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BCL2L2 promotes MK numbers and PPF
nists (Figure 2A-C). It is worth noting that although PS- positivity marked apoptotic SHG MK, some fraction of LLG MK were also capable of PS exposure in response to thrombin stimulation (Online Supplementary Figure S7), perhaps akin to collagen and thrombin activated (COAT) platelets.31-33 Thus, simple gating on flow cytometric for- ward and side scatter measurements, without the need for fluorescently-labeled antibodies, identified functional MK and can improve signal-to-noise read-outs that test the functionality of candidate MK/platelet genes.
Apoptosis was established as a mechanism for platelet lifespan by the Kile laboratory when they showed that the balance between Bcl-xL and Bak regulate murine lifespan in vivo.20 The pro-survival Bcl-2 family member Bcl-xL degrades in aging platelets, thus allowing apoptosis to pro- ceed. In addition to Bcl-xL, the Bcl-2 family includes Bcl- 2, Mcl-1 and Bcl-w, which are encoded by BCL2, MCL1 and BCL2L2, respectively. Altered murine expression of pro-survival Bcl-2 family members has shown variable effects on platelet number. Global chimeric vav-Bcl2 over- expression resulted in normal MK numbers but an approx- imately 50% reduction in platelet counts,34 whereas MK- specific deletion of Bcl2 had no effect on platelet number.35 MK-specific deletion of Bcl2l1 deletion caused MK apop- tosis, loss of platelet shedding, and a macrothrombocy- topenia.12 MK-specific deletion of Mcl1 did not affect MK number or morphology, or platelet count or volume.13 Print et al. globally inactivated Bcl2l2 to study its impor- tance in murine physiology.36 Although Mkpoiesis was not a major focus of their study, they reported that three
Bcl2l2-/- mice displayed numbers of MK colony-forming cells and platelets comparable to three wild-type litter- mates. This latter report appears to differ from our finding in primary human MK and platelets, where BCL2L2 increased during MK differentiation, overexpression of BCL2L2 increased the numbers of both the viable CD41a+ LLG MK and MK PPF (Figure 4E and F), and BCL2L2 levels correlated with platelet number (Figure 5A). This apparent discrepancy between human and mouse regarding the effect of BCL2L2 on MKpoiesis and platelet production could be due to a compensatory upregulation of other Bcl- 2 family members in the Bcl2l2 null mice. Alternatively, small numbers of mice and different conditions may have led to a chance finding in the Print et al. study,36 but there also may be species differences in the relative importance of BCL2L2 because human platelets contain 3.6-fold high- er levels of BCL2L2 transcripts than mouse platelets.37
We showed that the SHG MK were derived from the LLG MK (Figure 2D and E), supporting a process by which viable LLG avoid apoptosis long enough to mature and acquire mature MK markers, but an unknown trigger induces some cells to become dying SHG. The day 9 to day 13 increase in BCL2L2 is expected to restrain apopto- sis in LLG cells, whereas BCL2L2 reduction should enable apoptosis to proceed in SHG cells (Figure 3C). Perhaps a yet-to-be-defined switch regulates BCL2L2 levels after day 9 in cultured cells. CB-derived LLG MK began to develop proplatelet extension around day 12 and peaked at days 13-15, similar to what had been reported by Balduini et al.38 Because SHG appear days before PPF MK (Online
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Figure 6. Relationship of BCL2L2 to blood platelet number and cultured platelet-like particles (PLP). (A) BCL2L2 is positively associated with peripheral blood platelet counts in 154 healthy individuals in the PRAX1 study. (B) Forward scatter and side scatter flow cytometric analysis of human peripheral blood platelets (Peripheral Plts), day 13 PLP and the merged image. (C-E) Flow cytometric assessment of cultured PLP after MK transduction with empty lentiviral vector (Ctrl lenti) or lentiviral vector containing BCL2L2. PLP were defined by forward scatter, side scatter and presence of platelet-specific surface marker (see Methods). (C) The per- centages of day 13 PLP that were CD41a-positive (+) (n=6). (D) The percentages of day 13 PLP that bound PAC1 after stimulation with 250 nM thrombin (n=5). (E) The percentages of day 13 PLP that expressed P-selectin after stimulation with 250 nM thrombin (n=5).
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