Page 329 - Haematologica Vol. 109 - July 2024

LETTER TO THE EDITOR
made proplatelets) or 12 hr (1:10 dilution: 4.0+2.6% of mega- karyocytes made proplatelets; 1:100 dilution: 3.5+2.0% of megakaryocytes made proplatelets).
To begin to define PPF, we carried out a detailed charac- terization of the proplatelet-promoting activity contained in the S100 cytosol. We adapted a strategy used to char- acterize prions9 and systematically added chemical agents and detergents to PPF to determine whether they altered its ability to induce proplatelet formation (Figure 3A). We found that the proplatelet-triggering activity of the crude S100 extract was stable for months at -80°C, and for at least three weeks at 4°C. However, it was unstable at higher temperatures; at 25°C and 37°C, the activity was lost after 5 hr. Further, PPF lost its ability to stimulate proplatelet production when exposed to protein-destroying treatments such as phenol, proteinase K, or trypsin for 1 hr at 25°C. However, pre-incubation of proteinase K with PMSF restored PPF activity. Notably, there was no loss in PPF activity after nucleic acid-destroying treatments including UV radiation, ribonuclease, or deoxyribonuclease. Non-denaturing, ionic detergents (at 2.5%) such as Triton X-100, Nonidet P-40,
octyl glucoside, and Tween-20 also did not inactivate the agent. In contrast, the denaturing detergent SDS inactivated PPF at a concentration of 1%. High-speed centrifugation to remove membranes from the S100 also failed to inactivate PPF. There was also no loss in PPF activity when S100 was dialyzed (molecular weight cutoff 5,000 kDa), suggesting PPF is not a small molecule.
Given that our previous studies demonstrated that inhib- itors of protein synthesis blocked proplatelet initiation,10 we wondered whether injection of PPF could rescue this phenotype and overcome the loss of proplatelet formation with protein synthesis inhibition. Indeed, injection of PPF caused megakaryocytes treated with puromycin to avidly begin proplatelet production (Figure 3B-D). Together, these data provide evidence for a megakaryocyte-intrinsic protein or multiple proteins, PPF, that is a master trigger for plate- let production. Further, they suggest that PPF is present in megakaryocytes as they undergo proplatelet formation and justify future studies examining its potential existence in platelets. While PPF is sufficient to trigger proplatelet formation, we recognize that external factors in the bone
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E
Figure 2. Data substantiating a proplatelet-promoting factor. (A) Representative images of megakaryocytes (MK) 12-hours (hr) post microinjection showing that round MK injected with S100 from proplatelet-producing (PP) MK continue to develop classic beads- on-string proplatelets. Scale bar = 10 μm. Original magnification = 40x. (B) Percentage of MK making proplatelets 12-hr post micro- injection. N=3 biological replicates. Data are presented as mean + Standard Deviation (SD). (C) Percentage of MK making proplatelets 24-hr post microinjection. N=3 biological replicates. Data are pre- sented as mean + SD. (D) Graph showing that the frequency of proplatelet initiation is proportional to the volume of injected S100 from PP MK. Volume is in nL and cells assayed 1 hr after injection. N=3 biological replicates. Data are presented as mean + SD. Round MK were injected with indicated volume of S100 from PP MK. (E) Representative images showing the effect of the addition of cyto- sol on round MK. Cytosol (S100 supernatant) was placed in the culture medium at dilutions of 1:10 and 1:100. (Top) Representative images of round MK cultured with cytosol from PP MK. (Bottom) Representative images of round MK cultured with cytosol from round MK. N=3 biological replicates. Scale bar = 80 μm. Original magnification = 10x. P<0.0001.
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