Figure 3.
POmAb exerts an anticoagulant effect in human plasma by forcing prothrombin (PT) to open. (A) Antibody-stimulated binding of prothrombin wild type (WT; 250 nM; red) and variant S91A/Y93A (250 nM; blue) to negatively charged liposomes (POPC:POPS 80:20) monitored by SPR. IgG and Fab POmAb were used at 2.5 μg/mL. Values of fold increase were determined using the formula: RUIgG/Fab/RUfree at 115 seconds. The horizontal dotted line set at y = 1.0 indicates there is no effect of the treatment. Raw data are shown in supplemental Figure 7. (B) Activated partial thromboplastin time of human plasma measured at increasing concentrations of POmAb IgG (blue) and Fab (red) (0-5 μM). (C) Dilute Russell’s viper venom time expressed as normalized LA ratio, as per manufacturer’s instructions. Shown are normal control (CTRL) plasma (negative [-ve] CTRL), LA-control plasma (positive [+ve] CTRL), and normal control plasma supplemented with 1 μM POmAb. The horizontal dotted line set at y = 1.2 indicates the threshold provided by the manufacturer for considering a specimen LA positive (above threshold) or negative (below threshold). (D) Clotting time measured prothrombin-deficient plasma supplemented with prothrombin wild type or the variant S91A/Y93A. (E) Continuous assay monitoring of the conversion of prothrombin to thrombin by prothrombinase complex. The assay started with the addition of 2.5 pM factor Xa to 25 nM prothrombin that was preincubated for 20 minutes with 20 μM phospholipids, 2 nM cofactor Va, and increasing concentrations of Fab (0-2 μM). The release of p-nitroaniline on hydrolysis of FPF (H-D-Phe-Pro-Phe-p-nitroanilide) was monitored at 405 nm.19 (F) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the conversion of prothrombin (1.25 μM) to thrombin by the prothrombinase complex (0.2 nM factor Xa, 20 μM phospholipids, and 10 nM cofactor Va) in the absence and presence of 1.5 μM Fab POmAb, which shows as 2 bands at 25 kDa because of the presence of reducing agent. The disappearance of PT (72-kDa band) is slower in the presence of Fab and is linked to lower accumulation of B-chain (28 kDa) and A-chain (6 kDa), which are signatures of thrombin. Moreover, in the presence of Fab, there is significantly more accumulation of a band at 50 kDa (black arrow), compatible with cleavage at R155 and the formation of prethrombin-1.9,19 (G) Proposed mechanism of action of POmAb resulting in anticoagulation. Note that prothrombin circulates mostly in closed form. Our SPR data with Fab in A suggest that, on binding to phospholipids, prothrombin does spontaneously open. Thus, it requires POmAb to decisively shift the equilibrium toward the open form.∗∗P < .01, ∗∗∗P < .001. ns, not significant; PTC, prothrombinase complex.

POmAb exerts an anticoagulant effect in human plasma by forcing prothrombin (PT) to open. (A) Antibody-stimulated binding of prothrombin wild type (WT; 250 nM; red) and variant S91A/Y93A (250 nM; blue) to negatively charged liposomes (POPC:POPS 80:20) monitored by SPR. IgG and Fab POmAb were used at 2.5 μg/mL. Values of fold increase were determined using the formula: RUIgG/Fab/RUfree at 115 seconds. The horizontal dotted line set at y = 1.0 indicates there is no effect of the treatment. Raw data are shown in supplemental Figure 7. (B) Activated partial thromboplastin time of human plasma measured at increasing concentrations of POmAb IgG (blue) and Fab (red) (0-5 μM). (C) Dilute Russell’s viper venom time expressed as normalized LA ratio, as per manufacturer’s instructions. Shown are normal control (CTRL) plasma (negative [-ve] CTRL), LA-control plasma (positive [+ve] CTRL), and normal control plasma supplemented with 1 μM POmAb. The horizontal dotted line set at y = 1.2 indicates the threshold provided by the manufacturer for considering a specimen LA positive (above threshold) or negative (below threshold). (D) Clotting time measured prothrombin-deficient plasma supplemented with prothrombin wild type or the variant S91A/Y93A. (E) Continuous assay monitoring of the conversion of prothrombin to thrombin by prothrombinase complex. The assay started with the addition of 2.5 pM factor Xa to 25 nM prothrombin that was preincubated for 20 minutes with 20 μM phospholipids, 2 nM cofactor Va, and increasing concentrations of Fab (0-2 μM). The release of p-nitroaniline on hydrolysis of FPF (H-D-Phe-Pro-Phe-p-nitroanilide) was monitored at 405 nm.19 (F) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis of the conversion of prothrombin (1.25 μM) to thrombin by the prothrombinase complex (0.2 nM factor Xa, 20 μM phospholipids, and 10 nM cofactor Va) in the absence and presence of 1.5 μM Fab POmAb, which shows as 2 bands at 25 kDa because of the presence of reducing agent. The disappearance of PT (72-kDa band) is slower in the presence of Fab and is linked to lower accumulation of B-chain (28 kDa) and A-chain (6 kDa), which are signatures of thrombin. Moreover, in the presence of Fab, there is significantly more accumulation of a band at 50 kDa (black arrow), compatible with cleavage at R155 and the formation of prethrombin-1.9,19 (G) Proposed mechanism of action of POmAb resulting in anticoagulation. Note that prothrombin circulates mostly in closed form. Our SPR data with Fab in A suggest that, on binding to phospholipids, prothrombin does spontaneously open. Thus, it requires POmAb to decisively shift the equilibrium toward the open form.∗∗P < .01, ∗∗∗P < .001. ns, not significant; PTC, prothrombinase complex.

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