Fibronectin inhibition of platelet thrombus formation in an in vivo porcine model of vascular injury.

Platelets adhere and aggregate in response to exposed subendothelial matrix during vascular injury. The present study examines the effect of plasma fibronectin on platelet deposition at a site of vascular injury in an in vivo porcine model. The internal carotid arteries in anesthetized Yorkshire pigs were bilaterally exposed and the distal half of each vessel stripped of endothelium. Following stripping, one in situ carotid artery preparation was filled with 0.5 mg/ml porcine plasma fibronectin and the other artery filled with vehicle solution, to serve as a control. After five minutes, 6-7 x 10(9) 111Indium-labeled autologous platelets were infused via a femoral vein cannula, and carotid blood flow was re-established for 20 minutes. The vessel segments were excised and deposition of platelets determined. Vascular stripping increased platelet deposition 52-fold, as compared to unstripped vessel segments. Fibronectin pretreatment did not affect platelet deposition in control vessel segments but decreased platelet deposition by 77% in stripped vessel segments. Transmission and scanning electron microscopy indicated that reduced platelet deposition in the fibronectin treated group was due to decreased platelet aggregation rather than decreased adhesion.

Platelet cGMP, but not cAMP, inhibits thrombin-induced platelet adhesion to pulmonary vascular endothelium.

We have compared the effects of intracellular pathways initiated by nitric oxide and prostacyclin on thrombin-induced platelet adhesion to endothelial cells. Platelet aggregate adhesion was enhanced when endothelial monolayers were pretreated with NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide production. In addition, decreased platelet aggregate adhesion was seen when platelets were pretreated with 8-bromoadenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) or 8-bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP). Single platelet adhesion in isolated perfused lungs under flow conditions in the presence of shear was also assessed. Pretreatment of platelets with either Iloprost, in a dose sufficient to decrease platelet aggregation, or 8-bromo-cAMP did not affect platelet adhesion. However, pretreatment of platelets with 8-bromo-cGMP significantly reduced single platelet adhesion to endothelium. These studies illustrate that nitric oxide inhibits platelet adhesion to endothelium in the presence of shear. They further indicate that prostacyclin is also a regulator of this response but has effects more specifically related to the inhibition of platelet aggregation than platelet-endothelium interactions.

Fibronectin dependent macrophage fibrin binding.

Plasma fibronectin has been shown to increase the binding of fibrin monomer to macrophages in vitro. In the present study we began characterization of the mechanism underlying this fibronectin activity. Fragments of fibronectin containing the amino terminus enhanced macrophage fibrin binding to the same extent as intact fibronectin on an equimolar basis. However, fibronectin fragments containing the gelatin-binding domain or the cell-binding domain, but lacking the amino terminus, had no effect on fibrin binding. Fibronectin enhanced fibrin binding was not affected by the addition of synthetic peptides containing the RGD adhesion sequence. The ability of fibronectin to augment fibrin binding remained after paraformaldehyde fixation of macrophage monolayers. Fixation did not alter the basal levels of fibrin binding by macrophages. Preincubation of macrophages with exogenous fibronectin did not increase the binding of fibrin. Fibronectin enhanced fibrin binding remained unaltered after the removal of endogenous cell surface fibronectin by capping with F(ab')2 fragments of antibodies to fibronectin. These results suggest that the amino terminus of fibronectin supports the attachment of fibrin to macrophages by an initial fluid-phase interaction that precedes cellular binding and does not require a cellular response.

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.

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.

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.

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.

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.

Influence of prostaglandin I2 on fibronectin-mediated phagocytosis in vivo and in vitro.

This study evaluated the effect of prostaglandin I2 (PGI2) on fibronectin-mediated macrophage phagocytosis in vivo and in vitro. Phagocytosis measured in vivo in rats by the vascular clearance rate and hepatic localization gelatinized sheep erythrocytes was inhibited in a dose-dependent manner after intravenous administration of PGI2. Phagocytosis was assessed in vitro in terms of uptake of fibronectin-dependent gelatinized sheep erythrocytes by monolayers of casein-elicited rat peritoneal macrophages. Concentrations of 1 ng/ml PGI2 or greater resulted in inhibition of particle internalization but not attachment to macrophages. This inhibitory effect was enhanced by aminophylline, a phosphodiesterase inhibitor. PGI2 increased cAMP levels and these were further increased in the presence of aminophylline. These data indicate that PGI2 inhibits macrophage uptake of gelatinized particles and support the idea that this is mediated by increased intracellular levels of cyclic AMP. PGI2 should thus be considered a potential etiologic factor in the phagocytic depression observed in association with thrombosis.