M J Metzelaar

To clone the CD63 antigen, originally described as a blood platelet activation marker, we adapted the expression cloning procedure of Seed and Aruffo (Seed, B., and Aruffo, A. (1987) Proc. Natl. Acad. Sci. U.S. A. 84, 3365-3369) to allow cloning of intracellular antigens. A megakaryocyte expression cDNA library was transiently transfected into MOP-8 mouse fibroblasts cultured on polyvinylidene difluoride membranes. Individual cells expressing intracellular CD63 were identified by autoradiography. cDNA was extracted from positive spots and reintroduced into Escherichia coli. After two screening rounds, a CD63 cDNA clone was isolated as assessed by immunofluorescence and Western blot analysis. The single long open reading frame of 238 amino acids contained four putative transmembrane regions and three N-glycosylation sites. The CD63 gene was expressed in a wide variety of cells. Surprisingly, CD63 was identical to ME491, an antigen reported as a melanoma-associated antigen (Hotta, H., Ross, A. H., Huebner, K., Isobe, M., Wendeborn, S., Chao, M. V., Ricciardi, R. P., Tsujimoto, Y., Croce, C. M., and Koprowski, H. (1988) Cancer Res. 48, 2955-2962). By immunoelectron microscopy, co-localization with the lysosomal glycoproteins lamp-1 and -2 identified CD63 as a novel lysosomal membrane glycoprotein. CD63 was not related to the lysosomal glycoprotein family but contained the putative lysosomal targeting signal Gly-Tyr in its short cytoplasmic tail.

We have characterized the mechanisms by which thrombin enhances neutrophil leukocyte (PMN) adhesion to human endothelial cells in vitro. Thrombin rapidly and transiently increased PMN adhesion by an action on the endothelial cells. The transience of the response was due to at least two factors: desensitization of the endothelial cell responsiveness to thrombin in the continued presence of the agonist; and the lability (t1/2 less than 15 min) of the effector molecules expressed by the endothelium. Experiments with exogenous platelet-activating factor (PAF) and with PAF antagonists demonstrated that PAF production, although it may facilitate the enhanced PMN adhesion seen in response to thrombin, is not sufficient to explain the reaction. By using a variety of antibodies directed against cell surface ligands, and comparing adhesion of PMN to endothelium and to protein-coated surfaces, we deduce that several endothelial ligands not previously reported as playing a role in PMN adhesion are involved in these interactions. Of particular interest was the finding that antibodies recognizing two thrombin-regulated endothelial cell surface ligands, GMP-140 and the CD63-related Ag, both inhibited adhesion of PMN to thrombin- or LPS-pretreated endothelium. We conclude that thrombin acts to enhance PMN adhesion to endothelium at least in part by transiently altering the conformation or level of expression of these ligands.

Although gamma delta T lymphocytes were identified several years ago, the functional importance of these cells remains to be established. gamma delta T cells of ruminants are unique in two respects. First, they are present at much higher levels compared to man and rodents. Second, ruminant CD4-CD8- gamma delta T cells uniquely express a 220 kD surface Ag recognized by a panel of mAb, recently clustered as WC1. WC1 has been most extensively studied in sheep with the use of the mAb T19. Here, we report on the isolation of a full length cDNA clone, encoding the WC1 Ag, from a COS cell cDNA expression library prepared from a bovine gamma delta T cell line. The protein encoded by the pWC1 cDNA clone was reactive with the bovine mAb CC15 and IL.A29, and with T19. The cDNA clone consisted of 4475 bp and contained a single long open reading frame of 1436 amino acids. The pWC1 cDNA clone encoded a type 1 integral membrane protein with an extracellular domain consisting of 11 scavenger receptor cysteine-rich-repeats with homology to CD5 and CD6. Southern blotting suggested that the bovine genome contained multiple sequences highly related to the isolated WC1 cDNA. Furthermore, WC1-like sequences were present in the genomes of all mammals tested including mouse and man. The molecular characterization of the WC1 Ag as reported here provides a starting point for the definition of its role in gamma delta T cell biology.

CD4-CD8- gamma delta T cells of ruminants uniquely express a 220-kDa surface Ag recognized by several mAbs clustered as WC1. We recently reported the isolation of a cDNA clone encoding a WC1 Ag. Southern blotting suggested that the bovine genome contains multiple sequences highly related to the isolated WC1 cDNA. Here, we demonstrate that some of the clustered WC1 mAbs stain predominantly nonoverlapping subsets of bovine CD4-CD8- gamma delta T cells. By the isolation of two additional cDNA clones encoding molecules highly related to the original WC1 Ag, we provide a molecular basis for this phenomenon. Cells transfected with cDNAs encoding individual WC1 Ags are differentially recognized by various WC1 mAbs. Thus, expression of members of the WC1 gene family divides bovine CD4-CD8- gamma delta T cells into phenotypical subsets. Field inversion gel electrophoresis revealed that all WC1 genes map to a single, large (> 1 Mbp) Notl fragment. Although the function of WC1 remains unknown, it likely involves interaction with ligands that originate from a similarly complex genetic system.

Changes in the platelet plasma membrane during activation were investigated by flow cytometry in a comparative study of in vitro platelet activation during platelet storage and cardiopulmonary bypass surgery. We studied changes in the expression of the plasma membrane glycoproteins lb and llla and CD31 antigen (PECAM-1), the alpha-granule membrane proteins GMP-140 (PADGEM, CD62 antigen) and GMP-33, and lysosomal integral membrane protein-CD63. A simultaneous change in the expression of the various glycoproteins induced by platelet activation was seen after thrombin stimulation in vitro and during platelet storage. Platelet activation in vivo in patients showed a more complex change in the expression of membrane glycoproteins. During cardiopulmonary bypass the mean fluorescence values for glycoprotein llla, GMP-33, and the percentage of GMP-140 and lysosome integral membrane protein-CD63 expressing platelets increased significantly. CD31 antigen expression was significantly decreased, whereas glycoprotein lb expression did not change. We conclude that flow cytometry is useful for the detection of changes in the expression of membrane glycoproteins induced by platelet activation in vitro and during platelet storage. Application of flow cytometry as clinical tool for screening platelet activation in patients or for identification of a prethrombotic state requires evaluation of a panel of platelet membrane glycoproteins because the changes in membrane expression may be different in various clinical situations.