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. 2018 May 8;2(9):1040–1045. doi: 10.1182/bloodadvances.2017014001

Figure 1.

Figure 1.

Pfn1 deficiency impairs platelet integrin function. Activation of platelet αIIbβ3- (JON/A-phycoerythrin [PE]) (A) and β1-integrins (9EG7-FITC) (B), as well as α-granule release (anti-P-selectin-FITC) (C) in response to different agonists were assessed by flow cytometry after 15 minutes. FITC, fluorescein isothiocyanate. Antibodies were present throughout the stimulation to assess maximal integrin activation at the respective time points. Values are mean ± standard deviation (SD; n = 6 vs 6). Impaired platelet αIIbβ3- and α2β1-integrin function was further revealed by flow cytometry assessing platelet fibrinogen binding (samples were stimulated for 10 minutes and Mn2+ served as activation-independent positive control) (D) and aggregation responses to different agonists (E). Values are mean ± SD (n = 6 vs 6). Aggregation traces are representative of at least 6 animals per group. Assessment of platelet (F) adhesion and aggregate formation under flow (1000/s) on collagen I (70 μg/mL) (G) of WT and platelet count-adjusted Pfn1fl/fl-Pf4Cre samples. Values are mean ± SD (n = 12 vs 12). (H) Platelet clot retraction was determined over time in response to 5 U/mL thrombin and residual serum volume was quantified after 180 minutes at the end of the observation period. Values are mean ± SD (n = 6 vs 6). In vivo, Pfn1 deficiency resulted in impaired hemostasis as assessed by a tail bleeding time assay (n = 9 vs 9) (I) and a protection from arterial thrombosis upon induction of a mechanical injury in the abdominal aorta (n = 7 vs 6) (J). Basal [Ca2+]i (n = 17 vs 19) (K) and maximal increase of [Ca2+]i after stimulation with thrombin (Thr), collagen-related peptide (CRP), or the sarco/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA) inhibitor thapsigargin (TG) of at least 9 vs 11 animals (L). Experiments were performed in the presence of 1 mM extracellular Ca2+. Each symbol in panels I and J represents 1 animal. Horizontal lines in panel I represent mean. ADP, adenosine diphosphate; CVX, convulxin; Mn2+, manganese; Rest, resting; Rhd, rhodocytin; U46, stable thromboxane A2 analog U46619. Unpaired Student t test (A-D, F-I, and K-L) and Fisher’s exact test (J) were used to assess statistical differences between the groups: ***P < .001; **P < .01; *P < .05. NS, nonsignificant.