Generation of platelet-derived microparticles through the activation of the toll-like receptor 4

Abstract

Introduction: Infection from different bacterial may increase the risk of thrombosis and atherosclerosis risk by production and secretion of many proinflammatory factors. Human platelets have toll-like receptor 4 (TLR4), the principal receptor for lipopolysaccharide (LPS). The activation of platelet produces Platelet-derived Microparticles (PDMPs) measuring less than 1.0 micron (that are very abundant in circulation >90%), which are associated with the development of Cardiovascular Diseases (CVDs), the leading cause of death in the world.

Objectives: Experiments were designed to evaluate the generation of pro-thrombogenic microparticles in vitro on platelets via TLR4 activation.

Methods: Platelet-rich plasma and washed platelets from healthy volunteers were incubated for the generation of PDMPs. The best source for the generation of microparticles was washed platelets. Then the washed platelets were incubated for 15 minutes with ultrapure Escherichia coli LPS (0-9 μg/mL) followed by activation with ADP (1 μM, subaggregant concentration), centrifuged for 60 minutes and analyzed by flow cytometry.

Results: Incubating platelets with LPS (9 μg/mL) and ADP (1 μM) produced a 34-fold increase in PDMPs generation. Finally, we evaluated this protocol to detect the inhibition of PDMPs generation, washed platelets were incubated with acetylsalicylic acid (10 μM) and an inhibition of 7.7-fold in PDMPs generation for activation of TLR4 was found.

Conclusion: A new and easy protocol for PDMPs generation and analysis by Flow Cytometry is established. In the future it could be used to determine the association of PDMPs with different pathologies.

Keywords: Biochemistry; Immunology.

Fig. 1

Generation of microparticles from a platelet-rich plasma. (A) Construction of the PDMPs region according to size (FSC-A) and complexity (SSC-A), the region is limited in the upper part of the 1.0 μm bead (B–D) Representative Histogram of PDMPs (CD61 positive events). The fluorescence intensity (x-axis) versus the PDMPs (y-axis) count is shown, solid line divides the x-axis (positive to CD61) into two quartiles (Q1 and Q2), and the percentage of positivity for PDMPs is represented in Q2. (E) The basal PDMPs were compared with the stimulating platelets by ADP (4 μM) for 30 and 60. ns, not significant and ***, p < 0.0001; each in triplicate.

Fig. 2

Generation of microparticles from washed platelets (A–C) As in the previous figure shows the representative Histogram of PDMPs (CD61 positive events) by fluorescence intensity (x-axis) versus the percentage of PDMPs (y-axis), of quartile 2 (Q2) represents the percentage of positivity for PDMPs. (D) The sample stimulated with ADP (4 μM) for 30 and 60 was compared with the basal. ***, p < 0.0001 analyzed by ANOVA using Bonferroni's post-hoc test.

Fig. 3

Standardization and Generation of PDMPs in response to the stimulation with Lipopolysaccharide (LPS) and Adenosine diphosphate (ADP) (A–F) Representative Histogram of PDMPs (CD61 positive events). Shows the fluorescence intensity (x-axis) versus the PDMPs (y-axis) and quartile (Q2) is the percentage of PDMPs (positive to CD61). (G) The generated PDMPs were compared by stimulating with LPS (0–9 μg/mL) and ADP (1 μM, subaggregant concentration) with the basal. ***p < 0.0001 analyzed by ANOVA using Bonferroni's post-hoc test.

Fig. 4

Effects of acetylsalicylic acid (ASA) in the generation of PDMPs induced by stimulation of washed platelets with Lipopolysaccharide (LPS) and Adenosine diphosphate (ADP). (A) Representative Histogram of the PDMPs generation induced by LPS (0–9 μg/mL) and ADP (1 μM, subaggregant concentration) the washed platelets were previously incubated for 10 minutes with ASA (10 μM). The fluorescence intensity (x-axis) versus the percentage of PDMPs (y-axis) is shown, the quartile 2 (Q2) is the region positive for CD61. (B) The Graphs show the effects of ASA (10 μM) on platelet microvesiculation induced by LPS (0–9 μg/mL) and ADP (1 μM, subaggregant concentration), we contrast the different PDMPs levels, where the ASA samples have a 7.7-fold decrease compared with the stimulated sample. ***p < 0.0001; each of them in triplicate.