Desialylation and Apoptosis Crosstalk to Modulate Platelet Clearance

Our understanding of cell biological processes involved in aging has advance greatly over the past decades. Platelets are small cells that circulate for 4-5 days in mice and 7-10 days in humans. And even though, platelets are anucleated cells, a growing body of evidence shows that platelet clearance is a well-regulated mechanism. We have recently demonstrated that platelets lose sialic acid as they circulate and age in blood and are rapidly cleared by the hepatic Ashwell-Morell receptor (AMR). And others have shown, in a series of studies using genetically modified mice or pharmacological inhibitors that platelets undergo apoptosis by triggering the intrinsic mitochondrial apoptotic machinery. Here, we investigate if desialylation and apoptosis are related events.

First, using a newly developed state-of-the-art technique called dynamic BH3 profiling (DBP), we investigated the mitochondria readiness to undergo apoptosis on platelets derived from WT and AMR deficient (Asgr2^-/-) mice. In our assay, digitonin-permeabilized platelets were exposed to activators signaling peptides (such as Bim, Bid and PUMA), and as cells undergo apoptosis due to peptide treatment, they released Cytochrome C. Our data showed that desialylated platelets derived from Asgr2^-/-mice have high background levels of Cytochrome C release when compared to WT platelets in the presence of all activator peptides, indicating that desialylated platelets are highly primed to apoptosis. We also tested the level of dependence on pro-survival protein, by using sensitizer peptides (Bad, Hrk and MS1). We observed that desialylated platelets (Asgr2^-/-platelets), and to a certain degree, WT platelets, are extremely sensitive to BCL-xL inhibition, as indicated by the extremely high response to Bad and Hrk peptides even at lower concentrations (0.1 and 1uM). Surprisingly, WT and Asgr2^-/-platelets show very little response to the MS1 peptide, indicating that they are not dependent on MCL1 for survival, as otherwise suggested.

Flow cytometry analysis revealed desialylated platelets from Asgr2^-/-mice have a ~2-fold increase in Phosphatidylserine (PS) surface exposure when compared to WT platelets. In addition, western blot analysis showed increased expression of cleaved caspase 3 in Asgr2^-/-platelets, but no changes in Bcl-xL protein expression between WT and Asgr2^-/-platelets.

Next, WT and Asgr2^-/-mice received a single dose of the BH3 mimetic, ABT-737, which binds and inhibits pro-survivor proteins (Bcl-2, Bcl-xL and Bcl-w) inducing apoptosis in vivo. Approximately 2 hours after the injection of ABT-737, we observed a big drop on platelets counts in both WT (~42%) and Asgr2^-/-(~59%) mice. Importantly, platelets from Asgr2^-/-mouse were cleared more efficiently (~20%) from the circulation when compared to those in WT mice, consistent with the ~20% increment in platelet number observed in this mouse model and supporting the notion that the platelets that circulate longer in the Asgr2^-/-mice are more sensitive to apoptotic events.

To investigate if apoptosis could be triggering platelet desialylation, WT mice were treated with ABT-737 and 1hour later (time point before platelet count drop), platelets were collected and analyzed by flow cytometry. Interestingly, analysis of galactose exposure by RCA-I lectin showed no differences in desialylation between ABT-737 and PBS control groups. On the other hand, Phosphatidylserine (PS) exposure was significantly elevated on ABT-737 group, indicating that platelets were undergoing apoptosis without changing their sialylated status. To confirm our in vivodata, freshly isolated washed WT platelets were treated with ABT-737 to induce apoptosis or Neuraminidase (NA) to desialylated platelets. NA treatment induced platelet desialylation (increased RCA-I binding) in WT platelets, as expected, and interestingly triggered apoptosis, judge by increased PS exposure in both ABT-737 and NA treated groups. However, ABT-737 treatment wasn't able to induce desialylation as levels of RCA-I binding to platelets was the same when compared to PBS control platelets.

Taken together, our data shows that desialylated platelets in circulation are prone to apoptosis. In addition, our findings strongly support the hypothesis that desialylation of platelet surface glycoproteins trigger the intrinsic apoptotic pathway in platelets in vivo.