Figure 2.
Natural IgM antibodies inhibit MV-sensitive plasma coagulation and factor Xa (FXa) binding to MVs. (A) Effect of the MDA-specific natural IgM antibody (ab) LR04 on TG. TG curves of MV-depleted plasma, triggered by platelet-derived MVs (2 µg/mL), which were preincubated with 2 concentrations of LR04 or an isotype control ab (0, 25, or 50 µg/mL). (B-C) Inhibitory effect of the LR04-specific peptide mimotope P2. Flow cytometry histograms depict binding of LR04 to platelet-derived MVs in the presence or absence of P2 (100 µg/mL) (B); and TG curves of MV-depleted plasma triggered by platelet-derived MVs (2 µg/mL) incubated with LR04 (25 µg/mL) in the presence of increasing concentrations of P2 (0, 12, 25, 50, or 100 µg/mL) (C). (D) TG curves of MV-depleted plasma triggered by platelet-derived MVs (2 µg/mL), which were preincubated with 25 µg/mL of the MDA-specific natural IgM LR04 or NA17, the phosphocholine-specific natural IgM E06, or an isotype control ab. (E) TG curves of MV-depleted plasma triggered by THP-1 cell-derived MVs (2 µg/mL). MVs were preincubated with LR04 or an isotype control ab (25 or 50 µg/mL) or a TF-blocking ab (HTF-1, 10 µg/mL). (F) Activated partial thromboplastin time (aPTT) and prothrombin time (PT) of platelet-poor plasma that was preincubated with increasing amounts of LR04 or an isotype control ab (0, 12, 25, or 50 µg/mL). (G) Effect of LR04 on THP-1/MV–triggered (2 µg/mL) TG of MV-depleted plasma deficient for single coagulation factors (FVII, FVIII, FIX, FXI, and FXII). Shown is the relative reduction of peak height after preincubation of MVs with LR04 (25 µg/mL) compared with the peak height of each respective plasma. (H) Flow cytometry–based size profiles of platelet-derived MVs after incubation (30 minutes, 37°C) with LR04 (25 µg/mL), isotype control ab (25 µg/mL), or concanavalin A (ConA) (5 µg/mL). (I) Inhibition of the binding of plasma-derived factor X/Xa to MVs by increasing concentrations of LR04. Symbols represent the mean ± SEM of 4 independent experiments. *P < .05, **P < .01, two-way analysis of variance, Bonferroni’s multiple comparisons test. In panels A through G, plots depict representative experiments of least 4 experiments. APC-H, allophycocyanin; FSC-H, forward scatter.

Natural IgM antibodies inhibit MV-sensitive plasma coagulation and factor Xa (FXa) binding to MVs. (A) Effect of the MDA-specific natural IgM antibody (ab) LR04 on TG. TG curves of MV-depleted plasma, triggered by platelet-derived MVs (2 µg/mL), which were preincubated with 2 concentrations of LR04 or an isotype control ab (0, 25, or 50 µg/mL). (B-C) Inhibitory effect of the LR04-specific peptide mimotope P2. Flow cytometry histograms depict binding of LR04 to platelet-derived MVs in the presence or absence of P2 (100 µg/mL) (B); and TG curves of MV-depleted plasma triggered by platelet-derived MVs (2 µg/mL) incubated with LR04 (25 µg/mL) in the presence of increasing concentrations of P2 (0, 12, 25, 50, or 100 µg/mL) (C). (D) TG curves of MV-depleted plasma triggered by platelet-derived MVs (2 µg/mL), which were preincubated with 25 µg/mL of the MDA-specific natural IgM LR04 or NA17, the phosphocholine-specific natural IgM E06, or an isotype control ab. (E) TG curves of MV-depleted plasma triggered by THP-1 cell-derived MVs (2 µg/mL). MVs were preincubated with LR04 or an isotype control ab (25 or 50 µg/mL) or a TF-blocking ab (HTF-1, 10 µg/mL). (F) Activated partial thromboplastin time (aPTT) and prothrombin time (PT) of platelet-poor plasma that was preincubated with increasing amounts of LR04 or an isotype control ab (0, 12, 25, or 50 µg/mL). (G) Effect of LR04 on THP-1/MV–triggered (2 µg/mL) TG of MV-depleted plasma deficient for single coagulation factors (FVII, FVIII, FIX, FXI, and FXII). Shown is the relative reduction of peak height after preincubation of MVs with LR04 (25 µg/mL) compared with the peak height of each respective plasma. (H) Flow cytometry–based size profiles of platelet-derived MVs after incubation (30 minutes, 37°C) with LR04 (25 µg/mL), isotype control ab (25 µg/mL), or concanavalin A (ConA) (5 µg/mL). (I) Inhibition of the binding of plasma-derived factor X/Xa to MVs by increasing concentrations of LR04. Symbols represent the mean ± SEM of 4 independent experiments. *P < .05, **P < .01, two-way analysis of variance, Bonferroni’s multiple comparisons test. In panels A through G, plots depict representative experiments of least 4 experiments. APC-H, allophycocyanin; FSC-H, forward scatter.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal