A Small Molecule Inhibitor of the Gas6/Mer Pathway Inhibits Platelet Activation and Thrombosis with Equal Efficacy to, but Greater Potency Than, iMer, the Novel MerTK Splice Variant
Authors: Branchford Brian R. , Law Luke , Sather Susan , Brodsky Gary , Earp H. Shelton , Wang Xiaodong , Frye Stephen , Graham Douglas K. , Di Paola Jorge A 
Published: October 2019

Abstract
Abstract 3303

Background:
Growth Arrest Specific gene 6 (Gas6) signals through platelet-surface Mer receptors, leading to platelet activation and thrombus stabilization via activation of PI3K and Akt, and β3 integrin phosphorylation. This amplifies outside-in signaling via αIIb β3, a necessary step for stable platelet aggregation. iMer is a truncated form of the Mer receptor tyrosine kinase's extracellular domain, produced by alternative splicing, that inhibits Gas6 signaling. A selective UNC Mer small molecule inhibitor (UNC Mer TKI) inhibits signaling by inhibiting Mer tyrosine phosphorylation.

Objectives:
We hypothesized that inhibiting the Gas6/Mer pathway with UNC Mer TKI would decrease platelet activation responses and thrombus formation to a greater degree than iMer. So, we comparatively evaluated iMer's and UNC Mer TKI's inhibition of Gas6/Mer signaling in vitro and in vivo.

Methods:
We measured the effect on platelet inhibiton of iMer and UNC Mer TKI using lab assays of human platelet function and murine thrombosis models. In vitro studies included standard aggregometry and aggregate formation on collagen surfaces in a microfluidic flow chamber. In vivo studies included a FeCl3-induced model of carotid artery injury and a collagen/epinephrine-induced pulmonary embolism (PE) model to compare thrombosis protection between littermate C57BL/6 mice treated with inhibitors or vehicle control. A paired t-test was used to compare samples in aggregation, microfluidic flow surface area coverage, as well as elapsed time to initial and stable occlusions in the FeCl3model and survival time in the PE model.

Results/Discussion:
Inhibitor-treated platelets exhibited significantly decreased aggregation. Collagen-stimulated samples treated with 1.2 μM iMer had mean aggregation of 45 +/− 13% (Stand Dev), compared to 72 +/− 8% in controls (n=6, p=0.02). 1.2 μM UNC Mer TKI-treated samples had mean aggregation of 56 +/−15%, compared to 74 +/−10% in controls (n=5, p<0.01).
Adhesion of platelets to collagen under physiologic flow conditions resulted in 1.9% (+/− 1%) coverage in 1.2 μM iMer-treated samples compared to 8.7 +/− 2% mean surface area coverage in controls (n=7, p<0.001). 1.2 μM UNC Mer TKI-treated samples exhibited 8.5 +/−8.8% surface area coverage, compared to 19.2 +/− 15.7% for control-treated samples (n=5 in quadruplicate, p<0.01), and 2.4 +/−1.7% for abciximab-treated positive controls. Both iMer- and UNC Mer TKI-treated samples have a higher average percentage of small platelet aggregates (1–10 platelets/aggregate) compared to controls, which had a higher average percentage of large aggregates (>10 platelets/aggregate).
Following 6% FeCl3-induced carotid artery injury for analysis of iMer's thrombosis protection, vehicle-treated control mice (n=6) had a hazard ratio for stable artery occlusion of 7.9 (95%CI 1.7–36.3) compared to 30mg/kg iMer-treated mice (n=8, p<0.01). The 1.2 μM iMer treated mice also demonstrated significantly prolonged time to both initial (p=0.03) and stable occlusion (p=0.02), suggesting decreased thrombus stability. In analysis of UNC Mer TKI's thrombosis inhibition, vehicle-treated control mice (n=10) had a hazard ratio for stable artery occlusion of 4.8 (95%CI 1.4–15.8) compared 3 mg/kg UNC Mer TKI-treated mice (n=9, p=0.02). Times to initial (p=0.06) and stable (p<0.01) occlusions were also prolonged in the UNC Mer TKI-treated mice compared to controls.
In the PE model, 1.2 μM iMer-treated mice (n=5) had longer mean survival (14.1 +/− 14.5 min) following collagen/epinephrine injection than controls (n=5, 1.78 +/12.4 min, p=NS), with 2 iMer–treated mice and no controls surviving for 30 min. A much greater effect was seen with the 3 mg/kg UNC Mer TKI-treated mice (n=5) who had a mean survival time of 37.24 +/−31.2 min, with 3 surviving for a full 60 min, compared to 1.63 +/− 0.6 min (p=0.03) for controls (n=5), of which only 1 survived for 60 min.

Conclusions:
iMer and UNC Mer TKI are Gas6/Mer inhibitors that decrease platelet activation and protect mice from thrombosis, and may have translational applications as novel anti-platelet agents. UNC Mer TKI has similar efficacy, and higher potency, especially in the animal models where similar (FeCl3 model) or superior (PE model) results were seen with a 10-fold lower concentration. UNC Mer TKI also has the advantage of possible oral preparation, which may increase its potential therapeutic appeal.

Disclosures:
Branchford: University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties. Sather:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties. Brodsky:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties. Graham:University of Colorado: This author has provisional patent considerations for iMer, This author has provisional patent considerations for iMer Patents & Royalties.