Isolation and characterization of lipid-protein particles containing platelet factor 3 released from human platelets.
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ABSTRACT:
SUBMITTER: Sandberg H
PROVIDER: S-EPMC1158224 | biostudies-other | 1982 Apr
REPOSITORIES: biostudies-other
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Project description:The role of platelets in hemostasis and thrombosis is clearly established; however, the mechanisms by which platelets mediate inflammatory and immune pathways are less well understood. Platelets interact and modulate the function of blood and vascular cells by releasing bioactive molecules. Although the platelet is anucleate, it contains transcripts that may mirror disease. Platelet mRNA is only associated with low-level protein translation; however, platelets have a unique membrane structure allowing for the passage of small molecules, leading to the possibility that its cytoplasmic RNA may be passed to nucleated cells. To examine this question, platelet-like particles with labeled RNA were cocultured with vascular cells. Coculture of platelet-like particles with activated THP-1, monocytic, and endothelial cells led to visual and functional RNA transfer. Posttransfer microarray gene expression analysis of THP-1 cells showed an increase in HBG1/HBG2 and HBA1/HBA2 expression that was directly related to the transfer. Infusion of wild-type platelets into a TLR2-deficient mouse model established in vivo confirmation of select platelet RNA transfer to leukocytes. By specifically transferring green fluorescent protein, we also observed external RNA was functional in the recipient cells. The observation that platelets possess the capacity to transfer cytosolic RNA suggests a new function for platelets in the regulation of vascular homeostasis.
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Project description:Platelets are the sole source of EGF in circulation, yet how EGF is stored or released from stimulated cells is undefined. In fact, we found platelets did not store EGF, synthesized as a single 6-kDa domain in pro-EGF, but rather expressed intact pro-EGF precursor on granular and plasma membranes. Activated platelets released high-molecular-weight (HMW)-EGF, produced by a single cleavage between the EGF and the transmembrane domains of pro-EGF. We synthesized a fluorogenic peptide encompassing residues surrounding the putative sessile arginyl residue and found stimulated platelets released soluble activity that cleaved this pro-EGF1020-1027 peptide. High throughput screening identified chymostatins, bacterial peptides with a central cyclic arginyl structure, as inhibitors of this activity. In contrast, the matrix metalloproteinase/TACE (tumor necrosis factor-?-converting enzyme) inhibitor GM6001 was ineffective. Stimulated platelets released the soluble protease ADAMDEC1, recombinant ADAMDEC1 hydrolyzed pro-EGF1020-1027, and this activity was inhibited by chymostatin and not GM6001. Biotinylating platelet surface proteins showed ADAMDEC1 hydrolyzed surface pro-EGF to HMW-EGF that stimulated HeLa EGF receptor (EGFR) reporter cells and EGFR-dependent tumor cell migration. This proteolysis was inhibited by chymostatin and not GM6001. Metabolizing pro-EGF Arg1023 to citrulline with recombinant polypeptide arginine deiminase 4 (PAD4) abolished ADAMDEC1-catalyzed pro-EGF1020-1027 peptidolysis, while pretreating intact platelets with PAD4 suppressed ADAMDEC1-, thrombin-, or collagen-induced release of HMW-EGF. We conclude that activated platelets release ADAMDEC1, which hydrolyzes pro-EGF to soluble HMW-EGF, that HMW-EGF is active, that proteolytic cleavage of pro-EGF first occurs at the C-terminal arginyl residue of the EGF domain, and that proteolysis is the regulated and rate-limiting step in generating soluble EGF bioactivity from activated platelets.
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Project description:BackgroundOvarian cancer patients have a high risk of developing venous thrombosis. The membrane lipid bilayer of platelets and platelet-derived microparticles (PMP) provides a platform for assembly of coagulation proteins and generation of blood clots.MethodsWe compared the lipid composition of platelets and PMPs in patients with ovarian cancer to those in healthy subjects. We used shotgun lipidomics to quantify 12 classes and 177 species of lipids.ResultsWe found a significant change in 2 classes of lipids in platelets and PMPs isolated from ovarian cancer patients: higher phosphatidylinositol and lower lyso-phosphatidylcholine. The level of 28 species of lipids was also significantly altered in the direction of an increase in the pro-coagulant and a reduction in the anticoagulant lipids. We found that cancer platelets expressed less lipid phosphate phosphatase 1 (LPP1), a key enzyme in phospholipid biosynthesis pathways, than normal platelets. The reduction in LPP1 might contribute to the changes in the lipid profile of cancer platelets.ConclusionOur results support a procoagulant lipid profile of platelets in ovarian cancer patients that can play a role in the increased risk of venous thrombosis in these patients.General significanceAs far as we are aware, our study is the first study on platelet lipidomics in ovarian cancer. The importance of our findings for the future studies are: 1) a similar change in lipid profile of platelets and PMP may be responsible for hypercoagulability in other cancers, and 2) plasma level of high-risk lipids for venous thrombosis may be useful biomarkers.
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Project description:Platelets not only play an essential role in hemostasis after vascular injury but are also involved in the development of coronary artery disease (CAD) and cerebrovascular lesions. Patients with CAD and cerebral ischemia are recommended to undergo antiplatelet therapy, but they have an increased incidence of major bleeding complications. Both assessment of the platelet activation status and response to antiplatelet therapy in each patient are highly desired. β-Amyloid precursor protein (APP) 770 is expressed in vascular endothelial cells, and its extracellular region, a soluble form of APP770 (sAPP770, also called nexin-2), is proteolytically cleaved for shedding. Abundant sAPP770 is also released from activated platelets. In this study, we used peripheral blood samples from patients with CAD and control subjects and evaluated sAPP770 as a specific biomarker for platelet activation. First, the plasma levels of sAPP770 correlated well with those of the soluble form CD40 ligand (CD40L), an established biomarker for platelet activation. Additionally, flow cytometry analysis using peripheral blood cells showed that CD40L expression is up-regulated in activated T cells, whereas APP770 expression is negligible in all blood cell types except platelets. Following stimulation with collagen or ADP, aggregating platelets immediately released sAPP770. Finally, patients with dual antiplatelet therapy showed significantly lower levels of plasma sAPP770 than those with no therapy. Taken together, our data show that plasma sAPP770 could be a promising biomarker for platelet activation.
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Project description:We profiled gene expression of pelleted material containing platelets from frozen plasma of healthy controls and ME/CFS cases for bulk RNA-seq, collected before and 24h after participants conducted a cardiopulmonary exercise test (CPET).
2024-01-16 | GSE236402 | GEO
Project description:Objective: Recent genome-wide association studies (GWAS) identified a variant rs7575840 in the apolipoprotein B (APOB) gene region to be associated with LDL-C. However, the underlying functional mechanism of this variant that resides 6.5 kb upstream of APOB has remained unknown. Our objective was to investigate rs7575840 for association with refined apoB containing lipid particles; for replication in a non-Caucasian Mexican population; and for underlying functional mechanism. Methods and Results Our data show that rs7575840 is associated with serum apoB levels (P=4.85x10-10) and apoB containing lipid particles, very small VLDL, IDL and LDL particles (P=2x10-5 - 9x10-7) in the Finnish METSIM study sample (n=7,710). Fine mapping of the APOB region using 43 SNPs replicated the association of rs7575840 with apoB in a Mexican study sample (n=2,666, P=3.33x10-05). Furthermore, our transcript analyses of adipose RNA samples from 175 Finnish METSIM subjects indicate that rs7575840 alters expression of APOB (P=1.13x10-10) and a regional non-coding RNA (BU630349) (P=7.86x10-6) in adipose tissue. Conclusions It has been difficult to convert GWAS associations into mechanistic insights. Our data show that rs7575840 is associated with serum apoB levels and apoB containing lipid particles as well as influences expression of APOB and a regional transcript BU630349 in adipose tissue. We thus provide evidence how a common genome-wide significant SNP rs7575840 may affect serum apoB, LDL-C, and TC levels. 175 samples, no replicates
2011-08-31 | E-GEOD-27666 | biostudies-arrayexpress
Project description:Objective: Recent genome-wide association studies (GWAS) identified a variant rs7575840 in the apolipoprotein B (APOB) gene region to be associated with LDL-C. However, the underlying functional mechanism of this variant that resides 6.5 kb upstream of APOB has remained unknown. Our objective was to investigate rs7575840 for association with refined apoB containing lipid particles; for replication in a non-Caucasian Mexican population; and for underlying functional mechanism. Methods and Results Our data show that rs7575840 is associated with serum apoB levels (P=4.85x10-10) and apoB containing lipid particles, very small VLDL, IDL and LDL particles (P=2x10-5 - 9x10-7) in the Finnish METSIM study sample (n=7,710). Fine mapping of the APOB region using 43 SNPs replicated the association of rs7575840 with apoB in a Mexican study sample (n=2,666, P=3.33x10-05). Furthermore, our transcript analyses of adipose RNA samples from 175 Finnish METSIM subjects indicate that rs7575840 alters expression of APOB (P=1.13x10-10) and a regional non-coding RNA (BU630349) (P=7.86x10-6) in adipose tissue. Conclusions It has been difficult to convert GWAS associations into mechanistic insights. Our data show that rs7575840 is associated with serum apoB levels and apoB containing lipid particles as well as influences expression of APOB and a regional transcript BU630349 in adipose tissue. We thus provide evidence how a common genome-wide significant SNP rs7575840 may affect serum apoB, LDL-C, and TC levels.
2011-09-01 | GSE27666 | GEO
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Project description:Recent genome-wide association studies identified a variant rs7575840 in the apolipoprotein B (APOB) gene region as associated with low-density lipoprotein (LDL) cholesterol. However, the underlying functional mechanism of this variant, which resides 6.5 kb upstream of APOB, has remained unknown. Our objective was to investigate rs7575840 for association with refined apoB-containing lipid particles, for replication in a Mexican population, and for its underlying functional mechanism.Our data show that rs7575840 is associated with serum apoB levels (P=4.85×10(-10)) and apoB-containing lipid particles, very small very-low-density lipoprotein, intermediate lipoprotein, and LDL particles (P=2×10(-5) to 9×10(-7)) in the Finnish Metabolic Syndrome in Men study sample (n=7710). Fine mapping of the APOB region using 43 single-nucleotide polymorphisms replicated the association of rs7575840 with apoB in a Mexican study sample (n=2666, P=3.33×10(-5)). Furthermore, our transcript analyses of adipose RNA samples from 175 subjects in the Finnish Metabolic Syndrome in Men study indicate that rs7575840 alters expression of APOB (P=1.13×10(-10)) and a regional noncoding RNA (BU630349) (P=7.86×10(-6)) in adipose tissue.It has been difficult to convert genome-wide association study associations into mechanistic insights. Our data show that rs7575840 is associated with serum apoB levels and apoB-containing lipid particles, as well as influencing expression of APOB and a regional transcript BU630349 in adipose tissue. We thus provide evidence how a common genome-wide significant single-nucleotide polymorphism, rs7575840, may affect serum apoB, LDL cholesterol, and total cholesterol levels.
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