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The influence of variations in plasma fibrinogen concentration on platelet adhesion to immobilized fibrinogen was investigated in a parallel-plate perfusion chamber. At a shear rate of 1600 s-1 platelet adhesion decreased when increasing concentrations of purified fibrinogen were added to the plasma (IC50 = 1.5 +/- 0.2 g/L fibrinogen, n = 24). Washed platelets reconstituted in a human albumin solution with red blood cells were more sensitive for soluble fibrinogen (IC50 = 0.4 +/- 0.1 g/L, n = 5, P < .05). When platelet activation during circulation of the blood was minimized by using a single-passage perfusion system, an IC50 of 2.0 +/- 0.2 g/L was found (n = 9). To exclude the possibility that the inhibition of fibrinogen was caused by irreversible changes in the fibrinogen molecule during the purification procedure, normal plasma was mixed in different ratios with plasma from a patient with congenital afibrinogenemia. Under these conditions, the plasma fibrinogen IC50 was 1.5 +/- 1.1 g/L. Absence of endogenous fibrinogen in the platelets of the patient resulted in an IC50 of 1.2 +/- 0.5 g/L for plasma fibrinogen. These results demonstrate that increased plasma fibrinogen concentrations inhibit platelet adhesion to fibrinogen under flow.

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Platelet adhesion to fibrin at high shear rates depends on both the glycoprotein (GP) IIb:IIIa complex and a secondary interaction between GPIb and von Willebrand factor (vWF). This alternative link between platelets and vWF in promoting platelet adhesion to fibrin has been examined in flowing whole blood with a rectangular perfusion chamber. Optimal adhesion required both platelets and vWF, as shown by the following observations. No binding of vWF could be detected when plasma was perfused over a fibrin surface or when coated fibrinogen was incubated with control plasma in an enzyme-linked immunosorbent assay. However, when platelets were present during perfusion, interactions between vWF and fibrin could be visualized with immunoelectron microscopy. Exposure of fibrin surfaces to normal plasma before perfusion with severe von Willebrand's disease blood did not compensate for the presence of plasma vWF necessary for adhesion. vWF mutants in which the GPIIb:IIIa binding site was mutated or the GPIb binding site was deleted showed that vWF only interacts with GPIb on platelets in supporting adhesion to fibrin and not with GPIIb:IIIa. Complementary results were obtained with specific monoclonal antibodies against vWF. Thus, vWF must first bind to platelets before it can interact with fibrin and promote platelet adhesion. Furthermore, only GPIb, but not GPIIb:IIIa is directly involved in this interaction of vWF with platelets.

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The effect of sera and IgG from 12 patients with systemic lupus erythematosus (SLE) on the endothelial cell (EC) procoagulant activity (PCA) was investigated in an in vitro thrombosis model. Six of the 12 SLE sera contained antiphospholipid antibodies (aPL). EC were stimulated for 8 h at 37 degrees C with or without 50 pM tumor necrosis factor (TNF) in culture medium containing 20% patient or control serum. Then the endothelial cell matrix (ECM) was isolated and subsequently exposed in a perfusion chamber to circulating normal whole blood, anticoagulated with low molecular weight heparin (LMWH). The PCA of the ECM was determined as the amount of generated fibrinopeptide A (FPA) in samples taken before and after perfusion. Furthermore, cross sections were made of the perfused matrix and analyzed for platelet adhesion and aggregate formation. All six aPL containing sera induced a small, but significant increase of ECM procoagulant activity. When added in combination with a low dose of TNF (50 pM), a synergistic enhancement of ECM procoagulant activity was found. The FPA generation was increased to 150-614% from the values obtained after stimulation with TNF and control serum. Also a shift towards the formation of larger platelet thrombi was observed. After stimulation with TNF and patient serum the surface of ECM covered with large aggregates (greater than 5 microns) was increased by 124-329% compared to the results obtained after stimulation with control serum and TNF. When patient sera were depleted from IgG the effects were strongly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

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The interrelationship of antibodies directed against cardiolipin (CL), double stranded DNA (dsDNA), endothelial cells (EC) and blood platelets was investigated. IgG fractions, reactive with these antigens, were isolated from the plasmas from 8 patients with systemic lupus erythematosus and tested for crossreactivity with anionic phospholipids (CL, phosphatidylserine, phosphatidylinositol), zwitterionic phospholipids (phosphatidylethanolamine, phosphatidylcholine), dsDNA, EC and platelets by enzyme-linked immunosorbent assays and for lupus anticoagulant (LAC) activity with a coagulation assay. Our results demonstrate the frequent occurrence of crossreactivity between antibodies to anionic phospholipids, EC and platelets. Crossreactivities between these antibodies and antibodies to dsDNA or zwitterionic phospholipids are exceptional. LAC activity was found in the anti-CL, anti-EC and anti-platelet fractions of only one patient. These findings support the hypothesis that subpopulations of antibodies directed against negatively charged phospholipids can bind to EC and blood platelets, which may have implications for the pathogenetic potential of antiphospholipid antibodies.

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We found that levels of antiphospholipid antibodies (aPLA), measured with an ELISA increase if serum or plasma samples are heated. The phenomenon is dependent on duration and degree of heating, optimum levels being reached at 3 h at 56 degrees C. Negative samples become positive after heating. The heating effect is more pronounced for IgG-aPLA than for IgM-aPLA and is not observed for adsDNA, atetanus or lymphocytotoxic antibodies. The presence of serum/plasma components in addition to IgG is essential for the phenomenon to occur. Ultracentrifugation and mixing experiments with isolated IgG did not enable us to explain our observations. Nevertheless, knowledge of this phenomenon is of practical importance.

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Lupus anticoagulant, concentrations of anticardiolipin antibodies, antithrombin III, plasminogen, (free) protein S, protein C, prothrombin, platelet counts, and bleeding times were determined in 74 lupus patients (58 with systemic lupus erythematosus; 16 with lupus-like disease) to establish the presence of risk factors for thrombosis in these patients. Of the variables evaluated, lupus anticoagulant had the strongest association with a history of thrombosis. Both positive anticardiolipin antibody concentrations and the presence of (mild) thrombocytopenia were significantly associated with a history of thrombosis and the presence of lupus anticoagulant. Reduced concentrations of antithrombin III, plasminogen, (free) protein S, and protein C were found in some patients but were not associated with either thrombosis or lupus anticoagulant. Mean concentrations of total protein S were significantly lower in patients with thrombosis than in those without and in patients with lupus anticoagulant than in those without. The antigenic concentration of prothrombin was reduced in 3/74 (4%) lupus patients. These three patients had lupus anticoagulant but no history of thrombosis, which suggests that a low prothrombin concentration protects patients with lupus anticoagulant from the development of thrombosis. A prolonged bleeding time was associated with the presence of lupus anticoagulant but not with a history of thrombosis. Analysis by stepwise logistic regression did not disclose additional risk factors for thrombosis in lupus patients with lupus anticoagulant. Increased antithrombin III concentrations and decreased free protein S concentrations are often found in lupus patients, unrelated to lupus anticoagulant or thrombosis.

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The effect of 15 antiphospholipid antibody (APA) positive SLE sera, 13 APA negative SLE sera, 10 APA negative sera from patients with other connective tissue diseases (CTD) and 15 control sera on the expression of endothelial procoagulant activity (PCA) was studied. Endothelial cells (EC) were stimulated with tumor necrosis factor (TNF) and 20% serum for 4 hr and the surface PCA expression was measured. Without TNF, none of the sera stimulated PCA expression. With suboptimal TNF stimulation, 14/15 APA positive SLE sera, 7/13 APA negative SLE sera, 2/10 CTD sera and 2/15 control sera enhanced PCA expression. This stimulating effect resided in the IgG fraction and was associated with the presence of APA, but not with a history of thrombosis. Purified APA had no PCA stimulating activity. PCA expression was inhibited by cycloheximide and heat treatment (30 min, 56 degrees C) of serum. In conclusion, 21/28 (75%) SLE sera increase the TNF-induced endothelial PCA expression. Although this effect predominantly occurs with APA positive serum, a causative role of APA was not demonstrated.

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In 111 lupus patients we compared the potential of the IgG and IgM anticardiolipin antibody (ACA) enzyme linked immunosorbent assay (ELISA) and four different lupus anticoagulant (LAC) assays (partial thromboplastin time (PTT) of a 1:1 mixture of patient and control plasma with phospholipids from animal (PTT-st) or human brain (PTT-HB); PTT with dilutions of human brain phospholipids (PL dilution); and kaolin clotting time of mixtures of patient and control plasma (KCT] to identify patients with thrombosis (26/111), fetal loss (19/46), and/or thrombocytopenia (11/106). The highest specificity for thrombosis (87%) was found with PTT-HB and PL dilution (sensitivity 65%, detection rate 61%); for fetal loss (93%) with PL dilution (sensitivity 47%; detection rate 82%), and for thrombocytopenia (83%) with KCT (sensitivity 82%; detection rate 36%). Compared with LAC assays, the sensitivity of ACA-ELISA was high (greater than or equal to 77%), but specificity (less than or equal to 51%) and detection rate (less than or equal to 52%) were low. So, a panel of three LAC assays (PTT-HB, PL dilution, and KCT) can identify lupus patients apparently at risk for thrombosis, fetal loss, and/or thrombocytopenia, whereas the ACA-ELISA is insufficiently specific.

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The effect of 23 antiphospholipid antibody positive SLE sera, 4 antiphospholipid antibody negative SLE sera and 17 control sera on endothelial prostacyclin and platelet thromboxane A2 production was studied. Endothelial cells and platelets were stimulated with different agonists. Depending on the stimulus used, 4-19% of the SLE sera inhibited the prostacyclin release, whereas 4-28% enhanced prostacyclin production. Our data suggest that the pathophysiological mechanisms underlying decreased prostacyclin production are heterogeneous. Follow-up of two patients showed that prostacyclin inhibitory activity was variable in time. Platelet thromboxane production was normal or increased, but never decreased in the presence of the SLE sera. An imbalance in thromboxane A2/prostacyclin ratio was present in some patients, but did not correlate with a history of thrombosis. We conclude that, in general, interference of antiphospholipid antibodies with endothelial or platelet prostanoid synthesis does not explain the occurrence of thromboembolic manifestations in antiphospholipid antibody positive SLE patients.

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