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PubMed1993

Hydrophobic affinity chromatography of human thrombin.

Hydrophobic affinity chromatography on p-chlorobenzylamido-agarose (p-CBA-agarose) was used to characterize various modified forms of human thrombin. Native alpha-thrombin bound tightly to the column and was eluted with either acetonitrile or 1,4-dioxane, while the catalytically inactive prethrombin 2 did not bind to the matrix. Site-specific chemical modification with pyridoxal 5'-phosphate resulted in the loss of at least 80% of fibrinogen clotting activity but did not influence the binding of thrombin to p-CBA agarose. Modification of thrombin with pyridoxal 5'-phosphate is thought to occur at the fibrinogen-binding site and the heparin-binding site. In contrast, binding of thrombin to p-CBA agarose was eliminated by modification of the active site histidine using either H-D-phenylalanyl-L-prolyl-L-arginine chloromethylketone or dansyl-L-glutamyl-glycyl-L-arginine chloromethylketone but not with tosyl-L-lysine chloromethylketone. The presence of either hirudin or heparin blocked the binding of thrombin to p-CBA-agarose but dansyl-arginine-N-(3-ethyl-1,5-pentanediyl)amide had no effect. These results indicate that p-CBA agarose binds to thrombin outside of the enzyme active site and its use should be valuable in characterizing site-specific modified thrombins obtained by either protein engineering or chemical modification.

Acetonitriles↗

PubMed1994

Thrombin receptor activating peptide does not stimulate platelet procoagulant activity.

Platelets after challenge with alpha-thrombin alone, collagen alone or thrombin/collagen mixture were observed to increase the rate of activation of prothrombin by factor Xa in the presence of factor Va and calcium ion (platelet procoagulant activity) by a maximum of 25, 45 and 110 fold, respectively. The increase in platelet procoagulant activity due to these agonists has been described previously and arises from increased expression of phosphatidylserine on the platelet surface. When platelets were treated with the thrombin receptor activating peptide (TRAP) (SFLLRNPNDKYEPK), alone or in the presence of collagen or thrombin, no change in platelet procoagulant activity was observed at concentrations of TRAP sufficient to cause increased intracellular calcium levels and protein phosphorylation in a manner similar to that of thrombin. In addition, no increase in platelet procoagulant activity was seen upon treatment with TRAP in the presence of inactivated thrombin (PPACK-thrombin). These results suggest that the thrombin-mediated increase in procoagulant activity may be due to activation of a thrombin receptor distinct from the recently cloned G-protein-coupled receptor, or to other proteolytic events on the platelet surface.

Amino Acid Sequence↗

PubMed1994

Thrombin-induced Ca2+ influx and protein tyrosine phosphorylation in endothelial cells is inhibited by herbimycin A.

During endothelial cell activation, alpha-thrombin elicits both Ca2+ release from internal stores and influx of external Ca2+ across the plasma membrane. The mechanisms of alpha-thrombin-induced Ca2+ entry into endothelial cells are unclear. Therefore, effects of the specific tyrosine kinase inhibitor herbimycin A on protein tyrosine phosphorylation and on intracellular Ca2+ transients were studied in alpha-thrombin-stimulated human umbilical vein endothelial cells. alpha-Thrombin caused significant tyrosine phosphorylation of mainly two proteins and evoked typical biphasic changes of free cytosolic Ca2+ concentration. We show that 24 h pretreatment with herbimycin A inhibited alpha-thrombin-induced endothelial protein tyrosine phosphorylation. Moreover, herbimycin A significantly attenuated alpha-thrombin-induced Ca2+ influx but not release from internal stores. The data suggest that protein tyrosine phosphorylation by alpha-thrombin is involved in the regulation of alpha-thrombin-induced Ca2+ influx into endothelial cells.

Benzoquinones↗

PubMed1994

Involvement of NF-kappa B activation in thrombin-induced human vascular smooth muscle cell proliferation.

A thrombin receptor has been cloned and is thought to mediate a variety of thrombin-induced responses. However, the transcription factors important for postreceptor signaling have been little clarified. The post-receptor signals are mediated by several protein kinases responsible for NF-kappa B activation, and most thrombin-inducible genes have the kappa B sequence in the regulatory elements. The possibility that NF-kappa B may participate in thrombin signaling was therefore investigated in cultured human vascular smooth muscle cells (VSMCs). Thrombin receptor stimulation resulted in activation of NF-kappa B. Furthermore, treatment of cells with antisense p65 ODNs of NF-kappa B inhibited thrombin-stimulated growth of VSMC in vitro. Results indicate that the activation of NF-kappa B is involved in thrombin signaling and that this pathway causes the proliferation of VSMC induced by thrombin. Therapeutic potential of antisense NF-kappa B ODNs for the treatment with atherosclerosis and restenosis is also indicated.

Acid Phosphatase↗

PubMed1995

Endothelin-1 induces rapid and long lasting internalization of the thrombin receptor in human glomerular epithelial cells.

Endothelin-1 acts as a potent autocrine and paracrine mediator in the kidney. Glomeruli and renal arterioles also express functional thrombin receptors which mediate the cellular effects of thrombin. Using the binding on glomerular epithelial cells of 125I-labeled ATAP2, a monoclonal antibody raised against the functional thrombin receptor, we demonstrate here that endothelin-1 induces thrombin receptor internalization in a dose- and a time-dependent manner. The maximal effect is observed at 10(-7) M endothelin-1 corresponding to internalization of approximately 25% of thrombin receptors. It is maximal at 20 min and persists for at least 24 h. This effect is mediated by the endothelin receptor subtype A (ETA), and involves a pertussis toxin-sensitive G-protein and protein kinase C activation. Endothelin-1 does not induce thrombin receptor degradation nor change in thrombin receptor mRNA level after 2 h and 24 h of incubation. These results indicate that partial heterologous internalization of the functional thrombin receptor is induced by endothelin-1.

Cells, Cultured↗

PubMed1995

Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin.

The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

Amino Acid Sequence↗

PubMed1994

Molecular recognition by thrombin. Role of the slow-->fast transition, site-specific ion binding energetics and thermodynamic mapping of structural components.

The interaction of thrombin with the potent natural inhibitor hirudin is controlled in a complex fashion by the binding of Na+ and Cl- to the enzyme and allosteric transitions. Binding of hirudin is positively linked to Na+ binding, but is opposed in a competitive fashion by the binding of Cl-. Since Na+ binding induces the slow-->fast transition of thrombin, it follows from linkage principles that hirudin binds to the fast form with higher affinity. Hence, the slow-->fast transition is a key component of molecular recognition of hirudin by thrombin. We propose a three-step mechanism for molecular recognition of hirudin by thrombin, which is also relevant for recognition of fibrinogen and possibly the platelet receptor and thrombomodulin. First, the C-terminal acidic tail of hirudin binds to the fibrinogen recognition site of thrombin displacing one Cl ion from the thrombin surface. Then, the enzyme undergoes a conformational transition that gives rise to increased accessibility of the catalytic pocket to small synthetic substrates through movement of the Trp148 loop. The changes in the catalytic moiety triggered allosterically by binding to the fibrinogen recognition site are linked to the uptake of Na+ and are similar to, if not identical with, those observed in the Na(+)-induced slow-->fast transition. Finally, the compact N-terminal domain is accommodated in the region surrounding the catalytic pocket. Hirudin binding is also used as a probe of site-specific ion-binding interactions of Na+ and Cl- with the enzyme, characterized by cooperativity between the Na+ and Cl- binding domains. The structural components directly involved or linked to Na+ and Cl- binding have been explored in terms of free energy perturbations of the binding of hirudin and a number of ligands. The fibrinogen recognition site stores most of the free energy of coupling with Cl- binding, while regions surrounding the access to the catalytic pocket provide most of the free energy of coupling with Na+ binding and the slow-->fast transition of thrombin. It is concluded that thrombin is an allosteric enzyme that exists in two forms, slow and fast, and that the allosteric transition slow-->fast represents the key element of molecular recognition of important physiological substrates. The allosteric nature of the enzyme also enables different structural domains to communicate and give rise to biological function.

Amino Acid Sequence↗

PubMed1997

Oligonucleotide inhibitors of human thrombin that bind distinct epitopes.

Thrombin, a multifunctional serine protease, recognizes multiple macromolecular substrates and plays a key role in both procoagulant and anticoagulant functions. The substrate specificity of thrombin involves two electropositive surfaces, the fibrinogen-recognition and heparin-binding exosites. The SELEX process is a powerful combinatorial methodology for identifying high-affinity oligonucleotide ligands to any desired target. The SELEX process has been used to isolate single-stranded DNA ligands to human thrombin. Here, a 29-nucleotide single-stranded DNA ligand to human thrombin, designated 60-18[29], with a Kd of approximately 0.5 nM is described. DNA 60-18[29] inhibits thrombin-catalyzed fibrin clot formation in vitro. Previously described DNA ligands bind the fibrinogen-recognition exosite, while competition and photocrosslinking experiments indicate that the DNA ligand 60-18[29] binds the heparin-binding exosite. DNA 60-18[29] is a quadruplex/duplex with a 15-nucleotide "core" sequence that has striking similarity to previously described DNA ligands to thrombin, but binds with 20 to 50-fold higher affinity. The 15-nucleotide core sequence has eight highly conserved guanine residues and forms a G-quadruplex structure. A single nucleotide within the G-quadruplex structure can direct the DNA to a distinct epitope. Additional sequence information in the duplex regions of ligand 60-18[29] contribute to greater stability and affinity of binding to thrombin. A low-resolution model for the interaction of DNA 60-18[29] to human thrombin has been proposed.

Aptamers, Nucleotide↗

PubMed2001

Solvent accessibility of the thrombin-thrombomodulin interface.

The kinetics of solvent accessibility at the protein-protein interface between thrombin and a fragment of thrombomodulin, TMEGF45, have been monitored by amide hydrogen/deuterium (H/2H) exchange detected by MALDI-TOF mass spectrometry. The interaction is rapid and reversible, requiring development of theory and experimental methods to distinguish H/2H exchange due to solvent accessibility at the interface from H/2H exchange due to complex dissociation. Association and dissociation rate constants were measured by surface plasmon resonance and amide H/2H exchange rates were measured at different pH values and concentrations of TMEGF45. When essentially 100% of the thrombin was bound to TMEGF45, two segments of thrombin became completely solvent-inaccessible, as evidenced by the pH insensitivity of the amide H/2H exchange rates. These segments form part of anion-binding exosite I and contain the residues for which alanine substitution abolishes TM binding. Several other regions of thrombin showed slowing of amide exchange upon TMEGF45 binding, but the exchange remained pH-dependent, suggesting that these regions of thrombin were rendered only partially solvent-inaccessible by TMEGF45 binding. These partially inaccessible regions of thrombin form both surface and buried contacts into the active site of thrombin and contain residues implicated in allosteric changes in thrombin upon TM binding.

Allosteric Site↗

PubMed2001

Bimodal concentration-dependent effect of thrombin on endothelial cell proliferation and growth factor release in culture.

BACKGROUND: The role of thrombin in the stimulation of endothelial cell (EC) proliferation is controversial. The aim of this study was to investigate if thrombin regulates cell proliferation and production of platelet-derived growth factor (PDGF), bovine fibroblast growth factor (bFGF), and transforming growth factor beta(1) (TGF-beta(1)) by bovine aortic ECs. METHODS: ECs, obtained from thoracic aortas of calves, were stimulated with thrombin at various concentrations (from 0.05 to 1.0 IU/ml) in serum free culture. Mitogenic activity of thrombin on ECs was determined by tritiated thymidine uptake. The release of PDGF, bFGF, and TGF-beta(1) was assessed by ELISA. PDGF release was confirmed by Western blot and bFGF and TGF-beta(1) mRNA expression was determined by polymerase chain reaction (PCR). RESULTS: Thrombin at high concentrations did not cause any increase in EC proliferation after 72 h of culture and induced inhibition of EC proliferation after 96 h and 8 days of culture. It induced a decrease in PDGF release and an increase in TGF-beta(1) release. Thrombin at low concentrations induced a significant increase in EC proliferation at 72 h, 96 h, and 8 days of culture. It induced an increase in PDGF release and a decrease in TGF-beta(1) release. bFGF release was higher than control at all thrombin concentrations. These data were confirmed by Western blot and PCR studies. CONCLUSIONS: Thrombin regulates EC growth through the inhibition of EC proliferation at high concentrations and through the stimulation of EC proliferation at low physiological concentrations. EC proliferation is partially mediated by autocrine production of PDGF, bFGF, and TGF-beta(1).

Animals↗

PubMed2006

Up-regulation of brain ceruloplasmin in thrombin preconditioning.

Pretreatment with low-dose thrombin attenuates brain edema induced by iron or intracerebral hemorrhage (ICH). Ceruloplasmin is involved in iron metabolism by oxidizing ferrous iron to ferric iron. The present study examines whether thrombin modulates brain ceruloplasmin levels and whether exogenous ceruloplasmin reduces brain edema induced by ferrous iron in vivo. In the first set of experiments, rats received intracerebral infusion of saline or 1 U thrombin into the right basal ganglia. Rats were killed 1, 3, or 7 days later for Western blot analysis and RT-PCR analysis. In the second set of experiments, rats received either ferric iron, ferrous iron, or ferrous iron plus ceruloplasmin, then were killed 24 hours later for brain edema measurement. We found that ceruloplasmin protein levels in the ipsilateral basal ganglia increased on the first day after thrombin stimulation and peaked at day 3. Brain ceruloplasmin levels were higher after thrombin infusion than after saline injection. RT-PCR showed that brain ceruloplasmin mRNA levels were also up-regulated after thrombin injection (p < 0.05). We also found ipsilateral brain edema after intracerebral infusion of ferrous iron but not ferric iron at 24 hours. Co-injection of ferrous iron with ceruloplasmin reduced ferrous iron-induced brain edema (p < 0.05). Our results demonstrate that thrombin increases brain ceruloplasmin levels and exogenous ceruloplasmin reduces ferrous iron-induced brain edema, suggesting that ceruloplasmin up-regulation may contribute to thrombin-induced brain tolerance to ICH by limiting the injury caused by ferrous iron released from the hematoma.

Animals↗

PubMed1992

Generation of thrombin activity in relation to factor VIII:C concentrations and vascular complications in type 1 (insulin-dependent) diabetes mellitus.

A possible association between plasma coagulant activity and the presence of vascular complications in patients with diabetes mellitus was studied by measuring the generation of thrombin in plasma of 20 control subjects and 50 diabetic patients classified according to the presence or absence of microvascular complications. Thrombin production was determined in defibrinated plasma using a semi-automated technique with measurement of thrombin activity using chromogenic peptide S2238. Values determined were the lag time to appearance of thrombin activity and time taken to generate 50% maximal thrombin activity. Thrombin activity was related to concentrations of coagulant factor VIII activity and fibrinopeptide A and these were correlated with HbA1C levels. The median time to generate 50% maximal thrombin activity was not significantly reduced in diabetic patients compared with control subjects (53 vs 54 s, p = 0.076) and there were no significant differences between patients with and without microvascular complications. There were no differences in median fibrinopeptide A concentrations between the diabetic and control subjects (1.5 vs 2.2 nmol/l, p = 0.169). Time to 50% maximal thrombin activity correlated inversely with factor VIII:C concentrations in diabetic patients (r = -0.344, p = 0.015, n = 50) and both this and lag time correlated with factor VIII:C in diabetic patients and control subjects combined (r = -0.395, p less than 0.01; r = -0.327, p = 0.006, n = 70). Factor VIII:C concentrations increased with age of the subject and with HbA1C concentrations. The results failed to show enhancement of coagulation in contact-activated diabetic plasma compared with control plasma and suggest that a relationship between high levels of factor VIII:C in diabetes and the development of mcirovascular complications is unlikely to be mediated through procoagulant activity in plasma.

Adult↗

PubMed2000

Mental stress and physical exercise increase platelet-dependent thrombin generation.

Thrombin generation is an important factor in the pathogenesis of thrombogenic disorders and acute coronary syndromes. Increase in mental stress has been associated with the initiation of the acute coronary syndromes, but the exact mechanism is not known. The present study examined the effects of physical exercise and mental stress on platelet-dependent thrombin generation. Twelve healthy men (mean age 34.2 +/- 2.4 years) underwent a treadmill exercise test and a mental stress test by performing mental arithmetic. Platelet-dependent thrombin generation and plasma concentrations of catecholamines, thrombin-antithrombin III complex (TAT), plasmin-alpha2 plasmin inhibitor complex (PIC), and plasminogen activator inhibitor-1 (PAI-1) were measured before, immediately after, and at 10 and 30 min after stress. Thrombin generation increased significantly immediately after exercise, followed by rapid normalization. Mental stress caused a significant increase in thrombin generation 10 min after stress. While plasma concentrations of epinephrine, norepinephrine, and dopamine were elevated immediately after exercise, and rapidly returned to baseline, only plasma norepinephrine increased immediately after mental stress. TAT and PIC concentrations did increase immediately after exercise; however, PAI-1 remained unchanged. The increase in thrombin generation with exercise and mental stress was unaffected by treatment with 81 mg/day of aspirin of 7 days. However, it was inhibited by a single oral 40-mg dose of metoprolol. Both exercise and mental stress cause an increase in platelet-dependent thrombin generation, which was suppressed by beta-blocker therapy, but not by aspirin.

Adrenergic beta-Antagonists↗

PubMed1997

Activity of protein kinase C is necessary for sustained thrombin-induced [Ca2+]i oscillations in rat glioma cells.

The aim of the present study was to examine the possible role of protein kinase C (PKC) in thrombin-induced Ca2+ signalling. As shown before, continuous superfusion of rat glioma cells with thrombin caused sustained [Ca2+]i oscillations through activation of cell surface receptors [Czubayko U, Reiser G (1995) Neuroreport 6: 1249]. These oscillations were inhibited by protease nexin-1. Addition of PKC inhibitors, i. e. staurosporine (0.2-20 microM), bisindolylmaleimide (1 microM) or chelerythrine (1 microM), irreversibly suppressed thrombin-induced [Ca2+]i oscillations. Thereafter application of 2,5-di(tert-butyl)-1,4-benzohydroquinone (t-BuBHQ, 20 microM) or thapsigargin (1 microM) (inhibitors of sarco/endoplasmic reticulum Ca2+-ATPase) caused no [Ca2+]i response, indicating that intracellular Ca2+ stores were completely empty. We tested whether PKC affects the refilling of internal Ca2+ stores in thrombin-stimulated cells, by monitoring the amount of Ca2+ release caused by t-BuBHQ in the presence or absence of PKC inhibitors or activators. The amount of Ca2+ released by t-BuBHQ, which was normalized by comparison with the thrombin-induced Ca2+ response, was decreased by simultaneous incubation with staurosporine or chelerythrine, but enhanced with the PKC activator oleoyl acetyl glycerol. Furthermore, the capacitative Ca2+ entry was reduced by inhibition or downregulation, and increased by activation, of PKC. Capacitative Ca2+ entry was induced in these experiments by depletion of Ca2+ stores by the addition of thapsigargin or t-BuBHQ. In contrast, the inhibition of PKC during thrombin-induced depletion of intracellular stores did not influence the Ca2+ entry but nearly completely abolished the refilling of the internal stores. Thus we conclude that during thrombin receptor stimulation activation of PKC is required to maintain the refilling of intracellular Ca2+ stores for sustained [Ca2+]i oscillations. Thus, the control by PKC of the capacitative Ca2+ entry is apparently different depending on whether it is induced by sarco/endoplasmic reticulum Ca2+-ATPase inhibition or by activation of the thrombin receptor.

Animals↗

PubMed1988

Intracameral thrombin and the corneal endothelium.

We perfused the endothelia of isolated human corneas mounted in the specular microscope with BSS Plus containing 1,000-U/ml or 100-U/ml dilutions of two commercially available topical thrombin preparations. Corneas perfused with thrombin at 1,000 U/ml showed intracellular and intercellular vacuole formation and altered junctional complexes. As listed on the package inserts, the thrombin preparations contained preservatives and other additives that present a significant osmotic load in 1,000-U/ml preparations. Corneas perfused with 100-U/ml thrombin solutions showed a significant attenuation in their deswelling rate but no ultrastructural alterations. One available thrombin preparation when diluted to 100 U/ml had a glycine concentration associated with previous retinal electroretinography changes. Polyacrylamide gel electrophoresis of one manufacturer's thrombin solution showed multiple high and low molecular weight constituents. Analysis of particulate contamination showed one 100-U/ml thrombin preparation to have a large quantity of particulates. Although thrombin may be useful when applied topically as an aid in surgical hemostasis, its use intraocularly presents substantial concern regarding the preparation's purity, additives, contaminants, and adverse effects on ocular tissues.

Aged↗

PubMed1984

Kinetics of hydrolysis of peptide thioester derivatives of arginine by human and bovine thrombins.

Several peptide thioester substrates have been synthesized and tested with human thrombins (alpha, gamma, and nitrated), bovine thrombin, trypsin, and Factor X alpha beta. The substrates include various thioalkyl esters, thiobenzyl esters with substitution in the 4-position, substrates containing additional residues on the amino-terminal side of the scissile bond (P extended substrates) and one substrate containing additional residues on the carboxyl-terminal side (P' extended substrate). Neither the P nor the P' extensions resulted in significantly increased specificity; however, with one P extended substrate, D-Phe-Pro-Arg-SBzl, the KM with bovine thrombin (0.72 microM) was the second lowest KM yet reported. The results of this study underscore the importance of P vs. P' extension for thrombin substrates. The kinetic constants of the thiobenzyl esters were found to be little affected by the 4-position substitutions. A comparison of gamma-thrombin and nitrothrombin shows them to be quite similar kinetically, while both are significantly less reactive than alpha-thrombin. With these substrates, trypsin, bovine thrombin, and Factor X alpha beta have kcat/KM values in the approximate ratio of 35:10:1, respectively. The results presented here should be of value in the future design of reactive yet specific substrates for thrombin. The comparisons between the various enzymes could be helpful in clarifying the nature of their active sites.

Animals↗

PubMed1990

Persistent activation of platelet membrane phospholipase C by proteolytic action of trypsin and thrombin.

Trypsin causes rapid activation of intact platelets that mimics many actions of thrombin, including the stimulation of phospholipase C (PLC). We have examined the effects of thrombin and trypsin on PLC in a platelet membrane preparation using exogenous [3H]-phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. Trypsin induced PIP2 breakdown, which was maximal at 20 micrograms/ml, but was reduced at higher concentrations. alpha- and gamma-Thrombins also stimulated PLC-induced hydrolysis of PIP2 in membranes. This effect was inhibited by leupeptin. Exogenous [3H]phosphatidylinositol 4-monophosphate (PIP) was hydrolyzed in response to both thrombin and trypsin in the same ratio as PIP2. Activation of membrane-bound PLC persisted after removal of thrombin and trypsin. The hydrolysis of [3H]phosphatidylinositol was not activated by alpha-thrombin and trypsin. We examined the question of whether calpain was involved in the observed PLC activation by thrombin and trypsin. Although dibucaine activated a Ca2(+)-dependent protease as judged by the hydrolysis of actin-binding protein and by the activation of phosphoprotein phosphatases, it failed to stimulate the generation of phosphatidic acid in 32P-prelabeled platelets. Moreover, when PLC was assayed in the membranes, the addition of Ca2(+)-activated neutral proteinases did not increase the rate of hydrolysis of either PIP or PIP2. Our results show that proteases such as trypsin and thrombin are able to stimulate membrane-bound PLC, but this activation does not seem to be related to calpain.

Blood Platelets↗

PubMed1986

Elongation of arachidonic and eicosapentaenoic acids limits their availability for thrombin-stimulated release from the glycerolipids of vascular endothelial cells.

This study has examined the thrombin-stimulated release of polyunsaturated fatty acids from endothelial glycerolipids. Human umbilical vein endothelial cells were incubated with 1.25 microM [14C]arachidonate or [14C]eicosapentaenoate and then exposed to thrombin in buffered saline plus albumin. After an incorporation period of 0.5 h, the thrombin-stimulated release of the two radiolabeled fatty acids was quite similar. By contrast, after 24 h of fatty acid incorporation, the thrombin-stimulated release of radiolabeled fatty acid from cells incubated with [14C]eicosapentaenoate was only 25-30% of that from cells with [14C]arachidonate. Analysis of cellular glycerolipids indicated that 23 and 72%, respectively, of the incorporated [14C]arachidonate and [14C]eicosapentaenoate had been elongated to 22-carbon fatty acids in 24 h. Both 20- and 22-carbon 14C-labeled fatty acids were released to albumin in the medium in control incubations. Addition of thrombin stimulated the release of [14C]arachidonate and [14C]eicosapentaenoate, but not of their respective elongation products. Furthermore, endothelial cells incorporated exogenous [14C]docosatetraenoate into cellular glycerolipids but did not release it in response to thrombin. Thus, the thrombin-stimulated release of polyunsaturated fatty acids from vascular endothelial cells is highly selective for arachidonate and eicosapentaenoate. These results suggest that the extensive elongation of eicosapentaenoate by these cells serves to remove n - 3 polyunsaturated fatty acids from the pool of cellular acyl groups which are released in response to thrombin and are thus made available for metabolism by cyclooxygenase and lipoxygenase enzymes.

Arachidonic Acid↗