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In vivo role and mechanism of platelet migfilin
important integrin-binding proteins such as kindlins and filamin A, which can potentially profoundly influence the function of aIIbβ3.5-7 Thus, kindlin-3 co-operates with talin and positively regulates the activation of integrin
Migfilin gene expression was quantified by real-time quantitative PCR based on SYBR Green, using -actin RNA as the loading con- trol.16 The sequence of the primers used were as follows: migfilin forward primer (mFBLIM1-qRCR-F): TAGCCGTGAGTGAG- GAAGTG, reverse primer (mFBLIM1-qRCR-R): CAGAGAGT- GAGGCATTGGTCT.
Migfilin peptides
As previously reported,13 WT migfilin (4KPEKRVASSVFITLAP19C) and mutant (MT) peptides (4KPEKR- VADSAFITLAP19C) with an additional C-terminal cysteine residue were synthesized by ChinaPeptides (Suzhou, China). WT- migfilin peptide harbors the filamin A binding sequence and the MT-migfilin peptide contains two point mutations (Ser11 was replaced by Asp, and Val13 was replaced by Ala, bold type in the sequence) with substantially reduced ability to bind filamin A (at least 12.5-times weaker than the WT peptide).13 To render them cell permeable, a CR7 transport peptide was conjugated to the migfilin peptides through a disulfide bond.17 The conjugated pep- tides were purified by high pressure liquid chromatography and conjugation confirmed by electrospray ionization mass spectrom- etry.
Tail bleeding assay
As previously described,18 tails of anesthetized mice were cut 0.5 cm from the tip and immediately immersed in microtubes filled with 1.7 ml of saline at 37°C. The time for the bleeding to stop (no blood flow for 1 min) and the weight of blood loss were recorded. Tail bleeding assays were stopped at 900 seconds if the bleeding did not stop. The same procedure was used for the eval- uation of migfilin peptides in hemostatic function after their intra- venous injection.
Platelet preparation
Whole blood was collected from the inferior vena cava into 0.1 vol of ACD buffer (75 mM sodium citrate, 39 mM citric acid, and 135 mM dextrose, pH 6.5), and was diluted 1:2 with modified Tyrode’s buffer (in mM: 20 HEPES, 137 NaCl, 13.8 NaHCO3, 2.5 KCl, 0.36 NaH2PO4, 5.5 glucose, pH 7.4). Diluted whole blood was centrifuged at 180×g for 10 minutes (min) at room temperature, and platelet-rich-plasma was collected into a fresh tube. The platelet-rich-plasma was diluted in ACD buffer, and centrifuged at 700×g for 10 min. Platelet pellet was then re-suspended in modi- fied Tyrode’s buffer.
Detailed information of the reagents, platelet function measure- ments, Western blotting and statistical analysis is provided in the Online Supplementary Materials and Methods.
Results
Migfilin deficiency impairs hemostasis and thrombosis.
The truncation of exon 7 of migfilin in platelets was val- idated by PCR measurement of migfilin messenger RNA (Figure 1A). Multiple commercially available antibodies (Santa Cruz Biosciences sc-162823, sc-162822, sc-134724; ABclonal A15850; GeneTex GTX116584) were used to detect migfilin. These antibodies did not clearly reveal the expression of migfilin protein in murine platelets, although they did confirm the presence of migfilin in murine hearts as previously reported (Figure 1B). Real- time quantitative PCR experiments were performed as a surrogate method to quantify migfilin expression in murine platelets. The results not only confirmed the loss of migfilin mRNA in the platelets from KO mice, but also showed
aIIbβ3,3 whereas filamin A competitively blocks the talin-integrin interaction5 and negatively regulates aIIbβ3.3,8 Intriguingly, the interactions between integrin β3 and integrin-binding proteins are highly dynamic, even the same pair of interactive partners may serve distinctive roles in different phases of the platelet activation pathway. For example, talin-β3 binding is the essential step for aIIbβ3 activation for inside-out signaling, whilst the subsequent G13-regulated dissociation and re-association between talin and β3 are not only pivotal steps for out- side-in signaling but are also the crucial events that differentiate thrombosis from hemostasis.9 This example highlights the importance of clarifying the dynamic inter- action between integrin and integrin-binding proteins during aIIbβ3 activation. However, the spatial temporal dynamics for most other integrin-binding proteins remain largely unknown.
Migfilin, also known as FBLIM1/FBLP-1,10 is a putatively expressed protein consisting of an N-terminal filamin- binding domain, a central proline rich domain and three C-terminal LIM domains. Through participation in cytoskeleton reorganization, migfilin is involved in cellu- lar functions such as adhesion, morphological change, and motility.11 Migfilin has also been reported to promote car- diomyocyte differentiation.12 There are multiple binding partners of migfilin, including mitogen inducible gene-2 (MIG-2), VASP, and transcriptional factor CSX/NKX2-5.10 Particularly, migfilin is capable of strong interaction with filamin A, which raises the possibility that migfilin could competitively dissociate filamin A from integrin and thus promote talin-aIIbβ3 interaction.13 Indeed, migfilin pep- tides are capable of inducing PAC-1 binding in human platelets.14 However, these findings were obtained in in vitro conditions and neither the contribution of migfilin to thrombosis and hemostasis in vivo nor to integrin dynamics during platelet activation are clear.
With migfilin-/- mice, the current study investigated the role of migfilin in platelet activation, hemostasis and thrombosis. The phenotype exhibited by migfilin-/- mice indicated that migfilin is an important positive regulator for hemostasis and thrombosis. Mechanistically, migfilin promotes early outside-in signaling of platelet aIIbβ3, pos- sibly through binding to filamin A and sequestering the aIIbβ3-inhibiting effect of filamin A.
Methods
Generation of migfilin deficient mice
Migfilin-/- mice were bred as described,15 ablation of migfilin was achieved by the deletion of exon 7. The loss of exon 7 of migfilin in migfilin-/- platelets was confirmed by mRNA expression, using littermate wild-type (WT) mice platelets as a control. All experimental procedures were reviewed and approved by the Animal Care and Use Committee of the Zhejiang University School of Medicine. Total RNA of platelets and heart was extract- ed using Trizol (Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions. RNA was reversely transcribed to cDNA using the ReverTraAce qPCR RT kit (Toyobo, Osaka, Japan). PCR was performed using the primers P1: GCTGTTGAG- GCCATGAAGAG and P2: TCCTTCCCATGCACTCGATT.
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