Platelet Response to Allergens, CXCL10, and CXCL5 in the Context of Asthma

Asthma is a chronic respiratory disease initiated by a variety of factors, including allergens. During an asthma attack, the secretion of C-X-C-motif chemokine 10 (CXCL10) and chemokine ligand 5 (CCL5) causes the migration of immune cells, including platelets, into the lungs and airway. Platelets, which contain three classes of chemical messenger-filled granules, can secrete vasodilators (adenosine diphosphate and adenosine triphosphate), serotonin (a vasoconstrictor and a vasodilator, depending on the biological system), platelet-activating factor, N-formylmethionyl-leucyl-phenylalanine ((fMLP), a bacterial tripeptide that stimulates chemotaxis), and chemokines (CCL5, platelet factor 4 (PF4), and C-X-C-motif chemokine 12 (CXCL12)), amplifying the asthma response. The goal of this work was threefold: (1) to understand if and how the antibody immunoglobulin E (IgE), responsible for allergic reactions, affects platelet response to the common platelet activator thrombin; (2) to understand how allergen stimulation compares to thrombin stimulation; and (3) to monitor platelet response to fMLP and the chemokines CXCL10 and CCL5. Herein, high-pressure liquid chromatography with electrochemical detection and/or carbon-fiber microelectrode amperometry measured granular secretion events from platelets with and without IgE in the presence of the allergen 2,4,6-trinitrophenyl-conjugated ovalbumin (TNP-Ova), thrombin, CXCL10, or CCL5. Platelet adhesion and chemotaxis were measured using a microfluidic platform in the presence of CXCL10, CCL5, or TNP-OVA. Results indicate that IgE binding promotes δ-granule secretion in response to platelet stimulation by thrombin in bulk. Single-cell results on platelets with exogenous IgE exposure showed significant changes in the post-membrane–granule fusion behavior during chemical messenger delivery events after thrombin stimulation. In addition, TNP-Ova allergen stimulation of IgE-exposed platelets secreted serotonin to the same extent as thrombin platelet stimulation. Enhanced adhesion to endothelial cells was demonstrated by TNP-Ova stimulation. Finally, only after incubation with IgE did platelets secrete chemical messengers in response to stimulation with fMLP, CXCL10, and CCL5

Stability of candidate housekeeping gene expression <i>in P. yezoensis</i> (from smallest to largest difference) determined by difference between the gametophytes and the conchospores.

<p>Results are calculated as maximum/minimum. Difference (RNA) is determined by transcript number normalized to total RNA quantity and Difference (DNA) is determined by transcript number normalized to genomic DNA quantity.</p

The standard curves constructed for <i>18S</i> (A), <i>Act3</i> (B), <i>EF1alpha</i> (C), <i>GAPDH</i> (D), <i>PUB-2</i> (E), <i>RPS8</i> (F), <i>TubB</i> (G).

<p>The results showed that amplification efficiency was between 96% and 103%, and linear correlation coefficient was >0.99.</p

Primer names, sequences and PCR product size of selected candidate housekeeping genes.

<p>Primer names, sequences and PCR product size of selected candidate housekeeping genes.</p

Yields of total RNA and genomic DNA of various sample groups.

<p>Yields of total RNA and genomic DNA of various sample groups.</p

Stability of candidate housekeeping gene expression <i>in P. yezoensis</i> (from smallest to largest difference) determined by difference across all samples.

<p>Results are calculated as maximum/minimum. Difference (RNA) is determined by transcript number normalized to total RNA quantity and Difference (DNA) is determined by transcript number normalized to genomic DNA quantity.</p

The melting curve analysis for <i>18S</i> (A), <i>Act3</i> (B), <i>EF1alpha</i> (C), <i>GAPDH</i> (D), <i>PUB-2</i> (E), <i>RPS8</i> (F), <i>TubB</i> (G).

<p>Melting peaks were examined with standard samples and unkown samples (sporophytes, gametophytes and conchospores). The melting curve for each gene had only one peak.</p

The OD 260/280 ratios of extracted nucleic acid.

<p>The OD 260/280 ratios of extracted nucleic acid.</p

Transcript numbers of candidate housekeeping genes in <i>P. yezoensis</i> determined by absolute quantitative analysis normalized to total RNA quantity (copies/μg).

<p>Transcript numbers of candidate housekeeping genes in <i>P. yezoensis</i> determined by absolute quantitative analysis normalized to total RNA quantity (copies/μg).</p

Stability of candidate housekeeping gene expression <i>in P. yezoensis</i> (from smallest to largest difference) determined by difference between the sporophytes and the conchospores.

<p>Results are calculated as maximum/minimum. Difference (RNA) is determined by transcript number normalized to total RNA quantity and Difference (DNA) is determined by transcript number normalized to genomic DNA quantity.</p