Figure 3
Figure 3. HCQ reduced bindings of aPL IgG, increased quantity of AnxA5, and reversed the acceleration of plasma coagulation on cultured HUVECs and STCs. (A) HCQ reduced binding of aPL IgGs to cultured cells. In the absence of HCQ, HUVECs (left panel) exposed to human aPL IgGs (0.5 mg/mL) bound significantly more IgG than with control IgGs (44.2 ± 0.7 AU/well vs 26.9 ± 1.1 AU/well; P < .001); HCQ significantly reduced the binding of aPL IgGs to the surfaces of HUVECs (31.5 ± 5.2 AU/well for HCQ at 1 μg/mL and 27.8 ± 1.8 AU/well for HCQ at 1 mg/mL) to levels that were not significantly different from control IgGs (26.9 ± 1.1 AU/well;P = .19 and P = .50, respectively). Similarly, STCs (right panel) exposed to human aPL IgGs (0.5 mg/mL) bound significantly more IgG than with control IgGs (53.0 ± 4.0 AU/well vs 29.8 ± 1.8 AU/well; P < .001); HCQ significantly reduced the binding of aPL IgGs to the surfaces of STCs (36.2 ± 7.0 AU/well for HCQ at 1 μg/mL and 35.0 ± 6.6 AU/well for HCQ at 1 mg/mL) to levels that were not significantly different from control IgGs (29.8 ± 1.8 AU/well; P = .20 and P = .26, respectively). There were no significant differences between control IgGs in the absence or presence of both concentrations of HCQ (data not shown). As described in “Human umbilical vein endothelial cell cultures” and “STC cultures,” 3 pairs of IgGs from APS patients and controls were used for all of these experiments, done in quadruplicate for each IgG. (B) HCQ increased AnxA5 levels on cultured HUVECs and STCs exposed to aPL IgGs. In the absence of HCQ, aPL reduced AnxA5 levels compared with control IgG (1.2 ± 0.1 ng/well vs 1.9 ± 0.2 ng/well; P = .003); HCQ increased AnxA5 levels on the aPL IgG-treated cells to levels similar to control IgGs (1.8 ± 0.4 ng/well for 1 μg/mL HCQ and 1.9 ± 0.1 ng/well for 1 mg/mL HCQ vs 1.9 ± 0.2 ng/well for control IgG; P = .85 and P = .88, respectively). The same effect was seen with STCs, wherein aPL IgG reduced AnxA5 (2.5 ± 0.4 ng/well vs 4.1 ± 0.7 ng/well for control IgGs; P = .02), and HCQ increased the AnxA5 to levels similar to control IgGs (4.6 ± 1.1 ng/well for 1 μg/mL HCQ and 5.7 ± 0.8 ng/well for 1 mg/mL HCQ vs 4.1 ± 0.7 ng/well; P = .57 and P = .06, respectively). There were no significant differences between control IgGs in the absence or presence of both concentrations of HCQ (data not shown). (C) HCQ reversed the acceleration of coagulation times of plasmas overlaid on cultured HUVECs and STCs exposed to aPL IgGs. The coagulation times of plasma on aPL IgG-treated HUVECs (left panel) were significantly accelerated compared with control IgGs (101 ± 11 seconds vs 135 ± 4 seconds; P = .007). HCQ significantly prolonged them to coagulation times that were not significantly different from control IgGs (130 ± 3 seconds for 1 μg/mL HCQ and 134 ± 13 seconds for 1 mg/mL HCQ vs 135 ± 4 seconds for control IgGs; P = .15 and P = .90, respectively). The same effect was seen with STCs (right panel), wherein the coagulation times of plasma overlaid on aPL IgG-treated cells were significantly accelerated (172 ± 14 seconds vs 231 ± 7 seconds for control IgGs; P = .003). HCQ significantly prolonged them to coagulation times that were not significantly different from control IgGs (256 ± 17 seconds for 1 μg/mL HCQ and 242 ± 3 seconds for 1 mg/mL HCQ vs 231 ± 7 seconds for control IgG; P = .08 and P = .07, respectively). There were no significant differences between control IgGs in the absence or presence of both concentrations of HCQ (data not shown).

HCQ reduced bindings of aPL IgG, increased quantity of AnxA5, and reversed the acceleration of plasma coagulation on cultured HUVECs and STCs. (A) HCQ reduced binding of aPL IgGs to cultured cells. In the absence of HCQ, HUVECs (left panel) exposed to human aPL IgGs (0.5 mg/mL) bound significantly more IgG than with control IgGs (44.2 ± 0.7 AU/well vs 26.9 ± 1.1 AU/well; P < .001); HCQ significantly reduced the binding of aPL IgGs to the surfaces of HUVECs (31.5 ± 5.2 AU/well for HCQ at 1 μg/mL and 27.8 ± 1.8 AU/well for HCQ at 1 mg/mL) to levels that were not significantly different from control IgGs (26.9 ± 1.1 AU/well;P = .19 and P = .50, respectively). Similarly, STCs (right panel) exposed to human aPL IgGs (0.5 mg/mL) bound significantly more IgG than with control IgGs (53.0 ± 4.0 AU/well vs 29.8 ± 1.8 AU/well; P < .001); HCQ significantly reduced the binding of aPL IgGs to the surfaces of STCs (36.2 ± 7.0 AU/well for HCQ at 1 μg/mL and 35.0 ± 6.6 AU/well for HCQ at 1 mg/mL) to levels that were not significantly different from control IgGs (29.8 ± 1.8 AU/well; P = .20 and P = .26, respectively). There were no significant differences between control IgGs in the absence or presence of both concentrations of HCQ (data not shown). As described in “Human umbilical vein endothelial cell cultures” and “STC cultures,” 3 pairs of IgGs from APS patients and controls were used for all of these experiments, done in quadruplicate for each IgG. (B) HCQ increased AnxA5 levels on cultured HUVECs and STCs exposed to aPL IgGs. In the absence of HCQ, aPL reduced AnxA5 levels compared with control IgG (1.2 ± 0.1 ng/well vs 1.9 ± 0.2 ng/well; P = .003); HCQ increased AnxA5 levels on the aPL IgG-treated cells to levels similar to control IgGs (1.8 ± 0.4 ng/well for 1 μg/mL HCQ and 1.9 ± 0.1 ng/well for 1 mg/mL HCQ vs 1.9 ± 0.2 ng/well for control IgG; P = .85 and P = .88, respectively). The same effect was seen with STCs, wherein aPL IgG reduced AnxA5 (2.5 ± 0.4 ng/well vs 4.1 ± 0.7 ng/well for control IgGs; P = .02), and HCQ increased the AnxA5 to levels similar to control IgGs (4.6 ± 1.1 ng/well for 1 μg/mL HCQ and 5.7 ± 0.8 ng/well for 1 mg/mL HCQ vs 4.1 ± 0.7 ng/well; P = .57 and P = .06, respectively). There were no significant differences between control IgGs in the absence or presence of both concentrations of HCQ (data not shown). (C) HCQ reversed the acceleration of coagulation times of plasmas overlaid on cultured HUVECs and STCs exposed to aPL IgGs. The coagulation times of plasma on aPL IgG-treated HUVECs (left panel) were significantly accelerated compared with control IgGs (101 ± 11 seconds vs 135 ± 4 seconds; P = .007). HCQ significantly prolonged them to coagulation times that were not significantly different from control IgGs (130 ± 3 seconds for 1 μg/mL HCQ and 134 ± 13 seconds for 1 mg/mL HCQ vs 135 ± 4 seconds for control IgGs; P = .15 and P = .90, respectively). The same effect was seen with STCs (right panel), wherein the coagulation times of plasma overlaid on aPL IgG-treated cells were significantly accelerated (172 ± 14 seconds vs 231 ± 7 seconds for control IgGs; P = .003). HCQ significantly prolonged them to coagulation times that were not significantly different from control IgGs (256 ± 17 seconds for 1 μg/mL HCQ and 242 ± 3 seconds for 1 mg/mL HCQ vs 231 ± 7 seconds for control IgG; P = .08 and P = .07, respectively). There were no significant differences between control IgGs in the absence or presence of both concentrations of HCQ (data not shown).

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