Differentiation Route Generates Functional Biased Platelets and Responses Distinctly to Varying Physiological Demands

Platelets, small circulating anucleated cells generated by megakaryocytes, are essential for hemostasis and immunity. Our previous study (Immunity, 2024) developed a fate-mapping system (Cd48^dre; R26^rox-tdTomato mouse model) to reveal that megakaryocytes can differentiate via both direct and stepwise routes, and both the routes contribute equally to platelet production under physiological condition. However, the functional outputs of platelets derived from these distinct differentiation routes, as well as their responses to varying biological demands, have yet to be fully elucidated.

Given the crucial role of platelets in hemostasis and immune response, we first investigated whether there is the functional bias of platelets derived from distinct differentiation routes under physiological conditions. Using the Cd48^dre; R26^rox-tdTomato mouse model, we could distinguish platelets derived from these routes via Tomato expression. Platelets developed through the stepwise route expressed Tomato, while those from the direct differentiation route were Tomato^-. Upon thrombin activation, platelets derived from both routes exhibited adhesion to fibrinogen, but the Tomato^- platelets demonstrated superior adhesion ability and higher expression of integrin GPIIb/IIIa, which mediates platelet adhesion and aggregation, compared to Tomato⁺ platelets. Moreover, we cocultured platelets from different routes with DQ-OVA, a self-quenched conjugate of OVA that emits fluorescence upon proteolysis. We found that platelets capable of internalizing and proteolyzing exogenous antigens were mostly Tomato+ platelets. Incubating purified neutrophils with activated platelets further revealed significant interactions between Tomato⁺ platelets and neutrophils. Additionally, we observed that the expression of P-selectin (CD62P) on Tomato⁺ platelets was three times higher than Tomato^- platelets after stimulation, suggesting a greater interaction of Tomato⁺ platelets with immune cells. These data suggest that although platelets derived from both routes are capable of adhesion, the direct differentiation route endows platelets with enhanced coagulation ability, while the stepwise differentiation route equips platelets with potent immune responses.

Next, we investigated how the direct and stepwise differentiation routes respond to demands for rapid platelet production. We established acute thrombocytopenia in Cd48^dre;R26^rox-tdTomato mice by administering a single intraperitoneal injection of anti-GPIbα (2 μg/g body weight), which caused a drastic drop in platelet count that gradually recovered after ~3 days. Interestingly, during platelet recovery, the vast majority of newly produced platelets were Tomato⁺ (up to 70% at day 5), suggesting that the stepwise route may respond more rapidly instead of the direct route. This increased proportion was also observed in megakaryocytes and megakaryocyte progenitors (MkPs). In vitro expansion of MkPs revealed that stepwise-derived MkPs exhibited significantly greater proliferation capacity following platelet depletion. Conversely, when 5-Fluorouracil (5-FU) was used to induce myeloablation and assess platelet recovery after chemotherapy, Tomato^- platelets, derived from the direct differentiation route, exhibited rapid production initially, reaching up to 80% during recovery at day 7. To explore how the direct and stepwise differentiation routes respond to inflammation, we injected LPS intraperitoneally into Cd48^dre;R26^rox-tdTomato mice. The percentage of Tomato⁺ platelets increased within 12 hours, accompanied by elevated expression of MHCII, indicating that the stepwise route confers stronger immune characteristic to meet the inflammatory stress.

This study establishes the link between differentiation route and functions of platelets, and characterizes the two different routes can generate functional biased platelet output with differential kinetics in responding to varying physiological demands.