Identification and characterization of LRP8 (apoER2) in human blood platelets

Abstract

Recently, we reported that apoE inhibits platelet reactivity by stimulating NO release and postulated apoE-receptor activation of intracellular NO synthase (eNOS). Here, we implicate a low density lipoprotein receptor (LDL-R) family member by studying ligand requirements using purified apoE isoforms, synthetic peptides, and the receptor antagonist, receptor-associated protein (RAP). Then, using a homology cloning approach and degenerate PCR primers to amplify the conserved Cys-rich binding domain of the LDL-R family, this receptor was identified as LRP8 (formerly termed, apoER2), a newly described brain protein with several splice variants. Immunoprecipitation of platelet membranes with anti-peptide antisera confirmed protein expression, while analysis of RNA from platelets and two megakaryocytic cell lines (Meg-01 and HEL) disclosed that the major LRP8 transcript lacked binding repeats 4–6 (LRP8Δ4–6) but contained the full-length cytoplasmic tail. Sequence analysis of cytoplasmic LRP8 revealed several peptide motifs with potential for cellular signaling and we propose this as a rational mechanism through which apoE inhibits platelet aggregation.—Riddell, D. R., D. V. Vinogradov, A. K. Stannard, N. Chadwick, and J. S. Owen. Identification and characterization of LRP8 (apoER2) in human blood platelets. J. Lipid Res. 1999. 40: 1925–1930. Recently, we reported that apoE inhibits platelet reactivity by stimulating NO release and postulated apoE-receptor activation of intracellular NO synthase (eNOS). Here, we implicate a low density lipoprotein receptor (LDL-R) family member by studying ligand requirements using purified apoE isoforms, synthetic peptides, and the receptor antagonist, receptor-associated protein (RAP). Then, using a homology cloning approach and degenerate PCR primers to amplify the conserved Cys-rich binding domain of the LDL-R family, this receptor was identified as LRP8 (formerly termed, apoER2), a newly described brain protein with several splice variants. Immunoprecipitation of platelet membranes with anti-peptide antisera confirmed protein expression, while analysis of RNA from platelets and two megakaryocytic cell lines (Meg-01 and HEL) disclosed that the major LRP8 transcript lacked binding repeats 4–6 (LRP8Δ4–6) but contained the full-length cytoplasmic tail. Sequence analysis of cytoplasmic LRP8 revealed several peptide motifs with potential for cellular signaling and we propose this as a rational mechanism through which apoE inhibits platelet aggregation.—Riddell, D. R., D. V. Vinogradov, A. K. Stannard, N. Chadwick, and J. S. Owen. Identification and characterization of LRP8 (apoER2) in human blood platelets. J. Lipid Res. 1999. 40: 1925–1930. Apolipoprotein E (apoE) is a 34-kDa polymorphic protein in the surface of circulating lipoproteins (1Weisgraber K.H. Apolipoprotein E: structure–function relationships.Adv. Protein Chem. 1994; 45: 249-302Google Scholar). Of the three major human alleles (ε2, ε3, ε4) the rarest variant, apoE2, is associated with recessive forms of type III hyperlipidemia and differs from apoE3, the common or wild-type form, by an Arg158Cys substitution. The apoE4 allele (Cys112Arg) produces a dominant hyperlipidemia and is a risk factor for restenosis (2de Knijff P. Havekes L.M. Apolipoprotein E as a risk factor for coronary heart disease: a genetic and molecular biology approach.Curr. Opin. Lipidol. 1996; 7: 59-63Google Scholar) and neurodegenerative diseases, including Alzheimer's disease. ApoE mediates hepatic clearance of lipoproteins through two receptors, the low density lipoprotein receptor (LDL-R) and LRP1 (LDL receptor-related protein 1), although there is a significant allelic influence (E4>E3>>E2) (1Weisgraber K.H. Apolipoprotein E: structure–function relationships.Adv. Protein Chem. 1994; 45: 249-302Google Scholar). Plasma apoE is largely liver-derived, but apoE released by macrophages has important local effects (3Linton M.F. Fazio S. Macrophages, lipoprotein metabolism, and atherosclerosis: insights from murine bone marrow transplantation studies.Curr. Opin. Lipidol. 1999; 10: 97-105Google Scholar). Low apoE is a risk factor for coronary heart disease and severe hyperlipidemia and atherosclerosis ensue in humans or animals if apoE is dys-functional or absent (reviewed in ref. 2de Knijff P. Havekes L.M. Apolipoprotein E as a risk factor for coronary heart disease: a genetic and molecular biology approach.Curr. Opin. Lipidol. 1996; 7: 59-63Google Scholar). Recently, we discovered that apoE potently inhibits platelet reactivity by stimulating intracellular nitric oxide synthase (eNOS or NOSIII), the NO released acting on soluble guanylate cyclase to elevate levels of anti-aggregatory cGMP (4Riddell D.R. Graham A. Owen J.S. Apolipoprotein E inhibits platelet aggregation through the L-arginine:nitric oxide pathway. Implications for vascular disease.J. Biol. Chem. 1997; 272: 89-95Google Scholar). A receptor-mediated process was postulated as platelets bound both HDL-E (5Desai K. Bruckdorfer K.R. Hutton R.A. Owen J.S. Binding of apoE-rich high density lipoprotein particles by saturable sites on human blood platelets inhibits agonist-induced platelet aggregation.J. Lipid Res. 1989; 30: 831-840Google Scholar) and apoE–phospholipid vesicles (6Higashihara M. Kinoshita M. Teramoto T. Kume S. Kurokawa K. The role of apoE in inhibitory effects of apoE-rich HDL on platelet function.FEBS Lett. 1991; 282: 82-86Google Scholar). ApoE binds to members of the LDL-R family (7Gliemann J. Receptors of the low density lipoprotein (LDL) receptor family in man. Multiple functions of the large family members via interaction with complex ligands.Biol. Chem. 1998; 379: 951-964Google Scholar), but none have yet been detected in platelets. Here, we identify a splice variant of LRP8 (the HUGO-NC approved gene symbol for apoE receptor 2 (apoER2), also termed LR7/8B) in platelets, which contains several cytoplasmic peptide motifs with potential for cellular signaling, and speculate that binding of apoE by this receptor inhibits platelet aggregation. Recombinant human apoE4, apoE3, and RAP were generous gifts from Dr. Weisgraber (Gladstone Institute) and Dr. Gliemann (University of Aarhus). ApoE2 was purified from VLDL obtained from type III hyperlipidemic patients (8Innerarity T.L. Pitas R.E. Mahley R.W. Lipoprotein-receptor interactions.Methods Enzymol. 1986; 129: 542-565Google Scholar). Synthetic peptides corresponding to sequences within apoE (E263–286, E141–155, and E(141–155)2) and LDL-R-deficient CHO cells expressing full-length LRP8 (CHOLRP8) were from Dr. Harmony (University of Cincinnati) and Drs. Soutar and Sun (Hammersmith Hospital), respectively. All chemicals, unless otherwise stated, were supplied by Sigma. ApoE isoforms and peptides were incorporated into small, unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and, as described previously (4Riddell D.R. Graham A. Owen J.S. Apolipoprotein E inhibits platelet aggregation through the L-arginine:nitric oxide pathway. Implications for vascular disease.J. Biol. Chem. 1997; 272: 89-95Google Scholar), used in platelet aggregation studies with ADP as an agonist. All experiments were repeated several times and were qualitatively reproducible. Results are expressed as means ± SEM, and statistical differences were determined by Student's two-tailed unpaired t test. Polyadenylated RNA was extracted from washed platelets (200 ml of fresh blood) and from HEL, Meg-01 and CHOLRP8 cells (107 cells) using a QuickPrep Micro mRNA Purification Kit (Amersham Pharmacia Biotech). One μg mRNA was converted to cDNA using the GeneAmp RNA PCR Kit (PE Biosystems). Oligonucleotide primers used for PCR amplification were based on alignment of published cDNA sequences corresponding to the highly conserved Cys-rich LDL-R class A binding domains from human LDL-R family members. The primer sequences incorporated restriction sites to facilitate cloning of PCR products. The sense primers (A1: 5′-GATCGGATCCTG(C/T)(C/G)(A/C)(G/T)GATGG(C/T)TC(T/C/A)GATGA-3′ and A2: 5′-GATCGGATCCTCTGGGGA(C/T)(G/A)(G/A)CAGTGA (C/T)-3′) contained a BamHI site (underlined), and the antisense primer (A3: 5′-GATCGAATTC(C/T)C(G/A/C)CC(A/G)TC(A/G)CA(G/T)(C/A)(G/T)CCA-3′) an EcoRI site (double underlined). Five μl of the reverse transcription reaction mixtures was subjected to “hot-start” PCR using the GeneAmp AmpliTaq Gold PCR DNA polymerase system (PE Biosystems) with 10 μm A1/A2 and 10 μm A3 in a total volume of 50 μl. Amplification proceeded for 40 cycles, with primer annealing at 53°C. One-tenth (5 μl) of the reaction mixture was then subjected to a second round of PCR amplification to increase detection of rare transcripts. Products of interest were digested with EcoRI and BamHI, gel purified, and cloned into pUC18 for automated fluorescent sequencing. To confirm the presence of LRP8, a specific sequence from the O-linked sugar domain to the cytoplasmic insert was amplified, carrying out “hot-start” PCR on platelet, HEL and Meg-01 cDNA as outlined above (sense primer: 1 μm of oligonucleotide 2092 5′-GGAGG(C/A)TGTGAATACCT(G/A)TGC-3′; antisense primer: 1 μm of oligonucleotide 2696 5′-CGATCAAAGCTGCTG ATTGC-3′). To detect LRP8 splice variants, the region corresponding to the ligand-binding domain was amplified using the conditions and primers (oligonucleotide 24: 5′-TCTCCGGCT TCTGGCGCT-3′ and oligonucleotide 1114: 5′-TCTGGTCCAGG AGCTGGAA-3′) described elsewhere (9Kim D.H. Magoori K. Inoue T.R. Mao C.C. Kim H.J. Suzuki H. Fujita T. Endo Y. Saeki S. Yamamoto T.T. Exon/intron organization, chromosome localization, alternative splicing, and transcription units of the human apolipoprotein E receptor 2 gene.J. Biol. Chem. 1997; 272: 8498-8504Google Scholar). Because multiple products of unexpected length were obtained due to mispriming, the ligand-binding domain PCR products were subjected to Southern blotting using 10 fmol/ml of an internal oligo 5′-32P-labeled probe (oligonucleotide 71: 5′-TGCGGCTCCAGCATCTTG-3′) for hybridization. Finally, to amplify the full-length open reading frame of LRP8, the Advantage-GC PCR kit (Clontech) was used (sense primer: oligonucleotide 24; antisense primer: oligonucleotide 2918 5′-GAGGCACGAAGGGGGTGAT-3′, both at 300 nm). Reaction products were cloned into pCRII (Invitrogen) and restriction mapped using BlpI, EcoRI, BstYI, HindIII, SmaI, AvaII, SacI, HincII, PstI, BbsI and XhoI; appropriate fragments were sub-cloned into pUC18 and sequenced. A 17-amino acid sequence (residues 865–881: clgetrepedpa palke) within the cytoplasmic insert of human LRP8 was conjugated to KLH and used to immunize rabbits for production of antipeptide antisera. Platelets (109 cells) and CHOLRP8 (106 cells) were surface labeled using the ECL protein biotinylation module (Amersham Pharmacia Biotech), lysed with 1% Triton X-100 and subjected to immunoprecipitation. Precipitated proteins were analyzed by Western blotting and any biotin-labeled, cell-surface proteins detected with streptavidin-horseradish peroxidase and chemiluminescence. Several approaches have shown that an Arg/Lys-rich sequence within apoE (residues 140–150) is bound by the “class A repeats” at the N-terminus of LDL-R family members (reviewed in ref. 1Weisgraber K.H. Apolipoprotein E: structure–function relationships.Adv. Protein Chem. 1994; 45: 249-302Google Scholar). To assess whether the anti-platelet action of apoE requires a similar interaction, we studied ligand requirements. RAP, the 39-kDa intracellular chaperone glycoprotein that facilitates folding and processing of LDL-R family members, competes with apoE for receptor binding (7Gliemann J. Receptors of the low density lipoprotein (LDL) receptor family in man. Multiple functions of the large family members via interaction with complex ligands.Biol. Chem. 1998; 379: 951-964Google Scholar, 10Willnow T.E. Receptor-associated protein (RAP): a specialized chaperone for endocytic receptors.Biol. Chem. 1998; 379: 1025-1031Google Scholar). When apoE–DMPC vesicles and RAP were coincubated, the anti-platelet action of apoE was significantly reduced (30.6 ± 6.6 vs. 62.5 ± 2.9% inhibition; P < 0.001, n = 3) (Fig. 1A,B). By contrast, RAP alone had no effect on aggregation compared to untreated controls (9.8 ± 6.6 vs. 0.0 ± 2.8% inhibition; P > 0.05, n = 3) (Fig. 1A), implying that the receptor–ligand interaction stimulating release of NO (4Riddell D.R. Graham A. Owen J.S. Apolipoprotein E inhibits platelet aggregation through the L-arginine:nitric oxide pathway. Implications for vascular disease.J. Biol. Chem. 1997; 272: 89-95Google Scholar) was specific to apoE. The monomeric peptide (E141–155; lrklrkrllrdaddl) and its tandem (E(141–155)2; lrklrkrllrdaddl-lrklrkrll rdaddl) both inhibited ADP-induced aggregation in a dose-dependent manner (26.5 ± 2.9% and 26.4 ± 5.8% inhibition at 3.0 μm peptide, respectively; both P < 0.05, n = 3; Fig. 1C), although less potent than full-length apoE (70.1 ± 7.3% inhibition; P < 0.001, n = 3). By contrast, a control peptide within the carboxyl-terminal 10-kDa of apoE (E263–286; swfeplvedmqrqwaglvekvqaa) lacked anti-platelet activity (4.2 ± 3.1% inhibition at 3.0 μm peptide, P > 0.05, n = 3). The three major apoE isoforms, E3, E2, and E4, suppressed ADP-induced aggregation of washed platelets to a similar degree (70.0 ± 8.3%, 81.8 ± 8.4%, and 74.9 ± 7.7% inhibition, respectively, at 50 μg (1.47 μm) protein/ml of apoE–DMPC; P > 0.05, n = 3; Fig. 1D). This suggests that the LDL-R or LRP1 are not involved as these bind apoE2 inefficiently (11Mahley R.W. Angelin B. Type III hyperlipoproteinemia: recent insights into the genetic defect of familial dysbetalipoproteinemia.Adv. Intern. Med. 1984; 29: 385-411Google Scholar, 12Kowal R.C. Herz J. Weisgraber K.H. Mahley R.W. Brown M.S. Goldstein J.L. Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein.J. Biol. Chem. 1990; 265: 10771-10779Google Scholar). By contrast, the VLDL-R, which has similar affinities for each apoE isoform (13Takahashi S. Oida K. Ookubo M. Suzuki J. Kohno M. Murase T. Yamamoto T. Nakai T. Very low density lipoprotein receptor binds apolipoprotein E2/2 as well as apolipoprotein E3/3.FEBS Lett. 1996; 386: 197-200Google Scholar), or one of the newly recognized apoE receptors, LRP2-LRP8 or LR11 (7Gliemann J. Receptors of the low density lipoprotein (LDL) receptor family in man. Multiple functions of the large family members via interaction with complex ligands.Biol. Chem. 1998; 379: 951-964Google Scholar, 14Schneider W.J. Young S.G. Genetics and molecular biology.Curr. Opin. Lipidol. 1999; 10: 85-87Google Scholar), remain The above implicate an LDL-R family member as the through which apoE platelet aggregation. A homology cloning approach was used to detect a or platelet receptor of this gene family, the conserved Cys-rich binding degenerate sense primers and and one antisense degenerate primer were with the potential to members of the LDL-R family human blood platelets are fragments of and of two human cell HEL and Meg-01 D. receptor on three human cell Scholar), were also of the and primer and HEL mRNA not detect VLDL-R, or a with to human LRP8, a receptor expressed in brain D.H. H. K. J. H. Kim H.J. Suzuki H. H. Saeki S. Yamamoto T. apolipoprotein E receptor A lipoprotein receptor of the low density lipoprotein receptor family expressed in Biol. Chem. 1996; Scholar, S. T. W.J. J. A low density lipoprotein receptor with ligand binding repeats in brain of and Biol. Chem. 1996; Scholar). To confirm the presence of LRP8 in cells of the megakaryocytic was on mRNA from platelets, HEL, and Meg-01 cells using specific the antisense primer the sequence of in cytoplasmic LRP8 (Fig. a was amplified from each cell type (Fig. and confirmed this was from human LRP8 and cDNA studies of brain LRP8 several splice variants, including binding repeats 4–6 or the cytoplasmic insert (9Kim D.H. Magoori K. Inoue T.R. Mao C.C. Kim H.J. Suzuki H. Fujita T. Endo Y. Saeki S. Yamamoto T.T. Exon/intron organization, chromosome localization, alternative splicing, and transcription units of the human apolipoprotein E receptor 2 gene.J. Biol. Chem. 1997; 272: 8498-8504Google Scholar). To which variant megakaryocytic cells was out on HEL and Meg-01 A was obtained from both than the of full-length LRP8 in CHOLRP8 (Fig. restriction and sequence analysis identified this as repeats 4–6 in the binding domain but the acid cytoplasmic To confirm the of binding repeats was out on HEL, and platelet mRNA using published primers that the ligand-binding domain of previously reported (9Kim D.H. Magoori K. Inoue T.R. Mao C.C. Kim H.J. Suzuki H. Fujita T. Endo Y. Saeki S. Yamamoto T.T. Exon/intron organization, chromosome localization, alternative splicing, and transcription units of the human apolipoprotein E receptor 2 gene.J. Biol. Chem. 1997; 272: 8498-8504Google Scholar), multiple products of unexpected length were due to Southern using an internal probe with the major of (Fig. Identification of platelet LRP8 mRNA by the of not of while the of platelet mRNA of platelet was used to that receptor protein was A with reduced molecular of was detected using antisera a synthetic peptide within the cytoplasmic insert of LRP8 (Fig. that LRP8 was The in molecular CHO cell and platelet LRP8 vs. the 4–6 ligand binding domains in the platelet we reported that apoE platelet by intracellular NO in to an agonist-induced of that of specific by circulating apoE platelets to activation by or (4Riddell D.R. Graham A. Owen J.S. Apolipoprotein E inhibits platelet aggregation through the L-arginine:nitric oxide pathway. Implications for vascular disease.J. Biol. Chem. 1997; 272: 89-95Google Scholar). The that LRP8 is the means through which apoE inhibits platelet aggregation. apoE were by binding studies with (5Desai K. Bruckdorfer K.R. Hutton R.A. Owen J.S. Binding of apoE-rich high density lipoprotein particles by saturable sites on human blood platelets inhibits agonist-induced platelet aggregation.J. Lipid Res. 1989; 30: 831-840Google Scholar, M. Kinoshita M. Teramoto T. Kume S. Kurokawa K. The role of apoE in inhibitory effects of apoE-rich HDL on platelet function.FEBS Lett. 1991; 282: 82-86Google Scholar). aggregation studies an LDL-R family with the that apoE in or with both of which interaction with the LDL-R (1Weisgraber K.H. Apolipoprotein E: structure–function relationships.Adv. Protein Chem. 1994; 45: 249-302Google Scholar), was not inhibitory (4Riddell D.R. Graham A. Owen J.S. Apolipoprotein E inhibits platelet aggregation through the L-arginine:nitric oxide pathway. Implications for vascular disease.J. Biol. Chem. 1997; 272: 89-95Google Scholar). the receptor RAP T.E. Receptor-associated protein (RAP): a specialized chaperone for endocytic receptors.Biol. Chem. 1998; 379: 1025-1031Google Scholar), the apoE anti-platelet the synthetic peptide, E141–155, the apoE domain inhibited aggregation. the LDL-R or LRP1 are to the peptide which has a and for these of synthetic peptides of apolipoprotein E that bind the Lipid Res. Scholar), was no than the monomeric and apoE2, which with the LDL-R or LRP1 (1Weisgraber K.H. Apolipoprotein E: structure–function relationships.Adv. Protein Chem. 1994; 45: 249-302Google Scholar, 12Kowal R.C. Herz J. Weisgraber K.H. Mahley R.W. Brown M.S. Goldstein J.L. Opposing effects of apolipoproteins E and C on lipoprotein binding to low density lipoprotein receptor-related protein.J. Biol. Chem. 1990; 265: 10771-10779Google Scholar), was as anti-aggregatory as for an homology cloning approach to identify the platelet apoE than of specific primer as this amplify an LDL-R family This was there are recent to the LDL-R family (7Gliemann J. Receptors of the low density lipoprotein (LDL) receptor family in man. Multiple functions of the large family members via interaction with complex ligands.Biol. Chem. 1998; 379: 951-964Google Scholar, 14Schneider W.J. Young S.G. Genetics and molecular biology.Curr. Opin. Lipidol. 1999; 10: 85-87Google Scholar), while the multiple class A domains which bind apoE and also in several we primers the Cys-rich class A binding domains with to members of the LDL-R as the domains are highly conserved within each receptor (7Gliemann J. Receptors of the low density lipoprotein (LDL) receptor family in man. Multiple functions of the large family members via interaction with complex ligands.Biol. Chem. 1998; 379: 951-964Google Scholar), as well as LDL-R family members, primers several annealing sites and increase detection the receptor sequence amplified was that of LRP8, an unexpected as this receptor is in human brain and but reported absent from D.H. H. K. J. H. Kim H.J. Suzuki H. H. Saeki S. Yamamoto T. apolipoprotein E receptor A lipoprotein receptor of the low density lipoprotein receptor family expressed in Biol. Chem. 1996; Scholar, S. T. W.J. J. A low density lipoprotein receptor with ligand binding repeats in brain of and Biol. Chem. 1996; Scholar). LRP8 has domains (Fig. but while the has class A LRP8 and LDL-R the ligand-binding domain of LRP8 that of the class A repeats have acid homology and both bind RAP and lipoproteins with high D.H. H. K. J. H. Kim H.J. Suzuki H. H. Saeki S. Yamamoto T. apolipoprotein E receptor A lipoprotein receptor of the low density lipoprotein receptor family expressed in Biol. Chem. 1996; Scholar, Soutar in of of the RNA for human apolipoprotein E identified in human by J. 1999; Scholar). that apoE2 is an of platelet aggregation is with a (13Takahashi S. Oida K. Ookubo M. Suzuki J. Kohno M. Murase T. Yamamoto T. Nakai T. Very low density lipoprotein receptor binds apolipoprotein E2/2 as well as apolipoprotein E3/3.FEBS Lett. 1996; 386: 197-200Google Scholar). of LRP8 protein in platelets and megakaryocytic cells was confirmed by anti-peptide LRP8 and, although the splice variant lacked binding repeats the of this is as and full-length LRP8 bind apoE-rich with (9Kim D.H. Magoori K. Inoue T.R. Mao C.C. Kim H.J. Suzuki H. Fujita T. Endo Y. Saeki S. Yamamoto T.T. Exon/intron organization, chromosome localization, alternative splicing, and transcription units of the human apolipoprotein E receptor 2 gene.J. Biol. Chem. 1997; 272: 8498-8504Google Scholar, Soutar in of of the RNA for human apolipoprotein E identified in human by J. 1999; Scholar). of the platelet apoE receptor as LRP8 a rational for inhibition of platelet LRP8, LDL-R family members, contains the cytoplasmic sequence is for via (7Gliemann J. Receptors of the low density lipoprotein (LDL) receptor family in man. Multiple functions of the large family members via interaction with complex ligands.Biol. Chem. 1998; 379: 951-964Google Scholar). this as a ligand for the binding domains of signaling including protein and B. Herz J. of proteins with apolipoprotein E and the protein.J. Biol. Chem. 1998; Scholar, M. T. J. J. R.E. Herz J. of in the VLDL receptor and apoE receptor 1999; Scholar) and also LRP1 to intracellular activity protein with a J. 1998; Scholar). the cellular role of LRP8 is to lipoprotein and Soutar in of of the RNA for human apolipoprotein E identified in human by J. 1999; Scholar), implying an alternative for this in have also identified three motifs within the cytoplasmic insert which sequences for homology the is a for binding as is by and an T. Protein and Scholar). Finally, and are potential for or protein by protein of a synthetic peptide corresponding to the site in the Biol. Chem. Scholar). is to speculate that the anti-aggregatory effect of apoE is through LRP8 and activity is by and at multiple sites K. T. and of nitric oxide Med. 1997; 7: Scholar). we have that the in platelets is by an LDL-R family member and, and a LRP8 variant in human blood platelets, have cellular signaling as a rational mechanism by which apoE inhibits aggregation. the of platelets to of LRP8 and and including of these proteins in are to several Dr. Weisgraber (Gladstone Dr. Gliemann (University of Dr. Soutar and Dr. Sun (Hammersmith and Dr. Harmony (University of for the generous of This was by a and from the and by a apolipoprotein E apoE receptor 2 dimyristoylphosphatidylcholine high density lipoprotein low density lipoprotein receptor receptor-related protein nitric oxide synthase receptor-associated protein reverse reaction low density lipoprotein