Infusing Mature Megakaryocytes Into Mice Yields Functional Platelets: Biological and Clinical Implications.

Abstract 225

Thrombopoiesis, the process by which circulating platelets (plts) arise from megakaryocytes (Megs) remains incompletely understood. Two models had been proposed: (1) release of plts produced from Megs within the marrow space and (2) release of plts by circulating Megs within the pulmonary vasculature. Recently, murine two-photon electron microscopy suggested that the actual process may involve an intermediate model, wherein Megs adjacent to vascular spaces within the marrow shed large cytoplasmic fragments into the circulation, which presumably go on to form plts in the vascular space. We have now tested whether infused cultured Megs can form circulating, functional plts. Cultured Megs from the fetal livers of Day 14 wildtype (WT) C57Bl6 murine embryos grown in the presence of thrombopoietin for 10 days were separated using a bovine serum albumin gradient into two cell fractions: (1) large, mature CD41+/CD42+ Megs (average ploidy = 16–32N) and (2) an admixture of proplatelets and small CD41+/CD42+ Megs (average ploidy = 2-4N). These fractions were separately infused into hαIIb mice that expressed human, but not mouse, αIIb on their platelets. Donor-derived circulating plts were detected using species-specific antibodies to distinguish them from recipient hαIIb+ plts. Infusions of isolated WT plts as a positive control resulted in immediately detectable donor plts, and these plts lasted 24–36 hrs. Infused large Megs produced plts with delayed kinetics. Few donor plts were detectable at 5 mins, followed by a peak at 30–60 min. These plts were normal in size as determined by side-scatter and lasted for ∼24 hrs. The peak yield was ∼50-100 plts/infused Meg. In these studies, these derived plt levels reached as high as 10% of the total circulating plts in these mice who had plt counts of >8×105/μl. Maximal tolerated dose of infused large Megs was not tested. Infusion of the proplatelets/small Megs fraction generated an immediate peak of circulating plts and these lasted <24 hrs. The yield of plts was approximately equal to the number of infused proplatelets/platelets in this admixture. Half-lives of the derived plts were unaffected by the inclusion of GM6001 or TAPI-1, both of which have been reported to block metalloproteinase MMP9/ADAM17 and lengthen the half-life of culture-derived plts. Functionality of the derived plts from both cell fractions was compared to infused plts in situ using the laser-injury cremaster system. Qualitatively, derived plts were incorporated as well as infused plts into growing thrombi. Remarkably, we also detected large CD41+ cells, which circulated and occasionally became incorporated into thrombi. These cells were much more common in the proplatelet/small Meg infusions than in the large Megs infusion. Thus, our studies show that whole, mature Megs shed a significant number of plts in circulation within mice. Plts level achievable in the recipient mouse approximate a clinically relevant level. These plts are normal in size and appear to be functional. Further studies to demonstrate where plts are released from these Megs and the basis for their shortened half-life remain to be performed; however, these studies suggest an alternative source of circulating platelets and a novel strategy for generating sufficient plts from cultured Megs for clinical use.