ABSTRACT
Essentials Signaling by Gas6 through Tyro3/Axl/Mer receptors is essential for stable platelet aggregation. UNC2025 is a small molecule inhibitor of the Mer tyrosine kinase. UNC2025 decreases platelet activation in vitro and thrombus formation in vivo. UNC2025's anti-platelet effect is synergistic with inhibition of the ADP receptor, P2Y12 . SUMMARY: Background Growth arrest-specific protein 6 signals through the TAM (TYRO-3-AXL-MERTK) receptor family, mediating platelet activation and thrombus formation via activation of the aggregate-stabilizing αIIb ß3 integrin. Objective To describe the antithrombotic effects mediated by UNC2025, a small-molecule MERTK tyrosine kinase inhibitor. Methods MERTK phosphorylation and downstream signaling were assessed by immunoblotting. Light transmission aggregometry, flow cytometry and microfluidic analysis were used to evaluate the impact of MERTK inhibition on platelet activation and stability of aggregates in vitro. The effects of MERTK inhibition on arterial and venous thrombosis, platelet accumulation at microvascular injury sites and tail bleeding times were determined with murine models. The effects of combined treatment with ADP-P2Y1&12 pathway antagonists and UNC2025 were also evaluated. Results and Conclusions Treatment with UNC2025 inhibited MERTK phosphorylation and downstream activation of AKT and SRC, decreased platelet activation, and protected animals from pulmonary embolism and arterial thrombosis without increasing bleeding times. The antiplatelet effect of UNC2025 was enhanced in combination with ADP-P2Y1&12 pathway antagonists, and a greater than additive effect was observed when these two agents with different mechanisms of inhibition were coadministered. TAM kinase signaling represents a potential therapeutic target, as inhibition of this axis, especially in combination with ADP-P2Y pathway antagonism, mediates decreased platelet activation, aggregate stability, and thrombus formation, with less hemorrhagic potential than current treatment strategies. The data presented here also demonstrate antithrombotic activity mediated by UNC2025, a novel translational agent, and support the development of TAM kinase inhibitors for clinical applications.
Subject(s)
Adenine/analogs & derivatives , Blood Platelets/drug effects , Piperazines/pharmacology , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Protein Kinase Inhibitors/pharmacology , Pulmonary Embolism/prevention & control , Thrombosis/prevention & control , c-Mer Tyrosine Kinase/antagonists & inhibitors , Adenine/pharmacokinetics , Adenine/pharmacology , Animals , Blood Platelets/enzymology , Disease Models, Animal , Drug Synergism , Drug Therapy, Combination , Female , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Male , Mice, Inbred C57BL , Phosphorylation , Piperazines/pharmacokinetics , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/pharmacokinetics , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Proto-Oncogene Proteins/metabolism , Pulmonary Embolism/blood , Pulmonary Embolism/enzymology , Purinergic P2Y Receptor Antagonists/pharmacology , Receptor Protein-Tyrosine Kinases/metabolism , Signal Transduction/drug effects , Thrombosis/blood , Thrombosis/enzymology , c-Mer Tyrosine Kinase/metabolism , Axl Receptor Tyrosine KinaseABSTRACT
Hemostasis is the process of sealing a vascular injury with a thrombus to arrest bleeding. The type of thrombus that forms depends on the nature of the injury and hemodynamics. There are many models of intravascular thrombus formation whereby blood is exposed to prothrombotic molecules on a solid substrate. However, there are few models of extravascular thrombus formation whereby blood escapes into the extravascular space through a hole in the vessel wall. Here, we describe a microfluidic model of hemostasis that includes vascular, vessel wall, and extravascular compartments. Type I collagen and tissue factor, which support platelet adhesion and initiate coagulation, respectively, were adsorbed to the wall of the injury channel and act synergistically to yield a stable thrombus that stops blood loss into the extravascular compartment in ~7.5 min. Inhibiting factor VIII to mimic hemophilia A results in an unstable thrombus that was unable to close the injury. Treatment with a P2Y12 antagonist to reduce platelet activation prolonged the closure time two-fold compared to controls. Taken together, these data demonstrate a hemostatic model that is sensitive to both coagulation and platelet function and can be used to study coagulopathies and platelet dysfunction that result in excessive blood loss.
ABSTRACT
Wistar rats immunized with an homologous testicular homogenate (TH) and complete Freund's adjuvant, followed by i.v. injection of Bordetella pertussis, developed an autoimmune orchitis (EAO). Animals were studied at 7, 16, 30, 50, and 80 days (d) after the first immunization. An important lesion of the testis only appeared at 50 d, increasing in severity and incidence (77%) at 80 d. Lesions were characterized by a prevalent aspermatogenesis with tubular atrophy and mild interstitial mononuclear infiltrates. Delayed-type hypersensitivity response (DTH) against TH was detected early at 7 d and, except for 16 d, it increased with time, reaching a maximum at 80 d. A good temporal relationship between DTH and histopathology was found. Circulating antibodies to TH, detected by ELISA, were only present in 64% of the animals with testis lesion, while no deposits of IgG or C3 in the seminiferous tubules were seen. We describe a sequence of immunological events, concomitant with pathological changes of the testis, during the development of a severe EAO in Wistar rats.
