Maresin 1 induces a novel pro-resolving phenotype in human platelets

Abstract

Essentials Specialized proresolving mediators (SPMs) promote the resolution of inflammation. This study sought to investigate the effects of SPMs on human platelet function. The SPM, Maresin 1, enhanced hemostatic, but suppressed inflammatory functions of platelets. SPMs uniquely regulate platelet function and may represent a new class of antiplatelet agents.

Summary: Background Antiplatelet therapy is a cornerstone of modern medical practice and is routinely employed to reduce the likelihood of myocardial infarction, thrombosis and stroke. However, current antiplatelet therapies, such as aspirin, often have adverse side-effects, including increased risk of bleeding, and some patients are relatively 'aspirin-resistant'. Platelets are intimately involved in hemostasis and inflammation, and clinical consequences are associated with excessive or insufficient platelet activation. Objectives A major unmet need in the field of hematology is the development of new agents that safely prevent unwanted platelet activation in patients with underlying cardiovascular disease, while minimizing the risk of bleeding. Here, we investigate the potential of endogenously produced, specialized pro-resolving mediators (SPMs) as novel antiplatelet agents. SPMs are a recently discovered class of lipid-derived molecules that drive the resolution of inflammation without being overtly immunosuppressive. Methods Human platelets were treated with lipoxin A4, resolvin D1, resolvin D2, 17-HDHA or maresin 1 for 15 min, then were subjected to platelet function tests, including spreading, aggregation and inflammatory mediator release. Results We show for the first time that human platelets express the SPM receptors, GPR32 and ALX. Furthermore, our data demonstrate that maresin 1 differentially regulates platelet hemostatic function by enhancing platelet aggregation and spreading, while suppressing release of proinflammatory and prothrombotic mediators. Conclusions These data support the concept that SPMs differentially regulate platelet function and may represent a novel class of antiplatelet agents. SPMs also may play an important role in the resolution of inflammation in cardiovascular diseases.

Keywords: antiplatelet agents; hemostasis; inflammation; platelet activation; platelets.

Figure 1. Human platelets and the megakaryocyte cell line, Meg01, express the SPM receptors, GPR32 and ALX

Washed human platelets were generated from leukoreduced whole blood from healthy human donors. Platelets were blocked with human Fc Receptor blocking reagent for 30 minutes, then stained with rabbit anti-human GPR32 followed by rabbit FITC secondary antibody (A–B) or rabbit anti-human fpr1-FITC (D–E) antibodies for one hour. Grey shaded histogram represents the isotype control. Mean +/− SEM n=3. The mean fluorescence intensity (MFI) for three independent human donors is shown for GPR32 (B) and ALX (E). The MFI for GPR32 was determined by subtracting the MFI from secondary only control. (C, F) Lysates were generated from washed human platelets, the megakaryocyte Meg01 cell line, A549 cells (GPR32 positive control), or HEK cells (ALX positive control). Proteins were separated on two separate 10% SDS-PAGE gels and probed for GPR32 or ALX. One representative donor is shown.

Figure 2. SPMs mitigate platelet aggregation

PRP was generated from healthy human donors and treated with 100 nM SPMs (Lipoxin A4, LXA4; Resolvin D1, RvD1; Resolvin D2, RvD2; 17HDHA; and Maresin 1, Mar1) for 15 minutes prior to initiation of aggregation by 5 μM ADP (A–D). Aggregation was measured for five minutes and the percent change in maximum amplitude was calculated for each SPM relative to vehicle control (0.1% EtOH) (E). Statistical significance was determined by One-way ANOVA with Dunnett’s multiple comparison post-test. *p<0.05, ***p<0.001

Figure 3. SPMs mitigate platelet spreading

Washed platelets were generated from healthy human donors and treated with vehicle or 100 nM SPMs for 15 minutes then were allowed to spread on fibrinogen-coated coverslips for 45 minutes. Coverslips were fixed and visualized by differential interference contrast (DIC) optics using an Olympus BX51 microscope at 100X and SPOT computer software. Scale bars = 10 microns. One representative donor of 5 is shown (A–F). The percentage of fully spread platelets per field of view was determined by manual counting four fields of view for 5 individual donors and the percent change in spreading was calculated for each SPM relative to vehicle control (0.1% EtOH). (E). Mean +/− SEM. n=5 Statistical significance determined by One-Way ANOVA with Dunnett’s multiple comparison post-test. *p<0.05, ***p<0.001

Figure 4. SPMs differentially affect mediator release from unactivated and activated human platelets

Freshly isolated washed platelets from healthy human donors were treated with vehicle (0.1% DMSO), SPMs for 30 minutes then were either left unactivated, or activated with 5 μM ADP or 0.1U/mL thrombin for 15 minutes. Supernatants were collected and analyzed for PF4 or sCD40L release by ELISA (A–B) or platelets were assessed for CD62P positivity by flow cytometry (C). Mean +/− SEM. n=4 Statistical significance was determined by Two-Way RM ANOVA with Dunnett’s multiple comparison post-test.

Figure 5. Maresin 1 enhances platelet spreading, aggregation, and dense granule release

PRP was generated from healthy human donors and treated with vehicle (0.1% EtOH) or maresin 1 for 15 minutes then were allowed to spread on fibrinogen-coated coverslips for 45 minutes. Coverslips were fixed and visualized by differential interference contrast (DIC) optics using an Olympus BX51 microscope at 100X and SPOT computer software. The percentage of fully spread platelets per field of view was determined by manual counting four fields of view for 5 individual donors and the percent change in spreading was relative to vehicle control (A). Aggregation was initiated by 5 μM ADP, then aggregation and ATP release were measured for five minutes. The maximum percent aggregation was calculated (B). One representative donor is shown for aggregation and ATP release in response to ADP (C). The inactive isomer of 7R-maresin 1, 7S-maresin 1, was added to platelets prior to aggregation with ADP (D). Maximum ATP release, the slope, lag time, and area under the curve were calculated for 4 independent donors (E–H). Statistical significance was determined by Kruskal-Wallis test with Dunns multiple comparison post-test. *p<0.05

Figure 6. Maresin 1 dampens inflammatory mediator release from activated platelets

Freshly isolated PRP from healthy human donors was treated with vehicle (0.1% DMSO) or maresin 1 for 15 minutes then were either left unactivated, or activated with 5 μM ADP, 5 μg/mL Collagen, or 0.1 U/mL thrombin for 15 minutes. Supernatants were collected and analyzed for mediator release by ELISA (A–C) or platelets were assessed for CD62P positivity by flow cytometry (D). The percentage of platelet microparticles (PMPs) was determined by flow cytometry (E). Mean +/− SEM. n=4 Statistical significance was determined by Two-Way RM ANOVA with Dunnett’s multiple comparison post-test.

Figure 7. Maresin 1 differentially affects mediator release from activated platelets

Freshly isolated washed platelets from healthy human donors were treated with vehicle (0.1% DMSO) or maresin 1 for 15 minutes then were either left unactivated (A), or activated with 5 μM ADP (B), or 0.1 U/mL thrombin (C) for 15 minutes. Supernatants were collected and analyzed for mediator release by Luminex Assay. Mean +/− SEM. n=4 Statistical significance was determined by One-Way RM ANOVA with Dunnett’s multiple comparison post-test.

Figure 7. Maresin 1 differentially affects mediator release from activated platelets

Freshly isolated washed platelets from healthy human donors were treated with vehicle (0.1% DMSO) or maresin 1 for 15 minutes then were either left unactivated (A), or activated with 5 μM ADP (B), or 0.1 U/mL thrombin (C) for 15 minutes. Supernatants were collected and analyzed for mediator release by Luminex Assay. Mean +/− SEM. n=4 Statistical significance was determined by One-Way RM ANOVA with Dunnett’s multiple comparison post-test.

Figure 7. Maresin 1 differentially affects mediator release from activated platelets

Freshly isolated washed platelets from healthy human donors were treated with vehicle (0.1% DMSO) or maresin 1 for 15 minutes then were either left unactivated (A), or activated with 5 μM ADP (B), or 0.1 U/mL thrombin (C) for 15 minutes. Supernatants were collected and analyzed for mediator release by Luminex Assay. Mean +/− SEM. n=4 Statistical significance was determined by One-Way RM ANOVA with Dunnett’s multiple comparison post-test.