM imaging, we observed significant differences in the pattern of adherent platelets between JAK2 mutated patients, CALR mutated patients and controls (Figure ).…”

Section: Resultsmentioning

confidence: 94%

“…Interference reflection microscopy (IRM) time‐lapse imaging of several fields per specimen was carried out using a 100x/1.3 oil objective, at 5 seconds between frames. Platelet activation elicits major cytoskeletal rearrangements, resulting in shape change; platelets round up, then extend filopodia and lamellipodia, finally spreading into a roughly‐circular shape . Fully spread platelets were manually recognized and counted at the end of the experiment (30 minute time point), using a 20X objective.…”

Section: Methodsmentioning

confidence: 99%

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Platelets from Calreticulin mutated essential thrombocythemia patients are less reactive than JAK2 V617F mutated platelets

et al. 2020

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Both JAK2V617F and calreticulin (CALR) mutated essential thrombocythemia (ET) patients have different clinical characteristics, with lower thrombosis risk in patients with CALR mutations. To elucidate the mechanism for this lower risk we studied platelet function in ET patients with either JAK2V617F or a CALR mutation. Platelet activation state was similar in ET and healthy controls at baseline using P‐selectin and PAC1 flow cytometry analysis. However, CALR mutated platelets were significantly less activated following ADP stimulation, compared to control or JAK2 mutated platelets (P < .001). In live‐cell imaging of platelet attachment to immobilized fibrinogen by Interference Reflection Microscopy (IRM), the number of attached CALR mutated platelets was lower compared to control and JAK2 mutated platelets, with lower fractions of platelets achieving the fully spread state (90%, 78% and 54% of adherent cells for control, JAK2 and CALR mutated subjects, respectively). Compared to controls, ET patients, regardless of the mutation type, had increased numbers of immature platelets (IP) and leukocyte platelet aggregates (LPA), as well as plasma sP‐selectin. These were all correlated with the platelet count and not to the state of platelet activation. We also found that intracellular free Ca2+ was increased in resting ET compared to control platelets. Note, CALR had a more dispersed localization in activated ET platelets compared to healthy controls, and mutated CALR interact physically with TpoR in CALR mutated platelets. We hypothesize that defects in platelet activation and spreading in CALR mutated patients can explain, at least in part, the lower thrombotic tendency in CALR mutated ET patients.

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Section: Resultsmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

Platelets from Calreticulin mutated essential thrombocythemia patients are less reactive than JAK2 V617F mutated platelets

et al. 2020

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“…After centrifuging at 1100 × g for 5 minutes to discard the supernatant, the pellet was washed twice in phosphate-buffered saline (PBS) containing EDTA (0.009 mol/l Na2EDTA,0.0264 mol/l Na2HPO4, 0.07 mol/l NaCl). The concentration of platelets in PBS-EDTA buffer was 1 × 10 7 /ml [24][25][26].…”

Section: Preparation Of Platelet Suspensionmentioning

confidence: 99%

Aberrant Expression of a Proliferation-Inducing Ligand Underlies Autoimmune Mechanisms in Immune Thrombocytopenia

Hao

Yang

Zhou

2018

Blood

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Background: Immunological thrombocytopenia (ITP) is an antibody-mediated autoimmune disease characterized by accelerated platelet destruction and suboptimal platelet production. The proliferation-inducing ligand (APRIL or TNFSF13), a member of the TNF superfamily, is structurally and functionally related to the TNF family of B cell activating factors (BAFF, TNFSF13b) and has been shown to regulate lymphocyte survival by interacting with its receptors. And activation. Transmembrane activators and calcium regulate cyclophilin ligand interactors (TACI) and B cell mature antigens (BCMA). APRIL is secreted by various cells as soluble factors, including inactive B cells, T cells, monocytes, neutrophils, macrophages and dendritic cells, as well as epithelial cells, osteoclasts and megakaryocytes. Recent studies have shown that APRIL not only participates in normal immune responses, but also plays an important role in the establishment and/or maintenance of autoimmune and inflammatory diseases. Aims: Based on the relationship between APRIL, which promotes proliferation and regulates immunity, and the development of autoimmunity, we hypothesize that APRIL may play a role in the pathogenesis of ITP. Methods:1. The EDTA anticoagulated whole blood was collected, and peripheral blood mononuclear cells (PBMC) were separated by Ficoll density gradient centrifugation. The APRIL levels on the surface of T cells, B cells, DC cells and platelets were detected by flow cytometry.Detection of plasma APRIL levels in patients with ITP by ELISA.Real time quantitative PCR were used for detecting the level of APRIL and its receptors BCMA and TACI from PBMC of healthy controls and ITP patients.Use soluble APRIL or BLyS protein and APRIL inhibitors to examine the effect of APRIL inhibition on IL-10 secretion by B cells. Flow cytometry and intracellular staining were used to evaluate B10 cells. Resoult: 1. The APRIL on the platelet surface of patients with ITP was significantly lower than that of the normal control group (p<0.01). In the ITP patients of 10 patients with complete remission, the content of APRIL on the platelet surface was significantly increased after treatment (p=0.02), and there was no significant change in the treatment-ineffective group. . The levels of APRIL and its receptors BCMA and TACI on B cells and DC cells in ITP patients were higher than those in normal controls, and the difference was statistically significant. APRIL is not expressed on CD4 + T cells, CD8 + T cells.The expression of APRIL mRNA in PBMNCs was significantly higher in ITP patients than in the normal control group (p <0.01). There was no difference in BCMA and TACI expression in PBMNC of ITP patients compared to normal controls.Plasma APRIL levels were significantly higher in ITP patients than in the normal control group, p = 0.04, and negatively correlated with platelet count, p = 0.029.In 10 patients with ITP, the percentage of CD19 + B cells remained similar between patients, and the results showed that the amount of B10 cells in the medium supplemented with APRIL was greater than that of B10 cells containing BLyS and control medium (p<0.01; p= 0.01), and the use of APRIL inhibitors resulted in a decrease in B10 cells. Conclusion: Our study shows that aberrant expression of APRIL is involved in the autoimmune response of ITP, and the effect of treatment can be assessed by measuring changes in the level of APRIL on the platelet surface. We also speculate that APRIL inhibits, rather than promotes, an immune-mediated inflammatory response in the pathogenesis of ITP. Our current observations support that the immunomodulatory effects of APRIL may be due, at least in part, to stimulation of IL-10 producing B cells. Disclosures No relevant conflicts of interest to declare.

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Aberrant Expression of a Proliferation-Inducing Ligand Underlies Autoimmune Mechanisms in Immune Thrombocytopenia

et al. 2021

Journal of Immunology Research

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Purpose. To study the relationship between surface membrane-bound APRIL and ITP. Methods. The peripheral blood of all subjects, 50 patients diagnosed with ITP and 25 healthy controls, was collected. Flow cytometry was used to detect the expression of membrane-bound APRIL on immune cells and platelets. ELISA was used to detect the content of soluble APRIL in plasma. Results. Membrane-bound APRIL was only expressed on the surface of platelets in both ITP patients and controls. APRIL expression on the platelet surface was significantly lower in newly diagnosed ( P < 0.001 ) and chronic ( P < 0.001 ) ITP patients than in controls. Platelet surface APRIL level was significantly enhanced in patients with complete remission after treatment ( P = 0.02 ) but not in those with no response after treatment. Platelet surface APRIL level in ITP patients was negatively correlated with serum APRIL level ( r = − 0.09765 , P = 0.0424 ). Conclusions. Platelet surface APRIL may play a key immunoregulative role. Platelet surface APRIL is likely to be one source of the excessive serum APRIL in ITP patients. The effectiveness of treatment may be measured by determining the platelet surface APRIL levels in ITP patients.

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In Vitro Methods to Characterize the Effects of Tobacco and Nontobacco Products on Human Platelet Function

Cited by 3 publications

References 14 publications

Platelets from Calreticulin mutated essential thrombocythemia patients are less reactive than JAK2 V617F mutated platelets

Platelets from Calreticulin mutated essential thrombocythemia patients are less reactive than JAK2 V617F mutated platelets

Aberrant Expression of a Proliferation-Inducing Ligand Underlies Autoimmune Mechanisms in Immune Thrombocytopenia

Aberrant Expression of a Proliferation-Inducing Ligand Underlies Autoimmune Mechanisms in Immune Thrombocytopenia

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