![Figure 2. Reactivity of aPS/PT toward closed and open conformations of prothrombin in solution. (A) IgG aPS/PT were researched in 27 APS triple-positive patients (P1-P27). Two healthy donors (HD1 and HD2), one negative control (– CTRL), and 1 positive control (+ CTRL) were included. The dashed red line (optical density at 450 nm [OD450nm] = 0.32 or 30 units) identifies the cutoff value. (B-D) Competition experiments. (B) Plasma samples (50 μL, 1:50 vol/vol) from 24 APS patients positive for aPS/PT were incubated side-by-side with a solution (50 μL, 0.72 mg/mL, 10 μM) of proTCC, proTY93A and proTΔ154-167 for 30 minutes at room temperature. The residual levels of aPS/PT were quantified by using ELISA assays. The effect of each competitor is reported as the percentage of inhibition calculated by using the following: 100 × ((OD1 – OD2)/OD1), where optical density 1 (OD1) and OD2 are the values of absorbance in the absence and presence of competitor, respectively. (C) Dose-dependent effect of proTCC (blue circles) and proTY93A (red circles) (0-15 μM) as shown for a representative patient (P7). Data were analyzed with a simple binding equation, S = S0 + (S∞ I/IC50)/(1 + I/IC50), and the values of IC50 are reported in Table 2. (D) Plasma samples (50 μL, 1:50 vol/vol) from 24 APS patients positive for aPS/PT were incubated side-by-side with a solution (50 μL, 0.72 mg/mL, 10 μM) of proTWT in the absence or presence of 200 μM argatroban (Arg) for 30 minutes at room temperature. The residual levels of aPS/PT were quantified by using ELISA assays as described before. (E-F) Reactivity of aPT-A toward closed and open conformations of prothrombin in solution. (E) Diluted plasma samples (50 μL, 1:50 vol/vol) from 13 APS patients positive for aPT-A were incubated side-by-side with a solution (50 μL, 0.72 mg/mL, 10 μM) of proTWT, proTCC, proTY93A, or proTΔ154-167 for 30 minutes at room temperature. The residual levels of aPT-A were calculated as described above. (F) Dose-dependent effect of proTCC (blue circles) and proTY93A (red circles) (0-15 μM) as shown for a representative patient (P7). IC50 values are reported in Table 2. (G) Subpopulations of aPS/PT. Two-dimensional representation of the inhibitory effect determined in Figure 2B for proTCC (closed, x-axis) and proTY93A (open, y-axis) in the 24 APS patients positive for aPS/PT. Dashed black lines divide the plot into 4 quadrants. Group A patients (red box) are mostly located in the upper left quadrant, with a few outliers. Group B patients falls into the upper right quadrant. No aPS/PT were detected in the lower right quadrant.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/3/11/10.1182_bloodadvances.2019030932/2/advances030932f2.png?Expires=1724147422&Signature=ztiUNgnpwhH9SGhjt38qOyoJ5czPDKApZbN5wC18P0a8cr4hzQ8M3HMOrxsoLiAONNIFkNm5z--D768FKUrDDvvSrJ-SEHRSgfXDgoEMoZIJFvpvAdQyC9mS1VXw4E5cgIbPsPsBdTJabEWjQpAX9xhDDFmutCLiB0~YRHshOc0t0q7gMJ~SmBAw2FF4-PYktlxBv6YrsL2NddR5TWQO1HduBvPdRSKRfktV~cXmmFd~NEBHUXXta2jBKOaB65hWH-5~N4QImivcWFgVUhTFUA5u0mHxZFK3p~HC5k0sxPUrYoVhrwgWY7IjS15OzgGy9I1VOzeopMefC1kIiL0wuw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Reactivity of aPS/PT toward closed and open conformations of prothrombin in solution. (A) IgG aPS/PT were researched in 27 APS triple-positive patients (P1-P27). Two healthy donors (HD1 and HD2), one negative control (– CTRL), and 1 positive control (+ CTRL) were included. The dashed red line (optical density at 450 nm [OD450nm] = 0.32 or 30 units) identifies the cutoff value. (B-D) Competition experiments. (B) Plasma samples (50 μL, 1:50 vol/vol) from 24 APS patients positive for aPS/PT were incubated side-by-side with a solution (50 μL, 0.72 mg/mL, 10 μM) of proTCC, proTY93A and proTΔ154-167 for 30 minutes at room temperature. The residual levels of aPS/PT were quantified by using ELISA assays. The effect of each competitor is reported as the percentage of inhibition calculated by using the following: 100 × ((OD1 – OD2)/OD1), where optical density 1 (OD1) and OD2 are the values of absorbance in the absence and presence of competitor, respectively. (C) Dose-dependent effect of proTCC (blue circles) and proTY93A (red circles) (0-15 μM) as shown for a representative patient (P7). Data were analyzed with a simple binding equation, S = S0 + (S∞ I/IC50)/(1 + I/IC50), and the values of IC50 are reported in Table 2. (D) Plasma samples (50 μL, 1:50 vol/vol) from 24 APS patients positive for aPS/PT were incubated side-by-side with a solution (50 μL, 0.72 mg/mL, 10 μM) of proTWT in the absence or presence of 200 μM argatroban (Arg) for 30 minutes at room temperature. The residual levels of aPS/PT were quantified by using ELISA assays as described before. (E-F) Reactivity of aPT-A toward closed and open conformations of prothrombin in solution. (E) Diluted plasma samples (50 μL, 1:50 vol/vol) from 13 APS patients positive for aPT-A were incubated side-by-side with a solution (50 μL, 0.72 mg/mL, 10 μM) of proTWT, proTCC, proTY93A, or proTΔ154-167 for 30 minutes at room temperature. The residual levels of aPT-A were calculated as described above. (F) Dose-dependent effect of proTCC (blue circles) and proTY93A (red circles) (0-15 μM) as shown for a representative patient (P7). IC50 values are reported in Table 2. (G) Subpopulations of aPS/PT. Two-dimensional representation of the inhibitory effect determined in Figure 2B for proTCC (closed, x-axis) and proTY93A (open, y-axis) in the 24 APS patients positive for aPS/PT. Dashed black lines divide the plot into 4 quadrants. Group A patients (red box) are mostly located in the upper left quadrant, with a few outliers. Group B patients falls into the upper right quadrant. No aPS/PT were detected in the lower right quadrant.