Figure 2.
Platelet FXIII-A is not synthesized during activation of human platelets. (A-C) Washed human platelets were unstimulated (Unstim) or stimulated with thrombin (IIa), CVX plus IIa, or A23187. The platelet pellet (P) and releasate (R) were separated by centrifugation. FXIII-A was visualized by immunoblotting and quantified by densitometry. (A) Representative immunoblots of FXIII-A from the pellet (1.5 × 105 platelets) or releasate (7.5 × 105 platelets). (B-C) Quantification of total FXIII-A in the pellet and releasate, respectively (mean ± SEM; n = 5 separate donors; same symbols in both panels; P values vs unstimulated platelets at the same time point are indicated). (D-F) Washed human platelets were unstimulated (Unstim) or stimulated with CVX plus IIa in the presence of cycloheximide (cyclo) or vehicle (Veh [ethanol]). Samples were processed as those in panels A-C. (D) Representative immunoblots of FXIII-A. For analysis of the pellet, proteins from ∼2 × 105 platelets were loaded. For analysis of the releasate, supernatant from 1 × 106 platelets were loaded. (E-F) Quantification of total FXIII-A in the pellet and releasate, respectively (mean ± SEM; n = 6 separate donors; same symbols in both panels; P = .05 vs convulxin plus thrombin in the absence of cycloheximide at the same time point; the slight increase in FXIII-A in the pellet in the absence of cycloheximide at 30 minutes is likely an artifact because this is too short of a time frame for significant protein synthesis). For both immunoblots, rhFXIII-A is recombinant human FXIII-A loading control, and the band in the MWM lane indicates 100 kDa. FXIII-A species are labeled on the right of the immunoblot as (O) (representing zymogen FXIII-A or nonproteolytically activated FXIII-A°) and ∗ (representing FXIII-A∗).

Platelet FXIII-A is not synthesized during activation of human platelets. (A-C) Washed human platelets were unstimulated (Unstim) or stimulated with thrombin (IIa), CVX plus IIa, or A23187. The platelet pellet (P) and releasate (R) were separated by centrifugation. FXIII-A was visualized by immunoblotting and quantified by densitometry. (A) Representative immunoblots of FXIII-A from the pellet (1.5 × 105 platelets) or releasate (7.5 × 105 platelets). (B-C) Quantification of total FXIII-A in the pellet and releasate, respectively (mean ± SEM; n = 5 separate donors; same symbols in both panels; P values vs unstimulated platelets at the same time point are indicated). (D-F) Washed human platelets were unstimulated (Unstim) or stimulated with CVX plus IIa in the presence of cycloheximide (cyclo) or vehicle (Veh [ethanol]). Samples were processed as those in panels A-C. (D) Representative immunoblots of FXIII-A. For analysis of the pellet, proteins from ∼2 × 105 platelets were loaded. For analysis of the releasate, supernatant from 1 × 106 platelets were loaded. (E-F) Quantification of total FXIII-A in the pellet and releasate, respectively (mean ± SEM; n = 6 separate donors; same symbols in both panels; P = .05 vs convulxin plus thrombin in the absence of cycloheximide at the same time point; the slight increase in FXIII-A in the pellet in the absence of cycloheximide at 30 minutes is likely an artifact because this is too short of a time frame for significant protein synthesis). For both immunoblots, rhFXIII-A is recombinant human FXIII-A loading control, and the band in the MWM lane indicates 100 kDa. FXIII-A species are labeled on the right of the immunoblot as (O) (representing zymogen FXIII-A or nonproteolytically activated FXIII-A°) and ∗ (representing FXIII-A∗).

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