i[VREY]₄ blocks CXCL12-induced phosphorylation of Btk (pBTK) CXCR4 dependently without affecting CXCR4 internalization. (A) pBTK in human platelets was analyzed by using flow cytometry. Platelets were treated with CXCL12 (1 µg/mL) and as indicated with i[VREY]₄ (n = 3). (B) Changes in CXCR4 expression on human platelets was analyzed by flow cytometry after treatment with recombinant CXCL12 (0.1 µg/mL), collagen (1 µg/mL), and i[VREY]₄ (5 µM). (C-F) CXCL12 on human platelets was detected by flow cytometry. Human blood was treated with CXCL12 (0.1 µg/mL) (C,E) or collagen (1 µg/mL) (D,F), and detection was conducted with directly conjugated monoclonal antibodies (panels C and D, clone K15C, n = 10; panels E and F, clone 79018, n = 8). Binding of i[VREY]₄-biot to platelets from tamoxifen-injected CreErt^wt/wt Cxcr4^flox/flox (wild type [WT]) or CreErt^tg/wt Cxcr4^flox/flox (CXCR4 knockout [KO]) mice was measured by flow cytometry under resting conditions or stimulated with 10 µg/mL collagen (G) (n = 3-5). Data represent mean ± standard deviation from the indicated numbers of independent experiments or mice. *P ≤ .05, **P ≤ .01, as analyzed by one-way analysis of variance with Tukey’s multiple comparison test (panels A-F) or unpaired t test (panel G). MFI, mean fluorescence intensity; ns, not significant.