iMer, a naturally occurring MERTK splice variant, binds to GAS6 to decrease platelet activation and thrombus formation

Abstract

Unopposed platelet activation can be associated with pathologic thrombosis. An intact growth arrest-specific gene 6 (GAS6)/Mer receptor tyrosine kinase (MERTK) signaling pathway contributes importantly to potentiating platelet activation triggered by molecular agonists ex vivo and thrombus stabilization in vivo. We describe, herein, the inhibition of platelet function and stable thrombus formation conferred by iMer, a naturally occurring MERTK splice variant, that acts as a GAS6 decoy receptor and decreases phosphorylation of MERTK. Human and murine platelets incubated with this truncated protein demonstrate reduced activation in ex vivo assays including aggregometry (similar to treatment with anti-GAS6 antibody), expression of P-selectin, spreading on collagen, and accumulation on collagen at a venous shear rate. Wild-type C57BL/6 mice treated with iMer had improved survival in a collagen/epinephrine-induced pulmonary embolism model, without increase in tail bleeding time on preliminary analysis. Taken together, these findings confirm previous data suggesting the importance of GAS6-MERTK signaling in platelet activation and thrombus formation and highlighting the potential therapeutic implications of targeting this pathway as a means of treating or preventing thrombosis.

Graphical abstract

Figure 1.

iMer is expressed in gastrointestinal, genitourinary, endocrine, and lymphatic tissues, and inhibits phosphorylation of MERTK induced by GAS6. (A) iMer and full-length MERTK transcripts were detected by reverse transcription PCR, with G3PDH as loading control, from C57BL/6 wild-type mouse tissue. (B) A total of 697 cells treated with vehicle control or with iMer for 10 or 20 minutes as indicated were cultured with or without 200 nM GAS6 to activate MERTK receptor. MERTK was then immunoprecipitated. Phosphorylated (denoted by p-MERTK) and total MERTK proteins were detected by immunoblot. Images shown are representative of 2 independent experiments. (C) Mer isoform prevalence in platelets varies among individuals. Immunoblot of platelet lysates collected from 3 donors, 1 of whom had multiple samples collected across different days. The blots demonstrate full-length MERTK (cMer), soluble MERTK extracellular domain (sMer), and iMer. G3PDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2.

iMer and GAS6 bind directly in coimmunoprecipitation. Brackets depict protein added and blots are labeled by detection antibody as anti-MERTK (MERTK detect) or anti-GAS6 (GAS6 detect) as indicated. (A) Glutathione S-transferase–tagged GAS6/iMer complexes were detected by immunoprecipitating with anti-human MERTK antibody and immunoblotting with an anti-human GAS6 antibody. (B) Respectively, GAS6/iMer complexes were also detected when immunoprecipitated with anti-human GAS6 antibody and immunoblotting with anti-human MERTK antibody. Also shown are the input protein and same protein immunoprecipitation/immunoblot controls. (C) His-tagged GAS6 (200 nM) or 200 nM each GAS6 and iMer were immunoprecipitated with anti-human GAS6 antibody. iMer coimmunoprecipitates with the GAS6 and is detected with anti-human MERTK antibody. Also shown are control immunoprecipitations with anti-human MERTK and input iMer. (D) Respectively, 200 nM iMer or 200 nM each iMer and GAS6 were immunoprecipitated with anti-human MERTK antibody. GAS6 coimmunoprecipitates with the iMer and is detected with anti-human GAS6 antibody. Also shown are the control immunoprecipitations with anti-human GAS6 and input GAS6. Images are representative of 3 independent assays. IP, immunoprecipitation.

Figure 3.

Removal of circulating GAS6 by addition of either iMer or anti-GAS6 antibody dampens ADP-induced platelet aggregation. (A) Representative light transmission aggregometry tracings showing inhibition of 1 to 2 mM ADP-induced platelet aggregation in human PRP in the presence of 5 μM iMer (red) or vehicle control (green). Tracings are representative of 2 to 4 independent experiments. (B) Quantitation of 4 independent experiments, median value and interquartile range are shown; ∗∗P < .01, Wilcoxon signed pairs rank test. (C) Representative tracings showing ADP-induced platelet aggregation in the presence of 5 μM iMer (red), vehicle control (green), or 5 μM iMer plus rhGAS6 (blue). Addition of excess GAS6 can partially overcome iMer-mediated inhibition. (D) Summary of 3 experiments; ∗P <.05 for control vs iMer-treated; nsfor other comparisons. (E) Representative tracings of ADP-induced platelet aggregation in the presence of GAS6 antibody (red), vehicle control (green), and GAS6 antibody plus rhGAS6 (blue). (F) Summary of 4 separate experiments, P < .05 for control vs anti-GAS6 antibody treatment; P = ns for other comparisons, Friedman test.

Figure 4.

Platelet aggregation is not affected by treatment with iMer in the absence of GAS6. Human PRP was treated with antibody to deplete GAS6 and then incubated with 5 μM iMer (red) or vehicle control (green) before platelet activation was stimulated with 1 to 2 mM ADP. Median values and error bars denoting IQR from 3 experiments are shown (P = ns, Wilcoxon signed pairs rank test).

Figure 5.

iMer inhibits platelet granule release stimulated by thrombin. Human WPs were exposed to 0.5 U/mL thrombin for 10 minutes after incubation with 5 μM iMer (red) or vehicle control (green) for 10 minutes. Median values and IQR from 4 experiments are shown (n = 4; P < .05, Wilcoxon signed pairs rank test). MFI, mean fluorescense intensity.

Figure 6.

iMer inhibits stable aggregate formation under conditions of physiologic shear. (A) Human WB was pulled vertically (bottom to top) across horizontally oriented fibrillar collagen by vacuum syringe at a venous shear rate of 750 s⁻¹. Plts were stained with anti-CD41 antibody (blue), then counterstained with anti–P-selectin antibody (green). (B) Surface area coverage was calculated by densitometry and circularity measurements and was significantly higher in vehicle-treated human platelets (green, n = 7), compared with iMer-treated platelets (red, n = 7 independent experiment samples; P < .05, Wilcoxon signed pairs rank test). (C) Quantitation of brightfield images of platelets showing a higher proportion of small aggregates (1-2 and 3-10 platelets per aggregate) after pretreatment with iMer than samples pretreated with vehicle control, which contained higher proportions of large aggregates (11-50, 50-100, and >100 platelets per aggregate). plts, platelets.

Figure 7.

iMer protects against thrombus formation without increasing bleeding in preliminary studies. (A) C57BL/6 mice were injected with collagen/epinephrine to induce systemic venous thrombosis resulting in pulmonary embolism. WT mice treated with 60 mg/kg iMer (red, n = 9) and Gas6^−/− KO mice (blue, n = 6) exhibited a significant increase in survival compared with vehicle-treated controls (green line; n = 7; ∗∗P < .01 by log-rank [Mantel-Cox] test). (B) Tail-clip bleeding times did not differ significantly among WT mice treated with 60 mg/kg iMer (red, n = 3) or vehicle control (green, n = 8), or Gas6^−/− KO mice (blue, n = 5; ∗∗P < .01 by log-rank [Mantel-Cox] test). KO, knockout; WT, wild-type.