SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics.

BACKGROUND
Reorganisation of the actin cytoskeleton is required for proper functioning of platelets following activation in response to vascular damage. Formins are a family of proteins which regulate actin polymerisation and cytoskeletal organisation via number of domains including the FH2 domain. However, the role of formins in platelet spreading has not been studied in detail.


OBJECTIVES
Several formin proteins are expressed in platelets and so we used an inhibitor of FH2 domains (SMIFH2) to uncover the role of these proteins in platelet spreading and in maintenance of resting platelet shape.


METHODS
Washed human and mouse platelets were treated with various concentrations of SMIFH2 and the effect on platelet spreading, platelet size, platelet cytoskeletal dynamics and organisation and were analysed using fluorescence and electron microscopy.


RESULTS
Pre-treatment with SMIFH2 completely blocks platelet spreading in both mouse and human platelets through effects on the organisation and dynamics of actin and microtubules. However, platelet aggregation and secretion are unaffected. SMIFH2 also caused a decrease in resting platelet size and disrupted the balance of tubulin post-translational modification.


CONCLUSIONS
These data therefore demonstrated an important role for formin mediated actin polymerisation in platelet spreading and highlighted the importance of formins in cross talk between the actin and tubulin cytoskeletons.


| INTRODUC TI ON
Platelets require a dynamic actin and tubulin cytoskeleton for proper maintenance of their resting size and shape, and for their response to vascular damage. Formins are a family of 15 actin-nucleating factors that promote the assembly of linear actin filaments downstream of the Rho family of small GTPases. 1 Formin proteins contain a number of domains including FH1 and FH2 1 and are known to function as homodimers. 2 Profilin-bound actin monomers associate with the FH1 domain of the formin dimer and are added to the growing actin filament by the action of FH2 domains, which act to both elongate the filament and protect it from capping proteins. [3][4][5] In addition to their role in nucleating actin filaments, formins have been shown to play an important role in microtubule organization and dynamics, including alignment of actin and microtubules, microtubule stabilization, and microtubule bundling 6,7 indicating a role for formin proteins in coordination of actin and microtubule cytoskeletons in cells. Furthermore, changes in formin protein activity have been associated with human disease including hearing loss, cancer invasion, and neuropathy. [8][9][10][11][12] We have previously reviewed expression databases for the 15 formin proteins in developing megakaryocytes and platelets at both the DNA and protein level. 7 We reported that only four of the 15 formins were expressed in human or mouse platelets; these being DAAM1, mDia1, FHOD1, and INF2 (see table 1 of Zuidscherwoude et al 7 for relative expression levels). The presence of DAAM1, mDia1, and FHOD1 has been confirmed in platelets by western blotting. 13 Studies on the mDia1 knockout mouse indicated no major phenotype in terms of platelet activation, aggregation, or spreading, possibly due to functional redundancy between the expressed formin members; 13 however, blocking mDia1 function in platelets using anti-mDia1 antibodies was shown to reduce platelet spreading. 14 mDia1 has been shown to play a role in megakaryocyte development and proplatelet formation (PPF) as knockdown of mDia1 using shRNA resulted in increased PPF. 15 Further studies on gain of function mutations in mDia1 have shown that constitutively active mDia1 leads to reduced PPF, and patients expressing gain of function mutations display (macro) thrombocytopenia. [15][16][17] Despite these studies, the contribution of formin proteins to platelet actin and microtubule organization remains unclear.
SMIFH2 was identified in 2008 by screening small molecules for their ability to inhibit actin assembly by mDia1 and mDia2. 18 This activity was attributed to the FH2 domain, as the ability to inhibit actin polymerization persisted when profilin and the FH1 domain were absent, and therefore SMIFH2 is a useful tool to block the FH2 domains of all expressed formins. In mammalian fibroblasts, SMIFH2 inhibited formin-dependent migration and reduced membrane integrity, but had little effect on Arp2/3 complex-dependent structures such as lamellipodia. 18 However, the effects of FH2 domain inhibition on the platelet cytoskeleton are not defined.
In the current study, human and mouse platelets were visualized with fluorescence and electron microscopy to observe changes to morphology and actin and tubulin dynamics in response to SMIFH2 treatment. Assays were performed with resting platelets, platelets undergoing spreading in response to activation by fibrinogen, and in platelet aggregate formation under flow conditions. Furthermore, the stability of microtubules following SMIFH2 treatment was investigated by fluorescence microscopy and western blotting to detect post-translationally modified populations of α-tubulin in resting platelets, indicative of stable and dynamic forms of the protein.

| ME THODS
Full detailed methods are provided in the Supporting Information.

| Effect of global formin inhibition on platelet function
We had previously hypothesized that the lack of a phenotype in the mDia1 knockout (KO) mice was due to a redundancy with other formin proteins expressed in platelets. 13 We tested this by treating both mouse and human platelets with a global formin inhibitor (SMIFH2) that blocks the FH2 domain of formins 18 to establish if formin activity was required for proper platelet function.

| Mouse platelets
In mouse platelets treated with increasing concentrations of SMIFH2, platelet spreading on fibrinogen is significantly reduced ( Figure S1C in supporting information). Control samples have more than 80% of platelets displaying a nodule/filopodia phenotype, characteristic of mouse platelets spreading on fibrinogen. 19 With increasing concentrations of SMIFH2 this decreases so that at 5 µmol/L approximately 90% of platelets are unspread (Figure 1Ai). Accordingly, platelet surface area was significantly reduced at both 4 µmol/L (P = .027) and 5 µmol/L (P = .013) SMIFH2 compared to controls (Figure 1Aii).

Essentials
• Actin dynamics are critical for proper platelet function.
• We established the contribution of formin proteins in platelets using an FH2 domain inhibitor.
• Formins are required for spreading and organization of the actin and microtubule cytoskeletons.
• Formin function is needed for maintenance of resting platelet size and tubulin ring acetylation.

| Human platelets
A similar effect on spreading is seen in human platelets to that observed in mouse platelets in that spreading is reduced ( Figure S1D).
To test the effect of formin inhibition on the F-actin content of platelets after activation, human platelets were incubated with SMIFH2 and F-actin content measured by flow cytometry. Data indicate that SMIFH2 reduced, but did not completely abrogate F-actin polymerization after activation of platelets with thrombin ( Figure   S2 in supporting information). To visualize these effects, staining of human platelet F-actin with Alexa488-phalloidin and imaging using SR-SIM reveals that SMIFH2 causes disruption to F-actin organization. Figure 2A shows control platelets at various stages of spreading displaying characteristic F-actin organization including nodules, filopodia, lamellipodia, and stress fibers. Pretreatment with SMIFH2 prior to spreading causes disruption to this organization so that many platelets display poorly organized F-actin and platelets fail to spread properly ( Figure 2B). Tubulin organization is also disrupted; at 5 µmol/L SMIFH2 platelets show neither microtubule coils, nor spread cell microtubule networks ( Figure S1D). To establish whether this effect was restricted to platelets spreading on fibrinogen, we also tested the effect of SMIFH2 on platelets spreading on fibrinogen following pre-activation with thrombin (0.1 U/mL) and on collagen-coated surfaces. In the presence of thrombin, spreading is completely inhibited by 5 µmol/L SMIFH2 (surface area P = .0001; Figure S3A in supporting information). On collagen surfaces SMIFH2 completely blocked spreading in both mouse and human platelets at 2 µmol/L ( Figure   S3B,C). To further visualize the cytoskeleton, mouse platelets treated with 5 µmol/L SMIFH2 or dimethyl sulfoxide (DMSO) were imaged using electron microscopy. In control-treated cells, the actin formed the characteristic pattern for fully spread platelets ( Figure 2C), whereas in platelets treated with SMIFH2, the majority of platelets failed to spread, displayed poorly organized actin, and microtubule coils ( Figure 2D). To confirm that the observed effects were not due to nonspecific inhibition of platelet function, human platelets pretreated with SMIFH2 were tested for aggregation and secretion responses to collagen and thrombin. No significant difference was observed for platelets treated with 5 µmol/L SMIFH2 compared to control ( Figure   S4A-D in supporting information). We also tested α-granule secretion and integrin activation in platelets treated with 5 µmol/L SMIFH2 by flow cytometry and although small reductions were observed, platelets were still able to express P-selectin on their surfaces and to bind fibrinogen ( Figure S4E,F). Together these data indicate that the observed effects on spreading are not due to nonspecific inhibition on platelet signalling or due to death of the platelets.

| Live platelet spreading
To determine the effect of FH2 inhibition on cytoskeletal and spreading dynamics, platelet spreading on fibrinogen was followed in real time using differential interference contrast (DIC) microscopy for both human and mouse platelets and spread platelet area measured at a range of time points in an approach similar to that of Paknikar et al. 20 In control mouse platelets, mean surfaces area remained fairly constant for the first 60 seconds after attachment as the platelets began spreading and extended and retracted filopodia. From about 60 seconds onward, the platelets started to form lamellipodia and increased in size ( Figure 1C). A similar pattern was observed for control human platelets, although the formation of lamellipodia happened faster than in mouse platelets ( Figure 1D). In the presence of 5 µmol/L SMIFH2, both mouse and human platelets failed to spread and remained in this state for the duration of the imaging ( Figure 1C To investigate this effect in more detail, platelets from the Lifeact-GFP mouse were loaded with SiR-Tubulin 21 to allow visualization of the F-actin and microtubule dynamics. Platelets were then allowed to spread on fibrinogen-coated coverslips in the presence or absence of 5 µmol/L SMIFH2. Still images of representative platelets can be found in Figure 3A,B. Example videos can be found in Videos S1-S4 in supporting information. In control cells, the characteristic spreading stages are observed including the generation of filopodia, actin nodules, lamellipodia, and stress fibers as well as the twisting and contraction of the microtubule coil ( Figure 3A). In platelets pretreated with 5 µmol/L SMIFH2, spreading dynamics and cytoskeletal organization are completely disrupted. Occasional small protrusions can be observed in treated platelets; however, these seemed to form by blebbing rather than by the conventional actin-mediated extension. No evidence of "normal" filopodia or lamellipodia formation was observed. Actin nodules appeared, but only one or two were seen per platelet and these did not turn over as observed in control platelets; in addition, the platelets did not form stress fibers. Tubulin dynamics were also disrupted in SMIFH2-treated cells; upon adhesion/ activation, tubulin rings were less obvious than in control platelets and the characteristic twisting and coiling of the ring was not observed. The tubulin ring also seemed to depolymerize much more quickly than in control cells, which often displayed a remnant ring even when fully spread ( Figure 3A,B; Videos S1-S4).
The above data indicate that platelet spreading requires formin proteins, as global inhibition of FH2 domains blocks platelet spreading. To establish if continued formin activity is required after platelet spreading, mouse or human platelets were allowed to spread for 45 minutes on fibrinogen before addition of SMIFH2 for 30 minutes (Figure 4). In both human ( Figure 4C) and mouse ( Figure 4A) platelets, incubation with DMSO vehicle control for 30 minutes had no effect on platelet spreading. However, in human platelets incubated with 2 µmol/L SMIFH2, many platelets significant reduction in spread platelet area (P = .04; Figure 4D).
A similar situation was observed in mouse platelets; however, the morphological changes were less clear due to the reduced spreading of mouse platelets on fibrinogen ( Figure 4C). Despite this, a significant reduction in spread platelet area (2 µmol/L, P = .006; 5 µmol/L, P = .008) was observed ( Figure 4B). These data indicate that continued formin activity is required once platelets have spread to maintain stress fibers and lamellipodia and hence spreading. Taken together these data show that global inhibition of formin proteins in mouse and human platelets disrupts actin and tubulin dynamics and prevents platelet spreading and that continued formin activity is required to maintain this spread morphology.

| Flow data
To establish the effect of inhibiting formin activity on platelet thrombus formation under shear conditions, whole human blood was treated with SMIFH2 prior to flow assays. Surprisingly, no effect of inhibition of FH2 domains was observed on the formation of Video S5 in supporting information). In addition, no effect was seen when the concentration of SMIFH2 was increased to 50 µmol/L.
To test the hypothesis that the inhibitor was being strongly bound by plasma proteins, washed platelets and platelet-rich plasma (PRP) were treated with either 0, 5, or 50 µmol/L SMIFH2 before spreading on fibrinogen and staining for F-actin. The effect of SMIFH2 was completely abrogated in PRP, even when the concentration of inhibitor was increased 10 times ( Figure S5C).
As we have demonstrated that continued formin activity is required for maintenance of platelet spreading (Figure 4), we performed experiments in which thrombi were allowed to form for 10 minutes and then were washed for 20 minutes in buffer containing 0, 5, or 25 µmol/L SMIFH2. No effect of formin inhibition was observed on the size of these preformed aggregates when measured as total surface area coverage at the end of the experiment ( Figure S5D) or when assessing the change in individual thrombi size pre-and postwashing ( Figure S5E).
In addition to the change in diameter of resting platelets, superresolution structured illumination microscopy (SR-SIM) imaging of the resting actin cytoskeleton demonstrates that inhibition of FH2 domains causes the loss of the F-actin network identified in control resting platelets ( Figure 6A; Videos S6 and S7 in supporting information). This is accompanied by rounding of the cells (as can be observed by visualizing orthogonal projections of SR-SIM z stacks), compared to control platelets, which retain their discoid shape ( Figure 6B). To further establish the effect of FH2 inhibition, the cytoskeleton of resting platelets treated with either DMSO or 5 µmol/L SMIFH2 was visualized by electron microscopy ( Figure 6C).
Platelets treated with inhibitor for 10 minutes were smaller than controls and the extensive, filamentous F-actin network of the platelets was disrupted ( Figure 6C, upper panel). This effect was even more prominent after 3 hours of SMIFH2 treatment and in addition, resting platelets were considerably smaller and displayed microtubule coils which seemed to display more coils ( Figure 6C, lower panel).
These data therefore indicate that the FH2 domains of formin proteins are important for the maintenance of resting platelet size and cytoskeletal organization.

| Effect of FH2 inhibition on microtubule posttranslational modifications
Formins can bind to microtubules via their FH2 domain and have been shown to regulate microtubule dynamics independently of effects on actin dynamics. 22 Formins have been implicated in the acetylation of microtubules, a modification which, in combination with detyrosination, marks stable microtubules. 23 We therefore

| D ISCUSS I ON
The data presented in this study indicate that the FH2 domain of formin proteins is critical for normal cytoskeletal organization and dynamics of platelets, as a dose-dependent inhibition of platelet spreading by SMIFH2 was observed in both human and mouse platelets. While the data are generated using a single pharmacological inhibitor of formin function, it is well established that the FH2 domain is highly conserved across species and is absolutely required for the actin polymerization action of formin proteins. However, data from models of reduced Arp2/3 complex expression show that full spreading is lost, but platelets are still able to form filopodia, 26,27 presumably via the action of formins and the presence of preformed actin filaments. The observation that adding SMIFH2 to platelets that had already spread resulted in the loss of stress fibers and lamellipodia collapse (Figure 4) would seem to support the hypothesis that lamellipodia formation and persistence requires formin-mediated actin filaments. It is interesting that adding SMIFH2 to preformed platelet thrombi had no effect on their size or stability, even when added at five times the concentration that blocks platelet spreading ( Figure S5).
Whether this reflects that actin filaments/lamellipodia are not required for aggregate stability at this stage, as has recently been reported by Schurr et al, 28 is an intriguing possibility.
The and appearing to depolymerize more rapidly than in controls.
Thus, it would appear that there is an effect of SMIFH2 on microtubules in addition to the inhibition of actin polymerization.
There is substantial evidence that formins can regulate microtu-  In conclusion, we demonstrate that the FH2 domain of formin proteins plays a key role in the organization and dynamics of the platelet actin and tubulin cytoskeletons ( Figure 8). The fact that mDia1 knockout platelets show no spreading phenotype would seem to support the hypothesis that multiple formin proteins can fulfil this role during platelet activation and thus the challenge is now to identify the contribution that each of the platelet-expressed formin proteins makes to this process.

CO N FLI C T S O F I NTE R E S T
The authors declare no competing financial interests.