Human apolipoprotein E (apoE) is a 34 kDa protein present in the surface of circulating lipoproteins. ApoE is synthesized predominantly by the liver and brain. Its major function is to mediate cholesterol transport, uptake and redistribution through interaction with members of the LDLreceptor super family. When the apoE polypeptide is dysfunctional or absent, neurological disorders and atherosclerosis in humans or animal models ensue.We have recently proposed a new cell-signalling role for apoE (I). We discovered that the potent anti-platelet effect of apoE (2) was due to enhanced production of endogenous nitric oxide (NO); apoE markedly elevated platelet NO synthase activity and intra-platelet levels of cGMP. The initial step was an apoE-receptor interaction; apoE bound saturably to platelets (3), while neutralizing the arginine residues of apoE abolishes the effect (I). Further support for this concept is provided by our observation that lactofenin, though less potent than apoE, also exhibits anti-platelet properties (2). This glycoprotein is also bound by saturable sites in platelet plasma membranes (4) and has sequence homology to the arginine and lysine enriched binding domain of apoE (5). Interestingly, both proteins are recognised by the well-characterised LDL-receptor-related protein (LRP).The LRP is a ubiquitously expressed endocytic receptor that binds a diverse p u p of ligands including lipoprotein lipase, proteases, protease inhibitors, bacterial toxins and viruses, as well as apoE containing lipoproteins and lactoferrin. LRP is synthesized as a 600 kDa precursor that is cleaved to generate an amino-terminal 515 kDa (LRP 515) fragment and a carboxyl-terminal 85 kDa (LRP 85) fragment. LRP 515 harbours all known ligand binding sites and remains noncovalently associated with LRP 85, which contains the membrane anchor and the cytoplasmic domain (6). The LRP is considered the ancestral prototype of multifunctional endocytic receptors within the LDL-receptor gene family and since platelets lack conventional LDL-receptors (7), it may be a candidate cell-surface protein for mediating the anti-platelet effects of apoE. Therefore, in the present study we have investigated whether platelets contain LRP both by Western blotting and by reverse transcriptmepolymerase chain reaction (RT-PCR).Recently, a monoclonal antibody has been raised against LRP 5 15, which has been used to investigate the expression of the LRP on a variety of cell types (8). This antibody designated a2MR2, was used to probe membrane extracts from both platelets and the megakaryocytic cell line, HEL, for the presence of LRP.Membrane extracts of the hepatocarcinoma cell line, HepG2, and purified placental LRP served as controls.As shown in Figure I, although both HepG2 extracts and purified LRP readily stained a 500 kDa species, HEL cell and platelet extracts were devoid of staining. Thus, these results indicate the absence of the LRP 5 15 polypeptide by Western blotting.
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