Novel Monoclonal Antibody Reactive with Thrombin-Sensitive 74-kDa Glycoproteins Present on Platelets and Megakaryocytes Both from Mouse and Rat

Abstract

A monoclonal antibody (designated 1C2) that reacts only with mouse platelets and megakaryocytes among hematopoietic cells was established by immunizing mouse platelets to an Armenian hamster. 1C2 reactive mouse molecule (1C2 antigen) was a surface glycoprotein with molecular weight of 74 kDa. Side by side comparison revealed that 4A5, a rat monoclonal antibody against mouse platelet, immunoprecipitated the identical molecule to 1C2 antigen. Of particular interest, 1C2 also labeled rat tissues with an identical pattern to that of mouse tissues and recognized a 74-kDa protein from rat platelets. Reactivity of 1C2 to mouse and rat platelets decreased when they were treated with thrombin. Following thrombin treatment of mouse platelets, 1C2 reactive 69-kDa protein appeared in the supernatants. Mouse and rat 1C2 antigens purified on 1C2-coated beads were cleaved by thrombin to generate 69-kDa fragments, establishing that 1C2 antigen is a direct substrate for thrombin. 1C2 is the first antibody to platelets and megakaryocytes of mouse and rat whose reactive molecule is well characterized, i.e., substrate for thrombin. 1C2 can be a useful tool in studying megakaryocytopoiesis and thrombopoiesis in rodent systems.

Multipotential differentiation ability of GATA-1-null erythroid-committed cells

“…PE-conjugated anti-CD71 (e-Bioscience), biotinylated anti-GPV (Seikagaku Corp.) (Takada et al 1995), anti-GPIIb, biotinylated Mac-1, biotinylated or PE-conjugated GR-1, FITC-conjugated FcR␥, biotinylated B220, biotinylated Thy-1.2, biotinylated or APC/Cy7-conjugated anti-c-KIT, PE-conjugated anti-CD34, and biotinylated or APC-conjugated SCA-1 antibodies (Pharmingen) were used for FACS analysis. The biotinylated antibodies were visualized by PE-or Cychrome-conjugated streptavidin (Pharmingen), and the GPIIb antibody was visualized by PE-conjugated anti-Rat IgG antibody (Pharmingen).…”

An Expandable, Inducible Hemangioblast State Regulated by Fibroblast Growth Factor

“…When differentiated in conditions similar to those previously employed to generate macrophages ( Choi et al., 2011 ), the eHBs produced CD45 + /CD11B + (Mac-1) macrophages ( Figures 2 F and S1 G). When differentiated in Stemline II medium (Sigma) supplemented with SCIF and the megakaryocyte cytokine TPO, the cells gave rise to megakaryocytes and proplatelets, as indicated by the dual expression of the markers CD42D ( Takada et al., 1995 ) and CD41 ( Figure 2 G) and the presence of multinucleated cells ( Figure S1 H). Finally, when differentiated in medium supplemented with SCIF, the eHBs also produced, albeit quite inefficiently, BFU-Es (erythrocyte progenitors), CFU-G/M/GMs (granulocyte and/or macrophage progenitors), and CFU-GEMMs (granulocyte, erythrocyte, macrophage, and megakaryocyte progenitors) ( Figure S1 I).…”

GATA-2 and GATA-2/ER display opposing activities in the development and differentiation of blood progenitors

“…Total cell numbers at days 8 and 11 in the presence of EPO and those at day 8 in the presence of TPO are shown in Figure 4A–C. Cell surface expression patterns of an erythroid marker, TER‐119 (Ikuta et al ., 1990), and a megakaryocyte marker, platelet glycoprotein V (GPV) (Takada et al ., 1995; Sato et al ., 2000), on the cells induced in the presence of EPO and TPO, respectively, are shown in Figure 4D–F. Enforced expression of GATA‐2 increased the number of primitive erythrocytes, definitive erythrocytes and megakaryocytes by ∼4‐, ∼7‐ and ∼7‐fold, respectively.…”