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

Abstract

Background Reorganization of the actin cytoskeleton is required for proper functioning of platelets following activation in response to vascular damage. Formins are a family of proteins that regulate actin polymerization and cytoskeletal organization via a 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 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 effects on platelet spreading, platelet size, platelet cytoskeletal dynamics, and organization were analyzed using fluorescence and electron microscopy. Results Pretreatment with SMIFH2 completely blocks platelet spreading in both mouse and human platelets through effects on the organization 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 polymerization in platelet spreading and highlighted the importance of formins in cross‐talk between the actin and tubulin cytoskeletons.

Loss of mDia1 and Fhod1 impacts platelet formation but not platelet function

“…It is probable that 40% formin mediated-actin polymerizing activity, alongside redundancy between actin polymerization regulatory pathways, is sufficient for “normal” platelet function. Indeed, there are examples of formin redundancy recorded in the literature [ 46 ], and this hypothesis fits with our observation of a dose-dependent effect on platelet spreading of the FH2 domain inhibitor SMIFH2 [ 17 ]. It is interesting to note that in patients with a DIAPH1 gain of function mutation, a similar effect was observed; namely, disrupted platelet production, but a relatively mild platelet functional defect [ 14 ], and in Arp2/3 knockout mice, twinfilin/cofilin1 double knockout mice and ADF/cofilin knockout mouse, similar discrepancies between alterations to platelet production and mildly affected hemostasis are observed [ 47–49 ].…”

“…However, reports from shRNA knockdown of mDia1 in human CD34 + cells and patients expressing a gain of function mutation in mDia1 demonstrate an effect on platelet formation [ 14 , 24 ]. Furthermore, we have recently demonstrated the importance of formin FH2 domains for platelet function through use of a global FH2 domain inhibitor [ 17 ]. Therefore, we generated a transgenic mouse model for the most abundant platelet formin, Fhod1 (Fhod1 KO), which we have previously shown is activated upon platelet stimulation [ 16 ], and compared platelet function and formation to wild type (WT) mice.…”

“…We have recently showed that blocking formin-mediated actin polymerization using a selective inhibitor blocks platelet spreading and causes increased coiling of the platelet microtubule coil [ 17 ]. To establish the effect that loss of specific formin proteins have on the organization of the resting platelet cytoskeleton, we stained fixed, resting platelets with α-tubulin antibodies and used SIM imaging to visualize the microtubule coil.…”

“…Formins are actin nucleation-promoting factors and we have previously demonstrated the role of FH2 domains in regulating platelet spreading [ 17 ]. Therefore, we monitored the effect of the loss of mDia1, Fhod1, or both proteins on platelets spreading.…”

“…We have previously shown that platelets from an mDia1 knockout mouse were functionally normal and hypothesize that this was due to redundant function with other platelet/megakaryocyte expressed formin family members [ 16 ]. In support of this, we have recently reported that blocking formin FH2 domains using a small molecule inhibitor (SMIFH2) completely abolishes platelet spreading and disrupts microtubule dynamics [ 17 ], highlighting the role of formins in regulating both actin and microtubule platelet cytoskeletons.…”