Homogenization by nitrogen cavitation technique applied to platelet subcellular fractionation.
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Blood Platelets↗
Biomedical subjects
M J Broekman
Publications and source records attributed to M J Broekman.
Inhibition of human platelet reactivity by endothelium-derived relaxing factor from human umbilical vein endothelial cells in suspension: blockade of aggregation and secretion by an aspirin-insensitive mechanism.
To determine a role for endothelium-derived relaxing factor/nitric oxide (EDRF/NO) in regulation of human platelet reactivity by human endothelial cells (EC), we studied combined suspensions of human umbilical vein endothelial cells (HU-VEC, passage 2 through 3) and washed human platelets. Confluent HUVEC monolayers were treated with aspirin (1 mmol/L) to prevent prostacyclin (PGI2) formation, washed, and harvested. Aspirin-treated platelets alone (58 x 10(6)) were fully aggregated by thrombin at 0.05 U/mL or more. In the presence of 10(6) HUVEC, however, platelet serotonin release and aggregation in response to thrombin at doses as high as 0.5 U/mL were blocked. We demonstrated for the first time that inhibition of aggregation and serotonin release, due to EDRF/NO, occurred in parallel. HUVEC-dependent inhibition of platelet responsiveness was enhanced by superoxide dismutase (SOD) and reversed by hemoglobin. The inhibitory effect was also reversed by preincubation of HUVEC with NG-monomethyl-L-arginine (NMA) or NG-nitro-L-arginine (NNA) through competitive blockade of arginine metabolism. Pretreatment of platelets with methylene blue indicated that EC-dependent inhibition of platelet reactivity occurred through activation of platelet soluble guanylate cyclase. When platelets and HUVEC were separated by a permeable membrane and both cells were stimulated by thrombin, platelets remained unresponsive. This indicated that inhibition was induced by a fluid-phase mediator, independent of direct cell-cell contact. These data demonstrate that EDRF/NO formation from L-arginine by human EC plays an important role as an aspirin-insensitive fluid-phase inhibitor of human platelet reactivity.
Arginine↗
Erythrocytes metabolically enhance collagen-induced platelet responsiveness via increased thromboxane production, adenosine diphosphate release, and recruitment.
Erythrocytes promoted platelet reactivity in a plasma medium, as demonstrated in an in vitro system that independently evaluated the biochemistry of platelet activation and recruitment. The prothrombotic erythrocyte effects were metabolically regulated, as evidenced by lack of activity of ATP-depleted or glutaraldehyde-fixed erythrocytes. They occurred in the absence of cell lysis as verified by lactate dehydrogenase assays, and had an absolute requirement for platelet activation. The presence of erythrocytes induced a twofold increase in platelet thromboxane B2 (TXB2) synthesis upon collagen stimulation, indicating that erythrocytes modulated platelet eicosanoid formation. Cell-free releasates from stimulated platelet-erythrocyte suspensions, which exhibited increased recruiting capacity, contained 6.9-fold more ADP and 4.9-fold more ATP than releasates from stimulated platelets alone. Following aspirin ingestion, TXB2 formation was blocked, but erythrocyte promotion of platelet reactivity persisted at those doses of collagen that reinduced platelet activation. Moreover, when platelet mixtures consisted of as little as 10% obtained before aspirin plus 90% obtained post-aspirin ingestion, significant erythrocyte enhancement of platelet reactivity occurred, even at low agonist concentrations. These erythrocyte effects would decrease the therapeutic potential of inhibition of platelet cyclooxygenase by aspirin. The erythrocyte-induced modulation of platelet biochemistry and function emphasizes the importance of cell-cell interactions in stimulus-response coupling.
Adenosine Diphosphate↗
Enhancement of platelet reactivity and modulation of eicosanoid production by intact erythrocytes. A new approach to platelet activation and recruitment.
Erythrocytes are known to influence hemostasis. Bleeding times are prolonged in anemia and corrected by normalizing the hematocrit. We now demonstrate that intact erythrocytes modulate biochemical and functional responsiveness of activated platelets. A two-stage procedure, permitting studies of cell-cell interactions and independently evaluating platelet activation and recruitment within 1 min of stimulation, was developed. Erythrocytes increased platelet serotonin release despite aspirin treatment, enzymatic adenosine diphosphate removal, protease inhibition, or combinations thereof. The data suggested that erythrocyte enhancement of platelet reactivity can reduce the therapeutic effectiveness of aspirin. Erythrocytes metabolically modified platelet arachidonate or eicosapentaenoate release and eicosanoid formation. They promoted significant increases in cyclooxygenase and lipoxygenase metabolites upon platelet stimulation with collagen or thrombin. However, with ionophore, erythrocytes strongly reduced platelet lipoxygenation. These erythrocyte modulatory effects were stimulus-specific. Activated platelet-erythrocyte mixtures, with or without aspirin, promoted 3-10-fold increases in extracellular free fatty acid, which would be available for transcellular metabolism. Erythrocyte-induced increases in free eicosapentaenoate may contribute to antithrombotic and anti-inflammatory effects of this fish oil derivative. These results provide biochemical insight into erythrocyte contributions to thrombosis and hemostasis, and support the concept of thrombus formation as a multicellular event.
Adenosine Diphosphate↗
Inhibition of platelet function by an aspirin-insensitive endothelial cell ADPase. Thromboregulation by endothelial cells.
We previously reported that platelets become unresponsive to agonists when stimulated in combined suspension with aspirin-treated human umbilical vein endothelial cells. Inhibition occurred concomitant with metabolism of platelet-derived endoperoxides to prostacyclin by endothelial cells. We now demonstrate that if aspirin-treated platelets which fully respond to appropriate doses of agonists are exposed to aspirin-treated endothelial cells, they remain unresponsive despite absence of prostacyclin. Platelet inhibition is due in large part to ecto-ADPase activity on the endothelial cells. This was established by incubating aspirin-treated endothelial cells with 14C-ADP. Radio-thin layer chromatography and aggregometry demonstrated that 14C-ADP and induction of platelet activation decreased rapidly and concurrently. AMP accumulated transiently, was further metabolized to adenosine, and deaminated to inosine. The apparent Km of the endothelial cell ADPase was 33-42 microM and the Vmax 17-43 nmol/min per 10(6) cells, values in the range of antithrombotic potential. Thus, at least three complementary systems in human endothelial cells control platelet responsiveness: a cell-associated, aspirin-insensitive ADPase which functions in parallel with fluid phase autacoids such as the aspirin-inhibitable eicosanoids, and the aspirin-insensitive endothelium-derived relaxing factor.
Adenosine Diphosphate↗
Platelet-neutrophil interactions. 12S,20- and 5S,12S-dihydroxyeicosapentaenoic acids: two novel neutrophil metabolites from platelet-derived 12S-hydroxyeicosapentaenoic acid.
Dietary marine n-3 polyunsaturated fatty acids have demonstrated an antiinflammatory potential in epidemiologic and intervention studies in humans. Proposed mechanisms, involving only leukocytes, fall short of explaining this potential completely. Enriched by dietary means with eicosapentaenoic acid (EPA), stimulated human platelets release substantial amounts of eicosapentaenoic acid and 12S-hydroxyeicosapentaenoic acid (12S-HEPE) in addition to 12S-hydroxyeicosatetraenoic acid (12S-HETE) derived from arachidonic acid. Human neutrophils metabolize 12S-HETE to 5S,12S-DiHETE when stimulated, whereas unstimulated neutrophils produce 12S,20-DiHETE. This study was undertaken to characterize metabolism of 12S-HEPE in human neutrophils. We demonstrate herein for the first time that 12S-HEPE is metabolized by human neutrophils. In unstimulated neutrophils 20-hydroxylation to 12S,20-DiHEPE occurs, whereas in stimulated neurtrophils 5-lipoxygenation to 5S,12S-DiHEPE takes place. The structures of these metabolites were characterized by their relative retention times on reversed-phase high pressure liquid chromatography, by their UV absorbance spectra, and by gas-liquid chromatography-mass spectrometry. With increasing amounts of 12S-HEPE, stimulated neutrophils produced increasing amounts of 5S,12S-DiHEPE, which is virtually inactive biologically. Concomitantly, production of the potent chemokinetic and chemoattractant arachidonic acid derivative leukotriene B4 decreased. Thus, 12S-HEPE can compete with endogenous arachidonic acid for 5-lipoxygenation in stimulated human neutrophils. 12,20-DiHEPE, LTB5, and 5S,12S-DiHEPE were detectable after coincubating EPA-enriched platelets with unenriched neutrophils, and arachidonic acid-derived 5-lipoxygenase products were decreased. We conclude that 12S-HEPE can participate in platelet-neutrophil interactions in a manner similar to 12S-HETE. By providing competing substrates for neutrophil 5-lipoxygenase, platelets might contribute to the antiinflammatory potential of dietary n-3 fatty acids through platelet-neutrophil interaction.
Anti-Inflammatory Agents, Non-Steroidal↗
Occupancy of adenosine receptors on human neutrophils inhibits respiratory burst stimulated by ingestion of complement-coated particles and occupancy of chemoattractant but not Fc receptors.
Chemoattractants are generated at inflammatory loci that not only induce neutrophils (PMNs) to leave the vasculature but also stimulate PMNs to release potentially toxic agents (e.g., H2O2, O2- or OH). We have recently demonstrated that endothelium releases adenosine which, when bound to a specific receptor on the PMN surface, inhibits release of toxic oxygen metabolites from stimulated PMN. To determine whether occupancy of adenosine receptors modulates generation and release of oxygen metabolites, we have studied the effect of 2-chloroadenosine on O2- generation and O2 consumption in response to opsonized zymosan particles (STZ) and immune complexes (IC). 2-Chloroadenosine inhibits, in a dose-dependent fashion, O2- generation by neutrophils that have been exposed to C3b-coated particles (STZ). Inhibition of O2- generation is similar in the presence or absence of cytochalasin B (IC50 = 53 +/- 19 and 16 +/- 5 nM, respectively, P = NS). Since occupancy of adenosine receptors might inhibit only externalization but not generation of oxygen metabolites, we studied the effect of 2-chloroadenosine on oxygen consumption by activated neutrophils. 2-Chloroadenosine inhibited O2 consumption stimulated by STZ and the surrogate bacterial chemoattractant FMLP; however, inhibition of O2 consumption varied with the presence or absence of cytochalasin B. In contrast, when neutrophils were stimulated by immune complexes, 2-chloroadenosine only minimally inhibited O2- release and O2 consumption (10 +/- 5 and 5 +/- 4% inhibition, respectively). Thus, occupancy of adenosine receptors inhibits O2 consumption in parallel with inhibition of O2- release.(ABSTRACT TRUNCATED AT 250 WORDS)
2-Chloroadenosine↗
Endogenous phosphatidylinositol 4,5-bisphosphate, phosphatidylinositol, and phosphatidic acid in stimulated human platelets.
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Blood Platelets↗
Interactions between platelets and neutrophils in the eicosanoid pathway.
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Blood Platelets↗
Albumin redirects platelet eicosanoid metabolism toward 12(S)-hydroxyeicosatetraenoic acid.
Albumin is a major determinant of eicosanoid formation, affecting autacoids important in cell-cell interactions. We delineated three mechanisms by which albumin controlled platelet eicosanoid formation: 1) Albumin diverted free arachidonate toward 12-lipoxygenation. 2) Albumin enhanced release of arachidonate from phospholipids. 3) Albumin inhibited incorporation of arachidonate from the medium into platelet phospholipids. 12(S)-Hydroxyheptadecatrienoic acid (12-HHTrE) formation was reduced 70% by albumin as compared to that formed in albumin-free medium. In sharp contrast, formation of 12(S)-hydroxyeicosatetraenoic acid (12-HETE), the platelet lipoxygenase product, was much less influenced by albumin. Moreover, 12-HETE production in the presence of albumin was markedly increased and prolonged after aspirin treatment. These data suggested that albumin redirected released endogenous arachidonate from cyclooxygenase to lipoxygenase. Therefore, the metabolic fate of arachidonate present in the medium of stimulated platelets was studied by adding tracer [3H]arachidonate 30 sec before thrombin. Albumin increased arachidonate metabolism by lipoxygenase 7-fold as compared to albumin-free controls, while cyclooxygenation increased 2.7-fold. Redirection of eicosanoid metabolism by albumin toward lipoxygenase products constitutes a heretofore undescribed and potentially important physiological role for albumin. In vitro utilization of albumin may reflect in vivo events in thrombosis and hemostasis more accurately than previous studies without albumin could appreciate.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Platelet-neutrophil interactions. (12S)-hydroxyeicosatetraen-1,20-dioic acid: a new eicosanoid synthesized by unstimulated neutrophils from (12S)-20-dihydroxyeicosatetraenoic acid.
In the course of a cell-cell interaction, 12-HETE (12-hydroxy-5,8,10,14-eicosatetraenoic acid), the arachidonic acid lipoxygenase product released from stimulated platelets, is metabolized by a cytochrome P-450 enzyme system in unstimulated neutrophils to 12,20-DiHETE (12,20-dihydroxy-5,8,10,14-eicosatetraenoic acid). This report describes time-dependent formation of a new eicosanoid by unstimulated neutrophils exposed to 12-HETE, which is more polar than 12,20-DiHETE (reversed-phase high performance liquid chromatography). Time course studies indicated that the precursor compound of this new eicosanoid was 12,20-DiHETE. This was determined by incubation of purified 12,20-DiHETE with neutrophils, which resulted in a progressive decrease in 12,20-DiHETE as formation of the polar metabolite increased. In the absence of neutrophils, 12,20-DiHETE was quantitatively unchanged. The new metabolite of 12,20-DiHETE was identified as 12-hydroxyeicosatetraen-1,20-dioic acid, based upon its UV spectrum, co-chromatography with a chemically synthesized standard in both high performance liquid chromatography and thin layer chromatography systems, and gas chromatography-mass spectrometry. Formation of 12-HETE-1,20-dioic acid was partially inhibited by 20-hydroxy-LTB4. This indicated that the neutrophil dehydrogenase responsible for further metabolism of 12,20-DiHETE may also be involved in conversion of 20-hydroxy-LTB4 to 20-carboxy-LTB4. The 12,20-DiHETE dehydrogenase enzyme system specifically requires NAD as cofactor and has subcellular components in both cytosolic and microsomal fractions which are synergistic in their activity. These results provide additional evidence for the occurrence of multicellular metabolic events during hemostasis, thrombosis, and the inflammatory response.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Platelet-neutrophil interactions in the eicosanoid pathway.
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12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Cell-cell interactions in the eicosanoid pathways.
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Cell Communication↗
Cell-cell interactions in the eicosanoid pathway.
In vitro experiments carried out in several laboratories indicate that cell components of hemostatic plugs, thrombi, and inflammatory lesions are capable of sharing precursors and intermediates of both the lipoxygenase and cyclooxygenase systems. These cells produce new eicosanoids in a stimulus-specific manner. It is therefore important to further elucidate mechanisms by which eicosanoids are formed during cell-cell interactions and the functional implications thereof.
Animals↗
Studies on the mechanism of omega-hydroxylation of platelet 12-hydroxyeicosatetraenoic acid (12-HETE) by unstimulated neutrophils.
Stimulated platelets, in the presence or absence of aspirin, synthesize significant quantities of 12-hydroxyeicosatetraenoic acid (12-HETE), which is chemotactic and chemokinetic, and enhances mononuclear cell procoagulant activity. During a cell-cell interaction between stimulated platelets and unstimulated neutrophils, platelet 12-HETE is metabolized to 12,20-dihydroxyeicosatetraenoic acid (12,20-DiHETE) by neutrophils. Characteristics of the enzyme system in unstimulated neutrophils responsible for this omega-hydroxylation were investigated. A broad range of cytochrome P-450 inhibitors, as well as leukotriene B4, blocked formation of 12,20-DiHETE. Owing largely to released proteases, neutrophil homogenization abolished activity. Pretreatment with diisopropylfluorophosphate preserved activity in neutrophil homogenates. omega-Hydroxylation of 12-HETE was confined solely to the microsomal fraction. Specific activity increased 6.6-fold compared with neutrophil sonicates. The electron donor NADPH was a required cofactor. These results indicate that the enzyme in unstimulated human neutrophils, which metabolizes 12-HETE from stimulated platelets to 12,20-DiHETE in this cell-cell interaction, is a cytochrome P-450 monooxygenase.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Phospholipid metabolism in human neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine. Degranulation is not required for release of arachidonic acid: studies with neutrophils and neutrophil-derived cytoplasts.
Neutrophils respond to chemoattractants by aggregating, degranulating, remodelling of phospholipids and releasing arachidonic acid. To determine whether ligand-induced remodelling of phospholipids depends on redistribution of intracellular organelles (degranulation), we compared phospholipid remodelling of human neutrophils with that of neutrophil-derived cytoplasts. Cytoplasts, organelle-depleted vesicles of cytosol surrounded by plasmalemma, cannot degranulate. Without a stimulus, [3H]arachidonate was incorporated preferentially into phosphatidylinositol (PI) and phosphatidylcholine (PC). Exposure of cytoplasts and neutrophils prelabelled with [3H]arachidonate or [14C]glycerol to fMet-Leu-Phe (10(-7) M) induced rapid changes in distribution of label and mass of individual phospholipids: [3H]arachidonate in phosphatidic acid (PA) increased 500% (120 s), [14C]glycerol incorporation and mass of PA approached 200% of unstimulated values, and [3H]arachidonate in PI decreased continuously; these data are compatible with activity of a PI/PA cycle. However, the mass of PI in both preparations and [14C]glycerol label in intact neutrophils increased initially (5 s), suggesting net synthesis and mobilization of more than one pool of PI. Heterogeneity of PC pools was also observed: [3H]arachidonate was lost from PC immediately upon addition of stimulus, whereas mass and [14C]glycerol values increased. Thus, net phospholipid synthesis, redistribution of arachidonate and activation of the PI/PA cycle are immediate responses of the neutrophil to receptor occupancy by chemoattractants. Furthermore, the similarity in response to fMet-Leu-Phe of neutrophils and granule-free cytoplasts indicates that these processes are independent of degranulation.
Arachidonic Acids↗
Enhancement of mononuclear procoagulant activity by platelet 12-hydroxyeicosatetraenoic acid.
Platelets induce generation of procoagulant tissue factor activity (TFa) by mononuclear leukocytes, and also enhance the TFa induced by endotoxin. Our present investigation demonstrated that arachidonic acid, which by itself had no effect on mononuclear TFa, greatly enhanced platelet-induced TFa. The effect was concentration dependent for both platelets and arachidonate (1-20 microM); other fatty acids tested were inactive. The enhancing effect of arachidonate was more pronounced if platelets were exposed to aspirin, suggesting lipoxygenase product involvement. Production of 12-hydroxyeicosatetraenoic acid (12-HETE) was demonstrated biochemically in aspirin-treated platelet/arachidonate/mononuclear cell preparations that generated high levels of TFa. The enhancing role of 12-HETE was verified as follows. Addition of platelet-derived or synthetic 12-HETE amplified endotoxin-induced TFa more than threefold. Other lipoxygenase products were inactive. Enhancement of mononuclear cell TFa by 12-HETE represents a newly described biological function for this eicosanoid in cell-cell interactions between platelets and mononuclear cells.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
Stimulated platelets release equivalent amounts of arachidonate from phosphatidylcholine, phosphatidylethanolamine, and inositides.
Thrombin-induced changes in arachidonate content of platelet phospholipids were quantitated to establish the ultimate origins of this eicosanoid precursor. Fifteen seconds following thrombin addition (15 U/5 X 10(9) platelets), phosphatidylcholine lost 11.8 nmol of arachidonate and phosphatidylethanolamine lost 10.5 nmol. Arachidonate in phosphatidate, phosphatidylinositol, and phosphatidylinositol-4,5-bisphosphate combined decreased by 11.0 nmol. Increases in free and oxygenated arachidonate (41 nmol) exceeded decreases in inositides. Thus phospholipase A2 released at least twice as much arachidonate as phospholipase C-diglyceride lipase. Phosphatidylinositol-4-phosphate levels remained unchanged upon stimulation. Therefore, increases in phosphatidylinositol-4,5-bisphosphate indicated the minimum rate of phosphorylation of phosphatidylinositol to resynthesize phosphatidylinositol-4,5-bisphosphate, following stimulus-induced breakdown by phospholipase C. Phosphatidylinositol-4, 5-bisphosphate increased 1.4 nmol between 10 and 15 sec following thrombin, markedly less than phosphatidylinositol decreased (2.1 nmol). This could be due to phospholipase A2, in addition to phospholipase C, acting directly on phosphatidylinositol to a greater extent than estimated by accumulation of lysophosphatidylinositol, degraded rapidly by lysophospholipase. Thus, upon high-dose thrombin stimulation of human platelets inositide metabolism via phospholipase C directs initial formation of intracellular second messengers, and sequentially, or in parallel, arachidonate release by phospholipase A2 supplies the larger proportion of arachidonate for syntheses of eicosanoids involved in intercellular communication.
Arachidonic Acid↗