Cellular characterization of thrombocytes in Xenopus laevis with specific monoclonal antibodies

Platelets are produced from megakaryocytes (MKs) in the bone marrow. In contrast, most nonmammalian vertebrates have nucleated and spindle-shaped thrombocytes instead of platelets in their circulatory systems, and the presence of MKs as thrombocyte progenitors has not been verified. In developing a new animal model in adult African clawed frog (Xenopus laevis), we needed to distinguish nucleated thrombocytes and their progenitors from other blood cells, because the cellular morphology of activated thrombocytes resembles lymphocytes and other cells. We initially generated two monoclonal antibodies, T5 and T12, to X. laevis thrombocytes. Whereas T5 recognized both thrombocytes and leukocytes, T12 specifically reacted to spindle-shaped thrombocytes. The T12+ thrombocytes displayed much higher DNA ploidy than nucleated erythrocytes, and they expressed CD41 and Fli-1. In the presence of CaCl2, adenosine diphosphate, thrombin, or various collagens, T12+ thrombocytes exhibited aggregation. These thrombocytes were located predominantly in the hepatic sinusoids and the splenic red pulp, suggesting that both organs are the sites of thrombopoiesis. Notably, circulating thrombocytes exhibited lower DNA ploidy than hepatic thrombocytes. Intraperitoneal administration of T12 produced immune thrombocytopenia in frogs, which reached a nadir 4 days postinjection, followed by recovery, suggesting that humoral regulation maintained the number of circulating thrombocytes. Although differences between MKs and thrombocytes in X. laevis remain to be defined, our results provide further insight into MK development and thrombopoiesis in vertebrates.