Comparative biochemical and ultrastructural studies of P-selectin in rabbit platelets

Abstract

The role of platelets in thrombotic vascular disease has been widely studied in rabbits. Yet, in rabbit platelets, there is little known about the α-granules, which contain many of the key effector molecules for thrombosis. In this comparative study of rabbit platelets, we have characterized the structure and expression of P-selectin, an α-granule membrane protein that mediates leukocyte adhesion and thrombus propagation. The sequences of tryptic peptides of rabbit P-selectin show an overall sequence identity of 74% with human P-selectin, and 69–77% identity with cow, dog, mouse, rat and sheep P-selectins. The mean (±S.D.) apparent molecular mass of reduced rabbit P-selectin is 117±7 kDa which is ∼8 kDa larger than the unreduced protein (109±5 kDa). Rabbit P-selectin appears smaller than human P-selectin, but is comparable to other species P-selectins, that have fewer ‘complement regulatory protein’ repeat domains. Cell membrane labeling experiments and antibody binding studies indicate that rabbit P-selectin is nearly absent from the surface of resting platelets (290±30 molecules cell −1). However, cellular activation with thrombin causes nearly a 30-fold increase in expression to 14 200±1100 molecules cell −1. P-selectin is also be expressed on the surface of rabbit platelets activated by other agonists like ADP, A23817 and epinephrine. This selective expression is explained by immunoelectronmicroscopic studies, which show that rabbit P-selectin is sequestered in the intracellular granules of resting platelets. After cell activation by thrombin, P-selectin is found decorating the external membranes of platelet pseudopodia and the surface connected canalicular system. In summary, these studies of P-selectin in rabbit platelets indicate that it is similar in structure, cell localization and expression to human and other species P-selectins. This suggests that studies of P-selectin in thrombosis in rabbits are likely to provide useful insights into the role of this molecule in human thrombotic vascular disease and related conditions.