Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Phosphatidylserine-blocking nanoparticles inhibit thrombosis without increased bleeding in mice

doi: 10.1016/j.jtha.2024.10.007

Figure Lengend Snippet: ( A ) Whole blood cells from freshly drawn citrated human blood were precipitated and washed to deplete fibrinogen, and DPAL was added to cell suspensions at 6 x 10 11 particles/mL, followed by stimulation with thrombin (Thr) and/or convulxin (CVX) in the presence of 2 mM CaCl 2 as indicated. Platelets were identified in a flow cytometer by forward and side scatter, and counterstaining with AlexaFluor ® 488- or AlexaFluor ® 647-conjugated anti-CD41 antibodies. (i) Liposome binding was assessed by Cy3 fluorescencein CD41+ platelets, and are shown as percent Cy3-positive (DPAL+) platelets ± S.E.M. (ii) PS exposure was assessed with Annexin V-FITC (AXV) in separate tubes containing aliquots from the treated samples from (i), shown as mean fluorescence intensities (MFI) ± S.E.M. p < 0.01 for all samples compared to unstimulated. ( B ) (i) Platelets as in (A) were stimulated with thrombin with or without 10 μL AXV added as indicated, and DPAL binding is shown as fold change from unstimulated ± S.E.M. (ii) AXV MFI from the samples in (i) are shown ± S.E.M. ( C ) Washed platelets from WT or Ano6 KO mice were suspended in Tyrode’s buffer in the presence of DPAL at 6 x 10 11 particles/mL, stimulated with 0.1 U/mL thrombin as indicated, and assessed for (i) liposome binding as in (B), using FITC-conjugated anti-Cd41 antibodies to identify murine platelets, or (ii) AXV MFI, shown ± S.E.M. ( D ) Human platelets treated as in (A) were counterstained with FITC-conjugated PAC-1; FITC-PAC-1 MFI are shown ± S.E.M. n.s., no significant differences between groups. (E) Aggregation of human platelets suspended in buffer at 2x10 8 platelets/mL, following stimulation with thrombin at the indicated concentrations was measured in the absence (control, CTL) or presence of DPAL added as in (D). Representative of 4 independent experiments. ( F ) Dilute prothrombin time in pooled normal plasma with added rabbit brain tissue thromboplastin, in the presence or absence of DPAL as indicated, and shown as mean ± S.E.M.

Article Snippet: Rat-anti-mouse Cd41 FITC-conjugated antibodies and dry chemicals were from Fisher Scientific (Pittsburgh, PA, USA).

Techniques: Binding Assay, Flow Cytometry, Fluorescence, Control, Clinical Proteomics