Platelet Progenitors after Leaving Bone Marrow
Authors: Kowata Shugo , Togawa Ryo , Okano Yoshiaki , Isogai Sumio , Hitomi Jiro , Murai Kazunori , Ito Shigeki , Ishida Yoji 
Published: October 2019

Abstract
Background: In vitro observations have indicated that preplatelets, which are anucleate discoid particles that can reversibly into proplatelet fragments, are one of platelet progenitos from cultured megakaryocytes (MKs) (Thon JN, et al. JBC 2010). Recently, we identified that bone marrow (BM) MKs form and release two types of platelet progenitors, using intra-vital imaging and ultra-structural analysis of mice BM (Kowata S, et al. Thrombosis and Haemostasis 2014). However, in vivo, how the platelet progenitors translate into individual platelets after leaving BM remains poorly understood. To elucidate whether platelet progenitors have an ability of conversion into individual platelets, we performed following experiments.
Methods: We isolated platelet progenitors rich fraction from whole blood of enhanced green fluorescence protein (EGFP) transgenic mice by the centrifugation (100 g, 15 min) and bovine serum albumin gradient. Mature platelets were isolated from whole blood of EGFP mice by the centrrifugation (100 g, 15 min). In vivo: The freshly enriched progenitor rich fraction or mature platelets, both of which were positive for EGFP, was transfused into wild type mice. The peripheral blood was obtained and analyzed by flow cytometry continually. The EGFP positive mature platelets in platelet gate were counted. In vitro: The freshly enriched progenitor isolates were cultured in IMDM medium at 37°C. The continual change of their characteristics in the shape and size were assessed using fluorescent microscopy with high resolution. Time-lapse images of the processes were also captured. All animal procedures were approved by the Institutional Animal Care and Use Committee of Iwate Medical University.
Results: The length and size of platelet progenitors were extremely varied (Fig. 1). The red, yellow, green, and dark blue segments in the bar chart indicate the platelet progenitors (5um<), while blue and light blue segments indicate the mature platelets (5um>). The frequency of platelet progenitor was 4% at normal condition (Fig. 1 graph on the left), but increased remarkably to 3-fold at recovery from acute thrombocytopenia (Fig.1 graphs in the middle). The maximum length of platelet progenitor was more than 200 µm (Fig. 1 picture in the top right). These data were consisted with the concept from our previous study of intra-vital imaging of BM MKs (Thrombosis and Haemostasis 2014). In vivo: Flow cytometric analysis showed that there was a time-dependent increase in EGFP platelet number in the gate of mature platelet, after the injection of EGFP positive platelet progenitor rich fraction. The control was carried out using EGFP positive mature platelets instead of platelet progenitors rich fraction. (mean ± SD, 0 h: 100%, 3h: 111 ± 6.8%, 6h: 105 ± 7.1% n=3, control 0 h: 100%, 3h: 101± 6.0%, 6h: 94 ± 5.2% n=3, Data were shown as % of the number of EGFP positive mature platelets at 0 h. P<0.01). These data suggest that the infused platelet progenitors convert into mature platelets as time passed. In vitro: The platelet morphogenesis from platelet progenitors was observed as highly dynamic process, shown as follows. (1) A discoid platelet progenitor, which was several times bigger in diameter than mature platelet, extended to a string with some swellings, a proplatelet. (2) A long proplatelet became shorter with a fusion of swellings, and changed into a berbel shape proplatelet. Eventually, it snapped to become two platelet-sized particles. These data suggest that platelet progenitors have an ability of conversion into individual platelets. Conclusion: These data indicated that, after leaving BM sinusoid, platelet progenitors flow into a central vein and convert into individual platelets in the blood stream.
Figure 1. Figure 1.

Disclosures
Ishida: Bristol-Myers Squibb: Honoraria.