Platelets,Endothelium-Dependent Responses and Atherosclerosis

Basal release of endothelium-derived relaxing factor (EDRF) and prostacyclin from intact vascular endothelium may inhibit continuously platelet aggregation. If local platelet aggregation occurs, platelet-derived adenine nucleotides stimulate the release of EDRF. Stimulated EDRF release may override the direct vasoconstrictor effects of other platelet products such as thromboxane and serotonin resulting in local vasodilatation. In addition, stimulation of EDRF release by adenine nucleotides may inhibit further platelet adhesion and aggregation by a feedback mechanism. Thus, intact vascular endothelium may play an important role in the defense against platelet deposition and vasospasm. In atherosclerosis, basal and stimulated release of EDRF is markedly reduced. Endothelial dysfunction will impair this protective mechanism and will favour vasoconstriction and further platelet disposition. Occurrence of occlusive thrombus formation in patients with coronary artery disease may be pathophysiologically related to this impairment of endothelial defense.     

Nerve and drug-induced release of adenine nucleosides and nucleotides from rabbit aorta

The purpose of the present study was to assess the possible sites which contribute to the nerve stimulation- and alpha-agonist-induced overflow of endogenous adenine nucleosides and nucleotides in vascular tissue. Particular attention was focused on the endothelium because it is known that endothelial cells have a high concentration of ATP and its metabolites. Segments of rabbit thoracic aorta, some denuded of endothelial cells by rubbing the lumen of the vessel, were incubated in organ baths and subjected to transmural nerve stimulation or stimulated with the alpha-1 adrenoceptor agonist methoxamine. A portion of the bathing solution was processed for the determination of norepinephrine by high-performance liquid chromatography with electrochemical detection and a portion for determination of ATP, ADP, AMP and adenosine by high-performance liquid chromatography with fluorescence detection. Transmural stimulation led to a significant release of both norepinephrine and the adenine nucleosides and nucleotides in a ratio of 1 to 350. Removal of the endothelium did not change the release of norepinephrine but reduced the release of adenosine and its derivatives by 90%. Methoxamine also caused the release of adenosine and the adenine nucleotides which was reduced by 93% by removal of the endothelium. Thus, the endothelium seems to be a major source of transmural nerve stimulation and alpha-agonist induced overflow of adenosine and adenine nucleotides. The endothelium is not the exclusive source of these purine congeners, however. In the case of transmural stimulation there is approximately 10% of the total which is independent of the endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelium-dependent relaxation is independent of arachidonic acid release.

Endothelium-dependent relaxation is mediated by the release from vascular endothelium of an endothelium-derived relaxing factor (EDRF). It is not clear what role arachidonic acid has in this process. Inhibition of phospholipase A2, and diacylglycerol lipase in cultured bovine aortic endothelial cells caused a marked reduction in agonist-induced arachidonic acid release from membrane phospholipid pools, and complete inhibition of prostacyclin production. EDRF release, assayed by measuring endothelium-dependent cGMP changes in mixed endothelial-smooth muscle cell cultures, was not inhibited under these conditions. In fact, EDRF release in response to two agonists, melittin and ATP, was actually increased in cells treated with phospholipase A2 inhibitors. In addition, pretreatment of rats with high-dose dexamethasone, an inhibitor of PLA2, did not attenuate endothelium-dependent relaxation in intact aortic rings removed from the animals, or depressor responses in anesthetized animals induced by endothelium-dependent vasodilators. In summary, inhibition of arachidonic acid release from membrane phospholipid pools does not attenuate endothelium-dependent relaxation in rats, or the release and/or response to EDRF in cultured cells.     

The inhibition of cancer cell stickiness by somatostatin

We employed a test model, which we had developed for the investigation of platelet adhesiveness and aggregation in vivo. Our experiments demonstrated that somatostatin is not only able to dose-dependently inhibit the stickiness of i.v. injected Walker 256 carcinosarcoma cells to the vascular endothelium of the rat mesentery and the drastic, immediate reduction of platelet count in venous blood, but also to significantly reduce the rate of instantly occurring terminal tumor cell embolism of the lung. These actions may be explained as being mediated via an inhibition of platelet adhesion and aggregation to circulating cancer cells. Because some oral antidiabetics showed a similar but weaker effect in our test system (Gastpar et al. 1982), it should be examined as to whether the in vivo inhibition of platelet adhesiveness and aggregation of the investigated compounds are mediated by a somatostatin release from the pancreas.     

Activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid

The objective of this study was to ascertain whether "endothelium-derived relaxing factor" (EDRF) released from bovine intrapulmonary artery and vein is capable of directly activating soluble guanylate cyclase, thereby accounting for elevated vascular levels of cyclic GMP during EDRF release. Isolated arterial and venous rings, after equilibration and depolarization in bath chambers, were transferred to reaction tubes and incubated with soluble guanylate cyclase that had been purified to homogeneity from bovine lung. Addition of test agents to either bath chambers or enzyme reaction mixtures enabled the determination of their sites of action. Arterial and venous rings caused an endothelium-dependent 2- to 3-fold enzyme activation that was inhibited by methylene blue. Endothelium-dependent enzyme activation in artery but not vein was enhanced several-fold by acetylcholine in an atropine-sensitive manner. Bradykinin, which relaxes both artery and vein when endothelium is intact, activated guanylate cyclase upon addition of endothelium-intact rings to enzyme reaction mixtures. Vasoactive intestinal peptide, which causes endothelium-dependent relaxation of artery but not vein, also activated guanylate cyclase in the presence of endothelium-intact artery but not vein. Arachidonic acid activated the enzyme directly as well as through EDRF release from artery but not vein. Atrial peptides, prostacyclin, isoproterenol and nitroglycerin were inactive. Methylene blue was a powerful inhibitor of EDRF-elicited activation of guanylate cyclase but was without effect when rings were merely pretreated with methylene blue in bath chambers with no further addition to enzyme reaction mixtures. Thus, methylene blue did not interfere with the formation, release or chemical stability of EDRF, but rather inhibited its influence on guanylate cyclase. No agent was found to inhibit EDRF generation or release.(ABSTRACT TRUNCATED AT 250 WORDS)     

The mechanisms and significance of the coupled release of endothelium-derived relaxing factor (EDRF) and prostacyclin (PGI2) from endothelial cells

Endothelium-derived relaxing factor (EDRF) and prostacyclin (PGI2) are co-released from endothelial cells by stimuli acting via membrane-bound receptors or via non-receptor mediated mechanisms. The receptor-mediated release of EDRF and PGI2 is calcium-dependent and seems to be under the negative feedback regulation of protein kinase C. Significant interactions between EDRF and PGI2 or between their respective second messengers within the endothelial cell have not yet been conclusively demonstrated. Furthermore, although EDRF and PGI2 synergize in the inhibition of platelet aggregation, there is little evidence for such synergism in smooth muscle relaxation. These observations indicate that EDRF and PGI2 release may be coupled primarily by their requirement for raised intracellular calcium levels and by their regulation through protein kinase C.     

Formation of an Intermediate in Prostaglandin Biosynthesis and Its Association with the Platelet Release Reaction

A compound that could be converted to prostaglandin F(2alpha) by mild chemical reduction was formed by human platelets in response to arachidonic acid, collagen, or L-epinephrine. It was present in maximal amounts at about 1 min after addition of arachidonic acid or collagen to platelet-rich plasma. Its initial formation appeared to precede platelet aggregation by these agents and was closely correlated with the release of adenine nucleotides and radioactive 5-hydroxytryptamine from platelets. Moreover, the compound was itself found outside the platelets. This compound is probably an endoperoxide intermediate in prostaglandin biosynthesis and may be a trigger for the platelet release reaction.     

Thrombin-Induced Increase in Intracellular Cyclic 3′,5′-Adenosine Monophosphate in Human Platelets

The present data disagree with earlier suggestions that thrombin's effect on platelets is to cause a decrease in intracellular cyclic 3',5'-adenosine monophosphate. Washed human platelets or platelet-rich plasma were incubated at 37 degrees C with human thrombin. After centrifugation, the supernates were assayed for nucleotides and calcium released. The platelet pellets, and in some experiments the supernates as well, were assayed by radioimmunoassay for intracellular cyclic AMP. In the washed platelet system, increasing doses of thrombin to 0.5 U/cc induced increasing release of nucleotides and calcium. This was accompanied by an average twofold increase in intracellular cyclic AMP levels. Prostaglandin E(1), which inhibited 30-50% of release, induced a four- to fivefold increase in cyclic AMP levels that was additive to the cyclic AMP-stimulatory effect of thrombin. Theophylline, which inhibited only 20-40% of nucleotide release, was synergistic with thrombin in the intracellular accumulation of cyclic AMP. The time-course of cyclic AMP accumulation in response to thrombin was slower than thrombin-induced nucleotide release. Similar findings were made in the platelet-rich plasma system where thrombin stimulation of nucleotide release also resulted in a marked accumulation of intracellular cyclic AMP. Thrombin did not appear to stimulate the release of intracellular cyclic AMP.The mechanism underlying these observations was not apparent. The thrombin had no measurable inhibitory effect on platelet phosphodiesterase activity in either intact washed cells or the platelet homogenate supernates. Furthermore, thrombin inhibited, rather than stimulated, platelet adenyl cyclase activity in both intact washed cells and washed platelet particulate fractions. Of note, however, was the finding that thrombin did not completely inhibit the adenyl cyclase activity of prostaglandin-stimulated cells. Further work is needed to clarify the significance of this observation.Nonetheless, the accumulation of intracellular cyclic AMP in response to thrombin observed in the present study suggests that the antagonistic actions of various agents on the platelet release reaction, thought to underlie platelet function, may depend upon a mechanism more intricate than a straightforward mediation through directly opposite effects on platelet cyclic AMP.     

Nitric oxide levels in patients with atheromatous carotid plaque.

Nitric oxide (NO), an unstable derived of nitrogen, is released by endothelium in response to physiological stimulus. Indeed, the endothelium is not only a barrier between the lumen and the inner side of the vessel wall but also a metabolically active organ with endocrine, paracrine and autocrine functions. Endothelial vascular cells play an important role in the regulating vasomotor tone, local homeostasis and vascular bed proliferation. NO mediates the vasodilation and inhibits platelet aggregation, expression of molecular adhesion of monocyte, neutrophils adhesion and smooth muscle growth. Atherosclerosis risk factors such as hypercholesteremia, high blood pressure, smoking and oxidative stress inhibit NO production, leading paradoxically to vasodilatation, which affects endothelial function and may lead to ischemic manifestations in patients with arterial pathology. Therapies that increase NO production may improve endothelial vasodilatation. To check whether a decrease or lower production of NO terminates with the initial formation of atheromatous plaque or whether it continues, we determined the content of NO in plasma and plaque of subjects undergoing carotid surgery and in plasma of control subjects.     

Emerging role of epoxyeicosatrienoic acids in coronary vascular function

The importance of endothelium-derived nitric oxide in coronary vascular regulation is well-established and the loss of this vasodilator compound is associated with endothelial dysfunction, tissue hypoperfusion and atherosclerosis. Numerous studies indicate that the endothelium produces another class of compounds, the epoxyeicosatrienoic acids (EETs), which may partially compensate for the loss of nitric oxide in cardiovascular disease. The EETs are endogenous lipids which are derived through the metabolism of arachidonic acid by cytochrome P450 epoxygenase enzymes. Also, EETs hyperpolarize vascular smooth muscle and induce dilation of coronary arteries and arterioles, and therefore may be endogenous mediators of coronary vasomotor tone and myocardial perfusion. In addition, EETs have been shown to inhibit vascular smooth muscle migration, decrease inflammation, inhibit platelet aggregation and decrease adhesion molecule expression, therefore representing an endogenous protective mechanism against atherosclerosis. Endogenous EETs are degraded to less active dihydroxyeicosatrienoic acids by soluble epoxide hydrolase. Pharmacological inhibition of soluble epoxide hydrolase has received considerable attention as a potential approach to enhance EET-mediated vascular protection, and several compounds have appeared promising in recent animal studies. The present review discusses the emerging role of EETs in coronary vascular function, as well as recent advancements in the development of pharmacological agents to enhance EET bioavailability.     

Intravascular filarial parasites inhibit platelet aggregation. Role of parasite-derived prostanoids.

The nematode parasites that cause human lymphatic filariasis survive for long periods in their vascular habitats despite continual exposure to host cells. Platelets do not adhere to blood-borne microfilariae, and thrombo-occlusive phenomena are not observed in patients with circulating microfilariae. We studied the ability of microfilariae to inhibit human platelet aggregation in vitro. Brugia malayi microfilariae incubated with human platelets caused dose-dependent inhibition of agonist-induced platelet aggregation, thromboxane generation, and serotonin release. As few as one microfilaria per 10(4) platelets completely inhibited aggregation of platelets induced by thrombin, collagen, arachidonic acid, or ionophore A23187. Microfilariae also inhibited aggregation of platelets in platelet-rich plasma stimulated by ADP, compound U46619, or platelet-activating factor. The inhibition required intimate proximity but not direct contact between parasites and platelets, and was mediated by parasite-derived soluble factors of low (less than 1,000 Mr) molecular weight that were labile in aqueous media and caused an elevation of platelet cAMP. Prior treatment of microfilariae with pharmacologic inhibitors of cyclooxygenase decreased both parasite release of prostacyclin and PGE2 and microfilarial inhibition of platelet aggregation. These results indicate that microfilariae inhibit platelet aggregation, via mechanisms that may include the elaboration of anti-aggregatory eicosanoids.     

Platelet thromboembolism

Abstract. Platelet-dependent thrombosis and subsequent embolization are major causes of cerebral ischaemia. Beside aspirin which irreversibly blocks platelet cyclo-oxygenase, several other substances interfere in different platelet metabolic pathways and block platelet adhesion and aggregation. We found in an experimental model using non-human primates that a specific peptide inhibitor blocking GP IIb/IIIa platelet receptor which binds fibrinogen completely, prevents the retention of embolized platelet aggregates in the cerebral circulation. As thrombin may play a key role for platelet activation in vivo leech-derived hirudin, a direct thrombin inhibitor as well as activated protein C which limits thrombin production and also prevents platelet dependent thrombus formation very effective. We demonstrated in the same non-human primate model of platelet embolization that the amount of retention of platelet emboli in the vascular bed depends on the nature of the vasculature. For example, platelet emboli were cleared very quickly from brain microcirculation, whereas platelet embolization into the lower limb via the femoral artery caused a significantly longer retention of the embolized material. Such specific mechanisms may be caused by different levels of local vasodilators as PGI₂ or EDRF.     

Endothelium-derived constricting factor(s): the last novelty—endothelin

The vascular endothelium is not merely a passive physical barrier between the blood and the tissue surrounding the blood vessel, but may actively participate in key processes of metabolic, secretory, and vasoregulatory character. In addition, the endothelium plays an important role in the control of platelet activation. Under certain conditions endothelial cells have been shown to produce powerful vasodilators, like endothelium-derived relaxing factor (EDRF) and prostacyclin (GPI₂), and vasoconstrictors like endothelium-derived constricting factor (EDCF) and endothelin (ET) (Griffith el al., 1988; Vanhoutte & Katusic, 1988). In contrast to the extensive studies performed to characterize the actions and nature of EDRF, recently identified chemically as nitric oxide (Moncada et al., 1988), relatively little is known about EDCF(s). This paper reviews recent data on EDCF, with special emphasis on the newly discovered vasoconstrictor peptide, endothelin (ET).     

Inhibition of platelet function by polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNs) were evaluated for their ability to modulate platelet response induced by collagen, thrombin, platelet-activating factor and the stable analog of cyclic endoperoxides U46619. Platelet aggregation was first evaluated in whole blood and in leukocyte-depleted whole blood by the impedance method. This novel approach highlighted the inhibitory role of leukocytes on platelet aggregation in whole blood. The inhibitory role of PMNs on platelet function was subsequently evaluated on washed cells. PMN inhibition of platelet aggregation and beta-thromboglobulin release was more evident with threshold concentrations of stimuli. The inhibition also depended on the number of PMNs incubated in mixed cellular suspensions. Higher concentrations of stimuli may overcome the PMN-dependent inhibition. Under this condition, preincubation of cells with N-formyl-methionyl-leucyl-phenylalanine (a specific PMN agonist) restored the inhibitory effect of PMNs on platelet aggregation in whole blood and in mixed cellular suspensions. Not only PMNs, but also PMN-derived supernatants, dose-dependently inhibited U46619-induced platelet aggregation, suggesting that the inhibition observed may be exerted by chemically stable compound(s). Cytoplasmic Ca2+ movement was measured in aequorin-loaded platelets exposed to thrombin or U46619 to see whether cytoplasmic Ca2+ levels were affected by PMN. Ca2+ levels were similar in the presence or absence of PMNs, suggesting that inhibition may be related to a subsequent platelet response step. A series of bioassay experiments showed that PMNs were able to remove and/or convert adenosine diphosphate available for platelet aggregation but not to reduce U46619 availability. Our findings suggest that (1) unstimulated PMNs may release factor(s) that inhibit platelet aggregation and beta-thromboglobulin release; (2) this in itself is sufficient to block the platelet response to a threshold concentration of stimuli; (3) release of the same or other inhibitory mediators from stimulated PMNs may have to be greater to inhibit platelet response to higher concentrations of stimuli. Data presented here suggest that adenosine diphosphatase activity and chemically stable, as yet unidentified, compounds besides previously well-characterized labile compounds such as nitric oxide and arachidonic acid metabolites are responsible for the PMN-dependent mechanism of inhibition of platelet response that could be relevant in physiopathologic conditions.     

Labile aggregation stimulating substance, free fatty acids, and platelet aggregation.

Labile aggregation stimulating substance (LASS), an intermediate produced during platelet biosynthesis of PGE2 and PGF2alpha, acts as a physiologic intercellular messenger to promote platelet aggregation and the release reaction. The activity is formed by intact cells after physiologic stimulation or can be generated from platelet membrane fractions after combination with arachidonate. In the present investigation, small amounts of polyunsaturated fatty acids added to an incubation mixture of platelet microsomes and arachidonate were found to significantly inhibit subsequent platelet aggregation. Saturated and mono-unsaturated fatty acids in the same concentrations were without effect. However, in higher concentrations mono-unsaturated fatty acids were found to be inhibitory and stearic acid was found to enhance subsequent platelet aggregation. The inhibition caused by the polyunsaturated fatty acid, linoleate, was shown to be the result of an effect on the production of LASS through an interaction with the platelet enzyme responsible for conversion of arachidonate to LASS. In contrast, stearic acid was found to enhance platelet aggregation by acting on the platelets and not directly on LASS production. The results suggest that small changes in the fatty acid composition of platelet phospholipids could significantly influence platelet reactivity.     

Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor

Removal of endothelial cells on rings of rat aorta increased the sensitivity to the selective alpha-1 adrenoceptor agonist phenylephrine, to the nonselective alpha adrenoceptor agonist norepinephrine and to the selective alpha-2 adrenoceptor agonist clonidine. In the case of the first two, which are strong agonists for the alpha-1 adrenoceptor-mediating contraction, removal of endothelium increased sensitivity 4- and 6-fold at the EC30 level, but produced little or no increase in maximum. In the case of clonidine, a partial agonist for the alpha-1 adrenoceptor, which gave only about 15% of the maximum given by phenylephrine on endothelium-containing rings, removal of the endothelium not only shifted the curve to the left but also increased the maximum to about 50% of that given by phenylephrine. The depression of sensitivity to these agonists in rings with endothelium appeared to be due to the vasodepressor action of endothelium-derived relaxing factor (EDRF), as hemoglobin, a specific blocking agent of EDRF, abolished this depression. It is unlikely that the endothelium-dependent depression was due to stimulation of release of EDRF, because clonidine did not produce endothelium-dependent relaxation in precontracted rings even when its contractile action was blocked by the alpha-1 adrenoceptor antagonist prazosin. Further evidence against alpha adrenoceptor agents stimulating release of EDRF was that neither phenylephrine nor clonidine induced a rise in cyclic GMP in aortic rings, whereas acetylcholine, which does release EDRF, caused a large rise in cyclic GMP content. The possibility that the muscle cells of intact rat aortic rings were under the tonic influence of released EDRF was supported by the finding that, in the absence of any contractile agent, hemoglobin induced a fall in the basal level of cyclic GMP in endothelium-containing rings. Also consistent with EDRF being released spontaneously was the finding that contraction induced by 5-hydroxytryptamine, like that by alpha-adrenergic agonists, was also depressed in endothelium-containing rings of aorta. When the efficacy of phenylephrine as an alpha-1 agonist was reduced to about the initial efficacy of clonidine by irreversible inactivation of a very large fraction of alpha-1 adrenoceptors of the smooth muscle cells by pretreatment with dibenamine, the concentration-contraction curves for phenylephrine for both endothelium-containing rings and for endothelium-denuded rings now became very similar to the corresponding curves obtained for clonidine before receptor inactivation.(ABSTRACT TRUNCATED AT 400 WORDS)     

A STUDY OF ALLOGRAFT KIDNEY REJECTION OCCURRING SIMULTANEOUSLY IN WHOLE ORGAN AND EAR CHAMBER GRAFTS IN THE RABBIT

When portions of adult renal tissue are allografted into the rabbit ear chamber, they usually survive for periods of up to several months (6). When a kidney from the same donor is grafted as a whole organ, the ear chamber grafts then reject with the whole organ in 7 days. During that time serial needle biopsies of the whole organ are compared with the in vivo appearance of the ear chamber grafts. This establishes that the changes occurring in the ear chamber grafts are monitoring the rejection process proceeding in the whole organ grafts. Dramatic vascular changes herald the earliest stages of unmodified rejection. A highly characteristic form of individual discrete platelet adhesion to both endothelium and adherent leukocytes is observed which is associated with the release reaction. At times as many as 20 such discrete platelets are clearly visible in profile in one high-power field. This demonstrates in vivo a mechanism whereby vascular and parenchymal damage may be produced by platelet contents, without previous aggregation or thrombus formation being necessary.     

Inactivation of endothelial derived relaxing factor by oxidized lipoproteins.

Endothelial cell derived relaxing factor (EDRF) mediated relaxation of blood vessels is impaired in vessels exposed to lipoproteins in vitro and in arteries of hyperlipidemic humans and animals. To investigate the mechanism by which lipoproteins impair the effects of EDRF, which is likely nitric oxide (NO) or a related molecule, we have bioassayed EDRF/NO activity by measuring its ability to increase cGMP accumulation in rat fetal lung cultured fibroblasts (RFL-6 cells). Low density lipoprotein modified by oxidation (ox-LDL) induced a concentration-dependent inhibition of EDRF activity that had been released from bovine aortic endothelial cells (BAEC) stimulated with bradykinin or the calcium ionophore A23187. In addition, lipoproteins directly impaired authentic NO-induced stimulation of cGMP accumulation in the detector cells; stimulation by sodium nitroprusside was unaffected. Ox-LDL or oxidized HDL3 were highly potent in blocking NO-stimulated cGMP accumulation with EC50's of approximately 1 microgram/ml. Lipid extracted from ox-LDL blocked NO-stimulated cGMP accumulation to about the same extent as intact ox-LDL, while the protein component of ox-LDL did not inhibit the cGMP response. These results suggest that the lipid component of oxidized lipoproteins inactivate EDRF after its release from endothelial cells.