Phospholipase D1 is a regulator of platelet-mediated inflammation

Accepted Manuscript Phospholipase inflammation

D1

is

a

regulator

of

platelet-mediated

Meike Klier, Nina Sarah Gowert, Sven Jäckel, Christoph Reinhardt, Margitta Elvers PII: DOI: Reference:

S0898-6568(17)30186-9 doi: 10.1016/j.cellsig.2017.07.007 CLS 8956

To appear in:

Cellular Signalling

Received date: Revised date: Accepted date:

9 March 2017 29 June 2017 10 July 2017

Please cite this article as: Meike Klier, Nina Sarah Gowert, Sven Jäckel, Christoph Reinhardt, Margitta Elvers , Phospholipase D1 is a regulator of platelet-mediated inflammation, Cellular Signalling (2017), doi: 10.1016/j.cellsig.2017.07.007

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ACCEPTED MANUSCRIPT Klier et al.

Phospholipase D and inflammation

Phospholipase D1 is a regulator of platelet-mediated inflammation

Meike Kliera, Nina Sarah Gowerta, Sven Jäckelb,c, Christoph Reinhardtb,c, Margitta Elversa

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Department of Vascular and Endovascular Surgery, Heinrich-Heine University Medical

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Center, Düsseldorf, Germany. 2

German Center for Cardiovascular Research (DZHK), Partner Site RheinMain, Mainz,

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Germany. 3

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Center for Thrombosis and Hemostasis (CTH), University Medical Center Mainz, Mainz,

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Germany

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Short title: Phospholipase D and inflammation

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Key words: Phospholipase D1, platelets, inflammation, Glycoprotein Ib, leukocytes, endothelial

Abstract word count: 234

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Number of Figures: 6

Correspondence to: Margitta Elvers, Ph.D., Department of Vascular and Endovascular Surgery, Heinrich-Heine University Medical Center, Moorenstr. 5, 40225 Duesseldorf, Germany. Phone: +49 (0)211 81-08851 Fax: +49 (0)211 81-17498. Email: [email protected]

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ABSTRACT Glycoprotein (GP)Ib is not only required for stable thrombus formation but for platelet-mediated inflammatory responses. Phospholipase (PL)D1 is essential for GPIb-dependent aggregate formation under high shear conditions while nothing is known about PLD1-induced regulation of GPIb in platelet-mediated inflammation and the underlying mechanisms. This study aimed

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to investigate the relevance of PLD1 for platelet-mediated endothelial and leukocyte recruitment and activation in vitro and in vivo.

stimulated endothelial cells (ECs)

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Pld1-/- platelets showed strongly reduced adhesion to TNF

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under high shear conditions ex vivo. Normal cytoskeletal reorganization of Pld1-/- platelets but reduced integrin activation after adhesion to inflamed ECs confirmed that defective integrin

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activation is responsible for reduced platelet adhesion to ECs. This, together with significantly reduced CD40L expression on platelets led to reduced chemotactic and adhesive properties

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of ECs in vitro. Under flow conditions, recruitment of leukocytes to collagen–adherent platelets was reduced. Under inflammatory conditions in vivo, reduced platelet and leukocyte

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recruitment and arrest to the injured carotid artery was observed in Pld1-/- mice. In a second in

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vivo model of venous thrombosis, platelet adhesion to activated endothelial cells was reduced while leukocyte recruitment was attenuated in PLD1 deficient mice. Mechanistically, PLD1 modulated PLC 2 phosphorylation and integrin activation via Src kinases without affecting

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vWF binding to GPIb. Thus, PLD1 is important for GPIb-induced inflammatory processes of

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platelets and might be a promising target to reduce platelet-mediated inflammatory responses.

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1. Introduction Hemostasis relies on adequate platelet activation and thrombus formation [11]. Besides, growing evidence supports a contribution of platelets to the pathology of disorders not only related to thrombosis but also to inflammation [11, 35] host-pathogen interaction [19, 35] and angiogenesis [11]. Platelets are able to bind to pathogens such as viruses, bacteria and parasites and release many immunoreactive substances including cytokines and chemokines. They express toll-like receptors and are able to secrete membrane microparticles to allow the

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transport of inflammatory load to leukocytes [17]. Inflammation is characterised by the distinct interaction of platelets, leukocytes and endothelial

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cells (EC) to trigger activation processes leading to leukocyte recruitment into the vascular wall [11]. Cell-cell communication of platelets and endothelial cells has been analysed in recent

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years and includes paracrine signalling, transient interactions and receptor-mediated cell-cell adhesion. Platelet adhesion to activated ECs is mediated by fibrinogen bound integrin IIb 3

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binding to a3 integrin [4, 11]. Moreover, the EC receptors intercellular adhesion molecule-1 (ICAM-1), a3 integrin and glycoprotein (GP)Ib on platelets are known to be involved in the

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binding of activated platelets to ECs [11]. Adherent platelets become activated and release potent inflammatory and mitogenic substances into the microenvironment that alter the chemotactic, adhesive, and proteolytic properties of ECs[11]. The receptor GPIb, exclusively

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expressed on platelets, plays a major role in these processes because GPIb is involved in the interaction with ECs via binding to selectins [33]. Cells expressing P-selectin are able to roll on

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immobilized GPIb that can be inhibited by antibodies against GPIb and P-selectin [28]. However, the interaction of P-selectin and GPIb is insufficient for stable adhesion and requires integrin IIb 3 for firm platelet-endothelial adhesion [4, 12, 21]. Platelet adhesion via integrin

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IIb3 leads to up-regulation of CD40 ligand (CD40L, CD154)

on platelets and induces

endothelial cells to release chemokines and to express adhesion molecules via binding to

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CD40 on endothelial cells [15, 22]. Moreover, CD40-CD40L interaction initiates proteolytic activity of endothelial cells, because CD40 ligation induces the expression and release of matrix degrading enzymes. The adherent platelets are able to create a platform on which leukocytes roll and establish firm adhesion via P-selectin–PSGL-1 and GPIbα–CD11b/CD18 interactions [11]. After endothelial cell activation with enhanced P-and E-selectin expression, leukocyte recruitment including capture and rolling mediated by EC P-selectin, PSGL1 and beta2-integrin (CD11/CD18) interaction with EC ICAM-1, can occur directly and leads to leukocyte activation, firm adhesion and transendothelial migration [11]. Platelet GPIb binding to leukocytes via 3

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CD11/CD18 initiates monocyte secretion of chemokines, cytokines, and pro-coagulant tissue factor, up-regulates and activates adhesion receptors and proteases and induces monocyte differentiation into macrophages [11, 24, 37]. This supports that GPIb plays a role in systemic inflammation [5], endotoxemia-induced thrombosis and mortality[38] as well as ischemic stroke [7]. In a cecal ligation and puncture (CLP)-induced sepsis model, GPIb was associated with anti-inflammatory properties suggesting a dual role of GPIb in thrombotic and inflammatory processes [5]. In contrast, mortality after lipopolysaccharide (LPS)-induced sepsis was

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decreased when mice were treated with a micellar peptide inhibitor against GPIb or when mice were expressing a functionally deficient mutant of GPIb [38]. Additionally, GPIb was shown to

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be an interesting target for therapy in ischemic stroke. Recent studies provided evidence for a pro-thrombolytic activity when the vWF-GPIb axis was inhibited, leading to improved cerebral

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reperfusion rates and reduced cerebral ischemia/reperfusion injury [7]. In line with these observations, Phospholipase (PL)D1 deficient mice were protected from

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arterial thrombosis and ischemic brain infarction that was related to a GPIb-dependent defect [9]. Lack of PLD1 in platelets resulted in impaired IIb3 integrin activation in response to major

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agonists and following GPIb–dependent aggregate formation under high shear conditions indicating that PLD1 may be a critical regulator of platelet activity in the setting of ischemic cardiovascular and cerebrovascular events. Besides, PLD1 plays an important role in

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inflammation because PLD1 is involved in the inflammatory response e.g. after acute myocardial infarction playing a role in leukocyte chemotaxis, cell migration and integrin-

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mediated cell adhesion [18, 27, 30]. PLD1 was also shown to modulate peritonitis [32], pancreatitis [3] and rheumatoid arthritis [10, 16]. However, the impact of PLD1 in the platelet-

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mediated inflammatory response remains elusive.

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2. Materials and methods 2.1 Animals Gene-targeted mice lacking PLD1 or PLD2 were described before [6, 25]. Pld mutant mice and the corresponding wild-type littermates were bred from breeder pairs. Experiments were performed with male and female mice aged 2-6 months. Mice were anesthetised with Ketamin (Ketavet®, Pfizer, 100 mg/kg) and Xylazin (2 % Bernburg, medistar, 5 mg/kg) by intraperitoneal

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(i.p.) injection before surgery. Euthanasia was performed by cervical dislocation. All animal experiments were performed conform the guidelines from Directive 2010/63/EU of the

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European Parliament on the protection of animals used for scientific purposes. The protocol was approved by Heinrich-Heine-University Animal Care Committee and by the district

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government of North Rhine-Westphalia (LANUV, NRW; Permit Number: 84-02.04.2013.A486; 84-02.05.20.12.284) and the local committee on legislation on protection of animals

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(Landesuntersuchungsamt Rheinland-Pfalz, Koblenz, Germany; 23177-07/G16-1-013).

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2.2 Cell culture

Mouse heart endothelial cell clone 5-Transformed (MHEC5-T) were cultured in DMEM-Medium (Gibco) containing 10% FCS, 1% Penicillin/Streptomycin and 1% L-Glutamine. For passaging

2.3 Platelet isolation

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0.05% Trypsin- EDTA (Life technologies) was used to detach the cells.

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Whole blood was acquired by puncturing the retrobulbar vein plexus of isofluran anesthetised donor mice. For anticoagulation blood was immediately collected in heparin solution (20 U/ml,

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Ratiopharm) or ACD solution. Platelet rich plasma (PRP) was acquired by centrifuging the whole blood two times for 6 min and 500 g. Platelets were obtained by centrifuging PRP 5 min at 650g and resuspending in Tyrode buffer (2.73 M NaCl, 53.6 mM KCl, 238 mM Na2HCO3, 8 mM Na2HPO4, 0.5 M Hepes, 0.1 M MgCl2, 10% glucose, 10% BSA, 0.1 M CaCl, pH 7.35). Platelet counts were determined with an automated hematology analyser (Sysmex-KX21N, Sysmex Corporation).

2.4 Static adhesion experiment 5

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Coverslips were coated with vWF-Antibody (1:500, Dako, 1h at 37°C), blocked with 1%BSA 1h at RT, and covered for 2h at 37°C with murine plasma to allow binding of murine plasma vWF to the antibody to generate an immobilized vWF matrix. Next, isolated platelets (80x103/µl) were allowed to adhere to the vWF matrix for 3 and 20 min, respectively. Platelets were fixed with PHEM-buffer (100 mM Pipes; 5.25 mM Hepes, 10mM EGTA, 20 mM MgCl2;

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pH 6.8), washed with PBS and three images per coverslip were counted.

2.5 Spreading experiment

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Washed platelets (8x104/µl) were thrombin stimulated (0.008 U/ml, Roche) and allowed to adhere to fibrinogen (0.2 mg/ml) coated coverslip (24 x 60 mm) and incubated for 20 or 60 min

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platelets and counted five times per visual field.

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at RT. The different spreading stated were scaled in lamellipodia, filopodia or adherent

A vWF matrix was generated as described above and incubated with isolated platelets (80x103/µl) for 3 and 20 min, respectively. Platelets were fixed with PHEM-buffer, washed with

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PBS and the different spreading states were scaled in small nodular structured, lamellipodia,

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filopodia and adherent platelets. Three images per coverslip were counted.

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2.6 Immunoprecipitation

Pooled, resting platelets of three mice were lysed and samples were incubated with PLD1 antibody (Cell Signalling, 1:50) 1h at 4°C. Samples were incubated with washed Protein G-

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Sepharose (GE Healthcare) overnight at 4°C. The Sepharose pellet was washed before addition of Laemmli sample buffer. Immunoblotting was performed as indicated. Antibody

platelets.

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against Src (Cell Signalling, 1:1000) was used to analyse co-localization of GPIb and Src in

2.7 Western blot We determined the expression level of p-Src (Polyclonal Phospho-SRC Familiy (try416) antibody Rabbit anti mouse, Cell Signalling, 1:1000) and p-PLCγ2 (Phospho-PLCγ2 (Tyr759) antibody Rabbit anti mouse, Cell Signalling, 1:1000) in lysates of collagen-related peptide (20 µg/ml) or Botrocetin (2 µg/ml) stimulated platelets and determined the expression of total protein expression of Src (SRC (36D10) rabbit mAb, Cell Signalling, 1:1000) and PLCγ2 6

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(PLCγ2 (Tyr759) antibody Rabbit anti mouse, Cell Signalling, 1:1000) as controls. The lysates were prepared with reducing sample buffer (Laemmli buffer), denatured at 95°C for 5 min and separated on SDS-polyacrylamide gel. After separation the proteins were transferred onto nitrocellulose blotting membrane (GE Healthcare). The membrane was blocked with approved blocking medium, antibody incubation was performed by following the equal manufacturer´s protocol and followed membranes were incubated with peroxidase-conjugated goat- ant rabbit IgGs (GE Healthcare, 1:2500). Protein bands were visualized by the use of Immobilon™

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Western Chemiluminescent HRP Substrate solution (BioRad).

2.8 Flow chamber experiment with whole blood and inflammatory MHEC5-T

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For dynamic flow chamber experiments, fibrinogen (0.5 mg/ml, Sigma, 30 min/37°C) coated coverslips (24 x 60 mm) were incubated overnight at 37°C with MHEC5-T (2x105/ml). The

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confluent monolayer of MHEC5-T was activated with TNFα (100 ng/ml, Peprotech) at 37°C overnight. Platelets of heparinized whole blood were labelled with a DyLight488-conjugated

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anti-mouse GPIbβ derivative antibody (1 µg/ml, Emfret, 5 min/37°C) and perfusion was performed with a shear rates of 1.700 s-1 (150 sec-1) for 5 min. Image analysis was performed with the Zen 2012 software. For analysis of platelet adhesion the mean percentage of total

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area covered by platelets and the mean of fluorescence intensity per square millimetre was

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consumed.

2.9 FACS analysis of adhesion molecules, CD40L exposure and vWF binding to GPIb

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MHEC5-T were cultured in 96-well plates (1x105 per well) over night. The confluent MHEC5-T were brought into close apposition with thrombin (0.1 U/ml, 0.02 U/ml, Roche), Ristocetin-(0.6

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mg/ml, VWF) and collagen-related peptide (5 µg/ml) stimulated platelets (1x108 per well) by centrifuging the 96-well plate at 600g for 5min. After co-incubation (3.5 h at 37°C) platelets were removed and MHEC5-T were treated with Accutase (Paa) for 20 min at RT and relieved by pipetting. The adhesion molecules E-Selectin (CD62E), ICAM (CD54), VCAM (CD106) and P-Selectin (CD62P) were analysed by flow cytometry with either FITC or PE conjugated monoclonal antibodies (PE-CD62E, PE-CD106, FITC-CD54, BD; FITC-CD62P, Emfret). In addition integrin activation of co-incubated platelets with MHEC5-T was measured by fibrinogen binding (fibrinogen-Alexa 488, Life Technologies). 7

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Moreover, we determined CD40L expression on the surface of activated platelets. Isolated platelets (30x103 plt/µl) were activated with classical agonists 0.1, 0.02 U/ml Thrombin and 5 µg/ml collagen-related peptide and additionally with Botrocetin (2 µg/ml) or Botrocetin/vWF (2/10 µg/ml), incubated with murine CD40L antibody (Amenian Hamster monoclonal [MR1] to CD40L (FITC), Abcam) and analysed by flow cytometry. To analyse the binding of vWF to GPIb we performed flow cytometric analyses. Isolated platelets were washed twice, pellet was resuspended in vWF-buffer (Tyrodes´s buffer; 1%

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BSA, 10mM EDTA, 2mM MgCl2) and platelets were stimulated with collagen-related peptide (5 µg/ml), Botrocetin (1.25 µg/ml) or Botrocetin/vWF (1.25/10 µg/ml). After fixation with 1%

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PFA and incubation with vWF-antibody (vWF-FITC, Emfret) flow cytometry was performed.

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2.10 IL-6 ELISA

To investigate chemotactic alterations induced by activated platelets, we measured IL-6 levels in the supernatant of co-cultures of MHEC5-T and thrombin (0.1; 0.02 U/ml) or collagen-related

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peptide (5 µg/ml) activated platelets. The measurement was performed by following the

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manufacturer´s protocol (IL-6 ELISA; R&D System).

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2.11 CD40L ELISA

Human platelets were isolated as reported previously [8]. Briefly, fresh ACD-anti-coagulated blood was obtained from healthy and centrifuged at 200 x g for 10 minutes. The platelet rich

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plasma was separated and added to PBS [pH 6.5, 2.5 U/ml apyrase (Sigma), 1 µM PGI 2] in 1:1 volumetric ratio and centrifuged at 1000 x g for 6 minutes. The platelet pellet was

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resuspended in Tyrode buffer [140 mM NaCl, 2.8 mM KCl, 12 mM NaHCO3, 0.5 mM Na2HPO4, 5.5 mM Glucose, 0.1% HIBSA, pH 7.4]. Platelets were treated with the PLD inhibitor 5-Fluoro2-indolyl des-chlorohalopemide (FIPI, 1 µM) for one hour at room temperature and stimulated with the indicated agonists. The supernatant was taken and analyzed for soluble CD40L according to the manufacturer’s protocol using a human CD40L ELISA (Affymetrix eBioscience).

2.12 Carotid ligation in mice and assessment of platelet and leukocyte adhesion by intravital microscopy 8

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To evaluate the effect of inflammation on platelets and leukocyte activation and adhesion in vivo, we used intravital fluorescence microscopy on a carotid ligation as described previously [13]. Prior to the experiment, platelets of donor mice were stained with Rhodamine 6G (0.2 mg/ml, Sigma) and endogenous leukocytes were stained with Acridine Orange (2 mg/kg bodyweight, Sigma).

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2.13 Preparation of platelets and leukocytes for intravital epifluorescence microscopy Murine platelets were isolated and labeled with Rhodamin B (20 μg/ml; Invitrogen Life

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Technologies GmbH, Carlsbad, CA) as reported earlier [36]. The DCF-labeled platelet suspension was adjusted to a final concentration of 150x103 platelets/μl and 250 µl suspension

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was injected i.v. via a jugular vein catheter. Adhesion of murine platelets was assessed by high-speed epifluorescence microscopy. Leukocytes were stained

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µg/µl; 100 µl per mouse; Sigma-Aldrich) in vivo.

by Acridine orange (50

2.14 In vivo model of venous thrombosis by flow restriction The mouse model of venous thrombosis has been described in detail previously [34]. Briefly,

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mice were anesthetised with Ketamin (Ketavet®, Pfizer, 100 mg/kg) and Xylazin (2 %

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Bernburg, medistar, 5 mg/kg) by intraperitoneal (i.p.) injection, a median laparotomy was performed and the inferior vena cava (IVC) was exposed carefully to avoid trauma to the vessel wall. A space holder (FloppyR II Guide Wire 0.36 mm; Guidant Corporation, Indianapolis, USA)

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was placed on the outside of the vessel and a permanent narrowing ligature (8.0 monofil polypropylene filament, Premilene; Braun, Tuttlingen, Germany) was placed exactly below the left renal vein. Subsequently, the wire was removed to avoid complete vessel occlusion. Side

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branches were not ligated or manipulated. Mice with bleedings or any injury of the IVC during surgery were excluded from further analysis. Cell adhesion was followed by intravital microscopy. Citrated whole blood for platelet isolation was collected by intracardial puncture. Euthanasia of mice was performed by cervical dislocation.

2.15 Intravital microscopy Adhesion of platelets and leukocytes in living mice was estimated by using high-speed realtime intravital fluorescence video microscopy (BX51WI; Olympus, Hamburg, Germany). We 9

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anesthetised mice via intraperitoneal injection of midazolame, medetomidine and fentanyl (5, 0.5, and 0.05 mg kg1 body weight, respectively). Mice were placed on a heating pad (Foehr Medical Instruments, Seeheim-Jugenheim, Germany) for maintenance of body temperature at 37° C. A polyethylene catheter (0.28 mm ID, 0.61 mm OD; Smiths Medical, Grasbrunn, Germany) was placed in the right jugular vein to permit administration of platelets and

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fluorescence dyes.

2.16 Statistical analysis

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All experiments were performed at least three times and data are presented as means ± SEM. Statistical analysis was performed using the two- or one-tailed Student´s t-test or ANOVA

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where indicated, whereat a P values less than 0.05 was set as significant. For all figures

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*P<0.05, **P<0.01 and ***P<0.001.

3. Results 10

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3.1 PLD1 is required for stable adhesion on inflammatory endothelial cells under high shear conditions Pld1-/- mice show impaired integrin IIb3 activation and reduced GPIb dependent thrombus formation under conditions of high shear leading to protection against arterial thrombosis [9]. To analyse if defective GPIb signalling of PLD1 deficient mice plays a role in platelet mediated inflammation, we investigated platelet adhesion to tumor necrosis factor- (TNF) stimulated murine endothelial cells (MHEC5-T) under flow conditions. Under low shear rates (150 sec-1),

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that model flow conditions in veins or large arteries, respectively, no significant differences in platelet adhesion to activated MHEC5-T were measured between wild-type and Pld1-/- platelets

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(Fig. 1A). However, under high shear rates (1.700 sec-1), strongly reduced adhesion of Pld1-/-

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platelets to activated MHEC5-T was observed (Fig. 1B-C). The impact of PLD in platelet adhesion to activated endothelial cells was confirmed by treating platelets with the PLD inhibitor 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), known to inhibit both isoforms, PLD1

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and PLD2, leading to strongly reduced platelet adhesion to activated MHEC5-T as well (Fig. 1B-C). Interestingly, treatment of platelets with FIPI did not lead to further reduction of platelet

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adhesion to MHECs under flow compared to experiments with PLD1 deficient platelets suggesting that PLD1 plays a major role under high shear conditions. Accordingly, adhesion of Pld2-/- platelets to activated MHEC5-T under high shear conditions was not significantly

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different compared to wild-type platelets (Suppl.-Fig. 1).

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It is well known that CD40L on activated platelets triggers an inflammatory reaction of endothelial cells that depends –at least in part- on integrin IIb 3 [15]. To elucidate the relevance of PLD1 in CD40L mediated signalling on MHEC5-T, we activated platelets with the

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indicated agonists and analysed CD40L expression. As shown in figure 1D, Pld1-/- platelets show reduced CD40L expression upon stimulation with thrombin (0.1 U/ml) and collagenrelated peptide (0.5 µg/ml) as determined by flow cytometric analysis. To analyse the impact

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of GPIb in this process we activated platelets with Botrocetin and human vWF and found CD40L expression on the platelet surface of wild-type platelets. In contrast, significantly reduced CD40L was exposed at the platelet membrane of Pld1-/- platelets compared to controls (Fig. 1D). However, treatment of human platelets with the PLD inhibitor FIPI revealed no alterations in the amount of soluble CD40L in the supernatant of agonist-stimulated platelets (Fig. 1E). Since PLD1 has been identified as a regulator of GPIb dependent integrin activation [9] we examined fibrinogen binding as marker for integrin activation of Pld1-/- platelets in response to

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inflamed MHEC5-T. As a result we found reduced integrin activation of PLD1 deficient platelets compared to wild-type controls (Fig. 1F). PLD1 deficient macrophages were shown to display altered cytoskeletal reorganization that underlies decreases in phagocytic capacity [2]. To analyse if defects in cytoskeletal reorganization of platelets are involved in defective adhesion on MHEC5-T under flow, we determined filopodia and lamellipodia formation and adhesion of Pld1-/- platelets to a fibrinogen matrix. As shown in figure 2, no major alterations were found neither in cytoskeletal

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reorganization nor in adhesion of Pld1-/- platelets compared to wild-type controls (Fig. 2A-C). Same results were obtained with PLD2 deficient platelets (Suppl.-Fig. 2) showing no significant

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alterations in cytoskeletal reorganization and adhesion to different extracellular matrix proteins.

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In contrast, adhesion of PLD1 deficient platelets to vWF that is mediated solely by GPIb and IIb3 [23] as well as lamellipodia formation on a vWF matrix, was reduced significantly after 3

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min compared to control platelets (Fig. 2D-E). However, no differences were observed after 20 min of platelet adhesion and spreading (Fig. 2D-E). Taken together, these data indicate that reduced adhesion of Pld1-/- platelets on activated MHEC5-T is a result of impaired GPIb

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signalling and GPIb mediated integrin activation.

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3.2 PLD1 is essential for platelet-mediated alterations of chemotactic and adhesive properties

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of ECs

CD40L on platelets [15] and platelet release of IL-1 [14] induce endothelial cells to secrete chemokines such as interleukin (IL)-6 and IL-8 and to express adhesion molecules to generate

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signals for the recruitment and extravasation of leukocytes at sites of inflammation [14, 15]. To examine the role of PLD1 in these processes we co-incubated resting and stimulated platelets

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with MHEC5-T for 3.5 h and analysed up-regulation of ICAM-1, VCAM-1, E-selectin and Pselectin. We found a significant reduction in the expression of ICAM-1 on MHEC5-T when platelets were stimulated with intermediate concentrations but not with high concentrations of thrombin. No differences were observed when platelets were activated with collagen-related peptide (Fig. 3A). Furthermore, no differences in the expression of VCAM-1 and E-selectin were detected (Fig. 3B-C). In contrast, up-regulation of P-selectin on MHEC5-T was reduced after co-incubation with thrombin activated Pld1-/- platelets (Fig. 3D). Activation of platelets with collagen-related peptide was shown not to lead to up-regulation of P-selectin on MHEC5-T. To study specifically the effect of PLD1 deficiency on vWF-induced signalling, we analysed upregulation of selectins, ICAM-1 and VCAM-1 induced by Botrocetin and human vWF stimulated 12

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platelets [4, 20]. However, stimulation of platelets with Botrocetin and human vWF did not induce up-regulation of adhesion molecules on MHEC5-T compared to resting platelets and MHEC5-T (Fig. 3A-D). To investigate chemotactic alterations induced by activated platelets, we measured IL-6 levels in co-cultures of MHEC5-T and activated platelets (Fig. 3E). IL-6 can be used as indicator of the endothelial inflammatory reaction [14] that is induced by the release of IL1- of platelet origin [11] and ligation of CD40 on endothelial cells [15], respectively. Co-incubation of

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MHEC5-T and platelets induced substantial secretion of IL-6 (Fig. 3E) following thrombin stimulation. However, stimulation of PLD1 deficient platelets with intermediate concentrations

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of thrombin led to significantly reduced IL-6 levels released from MHEC5-T. However, stimulation of platelets with collagen-related peptide did not induce IL-6 release from MHEC5-

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T. Again, stimulation of platelets with Botrocetin and human vWF did not modulate the response of MHEC5-T because we were not able to detect IL-6 in the supernatant of MHEC5-

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T that were co-incubated with Botrocetin/vWF stimulated platelets (data not shown).

3.3 PLD1 modulates phosphorylation of Src upon GPIb signalling in platelets To investigate how PLD1 modulates GPIb-mediated signalling in platelets leading to integrin

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IIb3 activation, we performed experiments to analyse the phosphorylation of target proteins

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that become activated by GPIb engagement. Tyrosine phosphorylation of Src kinase (Lyn) and PLCγ2 were described to play an essential role in vWF-GPIb signalling to induce integrin αIIbβ3 activation [26]. Western blot analysis showed clear phosphorylation of Src kinase and PLC2

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(Fig. 4A-B) after stimulation of wildtype platelets with Botrocetin and human vWF after 30 (pSrc) and 120 sec (p-PLC2) of stimulation while Src and PLC2 phosphorylation was reduced in PLD1 deficient platelets. Moreover, phosphorylation of Src was not only reduced but

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attenuated in PLD1 deficient platelets following Botrocetin/vWF stimulation (Fig. 4A). Platelet activation with collagen-related peptide induces the activation of GPVI leading to phosphorylation of Src and PLCγ2 as well. Interestingly, phosphorylation of Src was attenuated while phosphorylation of PLC2 was reduced after collagen-related peptide stimulation of PLD1 deficient platelets. To investigate whether PLD1 interacts with Src to stimulate phosphorylation of the protein after GPIb engagement, we performed immunoprecipitation studies. We detected Src by Western blot analysis in PLD1-immunoprecipitates of platelets suggesting a functional interaction between PLD1 and Src that is responsible for GPIb mediated integrin activation (Fig. 4C). 13

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To examine if PLD1 only affects GPIb intracellular signalling and not vWF binding to GPIb, we performed flow cytometric analysis and measured the binding of FITC-labeled vWF to platelets in response to different agonists. As shown in figure 4D, deficiency of PLD1 did not affect vWF binding to GPIb (Fig. 4D).

3.4 Reduced adhesion of PLD1 deficient platelets at sites of inflammation in vivo

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PLD1 deficient mice are protected against arterial thrombosis after FeCl3-induced injury of the carotid artery [9]. To examine the impact of PLD1 in shear-dependent adhesion of platelets at

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sites of vascular injury, platelet adhesion at the ligated carotid artery was analysed by in vivo fluorescence microscopy. For visualization and quantification of tethered (transiently adherent)

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and adherent platelets at the ligated carotid artery, platelets from donor mice were fluorescently labeled with Rhodamine. In contrast to control mice, platelet tethering and firm adhesion at

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sites of injury was strongly reduced in Pld1-/- mice (Fig. 5A-B, video S1 and S2). These results demonstrate that PLD1 is required for platelet adhesion at the inflammatory endothelium in

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vivo. We next investigated the impact of PLD1 in platelet adhesion under inflammatory conditions in a second in vivo model of venous thrombosis that was used as another inflammatory mouse model. The inferior vena cava (IVC) was exposed and a permanent

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narrowing ligature was placed below the left renal vein to induce flow restriction but no full occlusion of the vessel. As shown in figure 5C-D, platelet adhesion to the vessel wall was

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reduced significantly after 180, 240 and 300 min after flow reduction in PLD1 deficient mice.

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3.5 Reduced adhesion of PLD1 deficient platelets and leukocytes at sites of inflammation in

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To investigate if platelets modulate leukocyte adhesion, we performed flow chamber experiments to analyse leukocyte adhesion to collagen-adherent platelets under high shear conditions (1.700 s-1). Transient and stable leukocyte adhesion to platelets was strongly reduced when PLD1 deficient leukocytes were perfused through the chamber and allowed to adhere to PLD1 deficient collagen-adherent platelets compared to controls (Fig. 6A-B). To analyse if leukocyte recruitment at the injured vessel is altered upon PLD1 deficiency as well, we injected Acridine orange into mice and quantified leukocyte adhesion at the ligated carotid artery. As expected, leukocyte adhesion to the subendothelial layers was strongly reduced in Pld1-/- mice (Fig. 6C-D, video S3 and S4). Importantly, leukocyte adhesion to the IVC was significantly reduced after 180 min of flow restriction using PLD1 deficient mice compared to 14

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controls confirming that PLD1 is important for leukocyte adhesion under inflammatory conditions. Taken together, PLD1 is required for platelet and leukocyte adhesion at the injured vessel in

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vivo.

4. Discussion This study reveals that PLD1 is required for platelet-endothelial cell interactions and plateletmediated leukocyte recruitment upon inflammatory processes. Pld1-/- platelets showed a 15

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significant reduction in adhesion to activated MHEC5-T under high shear conditions but no adhesion defects under conditions of low shear. Furthermore, PLD1 is important for leukocyte arrest to collagen-adherent platelets under high shear conditions. This suggests that PLD1 facilitates GPIb dependent IIb 3 integrin activation and firm adhesion of platelets and leukocytes to the inflamed endothelium. Platelet-mediated alterations of chemotactic and adhesive properties of ECs are reduced when platelets lack PLD1. In vivo, adhesion of platelets and leukocytes at the injured carotid artery and at the flow-restricted IVC was reduced

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in PLD1 deficient mice suggesting that PLD1 is important for the recruitment of platelets and leukocytes to the vessel wall upon vascular injury and endothelial activation in mice.

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The contribution of PLD1 in GPIb dependent integrin activation was already shown in arterial thrombosis leading to protection against vessel occlusion in Pld1-/- mice [9]. GPIb is essential

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for stable platelet adhesion to collagen via vWF [29]. This is a process that is also relevant under inflammatory conditions because platelet-EC adhesion occurs even under high shear

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conditions and blocking of vWF or IIb 3 integrin by antibodies inhibits platelet adhesion to HUVECs [11]. Hence, PLD1 plays a dual modulatory role in both, the thrombotic and the

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inflammatory pathways.

The impact of PLD1 in inflammatory diseases was shown recently by different groups providing evidence for an involvement of PLD1 in peritonitis [32], and myocardial ischemia and

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reperfusion injury [30]. These studies provided strong evidence that PLD1 is coupled to TNF-

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 signalling leading to reduced inflammatory responses including defective leukocyte migration [30, 32]. Here we now report that PLD1 plays a prominent role in platelet-mediated inflammation. Thus it is tempting to speculate that reduced inflammatory responses after myocardial infarction in Pld1-/- mice might be the result at least in part of defects in platelet-

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mediated inflammation. The thrombus instability observed in Pld1−/− mice resulted in protection against neuronal damage after cerebral ischemia and reperfusion injury [9]. This additionally

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provides evidence that infarct development is largely dependent on GPIb-vWF–mediated platelet adhesion and activation. Since ischemic stroke is a thrombo-inflammatory disease [7] it is tempting to speculate that reduced platelet-mediated inflammation accounts for the protection against ischemic brain infarction beside reduced thrombus formation in PLD1 deficient mice. Although Pld1-/- macrophages showed altered cytoskeletal organization accompanied by altered macrophage phagocytosis [2], we found normal cytoskeletal reorganization of platelets on a fibrinogen matrix. However, at early time points adhesion and cytoskeletal reorganization on a vWF matrix was reduced when PLD1 deficient platelets were allowed to adhere and 16

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spread on immobilized vWF. Thus, we believe that altered GPIb dependent integrin activation is responsible for reduced platelet adhesion to ECs in vitro and in vivo. As shown previously, Pld1-/- platelets display normal degranulation of  and dense granules upon activation. However, Pld1-/- platelets showed reduced CD40L expression on the platelet membrane upon thrombin and collagen-related peptide stimulation, respectively (Fig. 1D). CD40L is an important trigger for platelet-mediated inflammatory reaction of endothelial cells.[15] Thus we provide strong evidence that reduced CD40L expression together with reduced expression of

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adhesion molecules (ICAM-1 and P-selectin) and IL-6 release account for reduced inflammatory properties of ECs. Accordingly, altered chemotactic and adhesive properties of

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ECs lead to reduced recruitment of leukocytes in vivo. However, a direct influence of GPIb on the recruitment of leukocytes is possible because GPIb binds to CD11/CD18 (Mac-1) on

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leukocytes important for adhesion and transendothelial migration of inflammatory cells.[11] Furthermore, leukocyte adhesion to collagen-adherent platelets was reduced under high flow

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conditions that further strengthen the hypothesis that reduced leukocyte recruitment in vivo might be a result of defective GPIb mediated recruitment of leukocytes.

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The impact of PLD1 in GPIb signalling of platelets was already shown several years ago [9]. We now analysed how PLD1 affects GPIb-mediated integrin activation. Our data point to a novel role of PLD1 as an activator of platelet Src kinases that become phosphorylated after

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GPIb engagement under high shear conditions [31]. In 2003, Ahn and colleagues showed that wildtype PLDs, but not catalytically inactive PLD mutants, increase c-Src kinase activity leading

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to enhanced cell proliferation. They provided evidence for a transmodulation between PLD and c-Src, because c-Src acts as a kinase of PLD and PLD acts as an activator of c-Src [1]. Our data reveal that PLD1 modulates Src activity in platelets after GPIb stimulation but also affects

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collagen-dependent Src activity, because phosphorylation of Src was attenuated after collagen-related peptide stimulation of platelets, although maximal Src activity was comparable

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between PLD1 deficient and control platelets. As a result of defective Src phosphorylation, the phosphorylation of PLC2 following GPIb stimulation was reduced as well that might be responsible for defective GPIb-mediated integrin activation. Inhibition of PLD1 in platelets using the PLD inhibitor FIPI was shown to protect mice from arterial thrombosis [34]. In line with these results, we report reduced platelet adhesion to ECs upon pharmacological inhibition of PLD1 with FIPI suggesting a therapeutic potential for PLD1 in platelet-associated inflammatory diseases as well.

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Altogether, our data point to an important contribution of the PLD1-GPIb axis in the modulation of adhesive and chemotactic properties of ECs and recruitment of leukocytes upon platelet-

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mediated inflammation.

Acknowledgements and funding 18

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We thank Martina Spelleken for excellent technical assistance and Dr. Norbert Gerdes for support in CD40L measurements. This study was supported in part by grant from the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 1116/1 (A05) and 974/2 (A16), Düsseldorf and a grant by the Else Kröner-Fresenius-Stiftung to C.R. and S.J. (2014-A151).

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.

Figure legends 19

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Fig. 1. Significantly reduced adhesion of platelets on inflammatory ECs at high shear. (A-C) Platelets of heparinized whole blood were labelled and perfused through the dynamic flow -/-

chamber system , Adhesion of Pld1 platelets on TNF- stimulated MHEC5-T under low (A, -1

+/+

-1

150 sec ) and high shear conditions (C, 1.700sec ) ex vivo. Where indicated, Pld1

platelets

were incubated with the PLD inhibitor FIPI (1 µM). (B) Representative pictures of adherent platelets under high shear conditions (green). (D) Isolated platelets were activated by classical agonists and CD40L – exposure at the platelet membrane was measured by flow cytometry. platelets following GPIb and GPVI stimulation,

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-/-

Reduced expression of CD40L on Pld1

respectively, was shown. (E) Human platelets were treated with the PLD inhibitor FIPI (1 µM),

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stimulated with different agonists as indicated and CD40L in the supernatant was measured by ELISA. (F) Isolated resting platelets were co-incubated with (TNF-α stimulated) MHEC5-T

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and fibrinogen binding of platelets was measured by flow cytometry. Bar graphs represent

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mean ± SEM. N = 5 (A-C) N = 6 – 8 (D) N = 6 (E-F); * p<0.05, ** P < 0.01. plt = platelet. -/-

Fig. 2. Unaltered cytoskeletal reorganization of Pld1

platelets. Spreading of resting and

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thrombin stimulated platelets after 20 and 60 min to fibrinogen. (A) Determination of filopodia and lamellipodia formation. (B-C) Platelet adhesion to fibrinogen. (B) Representative images of spread platelets after 60 min and (C) number of adherent platelets. (D) Statistical evaluation

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of lamellipodia, filopodia and small nodular structure formation of platelets spread on a vWF matrix and (E) numbers of adherent platelets on a vWF matrix after 3 and 20 min. Bar graphs

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depict mean ± SD (C). N=3 (A, B), N = 5 (C, D, E); * p<0.05, ** P < 0.01, ***p<0.001. -/-

Fig. 3. Pld1 platelets induce altered chemotactic and adhesive properties of ECs. Activated

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platelets were co-incubated with MHEC5-T and platelet-induced expression of the adhesion molecules (A) ICAM-1, (B) VCAM-1, (C) E-selectin and (D) P-selectin on endothelial cells was

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measured by flow cytometry. MFI=mean fluorescence intensity. (E) After co-incubation platelet-induced IL-6 release of MHEC5-T was determined by Sandwich-ELISA analysis. Bar Graphs represent mean ± SEM. N = 10 (A), N = 11 (B), N = 7 (C), N = 8 (D), N = 5 (E); positive control (TNF-) N = 3; *p<0.05, **p<0.01, ***p<0.001. plt = platelet.

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Fig. 4. Reduced phosphorylation of SRC and PLCγ2 after Botrocetin/vWF stimulation of Pld1 /-

platelets. Western blot analysis of platelet (A) Src- and (B) PLCγ2 phosphorylation after

stimulation with Botrocetin/vWF or collagen-related peptide after 30, 60 and 120 min. (C) Src Western blot analysis of PLD1-immunoprecipitates of platelets. (D) After stimulation of 20

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platelets with collagen-related peptide or Botrocetin/vWF vWF-binding to GPIb of platelets was measured by flow cytometry. MFI= mean fluorescence intensity. Bar graphs represent mean ± SD. N = 3 (A, B), N = 7 (C) and N=8 (D).

Fig. 5. Reduced adhesion of platelets at sites of inflammation in vivo. (A) Reduced adhesion -/-

of platelets at the injured carotid artery of Pld1

mice at different time points after vessel

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ligation. (B) Representative images of adherent platelets at the vessel wall documented by intravital microscopy. (C) Quantification of Rhodamin B-stained adhering platelets to the V. -/-

(black) and Pld1 (red) mice at different time points after flow

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+/+

cava vessel wall in Pld1

reduction; (7-8 mice/group). Repeated measurements using ANOVA (mixed model), * P< 0.05.

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(D) Representative images of adherent platelets at the vena cava 180 min after flow reduction.

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Bar graphs depict mean ± SEM. N=5 (A, B), N = 8 (C, D); * p<0.05, ** P < 0.01, ***p<0.001.

Fig. 6. Reduced adhesion of leukocytes under inflammatory conditions. (A-B) Isolated

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leukocytes were perfused over collagen – activated, adherent platelets under high shear conditions ex vivo. Reduced rolling and adhesion of leukocytes at collagen-adherent platelets. (A) Statistical analysis and (B) representative images of adherent leukocytes. (C) Reduced

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-/-

adhesion of leukocytes at the injured carotid artery of Pld1 mice at different time points after

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vessel ligation. (D) Representative images of adherent leukocytes. (E) Quantification of +/+

Acridine Orange-stained adhering leukocytes to the vessel wall of the V. cava in Pld1 -/-

(black)

and Pld1 (red) mice after flow reduction; (11-12 mice/group). Repeated measurements using

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ANOVA (mixed model), * P< 0.05. (F) Representative images of adherent leukocytes after 180

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min. Bar graphs depict mean ± SEM. N=8 (A, B) N = 5 (C, D); * p<0.05, ** P < 0.01, ***p<0.001.

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Highlights ► Reduced adhesion of PLD1 deficient platelets to stimulated endothelial cells under high shear conditions. ► Integrin defects in PLD1 deficient platelets are responsible for reduced adhesion to inflamed endothelium. ► PLD1 is important for platelet mediated effects on chemotactic and adhesive properties of endothelial cells. ► Upon inflammation, the

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recruitment of leukocytes is modulated by PLD1 in platelets. ►These results point to a new

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role for PLD1 as an important regulator of platelet mediated inflammation.

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