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LIM kinase 2 (LIMK2) may play an essential role in platelet function
Journal Pre-proof LIM kinase 2 (LIMK2) may play an essential role in platelet function Juliana Antonipillai, Kevin Mittelstaedt, Sheena Rigby, Nicole Bassler, Ora Bernard PII:
S0014-4827(20)30010-0
DOI:
https://doi.org/10.1016/j.yexcr.2020.111822
Reference:
YEXCR 111822
To appear in:
Experimental Cell Research
Received Date: 19 July 2019 Revised Date:
7 January 2020
Accepted Date: 8 January 2020
Please cite this article as: J. Antonipillai, K. Mittelstaedt, S. Rigby, N. Bassler, O. Bernard, LIM kinase 2 (LIMK2) may play an essential role in platelet function, Experimental Cell Research (2020), doi: https:// doi.org/10.1016/j.yexcr.2020.111822. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Inc.
Juliana Antonipillai: Conceptualization, Methodology, Investigation, validation, Formal
analysis,
Writing-Draft
preparation,
Reviewing
and
Editing
Kevin
Mittelstaedt: Methodology, Investigation Sheena Rigby: Methodology, Investigation Nicole
Bassler:
Methodology,
Conceptualization, Writing-Editing.
Investigation
Ora
Bernard:
Supervision,
LIM kinase 2 (LIMK2) may play an essential role in platelet function
Juliana Antonipillai1,2,3, Kevin Mittelstaedt2, Sheena Rigby1, Nicole Bassler1, Ora Bernard2 1
Atherothrombosis and Vascular Biology, Baker IDI Heart & Diabetes
Institute, Melbourne, Victoria 3004, Australia 2
St. Vincent’s Institute, Fitzroy, Victoria 3065, Australia
3
College of Health and Biomedical Sciences, RMIT, Victoria 3083, Australia
Corresponding author: Dr Juliana Antonipillai [email protected]
1
Abstract: Actin filaments are highly dynamic structures involved in many cellular processes including cell-to-cell/substrate association and cell motility. The actin cytoskeleton is tightly regulated by actin-binding proteins, which include the members of the ADF (actin-depolymerizing factor)/cofilin family. The members of the LIM kinase family of proteins (LIMK1 and 2) regulate actin dynamics by controlling the binding affinity of ADF/cofilin towards actin. LIMK2 has two major splice variants, LMK2a and LIMK2b. We have generated mice lacking LIMK2a expression (LIMK2a KO), to study its specific role in the regulation of the actin cytoskeleton. The LIMK2a KO mice showed a significant prolonged bleeding complication upon injuries compared to wild type mice. This prolonged bleeding prompted us to check the expression of the LIMK2 protein in platelets as it was previously suggested that it is not expressed in platelets. We showed that human and mouse express LIMK2 in platelets and using our LIMK2a KO mice we have identified a potential key role for LIMK2 in platelet functions including platelet spreading, aggregation and thrombus formation.
Introduction Actin is an essential cytoskeletal protein expressed in all eukaryotic cells that is controlled by actin regulatory proteins including the members of the actindepolymerizing factors ADF/cofilin. The activity of cofilin is inhibited by its phosphorylation on serine 3 by the members of the LIMK family, LIMK1 and LIMK2, resulting in the accumulation of actin filaments within the cells [1, 2]. The activity of LIMK1 and LIMK2 is regulated by phosphorylation of threonine 508 and 505, respectively, by the effector proteins Rho kinases (ROCK1 and
2
2) and p21 activated kinases (PAK1 and 4) [3-5], which act downstream of the Rho GTPase family members. On the other hand, the cofilin phosphatase slingshot (SSH-1L) reactivates cofilin by dephosphorylation of both cofilin and LIMK [6, 7]. Disruption of this homeostasis has been implicated in many pathological conditions including cancer cell invasion [8], metastasis [9] [10] [11], intraocular hypertension [12, 13] and neurodevelopment disorders [1416]. The LIMK1 and LIMK2 proteins are widely expressed in all examined tissues [17] [18] and are structurally similar to each other, containing two double zinc finger LIM domains at the N-terminus followed by a PDZ domain and a catalytic kinase domain at the C-terminus [19]. LIMK1 contains several isoforms including full-length LIMK1a, LIMK1b, LIMK1c and dominant negative LIMK1s. The existing LIMK1 knockout (KO) mice exhibit significantly reduced bone mass [20], abnormalities in spine morphology and synaptic function including enhanced hippocampal long-term potentiation [21]. Several LIMK2 isoforms have been identified including the full-length LIMK2a, LIMK2b, which lacks half of the first N-terminal LIM domain, testis-specific LIMK2t, which contains a part of the PDZ domain and the kinase domain, and brain-specific LIMK2c, which contains an extra 6 amino acids within the kinase domain [22, 23] (Figure 1A). The LIMK2 KO mice generated by Takahashi et al. [24] do not exhibit a pathological phenotype and are fertile in spite of the degeneration of spermatogenic cells in their testis, suggesting that this phenotype may be attributed to the lack of the testis-specific isoform, LIMK2t [24] and not the full length. We have generated LIMK2a KO mice (Figure 1B) to identify the potential roles of LIMK2 in mice. Examination of these mice revealed that they have bleeding complications (Figure 1C). Pandey et al. previously investigated the
3
expression of LIMK1 and LIMK2 and concluded that only LIMK1 but not LIMK2 is expressed in human platelets [25]. However, we have recently demonstrated the expression of the LIMK2 protein in human platelets [26]. Here, we demonstrate that LIMK2 signalling is important in controlling platelet function. Platelets prepared from LIMK2a KO mice, which are selectively devoid of LIMK2 expression, displayed low levels of phospho-cofilin (P-cofilin) and showed prolonged tail bleeding times as well as carotid artery occlusion times compared to wild type mice. Using established LIMK inhibitors we have previously demonstrated that inhibition of LIMK activity results in inhibition of platelet function in vitro and in vivo and most notably improved thrombolysis with urokinase in vivo in mice [26]. Our new findings identify LIMK2 as a key modulator of platelet function in mice, further supporting our previous hypothesis that inhibition of LIMK-associated pathways may be a potential target for a novel anti-platelet therapy in particular in the context of thrombus destabilization in thrombolysis.
Materials and Methods: Animals The LIMK2a knockout mice were derived by injecting E14Tg2a.4 embryonic stem cells with one disrupted allele of the LIMK2 locus (BayGenomics Consortium, CA, USA) into blastocysts of C57BL/6 mice. Knockout and wild type mice were then obtained by breeding heterozygous mice for the interrupted allele. These mice were housed in a pathogen free facility at the BioResources Centre St Vincent’s Hospital, Fitzroy, Vic 3065 Australia and the Baker IDI Heart and Diabetic Institute, AMREP, Prahran, Vic. Care and use of laboratory animals followed the national guidelines and were approved
4
by the institutional animal care and ethics committees. Mixed background mice were used in this study. Tail bleeding time Mice were anaesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg) by intraperitoneal injection and the tip of their tail (5 mm) was cut and immediately immersed into saline, pre-warmed at 37°C. Bleeding time was monitored and recorded as the time needed for the cessation of visible blood stream, for at least 1 minute. Maximum bleeding time was defined as 20 minutes and mice bleeding for more than 20 minutes were sacrificed. Blood collection and platelet isolation Platelet rich plasma (PRP) from human blood collected by venepuncture from healthy volunteers taking no medications or anti-coagulants was prepared. Washed human platelets isolated from citrated PRP (1 mL) were passed through a pre-washed Sepharose CL-2B column (Sigma) and eluted by addition of 1 mL of modified Tyrode’s buffer (150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO3, 2 mM MgCl2, 2 mM CaCl2, 1 mg/mL BSA, 1 mg/mL dextrose; pH 7.4). After elution platelets were diluted (1:10) with modified Tyrode’s for staining and adhesion assay purposes. PRP from mouse blood was collected by cardiac puncture under anaesthetics into a syringe containing Heparin (20 U/mL) followed by centrifugation at 300g for 10 minutes. Washed platelets were prepared as previously described for human platelets. Briefly, PRP was acidified to pH 6.5 by addition of ACD buffer (1.32% w/v sodium citrate, 0.48% w/v citric acid and 1.47% w/v dextrose) followed by addition of 10 mM Theophylline to inhibit platelet activation during centrifugation. Acidified PRP was centrifuged at 720g for 10 minutes at 22°C and the platelet pellet was washed with 5 mL of PBS
5
containing 1 mM Theophylline. After centrifugation the platelet pellet was resuspended in PBS containing Ca2+/Mg2+. Immunoblotting Mouse platelets resuspended in PBS lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, Protease inhibitor cocktail (Roche Cat. #1 836 153), 10 mM NaF, 1 mM Na3VO4 and 10 mM NaP2O7] were sonicated for 10 seconds and then centrifuged at 12,000g for 10 minutes at 4°C. The supernatants (50 µg of platelet lysates) were subjected to Western blot analysis. Immunoblots were probed with rat anti-LIMK1 [17] (1:1000), rat antiLIMK2
mAbs
Biotechonology),
[18]
(1:1000),
rabbit
rabbit
anti-actin
anti-phospho-cofilin
(1:5000;
(1:1000;
Santa
R&D
Cruz
Systems,
Minneapolis, MN, USA), and rabbit anti-cofilin (1:5000; R&D Systems). Horseradish peroxidase-conjugated secondary antibodies against rat and rabbit (Santa Cruz Biotechnology) were used at their recommended dilutions and protein bands were detected with enhanced chemiluminescent substrate (Supersignal West Pico, Pierce, Rockford, IL, USA). RNA purification and qRT-PCR analysis Total RNA was prepared from human platelets and neuroblastoma BE cells (positive control) using the RNeasy Mini Kit (Qiagen) as instructed. Following treatment with DNase I, the total RNA was quantified using nano drop and 200 ng of RNA were reverse transcribed using the Superscript II First-Strand Synthesis Kit (Invitrogen) and random hexamer primers. Quantitative real time PCR was performed in triplicate using SYBR Green and sense and antisense primers specific for LIMK2a (5’-GGGTGAAGATGTCTGGAG-3’; 5’TCGTTGACAGTCCTGTACC-3’), LIMK2b (5’-ATGGGGAGTTACTTGTCAGTC-3’; 5’-CGAAACAGGTCTCTGGAG-3’) and the housekeeping gene, L32
6
(5’-CAGGGTTCGTAGAAGATTCAAGGG-3’; 5’-CTTGGAGGAAACATTGTGAGCGATC-3’). Real-time PCR and data collection were performed on a Stratagene MX3000P QPCR System. Immunostaining Cover slips coated with 30 µg/mL fibrinogen were blocked with 1% BSA for 1hour at room temperature (RT). Sepharose CL-2B purified platelets were diluted in modified Tyrode’s buffer (150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO3, 2 mM MgCl2, 2 mM CaCl2, 1 mg/mL BSA, 1 mg/mL dextrose; pH 7.4). Platelet (1:10 diluted) in a volume of 200 µL were allowed to adhere for 30 minutes at 37°C with ADP. For visualization of LIMK1 or LIMK2 or actin expression in platelets, platelets adhering on cover slips were first fixed with 1x Cellfix (BD) for 15 minutes, washed twice, and then permeablized with 0.1% Triton X-100 for 10 minutes. Following several washing steps, cover slips were stained with anti-LIMK1 or anti-LIMK2 mAbs for 1 hour at RT followed by incubation with a FITC-conjugated goat anti-rat IgG (1:100, Southern Biotechnology) or Alexa488 phalloidin (1:1000, Invitrogen) for 30 minutes for actin staining. Static adhesion assay Washed mouse platelets (1x107/L in PBS) were added onto fibrinogen-coated cover slips blocked with 1% BSA for 1 hour at RT. Platelets were allowed to adhere in the presence of 1U/mL thrombin at 37°C for 30 minutes and permeabilized with 0.1% Triton X-100 for 30 minutes at RT followed by staining with Alexa488 phalloidin for 30 minutes at RT. Platelets were visualized using a Nikon A1R confocal microscope (Japan) Apo x40 water 1.1n.a objective. Stress fibres (F-actin) were visualized under 2D mode NSIM structured illumination microscopy using a Nikon Ti inverted microscope
7
(Japan) with a Apo x100 oil 1.49n.a. objective and EMCCD camera (Andor Technology Ixon 3). The images were reconstructed using the NIS ELEMENTS software 4.10. F-actin/G-actin ratio assay Washed platelets were lysed (2x107/µL) in actin stabilisation buffer (0.1 M PIPES, pH 6.9, 30% glycerol, 5% DMSO, 1 mM MgSO4, 1 mM EGTA, 1% Triton X-100, 1 mM ATP and protease inhibitors) on ice for 10 minutes. Samples were centrifuged in ultra centrifuge at 100,000g for 1 hour and the supernatant containing G-actin was recovered. The pellets containing F-actin were solubilized with actin depolymerisation buffer (0.1 M PIPES, pH 6.9, 1 mM MgSO4, 10 mM CaCl2 and 5 µM cytochalasin D). Aliquots containing the supernatant and pellet fractions were analysed by immunoblotting with HRPconjugated anti-beta actin (Santa Cruz Biotechnology). Flow cytometry Washed mouse platelets (1x107/L in PBS) incubated with or without 0.1 U/mL thrombin for 10 minutes followed by incubation with FITC-labelled antifibrinogen antibody for 15 minutes. Samples were fixed with 1X Cell Fix and analyzed using a FACSCalibur flow cytometer (BD). PE anti-IgG and FITC anti-IgG were used to establish the background staining and the background noise were eliminated. For receptor expression, diluted mouse blood was incubated with either FITC-labelled anti-GPVI, PE-labelled anti-GPIb or PElabelled anti-GPIIb antibodies for 10 minutes, fixed and analyzed using a FACSCalibur flow cytometer. Visualization of platelet aggregation
8
Mouse blood was drawn by heart puncture and anticoagulated with 20 U/mL Heparin. Whole blood was centrifuged at 150g for 10 minutes and PRP was separated. 20 µM ADP was added to PRP and this mixture was spread onto a slide. Aggregates were visualized with a LUCPlanFL 40x 0.6 n.a. objective using an Olympus IX 81 microscope, Imaging Software for Life Science Microscopy. PRP aliquots of mouse were subjected to platelet aggregometry using an aggregometer (AggRam TM System) in the presence of 20 µM ADP. Carotid artery thrombosis Ferric chloride-induced injury in the carotid artery of mice was used as previously described [27]. Mice were anaesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg) by intraperitoneal injection. Anaesthetized mice were placed under a dissecting microscope in a supine position, and a midline incision of the skin extending from the mandible to the sternal notch was made. The fascia was bluntly dissected up to the left common carotid artery. Thrombosis was induced by applying a piece of filter paper (1×3 mm, GB003, Schleicher & Schuell) saturated with 10% ferric chloride (Sigma) under the left common carotid artery for 3 minutes. A piece of plastic sheet (3×6 mm) was laid under the filter paper to prevent the absorption of ferric chloride by surrounding tissue. After removing the filter paper, the artery was rinsed with saline, and a nano-Doppler flow probe (0.5 VB or 0.5 PBS, Transonic) was positioned over the artery. The blood flow was measured by a flow meter (T106 Transonic) and recorded with a PowerLab data acquisition unit (AD Instruments). Thrombotic occlusion was defined by a drop in blood flow to 0.03 mL/min for at least 20 seconds and reopening was defined by an increase in blood flow to 0.04 mL/min for at least 20 seconds. Maximum
9
occlusion time was defined as 60 minutes and mice that had not occluded after 60 minutes were sacrificed. Platelet counts Mouse platelets were counted using “HemaVet Auto Blood Analyzer”. Statistical analysis All the assays were repeated ≥3 times and the results were compared by ttest or one-way ANOVA and a value of p<0.05 was considered significant. Each figure legend indicated the repeated numbers and the statistical test details.
Results Deletion of the LIMK2a gene prolonged bleeding time but did not affect the expression of platelet receptors When the LIMK2a knockout (KO) mice were subjected to a tail bleeding time assay their bleeding time was significantly prolonged following tail transsection. The bleeding of wild type and heterozygous mice stopped within 3-6 minutes, whereas bleeding in the LIMK2a KO mice continued beyond 20 minutes, in 7 out of 8 mice (p< 0.01) (Figure 1C).
This suggested the
possibility that the LIMK2a KO mice had prolong bleeding due to a reduction in platelet number or receptor expression. However, platelet counts (determined by HemaVet Auto Blood Analyzer) and expression levels of the platelets receptors GPVI, GPIb and GPIIb (determined by FACS) were not significantly different in the KO mice in comparison with wild type and heterozygous mice (Table 1).
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Expression of cytoskeletal regulators in human platelets Previously, only LIMK1 but not LIMK2 expression has been detected in human platelets [25]. We have recently shown that both LIMK1 and LIMK2 proteins are expressed in human platelets [26]. Here, using rat anti-LIMK2 mAb [17], we confirmed our previous findings that both LIMK1 and LIMK2 are expressed in human platelets by immunofluorescence staining (Figure 2A). The two LIMK2 isoforms, LIMK2a and LIMK2b, which differ in their Nterminus, are expressed in most cell types [22]. As these two proteins have very similar molecular weights and are difficult to separate on SDS-PAGE, we performed RT-PCR to identify the two mRNA transcripts. While both LIMK2a and LIMK2b transcripts are expressed in BE neuroblastoma cells (positive control), in human platelets only LIMK2a is expressed at detectable level (Figure 2B). As these proteins are involved in the regulation of actin polymerization, it is highly suggestive that they are important for the regulation of human platelet function as previously suggested for LIMK1 and cofilin [25, 28]. We compared the level of LIMK2 and P-cofilin proteins in mouse platelets isolated from wild type and LIMK2a KO mice. Consistent with the RT-PCR results, Western blot analysis using anti-LIMK2 isoform nonselective antibodies, indicated that platelets from the LIMK2a KO mice do not express any detectable levels of LIMK2 protein compared to platelets isolated from wild type mice (Figure 2C). About 80% reduction in levels of P-cofilin was observed in the LIMK2a KO platelets (Figure 2C). These results confirm the expression of LIMK2a in platelets isolated from wild type but not in the KO mice.
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Platelet function is dependent on LIMK2a expression To demonstrate that LIMK2a is responsible for the regulation of the platelet actin cytoskeleton via cofilin phosphorylation, we determined the total Factin/G-actin ratio (insoluble and soluble fractions of actin) as a measure of actin depolymerization. The F-actin/G-actin ratio was significantly reduced in platelets from the LIMK2a KO mice compared to wild type and heterozygous mice (Figure 3A and 3B), which is consistent with the reduced level of Pcofilin in the LIMK2a KO platelets (Figure 2C). To show that loss of LIMK2 disrupts platelet function, platelets isolated from wild type, heterozygous and LIMK2a KO mice were allowed to adhere on a fibrinogen matrix for 30 minutes followed by staining for F-actin with Phalloidin Alexa488. Staining of thrombin stimulated platelets for F-actin revealed that platelets isolated from the LIMK2a KO mice had a reduced number of stress fibers as well as a reduced extent of spreading and a reduced size than platelets isolated from wild type and heterozygous mice (Figure 3C).
No
significant change in resting platelets size was observed when they were not stimulated by the agonist thrombin. Platelets isolated from the LIMK2a KO mice do not adhere well due to a defect in the formation of stress fibres (data not included). To investigate whether LIMK2a can regulate not only platelet adhesion but also platelet activation, we measured the activation of integrin αIIbβ3 by its capacity to bind its ligand fibrinogen, using flow cytometry of platelets prepared from wild type, heterozygous and LIMK2a KO mice. Fibrinogen binding to platelets stimulated with thrombin was significantly reduced in the LIMK2a KO platelets compared to wild type platelets (Figure 4A).
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Furthermore, ADP-induced platelet aggregation was also significantly reduced in the LIMK2a KO mice compared to wild type and heterozygous mice (Figure 4B and 4C). Consistent with this, visual microscopic inspection revealed that platelets prepared from wild type and heterozygous mice formed aggregates whereas platelets from LIMK2a KO mice lost their capacity to aggregate (Figure 4D). No differences were observed in clot retraction (data not included). LIMK2a knockout mice exhibit prolonged occlusion times During vessel wall injury, inhibition of platelet aggregation results in prolonged bleeding times. To further confirm our findings, we tested the role of LIMK2a in arterial thrombus formation, by using a carotid artery injury model. Vessel wall injury was induced by topical application of ferric chloride as described in Materials and Methods. Carotid artery occlusion times in the LIMK2a KO mice were significantly prolonged compared to wild type (p< 0.01) (Figure 4E).
Discussion In this study we investigated the mechanisms involved in LIMK2-dependent regulation of the platelet actin cytoskeleton and its role in thrombus formation and stabilization. Both LIMK1 and 2 are activated via phosphorylation by the protein kinases ROCK and PAK, the effectors of the Rho GTPases. Using LIMK2a knockout mice, which are devoid of expression of all LIMK2 isoforms in platelets, we demonstrate that LIMK2 plays an important role in the regulation of platelet function through cofilin phosphorylation (Figure 5).
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In agreement with the findings of Pandey et al. [25] we have been previously confirmed the expression of LIMK1. However, in addition to our previous findings of LIMK2 expression in resting human platelets [26] we show here that both LIMKs are also expressed in mouse platelets. Interestingly, our RTPCR results demonstrate that only LIMK2a, but not LIMK2b mRNA, is expressed at detectable levels in human platelets and that in mice lacking LIMK2a, expression of LIMK2b is not detectable (Figure 2). Aslan et al. demonstrated a reduced level of LIMK phosphorylation in platelets treated with the PAK specific inhibitors, IPA-3 and PF [29]. We have further demonstrated [26] a reduction in the phosphorylation level of the LIMKs substrate cofilin in platelets treated with the LIMK specific inhibitors, BMS3 [30], Pyr1 [31] and 22j [12]. Our immunofluorescence studies and the determination of F-actin levels in platelets isolated from LIMK2a KO mice showed a reduced amount of polymerized F-actin (Figure 3). Our studies of the LIMK2 KO mice further confirmed our previous findings of reduced F-actin in platelets treated with LIMK specific inhibitors [26]. Taken together, our data indicate that LIMKs, the PAK and ROCK substrates, via their regulation of ADF/cofilin activity, control F-actin levels in platelets. Interestingly Bender et al demonstrated increased platelet size and stress fibres in Cofilin KO mice [32]. This finding strongly supports our results which demonstrates reduction in platelet size and stress fibres in LIMK2a KO mice when they adhered on fibrinogen coated surface. Cofilin promotes actin depolymerization where LIMK inactivates cofilin and promotes actin polymerization. The studies conducted by Estevez et al. reported that LIMK1 is involved in arterial thrombosis and that LIMK1 KO mice show no sign of bleeding
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complications [28]. However, our studies with the LIMK2a KO mice did show significant bleeding prolongations (Figure 1C) which supports our previous findings with platelets treated with the LIMK inhibitors, which inhibit both LIMK1 and LIMK2 [26]. To date, LIMKs are the only established cofilin kinases. Estevez et al. found relatively high levels of P-cofilin protein in the LIMK1 KO platelets [28], strongly supporting our findings that an additional LIMK protein namely LIMK2, is expressed in platelets and regulate cofilin activity. In contrast to Pandey et al. [25] we have clearly demonstrated the expression of LIMK2 in human and mouse platelets. The genetic disruption of the LIMK2 gene in mice resulted in increased carotid artery occlusion and tailbleeding times, which is in agreement with our previous studies with the LIMK inhibitors [26] showing that inhibition of LIMK (LIMK1 and LIMK2) impair platelet function and reduce P-cofilin levels. However, endothelial dysfunction may also contribute to the increased carotid artery occlusion and tail bleeding times. Actin cytoskeletal network is essential to maintain proper function of endothelial cells found in the vessel wall. Stress fibre formation is essential to maintain cortical actin and cell to matrix adhesion [33]. Reducing level of stress fibres in LIMK2a KO mice may contribute to vessel wall dysfunction. This possibility has to be confirmed in the near future. In summary, the present findings provide a role for the LIMK2 proteins, in platelet function via cofilin phosphorylation and the regulation of actin dynamics. We have demonstrated that mice lacking LIMK2 are selectively impaired in platelet function, such as platelet adhesion and aggregation, indicating that LIMK2 is responsible for the regulation of the cytoskeleton in platelets. Our results indicated that inhibition of LIMK2 activity resulted in a
15
significant down-regulation of P-cofilin levels causing inhibition of platelet function in mice. Estevez et al. demonstrated that LIMK1 activity is also involved in regulation of platelet function in mice [28]. These findings support our previous hypothesis, that inhibition of the LIMK-associated pathway in platelets represents a novel strategy for anti-platelet therapy specifically targeting thrombus growth and stabilization.
Acknowledgement We thank Ishka Carmeal (Monash Micro Imaging) for excellent technical assistance with fluorescence microscopy. We thank Professor Karlheinz Peter (Baker IDI) for support during this study.
Funding: This work was supported by a NHMRC Principal Research Fellowship to OB.
Conflict of Interest: The authors declare that they have no financial conflict of interest.
Figure Legends: Figure 1: Schematic diagram of the LIMK genes and the LIMK2a KO construct (A) and the prolong bleeding time of the LIMK2a knockout mouse (B). (A) Schematic diagram of the structure of the LIMK1 and LIMK2 proteins. (a) The LIMK1 and LIMK2 proteins are composed of N-terminal two LIM domains, a PDZ domain and a C-terminal kinase domain. The LIMK2 subfamily contains several isoforms: (b) LIMK2a, the full-length LIMK2 protein. (c) LIMK2b, missing half of the first LIM domain and (d) LIMK2t, testis specific protein missing the 2 LIM domains and part of the PDZ domain. (B) The LIMK2a KO mice were generated by insertion of a plasmid (red arrow)
16
between exon 2 and 2b of the LIMK2 gene. The initiation codons for LIMK2a and LIMK2b are labelled “ATG” and differential splicing of the 2 transcripts is indicated by thin blue arrows. The 5’-terminal region of the LIMK2a and LIMK2b transcripts is shown in the middle and lower panels. Adapted from (Ikebe et al.,1997) [22, 23]. (C) Bleeding time was monitored following tail trans-section at 5 mm from the tip of the tail of LIMK2a knockout, heterozygous and wild type mice (n≥7). Data are expressed as mean ± S.E.M. and p<0.01, analysed by one-way ANOVA.
Figure 2: Expression of LIMK and LIMK-associated proteins in platelets. (A) Immunofluorescence analysis of LIMK1 and LIMK2 expression in activated platelets. Human platelets were allowed to adhere on fibrinogen coated cover slips. Adherent platelets were fixed, probed with anti-LIMK1 or LIMK2 mAbs and stained with goat anti-rat-FITC secondary antibodies, then visualized by florescence microscopy (top panels). Goat anti-rat-FITC secondary antibodies alone were used as a negative control (bottom panel). Bar = 10 µm. (B) Expression of LIMK2 mRNA isoforms in human platelets. RT-PCR of human platelet RNA demonstrates that the LIMK2a but not the LIMK2b isoform is expressed at detectable level in human platelets. Ribosomal L32 RNA was used as a positive control and omission of the cDNA represents negative control for the RT-PCR reaction (right panel). cDNA from neuroblastoma cells (BE), expressing both LIMK2a and LIMK2b isoforms, was used as a positive control for the oligonucleotides. (C) Western blot analysis of LIMK1, LIMK2 and related proteins in platelets isolated from wild type and LIMK2a KO mice.
Figure 3: Expression of Filamentous actin (F-actin) and stress fibres in platelets. (A) The F-actin/G-actin ratio is lower in platelets from LIMK2a KO
17
mice. Platelets were isolated from pooled blood of 5 different wild type, heterozygous and LIMK2a KO mice. F-actin assays were performed as described in Materials and Methods and total F-actin and G-actin were analysed by western blotting. (B) F-actin/G-actin ratio was calculated from 3 independent assays by measuring the band intensity using Bio-Rad software, p<0.01, analysed by one-way ANOVA. (C) Platelets isolated from wild type, heterozygous and LIMK2a KO mice were allowed to adhere on fibrinogencoated cover slips. Adherent platelets were fixed, permeablized and then stained with Alexa488 phalloidin for F-actin. Stained platelets were visualised under 2D mode Nikon N-SIM structured illumination microscope. Bar =10 µm.
Figure 4: LIMK2 is involved in platelet function. (A) Fibrinogen binding to platelets prepared from wild type and LIMK2a KO mice was measured during incubation with thrombin (0.1 U/mL). Flow cytometry was performed following incubation with FITC-conjugated mouse anti-fibrinogen mAb. Bars represent mean ± SEM, p<0.01, analysed by one-way ANOVA, n≥3. (B) LIMK2 is involved in platelet aggregation. Mouse PRP was collected from wild type, heterozygous and LIMK2a KO mice and platelet aggregation was initiated with 20 µM ADP. (C) The maximum percentage of platelet aggregation, from more than 10 mice at 10 minutes as depicted. *P<0.05. (D) PRP was incubated with 20 µM ADP and the formation of platelet aggregates was visualized under light microscope. Representative images of platelets obtained from 5 individual wild type, heterozygous, and LIMK2a KO mice are shown. (E) The carotid artery occlusion times was measured following exposure to 10% ferric chloride for 3 minutes for more than 10 mice. *P<0.01
Figure 5: Schematic diagram of LIMK signal transduction pathway in platelets. LIMK promotes actin polymerization via inhibition of the actin depolymerizing factor, cofilin while slingshot phosphatase (SSH-1L) promotes actin depolymerization via re activation of cofilin. In the LIMK2 KO mice, only LIMK1 regulates cofilin activity whereas in wild type mice both LIMK1 and
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LIMK2 are responsible for cofilin phosphorylation resulting in increased stress fibre formation.
Table 1: Platelet counts and expression of platelet specific receptors in wild type, heterozygous and LIMK2a KO mice. Platelets from wild type, heterozygous and LIMK2a KO mice (more than 8) were counted and their average numbers were calculated. The expression level of the GPVI, GPIb, and GPIIb receptors was determined by FACS and the average intensity calculated.
References
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8. Yoshioka K, Foletta V, Bernard O, Itoh K. A role for LIM kinase in cancer invasion. Proc Natl Acad Sci U S A. 2003;100(12):7247-52. doi: 10.1073/pnas.1232344100. PubMed PMID: 12777619; PubMed Central PMCID: PMCPMC165861. 9. Li R, Doherty J, Antonipillai J, Chen S, Devlin M, Visser K, et al. LIM kinase inhibition reduces breast cancer growth and invasiveness but systemic inhibition does not reduce metastasis in mice. Clin Exp Metastasis. 2013;30(4):483-95. doi: 10.1007/s10585-012-9553-6. PubMed PMID: 23239465. 10. Bernard O. Lim kinases, regulators of actin dynamics. Int J Biochem Cell Biol. 2007;39(6):1071-6. doi: 10.1016/j.biocel.2006.11.011. PubMed PMID: 17188549. 11. Suyama E, Wadhwa R, Kawasaki H, Yaguchi T, Kaul SC, Nakajima M, et al. LIM kinase-2 targeting as a possible anti-metastasis therapy. J Gene Med. 2004;6(3):357-63. doi: 10.1002/jgm.491. PubMed PMID: 15026997. 12. Harrison BA, Whitlock NA, Voronkov MV, Almstead ZY, Gu KJ, Mabon R, et al. Novel class of LIM-kinase 2 inhibitors for the treatment of ocular hypertension and associated glaucoma. J Med Chem. 2009;52(21):6515-8. doi: 10.1021/jm901226j. PubMed PMID: 19831390. 13. Frangiskakis JM, Ewart AK, Morris CA, Mervis CB, Bertrand J, Robinson BF, et al. LIM-kinase1 hemizygosity implicated in impaired visuospatial constructive cognition. Cell. 1996;86(1):59-69. PubMed PMID: 8689688. 14. Hoogenraad CC, Akhmanova A, Galjart N, De Zeeuw CI. LIMK1 and CLIP-115: linking cytoskeletal defects to Williams syndrome. Bioessays. 2004;26(2):141-50. doi: 10.1002/bies.10402. PubMed PMID: 14745832. 15. Meng Y, Takahashi H, Meng J, Zhang Y, Lu G, Asrar S, et al. Regulation of ADF/cofilin phosphorylation and synaptic function by LIMkinase. Neuropharmacology. 2004;47(5):746-54. doi: 10.1016/j.neuropharm.2004.06.030. PubMed PMID: 15458846. 16. Tursun B, Schluter A, Peters MA, Viehweger B, Ostendorff HP, Soosairajah J, et al. The ubiquitin ligase Rnf6 regulates local LIM kinase 1 levels in axonal growth cones. Genes Dev. 2005;19(19):2307-19. doi: 10.1101/gad.1340605. PubMed PMID: 16204183; PubMed Central PMCID: PMCPMC1240040. 17. Foletta VC, Moussi N, Sarmiere PD, Bamburg JR, Bernard O. LIM kinase 1, a key regulator of actin dynamics, is widely expressed in embryonic and adult tissues. Exp Cell Res. 2004;294(2):392-405. doi: 10.1016/j.yexcr.2003.11.024. PubMed PMID: 15023529. 18. Acevedo K, Moussi N, Li R, Soo P, Bernard O. LIM kinase 2 is widely expressed in all tissues. J Histochem Cytochem. 2006;54(5):487-501. doi: 10.1369/jhc.5C6813.2006. PubMed PMID: 16399995. 19. Arber S, Caroni P. Specificity of single LIM motifs in targeting and LIM/LIM interactions in situ. Genes Dev. 1996;10(3):289-300. doi: 10.1101/gad.10.3.289. PubMed PMID: 8595880. 20. Kawano T, Zhu M, Troiano N, Horowitz M, Bian J, Gundberg C, et al. LIM kinase 1 deficient mice have reduced bone mass. Bone. 2013;52(1):7082. doi: 10.1016/j.bone.2012.09.024. PubMed PMID: 23017662; PubMed Central PMCID: PMCPMC3688839.
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21. Meng Y, Zhang Y, Tregoubov V, Janus C, Cruz L, Jackson M, et al. Abnormal spine morphology and enhanced LTP in LIMK-1 knockout mice. Neuron. 2002;35(1):121-33. PubMed PMID: 12123613. 22. Ikebe C, Ohashi K, Fujimori T, Bernard O, Noda T, Robertson EJ, et al. Mouse LIM-kinase 2 gene: cDNA cloning, genomic organization, and tissuespecific expression of two alternatively initiated transcripts. Genomics. 1997;46(3):504-8. doi: 10.1006/geno.1997.5060. PubMed PMID: 9441759. 23. Ikebe C, Ohashi K, Mizuno K. Identification of testis-specific (Limk2t) and brain-specific (Limk2c) isoforms of mouse LIM-kinase 2 gene transcripts. Biochem Biophys Res Commun. 1998;246(2):307-12. doi: 10.1006/bbrc.1998.8609. PubMed PMID: 9610354. 24. Takahashi H, Koshimizu U, Miyazaki J, Nakamura T. Impaired spermatogenic ability of testicular germ cells in mice deficient in the LIMkinase 2 gene. Dev Biol. 2002;241(2):259-72. doi: 10.1006/dbio.2001.0512. PubMed PMID: 11784110. 25. Pandey D, Goyal P, Bamburg JR, Siess W. Regulation of LIM-kinase 1 and cofilin in thrombin-stimulated platelets. Blood. 2006;107(2):575-83. doi: 10.1182/blood-2004-11-4377. PubMed PMID: 16219803; PubMed Central PMCID: PMCPMC1895613. 26. Antonipillai J, Rigby S, Bassler N, Peter K, Bernard O. Pharmacological inhibition of LIM kinase pathway impairs platelets functionality and facilitates thrombolysis. Exp Cell Res. 2019. doi: 10.1016/j.yexcr.2019.06.003. PubMed PMID: 31185194. 27. Stoll P, Bassler N, Hagemeyer CE, Eisenhardt SU, Chen YC, Schmidt R, et al. Targeting ligand-induced binding sites on GPIIb/IIIa via single-chain antibody allows effective anticoagulation without bleeding time prolongation. Arterioscler Thromb Vasc Biol. 2007;27(5):1206-12. doi: 10.1161/ATVBAHA.106.138875. PubMed PMID: 17322097. 28. Estevez B, Stojanovic-Terpo A, Delaney MK, O'Brien KA, Berndt MC, Ruan C, et al. LIM kinase-1 selectively promotes glycoprotein Ib-IX-mediated TXA2 synthesis, platelet activation, and thrombosis. Blood. 2013;121(22):4586-94. doi: 10.1182/blood-2012-12-470765. PubMed PMID: 23620575; PubMed Central PMCID: PMCPMC3668491. 29. Aslan JE, Itakura A, Haley KM, Tormoen GW, Loren CP, Baker SM, et al. p21 activated kinase signaling coordinates glycoprotein receptor VImediated platelet aggregation, lamellipodia formation, and aggregate stability under shear. Arterioscler Thromb Vasc Biol. 2013;33(7):1544-51. doi: 10.1161/ATVBAHA.112.301165. PubMed PMID: 23640496; PubMed Central PMCID: PMCPMC3938029. 30. Ross-Macdonald P, de Silva H, Guo Q, Xiao H, Hung CY, Penhallow B, et al. Identification of a nonkinase target mediating cytotoxicity of novel kinase inhibitors. Mol Cancer Ther. 2008;7(11):3490-8. doi: 10.1158/15357163.MCT-08-0826. PubMed PMID: 19001433. 31. {Li P, R., Vassal-Stermann E, Nguyen CH, Pillet C, Martinez A, Prunier C, et al. Pharmacological inhibition of LIM kinase stabilizes microtubules and inhibits neoplastic growth. Cancer Res. 2012;72(17):4429-39. doi: 10.1158/0008-5472.CAN-11-3342. PubMed PMID: 22761334. 32. Bender M, Eckly A, Hartwig JH, Elvers M, Pleines I, Gupta S, et al. ADF/n-cofilin-dependent actin turnover determines platelet formation and sizing. Blood. 2010;116(10):1767-75. doi: 10.1182/blood-2010-03-274340. PubMed PMID: 20530287.
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33. Prasain N, Stevens T. The actin cytoskeleton in endothelial cell phenotypes. Microvasc Res. 2009;77(1):53-63. doi: 10.1016/j.mvr.2008.09.012. PubMed PMID: 19028505; PubMed Central PMCID: PMCPMC2700738.
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S0014-4827(20)30010-0
DOI:
https://doi.org/10.1016/j.yexcr.2020.111822
Reference:
YEXCR 111822
To appear in:
Experimental Cell Research
Received Date: 19 July 2019 Revised Date:
7 January 2020
Accepted Date: 8 January 2020
Please cite this article as: J. Antonipillai, K. Mittelstaedt, S. Rigby, N. Bassler, O. Bernard, LIM kinase 2 (LIMK2) may play an essential role in platelet function, Experimental Cell Research (2020), doi: https:// doi.org/10.1016/j.yexcr.2020.111822. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier Inc.
Juliana Antonipillai: Conceptualization, Methodology, Investigation, validation, Formal
analysis,
Writing-Draft
preparation,
Reviewing
and
Editing
Kevin
Mittelstaedt: Methodology, Investigation Sheena Rigby: Methodology, Investigation Nicole
Bassler:
Methodology,
Conceptualization, Writing-Editing.
Investigation
Ora
Bernard:
Supervision,
LIM kinase 2 (LIMK2) may play an essential role in platelet function
Juliana Antonipillai1,2,3, Kevin Mittelstaedt2, Sheena Rigby1, Nicole Bassler1, Ora Bernard2 1
Atherothrombosis and Vascular Biology, Baker IDI Heart & Diabetes
Institute, Melbourne, Victoria 3004, Australia 2
St. Vincent’s Institute, Fitzroy, Victoria 3065, Australia
3
College of Health and Biomedical Sciences, RMIT, Victoria 3083, Australia
Corresponding author: Dr Juliana Antonipillai [email protected]
1
Abstract: Actin filaments are highly dynamic structures involved in many cellular processes including cell-to-cell/substrate association and cell motility. The actin cytoskeleton is tightly regulated by actin-binding proteins, which include the members of the ADF (actin-depolymerizing factor)/cofilin family. The members of the LIM kinase family of proteins (LIMK1 and 2) regulate actin dynamics by controlling the binding affinity of ADF/cofilin towards actin. LIMK2 has two major splice variants, LMK2a and LIMK2b. We have generated mice lacking LIMK2a expression (LIMK2a KO), to study its specific role in the regulation of the actin cytoskeleton. The LIMK2a KO mice showed a significant prolonged bleeding complication upon injuries compared to wild type mice. This prolonged bleeding prompted us to check the expression of the LIMK2 protein in platelets as it was previously suggested that it is not expressed in platelets. We showed that human and mouse express LIMK2 in platelets and using our LIMK2a KO mice we have identified a potential key role for LIMK2 in platelet functions including platelet spreading, aggregation and thrombus formation.
Introduction Actin is an essential cytoskeletal protein expressed in all eukaryotic cells that is controlled by actin regulatory proteins including the members of the actindepolymerizing factors ADF/cofilin. The activity of cofilin is inhibited by its phosphorylation on serine 3 by the members of the LIMK family, LIMK1 and LIMK2, resulting in the accumulation of actin filaments within the cells [1, 2]. The activity of LIMK1 and LIMK2 is regulated by phosphorylation of threonine 508 and 505, respectively, by the effector proteins Rho kinases (ROCK1 and
2
2) and p21 activated kinases (PAK1 and 4) [3-5], which act downstream of the Rho GTPase family members. On the other hand, the cofilin phosphatase slingshot (SSH-1L) reactivates cofilin by dephosphorylation of both cofilin and LIMK [6, 7]. Disruption of this homeostasis has been implicated in many pathological conditions including cancer cell invasion [8], metastasis [9] [10] [11], intraocular hypertension [12, 13] and neurodevelopment disorders [1416]. The LIMK1 and LIMK2 proteins are widely expressed in all examined tissues [17] [18] and are structurally similar to each other, containing two double zinc finger LIM domains at the N-terminus followed by a PDZ domain and a catalytic kinase domain at the C-terminus [19]. LIMK1 contains several isoforms including full-length LIMK1a, LIMK1b, LIMK1c and dominant negative LIMK1s. The existing LIMK1 knockout (KO) mice exhibit significantly reduced bone mass [20], abnormalities in spine morphology and synaptic function including enhanced hippocampal long-term potentiation [21]. Several LIMK2 isoforms have been identified including the full-length LIMK2a, LIMK2b, which lacks half of the first N-terminal LIM domain, testis-specific LIMK2t, which contains a part of the PDZ domain and the kinase domain, and brain-specific LIMK2c, which contains an extra 6 amino acids within the kinase domain [22, 23] (Figure 1A). The LIMK2 KO mice generated by Takahashi et al. [24] do not exhibit a pathological phenotype and are fertile in spite of the degeneration of spermatogenic cells in their testis, suggesting that this phenotype may be attributed to the lack of the testis-specific isoform, LIMK2t [24] and not the full length. We have generated LIMK2a KO mice (Figure 1B) to identify the potential roles of LIMK2 in mice. Examination of these mice revealed that they have bleeding complications (Figure 1C). Pandey et al. previously investigated the
3
expression of LIMK1 and LIMK2 and concluded that only LIMK1 but not LIMK2 is expressed in human platelets [25]. However, we have recently demonstrated the expression of the LIMK2 protein in human platelets [26]. Here, we demonstrate that LIMK2 signalling is important in controlling platelet function. Platelets prepared from LIMK2a KO mice, which are selectively devoid of LIMK2 expression, displayed low levels of phospho-cofilin (P-cofilin) and showed prolonged tail bleeding times as well as carotid artery occlusion times compared to wild type mice. Using established LIMK inhibitors we have previously demonstrated that inhibition of LIMK activity results in inhibition of platelet function in vitro and in vivo and most notably improved thrombolysis with urokinase in vivo in mice [26]. Our new findings identify LIMK2 as a key modulator of platelet function in mice, further supporting our previous hypothesis that inhibition of LIMK-associated pathways may be a potential target for a novel anti-platelet therapy in particular in the context of thrombus destabilization in thrombolysis.
Materials and Methods: Animals The LIMK2a knockout mice were derived by injecting E14Tg2a.4 embryonic stem cells with one disrupted allele of the LIMK2 locus (BayGenomics Consortium, CA, USA) into blastocysts of C57BL/6 mice. Knockout and wild type mice were then obtained by breeding heterozygous mice for the interrupted allele. These mice were housed in a pathogen free facility at the BioResources Centre St Vincent’s Hospital, Fitzroy, Vic 3065 Australia and the Baker IDI Heart and Diabetic Institute, AMREP, Prahran, Vic. Care and use of laboratory animals followed the national guidelines and were approved
4
by the institutional animal care and ethics committees. Mixed background mice were used in this study. Tail bleeding time Mice were anaesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg) by intraperitoneal injection and the tip of their tail (5 mm) was cut and immediately immersed into saline, pre-warmed at 37°C. Bleeding time was monitored and recorded as the time needed for the cessation of visible blood stream, for at least 1 minute. Maximum bleeding time was defined as 20 minutes and mice bleeding for more than 20 minutes were sacrificed. Blood collection and platelet isolation Platelet rich plasma (PRP) from human blood collected by venepuncture from healthy volunteers taking no medications or anti-coagulants was prepared. Washed human platelets isolated from citrated PRP (1 mL) were passed through a pre-washed Sepharose CL-2B column (Sigma) and eluted by addition of 1 mL of modified Tyrode’s buffer (150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO3, 2 mM MgCl2, 2 mM CaCl2, 1 mg/mL BSA, 1 mg/mL dextrose; pH 7.4). After elution platelets were diluted (1:10) with modified Tyrode’s for staining and adhesion assay purposes. PRP from mouse blood was collected by cardiac puncture under anaesthetics into a syringe containing Heparin (20 U/mL) followed by centrifugation at 300g for 10 minutes. Washed platelets were prepared as previously described for human platelets. Briefly, PRP was acidified to pH 6.5 by addition of ACD buffer (1.32% w/v sodium citrate, 0.48% w/v citric acid and 1.47% w/v dextrose) followed by addition of 10 mM Theophylline to inhibit platelet activation during centrifugation. Acidified PRP was centrifuged at 720g for 10 minutes at 22°C and the platelet pellet was washed with 5 mL of PBS
5
containing 1 mM Theophylline. After centrifugation the platelet pellet was resuspended in PBS containing Ca2+/Mg2+. Immunoblotting Mouse platelets resuspended in PBS lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, Protease inhibitor cocktail (Roche Cat. #1 836 153), 10 mM NaF, 1 mM Na3VO4 and 10 mM NaP2O7] were sonicated for 10 seconds and then centrifuged at 12,000g for 10 minutes at 4°C. The supernatants (50 µg of platelet lysates) were subjected to Western blot analysis. Immunoblots were probed with rat anti-LIMK1 [17] (1:1000), rat antiLIMK2
mAbs
Biotechonology),
[18]
(1:1000),
rabbit
rabbit
anti-actin
anti-phospho-cofilin
(1:5000;
(1:1000;
Santa
R&D
Cruz
Systems,
Minneapolis, MN, USA), and rabbit anti-cofilin (1:5000; R&D Systems). Horseradish peroxidase-conjugated secondary antibodies against rat and rabbit (Santa Cruz Biotechnology) were used at their recommended dilutions and protein bands were detected with enhanced chemiluminescent substrate (Supersignal West Pico, Pierce, Rockford, IL, USA). RNA purification and qRT-PCR analysis Total RNA was prepared from human platelets and neuroblastoma BE cells (positive control) using the RNeasy Mini Kit (Qiagen) as instructed. Following treatment with DNase I, the total RNA was quantified using nano drop and 200 ng of RNA were reverse transcribed using the Superscript II First-Strand Synthesis Kit (Invitrogen) and random hexamer primers. Quantitative real time PCR was performed in triplicate using SYBR Green and sense and antisense primers specific for LIMK2a (5’-GGGTGAAGATGTCTGGAG-3’; 5’TCGTTGACAGTCCTGTACC-3’), LIMK2b (5’-ATGGGGAGTTACTTGTCAGTC-3’; 5’-CGAAACAGGTCTCTGGAG-3’) and the housekeeping gene, L32
6
(5’-CAGGGTTCGTAGAAGATTCAAGGG-3’; 5’-CTTGGAGGAAACATTGTGAGCGATC-3’). Real-time PCR and data collection were performed on a Stratagene MX3000P QPCR System. Immunostaining Cover slips coated with 30 µg/mL fibrinogen were blocked with 1% BSA for 1hour at room temperature (RT). Sepharose CL-2B purified platelets were diluted in modified Tyrode’s buffer (150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO3, 2 mM MgCl2, 2 mM CaCl2, 1 mg/mL BSA, 1 mg/mL dextrose; pH 7.4). Platelet (1:10 diluted) in a volume of 200 µL were allowed to adhere for 30 minutes at 37°C with ADP. For visualization of LIMK1 or LIMK2 or actin expression in platelets, platelets adhering on cover slips were first fixed with 1x Cellfix (BD) for 15 minutes, washed twice, and then permeablized with 0.1% Triton X-100 for 10 minutes. Following several washing steps, cover slips were stained with anti-LIMK1 or anti-LIMK2 mAbs for 1 hour at RT followed by incubation with a FITC-conjugated goat anti-rat IgG (1:100, Southern Biotechnology) or Alexa488 phalloidin (1:1000, Invitrogen) for 30 minutes for actin staining. Static adhesion assay Washed mouse platelets (1x107/L in PBS) were added onto fibrinogen-coated cover slips blocked with 1% BSA for 1 hour at RT. Platelets were allowed to adhere in the presence of 1U/mL thrombin at 37°C for 30 minutes and permeabilized with 0.1% Triton X-100 for 30 minutes at RT followed by staining with Alexa488 phalloidin for 30 minutes at RT. Platelets were visualized using a Nikon A1R confocal microscope (Japan) Apo x40 water 1.1n.a objective. Stress fibres (F-actin) were visualized under 2D mode NSIM structured illumination microscopy using a Nikon Ti inverted microscope
7
(Japan) with a Apo x100 oil 1.49n.a. objective and EMCCD camera (Andor Technology Ixon 3). The images were reconstructed using the NIS ELEMENTS software 4.10. F-actin/G-actin ratio assay Washed platelets were lysed (2x107/µL) in actin stabilisation buffer (0.1 M PIPES, pH 6.9, 30% glycerol, 5% DMSO, 1 mM MgSO4, 1 mM EGTA, 1% Triton X-100, 1 mM ATP and protease inhibitors) on ice for 10 minutes. Samples were centrifuged in ultra centrifuge at 100,000g for 1 hour and the supernatant containing G-actin was recovered. The pellets containing F-actin were solubilized with actin depolymerisation buffer (0.1 M PIPES, pH 6.9, 1 mM MgSO4, 10 mM CaCl2 and 5 µM cytochalasin D). Aliquots containing the supernatant and pellet fractions were analysed by immunoblotting with HRPconjugated anti-beta actin (Santa Cruz Biotechnology). Flow cytometry Washed mouse platelets (1x107/L in PBS) incubated with or without 0.1 U/mL thrombin for 10 minutes followed by incubation with FITC-labelled antifibrinogen antibody for 15 minutes. Samples were fixed with 1X Cell Fix and analyzed using a FACSCalibur flow cytometer (BD). PE anti-IgG and FITC anti-IgG were used to establish the background staining and the background noise were eliminated. For receptor expression, diluted mouse blood was incubated with either FITC-labelled anti-GPVI, PE-labelled anti-GPIb or PElabelled anti-GPIIb antibodies for 10 minutes, fixed and analyzed using a FACSCalibur flow cytometer. Visualization of platelet aggregation
8
Mouse blood was drawn by heart puncture and anticoagulated with 20 U/mL Heparin. Whole blood was centrifuged at 150g for 10 minutes and PRP was separated. 20 µM ADP was added to PRP and this mixture was spread onto a slide. Aggregates were visualized with a LUCPlanFL 40x 0.6 n.a. objective using an Olympus IX 81 microscope, Imaging Software for Life Science Microscopy. PRP aliquots of mouse were subjected to platelet aggregometry using an aggregometer (AggRam TM System) in the presence of 20 µM ADP. Carotid artery thrombosis Ferric chloride-induced injury in the carotid artery of mice was used as previously described [27]. Mice were anaesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg) by intraperitoneal injection. Anaesthetized mice were placed under a dissecting microscope in a supine position, and a midline incision of the skin extending from the mandible to the sternal notch was made. The fascia was bluntly dissected up to the left common carotid artery. Thrombosis was induced by applying a piece of filter paper (1×3 mm, GB003, Schleicher & Schuell) saturated with 10% ferric chloride (Sigma) under the left common carotid artery for 3 minutes. A piece of plastic sheet (3×6 mm) was laid under the filter paper to prevent the absorption of ferric chloride by surrounding tissue. After removing the filter paper, the artery was rinsed with saline, and a nano-Doppler flow probe (0.5 VB or 0.5 PBS, Transonic) was positioned over the artery. The blood flow was measured by a flow meter (T106 Transonic) and recorded with a PowerLab data acquisition unit (AD Instruments). Thrombotic occlusion was defined by a drop in blood flow to 0.03 mL/min for at least 20 seconds and reopening was defined by an increase in blood flow to 0.04 mL/min for at least 20 seconds. Maximum
9
occlusion time was defined as 60 minutes and mice that had not occluded after 60 minutes were sacrificed. Platelet counts Mouse platelets were counted using “HemaVet Auto Blood Analyzer”. Statistical analysis All the assays were repeated ≥3 times and the results were compared by ttest or one-way ANOVA and a value of p<0.05 was considered significant. Each figure legend indicated the repeated numbers and the statistical test details.
Results Deletion of the LIMK2a gene prolonged bleeding time but did not affect the expression of platelet receptors When the LIMK2a knockout (KO) mice were subjected to a tail bleeding time assay their bleeding time was significantly prolonged following tail transsection. The bleeding of wild type and heterozygous mice stopped within 3-6 minutes, whereas bleeding in the LIMK2a KO mice continued beyond 20 minutes, in 7 out of 8 mice (p< 0.01) (Figure 1C).
This suggested the
possibility that the LIMK2a KO mice had prolong bleeding due to a reduction in platelet number or receptor expression. However, platelet counts (determined by HemaVet Auto Blood Analyzer) and expression levels of the platelets receptors GPVI, GPIb and GPIIb (determined by FACS) were not significantly different in the KO mice in comparison with wild type and heterozygous mice (Table 1).
10
Expression of cytoskeletal regulators in human platelets Previously, only LIMK1 but not LIMK2 expression has been detected in human platelets [25]. We have recently shown that both LIMK1 and LIMK2 proteins are expressed in human platelets [26]. Here, using rat anti-LIMK2 mAb [17], we confirmed our previous findings that both LIMK1 and LIMK2 are expressed in human platelets by immunofluorescence staining (Figure 2A). The two LIMK2 isoforms, LIMK2a and LIMK2b, which differ in their Nterminus, are expressed in most cell types [22]. As these two proteins have very similar molecular weights and are difficult to separate on SDS-PAGE, we performed RT-PCR to identify the two mRNA transcripts. While both LIMK2a and LIMK2b transcripts are expressed in BE neuroblastoma cells (positive control), in human platelets only LIMK2a is expressed at detectable level (Figure 2B). As these proteins are involved in the regulation of actin polymerization, it is highly suggestive that they are important for the regulation of human platelet function as previously suggested for LIMK1 and cofilin [25, 28]. We compared the level of LIMK2 and P-cofilin proteins in mouse platelets isolated from wild type and LIMK2a KO mice. Consistent with the RT-PCR results, Western blot analysis using anti-LIMK2 isoform nonselective antibodies, indicated that platelets from the LIMK2a KO mice do not express any detectable levels of LIMK2 protein compared to platelets isolated from wild type mice (Figure 2C). About 80% reduction in levels of P-cofilin was observed in the LIMK2a KO platelets (Figure 2C). These results confirm the expression of LIMK2a in platelets isolated from wild type but not in the KO mice.
11
Platelet function is dependent on LIMK2a expression To demonstrate that LIMK2a is responsible for the regulation of the platelet actin cytoskeleton via cofilin phosphorylation, we determined the total Factin/G-actin ratio (insoluble and soluble fractions of actin) as a measure of actin depolymerization. The F-actin/G-actin ratio was significantly reduced in platelets from the LIMK2a KO mice compared to wild type and heterozygous mice (Figure 3A and 3B), which is consistent with the reduced level of Pcofilin in the LIMK2a KO platelets (Figure 2C). To show that loss of LIMK2 disrupts platelet function, platelets isolated from wild type, heterozygous and LIMK2a KO mice were allowed to adhere on a fibrinogen matrix for 30 minutes followed by staining for F-actin with Phalloidin Alexa488. Staining of thrombin stimulated platelets for F-actin revealed that platelets isolated from the LIMK2a KO mice had a reduced number of stress fibers as well as a reduced extent of spreading and a reduced size than platelets isolated from wild type and heterozygous mice (Figure 3C).
No
significant change in resting platelets size was observed when they were not stimulated by the agonist thrombin. Platelets isolated from the LIMK2a KO mice do not adhere well due to a defect in the formation of stress fibres (data not included). To investigate whether LIMK2a can regulate not only platelet adhesion but also platelet activation, we measured the activation of integrin αIIbβ3 by its capacity to bind its ligand fibrinogen, using flow cytometry of platelets prepared from wild type, heterozygous and LIMK2a KO mice. Fibrinogen binding to platelets stimulated with thrombin was significantly reduced in the LIMK2a KO platelets compared to wild type platelets (Figure 4A).
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Furthermore, ADP-induced platelet aggregation was also significantly reduced in the LIMK2a KO mice compared to wild type and heterozygous mice (Figure 4B and 4C). Consistent with this, visual microscopic inspection revealed that platelets prepared from wild type and heterozygous mice formed aggregates whereas platelets from LIMK2a KO mice lost their capacity to aggregate (Figure 4D). No differences were observed in clot retraction (data not included). LIMK2a knockout mice exhibit prolonged occlusion times During vessel wall injury, inhibition of platelet aggregation results in prolonged bleeding times. To further confirm our findings, we tested the role of LIMK2a in arterial thrombus formation, by using a carotid artery injury model. Vessel wall injury was induced by topical application of ferric chloride as described in Materials and Methods. Carotid artery occlusion times in the LIMK2a KO mice were significantly prolonged compared to wild type (p< 0.01) (Figure 4E).
Discussion In this study we investigated the mechanisms involved in LIMK2-dependent regulation of the platelet actin cytoskeleton and its role in thrombus formation and stabilization. Both LIMK1 and 2 are activated via phosphorylation by the protein kinases ROCK and PAK, the effectors of the Rho GTPases. Using LIMK2a knockout mice, which are devoid of expression of all LIMK2 isoforms in platelets, we demonstrate that LIMK2 plays an important role in the regulation of platelet function through cofilin phosphorylation (Figure 5).
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In agreement with the findings of Pandey et al. [25] we have been previously confirmed the expression of LIMK1. However, in addition to our previous findings of LIMK2 expression in resting human platelets [26] we show here that both LIMKs are also expressed in mouse platelets. Interestingly, our RTPCR results demonstrate that only LIMK2a, but not LIMK2b mRNA, is expressed at detectable levels in human platelets and that in mice lacking LIMK2a, expression of LIMK2b is not detectable (Figure 2). Aslan et al. demonstrated a reduced level of LIMK phosphorylation in platelets treated with the PAK specific inhibitors, IPA-3 and PF [29]. We have further demonstrated [26] a reduction in the phosphorylation level of the LIMKs substrate cofilin in platelets treated with the LIMK specific inhibitors, BMS3 [30], Pyr1 [31] and 22j [12]. Our immunofluorescence studies and the determination of F-actin levels in platelets isolated from LIMK2a KO mice showed a reduced amount of polymerized F-actin (Figure 3). Our studies of the LIMK2 KO mice further confirmed our previous findings of reduced F-actin in platelets treated with LIMK specific inhibitors [26]. Taken together, our data indicate that LIMKs, the PAK and ROCK substrates, via their regulation of ADF/cofilin activity, control F-actin levels in platelets. Interestingly Bender et al demonstrated increased platelet size and stress fibres in Cofilin KO mice [32]. This finding strongly supports our results which demonstrates reduction in platelet size and stress fibres in LIMK2a KO mice when they adhered on fibrinogen coated surface. Cofilin promotes actin depolymerization where LIMK inactivates cofilin and promotes actin polymerization. The studies conducted by Estevez et al. reported that LIMK1 is involved in arterial thrombosis and that LIMK1 KO mice show no sign of bleeding
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complications [28]. However, our studies with the LIMK2a KO mice did show significant bleeding prolongations (Figure 1C) which supports our previous findings with platelets treated with the LIMK inhibitors, which inhibit both LIMK1 and LIMK2 [26]. To date, LIMKs are the only established cofilin kinases. Estevez et al. found relatively high levels of P-cofilin protein in the LIMK1 KO platelets [28], strongly supporting our findings that an additional LIMK protein namely LIMK2, is expressed in platelets and regulate cofilin activity. In contrast to Pandey et al. [25] we have clearly demonstrated the expression of LIMK2 in human and mouse platelets. The genetic disruption of the LIMK2 gene in mice resulted in increased carotid artery occlusion and tailbleeding times, which is in agreement with our previous studies with the LIMK inhibitors [26] showing that inhibition of LIMK (LIMK1 and LIMK2) impair platelet function and reduce P-cofilin levels. However, endothelial dysfunction may also contribute to the increased carotid artery occlusion and tail bleeding times. Actin cytoskeletal network is essential to maintain proper function of endothelial cells found in the vessel wall. Stress fibre formation is essential to maintain cortical actin and cell to matrix adhesion [33]. Reducing level of stress fibres in LIMK2a KO mice may contribute to vessel wall dysfunction. This possibility has to be confirmed in the near future. In summary, the present findings provide a role for the LIMK2 proteins, in platelet function via cofilin phosphorylation and the regulation of actin dynamics. We have demonstrated that mice lacking LIMK2 are selectively impaired in platelet function, such as platelet adhesion and aggregation, indicating that LIMK2 is responsible for the regulation of the cytoskeleton in platelets. Our results indicated that inhibition of LIMK2 activity resulted in a
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significant down-regulation of P-cofilin levels causing inhibition of platelet function in mice. Estevez et al. demonstrated that LIMK1 activity is also involved in regulation of platelet function in mice [28]. These findings support our previous hypothesis, that inhibition of the LIMK-associated pathway in platelets represents a novel strategy for anti-platelet therapy specifically targeting thrombus growth and stabilization.
Acknowledgement We thank Ishka Carmeal (Monash Micro Imaging) for excellent technical assistance with fluorescence microscopy. We thank Professor Karlheinz Peter (Baker IDI) for support during this study.
Funding: This work was supported by a NHMRC Principal Research Fellowship to OB.
Conflict of Interest: The authors declare that they have no financial conflict of interest.
Figure Legends: Figure 1: Schematic diagram of the LIMK genes and the LIMK2a KO construct (A) and the prolong bleeding time of the LIMK2a knockout mouse (B). (A) Schematic diagram of the structure of the LIMK1 and LIMK2 proteins. (a) The LIMK1 and LIMK2 proteins are composed of N-terminal two LIM domains, a PDZ domain and a C-terminal kinase domain. The LIMK2 subfamily contains several isoforms: (b) LIMK2a, the full-length LIMK2 protein. (c) LIMK2b, missing half of the first LIM domain and (d) LIMK2t, testis specific protein missing the 2 LIM domains and part of the PDZ domain. (B) The LIMK2a KO mice were generated by insertion of a plasmid (red arrow)
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between exon 2 and 2b of the LIMK2 gene. The initiation codons for LIMK2a and LIMK2b are labelled “ATG” and differential splicing of the 2 transcripts is indicated by thin blue arrows. The 5’-terminal region of the LIMK2a and LIMK2b transcripts is shown in the middle and lower panels. Adapted from (Ikebe et al.,1997) [22, 23]. (C) Bleeding time was monitored following tail trans-section at 5 mm from the tip of the tail of LIMK2a knockout, heterozygous and wild type mice (n≥7). Data are expressed as mean ± S.E.M. and p<0.01, analysed by one-way ANOVA.
Figure 2: Expression of LIMK and LIMK-associated proteins in platelets. (A) Immunofluorescence analysis of LIMK1 and LIMK2 expression in activated platelets. Human platelets were allowed to adhere on fibrinogen coated cover slips. Adherent platelets were fixed, probed with anti-LIMK1 or LIMK2 mAbs and stained with goat anti-rat-FITC secondary antibodies, then visualized by florescence microscopy (top panels). Goat anti-rat-FITC secondary antibodies alone were used as a negative control (bottom panel). Bar = 10 µm. (B) Expression of LIMK2 mRNA isoforms in human platelets. RT-PCR of human platelet RNA demonstrates that the LIMK2a but not the LIMK2b isoform is expressed at detectable level in human platelets. Ribosomal L32 RNA was used as a positive control and omission of the cDNA represents negative control for the RT-PCR reaction (right panel). cDNA from neuroblastoma cells (BE), expressing both LIMK2a and LIMK2b isoforms, was used as a positive control for the oligonucleotides. (C) Western blot analysis of LIMK1, LIMK2 and related proteins in platelets isolated from wild type and LIMK2a KO mice.
Figure 3: Expression of Filamentous actin (F-actin) and stress fibres in platelets. (A) The F-actin/G-actin ratio is lower in platelets from LIMK2a KO
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mice. Platelets were isolated from pooled blood of 5 different wild type, heterozygous and LIMK2a KO mice. F-actin assays were performed as described in Materials and Methods and total F-actin and G-actin were analysed by western blotting. (B) F-actin/G-actin ratio was calculated from 3 independent assays by measuring the band intensity using Bio-Rad software, p<0.01, analysed by one-way ANOVA. (C) Platelets isolated from wild type, heterozygous and LIMK2a KO mice were allowed to adhere on fibrinogencoated cover slips. Adherent platelets were fixed, permeablized and then stained with Alexa488 phalloidin for F-actin. Stained platelets were visualised under 2D mode Nikon N-SIM structured illumination microscope. Bar =10 µm.
Figure 4: LIMK2 is involved in platelet function. (A) Fibrinogen binding to platelets prepared from wild type and LIMK2a KO mice was measured during incubation with thrombin (0.1 U/mL). Flow cytometry was performed following incubation with FITC-conjugated mouse anti-fibrinogen mAb. Bars represent mean ± SEM, p<0.01, analysed by one-way ANOVA, n≥3. (B) LIMK2 is involved in platelet aggregation. Mouse PRP was collected from wild type, heterozygous and LIMK2a KO mice and platelet aggregation was initiated with 20 µM ADP. (C) The maximum percentage of platelet aggregation, from more than 10 mice at 10 minutes as depicted. *P<0.05. (D) PRP was incubated with 20 µM ADP and the formation of platelet aggregates was visualized under light microscope. Representative images of platelets obtained from 5 individual wild type, heterozygous, and LIMK2a KO mice are shown. (E) The carotid artery occlusion times was measured following exposure to 10% ferric chloride for 3 minutes for more than 10 mice. *P<0.01
Figure 5: Schematic diagram of LIMK signal transduction pathway in platelets. LIMK promotes actin polymerization via inhibition of the actin depolymerizing factor, cofilin while slingshot phosphatase (SSH-1L) promotes actin depolymerization via re activation of cofilin. In the LIMK2 KO mice, only LIMK1 regulates cofilin activity whereas in wild type mice both LIMK1 and
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LIMK2 are responsible for cofilin phosphorylation resulting in increased stress fibre formation.
Table 1: Platelet counts and expression of platelet specific receptors in wild type, heterozygous and LIMK2a KO mice. Platelets from wild type, heterozygous and LIMK2a KO mice (more than 8) were counted and their average numbers were calculated. The expression level of the GPVI, GPIb, and GPIIb receptors was determined by FACS and the average intensity calculated.
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