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. 2024 Aug 10;8(19):5072–5085. doi: 10.1182/bloodadvances.2024012979

Figure 4.

Figure 4.

Released FXIII-A is mainly free protein. Washed human platelets were unstimulated (Unstim) or stimulated with thrombin (IIa), CVX plus thrombin (IIa), or A23187 for 30 minutes at 37°C. The reaction mixture was then quenched and centrifuged serially to separate the pellet and releasate and then large EVs, small EVs, and remaining soluble fraction (free proteins), as described in the “Methods” and illustrated in supplemental Figure 5. (A-D) Nanoparticle tracking analysis of size distribution and enumeration of large EVs (A-B) and small EVs (C-D). (E) Representative immunoblots of FXIII-A in large EVs (pellet of 20 000g, 15 minutes), small EVs (pellet of 100 000g, 70 minutes), and soluble fraction (supernatant of 100 000g, 70 minutes) from 4 of the donors (D) studied; rhFXIII-A is recombinant human FXIII-A loading control; 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∗). (F) Quantification of FXIII-A in large EVs (LEVs), small EVs (SEVs), and soluble proteins (Sol). The data show mean ± SEM of 5 separate donors; each dot represents a separate donor.