Discordant effects on eicosanoids and fibrin degradation products in two murine models of antiphospholipid antibody.

Two murine models of lupus were employed to challenge an hypothesized mechanism by which antiphospholipid antibodies (APLA) might promote thrombosis: altering prostacyclin (PGI2) and thromboxane (TX) production. PGI2 levels in mouse blood and the ex vivo release of PGI2 and TX from mouse kidney were measured. Since APLA have been reported to alter synthesis or activation of several molecules mediating fibrinolysis, murine plasma levels of the fibrin degradation product, D-dimer were also determined. Two murine strains, one prone to spontaneous "lupus-like" illness (MRL-lpr) and related strain (MRL-(+2)), were compared. The assays confirm that MRL-lpr mice have increased anticardiolipin antibody (ACA) and two-fold increased release of TX from renal tissues compared to MRL-(+2) mice. However, these mice have low levels of plasma D-dimer. NIH Swiss mice injected with IgG (containing APLA) from thrombosis-prone lupus patients had high blood ACA titers and D-dimer levels, but both ACA and D-dimer were low or non-detectable in Swiss mice injected with saline or normal IgG. Unlike mice with spontaneous lupus-like illness, healthy mice injected with APLA did not differ from controls with respect to plasma or tissue PGI2 or TX levels. The two murine models of lupus differ, because an altered PGI2-TX ratio is a finding in the chronic murine lupus strain MRL-lpr, but is not seen when APLA are injected into normal mice. It is unlikely that APLA alone has a direct effect on cellular production of eicosanoids in vivo.

Dysfibrinogenemia: a case with thrombosis (fibrinogen Richfield) and an overview of the clinical and laboratory spectrum.

Fibrinogen Richfield exemplifies a dysfibrinogen associated with a life-long thrombotic tendency. The evaluation of this novel case indicates that, like similar thrombotic dysfibrinogenemias, the abnormal protein polymerizes abnormally and demonstrates impaired clot dissolution. A survey of other cases of dysfibrinogenemia indicates that the relatively common abnormalities of Fibrinopeptide A release are generally asymptomatic or associated with bleeding, polymerization abnormalities are likely to be asymptomatic or associated with thrombosis (or occasionally bleeding), and complex abnormalities or additional, independent hemostatic defects are rather common. Thrombin and Reptilase clotting times are not helpful in distinguishing between the subsets, but clinical history, fibrinopeptide release, and polymerization studies may be useful. Abnormalities of fibrinogen function tend to correlate with changes in molecular domains related to binding and hydrolysis.

Some antiphospholipid antibodies inhibit phospholipase A2 activity.

Certain antiphospholipid antibodies, particularly those associated with arterial thrombosis, reduce vascular prostacyclin production. Studies were conducted to determine whether antibody-mediated inhibition of phospholipase A2 accounts for this effect. In this report we present evidence that purified antiphospholipid antibodies reduce phospholipase A2 activity toward phospholipid substrates, both in vitro and in a defined system. Purified immunoglobulin, obtained from patients at risk for thrombosis who had plasma antiphospholipid antibodies, impaired prostacyclin generation after endothelial stimulation with thrombin or the calcium ionophore A23187. The release of arachidonate in response to A23187 was reduced in endothelial cells pretreated with antibody; the metabolism of exogenous arachidonate to prostacyclin was normal. Thrombin-induced synthesis of platelet-activating factor, which follows phospholipase A2-mediated generation of lysophosphatidylcholine, was also inhibited in parallel with the inhibition of prostacyclin generation. Phospholipase A2 activity was determined in a defined test system with two phospholipases A2. The hydrolysis of fatty acid was less in the presence of patient immunoglobulin than in buffer alone or with normal immunoglobulin. Inhibition by antibody was present at a range of phospholipase concentrations. Antiphospholipid antibodies, purified from patient serum by adsorption to and subsequent elution from immobilized cardiolipin or phosphatidylserine, also inhibited phospholipase A2 activity. The data support our conclusions that purified antiphospholipid antibodies inhibit endothelial phospholipase A2 activity in response to thrombin or ionophore and that phosphatidylcholine in a common metabolic precursor of both prostacyclin and platelet-activating factor. In a defined enzyme assay, inhibition by antiphospholipid antibody of phospholipase A2 activity does not require additional cofactors.

Lupus anticoagulant inhibition of in vitro prostacyclin release is associated with a thrombosis-prone subset of patients.

PURPOSE: The effect of lupus anticoagulant-containing sera on endothelial prostacyclin generation (both basal and after thrombin stimulation) was determined. Subsets of patients who had experienced arterial, venous, or no thrombosis were compared with respect to the quantitation of antiphospholipid antibody and effects on prostacyclin production. PATIENTS AND METHODS: Serum antiphospholipid antibodies were detected in 26 patients by immunologic (enzyme-linked immunosorbent assay) and kinetic (anticoagulant) assays. Cultured human endothelial cells were exposed to patient or normal serum, and the release of prostacyclin was determined by radioimmunoassay of supernatants. Release was determined in the absence and presence of the secretagogue, thrombin (1 U/mL), corrected for interassay variation, and correlated with other clinical and laboratory variables. RESULTS: The normal prostacyclin response was a 2.5-fold increase after thrombin (1 U/mL) compared to basal production. Patients with a history of arterial thrombosis (Group 1, n = 10) had the highest IgG anticardiolipin antibody titers (449 +/- 115 [OD x 1,000]), most prolonged kaolin clotting times (140 +/- 15 seconds), and the least prostacyclin response to thrombin (1.36-fold). Patients with venous thrombosis (Group 2, n = 6) had lower titers (329 +/- 120), intermediate clotting times (125 +/- 19 seconds), and slightly impaired prostacyclin responses (2.18-fold). Patients with no history of thrombosis (Group 3, n = 10) had low antibody titers (220 +/- 20), mildly prolonged clotting times (108 +/- 6 seconds), and normal prostacyclin responses (2.33-fold). Patient serum did not alter basal or arachidonate-induced prostacyclin production. Group 1 had significantly lower platelet counts (99 +/- 19) compared to Group 2 (167 +/- 35) or Group 3 (167 +/- 34), but were similar in age and associated diagnoses. CONCLUSIONS: Inhibition of prostacyclin responses is commonly found in serum from patients with lupus anticoagulants, and is likely to be present in patients with high IgG anticardiolipin antibodies, strong lupus anticoagulants, low platelet counts, and a recent arterial thrombosis.

Effects of endothelium-derived relaxing factor and nitric oxide on rat mesangial cells.

We have investigated whether endothelium-derived relaxing factor (EDRF) and nitric oxide (NO), a substance proposed to be one of the EDRFs, could elicit biochemical and biological responses in rat glomerular mesangial cells (MC). In wells with MC alone, guanosine 3',5'-cyclic monophosphate (cGMP) levels were 2.6 +/- 0.6 fmol/microgram protein, and bradykinin did not affect these levels, whereas in coincubation experiments with bovine aortic EC and rat MC, cGMP levels in MC increased to 44.6 +/- 21 fmol/micrograms protein after bradykinin stimulation (P less than 0.05). This effect was potentiated by superoxide dismutase and inhibited by hemoglobin and L-NG-monomethyl arginine, a specific inhibitor of EDRF synthesis. Increases in cGMP were also observed when MC were incubated directly with NO and were potentiated by superoxide dismutase and inhibited by hemoglobin. We also tested whether NO could inhibit angiotensin II (ANG II)-induced reductions in cross-sectional area (CSA) of MC. When MC were exposed to ANG II only, 65% of the cells underwent a significant reduction in CSA, as measured by digital image analysis. However, when MC were incubated with ANG II and NO, only 10% of cells responded (P less than 0.04). These studies demonstrate that EDRF and NO induce significant biochemical and functional responses in rat glomerular MC and suggest that communication between EC and MC may be important in regulation of glomerular function.

Fish oil supplementation does not lower plasma cholesterol in men with hypercholesterolemia. Results of a randomized, placebo-controlled crossover study.

STUDY OBJECTIVE: To determine the effects of fish oil supplementation on plasma cholesterol in middle-aged men with isolated hypercholesterolemia. DESIGN: Randomized double-blind placebo-controlled (safflower oil) two-period crossover trial with 12-week treatment periods. SETTING: Outpatient general medicine clinic at a university-affiliated Veterans Affairs hospital. PATIENTS: Thirty-eight men with plasma cholesterol between 5.68 and 7.76 mmol/L (220 to 300 mg/dL), triglyceride levels less than 3.39 mmol/L (300 mg/dL), and free of coexisting diseases. INTERVENTIONS: Fish oil and placebo (safflower oil) supplementation. After basal measurements and a 4-week lead-in period, twenty 1-g capsules of either fish oil or placebo oil were provided for 12 weeks (period 1). After a 4-week washout phase participants then received the other oil for an additional 12 weeks (period 2). MEASUREMENTS AND MAIN RESULTS: Blood was drawn at the beginning and end of each study period and analyzed for levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, apolipoprotein A1, and apolipoprotein B. Low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald equation. Total and LDL cholesterol increased from the before treatment values by 4.8% and 9.1%, respectively, after ingestion of fish oil. Compared with placebo, LDL cholesterol was significantly higher (4.5 compared with 4.1 mmol/L, P = 0.01) and triglycerides lower (1.3 compared with 1.8 mmol/L, P = 0.01) after fish oil. Total and HDL cholesterol and apolipoprotein A1 and B levels did not differ. CONCLUSIONS: Fish oil supplements do not lower plasma cholesterol levels in middle-aged men with hypercholesterolemia without elevated triglycerides. They should not be recommended as a method to lower plasma cholesterol in these patients.

Interleukin 1 enhances arterial thrombogenicity in vitro.

An in vitro model of platelet adhesion, the annular perfusion chamber, was utilized to test the effects of Interleukin 1 on vascular thrombogenicity for platelets. De-endothelialized, everted human umbilical arteries were placed in cell culture media with or without Interleukin 1. The arteries were later perfused with citrated human blood and then fixed. Platelet adhesion and aggregate formation on artery segments was quantified by blinded morphometric analysis. A monolayer of contact platelets was seen on control artery segments, but arteries exposed to 100 Units/ml Interleukin 1 for 2-20 hours had increased numbers and size of platelet aggregates. Ultrastructurally, intact endothelial cells were not present on any segment. Both prostacyclin and thromboxane were released by vascular cells in the artery segments, but the quantity and ratio of these eicosanoids was not altered by artery exposure to Interleukin 1. Arterial thrombogenicity is modulated by non-endothelial vascular cells in response to Interleukin 1, and this does not appear to be mediated by changes in vascular production of thromboxane or prostacyclin.

Lupus anticoagulant induces a selective defect in thrombin-mediated endothelial prostacyclin release and platelet aggregation.

A patient with microvascular thrombosis and thrombocytopenia was found to have a high-titre lupus anticoagulant. The biological effects of the patient's lupus anticoagulant were studied using whole patient serum and plasma. Staph Protein A eluate, and affinity-purified lupus anticoagulant. The latter was isolated by immunoadsorption of serum onto cardiolipin/phosphatidylserine/cholesterol liposomes. Each source of lupus anticoagulant demonstrated 'anticoagulant' activity, defined as prolongation of a modified kaolin clotting time, and contained antibody which bound to endothelial monolayers. Each interfered with thrombin-mediated prostacyclin release from endothelial cells, but had no effect on arachidonate-induced prostacyclin release. In addition, the lupus anticoagulant selectively blocked platelet aggregation in response to thrombin, but not in response to arachidonate, ADP or epinephrine. Lupus anticoagulant also reduced thrombin-stimulated shifts in cytosolic calcium. Thrombin-mediated membrane inositol metabolism and total thrombin binding to endothelium were unaffected by lupus anticoagulant, and another endothelial anticoagulant function related thrombin binding. Protein C activation by thrombomodulin, was not altered. We conclude that the binding of lupus anticoagulant to endothelial cells and platelets does not prevent all thrombin signalling events, but does interrupt prostacyclin production.

Endothelial cell heterogeneity: antioxidant profiles determine vulnerability to oxidant injury.

Human umbilical vein endothelial cells were more sensitive to hydrogen peroxide lysis than cow pulmonary artery endothelial cells. Conversely, activated neutrophils which utilize hydrogen peroxide-mediated cell cytotoxicity cell mechanisms were more toxic to the cow pulmonary artery cells. This discordance was not related to neutrophil adhesion to either cell type or cell passage number. The antioxidant profiles of the endothelial cells revealed that cow pulmonary artery cells were rich in catalase to consume bolus hydrogen peroxide presented to them, while human umbilical vein endothelial cells utilize glutathione peroxidase-linked mechanisms to detoxify a slower more sustained release of hydrogen peroxide generated by neutrophils. Endothelial cells from different species and sites may utilize diversified antioxidant protective mechanisms.

Interleukin 1 or endotoxin increases the release of von Willebrand factor from human endothelial cells.

von Willebrand factor (vWF), a large adhesive glycoprotein, is synthesized by vascular endothelial cells (EC). Plasma levels of vWF manifest a broad normal range, and are elevated during sepsis and in inflammatory states. Since the inflammatory mediator, interleukin 1 (IL1) and bacterial endotoxin (LPS) both initiate procoagulant changes in vascular endothelium, we investigated the effect of these substances on endothelial cell release and residual endothelial cell content of vWF-antigen (vWFAg). Cultured human EC exposed to either IL1 or LPS released greater amounts of vWFAg compared to control EC. The augmented release could be detected within 1-2 h after exposure to IL1 or LPS and was not inhibited by cycloheximide, suggesting that de novo protein synthesis was not required for release to occur. Residual cellular vWFAg was reciprocally lower in IL1- or LPS-treated EC at 24 and 48 h, indicating that compensatory increase in synthesis of vWFAg did not occur during this time interval. Released vWF contained the higher molecular weight multimers observed in normal endothelial cells, and it possessed ristocetin cofactor activity. We propose that release of functional vWF from EC exposed to inflammatory mediators may be at a mechanism for localization of platelets and enhanced thrombogenicity at inflammatory foci.

Interleukin 1 stimulates endothelial cell tissue factor production and expression by a prostaglandin-independent mechanism.

Activation of coagulation occurs at inflammatory sites following the ingress of mononuclear cells, and may result from alterations in the vessel wall. Since the monokine, interleukin 1, initiates diverse responses to inflammation, its ability to enhance vascular procoagulant activity was studied. Interleukin 1-treated cultured human endothelial cells acquired elevated levels of the procoagulant, tissue factor. This required de novo protein synthesis, was maximal at 2 h after exposure to interleukin 1, and resulted in persistently elevated cellular procoagulant activity. Tissue factor was later expressed (6-24 h) on the surface of uninjured endothelial cells. Endothelial cell procoagulant production and expression in response to interleukin 1 could be dissociated from endogenous prostaglandin metabolism, being insensitive to hydrocortisone, indomethacin, eicosatetrayionic acid and exogenous arachidonic acid. In addition, no increase in prostaglandin synthesis occurred during the interval in which tissue factor was synthesized. We therefore conclude that interleukin 1 stimulates endothelial synthesis and surface expression of tissue factor by a prostaglandin-independent mechanism.

Structural features of endotoxin required for stimulation of endothelial cell tissue factor production; exposure of preformed tissue factor after oxidant-mediated endothelial cell injury.

Cultured human umbilical vein endothelial cells synthesize the procoagulant, tissue factor, after exposure to bacterial endotoxin. Wild-type lipopolysaccharide from Escherichia coli 0127:B8 stimulates a five- to 20-fold increase in cellular tissue factor. Similarly, rough or incomplete lipopolysaccharide subunits from mutant bacterial strains, or lipid A prepared by mild acid hydrolysis of whole endotoxin, are also stimulatory. In addition, a lipid A biosynthetic precursor, consisting of a phosphorylated glucosamine disaccharide substituted with four beta-hydroxymyristoyl residues, is stimulatory at nanomolar concentrations. Endothelial cell tissue factor is not detectable on the surface of undisrupted cells, but can activate clotting on the cell surface after oxidant-mediated cell injury. The procoagulant, tissue factor, is synthesized by endothelial cells after stimulation mediated by a moiety contained within the lipid A region of lipopolysaccharide. Exposure of clotting factors at the endothelial cell surface after cell injury suggests a mechanism for the microvascular thrombosis associated with disseminated intravascular coagulation with sepsis.

Oral contraceptive use alters the balance of platelet prostaglandin and thromboxane synthesis.

The ability of platelet microsomes to generate platelet aggregating activity on addition of arachidonic acid was evaluated in women taking oral contraceptives and in controls taking no medication but matched for age, sex, and family history. Oral contraceptive users generated significantly more platelet aggregating activity per 100 ug of platelet microsomal protein than controls. Variation in generation of platelet aggregating activity during the menstrual cycle was also observed with highest activity during the third week. These studies show an altered balance of platelet prostaglandin and thromboxane synthesis in oral contraceptive users which may contribute to their increased incidence of thromboembolic phenomena.