Despite many studies on the pathophysiology of antiphospholipid antibodies (aPL), the mechanism by which aPL causes thrombosis has not been established. We have tried to elucidate the paradox between the prolongation of the clotting time of phospholipid-dependent coagulation tests in vitro and the occurrence of thrombosis in vivo. The effect on endothelial cell-mediated prothrombinase activity of 30 IgG fractions, of which 22 prolong the aPTT of normal plasma, was investigated. Only 4 of 22 fractions (18%) inhibited prothrombinase activity when tested on this more physiologic phospholipid surface, indicating that in most patients with aPL the prolongation of clotting tests is predominantly as in vitro phenomenon. It was recently reported that in detection methods for aPL, two plasma proteins, beta 2-glycoprotein I and prothrombin, enhance the binding of aPL to phospholipids. We have studied the specificity of the 4 IgG fractions that inhibit the prothrombinase activity and found that they were directed against a combination of phospholipids and prothrombin. However, the involvement of prothrombin in binding of aPL leading to impaired thrombin generation could still result in both a bleeding and a thrombotic tendency. Therefore, we proposed a new thrombogenic mechanism for aPL in which aPL bind to complexes of phospholipids and coagulation proteins, thereby interfering in different coagulation reactions. We tested this new hypothesis by investigating the effect of IgG from the same 30 patients on the activated protein C (APC)-mediated factor Va inactivation in the absence and presence of protein S. Three IgGs that inhibited APC-mediated factor Va inactivation independent of protein S and 4 additional IgGs that inhibited in the presence of protein S were found. Furthermore, we could specifically adsorb the inhibitory IgG with cardiolipin vesicles to which APC with or without protein S was bound. In conclusion, these results suggest that subpopulations of aPL exist that are directed to complexes of phospholipids and different plasma proteins. The identity of the plasma proteins involved in the binding of aPL might determine which pathogenic mechanism causes thrombosis.

SummaryThe effect of sera and purified IgG isolated from plasma of 46 patients with systemic lupus erythematosus (SLE) and 9 healthy donors on the endothelial cell (EC) mediated protein C activation was investigated. Out of the 46 SLE sera used, 19 were antiphospholipid antibodies (aPL) positive. From 12 patients IgG was isolated, of which 6 contained aPL. EC were first incubated with IgG (7 mg/ml) or serum (1: 1 diluted) for 1 h and then tested for their ability to promote protein C activation by thrombin, with the cells either in a monolayer or in a suspension. The normal range (mean of control values ± 2 SD) of protein C activation was 80-120%. In contrast to others, we could not detect an inhibition of protein C activation by any of the patient IgG's or sera.The recently described cofactor for binding of antiphospholipid antibodies to phospholipids, β2-glycoprotein I, was purified and added to the purified IgG's. A combination of these two components did not inhibit the EC mediated protein C activation by thrombin.This study suggests that the inhibition of the protein C activation, mediated by EC, is not a general mechanism by which aPL related thrombosis can be explained.

SummaryThe effect of 23 antiphospholipid antibody positive SLE sera, 4 antiphospholipid antibody negative SLE sera and 17 control sera on endothelial prostacyclin and platelet thromboxane A? 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 nr 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 endoihelial or platelet prostanoid synthesis does not explain the occurrence of thromboembolic manifestations in antiphospholipid antibody positive SLE patients.

Binding of anti-phospholipid antibodies to circulating platelets and its consequences on platelet activation and aggregation was investigated in 11 patients with anti-phospholipid antibodies. Seven patients had mild thrombocytopenia. Nine healthy donors served as controls. Binding to platelets was investigated by performing enzyme- linked immunosorbent assays (ELISAs) with phospholipids as antigen on platelet eluates. Platelet activation was measured by flow cytofluorometry using monoclonal antibodies to an activation-specific lysosomal membrane protein. Findings in ELISA were compared with results of a conventional immunofluorescence method to detect platelet autoantibodies. In seven patients antibodies to negatively charged phospholipids were present in platelet eluates. In all thrombocytopenic patients and controls the platelets were not activated and aggregation was not impaired. There was a positive concordance of 50% between the results of immunofluorescence and ELISA. No apparent relation was found between the results of ELISA or immunofluorescence and platelet counts. It is concluded that anti-phospholipid antibodies can bind to circulating platelets. This binding is not associated with measurable aggregation abnormalities nor with platelet activation characterized by exposure of lysosomal membrane proteins. More studies are necessary to determine the exact role of anti-phospholipid antibodies in the pathogenesis of thrombocytopenia and thrombosis.