Novel Small Molecule Inhibitors Of The Gas6/TAM Signaling Pathway Inhibit Platelet Aggregation In Vitro and Protect Mice From Arterial and Venous Thrombosis In Vivo
Authors: Acevedo Gilbert , Branchford Brian R. , Brzezinski Christine , Sather Susan , Brodsky Gary , DeRyckere Deborah , Zhang Weihe , Liu Jing , Earp H. Shelton , Wang Xiaodong , Frye Stephen V , Graham Douglas K. , Di Paola Jorge 
Published: October 2019

Abstract

Background
Growth Arrest Specific gene 6 (Gas6) is a ligand for the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases found on the surface of platelets. Previous studies have shown that stimulation of these receptors results in amplification of platelet activation and thrombus stabilization via activation of phosphatidylinositol-3-kinase (PI3K) and Akt, leading to phosphorylation of the β3 integrin. Previous work (from our lab and others) demonstrated that inhibition of the Gas6/TAM pathway results in impaired platelet aggregation, reduced aggregate stability, and decreased platelet spreading. Additionally, knockout mice deficient in the receptor or ligand are protected from venous and arterial thrombosis, but retain normal tail bleeding times. Here, we describe development and characterization of novel Mer-selective small molecule inhibitors (SMIs) for thrombosis applications.

Objectives
To determine if Mer-selective SMIs can inhibit platelet aggregation and protect mice from thrombosis using in vitro and in vivo models

Methods
We used aggregometry and in vivo murine models of arterial and venous thrombosis to compare two Mer-selective SMIs (UNC Mer TKI1 and UNC Mer TKI2) and determine the most effective inhibitor of platelet aggregation and thrombus formation. The inhibitory effect of two doses (1µM and 5 µM) of the compounds were determined using standard light-transmission aggregometry after a 30 minute incubation with washed human platelets at 37 ¢ªC and compared to platelets treated with vehicle control or with a TKI control (UNC TKI Null), a SMI with similar structure but minimal anti-TAM activity. Both collagen/epinephrine-induced systemic venous thrombosis and FeCl3-induced carotid artery injury models were used to determine effects on thrombosis mediated by UNC TKIs. Wild type C57Bl/6 mice were treated with one of the two inhibitors and compared to mice treated with vehicle control. Mean values +/- SEM are shown and statistical significance (p<0.05) was determined using the student’s paired t-test.

Results
UNC Mer TKI1 exhibited more potent inhibition of platelet aggregation in vitro relative to UNC Mer TKI2, although both compounds mediated dose-dependent effects. At a concentration of 1uM, the maximum percent aggregation in UNC Mer TKI1-treated samples (n=7) was significantly greater than samples treated with UNC TKI Null (n=7), 20% DMSO vehicle (n=7), or UNC TKI2 (n=7), with mean values of 69 +/- 2.2%, 76.7 +/-1.8% (p<0.01), 76.9 +/- 2.1% (p=0.001), and 77 +/- 1.8% (p<0.001), respectively. At a concentration of 5 µM, UNC Mer TKI1-treated samples (n=7) exhibited a mean maximum percent aggregation of 23.7 +/- 2.4% compared to 50.4 +/- 4.8% for samples treated with UNC Mer TKI2 (n=7, p<0.001).
UNC Mer TKIs also mediated protection from thrombus formation in mice. Following FeCl3 injury to the carotid artery, vehicle-treated mice (n=11) developed stable vessel occlusions with a mean time of 6.77 +/- 0.25 min. In contrast, stable occlusion occurred at a mean time of 46.6 +/- 7.72 min (n=9, p=0.001) for UNC Mer TKI1-treated mice. Survival times following venous injection of collagen and epinephrine were also significantly increased in mice treated with either UNC Mer TKI relative to the UNC TKI Null or vehicle controls. Mice pre-treated with UNC Mer TKI1 (n=9, p=0.04 compared to vehicle alone) or UNC Mer TKI2 (n=9, p=0.03 compared to vehicle alone) survived for 19.84 +/- 4.4 and 21.25 +/- 4.65 minutes, respectively. In contrast, mice given UNC TKI Null (n=3) or vehicle (n=21), only survived for 3.21 +/- 2.4 min and 3.09 +/- 0.22 minutes, respectively.

Conclusion
UNC Mer TKIs mediate dose-dependent inhibition of platelet aggregation and protect mice from arterial and venous thrombosis. Their pronounced activity compared to an inactive scaffold protein with minimal anti-TAM activity suggest that Gas6/TAM pathway inhibition is the mechanism of action for these novel compounds. UNC Mer TKI1 has more potent anti-thrombotic properties than UNC Mer TKI2.

Disclosures:
Branchford: University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Sather:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. DeRyckere:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Zhang:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Liu:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Earp:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Wang:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Frye:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Graham:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties. Di Paola:University of Colorado: inventor on a patent application relevant to this work , inventor on a patent application relevant to this work Patents & Royalties.