Pharmacological inhibition of LIM kinase pathway impairs platelets functionality and facilitates thrombolysis

https://doi.org/10.1016/j.yexcr.2019.06.003Get rights and content

Highlights

  • Platelet actin polymerization is regulated by LIMK proteins.

  • LIMK inhibitors can control platelet functions.

  • Inhibition of LIMK can facilitate thrombolysis.

Abstract

Actin is highly abundant in platelets, and its function is dependent on its structure. Actin filaments (F-actin) are dynamic structures involved in many cellular processes including platelet shape changes and adhesion. The actin cytoskeleton is tightly regulated by actin-binding proteins, which include members of the actin depolymerising factor (ADF)/cofilin family. LIM kinase (LIMK) and its phosphatase slingshot (SSH-1L) regulate actin dynamics by controlling the binding affinity of ADF/cofilin towards actin. We hypothesised that the inhibition of LIMK activity may prevent the changes in platelet shape and their function during activation by controlling the dynamics of F-actin. Our results demonstrate that in platelet, inhibition of LIMK by small LIMK inhibitors controls the level of filamentous actin leading to decreased platelet adhesion and aggregation. These findings encourage further studies on controlling platelet function via the cytoskeleton.

Introduction

Changes in platelet shape and function are regulated by cytoskeletal rearrangements involving dynamic actin polymerisation and depolymerisation [1,2]. Platelets circulate in the blood as discs in an inactive form with distinct organisation of microtubules (MTs) and actin filaments (F-actin). Actin comprises ~20–30% of total platelet proteins [3]. During activation, platelets undergo dramatic changes including a rapid shape change from a disc to a sphere; formation of pseudopods; reorganization of the peripheral microtubules ring; migration of the dense bodies towards the centre of the cell; secretion from its granules; conformational change, cytoskeletal anchorage of the integrin receptor αIIbβ3 (GPIIb/IIIa, CD41/61) and finally aggregation. All of these changes require alterations in the membrane skeleton as well as actin and tubulin remodelling [2].

Changes in the actin cytoskeleton are important for platelet activity in many cardiovascular functions. The dynamic process of the actin cytoskeleton is regulated by actin regulatory proteins including members of the actin depolymerising factors ADF/Cofilin. The activity of cofilin is regulated by the members of the LIM kinase family, LIMK1 & 2, that phosphorylate its well known substrate cofilin on serine 3 resulting in its inactivation [4,5]. It was also shown that LIMK have some tyrosine kinase activity [6,7]. LIMK1 and LIMK2 are activated by phosphorylation of threonine 508/505, respectively, by the effector protein kinases belonging to the Rho GTPase family that includes Rho kinase (ROCK) and p21 activating kinases (PAK 1 and 4) [[8], [9], [10]]. Activated LIMKs phosphorylate and inactivate their substrate cofilin, resulting in the intracellular accumulation of actin filaments [4,5]. On the other hand, cofilin phosphatase slingshot (SSH-1L) (also known as LIMK1 phosphatase) reactivates cofilin by dephosphorylation of both cofilin and LIMK1 [11,12].

LIMK1 and SSH-1L play a major role in actin dynamics via the regulation of ADF/cofilin. Fig. 1A represents a schematic diagram of actin filament formation via the downstream of LIMK1 signal transduction pathway and the cofactors involved in the process.

Pandey et al. demonstrated that LIMK1 activity is important in thrombin-induced changes to platelet shape and aggregation, suggesting that LIMK1 may be a new therapeutic target to inhibit platelet aggregation [13]. Furthermore, these studies also demonstrated that ROCK activated LIMK1 during platelet shape change and aggregation. Notably, although LIMK1 was activated, no change in the level of P-cofilin was observed during shape change, while cofilin de-phosphorylation was observed during platelets aggregation [13]. Other previous studies demonstrated a role for thrombin in ROCK and LIMK1 activation. Addition of thrombin to human vein umbilical endothelial cells (HUVECs) resulted in ROCK activation followed by LIMK1 activation, increased P-cofilin and F-actin levels [14]. Notably, Aslan et al. demonstrated that also PAK, the other positive regulator of LIMK, regulates platelet shape change and aggregation [15].

Here we demonstrate that inhibition of LIMK by the small LIMK inhibitors, BMS3 [16,17], Pyr1 [18] and 22j [19], can control platelet function.

Section snippets

Blood collection and platelet isolation

Platelet rich plasma (PRP) from human blood collected by venepuncture from healthy volunteers taking no medications and anti-coagulants was prepared as previously described. Citrated PRP (1 ml) was 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 with modified Tyrode's buffer either 1:50

Expression of the cytoskeleton regulators in platelets

It has been previously demonstrated that LIMK1 protein is expressed in platelets but not LIMK2 [13]. Using rat anti-LIMK1 [21] or LIMK2 [22] monoclonal antibodies (mAb), we have confirmed both LIMK1 and LIMK2 expression in platelets by western blotting (Fig. 1B). Furthermore, we have shown that adult human platelets also express a number of other proteins involved in the regulation of actin dynamics including cofilin, phospho-cofilin (P-cofilin), PKD (Protein Kinase D) and slingshot-1L (SSH1L)

Discussion

Anti-platelet therapy is highly beneficial for patients with cardiovascular disease such as myocardial infarction and stroke. However, bleeding complications with current drug therapies can prevent their role in many patients. The aim of this study was to understand the basic mechanisms involved in the regulation of the platelet cytoskeleton in order to identify new targets and novel strategies for anti-platelet therapy, which preferentially target thrombus stabilisation.

Actin and tubulin

Declarations of interest

None.

Acknowledgements

We thank Dr Bryce Harrison (Lexicon) for his generous gift of 22j, Dr Laurence Lafanechere (Institut Albert Bonniot) for her generous gift of Pyr 1, Prof James Bamburg (Colorado State University) for his generous gift of the anti-SSH-1L Abs and Dr Iska Carmichael (Monash Micro Imaging) for excellent technical assistance with fluorescence microscopy.

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      In HBSMCs, phalloidin staining suggested that 5 μmol/L of SR7826 and LIMKi3 induce a breakdown of actin cytoskeleton organization. Our findings are in line with previous studies reporting a role of LIMK in actin dynamics in other cell types16,47. At 10 μmol/L of SR7826 and LIMKi3, phalloidin staining of actin was less, but still visible.

    • LIM kinase 2 (LIMK2) may play an essential role in platelet function

      2020, Experimental Cell Research
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      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.

    1

    Senior authors contributed equally to this work.

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