Structural and functional characterization of the mouse von Willebrand factor receptor GPIb-IX with novel monoclonal antibodies

Five novel monoclonal antibodies (mAbs; p0p 1-5) were used to characterize the structural and functional properties and the in vivo expression of the murine GPIb-IX complex (von Willebrand factor receptor). The molecular weig hts of the subunits are similar to the human homologs: GPIb alpha (150 kd), GPIb beta (25 kd), and GPIX (25 kd). Activation of platelets with thrombin or PMA predominantly induced shedding of glycocalicin (GC; 130 kd) but onl y low levels of receptor internalization. The GC concentration in normal mo use plasma was found to be at least 10 times higher than that described for human plasma (approximately 25 mu g/mL versus 1-2 mu g/mL), Two additional cleavage sites for unidentified platelet-derived proteases were found on G PIb alpha; as demonstrated by the generation of 3 N-terminal fragments duri ng in vitro incubation of washed platelets (GC, 60 kd, 45 kd). Occupancy of GPIb alpha with p0p mAbs or F(ab)(2)-fragments resulted in aggregate forma tion in vitro and rapid irreversible thrombocytopenia in vivo, irrespective of the exact binding epitopes of the individual antibodies. GPIb-IX was no t detectable immunohistochemically on endothelial cells In the major organs under normal or inflammatory conditions. The authors conclude that the mou se system might become an Interesting model for studies on GPIb-IX function and regulation. (C) 2000 by The American Society of Hematology.