Platelets are key coordinators of hemostatic and immune functions, rapidly responding to vascular perturbations. However, the ultrastructural signatures of platelet priming, including open canalicular system (OCS) remodeling and α-granule (αG) changes, remain poorly characterized. BNT162b2 vaccination induces a well-defined, self-limiting immune activation in healthy individuals, providing a controlled human model to investigate platelets and vascular responses. Using this model, we longitudinally investigated platelet ultrastructure, assessing alterations in OCS architecture and αG content and their association with circulating inflammatory and endothelial activation biomarkers. Healthy adults were evaluated at six time points across two BNT162b2 vaccine doses. Platelets were analyzed by TEM to quantify αG number and OCS area in a participant subcohort. Circulating levels of IL-6, CD40L, P-selectin and plasminogen activator inhibitor-1 (PAI-1) were measured by ELISA in the full cohort. Human umbilical vein endothelial cells (HUVECs) were stimulated with post-vaccination plasma to assess endothelial responses. Vaccination induced transient OCS expansion and αG depletion, accompanied by transient systemic immune activation, as reflected by changes in circulating IL-6, CD40L, and PAI-1, whereas P-selectin showed no sustained increase, consistent with a primed/intermediate activation state. Post-vaccination plasma induced dynamic changes in HUVECs IL-1Rs, MIP-3α, and IL-22 secretion, adhesion molecules and chemokine transcription, and 14–3–3γ, P2Y12, PAI-1, and ADAMTS13 signaling, consistent with transient endothelial activation. BNT162b2 vaccination thus elicits coordinated, non-pathological platelet remodeling alongside transient systemic and endothelial responses. OCS and αG may represent sensitive ultrastructural indicators of transient platelet activation, defining a novel morpho-functional signature of vaccine-induced immune activation at the vascular interface.