Identification of human megakaryocyte coagulation factor V.
Specific monoclonal and polyclonal antibody reagents and a double antigen indirect immunofluorescence microscopy technique were used to visualize coagulation factor V in human bone marrow. Marrow aspirates were smeared directly on glass slides, or washed and cytospun onto glass slides, or processed and plated into a plasma/methylcellulose cell culture system. Morphologically identifiable colonies of megakaryocytes, erythrocytes, granulocytes, or monocytes/macrophages were removed from 14- to 18-day marrow culture dishes by micropipette and streaked onto glass slides. Smears of marrow cell preparations were air-dried, fixed, washed, and incubated sequentially with primary IgG antibody reagents and with secondary anti-IgG antibody reagents conjugated with either fluorescein or rhodamine. Preparations were examined and photographed through a microscope suitably equipped for two-color fluorescence and phase contrast analysis. Cells of megakaryocytic lineage were identified by their immunofluorescent reactivity with murine monoclonal antibody HP1-1D, specific for human platelet plasma membrane glycoprotein IIb/IIIa (GP IIb/IIIa), or by their immunofluorescent reactivity with monoclonal or polyclonal antibodies specific for von Willebrand factor (vWF) or for platelet factor 4 (PF4). Coagulation factor V in bone marrow was detected by simultaneous immunofluorescent staining with polyclonal burro anti-human factor V antibody or with a panel of murine monoclonal anti-human factor V antibodies. The double antigen immunofluorescence staining technique, incorporating appropriate controls, revealed that coagulation factor V was principally located in marrow cells simultaneously identified as megakaryocytes by antibodies to GP IIb/IIIa, vWF, or PF4. The specific immunofluorescence of factor V in megakaryocytes and platelets was eliminated when excess purified factor V antigen was preincubated with anti-factor V antibody. Our observations establish the presence of human megakaryocyte coagulation factor V, confirm the presence of human platelet factor V, and indicate that human megakaryocyte/platelet coagulation factor V is a lineage-associated protein.
Nichols
W L
WL
Gastineau
D A
DA
Solberg
L A
LA
Mann
K G
KG
D000818:Animals; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000032:analysis; D001854:Bone Marrow Cells; D000939:Epitopes; D005165:Factor V / Q000032:analysis* / Q000276:immunology; D006041:Goats / Q000276:immunology; D006801:Humans; D007106:Immune Sera; D008533:Megakaryocytes / Q000032:analysis*; D051379:Mice; D008856:Microscopy, Fluorescence; D010978:Platelet Factor 4 / Q000032:analysis; D011817:Rabbits; D014841:von Willebrand Factor / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007106:Immune Sera; D014841:von Willebrand Factor; D010978:Platelet Factor 4; D005165:Factor V