Reelin modulates PLCγ2 phosphorylation upon platelet activation and integrin outside-in signaling Irena Krüger1, Nina Sarah Gowert1, Meike Klier1, Hans H. Bock2, Margitta Elvers1 1Clinic for Vascular and Endovascular Surgery, Heinrich Heine University Medical Center, Düsseldorf, Germany 2Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich Heine University Medical Center, Düsseldorf, Germany Aim of the Project Methods Reelin is known as an extracellular matrix protein which is produced In vitro and in vivo analysis of Reeler mice. by Cajal-Retzius neurons mediating cell migration in brain development. Recent studies provide evidence that Reelin is expressed in platelets, where it co-localizes to F-Aktin. Moreover, Reelin influences static adhesion on CRP and binding to GPVI Reelin binds to amyloid precursor protein (APP) and apolipoprotein E A B ´ Figure 5: Reelin is receptor 2 (ApoER2) and is important for thrombus formation under wt -JAQ1 high shear suggesting that Reelin is modulating glycoprotein (GP)Ib influencing adhesion on *** CRP and binding onto 350 *** 200 GPVI. (A) Wildtype and signaling in platelets. Consequently, defective thrombus formation 180 300 Reeler platelets were allowed protects Reelin-deficient mice (Reeler) against arterial thrombosis. 160 250 140 to adhere for 20 min onto a 120 This prompted to analyze the role and signaling mechanisms of 200 CRP matrix (1µg/ml), n=5. 100 reeler +JAQ1 (B) Resting wildtype platelets Reelin in platelet cytoskeletal reorganization, activation and 150 80 rest +Jaq1 -JAQ1 60 and with GPVI antibody 100 aggregation. 40 (JAQ1) treated wildtype cells cells per visual field cells cells per visual field 50 20 platelets were allowed to 0 0 adhere onto a Reelin matrix. wt reeler -JAQ1 +JAQ1 Reelin is present in platelets and released upon activation Bar graphs depict mean ± sem, *** P< 0.001. A B C Reelin 250 lysat rest supernatant 200 Reelin is influencing Integrin αIIbβIIIoutside-in signaling 150 F-Actin A 90 ** 80 100 ´ [pg/ml] +CRP 70 wt 6 min reeler wt 120 min reeler Volume 60 50 50 Mouse Reelin concentrationReelin Mouse merge 40 % % starting 30 0 20 wt 300 min reeler Thrombin Thrombin CRP CRP rest 10 10 min 60 min 10 min 60 min 0 wt 60 min reeler wt 300 min reeler Figure 1: Reelin is a platelet protein. (A) Reelin concentrations in the supernatant and lysates of platelets stimulated with thrombin (0.2 U/ml) and CRP (10 µg/ml) , respectively for 10 and 60 minutes were measured at indicated time points by sandwich ELISA assay. Rest = Figure 6: Reeler PRP shows impaired clot retraction thus Reelin enhances Integrin αIIbβIIIoutside-in signaling. (A) PRP from wt (black) resting platelets without stimulation, bar graphs depict mean values ± sem, n=3. (B) Platelets were isolated and stimulated with CRP (10 and reeler mice (grey) was stimulated with thrombin (5 U/ml) in the presence of CaCl2 (20mM) and photographed after several time points (0, µg/ml). Antibody: Anti-Reelin G10. (C) Reelin is present in platelets and co-localizes to F-actin. Platelets were washed and allowed to 60, 120 and 300 minutes). After 300 minutes the supernatant fluid was removed and its volume was measured and compared against the spread on immobilized human fibrinogen (100 µg/ml) for 60 min, fixed and stained for Reelin (clone G10, red) and for F-actin (Rhodamin- starting volume. Bar graphs depict mean values ± sem, n = 5-6, ** P ˂ 0.01. Phalloidin). Scale bar 10 µm. Reeler platelets show reduced activation upon GPVI stimulation Reeler mice are protected against arterial thrombosis * Figure 7: Reduced platelet A B Figure 2: Rl-/- are protected against * activation upon Reelin arterial thrombosis without affecting ** deficiency. (A) Blood was 1200 rl+/+ after 40 min bleeding time. (A) After injury of washed twice and incubated mesenteric arterioles with 20% FeCl , with 0.002, 0.02 and 0.1 U/ml 1000 3 platelet adhesion and thrombus thrombin; 1, 5 and 10 µg/ml 800 formation were monitored in vivo by CRP; 10 µM ADP; 10/3 µM fluorescence microscopy. Percent ´ ADP/U46 and 75, 100 µM 600 A rl-/- after 40 min distribution of irreversible occlusion PAR-4 as indicated for 15 ´ 400 (black), instable occlusion (dark grey) minutes in the presence of anti- Tail bleeding time (s) bleedingTail and no occlusion (light grey). Shear rate P-selectin and JON/A-PE 200 1.300 sec-1 (n=5). (B) Tail bleeding times antibodies. Platelets were +/+ -/- 0 for Rl and Rl mice. Each symbol gated by their forward and rl+/+ rl-/- represents one animal (n=15-17). (C) sideward characteristics. The *** Wildtype mice were injected with CR-50 ** mean fluorescence intensity *** C (20 µg/mice) and IgG (20 µg/mice) as (MFI) for each measurement is control. After injection of DCF the shown (n= 5). * P< 0,05, **P< mesenteric arterioles were injured with 0,01, ***P< 0,001. 20 % FeCl3 and thrombus formation was 0.002 0.02 0.1 1 5 10 10 10/3 75 100 monitored by fluorescence microscopy. Thrombin CRP ADP/U46 PAR-4 Representative pictures (left panel) and rest [U/ml] [µg/ml] ADP [µM] [µM] time to irreversible occlusion (right panel) was determined. Each symbol represents one individual mouse, n=3. Reelin modulates PLCγ2 phosphorylation A Reelin induces phosphorylation of the Rac1/Cdc42 effector PAK WT reeler A wt - p-PLCg2 CRP rest Reelin - PLCg2 1´ 2´ 1´ 5´ 15´ PAK- - p-Syk - Syk p-PAK- d ´ modified according to Bigalke et al., Current Drug Targets, 2011 - β-Tubulin b-tubulin- rest CRP rest CRP Figure 8: Intracellular Reelin is modulating PLCγ2 phosphorylation upon GPVI stimulation. (A) Wildtype platelets were stimulated Figure 3: Reelin is important for phosphorylation of Rho target Protein PAK1/2. (A) Wildtype platelets were stimulated with with CRP (5µg/ml) for 2 minutes, lysed and western blots were performed against phosphorylated PLCγ2 and PLCγ2 and CRP (10µg/ml) for 1 and 2 minutes and with recombinant Reelin (1:10) for 1, 5 and 15 minutes, lysed and western blots were Aslan et al., American Journal of Physiology, 2013 phosphorylated Syk and Syk. β-Tubulin was serving as loading control (n=4). performed against phosphorylated PAK and PAK. β-Tubulin was serving as loading control (n=3). Reelin supports lamellipodia formation on fibrinogen Conclusion A B 20 min wt reeler wt reeler Taken together these data provide first evidence for Reelin mediating signaling through GPVI and 100 90 modulating outside-in signaling suggesting that Reelin is a modulator for PLCγ2 phosphorylation in 80 lamellipodia 70 % platelets. 60 50 Our study reveals an important role for Reelin in hemostasis and arterial thrombosis, thus being a filopodia 40 Cell count in 30 promising therapeutic target for antithrombotic therapy. 20 adherent 10 cells 0 + thr + rec. Rln + thr/rec.Rln References 60 min 100 no treatment + thrombin 90 (1) Tseng WL, Huang CL, Chong KY, Liao CH, Stern A, Cheng JC, Tseng CP. Reelin is a platelet protein and functions as a positive 80 regulator of platelet spreading on fibrinogen. Cell Mol Life Sci. 2010 Feb;67(4):641-53. 70 in % 60 (2) Tseng WL, Chen TH, Huang CC, Huang YH, Yeh CF, Tsai HJ, Lee HY, Kao CY, Lin SW, Liao HR, Cheng JC, Tseng CP. Impaired 50 thrombin generation in Reelin-deficient mice: a potential role of plasma Reelin in hemostasis. Thromb Haemost. 2014 40 Dec;12(12):2054-64. Cell count 30 20 (3) Leemhuis J, Bouché, E, Frotscher, M, Henle, F, Hein, L, Herz, J, Meyer, DK, Pichler, M, Roth, G, Schwan, C, Bock, HH. Reelin 10 0 signals through Apoer2 and Cdc42 to increase growth cone motility and filopodia formation. J Neurosci. 2010 Nov; 30 (44):14759- + thr +rec. Rln + thr/rec.Rln 14772. (4) D'Arcangelo G, Nakajima K, Miyata T, Ogawa M, Mikoshiba K, Curran T. Reelin is a secreted glycoprotein recognized by the CR- Figure 4: Reelin is supporting lamellipodia formation. (A) Wildtype platelets were allowed to adhere on immobilized human fibrinogen 50 monoclonal antibody. The Journal of neuroscience : the official journal of the Society for Neuroscience. 1997;17(1):23-31. (1 mg/ml) without stimulation and stimulated with thrombin and recombinant Reelin. Graph bars depictmodified meanaccording to Goggsvalues, Williams etafter al., Biochemical20 Journal,and 201560 (5) 1. Archangelo GD, Miao GG, Chen S-C, Soares HD, Morgan JI, Curran T. A Protein Related to Extracellular-Matrix Proteins minutes, n=3-5. (B) Wildtype and Reeler platelets were allowed to spread on fibrinogen (1 mg/ml) and compared to spreaded wildtype and Deleted in the Mouse Mutant Reeler. Nature. 1995;374:719-23. Reeler platelets with thrombin stimulation (0.008U/ml). Bar graphs depict mean values ± s.e.m. after 20 and 60 min, n = 3-5. * P< 0.05. Disclosure Statement of Financial Interest Acknowledgments I, Irena Krüger, DO NOT have a financial interest/arrangement or affiliation with one or more organizations that could be We thank Martina Spelleken for providing outstanding technical assistance. perceived as a real or apparent conflict of interest in the context of the subject of this presentation. 1334--PB Platelet Signaling Poster presented at ISTH2017 on: 2017 Irena Krger DOI: 10.3252/pso.eu.ISTH2017.2017 Tuesday, July 11 ISTH.