Novel Small Molecule Inhibitors Of The Gas6/TAM Signaling Pathway Mediate Synergistic Inhibition Of Platelet Aggregation In Combination With ADP/P2Y Antagonists
Authors: Acevedo Gilbert , Branchford Brian R. , Law Luke , Brzezinski Christine , Sather Susan , Brodsky Gary , DeRyckere Deborah , Zhang Weihe , Earp H. Shelton , Frye Stephen V , Graham Douglas K. , Di Paola Jorge 
Published: October 2019

Abstract

Background
ADP activates platelets through P2Y1 and P2Y12 receptors. Various ADP/P2Y inhibitors are used clinically for arterial thrombosis prophylaxis. However, these agents exhibit inter-individual response variability and bleeding complications. A more recently described platelet activation signaling pathway is activated by Growth Arrest Specific gene 6 (Gas6), a ligand for the Tyro3/Axl/Mer (TAM) family of platelet surface receptor tyrosine kinases. Previous have shown that inhibiting this pathway decreases platelet activation responses and β3 integrin-mediated thrombus stabilization, and protects mice from arterial and venous thrombosis, without evidence of significant bleeding side effects. Here, we investigate the effects of a novel Mer-selective small molecule inhibitor (SMI) on platelet aggregation and murine models of induced thrombosis relative to ADP/P2Y pathway antagonists.

Objectives
We hypothesized that inhibition of the Gas6/TAM pathway with a novel SMI would decrease platelet aggregation and thrombosis, comparable to that seen with known ADP inhibitors. Additionally, we examined the effect of the combination of these 2 inhibitor types on platelet aggregation.

Methods
We compared the inhibitory effect of 2 known ADP inhibitors (1uM MRS2179 administered concurrently with 1uM 2-MeSAMP) to 1uM of a novel Mer-selective SMI (UNC Mer TKI), using standard light-transmission aggregometry with washed human platelets (30-minute incubation at 37 ¢ªC) and two murine thrombosis models (collagen/epinephrine-induced systemic venous thrombosis and FeCl3-induced carotid artery injury). Thrombosis studies were performed using WT C57BL/6 mice treated with either UNC Mer TKI or ADP/P2Y inhibitors compared to mice treated with vehicle only. Mean values +/- SEM are shown and statistical significance (p<0.05) was determined using the student’s paired t-test.

Results
ADP/P2Y antagonists and Gas6/TAM inhibitor both mediate protection from thrombosis in mice relative to vehicle-treated controls. Following FeCl3 –induced carotid artery injury, control mice (n=11) experienced stable vessel occlusion at a mean time of 6.77 +/- 0.25 minutes. In contrast, stable occlusion occurred at 46.6 +/- 7.72 minutes (n=9, p=0.001) in mice treated with UNC Mer TKI, and 18.74 +/- 4.35 minutes (n=3, p<0.001) for mice treated with the two ADP/P2Y inhibitors.
Survival times following venous injection of collagen and epinephrine significantly differed between mice treated with ADP/P2Y antagonists or UNC Mer TKI, compared to vehicle control. Mice pre-treated with UNC Mer TKI (n=9, p=0.04) or ADP/P2Y inhibitors (n=5, p<0.001) survived for 19.84 +/- 4.4 and 19.9 +/- 4.92 minutes, respectively. In contrast, mice treated with vehicle control (n=21), only survived for 3.21 +/- 2.4 minutes.
Both the ADP/P2Y antagonists and UNC Mer TKI also inhibit platelet aggregation. At 1uM doses, the maximum percent aggregation in UNC Mer TKI-treated samples (n=7) differed significantly from samples treated with vehicle alone, with mean values of 69 +/- 2.2% (p=0.04), 77 +/- 1.8% (n=7), and 76.9 +/- 2.1% (n=7), respectively. 1uM ADP/P2Y inhibitor-treated samples (n=7) exhibited a mean maximum aggregation of 62 +/- 5.2% (p=0.008), and the combination of ADP/P2Y inhibitors and UNC TAM TKI had a maximum percent aggregation of 31.3 +/- 7.7% (n=7, p=0.001). The Chou-Talalay Combination Index (a quantitative estimation of the effect of combined inhibitors) was 0.78, indicating a synergistic, rather than additive, effect. Similarly, using the Bliss additivity equation, 38.1 +/- 7.9% inhibition of aggregation was predicted for an additive interaction, but the actual % observed in samples treated with UNC Mer TKI combined with ADP/P2Y inhibitors was 64.3 +/- 8.9%, a statistically significant difference (p = 0.02), suggesting a synergistic effect of the combination therapy.

Conclusion
A novel Mer-selective SMI mediated inhibition of platelet aggregation and protection from arterial and venous thrombosis in a manner comparable to that seen with known ADP/P2Y inhibitors. Additionally, the two drugs mediate synergistic inhibition of platelet aggregation when used in combination. This observation suggests that combination therapies consisting of a Mer-inhibitor and an ADP/P2Y inhibitor could allow for dose reduction of one or both agents, thereby decreasing off-target effects and/or bleeding complications.

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. Earp: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.