Is dual targeting of FXII and Gas6 a good strategy to prevent thrombosis with no risks of bleeding?

Abstract Introduction: Preventing thrombosis without increasing bleeding remains a key clinical challenge. Safer anticoagulants are being explored by targeting early coagulation factors like FXII. Gas6, a TAM (Tyro3, Axl, Mertk) receptor ligand, supports hemostasis, and its inhibition prevents thrombosis in mice without bleeding. Since FXII and Gas6 act via distinct pathways, their combined targeting may enhance thrombosis prevention without raising bleeding risk.Aim: To evaluate whether dual targeting of FXII and Gas6 provides a safer, bleeding-free approach to thrombosis prevention. Additionally, it seeks to elucidate the mechanistic interplay between FXII and Gas6 pathways in hemostasis.Methods: A double knockout mouse lacking both FXII and Gas6 (F12-/-Gas6-/-) was generated, with F12-/-, Gas6-/- and wild-type (WT) as controls. Bleeding was assessed by tail bleeding assay. Thrombosis was evaluated using a platelet-dependent venous thromboembolism (VTE) model (collagen/epinephrine), ultrahigh-frequency ultrasound (UHFUS) monitoring of ferric chloride-induced vena cava thrombosis, and intravital confocal microscopy (IVM) of thrombus formation in mesenteric arterioles. Ex vivo assays included platelet aggregation (ADP or collagen) and thrombin-induced clot retraction. Clot ultrastructure was examined by scanning electron microscopy (SEM). Neutrophil extracellular trap (NET) formation was quantified by whole blood flow cytometry (WBFC) before and after stimulation with phorbol 12-myristate 13-acetate (PMA). Fibrinolysis was assessed using the ClotPro® TPA-test system. Plasmin generation (PG) was measured in plasma by a calibrated automated method.Results:F12-/-Gas6-/- mice were viable and phenotypically similar to WT mice. In a tail bleeding assay, they exhibited shorter bleeding times than WT mice (3 ± 2 min vs. 6 ± 3 min, n=8), indicating no bleeding tendency. After VTE induction, F12-/-(53%), Gas6-/- (73%, p<.05), and F12-/-Gas6-/-(60%, p<.05) mice had a better survival than in WT mice (20%), n=15. Lung histology of survivors showed fewer thrombi in WT mice (3 ± 2) than in F12-/- (14 ± 9), Gas6-/- (14 ± 9), and F12-/-Gas6-/- mice (6 ± 4), n=15, suggesting reduced thrombus stability and possible embolization in knockout models. UHFUS 30 min post-injury revealed less vena cava occlusion in F12-/- (4 ± 3%), Gas6-/- (30 ± 10%), and F12-/-Gas6-/- mice (24 ± 10%) compared to WT mice (37 ± 8%), n=2-5. IVM of mesenteric vessels showed prolonged vessel occlusion times in all knockout groups (>25 min) vs. WT mice (17 ± 2 min), n=2-4. Volume reconstruction of thrombi at 17 min (via Imaris 10.0) demonstrated higher volumes of fibrin, resting platelets, and activated platelets in WT mice compared to knockout mice. SEM of platelet-rich plasma (PRP) clots showed no differences among groups in fibrin coverage (F12-/-: 88 ± 6%; Gas6-/-: 71 ± 11%; F12-/-Gas6-/-: 86 ± 5%; WT: 85 ± 8%, n=3) or fibrin fiber diameter (F12-/-: 157 ± 23 nm; Gas6-/-: 155 ± 33 nm; F12-/-Gas6-/-: 161 ± 46 nm; WT: 149 ± 41 nm, n=3). However, clots from Gas6-/- and F12-/-Gas6-/- mice showed qualitative abnormalities in fibrin architecture, including disorganized fibers networks and absence of string-like structures in individual fibers. Platelet aggregation in response to ADP and collagen was similar across groups, indicating aggregation defects are unlikely to explain clot instability, n=4. WBFC revealed reduced NET formation in F12-/-Gas6-/- mice compared to WT (6.3 ± 2.8% vs. 11.8 ± 1.6%, n=4), indicating that decreased NETs may contribute to the observed thrombus instability in the VTE model. Fibrinolysis was faster in knockout mice, with shorter lysis time in F12-/- (24 ± 3 min), Gas6-/- (18 ± 1 min), and F12-/-Gas6-/- mice (21 ± 6 min) vs. WT mice (39 ± 8 min), n=2-3. In PG, F12-/-Gas6-/- showed a higher plasmin peak (72 ± 5 nM, p<.05) vs. WT (57 ± 11 nM) and a trend toward increased endogenous plasmin potential (295 ± 10 vs. 250 ± 34 nM·min), n=4, indicating elevated fibrinolytic activity and potential clot instability.Conclusions: Dual targeting of FXII and Gas6 effectively reduced bleeding but unexpectedly increased thrombus instability and embolism risk, likely due to reduced NET formation and enhanced fibrinolysis. These findings highlight challenges of combined inhibition and the need to clarify underlying mechanisms. Gas6 inhibition alone appears safer for thrombosis prevention and warrants further studies as a bleeding-free antithrombotic strategy.