Antithrombotic effects of PAR1 and PAR4 antagonists evaluated under flow and static conditions Kazuya Hosokawa, Tomoko Ohnishi, Naoki Miura, Hisayo Sameshima, Takehiko Koide, Kenichi A. Tanaka, Ikuro Maruyama PII: DOI: Reference:

S0049-3848(13)00512-4 doi: 10.1016/j.thromres.2013.10.037 TR 5271

To appear in:

Thrombosis Research

Received date: Revised date: Accepted date:

19 August 2013 9 October 2013 28 October 2013

Please cite this article as: Hosokawa Kazuya, Ohnishi Tomoko, Miura Naoki, Sameshima Hisayo, Koide Takehiko, Tanaka Kenichi A., Maruyama Ikuro, Antithrombotic effects of PAR1 and PAR4 antagonists evaluated under flow and static conditions, Thrombosis Research (2013), doi: 10.1016/j.thromres.2013.10.037

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ACCEPTED MANUSCRIPT Category: Original Article [Platelets and Cell Biology]

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Antithrombotic effects of PAR1 and PAR4 antagonists evaluated under flow and static conditions

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Koide2, Kenichi A Tanaka4, and Ikuro Maruyama2

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Kazuya Hosokawa1,2, Tomoko Ohnishi1, Naoki Miura3, Hisayo Sameshima1, Takehiko

Research Institute, Fujimori Kogyo Co., Ltd., Yokohama, Kanagawa, Japan;

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Department of System Biology in Thromboregulation, Graduate School of Medical and

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1

Dental Sciences, Kagoshima University, Kagoshima, Japan, 3Joint Faculty of Veterinary University,

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Kagoshima

Kagoshima,

Japan,

and

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Department

of

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Medicine,

Anesthesiology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania,

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USA

Running Title: Antithrombotic effects of PAR1 and PAR4 antagonism Word Count: Abstract, 234; Text, 4030

Address Correspondence to: Kazuya Hosokawa, Research Institute, Fujimori Kogyo Co., Ltd., 1-10-1, Sachiura, Kanazawa-ku, Yokohama, Kanagawa, 236-0003, Japan Tel: +81-45-769-0124

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ACCEPTED MANUSCRIPT Fax: +81-45-769-0131

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Email: [email protected]

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ACCEPTED MANUSCRIPT Abstract Introduction: Thrombin-mediated activation of human platelets involves the G-protein-

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coupled protease-activated receptors PAR1 and PAR4. Inhibition of PAR1 and/or PAR4

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is thought to modulate platelet activation and subsequent procoagulant reactions.

elucidated, particularly under flow conditions.

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However, the antithrombotic effects of PAR1 and PAR4 antagonism have not been fully

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Materials and Methods: A microchip-based flow chamber system was used to evaluate the influence of SCH79797 (PAR1 antagonist) and YD-3 (PAR4 antagonist) on thrombus

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formation mediated by collagen and tissue thromboplastin at shear rates simulating those experienced in small- to medium-sized arteries (600 s-1) and large arteries and small veins

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(240 s-1).

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Results: At a shear rate of 600 s-1, SCH79797 (10 μM) efficiently reduced fibrin-rich

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platelet thrombi and significantly delayed occlusion of the flow chamber capillary (1.44 fold of control; P5 nM) is reported to mediate prolonged platelet aggregation [2]. The dual activation of PAR1 and PAR4 collectively promotes platelet morphological

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changes, granule secretion, and glycoprotein (GP) IIb/IIIa receptor activation, resulting in stable platelet thrombus formation [1, 2, 8].

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PAR1 antagonism has potent antithrombotic and antiplatelet effects in animal models of arterial thrombosis [9-12], as well as in acute coronary syndrome (ACS) patients in

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phase II clinical trials [13, 14]. A recent phase III trial in ACS patients also demonstrated that combined PAR1 blockade with aspirin and/or clopidogrel reduces the incidence of myocardial infarction; however, the combination therapy increased the risk of fatal bleeding, including intracranial hemorrhage [15]. Therefore, further optimization of treatment strategies based on the assessment of total antiplatelet response to combination therapy may be necessary to achieve clinical benefits from PAR1 antagonism.

Platelet aggregometry is commonly used to assess platelet activation in response to a single exogenous agonist, such as collagen, thrombin, or adenosine 5’-diphosphate (ADP). However, in-vivo thrombus formation is a dynamic and complex process that

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ACCEPTED MANUSCRIPT involves multiple platelet activation and coagulation pathways under flow conditions [16-18]. In addition, the species specificity of platelet PARs often limits the direct

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application of animal models to investigate the roles of PAR1 and PAR4 in

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physiological haemostasis, and the influence of PAR antagonism on pathologic thrombus formations. To overcome these limitations, a microchip-based flow chamber

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system capable of evaluating fibrin-rich platelet thrombus formation under adjustable

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flow conditions is a potential alternative for modeling the pathogenesis of arterial thrombosis after plaque rupture [19].

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In the present study, we hypothesized that the antithrombotic mechanisms and efficacies of PAR antagonists can be accurately elucidated by analyzing fibrin-rich platelet

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thrombus formation patterns under flow conditions. Flow chamber experiments were

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conducted under shear conditions mimicking mid- to small-sized arteries (600 s-1) and

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large arteries and small veins (240 s-1) using human blood samples pre-treated with and

PAR4

antagonists.

Coagulation

assays,

including

whole

blood

viscoelastometry and thrombin generation (TG) assays, were also performed to comparatively evaluate PAR1- and PAR4-mediated platelet procoagulant activity under static conditions.

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ACCEPTED MANUSCRIPT Materials and Methods Materials

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SCH79797 [20] and AR-C66096 [21], specific antagonists of PAR1 and P2Y12 receptor,

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respectively, were purchased from Tocris Bioscience (Bristol, United Kingdom). YD-3, a specific antagonist of PAR4, was synthesized as previously reported [22].

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Acetylsalicylic acid was purchased from Wako Pure Chemicals (Osaka, Japan). PAR1-

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activating peptide (AP), SFLLRN, and PAR4-AP, AYPGKF, were purchased from Verum Diagnostica (Munich, Germany). The microchips used in the flow-chamber

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system were manufactured by Richell Corp. (Toyama, Japan). Porcine type I collagen was purchased from Nitta Gelatin, Inc. (Osaka, Japan). Tissue thromboplastin was

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purchased from Sysmex (Hyogo, Japan). Corn derived trypsin inhibitor (CTI), a specific inhibitor of factor XII, was prepared as reported previously [23]. For the TG assay, the

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tissue factor (TF)-based reagent for PRP, and FluCa-reagent, a fluorogenic substrate (ZGly-Gly-Arg-AMC) dissolved in HEPES buffer and calcium chloride, were purchased from Diagnostica Stago (Parsippany, NJ, USA). r-TF was purchased from Mitsubishi Chemical Medience (Tokyo, Japan). All other reagents were obtained from Wako Pure Chemicals (Osaka, Japan).

Blood samples Whole blood samples were collected in plastic tubes containing either 3.2% sodium citrate (Terumo, Tokyo, Japan) or 25 μg/ml hirudin (Verum Diagnostica, Munich, Germany). The subjects had not taken any medication that might have affected platelet function or coagulation in the two weeks preceding the blood draw, and had no history

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ACCEPTED MANUSCRIPT of coagulation disorders. The study protocol was approved by the institutional ethics committee of Kinki University (Osaka, Japan), and informed written consent was

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obtained from all subjects.

Effects of SCH79797 and YD-3 on platelet aggregation

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Antiplatelet effects of SCH79797 and YD-3 on platelet aggregation induced by

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SFLLRN and AYPGKF, respectively, were confirmed by whole blood platelet aggregometry using the MultiplateTM analyzer (Verum Diagnostica, Munich, Germany)

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as directed by the manufacturer. Briefly, 300 μl saline and 300 μl hirudinized blood were pipetted into a single-use cuvette, which was then incubated at 37 °C for 3 min.

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Either SFLLRN (final, 32 μM) or AYPGKF (final, 662 μM) was then added to the sample to initiate platelet aggregation. Platelet adhesion and aggregation were monitored

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for 6 min. The impedance change caused by platelet adhesion and aggregation was plotted against time and the area under the aggregation curve (AUC) to measure the aggregation response, which was quantified in arbitrary aggregation units (AU).

Effects of SCH79797 and YD-3 on thrombus formation measured under flow conditions (i) Microchip-based flow chamber analysis For the analysis of thrombus formation under various shear rates, we used the Total Thrombus-formation Analysis System (T-TAS; Fujimori Kogyo Co., Ltd., Tokyo, Japan), which is a microchip-based flow chamber system equipped with a pneumatic pump, a flow pressure sensor, and a videomicroscope, as described previously [19] (Fig. S1). Blood samples were used for assay within 1 – 3 hours after collection. Briefly,

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ACCEPTED MANUSCRIPT citrated whole blood (480 l) was mixed with 20l of 0.3 M CaCl2 containing 1.25 mg/ml CTI immediately before the sample was perfused over a microchip coated with

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collagen and tissue thromboplastin at flow rates of 4 and 10 μl/min, corresponding to

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initial wall shear rates of 240 and 600 s-1, respectively, as estimated by the FLUENT

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program (Ansys Co., Ltd., Tokyo, Japan). Flow pressure changes were monitored by the pressure transducer located upstream of the microcapillary during the perfusion

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experiments. Thrombus formation and breakdown within the microcapillary result in pressure increases and decreases, respectively. Based on the flow pressure pattern, the

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following parameters are used to analyze thrombus formation process. T10 (time to 10 kPa) is the lag time for the flow pressure to increase by 10 kPa from the baseline due to

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partial occlusion of the capillary. The T10 value represents initial thrombi formation. OT (occlusion time) is the lag time for the flow pressure to increase by 80 kPa from baseline

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owing to near complete occlusion of the capillary by thrombi. The OT value reflects the onset, growth, and stability of thrombi inside the capillary. Blood perfusion and flow pressure monitoring are programmed to stop when the flow pressure increases to 80 kPa. In addition to the flow pressure analyses, thrombus formation in the capillary was visually inspected using the built-in light microscope. The two-dimensional area covered by thrombi was analyzed using image analysis software (Zia; Fujimori Kogyo Co., Ltd., Tokyo, Japan), as previously described [19] (Fig. S2). The progression of thrombus formation within the microchip was recorded at 8 and 12 min after the start of blood perfusion.

(ii) Analysis of thrombi by confocal laser scanning microscopy

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ACCEPTED MANUSCRIPT Thrombi formed on the coated microchip surface were washed three times with phosphate-buffered saline (PBS) and then incubated with FITC-conjugated mouse anti-

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human CD41 (platelet GPIIb) IgG (1:5 dilution) for 15 min in the dark. After three

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washes with Tris-buffered saline containing 0.1% Triton X-100 (TBST), thrombi were immobilized with OptiLyse C (Immunotech, Marseille, France) for 15 min. After three

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washes with TBST, the sample was blocked for 1 h with Block Ace (Yukijirushi, Osaka,

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Japan) and then incubated for 30 min in the dark with rabbit anti-human fibrinogen IgG (1:99 dilution) labeled with Alexa 594. To test the immunospecificity of the primary

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antibodies, control experiments were conducted with isotype-matched IgG for primary antibodies against GPIIb and fibrinogen. Following antibody staining, the sample was

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Oberkochen, Germany).

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visualized using a LSM700 confocal laser microscope (Carl Zeiss Microscopy Co., Ltd.,

Effects of SCH79797 and YD-3 on thrombus formation measured under static conditions (i) Thromboelastometry measurements The ROTEMTM system (TEM International, Munich, Germany) was used to analyze the clot formation process in whole blood under static conditions. All ROTEMTM assays were performed in the presence of 50 μg/ml CTI to prevent contact phase activation in the analyzer cup, which was held at 37 °C. Briefly, citrated whole blood (300 μl) containing an appropriate concentration of antiplatelet agent(s) was mixed with 20 μl recombinant tissue factor (r-TF) (final concentration, 1 pM) and 25 μl CTI (final concentration, 50 μg/ml) dissolved in 165 mM CaCl2 (final concentration, 12 mM) in the analyzer cup. The following ROTEMTM measurements were performed over a 60-min

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ACCEPTED MANUSCRIPT period: clotting time (CT; sec), which corresponds to the lag time before clotting; clot formation time (CFT; sec), which reflects the initial rate of clot formation; and

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maximum clot firmness (MCF; mm), which is a measure of the maximal tensile strength

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(ii) Endogenous thrombin generation measurements

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of the clot.

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A calibrated automated thrombin generation test using a ThrombinoscopeTM (Thrombinoscope BV, Maastricht, Netherlands) was performed to evaluate the effects of

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SCH79797 and YD-3 on the rate and extent of thrombin generation according to the manufacturer’s protocol. All experiments were performed in the presence of CTI (50

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μg/ml) to prevent thrombin formation mediated by contact phase activation. Platelet-rich plasma (PRP) was obtained by centrifuging citrated whole blood at 125 x g for 10 min at

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room temperature, and platelet-poor plasma (PPP) was obtained by further centrifuging the PRP at 1,700 x g for 10 min. PRP was adjusted to a platelet count of 150,000 /μl by dilution with PPP. Thrombin generation was measured in PRP (80 µl) mixed with 20 μl PRP reagent containing r-TF (final concentration, 1 pM). SCH79797 (10 μM), YD-3 (20 μM), aspirin (100 μM), and AR-C66096 (1 μM), or their combinations were incubated with the PRP mixture at 37 °C for 5 min when appropriate. Thrombin generation was triggered by the automated addition of 20 μl FluCa-reagent (final, 416 μM fluorogenic substrate and 16 mM CaCl2). The reaction was monitored at 20-sec intervals for 60 min, and the acquired data were automatically processed using ThrombinoscopeTM software (Version 3.0.0.29; Thrombinoscope BV, Maastricht, Netherlands). The following

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ACCEPTED MANUSCRIPT parameters were collected: lag time (LT; min), time to peak (TTP; min), peak height

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(PH; nM), and endogenous thrombin generation potential (ETP; nM/min).

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Statistical analysis

Data are shown as the mean ± standard deviation (SD), unless otherwise indicated.

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Differences between groups were tested by one-way repeated ANOVA, followed by

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Tukey’s post-hoc test using Prism version 5.02® software (GraphPad Software, CA,

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USA). A P value of 1500 s) [30, 31]. These results, together with our present findings, suggest that shear-induced

platelet activation supports the growth and stabilization of platelet thrombi, even in the absence of thrombin-mediated platelet activation. The antithrombotic efficacy of PAR1 antagonism observed in the present study was relatively moderate, even when SCH79797 was combined with aspirin and AR-C66096. Notably, the thrombi that formed in the presence of the three antiplatelet agents contained numerous fibrin fibers (Fig. 4). Based on these findings, the inhibitory effect of triple antiplatelet therapy on fibrin deposition is likely limited, although the combined platelet inhibition appears to efficiently reduce platelet activation and deposition onto thrombi.

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ACCEPTED MANUSCRIPT The thrombi involved in acute coronary lesions consist of activated platelets and large amounts of fibrin due to exposure to collagen and TF after plaque rupture [34, 35]. The

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TF associated with ruptured plaques is one of the key factors determining

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thrombogenicity [36, 37]. In this regard, the enhanced efficacy of PAR-1 antagonism in combination with aspirin and AR-C66096 on fibrin-rich thrombus formation shown in

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the present study is in part concordant with the results of a recent phase III trial in ACS

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patients receiving dual antiplatelet therapy [15], which demonstrated that additional PAR1 blockade reduces the incidence of myocardial infarction. However, the treated

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ACS patients had increased fatal bleeding complications, including intracranial hemorrhage [15], suggesting that this combination therapy has a relatively narrow

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therapeutic window with respect to the balance between antithrombotic effect and bleeding risk. Taken together, these findings indicate that PAR1 antagonism may offer an

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additional therapeutic option for limiting the growth of intravascular thrombi in high-risk ACS patients [3, 5, 7, 38-40]. However, further optimization of treatment strategies, including other possible drug combinations, dose adjustment, and careful patient selection, are needed to achieve clinical benefits from PAR1 antagonism [41]. In contrast to PAR1 antagonists, our present results suggest that PAR4 antagonism under either flow or static conditions will have minimal antithrombotic effects in humans, even in combination with aspirin and a P2Y12 antagonist. There is a paucity of data on the antithrombotic efficacy of PAR4 antagonism in human blood under flow conditions, although platelet activation and aggregation via activation of PAR4 and downstream intracellular signaling pathways have been well studied [42, 43]. PAR4 activation mediates intracellular signaling via the G-protein subunits Gq and G12/13, but not via the

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ACCEPTED MANUSCRIPT Gi signaling pathway, whereas PAR1 activation leads to the induction of all of these signaling pathways [43]. Thus, combined platelet inhibition by PAR1 and P2Y12

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antagonists efficiently suppresses the Gi signaling pathway. In contrast, treatment with a

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PAR4 antagonist in combination with either a PAR1 or P2Y12 antagonist would induce Gi signaling, which may limit the antithrombotic efficacy of combined treatments

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involving PAR4 antagonism.

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Several limitations of the present study should be noted. First, due to the lack of endothelium in our assay system, our perfusion model may underestimate the

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antithrombotic efficacy of PAR1 antagonists, as PAR1 is an important modulator of vascular wall thrombogenicity in endothelial cells [6, 49, 50]. In addition, since CTI does

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not completely inhibit Factor XIIa, it cannot be denied that some residual activity of FXIIa might

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have promoted thrombin generation during perfusion assays. Second, as capillary occlusion is

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induced by fibrin-rich platelet thrombi, it was not feasible to separately analyze the effects of antiplatelet agents on fibrin and platelet-deposition from the flow pressure analysis parameters. In addition, the procoagulant activity of subendothelial TF is thought to influence thrombus components, including fibrin and platelets. Thus, additional studies are warranted to optimize the concentration of TF for the evaluation of antiplatelet agents using this flow chamber system. Third, because blood samples were perfused at a constant flow rate, the shear rate inside the microchip increases due to narrowing of the capillaries as a result of thrombi formation. In addition, rectangular capillary was used in the present study, which might have caused the increased wall shear rate and thrombi accumulation in the corner of the capillary. Lastly, our in-vitro results cannot be used to directly infer the clinical efficacy or hemorrhagic risk associated with PAR antagonist therapy. 20

ACCEPTED MANUSCRIPT Current techniques for the assessment of antiplatelet therapy are limited in their ability to simultaneously evaluate multiple agonists involved in platelet adhesion, aggregation, and

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procoagulant activity under physiological and pathological conditions [19, 44]. However,

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we have demonstrated here that it is possible to assess combined antiplatelet agents that possess distinct inhibitory mechanisms under physiological shear conditions using a shear

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rate-adjustable microchip-based flow chamber. Using this approach, we have shown that

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the antithrombotic activity of SCH79797-mediated PAR1 antagonism is shear-rate dependent and is also enhanced when combined with aspirin and a P2Y12 inhibitor. An

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increasing number of antiplatelet and anticoagulant agents are becoming available for clinical use [45-48], but few monitoring techniques exist for complex antithrombotic

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regimens. Thus, we intend to examine the suitability of our flow chamber analysis system

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for the clinical evaluation of new antithrombotic therapies and the assessment of bleeding

Conflict of interest statement K Hosokawa, T Ohnishi, and H Sameshima are employees of Fujimori Kogyo Co., Ltd.

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ACCEPTED MANUSCRIPT Tables Table 1. Effects of SCH79797 and YD-3 on thrombus formation under different shear

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conditions

SC

600 s-1 Treatment

OT (min)

7.7 ± 1.7

ASP + AR-C

9.8 ± 2.4*

SCH SCH + ASP + AR-C

14.2 ± 3.6***

OT (min)

9.7 ± 1.9

14.5 ± 2.5

12.1 ± 2.8*

11.0 ± 2.2

16.3 ± 3.2

11.7 ± 3.6***

14.1 ± 4.1***

10.6 ± 2.3

15.9 ± 3.5

16.4 ± 3.9***

13.4 ± 3.4*

19.0 ± 4.2***

8.8 ± 2.0

11.0 ± 2.4

9.9 ± 2.2

14.9 ± 3.0

YD-3 + ASP + AR-C

10.3 ± 2.4**

12.7 ± 2.8**

11.0 ± 1.9*

17.0 ± 3.3

SCH + YD-3

12.2 ± 3.5***

14.6 ± 3.7***

10.5 ± 2.2

17.0 ± 4.2

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AC CE P

YD-3

9.8 ± 2.0

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Control

T10 (min)

TE

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T10 (min)

240 s-1

AR-C, AR-C66096 (1 μM); ASP, aspirin (100 μM); SCH, SCH79797 (10 μM). Comparisons between the treatment and control groups were made by one-way repeated ANOVA. * P