Background —Antiphospholipid (aPL) antibodies are associated with thrombosis in patients diagnosed with antiphospholipid syndrome (APS) and enhance thrombus formation in vivo in mice, but the mechanism of thrombosis by aPL is not completely understood. Although aPL antibodies have been shown to inhibit protein C activation and activate endothelial cells (ECs) in vitro, no study has examined whether these antibodies activate ECs in vivo. Therefore, human affinity-purified aPL (ap aPL) antibodies from APS patients were tested in a mouse model of microcirculation using the cremaster muscle that allows direct microscopic examination of thrombus formation and adhesion of white blood cells (WBCs) to ECs as an indication of EC activation in vivo. Adhesion molecule expression on human umbilical vein endothelial cells (HUVECs) after aPL exposure was performed to confirm EC activation in vitro. Methods and Results —All 6 ap aPL antibodies significantly increased the expression of VCAM-1 (2.3- to 4.4-fold), with one of the antibodies also increasing the expression of E-selectin (1.6-fold) on HUVECs in vitro. In the in vivo experiments, each ap aPL antibody except for 1 preparation increased WBC sticking (mean number of WBCs ranged from 22.7 to 50.6) compared with control (14.4), which correlated with enhanced thrombus formation (mean thrombus size ranged from 1098 to 6476 versus 594 μm 2 for control). Conclusions —Activation of ECs by aPL antibodies in vivo may create a prothrombotic state on ECs, which may be the first pathophysiological event of thrombosis in APS.

The goal of the present study was to determine if voltage-sensitive calcium channels are present in bovine aortic endothelial cell plasmalemma and if they contribute to the rise in cytosolic calcium produced by bradykinin. After bradykinin (100 nM) exposure, endothelial cell associated fura-2 fluorescence peaked within 10-20 seconds and then declined to a steady level 2- to 3-fold above resting values. Pretreatment with lanthanum (20 microM) abolished the steady level produced by bradykinin but had little effect on the initial, transient rise in cytosolic calcium. Chelation of extracellular calcium with EGTA before addition of bradykinin resulted in a substantial decrease in the fura-2 transient and elimination of the long-lasting component. Nimodipine (3 microM) and nitrendipine (1 microM) were without effect on either phase of the bradykinin-induced response. Moreover, elevation of extracellular potassium failed to produce a rise in intracellular calcium. With the use of the tight seal technique to voltage clamp the cells, inwardly rectifying and calcium-activated potassium currents were found to exist in the endothelial cells. Addition of bradykinin (100 nM) elicited a calcium-activated potassium current that was eliminated in the absence of intracellular potassium. No voltage-sensitive calcium currents were activated when the cells were exposed to 10 mM or 110 mM calcium chloride in the presence or absence of bradykinin. The binding of [3H](+)PN200-110 to endothelial cell membrane preparations was 1-3 orders of magnitude lower than that observed in PC-12, GH3, or BC3H1 cell membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Objective. To characterize the binding and functional properties of antiphospholipid antibodies (aPL) induced by immunization with a viral peptide and to determine whether aPL are pathogenic in vivo.Methods. Ten murine monoclonal aPL were generated from spleen cells of PL/J mice immunized with TIFI, a phospholipid-binding peptide spanning Thr 101 -Thr 120 of ULB0-HCMVA from human cytomegalovirus (CMV), which shares structural similarity with the phospholipid-binding site of ␤ 2 -glycoprotein I (␤ 2 GPI).Results. The antibodies generated had aPL activity that was inhibited by cardiolipin liposomes, and this inhibition was enhanced in the presence of ␤ 2 GPI. Some of the antibodies exhibited binding to cultured endothelial cells in vitro, and some had lupus anticoagulant activity. Injection with 2 of the monoclonal aPL in mice resulted in a significant increase in the number of leukocytes adhering to endothelial cells and enhanced thrombus formation in vivo.Conclusion. These results indicate that aPL induced by immunization with a phospholipid-binding CMV peptide are pathogenic in vivo. The results also suggest a mechanism (molecular mimicry) by which pathogenic aPL may be generated in patients with antiphospholipid syndrome.

Summary. Antiphospholipid (aPL) antibodies, detected in patients with antiphospholipid syndrome (APS) are associated with thrombosis, pregnancy loss and thrombocytopenia. Studies have shown that aPL are thrombogenic in vivo, but the mechanism(s) involved are not completely understood. Several studies have demonstrated that aPL antibodies activate endothelial cells (ECs) in vitro, as determined by up-regulation of adhesion molecules: E-selectin (E-sel); intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and in vivo. The objectives of these study were to determine the effects of aPL antibodies on the expression of E-selectin on ECs, on the adhesion of monocytes to ECs and to study the role of E-selectin on aPL antibodies enhanced thrombus formation and activation of ECs in vivo. We demonstrated that the surface expression of E-selectin on HUVEC by ELISA was increased 400-fold when treated with tumor necrosis factor-alpha (TNF-a) and 421-fold when treated with aPL antibodies during 4 h. APL antibodies also induced activation of the nuclear factorkappa B (NF-kB). APL antibodies increased signi®cantly the number of adhering leukocytes to ECs in vivo in C57BL/ 6 J mice when compared to IgG-NHS treated mice. This effect was abrogated in E-selectin-de®cient mice. The thrombus size was signi®cantly increased in C57BL/6 J mice treated with aPL antibodies when compared to mice treated with IgG-NHS. This enhancement in thrombus size by aPL antibodies was abrogated in E-selectin-de®cient mice treated with aPL antibodies.