Platelet accretion at sites of vascular injury yields a neo-tissue comprising packed platelets and having an interstitial space not supplied with blood. Within growing thrombi platelet masses become anoxic and depolarize to yield interstitial cation concentrations characteristic of the more voluminous platelet cytosol, with extracellular [Ca2+] falling below that adequate to support the plasma clotting system. The platelet-associated clotting system reactivates during disaggregation of the thrombi in vitro, which proceeds with high yield of apparently basal, functional platelets when specific anticoagulants are included in the disaggregating media. The capacity of regulatory demand to lower extracellular [Ca2+] in the microenvironment of platelet aggregates provides a physiological basis for evolution of the highly cooperative calcium interactions of the hemostasis system.