Rudy Fuentes

Infusion of mature megakaryocytes into mice yields functional platelets

Thrombopoiesis, the process by which circulating platelets arise from megakaryocytes, remains incompletely understood. Prior studies suggest that megakaryocytes shed platelets in the pulmonary vasculature. To better understand thrombopoiesis and to develop a potential platelet transfusion strategy that is not dependent upon donors, of which there remains a shortage, we examined whether megakaryocytes infused into mice shed platelets. Infused megakaryocytes led to clinically relevant increases in platelet numbers. The released platelets were normal in size, displayed appropriate surface markers, and had a near-normal circulating half-life. The functionality of the donor-derived platelets was also demonstrated in vivo. The infused megakaryocytes mostly localized to the pulmonary vasculature, where they appeared to shed platelets. These data suggest that it may be unnecessary to generate platelets from ex vivo grown megakaryocytes to achieve clinically relevant increases in platelet numbers.

Challenges and promises for the development of donor-independent platelet transfusions

Platelet transfusions are often a life-saving intervention, and the use of platelet transfusions has been increasing. Donor-derived platelet availability can be challenging. Compounding this concern are additional limitations of donor-derived platelets, including variability in product unit quality and quantity, limited shelf life and the risks of product bacterial contamination, other transfusion-transmitted infections, and immunologic reactions. Because of these issues, there has been an effort to develop strategies to generate platelets from exogenously generated precursor cells. If successful, such platelets have the potential to be a safer, more consistent platelet product, while reducing the necessity for human donations. Moreover, ex vivo-generated autologous platelets or precursors may be beneficial for patients who are refractory to allogeneic platelets. For patients with inherited platelet disorders, ex vivo-generated platelets offer the promise of a treatment via the generation of autologous gene-corrected platelets. Theoretically, ex vivo-generated platelets also offer targeted delivery of ectopic proteins to sites of vascular injury. This review summarizes the current, state-of-the-art methodologies in delivering a clinically relevant ex vivo-derived platelet product, and it discusses significant challenges that must be overcome for this approach to become a clinical reality.

Comparative analysis of human ex vivo-generated platelets vs. megakaryocyte-generated platelets in mice: A cautionary tale

Thrombopoiesis is the process by which megakaryocytes release platelets that circulate as uniform small, disc-shaped anucleate cytoplasmic fragments with critical roles in hemostasis and related biology. The exact mechanism of thrombopoiesis and the maturation pathways of platelets released into the circulation remain incompletely understood. We showed that ex vivo-generated murine megakaryocytes infused into mice release platelets within the pulmonary vasculature. Here we now show that infused human megakaryocytes also release platelets within the lungs of recipient mice. In addition, we observed a population of platelet-like particles (PLPs) in the infusate, which include platelets released during ex vivo growth conditions. By comparing these 2 platelet populations to human donor platelets, we found marked differences: platelets derived from infused megakaryocytes closely resembled infused donor platelets in morphology, size, and function. On the other hand, the PLP was a mixture of nonplatelet cellular fragments and nonuniform-sized, preactivated platelets mostly lacking surface CD42b that were rapidly cleared by macrophages. These data raise a cautionary note for the clinical use of human platelets released under standard ex vivo conditions. In contrast, human platelets released by intrapulmonary-entrapped megakaryocytes appear more physiologic in nature and nearly comparable to donor platelets for clinical application.

Structural and therapeutic insights from the species specificity and in vivo antithrombotic activity of a novel αIIb-specific αIIbβ3 antagonist

We previously reported on a novel compound (Compound 1; RUC-1) identified by high-throughput screening that inhibits human alphaIIbbeta3. RUC-1 did not inhibit alphaVbeta3, suggesting that it interacts with alphaIIb, and flexible ligand/rigid protein molecular docking studies supported this speculation. We have now studied RUC-1s effects on murine and rat platelets, which are less sensitive than human to inhibition by Arg-Gly-Asp (RGD) peptides due to differences in the alphaIIb sequences contributing to the binding pocket. We found that RUC-1 was much less potent in inhibiting aggregation of murine and rat platelets. Moreover, RUC-1 potently inhibited fibrinogen binding to murine platelets expressing a hybrid alphaIIbbeta3 receptor composed of human alphaIIb and murine beta3, but not a hybrid receptor composed of murine alphaIIb and human beta3. Molecular docking studies of RUC-1 were consistent with the functional data. In vivo studies of RUC-1 administered intraperitoneally at a dose of 26.5 mg/kg demonstrated antithrombotic effects in both ferric chloride carotid artery and laser-induced microvascular injury models in mice with hybrid halphaIIb/mbeta3 receptors. Collectively, these data support RUC-1s specificity for alphaIIb, provide new insights into the alphaIIb binding pocket, and establish RUC-1s antithrombotic effects in vivo.

Pleiotropic platelet defects in mice with disrupted FOG1-NuRD interaction

Understanding platelet biology has been aided by studies of mice with mutations in key megakaryocytic transcription factors. We have shown that point mutations in the GATA1 cofactor FOG1 that disrupt binding to the nucleosome remodeling and deacetylase (NuRD) complex have erythroid and megakaryocyte lineages defects. Mice that are homozygous for a FOG1 point mutation (ki/ki), which ablates FOG1-NuRD interactions, have platelets that display a gray platelet syndrome (GPS)-like macrothrombocytopenia. These platelets have few α-granules and an increased number of lysosomal-like vacuoles on electron microscopy, reminiscent of the platelet in patients with GATA1-related X-linked GPS. Here we further characterized the platelet defect in ki/ki mice. We found markedly deficient levels of P-selectin protein limited to megakaryocytes and platelets. Other α-granule proteins were expressed at normal levels and were appropriately localized to α-granule-like structures. Treatment of ki/ki platelets with thrombin failed to stimulate Akt phosphorylation, resulting in poor granule secretion and platelet aggregation. These studies show that disruption of the GATA1/FOG1/NuRD transcriptional system results in a complex, pleiotropic platelet defect beyond GPS-like macrothrombocytopenia and suggest that this transcriptional complex regulates not only megakaryopoiesis but also α-granule generation and signaling pathways required for granule secretion.