db mice. The regulatory role of PAR-3

db mice. The regulatory role of PAR-3

Accepted Manuscript Flow cytometry analysis reveals different activation profiles of thrombin- or TRAP-stimulated platelets in db/db mice. The regulat...

912KB Sizes 0 Downloads 2 Views

Accepted Manuscript Flow cytometry analysis reveals different activation profiles of thrombin- or TRAP-stimulated platelets in db/db mice. The regulatory role of PAR-3

Hassan Kassassir, Karolina Siewiera, Marcin Talar, Tomasz Przygodzki, Cezary Watala PII: DOI: Reference:

S1079-9796(17)30003-7 doi: 10.1016/j.bcmd.2017.03.011 YBCMD 2174

To appear in:

Blood Cells, Molecules and Diseases

Received date: Accepted date:

3 January 2017 21 March 2017

Please cite this article as: Hassan Kassassir, Karolina Siewiera, Marcin Talar, Tomasz Przygodzki, Cezary Watala , Flow cytometry analysis reveals different activation profiles of thrombin- or TRAP-stimulated platelets in db/db mice. The regulatory role of PAR-3. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ybcmd(2017), doi: 10.1016/j.bcmd.2017.03.011

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Flow cytometry analysis reveals different activation profiles of thrombin- or TRAPstimulated platelets in db/db mice. The regulatory role of PAR-3. Hassan Kassassir1, Karolina Siewiera1, Marcin Talar1, Tomasz Przygodzki1, Cezary Watala1

of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences,

PT

1Department

RI

Medical University of Lodz, 6/8 Mazowiecka str., 92-250 Lodz, Poland

SC

Corresponding author: Hassan Kassassir, mailing address: Department of Haemostasis and Haemostatic Disorders, Chair of Biomedical Sciences, Medical University of Lodz,

AC

CE

PT E

D

MA

address: [email protected]

NU

Mazowiecka 6/8, 92-215 Lodz, Poland tel.: +48 42 2725720; fax: +48 42 2725730, e-mail

ACCEPTED MANUSCRIPT Abstract Introduction: Recent studies have shown that it may be the concentration of thrombin, which is discriminative in determining of the mechanism of platelet activation via protease activated receptors (PARs). Whether the observed phenomenon of differentiated responses of mouse

PT

platelets to various thrombin concentrations in non-diabetic db/+ and diabetic db/db mice depends upon the concerted action of various PARs, remains to be established.

RI

Results: We found elevated reactivity of platelets, as well as the enhanced PAR-3 expression

SC

in response to both the used concentrations of AYPGKF in db/db mice, as compared to db/+ heterozygotes. At low concentration of thrombin platelets from diabetic mice demonstrated

NU

hyperreactivity, reflected by higher expression of PAR-3. For higher thrombin concentration,

MA

blood platelets from db/db appeared hyporeactive, compared to db/+ mice, while no significant differences in PAR-3 expression were observed between diabetic and non-diabetic mice.

PT E

D

Conclusions: The novel and previously unreported finding resulting from our study is that the increased expression of PAR-3 in response to either TRAP for PAR-4 or low thrombin (when PAR-4 is not the efficient thrombin receptor) may be one of the key events contributing to

AC

CE

higher reactivity of platelets in db/db mice.

Key words: platelets; PARs; db/db mice; flow cytometry; diabetes;

ACCEPTED MANUSCRIPT Introduction Protease activated receptors (PARs) are transmembrane proteins with a structure characteristic for G protein-coupled receptors [1, 2]. The general mechanism of the activation for all PARs is similar and includes the binding a specific protease and cutting off the Nterminal fragment of the receptor at the first stage [3, 4]. In murine platelets signal

PT

transduction in response to thrombin involves activation of PAR-3 and PAR-4 [5]. However,

RI

the first acts only as a co-receptor and is responsible for effective binding of thrombin derived

SC

from the circulation and inflammatory cells. Upon cleavage by thrombin, PAR-3 rather than itself mediating transmembrane signaling, supports cleavage and activation of PAR-4. Recent

NU

reports show that in fact the concentration of thrombin is critical for platelet activation mechanism with the participation of these both receptors [6]. Above assumption was

MA

confirmed by the studies of Kahn and colleagues who demonstrated in PAR-4 -/- knockout mice the complete lack of response to stimulation with thrombin, while in PAR-3 -/- knockout

D

mice platelet response was significantly reduced at a low concentration of thrombin and

PT E

delayed at high concentrations of this agonist [7]. Our previous studies have shown that blood platelets derived from db/db mice

CE

appeared hyperreactive at low and hyporeactive at high concentrations of thrombin [8]. In the present study we aimed at verifying whether the observed variable responses of platelets to

AC

subliminal and maximum doses of thrombin in db/db and db/+ mice may be – to some extent, associated with the increased surface membrane expression of PAR-3, and - as a consequence, the increased PAR-4 activation in diabetic mice. We use flow cytometry to monitor platelet response to differentiating doses of thrombin or thrombin receptor activating peptides (TRAP) for PAR-3 (SFNGGP) and PAR-4 (AYPGKF). We also measured the extents of the surface expression of PAR-3 in resting and in vitro stimulated platelets derived from db/+ and db/db mice.

ACCEPTED MANUSCRIPT Materials and methods Drugs and chemicals Anaesthetics: sedazin (20 mg/ml xylazine) and ketamine (100 mg/ml ketamine hydrochloride) were obtained from Biowet (Biowet, Pulawy, Poland). Low molecular weight heparin

PT

(LMWH) was from Sanofi Aventis (Paris, France). FITC- or PE-conjugated rat antiCD41/61, PE-conjugated rat anti-CD62P, rat anti-CD42b/PE, PE-conjugated JON/A

RI

antibodies (rat anti-the active complex αIIbβ3), FITC-conjugated rat anti-von Willebrand factor

SC

and rat anti-fibrinogen antibodies were purchased from Emfret Analytics (Eibelstadt, Germany) and PE-conjugated rat anti-PAR-3 monoclonal antibodies were purchased from

NU

Santa Cruz Biotechnology (USA). PAR-3 and PAR-4 activating peptides (SFNGGP and

MA

AYPGKF, respectively) were obtained from American Peptide Company (USA). Thrombin from human plasma was purchased from Chronolog Co. (Havertown, PA, USA). Mouse exogenous FITC-labeled fibrinogen (a full length protein) was obtained from Abcam

PT E

D

(Cambridge, UK). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Water used for solutions preparation and glassware washing

USA)

AC

Animals

CE

was passed through an Easy Pure UF water purification unit (Thermolyne Barnstead, IA,

Male C57BL/6J strain BKS.Dock7(m)+/+Lepr(db)/J (n = 12) genetically diabetic mice (hereafter referred to as db/db and used as the animal model of type 2 diabetes) and their nondiabetic lean littermates (hereafter referred to as db/+) (n = 12), 20 weeks old at the time of blood collection/withdrawal, were provided by Charles Rivers Laboratory (Kisslegg, Germany). The db/db mice were obese (52.3 ± 3.7 g in db/db vs. 25.8 ± 2.6 g in db/+, P<0.0001) and characterized by significantly elevated markers of short- (blood glycaemia: 254 ± 55 mg/dL in db/db vs. 127 ± 46 mg/dL in db/+, P<0.001) and long-lasting

ACCEPTED MANUSCRIPT hyperglycemia (glycated haemoglobin: 8.2 ± 1.8% in db/db vs. 4.6 ± 0.7% in db/+, P<0.0001).

Animal anaesthesia, blood collection and preparation During experiments animals were housed in an isolated room with a 12 h light-dark cycle and

PT

were given free access to water and standard chow for rodents (Altromin Maintenance Diet). Mice were anaesthetized with an intramuscular injection of ketamine (100 mg/kg b.w.) and

RI

xylazine (23.32 mg/kg b.w.). Blood was collected from the inferior aorta on 10 U/ml LMWH

SC

in TBS buffer (20 mmol/l Tris-HCl, 137 mmol/l NaCl, pH 7.3), diluted (1:25) with modified Tyrode buffer (134 mmol/l NaCl, 0.34 mmol/l Na2HPO4, 2.9 mmol/l KCl, 12 mmol/l

NU

NaHCO3, 20 mmol/l HEPES, pH 7.0, 5 mmol/l glucose, 0.35% w/v bovine serum albumin)

MA

and centrifuged at 900 x g for 5 min in 37°C to obtain blood cells devoid of plasma. The pellet of blood cells was resuspended in 1.25 ml of modified Tyrode buffer and used in the

D

study as ‘washed blood’. Before the assay was started, CaCl2 was added to the washed blood

PT E

to a final concentration of 1 mmol/l. All experiments were performed in accordance with the guidelines formulated by the European Community for the Use of Experimental Animals (L358-86/609/EEC) and the Guide for the Care and Use of Laboratory Animals published by

CE

the US National Institute of Health (NIH Publication No. 85–23, revised 1985). All

AC

procedures used in these experiments were approved by local Ethical Committees on Animal Experiments (approval number 32/LB471/2009).

Flow cytometric analysis of platelet surface activation markers Activation of circulating platelets, as well as platelet reactivity in response to PARs activating peptides (SFNGGP for PAR-3 and AYPGKF for PAR-4, both at the final concentrations of 20 or 100 µM) or thrombin (0.025 or 0.25 U/ml) were evaluated on the basis of the measured expressions of specific surface membrane antigens: CD62P (P-selectin), activated GPIIb/IIIa

ACCEPTED MANUSCRIPT complex and CD42b (GPIbα), binding of endogenous von Willebrand factor and binding of endogenous fibrinogen to platelets surface. Apart from the use of monoclonal antibodies against the bound vWf or the bound Fg, we assayed the binding of the commercially available, exogenous mouse FITC-labeled fibrinogen to circulated and TRAP-stimulated platelets originating from db/db and db+ mice. Moreover, the expression of PAR-3 in resting

PT

and in vitro stimulated platelets was monitored. In order to study the possible interactions

RI

between PAR-3 and PAR-4, platelets were first incubated with SFNGGP (at the final

SC

concentration of 20 µM) and then with AYPGKF (20 µM). Platelets were gated on the basis of the binding of anti-CD41/61 antibodies. Flow cytometric measurements of platelet surface

NU

membrane antigens were performed using the FACS Canto II instrument (BD Biosciences). At least 10,000 cells were analyzed per sample. All data were processed using FACS/Diva

MA

ver. 6.0 software (Becton-Dickinson). The percent fractions of specific fluorescence-positive platelets were evaluated after subtracting of non-specific isotype mouse IgG1 binding (the

D

FL2 gate for isotype control set to 2%). Results were presented as the percent fraction and

Statistical analysis

PT E

MFI values for CD62P-, activated αIIbβ3-, vWf-, Fg- and PAR-3-positive platelets.

CE

The normal distribution of data was verified by the Shapiro-Wilks test. Homogeneity of

AC

variance was verified with Levene’s test. Normally distributed, homoscedastic data were analyzed with an unpaired Student’s t-test. These data are presented as arithmetic mean ± SD. Some data that were not normally distributed (and a simple transformation did not yield normal data distributions) were assessed using the nonparametric Mann-Whitney U test. Such data are presented as medians and interquartile ranges (lower quartile – LQ: 25% quartile, upper quartile – UQ: 75% quartile). For all the statistical tests used, we read the post-hoc P values; those lower than 0.05 were deemed statistically significant.

ACCEPTED MANUSCRIPT Results

PARs activation by TRAP or thrombin in db/+ and db/db mice Our results showed higher activation of circulating platelets in db/db mice compared to db/+ mice, as evidenced by the increased expression of CD62P, active form of GPIIb/IIIa, bound

PT

endogenous vWf and bound endogenous Fg to platelets (Fig. 1). When platelet reactivity was

RI

tested under in vitro conditions with PARs activating peptides or thrombin, we revealed differentiated patterns of altered platelet glycoprotein expressions/binding in response to these

SC

agonists used at different doses. Upon the stimulation of platelets with AYPGKF (PAR-4

NU

agonist), we obtained significantly higher platelet response in db/db as compared to db/+ mice, for all the tested markers of platelet activation (Fig. 1). This discrimination between

MA

diabetic and non-diabetic mice was apparent regardless of the concentration of PAR-4 activating peptide. Also, the binding of mouse exogenous FITC-labeled fibrinogen to resting

D

and AYPGKF-activated platelets was increased in db/db mice (Fig. 2). In contrast, the

PT E

differentiated, dose-dependent response in db/db and db/+ mice was observed for thrombinstimulated platelets (Fig. 3). At the highest concentration (0.25 U/ml), platelets from db/db

CE

mice expressed less CD62P, while for other platelet activation markers there were no statistical differences. For the lowest thrombin concentration (0.025 U/ml) we observed

AC

hyperreactivity of platelets from diabetic mice in comparison to their non-diabetic littermates, as evidenced by the increased expressions of CD62P, the active form of GPIIb/IIIa, the bound vWf and the bound Fg. Furthermore, there was no response in platelets stimulated with PAR3 activating peptide. When we first incubated platelets with 20 µM SFNGGP (the PAR-3 activating peptide) and then with 20 µM AYPGKF (the PAR-4 activating peptide), we observed no significant differences in platelet reactivity between db/+ and db/db mice.

ACCEPTED MANUSCRIPT Expression of PAR-3 in resting and in vitro stimulated platelets There were no differences between db/db and db/+ mice in the expression of PAR-3 in resting platelets (Fig. 4) Interestingly, PAR-3 expression raised after platelet activation with the increasing concentrations of PAR-4 activating peptide (AYPGKF) and the lower concentration of thrombin in both the tested groups of animals (Fig. 4). When platelets were

PT

stimulated with AYPGKF (20 or 100 µM), we found greater expression of PAR-3 in platelets

RI

from db/db compared to db/+ mice. However, for thrombin, the expression of PAR-3

SC

depended considerably upon the agonist concentration. At lower concentration (0.025 U/ml) we demonstrated higher expression of PAR-3 in diabetic compared to non-diabetic mice,

NU

while no significant differences were revealed at high thrombin dose (0.25 U/ml). Furthermore, there were no changes in PAR-3 expression in platelets stimulated with PAR-3

MA

activating peptide used at different doses.

D

Discussion

PT E

We demonstrated that higher platelet reactivity in diabetic mice, compared to nondiabetic animals, after their stimulation with low concentration of thrombin is likely to be

CE

associated with the increased expression of PAR-3. However, the mechanism(s) underlying this higher PAR-3 expression in response to low thrombin are not clear and should be

AC

thoroughly investigated in further studies. In turn, partially reduced platelet reactivity in response to high thrombin concentration in diabetic mice, without differences of PAR-3 expression between db/db and db/+ mice, may result from the fact, already confirmed and reported in a literature [9], that at high concentrations of thrombin PAR-4 is activated without the participation of PAR-3. This result may suggest that in diabetes PAR-3 acts as a compensation for the altered platelet response to low doses of thrombin, a natural, physiological PAR agonist. Interesting to these observations may be outcomes pointing to higher reactivity of diabetic platelets in response to low and high concentrations of PAR-4

ACCEPTED MANUSCRIPT activating peptide, AYPGKF. This synthetic peptide correspond to the tethered ligand for PAR-4 and specifically activates this receptor in the absence of a proteinase. The PAR-3 expression raised after platelet activation with the increasing concentrations of PAR-4 activating peptide and this occurred in both tested groups of animals. This indicates that regardless of the concentration of a direct agonist for PAR-4, AYPGKF, the activation of this

PT

receptor may be assisted by PAR-3, probably via intra- or extracellular interactions. The fact

RI

that we observed a higher expression of PAR-3 in diabetic mice suggests that such

SC

interactions between PAR-3 and PAR-4 may be intensified in diabetic states. Such an idea seems even more justified when concerning our results showing no significant differences in

NU

the reactivity of platelets between diabetic and non-diabetic mice following blood platelet stimulation with both PAR-3 and PAR-4 agonists. These results are also supportive for the

MA

following reasoning: although the expression of PAR-3 in stimulated platelets is on average higher in diabetic mice, the binding of tethered ligand for PAR-3 does not deepen such a

D

discrimination. In fact, such a binding does not stimulate platelet response, but it can rather

PT E

partially block the activation of PAR-4. Interestingly, some authors have proved that PAR-3 may affect PAR-4 signaling apart from a direct enhancing of PAR-4 activation. Nakanishi-

CE

Matsui et al. showed that the amount of the accumulated inositol phosphate (IP) in response to thrombin (10–100 nM) was 1.7-fold higher in COS7 cells, expressing mouse PAR-4 alone,

AC

compared to COS7 cells expressing both mouse PAR-3 and PAR-4 [6]. In another study by Mao and colleagues the authors revealed the increased intracellular calcium mobilization and platelet aggregation in response to plasmin in PAR-3 knockout mice, compared to wild-type animals [10]. Quite new information has been provided by the outcomes of the research by Arachiche et al., who demonstrated that PAR-3 negatively regulates PAR-4-mediated signaling pathway, involving Gq, by the reduction of the Ca2+ mobilization and the activation of PKC [11]. The effect of PAR-3 on PAR-4 was independent of the activation of PAR-4. The

ACCEPTED MANUSCRIPT authors demonstrated for the first time that PAR-3 forms a heterodimer with PAR-4, whereby it comes to such changes in signaling pathways mediated by PAR-4. The results of this study indicate that PAR-4 signaling may be modulated by other PAR subtypes and, viewed globally, the interaction between receptors coupled with G proteins creates a unique signal paths, which may be the target of antiplatelet agents. What seems to be interesting, the above-

PT

observed phenomenon of the PAR-3 impact on PAR-4 was observed not only in the case of

RI

the stimulation of platelets with thrombin, but also for PAR-4 agonist, the peptide AYPGKF.

SC

Thus, even in the case of a direct activation of PAR-4 by AYPGKF in subsequent signaling pathway is involved PAR-3.

NU

Diabetes is strongly associated with vascular complications and increased thrombin generation, leading to atherothrombosis [12, 13]. The relative contribution of PAR-type

MA

thrombin receptors has been defined in diabetes or diabetic conditions. Dangwal and colleagues showed that exposure of human vascular smooth muscle cells (SMCs) to high

D

glucose selectively induces PAR-4 expression, with no influence on other thrombin receptors,

PT E

like PAR-1 or PAR-3 [14]. In turn, another report indicated increased PAR-4 expression in aorta and carotid arteries of streptozotocin (STZ) diabetic mice [15]. These findings, together

CE

with a high PAR-4 abundance observed in diabetic versus nondiabetic human atherectomy and a saphenous vein specimen, highlight PAR-4 as an important glucose-regulated thrombin

AC

receptor in both mice and humans with a hitherto unsuspected role in the vascular complications of diabetes mellitus. It should be pointed out, that due to lack of a nucleus in platelets and their inability to synthesize the protein de novo, it is no possible for high glucose to impair the production of PARs in platelets. Indeed, we did not observe the differences in expression of PAR-3 in circulating platelets between db/+ and db/db mice. It is known that under resting conditions, all PARs expressed at the cell surface are available for stimulus, but on activation, these receptors would no longer be available for repeated application of the

ACCEPTED MANUSCRIPT same stimulus because of rapid uncoupling of the receptor from signaling or other desensitization mechanisms [16]. The fact that we could detect higher expression of PAR-3 on surface of diabetic platelets in response to PAR-4 agonist or low concentration of thrombin may indicate increased efficiency of shuttling PAR-3 copies to the cell surface or impairments in mechanisms of PAR-3 uncoupling or desensitization in db/db mice, in comparison to their

PT

control littermates. It also points to a unique role of PAR-3 in diabetic settings, which is likely

RI

to contribute to the exaggerated cardiovascular complications of diabetes.

SC

We monitored platelet functioning in db/+ and db/db mice since these laboratory animal models have been widely used in a variety of studies designed to evaluate diabetic

NU

complications, including the role of PAR receptors. Sakai et al. found up-regulated expression of PAR-1 receptor in kidneys in db/db mice, in comparison to db/+ mice, whereas another

MA

signal transduction receptor of thrombin, PAR-4, was not changed [17]. This study indicates the important role of PARs and their dysfunctional activity/expression in murine diabetic

D

models, however this aspect remains poor investigated regarding to specific thrombin

PT E

receptors in platelets. Enhanced PAR-4 sensitivity was reported in platelets from type 1 diabetic mice associated with increased susceptibility to arterial thrombosis [18]. Authors

CE

proved that platelets from diabetic mice were more sensitive to protease-activated receptor 4 (PAR-4) agonist-induced fibrinogen binding than platelets from non-diabetic mice These

AC

observations stay with agreement with our results showing increased platelet response to low thrombin or PAR-4 agonist in db/db, in comparison to db/+ mice. There is not so many examples in a literature of the described role of PARs in the phenomena of platelet hypo- and hyperreactivity in diabetes. While the functioning of the murine platelet thrombin receptors, PAR-3 and PAR-4, has been clarified, all the aspects of their interactions, especially in the case of db/db and db/+ mice, is not known. Our results suggest that the increased expression of PAR-3 in response to either TRAP for PAR-4 or low

ACCEPTED MANUSCRIPT thrombin (i.e. under conditions when PAR-4 is not the efficient thrombin receptor) may be one of the key events contributing to higher reactivity of platelets in db/db mice. This indicates that, regardless of the concentration of the used direct agonist for PAR-4 and at low concentrations of thrombin the activation of PAR-4 may be partly supported by PAR-3 in diabetic mice, as compared to their non-diabetic littermates. Under conditions when the PAR-

PT

3 receptor takes part in blood platelet activation to a lesser extent (i.e. at high thrombin

RI

concentrations), this activation is suppressed in db/db compared to db/+ mice. However, we

SC

are aware that our study is only a short preliminary report and the detailed mechanism(s) of the observed phenomena should certainlybe explained in further, more sophisticated

NU

experiments. We hope that the results will apply to other mouse models of various cardiovascular diseases, where the events of platelet hypo- and hyperreactivity are observed.

MA

These findings indicate that the increased expression of PAR-3 may be a valuable new biomarker for metabolic dysfunction and, further, that PAR-3 antagonism can be an effective

Acknowledgements

PT E

D

intervention for treating metabolic dysfunction and obesity.

CE

This work was supported by the grants from the Ministry of Science and Higher Education (N N401 265839), the National Science Centre (UMO-2012/06/A/NZ5/00069) and by the

AC

European Union funds from the resources of the European Regional Development Fund under the Innovative Economy Programme (POIG.01.01.02-00-069/09).

ACCEPTED MANUSCRIPT References 1. I. Kyriazis, J. Ellul, P. Katsakiori, G. Panayiotakopoulos, C. Flordellis, The multiple layers of signaling selectivity at protease-activated receptors, Curr Pharm Des. 18 (2012) 161-174.

PT

2. E. De Candia, Mechanisms of platelet activation by thrombin: a short history,

RI

Thromb Res. 129 (2012) 250-256.

SC

3. G. Cirino, N. Vergnolle, Proteinase-activated receptors (PARs): crossroads between innate immunity and coagulation, Curr Opin Pharmacol 6 (2006) 428–

NU

434.

MA

4. H. Lin, A.P. Liu, T.H. Smith, J. Trejo, Cofactoring and dimerization of proteinaseactivated receptors, Pharmacol Rev. 65 (2013) 1198-1213. Major, R.J.

Santulli, C.K.

Derian, P.

Andrade-Gordon,

Extracellular

D

5. C.D.

PT E

mediators in atherosclerosis and thrombosis: lessons from thrombin receptor knockout mice, Arterioscler Thromb Vasc Biol. 23 (2003) 931-939.

CE

6. M. Nakanishi-Matsui, Y.W. Zheng, D.J. Sulciner, E.J. Weiss, M.J. Ludeman et al,, PAR-3 is a cofactor for PAR-4 activation by thrombin, Nature. 404 (2000), 609–

AC

613.

7. M.L. Kahn, Y.W. Zheng, W. Huang, V. Bigornia, D. Zeng et al., A dual thrombin receptor system for platelet activation, Nature. 394 (1998) 690-694. 8. M. Rozalski, H. Kassassir, K. Siewiera, A. Klepacka, R. Sychowski et al., Platelet activation patterns are different in mouse models of diabetes and chronic inhibition of nitric oxide synthesis, Thromb Res. 133 (2014) 1097-1104.

ACCEPTED MANUSCRIPT 9. G.R. Sambrano, E.J. Weiss, Y.W. Zheng, W. Huang, S.R. Coughlin, Role of thrombin signalling in platelets in haemostasis and thrombosis, Nature. 413 (2001) 74-78. 10. Y. Mao, J. Jin, J.L. Daniel, S.P. Kunapuli, Regulation of plasmin-induced

PT

protease-activated receptor 4 activation in platelets, Platelets. 20 (2009) 191–198. 11. A. Arachiche, M. de la Fuente, M.T. Nieman, Calcium mobilization and protein

RI

kinase C activation downstream of protease activated receptor 4 (PAR-4) is

SC

negatively regulated by PAR-3 in mouse platelets, PLoS ONE. 8 (2013) e55740.

NU

12. J.A. Beckman, M.A. Creager, P. Libby, Diabetes and atherosclerosis:

MA

epidemiology, pathophysiology, and management, JAMA. 287 (2002) 2570–2581. 13. A. Undas, I. Wiek, E. Stepien, K. Zmudka, W. Tracz, Hyperglycemia is associated with enhanced thrombin formation, platelet activation, and fibrin clot resistance to

PT E

D

lysis in patients with acute coronary syndrome, Diabetes Care. 31 (2008) 1590 – 1595.

Dangwal, B.H.

Rauch, T.

Gensch, L.

Dai, E.

Bretschneider

CE

14. S.

High glucose enhances thrombin responses via protease

et

al.,

activated receptor

AC

4 in human vascular smooth muscle cells, Arterioscler Thromb Vasc Biol. 31 (2011) 624-633. 15. G.

Pavic, M.

Grandoch, S.

Dangwal, K.

Jobi, B.H.

Rauch

et

al.,

Thrombin receptor protease-activated receptor 4 is a key regulator of exaggerated intimal thickening in diabetes Circulation. 130 (2014) 1700-1711.

mellitus,

ACCEPTED MANUSCRIPT 16. SR. Coughlin, Thrombin signalling and protease-activated receptors, Nature. 407 (2000) 258 –264. 17. T.

Sakai, T.

Nambu, M.

Katoh, S.

Uehara, T.

Fukuroda

et

al.,

Up-

regulation of protease-activated receptor-1 in diabetic glomerulosclerosis,

Stolla, D.

Li, L.

Enhanced platelet activity and thrombosis in

Lu, D.S.

RI

18. M.C.

PT

Biochem Biophys Res Commun. 384 (2009) 173-179.

SC

a murine model of type I diabetes are partially insulin-like growth

NU

dependent and phosphoinositide 3-kinase-dependent,

AC

CE

PT E

D

MA

(2013) 919-29.

J

Thromb

Woulfe,

factor 1 Haemost. 11

ACCEPTED MANUSCRIPT Figure captions Fig. 1. Expressions of selected platelet surface membrane activation markers in resting and AYPGKF (PAR-4 agonist)-activated blood platelets in db/db and db/+ mice. Results are presented as median and interquartile range. The expressions of CD62P (A, B), the active form of αIIbβ3 (C, D) and binding of endogenous von Willebrand factor (vWf) (E, F) and endogenous fibrinogen (Fg) (G, H) in resting platelets and upon their in vitro stimulation

PT

with AYPGKF (at final concentrations of either 20 or 100 µM) were measured in non-fixed ‘washed blood’ in db/db mice (n = 12) (grey boxes) and respective control animals (db/+

RI

heterozygotes, n = 12 – open boxes) using flow cytometry. Results are expressed as the

SC

percent fraction and MFI value of CD62P- (A, B; respectively), activated αIIbβ3- (C, D; respectively), vWf- (E, F; respectively) and Fg- (G, H; respectively) positive platelets. For experimental details – see Materials and methods. Statistical significance of differences,

NU

estimated with two-tailed unpaired Student t test or lower-tail Mann-Whitney U test, was:

MA

CD62Prest, P1,α < 0,05, db/db > db/+; activated αIIbβ3rest, P1,α < 0,05, db/db > db/+; vWfrest, P1,α < 0,01, db/db > db/+; Fgrest, P1,α < 0,01, db/db > db/+; CD62PAYPGKF20, P1,α < 0.0001, db/db > db/+; CD62PAYPGKF100, P1,α < 0.0001, db/db > db/+; αIIbβ3AYPGKF20,

PT E

D

P1,α < 0.0001, db/db > db/+; αIIbβ3AYPGKF100, P1,α < 0.0001, db/db > db/+; vWfAYPGKF20, P1,α < 0.0001, db/db > db/+; vWfAYPGKF100, P1,α < 0.0001, db/db > db/+; FgAYPGKF20,

CE

P1,α < 0.0001, db/db > db/+; FgAYPGKF100, P1,α < 0.0001, db/db > db/+.

AC

Fig. 2. Binding of mouse exogenous FITC-labeled fibrinogen to resting or (PAR-4 agonist)-activated blood platelets in db/db and db/+ mice. Results are presented as median and interquartile range. The binding of mouse exogenous FITC-labeled fibrinogen to resting platelets (A) or upon their in vitro stimulation with AYPGKF (at final concentration of 100 µM) (B) were measured in non-fixed ‘washed blood’ in db/db mice (n = 12) (grey boxes) and respective control animals (db/+ heterozygotes, n = 12 – open boxes) using flow cytometry. Results are expressed as the percent fraction of FITClabeled fibrinogen-positive platelets. For experimental details – see Materials and methods. Statistical significance of differences, estimated with one-tailed unpaired Student t test or

ACCEPTED MANUSCRIPT lower-tail Mann-Whitney U test, was: Fg/FITCresting, P1,α < 0.05, db/db > db/+; Fg/FITC AYPGKF20,

P1,α < 0.0001, db/db > db/+.

Fig. 3. Expressions of selected platelet surface membrane activation markers in resting and thrombin-activated blood platelets in db/db and db/+ mice. Results are presented as median and interquartile range. The expressions of CD62P (A, B),

PT

the active form of αIIbβ3 (C, D) and binding of endogenous von Willebrand factor (E, F) and endogenous fibrinogen (G, H) in resting platelets and upon their in vitro stimulation with

RI

thrombin (at final concentrations of either 0.025 or 0.25 U/ml) were measured in non-fixed ‘washed blood’ in db/db mice (n = 12) (grey boxes) and respective control animals (db/+

SC

heterozygotes, n = 12 – open boxes) using flow cytometry. Results are expressed as the percent fraction and MFI value of CD62P- (A, B; respectively), activated αIIbβ3- (C, D;

NU

respectively), vWf- (E, F; respectively) and Fg- (G, H; respectively) positive platelets. For experimental details – see Materials and methods. Statistical significance of differences,

MA

estimated with one-tailed unpaired Student t test or lower-tail Mann-Whitney U test, was: CD62Prest, P1,α < 0,05, db/db > db/+; activated αIIbβ3rest, P1,α < 0,05, db/db > db/+; vWfrest, P1,α < 0,01, db/db > db/+; Fgrest, P1,α < 0,01, db/db > db/+; CD62Pthr0.025, P1,α < 0.0001, db/db >

D

db/+; CD62Pthr0.25, P1,α < 0.02, db/db < db/+; αIIbβ3thr0.025, P1,α < 0.0001, db/db > db/+;

PT E

vWfthr0.025, P1,α < 0.0001, db/db > db/+; Fgthr0.025, P1,α < 0.0001, db/db > db/+.

Fig. 4. Expressions of PAR-3 on platelet surface membrane in resting and AYPGKF

CE

(PAR-4 agonist)- or thrombin-activated blood platelets in db/db and db/+ mice. Results are presented as median and interquartile range. The expressions of PAR-3 in resting

AC

platelets and upon their in vitro stimulation with AYPGKF (at final concentrations of either 20 or 100 µM) (A, B) or thrombin (at final concentrations of either 0.025 or 0.25 U/ml) (C, D) were measured in non-fixed ‘washed blood’ in db/db mice (n = 12) (grey boxes) and respective control animals (db/+ heterozygotes, n = 12 – open boxes) using flow cytometry. Results are expressed as the percent fraction (A, C) and MFI value (B, D) of PAR-3-positive platelets. For experimental details – see Materials and methods. Statistical significance of differences, estimated with one-tailed unpaired Student t test or lower-tail Mann-Whitney U test was: PAR-3 PAR-3

AYPGKF20

, P1,α<0.0001, db/db > db/+; PAR-3 thr0.025,

P1,α<0.0001,

AYPGKF100,

db/db

, P1,α<0.0001, db/db > db/+; >

db/+.

AC

CE

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 1

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

PT E

D

MA

Figure 2

AC

CE

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 3

PT E

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

AC

CE

Figure 4