Imer Blocks Phosphorylation of the β3 Integrin, Decreasing Platelet Activation Responses and Protecting Mice From Arterial Thrombosis
Authors: Branchford Brian R. , Sather Susan , Brodsky Gary , White-Adams Tara C , DeRyckere Deborah , Neeves Keith B , Wilson Katina M , Lentz Steven R. , Graham Douglas K. , Di Paola Jorge A 
Published: October 2019

Abstract
Abstract 189

Background:
Growth Arrest Specific gene 6 (Gas6) signaling through platelet-surface Tyro3/Axl/Mer (TAM) receptors leads to platelet activation and thrombus stabilization. This occurs via activation of phosphatidylinositol-3-kinase (PI3K) and Akt, stimulating tyrosine phosphorylation of the β3 integrin. This process amplifies outside-in signaling via αIIbβ3, which is necessary for stable aggregate formation. iMer is a truncated form of the extracellular domain of the Mer receptor tyrosine kinase, produced by alternative splicing, that inhibits Gas6/TAM signaling, likely by acting as a decoy receptor for Gas6.

Objectives:
We hypothesized that inhibiting the Gas6/TAM pathway with a novel Gas6-sequestering protein would decrease platelet activation responses. We therefore evaluated iMer's inhibition of Gas6 signaling in human and murine platelets in vitro and in vivo.

Methods:
We measured the inhibitory effect of iMer on platelet activation using laboratory evaluations of platelet function and a murine carotid artery thrombosis model. In vitro studies included aggregometry, adhesion to collagen in a flow chamber, and platelet spreading. These platelet activation responses were tested in human platelets in the presence or absence of the inhibitor and also in wild type (WT) and Gas6/TAM −/− murine platelets. A ferric-chloride model of carotid artery injury was used to compare susceptibility to thrombosis between littermate C57BL/6 mice treated with either iMer or vehicle. Platelet aggregation data was evaluated by the Wilcoxon Signed Rank Test, and times to occlusion following carotid artery injury were compared using the Mann-Whitney Rank Sum Test.

Results/Discussion:
Western blot analysis demonstrated decreased β3 integrin phosphorylation in iMer-treated human platelets after addition of human Gas6 when compared to controls, consistent with decreased Mer signaling in the presence of iMer.
iMer-treated human platelets exhibited significant decreases in ADP- and collagen-induced platelet aggregation. ADP-stimulated samples treated ex vivo with iMer showed an aggregation mean of 74% (SD= +/− 3%), compared to 86% aggregation (+/− 3%) in controls (p=0.016). Collagen-stimulated samples treated ex vivo with iMer exhibited a mean of 70% aggregation (+/− 8%), compared to 88% aggregation (+/−2%) in controls (p=0.004).
Electron micrographs of adhered human platelets revealed that iMer delayed, but did not permanently abrogate, platelet spreading on fibrillar collagen (100 μg/mL).
Flow cytometric analysis of human platelets showed reduced expression of platelet-surface activation markers (P-selectin and PAC-1) despite stimulation with fibrillar collagen (1 μg/mL).
Microfluidic flow assay demonstrated that adhesion of untreated human platelets to collagen at a wall shear rate of 100s−1 resulted in 21.3% (SD=+/− 8%) mean surface area coverage, while ex vivo iMer-treated samples showed only 1.1% (+/− 0.9%) coverage. These results are consistent with those of WT mice compared to that of Gas6/TAM −/−mice in preliminary studies using the same system.
Following ferric chloride injury to the carotid artery, 71% of vehicle-treated control mice (n=7) had initial occlusions that remained stable, and only 14% remained patent. In contrast, only 25% of the iMer-treated mice (n=8) formed initial occlusions that remained stable, while 50% remained patent. The iMer treated mice also had a significant decrease (p=0.02) in the duration of first occlusion time (i.e. length of time the initial occlusion lasted), suggesting decreased thrombus stability.

Conclusions:
iMer is a novel inhibitor of the Gas6/TAM pathway that decreases platelet activation responses and protects mice from arterial thrombosis by decreasing phosphorylation of β3 integrin, which has been shown to be necessary for thrombus stabilization. This compound may, therefore, have translational applications as a novel anti-platelet agent.

Disclosures:
No relevant conflicts of interest to declare.