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Key Points

• The present study shows, for the first time, that Gas6 produced by endothelial cells contributes to venous thrombus formation.
• Gas6 is required for the expression of tissue factor in endothelial cells.

Abstract

Gas6 (growth-arrest specific gene 6) plays a role in thrombus stabilization. Gas6 null (−/−) mice are protected from lethal venous and arterial thromboembolism through platelet signaling defects induced only by 5μM ADP and 10μM of the thromboxane analog, U46619. This subtle platelet defect, despite a dramatic clinical phenotype, raises the possibility that Gas6 from a source other than platelets contributes to thrombus formation. Thus, we hypothesize that Gas6 derived from the vascular wall plays a role in venous thrombus formation. Bone marrow transplantation and platelet depletion/reconstitution experiments generating mice with selective ablations of Gas6 from either the hematopoietic or nonhematopoietic compartments demonstrate an approximately equal contribution by Gas6 from both compartments to thrombus formation. Tissue factor expression was significantly reduced in the vascular wall of Gas6−/− mice compared with WT. In vitro, thrombin-induced tissue factor expression was reduced in Gas6−/− endothelial cells compared with wild-type endothelium. Taken together, these results demonstrate that vascular Gas6 contributes to thrombus formation in vivo and can be explained by the ability of Gas6 to promote tissue factor expression and activity. These findings support the notion that vascular wall-derived Gas6 may play a pathophysiologic role in venous thromboembolism.

Introduction

Venous thromboembolism (VTE) is a common cause of morbidity and mortality in clinical medicine. The pathophysiology of VTE was first described by Virchow in 1853 and describes a triad of entities accounting for VTE. VTE could be triggered by alterations in the blood composition (thrombophilia), changes in blood flow (eg, stasis), and/or activation of the endothelium.1 Under normal conditions, the endothelial surface inhibits coagulation because of the presence of various proteins, such as tissue factor (TF) pathway inhibitor, thrombomodulin, and the endothelial cell protein C receptor.2 However, physical (eg, vascular damage) or functional (eg, hypoxia) perturbation of the endothelium promotes thrombosis because of reduced expression of anticoagulants and the induction of the expression of the transmembrane procoagulant protein TF.3

Gas6, the product of growth arrest specific gene 6 (Gas6), is a member of the vitamin K-dependent family of proteins, which includes the procoagulant factors II, VII, IX, and X and the anticoagulant factors, protein C and S, as well as protein Z.4Even though Gas6 was discovered as a homolog of protein S more than a decade ago, it plays no role in the generation of fibrin and its role in vivo remains incompletely characterized.5,6

Originally identified in fibroblasts, Gas6 is expressed in various cell types, including endothelial cells,7 smooth muscle,8 and bone marrow cells.9 Gas6 is a ligand for the TAM family of receptor tyrosine kinases, which include Axl, Tyro3 (Sky), and Mer.10Gas6 has been shown to have a wide range of biologic functions that is reflected by a broad tissue expression profile. For example, Gas6 is found in plasma11 as well as in hepatic,12 renal,13 and neural tissues.14,15 Gas6 and its receptors modify platelet activation and aggregation,16,17 but the role of Gas6 in the interplay between platelets and other cell types, such as endothelial cells, remains unclear. In vivo, Gas6-deficient mice are protected from lethal thromboembolism, suggesting a prothrombotic role for Gas6.16 This antithrombotic phenotype results from a loss of platelet signaling with consequent clot instability. Interestingly, these mice did not have a bleeding diathesis. Platelets from Gas6−/− mice showed defective platelet signaling when challenged with 5μM ADP or with 10μM of the thromboxane analog U46619 but not to other platelet agonists, such as thrombin or collagen, or even to higher concentrations of ADP.16 This subtle platelet defect suggests the existence of a discrepancy that could be explained by other mechanisms, such as the contribution of Gas6 from the vasculature to thrombus formation. Thus, we hypothesize that Gas6 from the vascular wall has a role in the pathophysiology of venous thrombosis.

Methods

Animals

All experiments performed on mice were approved by the Animal Care Committee of McGill University. The Gas6−/− mice (on a C57BL/6 background) were a generous gift from Dr Peter Carmeliet (Laboratory of Angiogenesis and Neurovascular Link, Vesalius Research Center, Leuven, Belgium). The colony was maintained by the continuous crossing of Gas6+/− mice. The mice used in all experiments were male and 8-12 weeks of age. Genotyping was performed by PCR amplification of the Gas6gene from total genomic DNA prepared by phenol-chloroform extraction of ear punch samples. Custom primers for Gas6 were from Invitrogen, and the nucleotide sequences are as follows: Gas6 sense, 5′-GAGTGCCGTGATTCTGGTC-3′; Gas6antisense, 5′-CCACTAAGGAAACAATAACTG-3′; and Gas6 “new,” 5′-ATCTCTCGTGGGATCATT-3′.

Thermal cycling was performed on a PerkinElmer GeneAmp PCR System (2400; PerkinElmer Life and Analytical Sciences). The thermal profile was as follows: 2 minutes at 94°C, 35 cycles of: 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds followed by a final incubation at 72°C for 10 minutes. PCR-amplified DNA was resolved on ethidium bromide-stained 1% agarose gels (Invitrogen). A single band that migrates at 500 bp corresponds to a wild-type (WT) mouse, whereas a single band of 350 bp corresponded to a Gas6−/− mouse. In heterozygous mice, both bands are present.