Fatty acids in animals: thrombosis and hemostasis.

Long-chain fatty acids, including stearic acid, can cause thrombosis when they are injected into the systemic circulation of animals. The toxicity is decreased if the fatty acids are bound to albumin. To date, the effect of individual fatty acid feeding is not known. In general, feeding animals diets high in saturated fat followed by the injection of a thrombogenic stimulus is associated with a greater incidence of thrombosis than when a normal diet or a diet high in unsaturated or polyunsaturated fat is fed. A high dietary intake of long-chain polyunsaturated fatty acids of the n-3 family may prevent thrombosis in animals. In assessing the effects of dietary fatty acids, the ability of the organism, be it animal or human, to convert fatty acids to less thrombogenic fatty acids (stearic to oleic) or create an antithrombotic fatty acid (linolenic to eicosapentaenoic) is a major attribute. Further studies should consider the storage of fatty acids in tissues and their release into the blood and the potential impairment of albumin binding and fatty acid transport mechanisms.

Stearic acid, clotting, and thrombosis.

Stearic acid causes hypercoagulability of the blood by activation of factor XII and by aggregation of blood platelets. Injection of unbound stearic acid (sodium salt) into the systemic circulation of dogs was followed by massive generalized thrombosis and sudden death. Similar infusions into birds, which are deficient in factor XII, did not cause hypercoagulability or thrombosis. The effects of the long-chain saturated fatty acids could be prevented by using albumin to bind the stearic acid at a molar ratio of free fatty acid (FFA) to albumin of < 2. The major issue is whether eating foods rich in stearic acid can cause a thrombogenic effect. We have no experimental evidence to support this concept. If a thrombogenic effect of long-chain saturated fatty acids exists in humans, it is most likely to occur as an aberration of fatty acid transport in which the FFA-albumin molar ratio exceeds 2 either as a result of very high plasma FFA concentrations from lipid mobilization or a low concentration of albumin in the blood as found in disease states such as the nephrotic syndrome.

Modulation of the fibrinolytic response of cultured human vascular endothelium by extracellularly generated oxygen radicals.

Clinical and experimental data indicate that activated oxygen species interfere with vascular endothelial cell function. Here, the impact of extracellular oxidant injury on the fibrinolytic response of cultured human umbilical vein endothelial (HUVE) cells was investigated at the protein and mRNA levels. Xanthine (50 microM) and xanthine oxidase (100 milliunits), which produces the superoxide anion radical (O2-) and hydrogen peroxide (H2O2), was used to sublethally injure HUVE cells. Following a 15-min exposure, washed cells were incubated for up to 24 h in serum-free culture medium. Tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen, and PAI-1 activity were determined in 1.25 ml of conditioned medium and t-PA and PAI-1 mRNA in the cell extracts of 2 x 10(6) HUVE cells. Control cells secreted 3.9 +/- 1.3 ng/ml (mean +/- S.D., n = 12) within 24 h. Treatment with xanthine/xanthine oxidase for 15 min induced a 2.8 +/- 0.4-fold increase (n = 12, p less than 0.05) of t-PA antigen secretion after 24 h. The t-PA antigen was recovered predominantly in complex with PAI-1. The oxidant injury caused a 3.0 +/- 0.8-fold increase (n = 9, p less than 0.05) in t-PA mRNA within 2 h. Total protein synthesis was unaltered by xanthine/xanthine oxidase. The oxidant scavengers superoxide dismutase and catalase, in combination, abolished the effect of xanthine/xanthine oxidase on t-PA secretion and t-PA mRNA synthesis. Xanthine/xanthine oxidase treatment of HUVE cells did not affect the PAI-1 secretion in conditioned medium nor the PAI-1 mRNA levels in cell extracts. Thus extracellular oxidant injury induces t-PA but not PAI-1 synthesis in HUVE cells.

Alterations in human vascular endothelial cell function by oxygen free radicals. Platelet adherence and prostacyclin release.

This study was directed to the ability of oxygen free radicals to cause reversible vascular endothelial cell dysfunction. A well-characterized system for the production of the superoxide anion radical (O2(-).) and hydrogen peroxide (H2O2), employing xanthine and xanthine oxidase, was used to sublethally injure human umbilical vein endothelial (HUVE) cells in vitro. We examined the effects of a 15-minute incubation of HUVE cells with xanthine (50 microM) and xanthine oxidase (2.5-100 munits) on platelet adherence and prostacyclin (PGI2) release. All experiments were conducted in a serum-free N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid)-Tyrode buffer (pH 7.4) incubation system. Exposure of HUVE cells to sublethal concentrations of oxygen free radicals caused significant enhancement of platelet adherence (65 +/- 6.3%) to injured endothelium. A 12-fold increase in PGI2 release resulted after a 15-minute treatment with xanthine and xanthine oxidase. The addition of exogenous PGI2 (150 mM) to platelet-endothelial systems did not completely prevent the enhanced platelet adherence, suggesting that a lack of PGI2 was not completely responsible for the adherence of platelets to O2(-).-injured cells. When superoxide dismutase (SOD) and catalase, scavengers of O2(-). and H2O2, were added in combination to treated cells, platelet adherence decreased by 42-77% and PGI2 release approached that of control cultures. No decrease in either platelet adherence or PGI2 release occurred when chemically inactivated forms of SOD and catalase or bovine serum albumin were added to oxidant-treated cultures.

Reduced prostacyclin formation after reoxygenation of anoxic endothelium.

Human umbilical vein endothelial cells subjected to 24 h of anoxia followed by reoxygenation released less prostacyclin (PGI2) in response to thrombin, calcium ionophore A23187, or arachidonic acid. This was associated with a substantial increase in stimulated platelet adherence. Increased lactate dehydrogenase and 51Cr release occurred after 1 h of reoxygenation, but the high rate of release did not persist during the subsequent 23 h of reoxygenation. The changes in platelet adherence and PGI2 release partially resolved over 24 h. PGI2 formation from prostaglandin H2 was not reduced, suggesting that cyclooxygenase activity, but not prostacyclin synthase, is affected by reoxygenation. A decrease in arachidonic acid release from cellular lipids also occurred. The reduction in cyclooxygenase activity, but not arachidonic acid release, was prevented by the presence of ibuprofen during reoxygenation. Addition of catalase or superoxide dismutase during reoxygenation increased PGI2 release but did not completely overcome the reduction relative to control cultures. These findings suggest that the increase in platelet adherence during reoxygenation may be mediated in part by a change in cyclooxygenase activity. This is only partly overcome by extracellular oxygen species scavengers but is prevented by the presence of a reversible cyclooxygenase inhibitor during reoxygenation.

The endothelium, platelets, and coronary vasospasm.

Significant advances have been made in our understanding of the role of the vascular endothelium in preventing thrombosis and in decreasing vascular spasm. The endothelium provides a surface receptor, thrombomodulin, that binds thrombin. In this form, thrombin loses its ability to clot fibrinogen or to aggregate platelets, but is able to activate protein C. In its activated state, protein C is able to act as an inhibitor of coagulation by virtue of its proteolytic destruction of Factors Va and VIIIa. Congenital deficiency of protein C is associated with early and recurrent thrombosis. The discovery that the endothelium is responsible for the production of a short-acting inhibitor of smooth-muscle contraction (EDRF) was a remarkable advance. One of the EDRF substances has been demonstrated to be NO, which has inhibitory effects on both smooth muscle and blood platelets. Activity of EDRF appears to be diminished or lost as a consequence of atherosclerosis, and stimuli that cause vasodilation via the EDRF pathway in normal vessels cause vasoconstriction in atherosclerotic arteries. Regression of atherosclerosis in experimental animals appears to be associated with restoration of EDRF activity.

Platelets and atherosclerosis.

This discussion has centered about the consequences of the loss of endothelial integrity and how the blood platelet behaves under these circumstances. It is clear that the platelet can function as a participant in the development of the atherosclerotic lesion, can be an important contribution to mechanisms that operate to produce vascular spasm, and ultimately can play a major role in the development of thrombosis at the site of the complicated end-stage atherosclerotic lesion.

Interaction of platelets and purified collagens in a laminar flow model.

Damage to the endothelial surface of the vessel wall can result in exposure of circulating blood components to collagen and other subendothelial structures. Collagen types I, III, IV, and V have been demonstrated in the vessel wall by chemical and immunohistological methods; type V is thrombin-sensitive, and is present on the endothelial cell surface. In an earlier study using a rocking model, both unstimulated and ADP-induced platelet adherence was reduced on wells coated with type V collagen in comparison to uncoated wells; and increased on plastic surfaces coated with types III and IV collagen in comparison to those coated with type V collagen. The present study was designed to determine the effect of erythrocytes and shear rate on platelet adherence to these purified collagen types in a laminar flow system. With platelet-rich plasma, adherence of labeled platelets was much lower in the laminar flow system compared with the rocking model. Erythrocytes significantly enhanced platelet adherence to surfaces that were untreated or absorbed with collagen types I, III, and IV. However, this enhancement was not seen in the presence of type V collagen. These studies provide additional evidence for the selectively nonthrombogenic nature of type V collagen.

Identification and characterization of an endothelial, cell-specific antigen with a monoclonal antibody.

The purpose of these studies was to use monoclonal antibodies to identify and characterize plasma membrane components unique to the vascular endothelium. Our assumption is that such components may perform some of the specialized functions of the endothelium and, by their identification with antibody probes, we may be able to study further their function and structure. Thus, primary cultures of human umbilical vein endothelium were used to immunize mice whose spleen cells were fused with the mouse myeloma cell NS-1. HEC-1 is a monoclonal antibody derived from such a fusion that appears to react with an antigen located only on endothelial cells. The antigen has been characterized by immunoprecipitation and polyacrylamide gel electrophoresis as a glycoprotein with a mol wt of 180,000 daltons under nonreducing conditions and 90,000 daltons under reducing conditions. Despite a close resemblance to a membrane component shown by others to be a receptor for transferrin, several lines of evidence reported in this paper indicate that this is not the function of the HEC-1 antigen. These data show that monoclonal antibodies can be used to identify and characterize membrane components of the vascular endothelium. Moreover, these probes can be used to inquire about the structure and function of the antigen with which they react.

Ocular neovascularization. Tissue culture studies.

The proliferative activity of a number of intraocular fluids, bovine retinal extract, and normal serum (from humans and cynomolgus monkeys) was investigated by in vitro tissue culture studies, with the use of tritiated thymidine incorporation by the cultured endothelial cells of human umbilical veins. There was increased tritiated thymidine incorporation by (1) the aqueous, vitreous, and intraocular fluid (IOF) (which filled the eye after lensectomy and vitrectomy) removed from cynomolgus monkey eyes with iris neovascularization or with neovascular glaucoma (NVG) that developed after experimental retinal vein occlusion, (2) by aqueous and vitreous removed from human eyes with NVG or proliferative diabetic retinopathy; (3) by the serum, and (4) by the bovine retinal extract. However, tritiated thymidine incorporation was not increased by the normal aqueous, vitreous, or IOF.

Induction and amplification of T-lymphocyte proliferative responses to periodate and soybean agglutinin by human adult vascular endothelial cells.

Human mononuclear phagocyte (M phi) populations were compared to adult human endothelial cells (HEC) for their respective abilities to influence the proliferative responses of purified human T lymphocytes to the mitogenic agents Na-m-periodate (IO-4), soybean agglutinin (SBA), or allogeneic cells. HEC and M phi were both capable of inducing proliferative responses of allogeneic T lymphocytes in mixed-lymphocyte culture. Under low cell density culture conditions, purified T-lymphocyte proliferative responses to IO-4 or SBA could be restored by addition of syngeneic M phi or HEC. At higher cell density culture conditions, proliferation of T cells to IO-4 could be amplified more by HEC than M phi. T-lymphocyte proliferative responses to SBA were amplified by addition of HEC but were suppressed by addition of M phi. These findings indicate that human adult HEC are unique and potent accessory cells for T lymphocytes. Furthermore, these findings demonstrate that accessory cell functions of HEC can be discriminated from those of M phi.

Eicosapentaenoic acid and prostacyclin production by cultured human endothelial cells.

Human umbilical vein endothelial cells incorporate eicosapentaenoic acid (EPA) when this fatty acid is present in the culture medium. From 30 to 70% of the uptake remains as EPA, and much of the remainder is elongated to docosapentaenoic acid. All of the cellular glycerophospholipids become enriched with EPA and docosapentaenoic acid, with the largest increase in EPA occurring in the choline glycerophospholipids. When this fraction is enriched with EPA, it exhibits a large decrease in arachidonic acid content. Cultures exposed to tracer amounts of [1-14C]linolenic acid in 5% fetal bovine serum convert as much as 17% of the radioactivity to EPA. The conversion is reduced, however, in the presence of either 20% fetal bovine serum or 50 microM linolenic acid. Like arachidonic acid, some newly incorporated EPA was released from the endothelial cells when the cultures were exposed to thrombin. However, as compared with arachidonic acid, only very small amounts of EPA were converted to prostaglandins. Cultures enriched with EPA exhibited a 50 to 90% reduction in capacity to release prostacyclin (PGI2) when subsequently stimulated with thrombin, calcium ionophore A23187, or arachidonic acid. The degree of inhibition was dependent on the time of exposure to EPA and the EPA concentration, and it was not prevented by adding a reversible cyclooxygenase inhibitor, ibuprofen, during EPA supplementation. EPA appears to decrease the capacity of the endothelial cells to produce PGI2 in two ways: by reducing the arachidonic acid content of the cell phospholipid precursor pools and by acting as an inhibitor of prostaglandin production. These findings suggest that regimens designed to reduce platelet aggregation and thrombosis by EPA enrichment may also reduce the capacity of the endothelium to produce PGI2.

Diminished platelet adherence to type V collagen.

Different types of collagen vary in their influence on platelet reactivity. Collagen Types III, IV, and V were obtained from human placental tissue, and Type I collagen was prepared from rat skin. Each collagen type was coated onto a plastic surface. Each collagen-coated surface or appropriate plastic surface control was studied using citrated human 51Cr-labeled platelet-rich plasma in both the presence and absence of 10 microM adenosine 5'-diphosphate (ADP). Both unstimulated and ADP-induced platelet adherence were: 1) reduced by Type V collagen coating in comparison to uncoated wells; and 2) increased by Types III and IV collagen coating in comparison to Type V coated or plastic surfaces. Addition of the fast-acting thrombin inhibitor dansylarginine (DAPA) had no significant effect on unstimulated and ADP-induced platelet adherence to Type III, IV or V collagen-coated surfaces. Type I collagen-coated surfaces, studied only in the presence of DAPA, caused greater platelet adherence than those coated with Types III, IV, or V collagen. We conclude that Type V collagen may be less thrombogenic than Types, I, III, or IV.

Prostacyclin biosynthesis in cultured vascular endothelium is limited by deactivation of cyclooxygenase.

Primary monolayer cultures of human umbilical vein endothelium produce prostacyclin (PGI2) in response to stimulation by thrombin, ionophore A23187, arachidonic acid, and the prostaglandin endoperoxide, PGH2. None of these treatments had a significant effect on the capacity of the endothelium to produce PGI2 in response to subsequent stimulation by PGH2. By contrast, endothelium initially exposed to thrombin, A23187, or arachidonic acid produced approximately 37, 68, and 84% less PGI2, respectively, upon subsequent stimulation by arachidonic acid. These findings suggest that PGI2 biosynthesis in cultured endothelium results in deactivation of cyclooxygenase-hydroperoxidase but not PGI2 synthetase. To test the hypothesis that PGI2 biosynthesis alone causes deactivation of cyclooxygenase, thrombin, A23187, and arachidonic acid were added to monolayers that had been preincubated with ibuprofen (250 microM), a rapidly reversible, competitive inhibitor of this enzyme. After removal of the ibuprofen and the initial stimulus, PGI2 production in response to subsequent stimulation by arachidonic acid was maximal. These findings suggest that the metabolism of arachidonic acid itself causes a direct deactivation of cyclooxygenase. After an initial exposure to arachidonic acid, PGI2 production in response to a second stimulation by arachidonic acid was restored to approximately 34, 69, and 74% of maximal, after recovery periods of 1, 24, and 48 h, respectively. We conclude that the regulation of PGI2 biosynthesis in normal vascular endothelium may be in part a function of the activity and biosynthesis of cyclooxygenase-hydroperoxidase and the deactivation of this enzyme may be a primary factor limiting the capacity of the endothelium to produce PGI2.

Arachidonic acid availability and prostacyclin production by cultured human endothelial cells.

When human umbilical vein endothelial cultures were grown in the presence of supplemental arachidonic acid, the cell phospholipids became enriched with arachidonic acid. Prostacyclin (PGI2) accumulated in the medium during supplementation with arachidonic acid. The capacity of these enriched cultures to produce PGI2 when subsequently incubated with either arachidonic acid or thrombin was reduced by as much as 90%, but release of arachidonic acid from the cell lipids in response to thrombin stimulation was not inhibited. Refractory cultures completely recovered the capacity to form PGI2 within 18 hours after removal of the medium containing supplemental arachidonic acid. However, recovery was prevented by cycloheximide. When enrichment with arachidonic acid was done in the presence of ibuprofen, a reversible cyclooxygenase inhibitor, PGI2 did not accumulate in the medium during supplementation, and the subsequent capacity of the cultures to produce PGI2 in response to thrombin increased by 70% to 240%. By contrast, the capacity of these supplemented cultures to convert added arachidonic acid to PGI2 did not increase. Therefore, the enhancement in thrombin-stimulated PGI2 production when the cultures are supplemented with arachidonic acid probably is due to the larger amount of arachidonic acid available in the intracellular lipid substrate pools, rather than to an activation of the PGI2 synthetic pathway. These findings suggest that changes in the arachidonic acid content of the endothelial cell lipids may modulate the capacity of the endothelium to produce PGI2 in response to stimulation.

Mechanisms of action: aspirin.

Aspirin, one of the oldest antiplatelet agents used for antithrombotic therapy, has been demonstrated to cause acetylation of the cyclo-oxygenase and irreversible inhibition of thromboxane synthesis for the life of the platelet. Aspirin has a similar effect upon the endothelial cyclo-oxygenase, but in contrast to that of the platelet, it is less sensitive and has the capacity to generate new cyclo-oxygenase activity if aspirin is removed from the system. Other non-steroidal anti-inflammatory agents such as ibuprofen react in a reversible manner with either the platelet or the endothelium and may actually "protect" the cyclo-oxygenase against aspirin or other agents which interact with the cyclo-oxygenase. Aspirin may act in other ways to exert an antithrombotic effect, but cause and effect relationships have not yet been demonstrated.