Palladin is involved in platelet activation and arterial thrombosis

    Palladin is involved in platelet activation and arterial thrombosis Xuejiao Chen, Xuemei Fan, Juan Tan, Panlai Shi, Xiyi Wang, Jinjin Wang, Ying Kuang, Jian Fei, Junling Liu, Suying Dang, Zhugang Wang PII: DOI: Reference:

S0049-3848(16)30623-5 doi:10.1016/j.thromres.2016.11.010 TR 6513

To appear in:

Thrombosis Research

Received date: Revised date: Accepted date:

24 May 2016 31 August 2016 9 November 2016

Please cite this article as: Chen Xuejiao, Fan Xuemei, Tan Juan, Shi Panlai, Wang Xiyi, Wang Jinjin, Kuang Ying, Fei Jian, Liu Junling, Dang Suying, Wang Zhugang, Palladin is involved in platelet activation and arterial thrombosis, Thrombosis Research (2016), doi:10.1016/j.thromres.2016.11.010

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ACCEPTED MANUSCRIPT Palladin is involved in platelet activation and arterial thrombosis Xuejiao Chen1,2＃, Xuemei Fan3＃, Juan Tan1,2,Panlai Shi3, Xiyi Wang1,2, Jinjin Wang2,

State Key Laboratory of Medical Genomics, Research Center for Experimental

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Ying Kuang2, Jian Fei2, Junling Liu3, Suying Dang3,2*, and Zhugang Wang1,2*

Medicine (SJTUSM), Shanghai 200025, China;

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Medicine, Rui-Jin Hospital Affiliated to Shanghai Jiao Tong University School of

Shanghai Research Center for Model Organisms, Shanghai 201203, China;

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Department of Biochemistry and Molecular Cell Biology, SJTUSM, Shanghai

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200025, China.

or Zhugang

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*Corresponding author: Suying Dang, [email protected]

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Wang, [email protected]; Building #17,197 Ruijin Road II, Shanghai, 200025, China. Tel & Fax: 021-54656097 or 021-58951591 ＃

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These authors contributed equally to this work.

Number of Figures and Tables: Figures (6); Tables (1) Number of Pages: 22 Words in Abstract: 183 Text Words: 4546 Number of Reference: 31 1

ACCEPTED MANUSCRIPT Abstract

The dynamics of actin cytoskeleton have been shown to play a critical

role during platelet activation. Palladin is an actin-associated protein, serving as a The functional

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cytoskeleton scaffold to bundle actin fibers and actin cross linker.

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role of palladin on platelet activation has not been investigated. Here, we characterized heterozygous palladin knockout (palladin+/-) mice to elucidate the platelet-related functions of palladin. The results showed that palladin was expressed

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in platelets and moderate palladin deficiency accelerated hemostasis and arterial

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thrombosis. The aggregation of palladin+/- platelets was increased in response to low levels of thrombin, U46619, and collagen. We also observed enhanced spreading of

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palladin+/- platelets on immobilized fibrinogen (Fg) and increased rate of clot

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retraction in platelet-rich plasma (PRP) containing palladin+/- platelets. Furthermore,

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the activation of the small GTPase Rac1 and Cdc42, which is associated with cytoskeletal dynamics and platelet activation signalings, was increased in the

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spreading and aggregating palladin+/- platelets compared to that in wild type platelets. Taken together, these findings indicated that palladin is involved in platelet activation and arterial thrombosis, implying a potent role of palladin in pathophysiology of thrombotic diseases.

Keywords: paladin; knockout mice; platelet activation; Rac1; Cdc42

Introduction Platelets are anucleate circulating blood cells. Activation of platelets is an 2

ACCEPTED MANUSCRIPT important part of the complex mechanism of thrombosis and haemostasis. Platelets shape change is the earliest event in the activation and it is accompanied by

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reorganization of the cytoskeleton. The main cytoskeletal component is actin. After

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activation, platelets undergo rapid changes in the amount of actin that is polymerized into filaments and these filaments get organized. The change from focal complexes into focal adhesion is accompanied by the actin network together with associated actin

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proteins result into stress fibers. This process is essential for the start of platelet

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aggregation. At the same time there are various stages of spreading of actin filaments on fibrinogen in the activated platelets. Finally, platelets retract the clot of fibrin that

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binds externally to the developing platelet aggregate[1].

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Palladin belongs to a small gene family which has similar Ig like domains at the

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C-terminus [2,3,4,5]. Palladin has been discovered co-localizing with actin-rich structures in a wide variety of cell types [6,7]. It is known that palladin localizes along

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filament actin at dense regions and takes part in the dynamic regulation of filament actin. Previously, to study the biological functions of palladin in vivo, our lab constructed a palladin gene knockout mouse model, which displayed embryonic lethality due to defects in the neural tube and body wall closure resulting in exencephaly and herniation of the intestine and liver, suggesting a crucial role of palladin during mouse embryogenesis [8,9]. Furthermore, we found that homozygous palladin knockout (palladin-/-) MEFs showed disorganized actin cytoskeleton architecture and decreased expression of β1-integrin, which imply that palladin is actively involved in the regulation of actin cytoskeleton dynamics and 3

ACCEPTED MANUSCRIPT cell-extracellular matrix (ECM) interactions [10]. The functional role of palladin in platelet activation has not been investigated. A

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study has shown that palladin acts as a gene variant associated with myocardial

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infarction (MI) and may associate with human cardiovascular diseases [11]. Myocardial infarction (MI), a complex disease, occurs when thrombosis, induced by a ruptured or eroded atherosclerotic plaque occludes coronary blood flow leading to

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ischemia and ultimately to necrosis of the myocardium[12]. Blood platelets play key

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roles in the process of thrombosis which causes blockage of blood vessels leading to ischemia [13,14,15]. We speculated that palladin would regulate platelet cytoskeleton

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dynamics and platelet activation.

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Here, we found that palladin was expressed in platelets and moderate palladin

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deficiency accelerated hemostasis and arterial thrombosis, increased agonist-induced platelet aggregation, enhanced platelet spreading, and increased rate of clot retraction.

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Furthermore, moderate palladin deficiency increased the activation of the small GTPase Rac1 and Cdc42, which are two important Rho family members and have shown to be important regulators of platelet cytoskeletal dynamics and platelet activation [1, 16]. In conclusion, this study demonstrated that palladin regulates platelet activation through affecting Rac1 and Cdc42 activation.

Materials and Methods Mice Mice containing a heterozygous deletion for palladin were maintained on 129SvJ 4

ACCEPTED MANUSCRIPT background. Genotypes of adult mice were analyzed as described previously [9]. All the procedures described were approved by the Animal Use and Care Committee of

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Shanghai Jiao Tong University School of Medicine.

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Real-time quantitative RT-PCR

Total RNA was isolated from platelets using the TRIzol reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA). DNaseI-treated (Promega,

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Madison, WI) total RNA was reverse transcribed to cDNA using AMV reverse

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transcriptase (Takara, Otsu, Japan). Different mouse palladin isoforms were amplified using specific primers as described previously[17]. RT-PCR primers were designed to

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previously[18]

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amplify mRNA of the Ig3 domain containing isoforms of human palladin as described

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Platelets preparation and aggregation Platelets were isolated and washed platelets were prepared as described [25].

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Aggregation was measured with the Lumi-Aggregometer (Chrono-Log, Havertown, PA) using washed platelets (300 μl) adjusted to approximately 106 platelets per microliter. The Rac1 inhibitor NSC23766 (Tocris Bioscience, Bristol, UK, soluble to 100 mM in water) was incubated at a 10μM final concentration with the platelets for 3 minutes before stimulation. Blood cell analysis For detection of hematologic parameters, blood samples were collected from palladin+/+ and palladin+/- mice using heparin-coated capillary tubes. The analysis of hematologic parameters was performed on a hemocytometer. 5

ACCEPTED MANUSCRIPT Bleeding time analysis Mice were anaesthetized and 0.5 cm tails were cut from the tip and immersed in 0.9%

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NaCl at 37°C immediately. Bleeding time was measured as described [19].

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Plasma Coagulation Assays

Mouse citrated platelet poor plasma (PPP) was prepared from blood collected from Palladin+/+ and Palladin+/- mice. Prothrombin time (PT) and activated partial

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thromboplastin time (aPTT) assays were performed were prepared as previously

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described [20]. PT or aPTT time was measured at 37°C with the Diagnostica Stago STart 4 Hemostasis Analyzer.

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Platelet spreading on immobilized Fg

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For detection of platelet spreading on immobilized Fg, the analysis was done as

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described [21]. Glass cover slips were coated with fibrinogen (100 μg mL-1) overnight at 4°C. Washed platelets (2 × 107 mL-1) spreading on immobilized Fg were stained by

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Rhodamin-conjugated Phalloidin. The different spreading time points of Images were captured with a Nikon E-600 microscope using a Plan Apo 60x/1.4 oil objective, and platelet size was quantified using the National Institutes of Health Image J software (http://rsbweb.nih.gov/ij ). Platelet-mediated clot retraction Clot retraction using mouse platelets was done as described [22]. Washed murine platelets to a concentration of 4×108/ml was mixed citrated human platelet-depleted plasma. The clots were allowed to retract at 37°C and were photographed at various times. NIH Image J software was used to quantify the two-dimensional sizes of 6

ACCEPTED MANUSCRIPT retracted clots on photographs. Quantification of clot at each time point was measured as retraction ratio [1- (final clot size/initial clot size)]. Statistical significance was

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determined using a Student t test.

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Ferric chloride–induced carotid artery injury

Carotid artery was separated from other tissue in isoflurane-anesthetized mice, and injury was induced by 10% FeCl3 as described [23]. Monitoring of carotid artery

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blood flow was initiated at the time of 10% FeCl3 treatment and continuously

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monitored for 13 minutes. Carotid artery blood flow < 0.06 mL/min was scored as occlusion, allowing the time to first occlusion to be determined.

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Western blot analysis

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Each platelet lysate was boiled at 100°C for 10 min, then resolved on a sodium

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dodecyl sulfate (SDS), 10% polyacrylamide gel and then transferred to PVDF membrane [24]. Western blots were performed using palladin antibody, Rac1 antibody,

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Cdc42 antibody. After detection, the membranes were stripped and incubated with anti-actin antibodies to demonstrate the amount of protein present in each lane. For measurements of Rac1 and Cdc42 activity, platelets were allowed to spread on Fg for 90 min, and protein lysates from the spreading platelets were prepared and performed using Rac/Cdc42 assay reagent (Upstate) according to the manufacturer’s instructions. Statistical analysis The two sets of the data generated from heterozygous palladin knockout mice and their wide type littermates were compared using the Student t test. P value < 0.05 was defined to be statistically significant. 7

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Palladin is expressed in both murine and human platelets.

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Results

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As we known, the palladin gene is expressed as four major isoforms, which are the 90–92kDa (isoform A and B), 140, and 200 kDa [17]. It remains unknown whether the palladin gene expresses in platelets. We detected palladin expression in the normal

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murine platelets by RT-PCR and real time PCR. The results showed that the 90-92kDa

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A was the dominant isoform in platelets, 90-92kDa B was detectable, and the 200kDa and 140kDa isoforms were nearly undetectable (Figure 1A and B). Western blotting

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results confirmed partial reduction of palladin expression in palladin+/- platelets

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compared to palladin+/+ platelets (Figure 1C). Overlapping distribution of filamentous

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actin and palladin was observed by immunofluorescent staining in mouse spreading platelets(Figure 1D). The results from RT-PCR revealed that palladin was also

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expressed in human platelets (Figure 1E), the 85-90kDa palladin isoform was detected in human platelets by western blot analysis (Figure 1 F), which was also colocalized with F-actin in spreading human platelets (Figure1G). Palladin regulates hemostasis and arterial thrombosis in vivo Palladin+/- mice exhibited no obvious abnormality in major hematologic parameters such as blood cell counts and hemoglobin levels. The mean platelet volume (MPV) and platelet counts of palladin+/- mice were similar to that of wild-type mice (Table 1). Furthermore，we found there were no significant differences in mouse activated partial thromboplastin time (APTT, WT vs HT: 35.01±2.20 vs 34.94±1.96 seconds, 8

ACCEPTED MANUSCRIPT p>0.05,n=6) and prothrombin time (PT, WT vs HT: 12.83±0.90 vs 12.69±0.63 seconds, p>0.05, n=6) between palladin+/+ and palladin+/- mice(data not shown). The

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role of palladin in hemostasis was evaluated by measuring bleeding times. The

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average bleeding time of palladin+/- mice was 108.29±9.24 seconds, significantly shorter than that (152.29±16.82 seconds) of the palladin+/+ mice (P < 0.05, n =7).Thus, moderate palladin deficiency leads to shortened bleeding time (Fig. 2A).

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In vivo, FeCl3-induced arterial injury creates stable thrombus formation, providing an

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end assessment for physiological effects of signaling molecules in hemostasis and thrombosis [23,25]. Therefore, thrombus formation in palladin+/- and wild-type mice

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was examined using the FeCl3-induced carotid artery thrombosis model. As shown in

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Fig. 2B and C, the average time to first occlusion in the palladin+/- mice was

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significantly reduced compared to WT mice (HT vs. WT, 8.8±0.87 vs. 11.17±0.72 minutes, P < 0.01, n=6). These results indicate that palladin plays important roles in

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hemostasis and arterial thrombus formation in vivo. The aggregation of palladin+/- platelets was enhanced in response to low doses of α-thrombin, U46619, and collagen Since palladin+/- mice showed shorter bleeding time and accelerated thrombus formation but normal platelet counts compared to palladin+/+ mice, platelet activation was investigated by stimulating palladin+/+ and palladin+/- platelets with decreasing concentrations of α-thrombin, U46619, and collagen. As shown in Fig.3, compared to the wild-type platelets, the aggregation of palladin+/- platelets was enhanced in response to low (0.05 U/mL) and medium (0.1 U/mL) concentrations of 9

ACCEPTED MANUSCRIPT α-thrombin; however, the aggregation of palladin+/- platelets in response to a high level of α-thrombin (0.2 U/mL) was not significantly different from the aggregation

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platelets (Figure 3A). As with α-thrombin, the aggregation of

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of palladin+/+

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palladin+/- platelets was enhanced in response to low concentrations of U46619(0.1 μg/ mL and 0.2μg/ mL) and collagen(1μg/ mL and 1.5μg/ mL), while high concentrations of U46619(0.3μg/ mL) and collagen(2μg/ mL) caused equivalent

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aggregation of both genotypes of platelets (Figure 3B and C). Taken together, these

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results demonstrate that palladin plays important roles in different agonist-induced platelet activation.

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Ehanced spreading of palladin+/- platelets on immobilized fibrinogen

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Platelet spreading on immobilized Fg is dependent on cytoskeletal reorganization

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driven by integrin outside-in signaling [15]. During spreading, the actin cytoskeleton undergoes marked remodeling and forms filopodia, lamellipodia and stress fibers. [1].

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Palladin, a cross-linker and actin-binding protein, dominates the reorganization of actin networks in different cell types [26,27,28]. Therefore, platelet spreading on immobilized Fg for 30, 60, and 90 minutes were assessed to characterize the role of palladin in integrin αIIbβ3 mediated outside-in signaling. The results in Figure 4A showed the average spreading area of platelets at each time points was significantly increased in the palladin+/- platelets compared to that of the wild type platelets (P <0 .005, Figure 4B). Accelerated clot retraction of palladin+/- platelets in vitro Outside-in signaling can also drive clot retraction[15]. We further investigated the 10

ACCEPTED MANUSCRIPT function of palladin in clot retraction. The results demonstrated the average ratio of clot retraction in platelet-rich plasma (PRP) containing palladin+/- platelets was

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significantly increased at both 1 hour (HT vs WT: 0.414±0.040 vs 0.240±0.030, P

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<0 .05, Figure 5A) and 3 hours (HT vs WT: 0.545±0.018 vs 0.337±0.033, P<0.005,5B) compared to the wild-type platelets. Therefore, we can conclude that moderate deficiency of palladin accelerates clot retraction.

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Rac1 and Cdc42 activation is increased in spreading and aggregating palladin+/-

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platelets

The early integrin outside-in signaling leading to cell spreading is thought to be

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mediated by small G proteins such as Rac1 and Cdc42 in many cells [15,16]. Few

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studies have identified that palladin plays a role in their activation [29]. Therefore, we

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evaluated the expression level and activation of Rac1 and Cdc42 in both palladin+/and palladin+/+ platelets spreading (90min) on immobilized Fg by western blot

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analyses. The results demonstrated that the expression of Rac1 and Cdc42 protein was not significantly different between palladin+/- platelets and palladin+/+ platelets, but both the GTP- Rac1 and the GTP-Cdc42 proteins (normalized to the amounts of total Rac1, Cdc42) in palladin+/- platelets were increased compared to those in palladin+/+ platelets (Rac1: 1.363±0.0824 fold, and Cdc42: 1.649±0.185 fold) (Figure 6A). Similar results were obtained from the western blot analysis of the lysates of platelets aggregating in response to thrombin (0.05U/ mL). The GTP- Rac1 (normalized to the amounts of total Rac1) in palladin+/- platelets aggregating in response to thrombin was increased compared to those in palladin+/+ platelets (Rac1: 1.530±0.127 fold) (Figure 11

ACCEPTED MANUSCRIPT 6 B). These findings suggest that moderate palladin deficiency in platelets apparently increased the activities of Rac1 and Cdc42. To further clarify the link between the

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phenotypes and the increased Rac1 activity in palladin+/- platelets, platelets were

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pre-incubated with a 10μM final concentration of the Rac1 inhibitor NSC23766 prior to being stimulated with 0.05U/mL α-thrombin. The results showed that the aggregation of both palladin+/+and palladin+/- platelets in response to 0.05U/ml

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α-thrombin were diminished to equivalent degree after Rac1 inhibitor treatment

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(Figure 6C). These observations suggest that palladin is associated with the regulation

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of Rac1 and Cdc42 activity in the dynamics of the actin cytoskeleton in platelets.

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Discussion

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In this study, we demonstrated for the first time that palladin plays important roles in platelet activation, thrombosis and hemostasis. It has been proved that palladin is

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required for normal cell motility and adhesion during embryogenesis [8,9]. Other studies have clearly indicated that palladin participates in actin-dependent behaviors in vitro [10,27]. The platelet cytoskeleton plays a key role in preventing blood loss after an injury. Wound healing and formation of thrombi begins with activation, adhesion, migration and aggregation of platelets. The cytoskeleton plays a prominent role in the all these processes [1]. Given that palladin regulates actin cytoskeleton organization, it is conceivable that palladin plays a role in platelet related functions. Here, we demonstrated that moderate deficiency of palladin in mice caused enhanced agonist-induced platelet aggregation, enhanced platelet spreading, and increased rate

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ACCEPTED MANUSCRIPT of clot retraction. Consistently, accelerated hemeostasis and arterial thrombosis were shown in palladin moderate deficient mice compared to wild-type mice, whereas the

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mean platelet volume (MPV) and counts of platelets in palladin+/- mice were similar

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to that of wild-type mice. Furthermore, we found there were no significant differences in mouse APTT and PT between palladin+/+ and palladin+/- mice. This demonstrates that the accelerated hemeostasis and thrombus formation in palladin+/- mice were not

regulator

of

platelet

activation.

However,

further

studies

using

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negative

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due to the coagulation factors. These findings suggest that palladin may play as a

megakaryocyte/platelet specific and specific isoform palladin knockout mice are

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needed to clarify the exact role of palladin in platelet activation.

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In this study, we identified a 90-92 kDa isoform A of palladin predominantly

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expressed in mouse platelets. The expression of different variants of palladin is regulated in a tissue specific manner [17], suggesting that the 90-92 kDa isoform A

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may be specialized for platelets functions. In the palladin knockout mice we used, all the four known isoforms of transcripts are missed. However, more palladin isoforms are predicted according to the NIH AceView database reports, including the N-terminal isoforms do not share the common C-terminus [8,30]. Whether other isoforms exist in mouse platelets and cause compensatory effects during the platelet activation still requires further study. Platelets are activated after adhesion to adhesive proteins or by soluble platelet agonists via different receptors. But the various platelet activation signaling pathways stimulate platelet shape change , granule secretion and ultimately induce the common 13

ACCEPTED MANUSCRIPT “inside-out” signaling process leading to ligand binding to integrin αIIbβ3 ,which mediates platelet adhesion and aggregation and triggers “outside-in” signaling,

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resulting in platelet spreading, additional granule secretion, stabilization of platelet

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adhesion and aggregation, and clot retraction[15]. During these processes, the platelet cytoskeleton is responsible for binding and positioning signaling molecules; and these signaling molecules are also important for different processes in the cytoskeleton

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reorganization [1]. As an actin-associated cytoskeleton scaffold and actin cross-linker,

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how palladin affects the signalings during platelet activation remains unknown. Here, we found that the activation of Rac1 and Cdc42 in the palladin+/- spreading

and

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aggregating platelets was increased compared to the wild-type platelets; while the

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aggregation of both palladin+/+and palladin+/- platelets in response to α-thrombin

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were diminished to equivalent degree after Rac1 inhibitor treatment. The Rho GTPases RhoA, Rac1 and Cdc42 are the main players in cytoskeletal dynamics of

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platelets and induce filopodia and lamellipodia formation and actin polymerization to strongly increase the platelet surface upon activation. Moreover, they are important for the signalings of platelet secretion, integrin activation and thrombus formation [16, 31]. Studies have shown that Rac1 activation is related to several steps of the platelet activation process, including integrin αIIbβ3 activation, secretion, aggregation and spreading events; while Cdc42 activation is associated with platelet spreading and secretion [16]. In our results, the increased activation of Rac1 and Cdc42 may account for the phenotypes of palladin moderate deficient platelets and mice. Thus, our results suggest that palladin might be associated with the regulation of Rac1 and Cdc42 14

ACCEPTED MANUSCRIPT activation in platelets. But it need further investigation that how palladin affects Rac1 and Cdc42 activation.

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We have reported that cell motility, adhesion, and actin organization were

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defective in palladin-deficient fibroblasts cultured from palladin knockout embryos [10]. Thus, we originally speculated that palladin would regulate platelet cytoskeleton dynamics in platelets as a way similar to that found in fibroblasts. Conversely,

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palladin moderate deficiency caused enhanced actin associated functions. This may

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due to different isoforms of palladin transcripts expression in fibroblasts and platelets. Our previous study showed that all the four major isoforms of palladin were

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expressed in fibroblasts. Thus, the special level and isoform of palladin in platelets

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might show different biological outcome.

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As one of four gene variants associated with myocardial infarction (MI), palladin has not been investigated in platelets. Our data showed that palladin moderate

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dificiency caused enhanced platelet activation and thrombus formation. This may provide further evidence for the pathological role of palladin in MI and suggest palladin acts as a useful marker for identifying individuals at a risk for thrombotic diseases.

Acknowledgements This work is partially supported by the grants from National Natural Science Foundation of China (81430028), the Ministry of Science and Technology of China (2011BAI15B02), the grants from Science and Technology Commission of Shanghai 15

ACCEPTED MANUSCRIPT Municipality

(13DZ2280600,

13DZ2293700,

15DZ2290800,

12ZR1421100,

16ZR1423700), and the E-Institutes of Shanghai Municipal Education Commission

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(E03003)

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formation in mice. J Thromb Haemost. 13(4):619-30.

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oligophrenin1 leads to uncontrolled Rho activation and increased thrombus

Figure legends

Palladin is expressed in both murine and human platelets. (A and B)

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Figure 1.

The 90-92kDa isoform A transcript was the dominant isoform of palladin in mouse

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platelets. RT-PCR and real time PCR were performed using palladin isoform-specific primers. RNA extracted from whole embryos at E10.5 day was used as the control. (C)

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Moderate deficiency of the 90-92kDa isoform A was confirmed in heterozygous palladin knockout mouse platelets by western blot analysis using a C-terminal

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palladin antibody. (D) Overlapping distribution of filamentous actin (red) and palladin (green) showed by immunofluorescent staining in mouse spreading platelets. (E)Total

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RNA was isolated from normal human platelets. RT-PCR was performed using palladin isoform-specific primers. (F) The 85-90kDa isoform was detected in human platelets by western blot analysis. (G) Overlapping distribution of filamentous actin (red) and palladin (green) showed by immunofluorescent staining in human spreading platelets. Figure 2. Accelerated hemostasis and thrombus formation in palladin+/- mice. (A)The bleeding time of palladin+/- mice was statistically different from wild-type mice ( *P < 0.05 n =7).(B-C)Monitoring of carotid artery blood flow at the time of 10% FeCl3 treatment for 13 minutes. (B)Representative traces of blood flow in the 20

ACCEPTED MANUSCRIPT carotid arteries of palladin+/- mice and wild-type mice were presented, respectively. (C)Carotid artery blood flow < 0.06 mL/min was scored as occlusion. The times from

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the beginning of 10% FeCl3 treatment to first occlusion were measured, showing

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thrombus formation was accelerated in palladin+/- mice compared to wild-type mice (n=6,**P <0 .01). Figure 3.

The aggregation of palladin +/- platelets was enhanced in response to

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low doses of thrombin, collagen, and U46619. Washed platelets from palladin+/- and

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wild-type mice were stimulated by doses of thrombin (A), collagen (B) or U46619 (C) and aggregation was recorded using a Lumi-Aggregometer at 37℃ under a stirring

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speed of 900 rpm. Representative tracings of stirred platelet aggregation from three

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experiments for each dose are shown. Mean ± SEM of platelet aggregation percentage

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from 3 individual experiments are plotted in the bars charts; *P <0.05, **P <0.01 as compared with the wild-type mice. Enhanced platelet spreading of palladin+/- platelets (A) Washed

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Figure 4.

platelets from palladin+/- and wild-type mice were layered over coverslips coated with immobilized Fg. Representative images at each time point showed increased spreading of palladin+/- platelets (B) Quantification of the areas (pixel number) of 4 random fields at each time point (mean ± standard error of mean), ***P <0.001.Statistical analyses were performed using the Student t test. Figure 5.

Enhanced clot retraction of palladin+/- platelets. Platelet-rich plasma

from palladin+/- and wild-type mice was induced with thrombin at 37℃ for clot retraction. Clot formation and retraction were continuously photographed at different 21

ACCEPTED MANUSCRIPT intervals. Representative images at each time point showed increased clot retraction in palladin+/- platelets compared to wild-type platelets. (B) Quantification of clot

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retraction at each time point was measured as the percentage of the final clot area as

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compared to the initial total area of the clot (*p<0.05, **p<0.01, n=4). Shown was the mean standard deviation. Figure 6.

Rac1 and Cdc42 activity was increased in palladin+/- spreading and

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aggregating platelets. (A) Western blot analysis of the lysates of platelets spreading

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(90min) on immobilized Fg. The activities of Rac1, Cdc42 were determined by GTPase activity assay. (B) Western blot analysis of the lysates of platelets stimulated

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with thrombin (0.05U/mL) with stirring at 900 rpm in an aggregometer at 37°C. In (A)

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and (B),the levels of active Rac1 and Cdc42 were quantified by densitometric analysis

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of western blots, and the amounts of active Rac1 and Cdc42 were normalized to the amounts of total Rac1, Cdc42. Shown was data from one representative experiment of

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three independent western blots tested. (C) Effects of Rac1 inhibitor on platelet aggregation. Washed platelets from palladin+/- and wild-type mice were stimulated with thrombin (0.05U/mL) after treatment with 10μM NSC23766. Representative aggregation traces of at least three individual experiments are depicted. *P <0.05**P <0.01

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Figure 4

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Figure 5

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Figure 6

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ACCEPTED MANUSCRIPT Table 1. Palladin+/- mice display normal hematologic parameters palladin+/-

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PLT X 105 /μL

8.93 ± 1.05

8.92 ± 0.87

n.s.

MPV (fL)

6.53 ± 0.26

6.50 ± 0.29

n.s.

WBC X 103 /μL

6.02 ±1.24

6.36 ± 1.93

n.s.

RBC X 103 / μL

11.45 ± 0.42

11.21 ± 0.38

n.s.

HGB [g/dL]

16.97 ± 0.43

16.8 ± 0.34

n.s.

HCT [%]

52.68 ±1.53

52.35 ± 1.36

n.s.

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palladin +/+

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Peripheral platelet counts (PLT), mean platelet volume (MPV), White blood cell (WBC) count, red blood cell (RBC) count, hemoglobin (HGB) and hematocrit (HCT) were determined with a

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hematologic analyzer (n = 10, n.s. = not significant ).

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ACCEPTED MANUSCRIPT Highlights 1. Moderate palladin deficiency leads to accelerated hemostasis and arterial

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thrombosis.

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2. Moderate palladin deficiency leads to enhanced agonist-induced platelet

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aggregation, enhanced platelet spreading, and increased rate of clot retraction. 3. Moderate palladin deficiency increases the activation of the small GTPase Rac1

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and Cdc42.

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