Multiphasic generation of diacylglycerol in thrombin-activated human platelets.
Ontology highlight
ABSTRACT:
SUBMITTER: Werner MH
PROVIDER: S-EPMC1130860 | biostudies-other | 1992 Mar
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Here, a group of specific lipids, comprising phosphatidylethanolamine (PE)- or phosphatidylcholine (PC)-esterified 12S-hydroxyeicosatetraenoic acid (12S-HETE), generated by 12-lipoxygenase was identified and characterized. 12S-HETE-PE/PCs were formed within 5 min of activation by thrombin, ionophore, or collagen. Esterified HETE levels generated in response to thrombin were 5.85 +/- 1.42 (PE) or 18.35 +/- 4.61 (PC), whereas free was 65.5 +/- 17.6 ng/4 x 10(7) cells (n = 5 separate donors, mean +/- S.E.). Their generation was stimulated by triggering protease-activated receptors-1 and -4 and signaling via Ca(2+) mobilization secretory phospholipase A2, platelet-activating factor-acetylhydrolase, src tyrosine kinases, and protein kinase C. Stable isotope labeling showed that they form predominantly by esterification that occurs on the same time scale as free acid generation. Unlike free 12S-HETE that is secreted, esterified HETEs remain cell-associated, with HETE-PEs migrating to the outside of the plasma membrane. 12-Lipoxygenase inhibition attenuated externalization of native PE and phosphatidylserine and HETE-PEs. Platelets from a patient with the bleeding disorder, Scott syndrome, did not externalize HETE-PEs, and liposomes supplemented with HETE-PC dose-dependently enhanced tissue factor-dependent thrombin generation in vitro. This suggests a role for these novel lipids in promoting coagulation. Thus, oxidized phospholipids form by receptor/agonist mechanisms, not merely as an undesirable consequence of vascular and inflammatory disease.
| S-EPMC2844139 | biostudies-literature
Project description:Because of the role of thrombin and platelets in myocardial infarction and other pathological processes, identifying and blocking the receptors by which thrombin activates platelets has been an important goal. Three protease-activated receptors (PARs) for thrombin -- PAR1, PAR3, and PAR4 -- are now known. PAR1 functions in human platelets, and the recent observation that a PAR4-activating peptide activates human platelets suggests that PAR4 also acts in these cells. Whether PAR1 and PAR4 account for activation of human platelets by thrombin, or whether PAR3 or still other receptors contribute, is unknown. We have examined the roles of PAR1, PAR3, and PAR4 in platelets. PAR1 and PAR4 mRNA and protein were detected in human platelets. Activation of either receptor was sufficient to trigger platelet secretion and aggregation. Inhibition of PAR1 alone by antagonist, blocking antibody, or desensitization blocked platelet activation by 1 nM thrombin but only modestly attenuated platelet activation by 30 nM thrombin. Inhibition of PAR4 alone using a blocking antibody had little effect at either thrombin concentration. Strikingly, simultaneous inhibition of both PAR1 and PAR4 virtually ablated platelet secretion and aggregation, even at 30 nM thrombin. These observations suggest that PAR1 and PAR4 account for most, if not all, thrombin signaling in platelets and that antagonists that block these receptors might be useful antithrombotic agents.
| S-EPMC408153 | biostudies-literature
Project description:The 1,2-diacylglycerol (DAG) mass content was measured in thrombin-stimulated human platelets. Thrombin stimulates a biphasic accumulation of DAG, with an early phase reaching a peak at 10 s and a later phase reaching a peak at 2-3 min. The time course of first-phase DAG production corresponded well to that of Ins(1,4,5)P3 formation, which was rapid and transient. The second phase of DAG accumulation occurred after the level of Ins(1,4,5)P3 returned to nearly basal. Thrombin stimulated the decrease in PtdIns and phosphatidylcholine contents. The source of second-phase DAG was examined in platelets prelabelled with three radioactive fatty acids, i.e. arachidonic, palmitic and myristic. Thrombin stimulated the increase in radioactivity of DAG with decline of PtdIns in platelets labelled with [3H]arachidonic acid or [3H]palmitic acid, in which PtdIns was considerably labelled. In contrast, significant accumulation of [3H]DAG was not observed in [3H]myristic acid-labelled platelets, in which PtdIns was poorly labelled. In platelets prelabelled with [3H]inositol, an increase in InsP in response to thrombin was seen for more than 5 min. In contrast, upon stimulation, significant increases in [3H]phosphocholine and [3H]choline were not observed in [methyl-3H]choline-labelled platelets. Thrombin induced a small production of phosphatidylethanol, when ethanol was present during stimulation. However, the formation of DAG and phosphatidic acid was not significantly affected by ethanol. These results suggest that thrombin stimulates a biphasic accumulation of DAG, initially from PtdInsP2 and later from PtdIns in human platelets.
| S-EPMC1150060 | biostudies-other
Project description:BackgroundThe direct oral anticoagulant rivaroxaban inhibiting specifically activated factor X (FXa) causes delayed thrombin generation (TG) as measured by calibrated automated thrombography (CAT). The implications of these changes for assessing bleeding or residual prothrombotic risks of patients are unclear in the absence of a better understanding of the underlying mechanism.MethodsWe compared platelet rich plasma (PRP) without or with prior collagen-induced platelet aggregation (agPRP) in the CAT assay to better characterize TG in the presence of rivaroxaban.ResultsIn the presence of rivaroxaban, TG curves in agPRP showed a distinct profile with a rapidly ascending phase followed with a protracted phase. Inhibition of tissue factor pathway inhibitor amplified the first phase of the curve which was also modulated by procoagulant phospholipids. Inhibition of FXIIa-dependent FXI activation revealed that aggregated platelets influenced the first phase by a combination of extrinsic and intrinsic coagulation pathway initiations. Thrombin-dependent amplification of TG (even prior collagen activation) was responsible for the second phase of the TG curve.ConclusionsAgPRP fully includes platelet ability to support TG and reveal distinct TG phases in the presence of direct FXa inhibitors highlighting its potential use in an anticoagulated setting.
| S-EPMC6912513 | biostudies-literature
Project description:BackgroundPlatelets are essential for maintaining haemostasis and play a key role in the pathogenesis of cardiovascular disease. Upon ligation of platelet receptors through subendothelial matrix proteins, intracellular reactive oxygen species (ROS) are generated, further amplifying the platelet activation response. Thrombin, a potent platelet activator, can signal through GPIbα and protease-activated receptor (PAR) 1 and PAR4 on human platelets, and recently has been implicated in the generation of ROS. While ROS are known to have key roles in intra-platelet signalling and subsequent platelet activation, the precise receptors and signalling pathways involved in thrombin-induced ROS generation have yet to be fully elucidated.ObjectiveTo investigate the relative contribution of platelet GPIbα and PARs to thrombin-induced reactive oxygen species (ROS) generation.Methods and resultsHighly specific antagonists targeting PAR1 and PAR4, and the GPIbα-cleaving enzyme, Naja kaouthia (Nk) protease, were used in quantitative flow cytometry assays of thrombin-induced ROS production. Antagonists of PAR4 but not PAR1, inhibited thrombin-derived ROS generation. Removal of the GPIbα ligand binding region attenuated PAR4-induced and completely inhibited thrombin-induced ROS formation. Similarly, PAR4 deficiency in mice abolished thrombin-induced ROS generation. Additionally, GPIbα and PAR4-dependent ROS formation were shown to be mediated through focal adhesion kinase (FAK) and NADPH oxidase 1 (NOX1) proteins.ConclusionsBoth GPIbα and PAR4 are required for thrombin-induced ROS formation, suggesting a novel functional cooperation between GPIbα and PAR4. Our study identifies a novel role for PAR4 in mediating thrombin-induced ROS production that was not shared by PAR1. This suggests an independent signalling pathway in platelet activation that may be targeted therapeutically.
| S-EPMC4656914 | biostudies-literature
Project description:BackgroundPlatelets are small anucleated blood particles that play a key role in the control of bleeding. Platelets need to be activated to perform their functions and participate in hemostasis. The process of activation is accompanied by vast protein reorganization and posttranslational modifications. The goal of this study was to identify changes in proteins in platelets activated by different agonists. Platelets were activated by three different agonists - arachidonic acid, collagen, and thrombin. 2D SDS-PAGE (pI 4-7) was used to separate platelet proteins. Proteomes of activated and resting platelets were compared with each other by Progenesis SameSpots statistical software; and proteins were identified by nanoLC-MS/MS.Results190 spots were found to be significantly different. Of these, 180 spots were successfully identified and correspond to 144 different proteins. Five proteins were found that had not previously been identified in platelets: protein CDV3 homolog, protein ETHE1, protein LZIC, FGFR1 oncogene partner 2, and guanine nucleotide-binding protein subunit beta-5. Using spot expression profile analysis, we found two proteins (WD repeat-containing protein 1 and mitochondrial glycerol-3-phosphate dehydrogenase) that may be part of thrombin specific activation or signal transduction pathway(s).ConclusionsOur results, characterizing the differences within proteins in both activated (by various agonists) and resting platelets, can thus contribute to the basic knowledge of platelets and to the understanding of the function and development of new antiplatelet drugs.
| S-EPMC2996359 | biostudies-literature
Project description:Specific oxidized phospholipids (oxPCCD36) promote platelet hyper-reactivity and thrombosis in hyperlipidemia via the scavenger receptor CD36, however the signaling pathway(s) induced in platelets by oxPCCD36 are not well defined. We have employed mass spectrometry-based tyrosine, serine, and threonine phosphoproteomics for the unbiased analysis of platelet signaling pathways induced by oxPCCD36 as well as by the strong physiological agonist thrombin. oxPCCD36 and thrombin induced differential phosphorylation of 115 proteins (162 phosphorylation sites) and 181 proteins (334 phosphorylation sites) respectively. Most of the phosphoproteome changes induced by either agonist have never been reported in platelets; thus they provide candidates in the study of platelet signaling. Bioinformatic analyses of protein phosphorylation dependent responses were used to categorize preferential motifs for (de)phosphorylation, predict pathways and kinase activity, and construct a phosphoproteome network regulating integrin activation. A putative signaling pathway involving Src-family kinases, SYK, and PLCγ2 was identified in platelets activated by oxPCCD36. Subsequent ex vivo studies in human platelets demonstrated that this pathway is downstream of the scavenger receptor CD36 and is critical for platelet activation by oxPCCD36. Our results provide multiple insights into the mechanism of platelet activation and specifically in platelet regulation by oxPCCD36.
| S-EPMC3882224 | biostudies-literature
Project description:Red blood cells (RBCs) and platelets contribute to the coagulation capacity in bleeding and thrombotic disorders. The thrombin generation (TG) process is considered to reflect the interactions between plasma coagulation and the various blood cells. Using a new high-throughput method capturing the complete TG curve, we were able to compare TG in whole blood and autologous platelet-rich and platelet-poor plasma to redefine the blood cell contributions to the clotting process. We report a faster and initially higher generation of thrombin and shorter coagulation time in whole blood than in platelet-rich plasma upon low concentrations of coagulant triggers, including tissue factor, Russell viper venom factor X, factor Xa, factor XIa, and thrombin. The TG was accelerated with increased hematocrit and delayed after prior treatment of RBC with phosphatidylserine-blocking annexin A5. RBC treatment with ionomycin increased phosphatidylserine exposure, confirmed by flow cytometry, and increased the TG process. In reconstituted blood samples, the prior selective blockage of phosphatidylserine on RBC with annexin A5 enhanced glycoprotein VI-induced platelet procoagulant activity. For patients with anemia or erythrocytosis, cluster analysis revealed high or low whole-blood TG profiles in specific cases of anemia. The TG profiles lowered upon annexin A5 addition in the presence of RBCs and thus were determined by the extent of phosphatidylserine exposure of blood cells. Profiles for patients with polycythemia vera undergoing treatment were similar to that of control subjects. We concluded that RBC and platelets, in a phosphatidylserine-dependent way, contribute to the TG process. Determination of the whole-blood hypo- or hyper-coagulant activity may help to characterize a bleeding or thrombosis risk.
| S-EPMC10651426 | biostudies-literature
Project description:The diacylglycerol kinase inhibitor R59022 (10 microM) potentiates secretion and aggregation responses in human platelets challenged with sub-maximal concentrations of thrombin. Potentiation correlates closely with increased formation of diacylglycerol, increased phosphorylation of a 40 kDa protein, a known substrate for protein kinase C, and with decreased formation of phosphatidic acid, the product of diacylglycerol kinase. Phosphorylation of myosin light chains, formation of inositol phosphates and the mobilization of Ca2+ by thrombin are not affected by R59022 (10 microM). These data support a role for protein kinase C in platelet aggregation and secretion, and provide further evidence that endogenous diacylglycerols bring about the activation of this enzyme. These data also add further argument against a role for phosphatidic acid in platelet activation.
| S-EPMC1147929 | biostudies-other
Project description:Thrombin inhibits adenylate cyclase and stimulates GTP hydrolysis by high-affinity GTPase(s) in membranes of human platelets at almost identical concentrations. Both of these thrombin actions are similar to those observed with agonist-activated alpha 2-adrenoceptors coupling to the inhibitory guanine nucleotide-binding protein N1. However, stimulation of GTP hydrolysis caused by adrenaline (alpha 2-adrenoceptor agonist) and by thrombin at maximally effective concentrations was partially additive, whereas with regard to adenylate cyclase inhibition no additive response was observed. Furthermore, treatment of platelet membranes with pertussis toxin, which inactivates Ni and largely abolishes thrombin- and adrenaline-induced adenylate cyclase inhibition and adrenaline-induced GTPase stimulation, decreased the thrombin-induced stimulation of GTP hydrolysis by only about 30%. Additionally, the thiol reagent N-ethylmalemide (NEM) at rather low concentrations abolished thrombin- and adrenaline-induced stimulation of GTP hydrolysis was decreased by only 30-40% by treatment of platelet membranes with even high concentrations of NEM. Treatment with cholera toxin, which inhibits GTPase activity of the Ns (stimulatory guanine nucleotide-binding) protein, has no effect on thrombin-stimulated GTP hydrolysis. The data suggest that thrombin interaction with its receptor sites in platelet membranes leads to stimulation of two GTP-hydrolysing enzymes. One of these enzymes is apparently Ni and is also activated by agonist-activated alpha 2-adrenoceptors and is inactivated by pertussis toxin and NEM treatment. The other GTP-hydrolysing enzyme activated by thrombin may represent a guanine nucleotide-binding protein apparently involved in the coupling of thrombin receptors to the phosphoinositide phosphodiesterase.
| S-EPMC1147043 | biostudies-other