modulates GPVI and outside-in signaling of

I. Krüger1, L. Mangels3, L. Gremer2,3, D. Willbold2,3, H. H. Bock4, Margitta Elvers1 1Experimental Vascular , Clinic of Vascular and Endovascular , University Medical Center, Heinrich-Heine-University Düsseldorf, Germany 2Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany. 3Institute of Biological Information Processing (IBI-7: Structural Biochemistry) & JuStruct, Forschungszentrum Jülich, 52425 Jülich, Germany 4Clinic for , and Infectiology, Heinrich-Heine-University Düsseldorf, Germany

Introduction Reelin is known as an mediating upon development1-5. Recent studies provide evidence for reelin being expressed in platelets6, co-localizing to F-, binding to amyloid precursor protein (APP)7 and receptor 2 (ApoER2)2, 4, 8 and being important for Ib (GPIb) mediated adhesion and formation under high shear7. Consequently, defective thrombus formation protects reelin-deficient mice () against arterial thrombosis7. Aim Defining the role and signaling mechanisms of reelin in platelet cytoskeletal reorganization and glycoprotein VI (GPVI) mediated platelet activation and aggregation.

Conclusions Taken together these data provide evidence for reelin to support GPVI signaling and integrin outside-in signaling of platelets. Thus, reelin affects platelet activation via different signaling pathways involving GPIb, GPVI and the classical reelin receptors APP and ApoER2 pointing to a promising role of reelin as new therapeutic target for .

Results Reelin induces weak activation of Rho-GTPases and Reduced platelet activation, ATP release and GPVI is important for platelet adhesion on supports cytoskeletal reorganization aggregation upon GPVI stimulation of reelin recombinant reelin deficient platelets

Figure 7. GPVI is important for platelet adhesion on recombinant reelin. A-B, Adhesion of WT platelets on recombinant reelin after 5, 20 and 60 min (A) and of WT and reelin deficient platelets on recombinant reelin after 20 min + JAQ1 or IgG treatment, bar graphs depict mean values + SEM n = 5, ** P < 0.01; *** P < 0.001.

No thrombus formation in reeler mice upon Figure 1. Reelin induces weak activation of Rho-GTPases and supports cytoskeletal reorganization. Increased activation of Rho GTPases RAC1 and RhoA following reelin GPVI-depletion after ferric chloride induced treatment. A, Detection of RAC1, and B, RhoA activation after treatment of WT platelets with CRP (20 µg/ml), (0.2 U/ml), recombinant reelin (5 nM) and mock for 15 min injury of the carotid artery by G-LISA assay in ∆ to resting = 0 and representative Western blotting examples for total Rac1 and RhoA, bar graphs depict mean values ± SEM, n = 8-16 (A) , n = 4-9 (B), * P < 0.05. C, Impaired RAC1 activation of reelin deficient platelets after GPVI stimulation. WT Figure 4. Reduced platelet activation, ATP release and aggregation upon GPVI and reeler platelets were treated with CRP and thrombin, lysed and analyzed in a G-LISA® stimulationReferencesof reelin deficient platelets. A, Flow cytometry measured P- (left) Assay including Western blot controls for total RAC1, bar graphs depict mean + SEM, n = and alphaIIb beta3 integrin (right) activation of thrombin, CRP, ADP, ADP/U46 and PAR4 5-16, * P < 0.05. D, Representative Western blots of PAK from WT and stimulated WT and reeler platelets at indicated concentrations, mean fluorescence intensity reeler platelets after CRP (5 µg/ml) treatment for indicated time periods. E-F, Spreading of (MFI) shown for each measurement + SEM, n = 4, *** P < 0.001. B, ATP release and WT platelets on after 20 min (E) and 60 min (f), bar graphs depict mean values + aggregation of WT and reeler platelets upon CRP treatment measured by standard SEM, n = 5. aggregometry, representative aggregometry curves are shown (E).

Reelin supports integrin α β outside-in signaling IIb III Reelin is important for phosphorylation of GPVI signaling target

Figure 2. Impaired clot retraction in PRP from reeler mice. A, WT and reeler mice PRP Figure 5. Reelin is important for phosphorylation of GPVI signaling target proteins. was stimulated with thrombin (5 U/ml) in the presence of CaCl (20 mM). After 300 min the 2 A, Representative Western blots of WT and reeler platelets after incubation with CRP Figure 8. No thrombus formation in reeler mice upon GPVI depletion after ferric supernatant was removed, its volume measured and compared to the starting volume and (5 µg/ml) and B, densitometric evaluation for pPLC gamma 2 (upper graph) and pSyk the clot was photographed, bar graphs depict mean values ± SEM, n = 5-6, *** P ˂ 0.001. chloride induced injury of the carotid artery. A-B, Occlusion time (A) and relative (lower graph), bar graphs depict mean relative density to resting = 1 + SEM, n = 4, *** P < maximum flow rate in the injured vessel of reelin deficient and GPVI depleted animals B, Rescue of impaired reeler clot retraction after the addition of recombinant reelin (5 nM), 0.001. mock served as control, bar graphs depict mean values + SEM, n = 5, * P ˂ 0.05, *** P ˂ at time of occlusion or 30 min after injury (break off point) normalized to mean flow in 0.001. carotid artery (B), bar graphs depict mean values ± SEM, n = 5-8. C-D, Flow cytometric Reelin interacts with GPVI determination of mean fluorescence intensity of anti-GPVI-FITC (C) and anti-GPIb-PE (D) , bar graphs depict mean values ± SEM, n = 12-16 (naive) and n = 5-9 (treated) (C) Reelin promotes adhesion and lamellipodia and n = 7-10 (naive) and n = 5-9 (treated) (D), * P < 0.05, *** P < 0.001. E, Representative pictures of hematoxylin-eosin stained thrombi after ferric chloride injury formation via GPVI of the carotid artery with 10% FeCl3 for 3 min, scale bar 20 µm. Reelin modulates platelet signaling

Figure 6. Reelin interacts with GPVI. A, Representative blot after immunoprecipitation using recombinant GPVI to precipitate platelet reelin from WT platelets, controlled by platelet lysates from reelin deficient mice (left), and densitometric analysis (right), bar graphs depict mean values ± SEM, n = 3-4, * P < 0.05. B, BLI response (nm) of different concentrations of recombinant reelin (left) and the corresponding mock control (right) plotted against the time (s). Figure 3. Reelin promotes and lamellipodia formation via GPVI. A, A reelin stock (235 nM) was diluted 1:5 (black), 1:10 (red), 1:20 (blue), 1:40 (green) and 1:60 -adherent WT and reeler platelets after 20 min, bar graphs depict mean values ± (purple) with Tyrode´s buffer, pH 7.35; measurements corrected by a Tyrode´s buffer control, Figure 7. Reelin modulates platelet signaling. Direct binding of collagen to GPVI (1) SEM, n = 5, *** P ˂ 0.001. B, Adhesion of WT and reeler platelets on CRP, bar graphs with association measured from 300 s to 1200 s and dissociation from 1300 s to 2400 s. initiates phosphorylation of Syk and PLC gamma 2 (2), leading to alpha and dense depict mean values + SEM, n = 5, ** P < 0,01, *** P < 0.001. C-D, WT and reeler platelets Comparison with mock control samples (right) indicate a concentration dependend binding granule (ATP, ADP, fibrinogen, reelin and other platelet granule derived were treated with recombinant reelin (5 nM) and allowed to spread on collagen for 20 min, behaviour of reelin. Association phase (left) was fitted with the following monoexponential mediators) (3), activation of RAC1 and RhoA, contraction of actin-myosin and stress 푘표푏푠푡 bar graphs depict mean values ± SEM, n = 5, * P < 0.05, ** P < 0.01, *** P < 0.001. equation: 푌 = 푌0 + 퐴푒 (white lines within the data points (left)). Measured kobs plotted fiber formation. Reelin and other mediators then interact with platelet receptors (4) and -5 against the respective reelin concentration (nM) (middle). The slope equals to –kon, i.e. -7.5E amplify signaling for cytoskeletal reorganization including GVI-dependent signaling and -1 -1 nM s . As dissociations were undetectable in all samples, the Kd is in the subnanomolar range. platelet receptor interaction (5).

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