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In vivo role and mechanism of platelet migfilin
that expression of migfilin mRNA is about 1,500-fold lower in platelets than that in heart of WT mice (normalized by actin mRNA) (Figure 1C). Migfilin-/- mice were viable and fertile, and did not exhibit any evident bleeding tendency or thrombotic events over their lifespan. Migfilin-/- mice did not differ significantly from their WT littermates in platelet counts, white cell counts, hematocrits, and hemo- globin concentrations (Table 1). Electron microscopy showed that migfilin-/- platelets had normal discoid mor- phology (Online Supplementary Figure S1A). No significant differences in the surface expression of the platelet glyco- proteins GPVI, CD41 (aIIb subunit) and CD42b (GPIb subunit) were found between WT and migfilin-/- platelets (Online Supplementary Figure S1C).
Notably, migfilin-/- mice exhibited tail-bleeding times twice as long as their WT littermates (Figure 1D), consis- tent with the comparison of the weight of blood loss between migfilin-/- and WT mice (Figure 1E). Moreover, 63.6% of the migfilin-/- mice had bleeding times exceeding 15 min, in comparison, only 14.3% of WT littermates had bleeding times over 15 min (Figure 1F). These data suggest that migfilin positively regulates hemostasis.
In a model of FeCl3-induced mesenteric arteriole throm- bosis, the time of forming stable occlusive thrombi in mesenteric arterioles were significantly longer in migfilin-/- mice than in WT mice (35.19±3.656 min vs. 20.63±4.422 min, P<0.01; Figure 1G). To confirm that the observed hemostatic functional defects are due to migfilin deficien- cy, we synthesized cell-deliverable migfilin peptides as described in the previous study.13 WT-migfilin-CCR7 pep- tide (range: 0.5-1.5 nM/kg) dose-dependently shortened the prolonged tail bleeding time in migfilin-/- mice (Online Supplementary Figure S2A), whereas MT-migfilin-CCR7 peptide did not show any effect on the bleeding time. WT- migfilin-CCR7 peptide restored the prolonged bleeding time of migfilin-/- mice to a comparable level of WT mice (Online Supplementary Figure S2B-C). WT-migfilin-CCR7, but not MT-migfilin-CCR7, also restored the occlusion time in Fecl3-induced mesenteric arterial injury in migfilin-/- mice (Online Supplementary Figure S2D).
Thrombus formation was also assessed using a microflu- idic whole-blood perfusion assay. After a 5-minute perfu- sion at a shear rate of 1000 s-1, thrombi formed on an immo- bilized collagen surface by migfilin-/- platelets were signifi- cantly smaller (average of a 50% reduction) than those by WT platelets (Figure 1H). In the meanwhile, as shown in the Online Supplementary Video S1, thrombi formed by migfilin-/- platelets displayed a severely compromised stabil- ity compared to WT platelets. In order to assess whether the observed phenotype of migfilin-/- platelets is due to a reduced ability of adhesion during the initiation of throm- bosis, a recombinant whole-blood system with a reduced concentration of platelets (107/mL) was employed in the perfusion assay. Without the interference from massive platelet aggregation, migfilin-/- and WT platelets had similar coverage area on the collagen surface (Online Supplementary Figure S3). These findings indicate that migfilin promotes thrombosis, possibly through influencing the extension and perpetuation of thrombi.
Migfilin-/- platelets have a decreased capability of aggregation due to a hampered dense granule secretion
To further evaluate the function of migfilin in platelets, aggregation and dense granule secretion in response to
Table 1. Hematologic analysis. WT
Migfilin-/-
10.96±1.63
14.93±8.92
562±268.03
49.40± 7.27
16.5±2.677
5.3±0.18
P
0.6974
0.8345
0.3570
0.4680
0.4883
0.4198
RBC×1012/L
WBC×109/L Platelets×109/L Hematocrit % Hemoglobin g/dL
MPV fL
10.72±1.01
14.26±4.51
676±271.26
47.40±4.45
15.8±1.62
5.2±0.33
Data are ± standard error of the mean (SEM). No abnormalities or significant differences between wild-type (WT) and migfilin-/- mice were found for hematologic parameters (n = 3-5; unpaired Student t test). RBC: red blood cells; WBC: white blood cells; MPV: mean platelet volume.
common platelet stimuli were measured. Compared to WT platelets, migfilin-/- platelets displayed a prominently decreased aggregation rates in response to low dose col- lagen (0.4 mg/mL), and a mildly decreased aggregation rates in response to thrombin (0.018 U/mL) (Figure 2A). Platelet aggregation induced by ADP (10 mM; 20 mM), U46619 (0.3 M; 0.6 M) or higher concentrations of thrombin (0.025 U/mL) and collagen (0.8 mg/mL) was not affected by migfilin deficiency. Notably, migfilin-/- platelets exhibited a markedly decreased ATP secretion in response to all stimuli including U46619, thrombin and collagen, even when the aggregation difference was no longer present (Figure 2A). Apyrase (0.25 U/mL) hydrolyzed secreted ADP and eliminated the aggregation difference between WT and migfilin-/- platelets induced by low concentrations of thrombin or collagen (Figure 2B). Supplementation with a low concentration of ADP (1 mM), insufficient to induce aggregation on its own, reversed the inhibitory effect of migfilin deficiency on collagen- and thrombin-stimulated platelet aggregation (Figure 2B). In addition, thrombin- and collagen-induced secretion of serotonin from migfilin-/- platelets was largely reduced compared to that of WT platelets, even though similar levels of serotonin were harbored inside resting migfilin-/- and WT platelets (Online Supplementary Figure S4). These data suggest that an impaired dense granule secretion is the central functional defect exhibited by migfilin-/- platelets.
WT-migfilin-CCR7 (5 mM), but not MT-migfilin-CCR7 (5 mM), rescued the impaired aggregation and ATP release of washed migfilin-/- platelets in response to thrombin or collagen, and eliminated the difference between WT and migfilin-/- platelets (Figure 3A-B). This result supports that the observed platelet phenotypes in the current study are bona fide migfilin effects.
Interestingly, an integrin aIIbβ3 inhibitor tirofiban (4 g/mL) eliminated the differences of aggregation and ATP secretion between migfilin-/- and WT platelets in response to thrombin and collagen (Online Supplementary Figure S6A-B), suggesting that migfilin-regulated platelet function is aIIbβ3-dependent. However, neither the secretion of -granules nor the conformational change of
aIIbβ3 on a single platelet, indicated by P-selectin expres- sion and JON/A binding, respectively, was affected by migfilin deficiency or exogenously applying of migfilin peptide (Online Supplementary Figure S7). These findings therefore suggest that migfilin may not be involved in inside-out signaling of aIIbβ3, but rather participate in the process post ligand-aIIbβ3 engagement.
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