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Biomedical subjects

D G Moon

Publications and source records attributed to D G Moon.

15 recordsLinked to original sources
PubMed1991

Effect of 13-hydroxyoctadeca-9,11-dienoic acid (13-HODE) on thrombin induced platelet adherence to endothelial cells in vitro.

The effect of exogenous 13-HODE on alpha-thrombin induced adherence of platelets to monolayers of cultured pulmonary artery endothelial cells was determined using homologous sheep cells. In a separate series of experiments, endogenous 13-HODE was demonstrated in sheep endothelial cells by reverse phase high pressure liquid chromatography. Levels of endogenous 13-HODE were decreased by alpha-thrombin preincubation. Exogenous 13-HODE (10 microM) reduced the augmented platelet adherence produced by coincubation of alpha-thrombin with platelets and endothelial monolayers, and eliminated the enhancement of platelet adherence produced by preincubation of alpha-thrombin with endothelial monolayers. 13-HODE also reduced the alpha-thrombin induced adherence of platelets to monolayers pretreated with aspirin and to fixed monolayers indicating a direct effect of 13-HODE as opposed to secondary effects mediated by the release of prostacyclin (PGI2) or endothelial derived relaxing factor (EDRF). Platelet adherence to subendothelial matrix was also reduced by 13-HODE. 13-HODE inhibited platelet aggregation initiated by 0.2 U/ml alpha-thrombin but did not affect aggregation initiated by 2.0 U/ml alpha-thrombin. These data provide evidence for the ability of exogenous 13-HODE to attenuate the interaction of thrombin activated platelets with endothelial cells as well as with other platelets.

Animals↗
PubMed1991

Thrombin structure and function: why thrombin is the primary target for antithrombotics.

Thrombin has both beneficial and harmful effects. In order of importance, at very low concentrations, alpha-thrombin firstly amplifies its own generation through the activation of factors V and VIII, which are the primary targets of antithrombotic agents. It secondly functions at the cellular level where, at low concentrations it activates platelets, and at higher concentrations, induces endothelial cell changes (e.g., shape changes, albumin transport release of plasminogen activators and other substances). It thirdly converts fibrinogen into clottable fibrin and becomes actively incorporated into the forming thrombus. In addition, it activates protein C, which in turn degrades factors V and VIII (and/or their activated forms) and causes the shutdown of thrombin generation. When compared to other serine proteinases of the blood coagulation and fibrinolytic systems, alpha-thrombin is unique in that it loses most of its proenzyme activation fragment and has developed multisite short-ranged bridge-binding interactions, which appear to explain thrombin specificity. To understand thrombin is to understand haemostasis.

Amino Acid Sequence↗
PubMed1991

Thrombin-induced leukopenia and thrombocytopenia are attenuated by PAF antagonist WEB 2086.

Thrombin has been shown to increase pulmonary transvascular permeability in vivo. This permeability change appears to be dependent on polymorphonuclear leukocytes (PMNs). In vitro, thrombin has been demonstrated to increase PMN adherence to endothelial cells coincident with generation of platelet activating factor (PAF) by endothelial cells. These observations have led to the suggestion that PAF mediates, in part, the attachment of PMNs to endothelial cells. We examined this hypothesis in vivo and in vitro with a specific PAF receptor antagonist, WEB 2086. Prior infusion of WEB 2086 into conscious sheep significantly attenuated the drop in peripheral blood PMN counts observed during and after infusion of alpha-thrombin (30 NIH U/kg). These data suggest that WEB 2086 prevented PMN margination on endothelial cells. WEB 2086 also attenuated the thrombocytopenia seen after thrombin infusion and ameliorated the thrombin-induced hypoxemia and hemoconcentration. WEB 2086 did not affect the thrombin-induced hemodynamic response, the degree of intravascular coagulation as assessed by fibrin degradation product generation, or thromboxane B2 generation. In vitro, WEB 2086 prevented the augmented adherence of sheep PMNs to sheep endothelial cell monolayers after thrombin stimulation. The results of the present study are consistent with the hypothesis that PAF mediates, at least in part, thrombin-induced leukopenia and thrombocytopenia in vivo.

Animals↗
PubMed1990

Platelet modulation of neutrophil superoxide anion production.

The effect of platelets on polymorphonuclear leukocytes (PMN) O2- production was examined using autologous sheep and human cell systems. Coincubation of sheep platelets with sheep PMNs in the absence of thrombin resulted in a significant inhibition in basal PMN O2- production. The platelet-derived inhibitory activity was released into the medium and could be destroyed by adenosine deaminase suggesting that the inhibitor was adenosine. Addition of alpha-thrombin or platelet activating factor (PAF) enhanced PMN O2- production but only when platelets were present. The enhancement of O2- production in response to thrombin was dependent upon the thrombin concentration and the platelet-PMN ratio. With a platelet: PMN ratio of 30: 1, addition of 10 nM thrombin to sheep cells resulted in a 5-fold increase in O2- production, whereas addition of 10 nM PAF caused a 2-fold increase in O2-. Addition of thrombin or PAF to either PMNs or platelets by themselves did not initiate an increase in O2- generation. The response of human cells was similar except that both thrombin and PAF triggered a 2-fold increase in PMN O2- production in the presence of platelets. The platelet-derived enhancement activity was not released into the medium and was not blocked by WEB 2086, NDGA, ETYA, aspirin or adenosine deaminase. The enhancement effect appeared to be localized to the platelet membrane and we believe requires platelet-PMN contact.

Adenosine↗
PubMed1990

Platelet activating factor and sheep platelets: a sensitive new bioassay.

The in vitro response of sheep platelets to platelet activating factor (PAF) was investigated. Sheep platelet-rich plasma aggregated in response to PAF with an EC50 of 10 nM. Platelets isolated via arabinogalactan density gradient centrifugation displayed an EC50 of 50 pM with a threshold response at 0.1 pM. PAF-induced release of 14C-serotonin from isolated sheep platelets was comparable with an EC50 of 50 pM and threshold release at 10 fM. PAF-induced aggregation was specific in that it could be blocked by the competitive receptor antagonists Alprazolam (Upjohn, IC50 = 40 microM), L-652,731 (MSD, IC50 = 6 microM), and WEB 2086 (Boehringer Ingelheim, IC50 = 0.8 microM). At micromolar concentrations, WEB 2086 did not inhibit ADP- or thrombin-induced aggregation nor thrombin-induced serotonin release. However, at higher concentrations of WEB 2086 some inhibition of thrombin-induced platelet aggregation and release was observed. Subsequent experiments demonstrated that high concentrations of WEB 2086 can inhibit thrombin-induced clotting (Ki = 866 microM) and cleavage of the chromogenic substrate Spectrozyme-TH (Ki = 842 microM). In summary, the response of isolated sheep platelets to PAF was specifically inhibitable and was 10 to 100 times more sensitive than washed rabbit platelets, the most popular bioassay currently in use.

Adenosine Diphosphate↗
PubMed1990

Electron microscopy of platelet interactions with heme-octapeptide-labeled fibrinogen.

Binding of fibrinogen to ADP-activated platelets was visualized by labeling the molecule with heme-octapeptide (microperoxidase) for direct cytochemical staining. Transmission electron microscopy of the platelet aggregates showed most of the fibrinogen distributed widely over the platelet surface in nonbridging rims of 7- to 9-nm thickness. Short peroxidase-positive bridges (less than 25 nm) were found in clusters in regions of close contact between the platelets, but 50-nm bridging corresponding to the length of the molecule was not seen by this method. Thus the fibrinogen appeared to be binding in a predominantly prone rather then upright orientation on the platelets. Abundant 50-nm bridging seen by nonspecific staining appeared unrelated to the length of the fibrinogen molecule because the bridging did not change when the length of the fibrinogen was more than doubled by end-to-end cross-linking with factor XIIIa. It is suggested that the observed binding and bridging of fibrinogen in a prone orientation is promoted by the existence of multiple platelet-binding domains on the molecule.

Blood Platelets↗
PubMed1990

Thrombin's enzymatic activity increases permeability of endothelial cell monolayers.

Human alpha-thrombin increases the permeability of bovine pulmonary artery endothelial cell (CCL-209) monolayers. To determine if this increase is via an enzymatic or receptor-mediated mechanism, enzymatically active forms of alpha-thrombin and enzymatically inactive forms with cell binding activity were incubated with the monolayers. Enzymatic forms included alpha-thrombin and two digestion products, zeta-thrombin (chymotryptic product with 89% clotting activity) and gamma-thrombin (tryptic product). Enzymatically inactive forms included D-Phe-Pro-Arg-chloromethylketone-(PPACK) alpha-thrombin and diisopropylphosphorofluoridate-(DIP) alpha-thrombin. Cell binding activity of alpha- and PPACK-alpha-thrombin was demonstrated to be similar to each other and comparable to that cited in the literature for DIP-alpha-thrombin. gamma-Thrombin, on the other hand, did not compete for binding of 125I-labeled alpha-thrombin. All enzymatic forms of alpha-thrombin increased endothelial permeability as assessed by the clearance of 125I-albumin across the monolayers. Coincubation of PPACK, an enzymatic site inhibitor, with alpha- or gamma-thrombin prevented the increase in permeability, further indicating that alpha-thrombin increased permeability by its enzymatic activity. Both enzymatically inactive forms of alpha-thrombin with high-affinity binding activity had no effect on permeability. To further examine whether cell binding activity of alpha-thrombin contributed to the increased permeability, a sulfated COOH-terminal fragment of hirudin (hirugen) that binds to the anion-binding site of alpha-thrombin but, unlike hirudin, does not interact with the catalytic site was coincubated with alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Chloromethyl Ketones↗
PubMed1989

Platelets decrease albumin permeability of pulmonary artery endothelial cell monolayers.

Since platelets may modulate endothelial cell permeability, we examined the effects of platelets on 125I-albumin permeability of cultured bovine pulmonary artery endothelial cell monolayers. The experimental system consisted of endothelial cells grown to confluence on a gelatinized polycarbonate filter. We quantified the diffusive flux of 125I-albumin from luminal chamber to the abluminal chamber. Washed human platelets added to the monolayers decreased the albumin flux in a concentration-dependent manner, with a 65% decrease occurring at the highest concentration of platelets (5 x 10(7) platelets) added to the 700-microliters luminal chamber. In contrast, neither paraformaldehyde-fixed platelets nor fresh red blood cells changed 125I-albumin permeability. Platelets had no effect on 125I-albumin permeability across the gelatinized filters without endothelial cells present. Supernatants of platelet lysates also reduced albumin flux. The effect produced by intact platelets or platelet lysate was not influenced by the presence of ketanserin (a serotonin receptor antagonist), propranolol (a beta-adrenergic receptor antagonist), or aspirin (an inhibitor of cyclooxygenase). Platelets activated by thrombin did not produce an effect that was different from the effect produced by intact platelets. The activity of the supernatant of platelet lysate remained in the aqueous phase after ether extraction. The results indicate that the platelet-mediated decrease in endothelial cell permeability to 125I-albumin is the result of a hydrophilic platelet-derived factor(s) and not secondary to mechanical obstruction of endothelial "leaks" by the platelets.

Albumins↗
PubMed1989

Endothelin-like pulmonary vasoconstrictor peptide release by alpha-thrombin.

The endothelial cells lining the vessel wall can modulate vasomotor tone by releasing vasoactive factors, such as endothelial-derived constricting factors. We observed that alpha-thrombin, but not catalytically inactivated alpha-thrombin, mediated the release of two pulmonary vasoconstrictor peptides into the venous effluent of guinea pig lungs. These peptides elicited a slow-onset, long-lasting pulmonary vasoconstriction similar to the effect of endothelin, an endothelial-derived 21-amino acid vasoconstrictor peptide previously isolated from cells in culture. One of the isolated peptides coelutes with endothelin upon reverse-phase HPLC with an acetonitrile gradient and has a molecular weight comparable to endothelin as determined by gel-permeation HPLC. The other vasoconstrictor peptide elutes earlier than endothelin on reverse-phase HPLC and exhibits a lower molecular weight. The studies show the release of endothelin-like pulmonary vasoconstrictor peptides in the intact lung by alpha-thrombin, a central regulatory enzyme in hemostasis.

Animals↗
PubMed1989

Platelets adhere to thrombin-treated endothelial cells in vitro.

Interaction of thrombin with vascular endothelial cells was investigated as a mechanism promoting platelet activation and adherence to endothelial monolayers. We found that pretreatment of endothelium with alpha-thrombin in the absence of platelets results in the attachment of platelets to endothelial cells after the removal of fluid-phase alpha-thrombin. This activity was eliminated by exposure of alpha-thrombin-pretreated endothelial cells to active site inhibitors of alpha-thrombin or by adding alpha-thrombin in the presence of excess diisopropyl fluorophosphate-inhibited thrombin, suggesting retention of active alpha-thrombin by a receptor-mediated mechanism. Morphological data and the results of [14C]serotonin release studies indicate that platelets are activated by alpha-thrombin-pretreated endothelium and that adherence represents aggregates of activated platelets as well as individual platelets. Adherence on alpha-thrombin-pretreated endothelium is dependent on divalent cations. Platelets also adhered to aortic segments pretreated with thrombin. The data of the current studies support the contention that alpha-thrombin can promote adherence of activated platelets to endothelial cells because of the binding and retention of alpha-thrombin to endothelial cells in a manner in which it remains active and available for platelet activation.

Animals↗
PubMed1989

Isoproterenol reduces thrombin-induced pulmonary endothelial permeability in vitro.

The ability of the beta-adrenergic agonist, isoproterenol, to attenuate the thrombin-induced increase in endothelial permeability was examined by measuring 125I-labeled albumin clearance across endothelial cell monolayers. Bovine pulmonary artery endothelial cells (CCL-209) were grown to confluence on gelatinized, polycarbonate micropore filters and mounted on modified Boyden chambers with Dulbecco's modified Eagle's medium (DMEM) and 0.5% bovine serum albumin. alpha-Thrombin at 0.2 nM to 2 microM produced a dose-related increase (P less than 0.01) in 125I-labeled albumin clearance from the DMEM control value. Light and electron microscopy revealed that the thrombin-induced increase in permeability correlated with changes in cell shape and rearrangement of filamentous actin. Coincubation of 2 microM isoproterenol with 2 microM alpha-thrombin reduced (P less than 0.01) the thrombin-induced increase in albumin clearance and the observed morphological changes. This attenuation was not caused by inhibition of thrombin's enzymatically active site, since isoproterenol did not impair thrombin's fibrinogen clotting activity nor its amidolytic cleavage of an artificial substrate (Spectrozyme-TH). Coincubation of 20 microM propranolol, a beta-adrenergic antagonist, with 2 microM isoproterenol and thrombin blocked the permeability-decreasing effect of isoproterenol. Both 2 microM isoproterenol and 2 pM alpha-thrombin alone decreased (P less than 0.01) albumin clearance below the DMEM control value. These results suggest that isoproterenol can reduce the thrombin-induced increase in endothelial permeability in vitro by directly maintaining actin filaments and the shape of endothelial cells.

Animals↗
PubMed1989

Fibronectin augments binding of fibrin to macrophages.

Because of the demonstrated ability of fibronectin to mediate particle uptake by macrophages and the demonstrated affinity of plasma fibronectin for fibrin, we investigated the ability of plasma fibronectin to augment macrophage binding of fibrin. Fibronectin significantly increased fibrin binding by elicited peritoneal macrophages and isolated hepatic Kupffer cells. The binding of fibrinogen was not augmented in the presence of fibronectin. The small amount of macrophage-associated fibrin observed in the absence of fibronectin was primarily internalized, whereas the increment in fibrin binding in the presence of fibronectin remained primarily surface bound, as indicated by susceptibility to removal by trypsin. An amino terminal fibrin-binding fragment of plasma fibronectin could similarly support binding of fibrin by peritoneal macrophages. Greater quantities of fibrin were associated with the macrophages in the presence of protease inhibitors, which inhibited elastase activity, but not in the presence of those that inhibited cathepsin activity, suggesting that an elastase-like protease may degrade surface-bound fibrin. Uptake of both fibrin and fibronectin was inhibited by prior treatment of cells with trypsin. Competitive binding studies suggested the presence of a high-affinity fibronectin receptor on peritoneal macrophages. Data from the current study thus support the conclusion that fibronectin augments binding of fibrin to the surface of mononuclear phagocytes.

Animals↗
PubMed1986

The inhibitory effect of plasma fibronectin on collagen-induced platelet aggregation.

Plasma fibronectin (Fn) has been proposed to have an antithrombotic effect, protecting against platelet and fibrinogen consumption after injury. The current study was designed to determine the effect of plasma fibronectin on collagen-induced platelet aggregation. In vitro aggregometry using an isolated homologous rat system, demonstrated a significant (P less than .05) inhibitory effect of 120 micrograms/mL Fn on platelet aggregation as induced by 60 micrograms/mL fibrillar collagen (type I). The inhibition was evidenced by a threefold increase in lag time and a significant decrease in the rate and extent of aggregation. The hypothesis was also tested using an in vivo model of collagen-induced platelet aggregation. The model used was intravenous injection of 2 mg/kg of homologous type I collagen into anesthetized Sprague-Dawley rats. Injection of collagen preincubated with 4 mg/kg Fn resulted in significantly less thrombocytopenia and fibrinogen consumption as compared with injection of collagen alone. The results of both the in vitro and in vivo studies are consistent with the proposed antithrombotic effect of plasma fibronectin.

Animals↗
PubMed1985

Reticuloendothelial-depressing substance: studies on the mechanism of action.

This study was carried out to evaluate the mechanism of action of a reticuloendothelial (RE)-depressing substance. This RE-depressing substance was obtained from the plasma of dogs subjected to 3 hr of intestinal ischemia. RE-depressing substance was partially purified by dialysis and reverse-phase column chromatography. The assay of RE-depressing activity was based on the depression of the rate of clearance of colloidal carbon from the blood of rats or mice. The effect of RE-depressing substance on three other RE system (RES) test particles (gelatinized lipid emulsion, formalinized sheep erythrocytes, and IgM-coated erythrocytes) was determined. RE-depressing substance did not affect the clearance rate or the organ localization of these three test particles. Therefore, RE-depressing substance affected only the clearance of colloidal carbon. Since platelet aggregation has been shown to contribute to the clearance of colloidal carbon, the effect of RE-depressing substance on platelet aggregation was evaluated. RE-depressing substance depressed in vitro platelet aggregation induced by ADP or collagen. It was concluded that the effect of RE-depressing substance on the clearance of colloidal carbon was due to a depression of platelet aggregation rather than to a depression of hepatic macrophage phagocytic function.

Animals↗