G-Protein Coupled and ITAM Receptor Regulation of the Formin FHOD1 Through Rho Kinase in Platelets

1648 Letters to the Editor G-protein coupled and ITAM receptor regulation of the formin FHOD1 through Rho Kinase in platelets

S. G. THOMAS,* S. D. J. CALAMINUS, L. M. MACHESKY, A. S. ALBERTSà andS. P. WATSON* *Centre for Cardiovascular Science, Institute for Biomedical Research, University of Birmingham, Edgbaston, Birmingham, UK; The Beatson Institute for Cancer Research, Bearsden, Glasgow, UK; and àCentre for Cancer and Cell Biology, Van Andel Research Institute, Grand Rapids, MI, USA

To cite this article: Thomas SG, Calaminus SDJ, Machesky LM, Alberts AS, Watson SP. G-protein coupled and ITAM receptor regulation of the formin FHOD1 through Rho Kinase in platelets. J Thromb Haemost 2011; 9: 1648–51.

Washed human platelets were prepared, stimulated and Rearrangements of the actin cytoskeleton downstream of many Western blotted as previously described [16] with antibodies signaling pathways are regulated by the Rho family of against FHOD1 (Santa Cruz Biotechnology, Santa Cruz, CA, guanosine triphosphate (GTP)-binding proteins [1]. In platelets, USA), pFHOD1 (Thr1141) and Daam1 (ECM Biosciences, the Rho GTP-binding proteins Rac1, cdc42 and RhoA mediate Versailles, KY, USA), mDia1 (Bethyl Labs, Montgomery, TX, platelet functional responses by controlling the organization of USA) and mDia2 (Provided by Art Alberts). Mouse platelets the cytoskeleton [2–6]. Furthermore, the RhoA – Rho kinase were prepared as described previously [17] from mDia1 pathway plays a key role in allowing full spreading of activated constitutive knockout mice [18] and from crosses between platelets [7,8] and in activation of myosin IIa, which provides the PF4-Cre [19] and Rac1 flox [20] mice. Quantitation of band contractile force required for stable thrombus formation [9]. intensity was performed using Adobe Photoshop CS. Formins are RhoA effector molecules that nucleate, elongate To determine which formins were likely to be present in and sometimes bundle actin filaments to support polarized cell platelets we analysed a Serial Analysis of Gene Expression division, adhesion and migration [10,11]. GTP-bound RhoA (SAGE) library for mouse megakaryocytes [21] and the binds to and directly activates formins by disrupting an HaemAtlas, which reports RNA expression in human megak- intramolecular autoinhibition mechanism that allows the aryocytes [22]. These searches revealed the presence of six of the FH1-FH2 cassette to bind directly to actin filaments and 15 mammalian formin proteins in mouse megakaryocytes microtubules to influence their assembly [12]. Furthermore, (mDia1, mDia2, Daam1, Fmnl1, Fmnl3 and FHOD1) with cooperation between the RhoA effector molecules Rho kinase mDia3 additionally being found in human megakaryocytes and the formins is required for the formation of stress fibres as (Fig. S1A). Furthermore, we have shown the presence of they require both F-actin polymerization and activation of mDia1, mDia2, Daam1 and FHOD1 in human platelets and myosin contractility for proper function [13,14]. mDia1, Daam1 and FHOD1 in mouse platelets using Western It has been previously shown that human platelets contain blotting (Fig. S1B), confirming previously published reports the formins mDia1 and Daam1, which are both effectors of for mDia1 and Daam1 [15] but reporting the presence of RhoA [15], yet little is known about the function of these mDia2 and FHOD1 for the first time. proteins or indeed other formin family members in platelets. The most thoroughly characterized mammalian formin is Additionally, the formin FHOD1 is known to be regulated by mDia1, which is present in mouse and human platelets. Rho kinase phosphorylation on three serine/threonine residues However, analysis of platelets from a knockout mouse model but to date its presence has not been reported in platelets. In for mDia1 [18] revealed no alteration in platelet count, tyrosine this study we aim to identify which formins are present in phosphorylation, fibrinogen binding, P-selectin surface expres- human and mouse platelets and megakaryocytes and report the sion, spreading or clot retraction in response to both G protein- analysis of platelets from a knockout mouse model for mDia1. coupled and tyrosine kinase-linked receptors (Fig. S2 and data Furthermore, we report that the formin FHOD1 is robustly not shown). Although no compensatory up-regulation of other phosphorylated downstream of the major platelet agonists in a formins was observed, a small decrease in Daam1 levels was Rho kinase-dependent manner and may be a key regulator of seen in mDia1)/) platelets (Fig S2B). However, the significance platelet stress fibre formation. of this is unclear. Additionally, even though platelet function was unaffected in the assays performed, there remains the Correspondence: Steven G. Thomas, Centre for Cardiovascular possibility that loss of mDia1 could affect aggregate stability Science, Institute for Biomedical Research, University of under conditions of shear stress. The reason for a lack of a Birmingham, Edgbaston, Birmingham B15 2TT, UK. functional role for mDia1 in mouse platelets is unclear and may Tel.: +44 121 414 8308; fax: +44 121 414 5925. E-mail: [email protected] be due to redundancy between the different formins expressed in platelets. Alternatively, the presence of mDia1 in platelets DOI: 10.1111/j.1538-7836.2011.04357.x may be a consequence of a role in megakaryocytes (for example in megakaryocyte migration or gene regulation via SRF/ Received 29 March 2011, accepted 9 May 2011 MAL), with protein being carried over to platelets during

2011 International Society on Thrombosis and Haemostasis Letters to the Editor 1649 platelet formation. However, no obvious difference was body (threonine residue 1141). Western blots of human observed in platelet number or function and so this potential platelet whole cell lysates stimulated with a range of agonists role of mDia1 in megakaryocytes requires further study. for 60 s were probed with apFHOD1 (Thr1141) to determine The most highly expressed of the formin proteins in mouse the extent of FHOD1 activation. In the presence of Integrilin, megakaryocytes, as indicated by number of SAGE tags, is which blocks platelet aggregation, marked phosphorylation of FHOD1. This formin is unique in that it is regulated by Ser/ FHOD1 was observed downstream of thrombin, which was Thr phosphorylation [23] and its activation can therefore be not altered in the presence of inhibitors of the secondary monitored using a phosphothreonine-speciﬁc FHOD1 anti- mediators ADP and TxA2 (Fig. 1A). In contrast, weak

A % of maximum B (i)Thrombin (ii) CRP phosphorylation 0 7 30 60 28 100 94 100 α pFHOD1 0 s 30 s60 s 90 s150 s300 s 0 s 30 s60 s 90 s150 s300 s α pFHOD1 α FHOD1 α FHOD1 Basal ADP (30U46619 µ ADP (3 µ+ CRPU46619 (10CRP +µg Indo (10mLThr µg (1 mLUThr mL (1 U mL

(iii) Thrombin M) + Indo –1 –1 + Integrilin – Integrilin M) + Apy + Indo –1 –1 ) + Apy) +– IndoApy – Indo ) + Apy) +– IndoApy – Indo

BasalThr 30Thr s 60Thr s 90Thr s 150Thr s 300Thr s30Thr s 60Thr s 90Thr s 150Thr s 300 s α pFHOD1

α FHOD1 C (i) (ii) (iii) 120 Thrombin + Integrilin 120 CRP120 Thrombin – Integrilin 100 100 100 80 80 80 60 60 60 40 40 40 20 20 (% of maximum) (% of maximum) (% of maximum) 20 0 0 FHOD1 phosphorylation FHOD1 FHOD1 phosphorylation FHOD1 FHOD1 phosphorylation phosphorylation FHOD1 0 0 30 60 90 150 300 0 30 60 90 150 300 0306090150300 Time after addition of agonist (s) Time after addition of agonist (s) Time after addition of agonist (s)

D E F –/– –/–

BasalBasal ThrombinThrombin CRP CRP Y27632 Wildtype Rac1 Wildtype Rac1 –1 – + – + – + –– + +Thrombin (0.1 U/mL ) α Basal Thr 60 s –Thr Y27632 60s + Y27632 pFHOD1 α pFHOD1 α pFHOD1 α FHOD1 α FHOD1 α FHOD1

α Rac1

Fig. 1. FHOD1 is present in human and mouse platelets and is threonine phosphorylated downstream of Rho kinase. (A) Representative Western blot and quantitation by densitometry of FHOD1 threonine phosphorylation in human platelets downstream of the major platelet agonists. All stimulations were performed in the presence of Integrilin. Although ADP and thromboxanes cause FHOD1 phosphorylation, phosphorylation downstream of thrombin is not dependent upon secondary mediator activity. (B) Temporal analysis of platelet stimulation with thrombin (Bi) and CRP (Bii) showed that FHOD1 phosphorylation occurred rapidly within 30 s of stimulation. For thrombin, phosphorylation peaked at 90 s and slowly declined although signiﬁcant phosphorylation was observed at 300 s after stimulation. For CRP stimulation, FHOD1 phosphorylation peaked at 60 s and thereafter declined rapidly so that at 150 and 300 s after stimulation FHOD1 phosphorylation was close to basal levels. Stimulation of platelets under aggregating conditions (Biii) appeared to a cause a reduction in FHOD1 phosphorylation at all time points measured, except for 30 s. (C) Quantitation of FHOD1 phosphorylation downstream of thrombin + Integrilin (Ci), CRP (Cii) and thrombin – Integrilin (Ciii) by densitometry. A signiﬁcant reduction in thrombin-induced phosphorylation under aggregating conditions was observed at 90, 150 and 300 s. (D) Phosphorylation of FHOD1 60 s after stimulation with thrombin or CRP is completely blocked by incubation with 20 lM of the Rho kinase inhibitor Y27632. (E) Mouse platelet FHOD1 is also phosphorylated downstream of thrombin in a Rho kinase-dependent manner. (F) FHOD1 phosphorylation in mouse platelets is independent of the presence of Rac1. All blots are representative of at least three independent experiments.

2011 International Society on Thrombosis and Haemostasis 1650 Letters to the Editor phosphorylation of FHOD1 was observed downstream of which unlike that in human platelets was sustained for up to GPVI by collagen related peptide (CRP) in the presence of 150 s after stimulation (data not shown). As for human apyrase and indomethacin, whereas stimulating platelets with platelets, FHOD1 phosphorylation in mouse was also depen- CRP in the absence of apyrase and indomethacin resulted in dent upon Rho kinase activity, being blocked by Y27632 strong FHOD1 phosphorylation (Fig. 1A). FHOD1 phos- (Fig. 1E). However, abrogation of Rac1 had no effect on the phorylation downstream of either ADP or the thromboxane phosphorylation of FHOD1 downstream of thrombin mimetic U46619 alone was weak, but these agonists acted (Fig. 1F), indicating that in platelets, Rac1 plays no role in synergistically to induce FHOD1 phosphorylation (Fig. 1A). FHOD1 activation. However, this does not exclude a role for There was no phosphorylation of FHOD1 in platelets spread Rac1 in ensuring correct localization of active FHOD1 as it has for 45 min on fibrinogen-coated dishes in the presence of been previously reported that Rac1 knockout platelets fail to apyrase and indomethacin (data not shown), although the spread fully [5]. non-synchronous nature of platelet spreading would mean In conclusion, using a serial analysis of gene expression that transient phosphorylation of FHOD1 would be missed. approach, we have identified FHOD1 as the most highly Analysis of the temporal dynamics of FHOD1 phosphor- expressed member of the formin family in mouse megakaryo- ylation following stimulation with thrombin or CRP showed cytes. Furthermore, we demonstrate that platelet FHOD1 that FHOD1 was strongly phosphorylated within 30 s of undergoes rapid phosphorylation in thrombin and CRP- agonist addition (Fig. 1Bi, ii). For thrombin, FHOD1 phos- stimulated platelets downstream of Rho kinase. FHOD1 is phorylation peaked at 90 s after stimulation with significant therefore a novel effector of Rho kinase in platelets and may be levels of phosphorylation being observed 300 s after stimu- involved in the formation of platelet stress fibres. Further lation (Fig. 1Bi,Ci). In contrast, stimulation with CRP peaked studies are required to establish the physiological role of at 60 s after stimulation and decreased rapidly so that by 150 FHOD1 in platelets downstream of Rho kinase. and 300 s after stimulation phosphorylation was only just above basal levels (Fig. 1Bii,Cii). To determine the effect of Acknowledgement integrin binding on FHOD1 phosphorylation, platelets were stimulated with thrombin and allowed to aggregate. Under This work was supported by the British Heart Foundation these conditions, maximum FHOD1 phosphorylation was (CH/03/003). observed at 30 s after stimulation before decreasing from 60 s onwards (Fig. 1Biii,Ciii). Statistical analysis showed that this Disclosure of Conflict of Interests reduction in phosphorylation was significantly different to The authors state that they have no conflict of interest. samples where aggregation was blocked at 90 s (P <0.05), 150 s and 300 s (P > 0.01). This is in contrast to the sustained phosphorylation observed in the presence of Supporting Information Integrilin, suggesting possible integrin regulation of serine/ Additional Supporting Information may be found in the online threonine phosphatases [24,25] or an increase in the speed of version of this article: platelet activation following engagement of the aIIbb3 integrin. Figure S1. Expression of formin proteins in megakaryocytes As FHOD1 has previously been shown to be phosphory- and platelets. lated downstream of Rho kinase [23], we investigated whether Figure S2. Analysis of platelets from the mDia1 knockout this was also true in platelets using a specific inhibitor of Rho mouse show no defects in platelet function. kinase, Y27632. Addition of 20 lM Y27632, a concentration Please note: Wiley-Blackwell are not responsible for the content previously shown to completely inhibit platelet Rho kinase [26], or functionality of any supporting materials supplied by the abrogated FHOD1 phosphorylation downstream of thrombin authors. Any queries (other than missing material) should be and CRP (Fig. 1D). Thus, FHOD1 is also regulated by Rho directed to the corresponding author for the article. kinase in platelets. Previous reports using GST fusions of Rac1 have suggested that FHOD1 interacts with Rac1 and that this may influence its References activity [27]. 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2011 International Society on Thrombosis and Haemostasis