![Most platelet FXIII-A is retained with the platelet after dual agonist stimulation. Washed human platelets were unstimulated (Unstim) or stimulated with convulxin (CVX) plus thrombin (IIa) for 30 minutes. The platelet pellet (P) and releasate (R) were separated by centrifugation. FXIII-A, TFPI, and von Willebrand factor (VWF) were visualized by immunoblotting and quantified by densitometry. For analysis of pellets, proteins from 1 × 105 and 2 × 105 platelets were loaded in the first and second lanes of each pairing, respectively. For analysis of releasates, supernatants from 0.5 × 106 and 1 × 106 platelets were loaded for the first and second lanes of each pairing, respectively. (A) Representative immunoblot for FXIII-A. Recombinant human FXIII-A loading control is labeled rhFXIII-A. 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∗). (B) Quantification of FXIII-A. (C) Representative immunoblot of VWF; each lane represents a separate donor. (D) Quantitation of VWF. (E) Representative immunoblot of TFPI (recombinant human TFPI [rhTFPI] loading control). (F) Quantification of TFPI. For all immunoblots, molecular weight marker (MWM) is indicated on the left. For the bar graphs, the data show mean ± standard error of the mean (SEM); each dot represents a separate donor.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/8/19/10.1182_bloodadvances.2024012979/2/blooda_adv-2024-012979-gr1.jpeg?Expires=1735843151&Signature=WwX7AhW7mR7MdS1eNYnJ7bxnnz~xPsxoNrG9fBfZDcuOS3le2ZUfnT3mn7nFaBF4AkPqzyV2p4QOtysK9Dsgo4CFnj71j1ZY2JNNEw-v4HZl8Io~L5MOqeWOad7WYbvIqyLlcCG2xwITFWytoH4MW-cS8UQ1PA9uSp~phSTU1Iyz9Gv~kiKhT6vyxlttUD23Iw9gKbFzMbBcj9mvgeA6K-BO-2P5~vIqH6hLynq-fqvXdNcQwjJiAk5OJCszIZYvP1O5U~SPiuNxEZxWicrRCLuwfbe1NBr5COf6TFA3tCWKktaO~u0SwYoqDX~MEE8oPfQobHjUC688cZoY71GBzQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Most platelet FXIII-A is retained with the platelet after dual agonist stimulation. Washed human platelets were unstimulated (Unstim) or stimulated with convulxin (CVX) plus thrombin (IIa) for 30 minutes. The platelet pellet (P) and releasate (R) were separated by centrifugation. FXIII-A, TFPI, and von Willebrand factor (VWF) were visualized by immunoblotting and quantified by densitometry. For analysis of pellets, proteins from 1 × 105 and 2 × 105 platelets were loaded in the first and second lanes of each pairing, respectively. For analysis of releasates, supernatants from 0.5 × 106 and 1 × 106 platelets were loaded for the first and second lanes of each pairing, respectively. (A) Representative immunoblot for FXIII-A. Recombinant human FXIII-A loading control is labeled rhFXIII-A. 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∗). (B) Quantification of FXIII-A. (C) Representative immunoblot of VWF; each lane represents a separate donor. (D) Quantitation of VWF. (E) Representative immunoblot of TFPI (recombinant human TFPI [rhTFPI] loading control). (F) Quantification of TFPI. For all immunoblots, molecular weight marker (MWM) is indicated on the left. For the bar graphs, the data show mean ± standard error of the mean (SEM); each dot represents a separate donor.
