Figure 2
Figure 2. Prostasomes trigger coagulation in a FXII-dependent manner. Comparison of (A-C, upper) seminal and (D-F, lower) PC3 cell prostasomes: TEM images show morphologies of isolated (A) seminal and (D) PC3 prostasomes (bar, 500 nm). Size distributions of (B) seminal and (E) PC3 prostasomes assessed from TEM images as shown in A and D, 6 fields each. Real-time thrombin generation stimulated with increasing concentrations (0-28 µg/mL) of (C) seminal and (F) PC3 prostasomes in platelet-free (solid lanes) and ultracentrifuged (dashed line) plasma. Representative curves of a series of n = 6 are shown. (G-H) Mechanism of prostasome-driven coagulation: real-time thrombin generation initiated by 14 µg/mL PC3 prostasomes in NP, NP supplemented with rHA-Infestin-4 (Inf-4; 500 µg/mL), ASIS (30 nM), or a combination of ASIS and Inf-4 (30 nM and 500 µg/mL) and plasma deficient in FXII (FXII def.) or FXI (FXI def.); n = 3 to 11. (I) PC3 prostasome (1.75 µg/mL)-stimulated recalcification clotting time in ultracentrifuged NP in the presence of inhibitors as in G and H or buffer or in FXII- or FXI-deficient plasma. **P < .01 and ***P < .001 vs NP, n = 5. (J-L) Contact activation was analyzed by conversion of the FXIIa/PK chromogenic substrate S-2302. Buffer stimulated plasma is shown as control. (J) Plasma was incubated with increasing concentrations of PC3 prostasomes (0-200 µg/mL). (K) PC3 prostasome (100 µg/mL)-induced S-2302 conversion in NP or plasma deficient in FXII (FXII def.), prekallikrein (PPK def.), or high-molecular-weight kininogen (HK def.). *P < .05 and ***P < .001 vs NP + buffer. Bars represent the absorbance at λ = 405 nm at 60 minutes; n = 6. (L) Normal (dark columns) or FXII-deficient (light columns) plasma was incubated with PC3 prostasomes and exosomes from various cancer cells (Panc1, BxPC3, Capan2, and HL-60; 100 µg/mL each). Absorbance of cleaved S-2302 in prostasome-treated plasma is given relative to buffer signal at 60 minutes. ***P < .001 NP vs FXII def., n = 3 to 6. P values were determined using 1-way ANOVA (I,K) or Student t test (L). Data are presented as mean ± SEM; n.s., nonsignificant.

Prostasomes trigger coagulation in a FXII-dependent manner. Comparison of (A-C, upper) seminal and (D-F, lower) PC3 cell prostasomes: TEM images show morphologies of isolated (A) seminal and (D) PC3 prostasomes (bar, 500 nm). Size distributions of (B) seminal and (E) PC3 prostasomes assessed from TEM images as shown in A and D, 6 fields each. Real-time thrombin generation stimulated with increasing concentrations (0-28 µg/mL) of (C) seminal and (F) PC3 prostasomes in platelet-free (solid lanes) and ultracentrifuged (dashed line) plasma. Representative curves of a series of n = 6 are shown. (G-H) Mechanism of prostasome-driven coagulation: real-time thrombin generation initiated by 14 µg/mL PC3 prostasomes in NP, NP supplemented with rHA-Infestin-4 (Inf-4; 500 µg/mL), ASIS (30 nM), or a combination of ASIS and Inf-4 (30 nM and 500 µg/mL) and plasma deficient in FXII (FXII def.) or FXI (FXI def.); n = 3 to 11. (I) PC3 prostasome (1.75 µg/mL)-stimulated recalcification clotting time in ultracentrifuged NP in the presence of inhibitors as in G and H or buffer or in FXII- or FXI-deficient plasma. **P < .01 and ***P < .001 vs NP, n = 5. (J-L) Contact activation was analyzed by conversion of the FXIIa/PK chromogenic substrate S-2302. Buffer stimulated plasma is shown as control. (J) Plasma was incubated with increasing concentrations of PC3 prostasomes (0-200 µg/mL). (K) PC3 prostasome (100 µg/mL)-induced S-2302 conversion in NP or plasma deficient in FXII (FXII def.), prekallikrein (PPK def.), or high-molecular-weight kininogen (HK def.). *P < .05 and ***P < .001 vs NP + buffer. Bars represent the absorbance at λ = 405 nm at 60 minutes; n = 6. (L) Normal (dark columns) or FXII-deficient (light columns) plasma was incubated with PC3 prostasomes and exosomes from various cancer cells (Panc1, BxPC3, Capan2, and HL-60; 100 µg/mL each). Absorbance of cleaved S-2302 in prostasome-treated plasma is given relative to buffer signal at 60 minutes. ***P < .001 NP vs FXII def., n = 3 to 6. P values were determined using 1-way ANOVA (I,K) or Student t test (L). Data are presented as mean ± SEM; n.s., nonsignificant.

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