Surface Ifitms on Megakaryocytes and Platelets Regulate Fibrinogen Endocytosis Under Inflammatory Conditions

Background

IFITM proteins (IFITM-1, -2, and -3) mediate cellular resistance to influenza, dengue, and other viruses. IFITM expression on human platelets has not been previously recognized.  Our laboratory recently demonstrated that IFITMs are robustly expressed by human platelets and megakaryocytes after stimulation by pathogens and inflammatory mediators and restrict viral infection. IFITMs, which are interferon inducible, also mediate clathrin localization and associated protein endocytosis.   Nevertheless, whether IFITMs regulate protein endocytosis by platelets and megakaryocytes remains unknown.  

Aims

We investigated IFITM expression on murine megakaryocytes and platelets and determined whether IFITMs regulate fibrinogen endocytosis under basal and inflammatory conditions.

Methods

We examined the expression of IFITMs and clathrin in bone-marrow derived murine megakaryocytes and platelets under basal conditions and following interferon-beta (IFN-β) stimulation.  To determine whether upregulation of IFITM causes increased fibrinogen endocytosis, megakaryocytes were stimulated ex vivo with IFN-β and treated with labeled fibrinogen.  Endocytosis of labeled fibrinogen was then measured by immunocytochemistry and flow cytometry.  To determine whether this response also occurred in vivo, C57Bl/6 mice were injected intraperitoneally (IP) with 50,000 units of IFN-β over four days.  On the fourth day, 100 μg of labeled fibrinogen was injected into the tail vein and the amount of endocytosed, labeled fibrinogen in platelets was determined the next day via flow cytometry.  Parallel experiments were performed in age and gender matched IFITM^-/-mice.

Results

Bone-marrow derived murine megakaryocytes and platelets basally express IFITMs.  Upon IFN-β stimulation, IFITM and clathrin expression significantly increased (p<0.05).  Fibrinogen endocytosis by murine megakaryocytes occurred under resting conditions and appeared to be punctate and granular in nature.   Upon IFN-b stimulation, fibrinogen endocytosis in megakaryocytes significantly increased compared to unstimulated conditions (p<0.004).  The increase in endocytosis appeared independent of changes in αIIbβ3 expression as IFN-β stimulation did not change αIIbβ3 surface protein.  Fibrinogen endocytosis after IFN-β stimulation did not increase in megakaryocytes from IFITM^-/- mice, suggesting that IFITMs regulate fibrinogen uptake under these conditions.  We next determined if fibrinogen endocytosis occurred in platelets isolated from IFITM^-/- mice.   Platelet counts and activation indices (assessed by JonA staining) were similar in C57Bl/6 mice (WT) and IFITM^-/- mice.  Nevertheless, the injection of IFN-β IP results in significant increases in fibrinogen endocytosis by platelets in vivo in WT but not IFITM^-/-mice (p<0.02).

Summary/Conclusions

These findings suggest IFITMs, in addition to their anti-viral roles, mediate fibrinogen endocytosis.  Further, in settings where inflammatory stimuli such as interferons are increased, enhanced IFITM expression may promote upregulation of fibrinogen endocytosis by platelets and megakaryocytes.