Signaling from collagen and G protein–coupled receptors leads to platelet adhesion and subsequent thrombus formation. Paracrine agonists such as ADP, thromboxane, and Gas6 are required for platelet aggregate formation. We hypothesized that thrombi are intrinsically unstable structures and that their stabilization requires persistent paracrine activity and continuous signaling, maintaining integrin α_IIbβ₃ activation. Here, we studied the disassembly of human and murine thrombi formed on collagen under high shear conditions. Platelet aggregates rapidly disintegrated (1) in the absence of fibrinogen-containing plasma; (2) by blocking or inhibiting α_IIbβ₃; (3) by blocking P2Y₁₂ receptors; (4) by suppression of phosphoinositide 3-kinase (PI3K) β. In murine blood, absence of PI3Kγ led to formation of unstable thrombi, leading to dissociation of multiplatelet aggregates. In addition, blocking PI3Kβ delayed initial thrombus formation and reduced individual platelet-platelet contact. Similarly without flow, agonist-induced aggregation was reversed by late suppression of P2Y₁₂ or PI3K isoforms, resulting in single platelets that had inactivated α_IIbβ₃ and no longer bound fibrinogen. Together, the data indicate that continuous outside-in signaling via P2Y₁₂ and both PI3Kβ and PI3Kγ isoforms is required for perpetuated α_IIbβ₃ activation and maintenance of a platelet aggregate. This novel concept of intrinsic, dynamic thrombus instability gives possibilities for the use of antiplatelet therapy.