Limited TALIN1-mediated integrin activation in platelets lacking both RAP1 isoforms is not mediated by RAP2 GTPase. (A) Flow cytometric analysis of αIIbβ3 integrin activation (JON/A-PE antibody binding) in Rap1a/b-mKO (Rap1a^fl/flRap1b^fl/flPf4-Cre⁺) and Talin1-mKO (Talin1^fl/flPf4-Cre⁺) platelets activated with high dose thrombin (HD Thr) or high dose convulxin (HD Cvx) (n = 5). (B) Aggregation response of Rap1a/b-mKO and Talin1-mKO platelets (representative of 4 independent experiments). For high-dose Par4p and convulxin (Cvx) aggregation, the residual aggregation response of Rap1a/b-mKO platelets was completely abolished by preincubation with 30 μg/mL of the αIIbβ3 blocking antibody Leo.H4. (C) Rap2-GTP pull-down assay. Washed platelets stimulated (S) in standard aggregometry with 0, 0.4 μg/mL, or 0.8 μg/mL the GPVI-agonist Cvx were lysed (L) after 10 minutes to measure the levels of active RAP2 by pull-down assay (n = 3). Total RAP2 was determined as loading control. (D) Platelet spreading on fibrinogen. Platelets were incubated in fibrinogen-coated wells and activated with ADP (100 µM), and the extent of platelet spreading was determined after 45 minutes. Representative images of phalloidin-stained platelets are shown (n = 3-5). Scale bars represent 10 μm. (E) Clot-contraction assay. Washed platelets from the indicated knockout mice were added to human platelet-poor plasma in ACD containing 5 mM Ca²⁺ and 0.2 U/mL thrombin in siliconized cuvettes (n = 3). Representative images of clots at time 0 and 120 minutes are shown (upper panel). The “no platelets” sample contained all components with the exception of washed platelets. *P < .05, **P < .01, ***P < .001. ns, not significant.