PRODUCTION OF A MONOCLONAL-ANTIBODY AGAINST CANINE GMP-140 (P-SELECTIN) AND STUDIES OF ITS VASCULAR DISTRIBUTION IN CANINE TISSUES

Rapid up regulation of the adhesion molecule GMP-140 (P-selectin) on e ndothelial cells is believed to play an important role in the initial binding of leukocytes to endothelium, a very early step in the inflamm atory response. Activated platelets that are involved in the coagulati on system and in inflammatory processes also express GMP-140 on their surfaces. The objectives of the present study were to develop a monocl onal antibody against this adhesion molecule in the dog and to use thi s antibody to study platelet-neutrophil interactions in whole blood an d to characterize the in vivo localization of GMP-140 in canine tissue s. Five Balb/c mice were immunized with thrombin-stimulated dog platel ets, and clones were screened using an enzyme-linked immunosorbent ass ay. The clone MD3 (IgG1) showed preferential binding to activated as c ompared with resting platelets. Flow cytometric analysis using MD3 rev ealed that 27% of circulating neutrophils in unstimulated blood had pl atelets bound to their surfaces; stimulation with platelet activating factor increased this percentage to 85%. Immunoblot analysis of solubi lized dog platelets resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the antibody MD3 recognized an appr oximately 140-kd protein. Immunohistochemical study of normal dog tiss ues with MD3 revealed that the antigen was present in endothelial cell s of arteries, capillaries, and veins, depending on the specific tissu e examined. Blood vessels staining positively with MD3 were most abund ant in the digestive system (liver, stomach, small and large intestine s), moderate in the lungs, kidneys, spleen, lymph nodes. and endocrine glands, and minimal in the brain, myocardium, skeletal system, and sk in. Based on its presence on stimulated but not resting platelets, its molecular weight, and its vascular distribution, the antigen recogniz ed by MD3 appears to be the selectin GMP-140 of the dog. This study do cuments that the cellular and tissue distribution of GMP-140 in dogs i s very similar to that in human beings.