Type 1 plasminogen activator inhibitor (PAI-1) is known to be synthesized in an active conformation but it is rapidly converted into an inactive conformation (t 1 h) upon incubation at 37 °C. This study was initiated to investigate the mechanism that account for the presence of active PAI-1 in anucleated platelets that have a mean life span of 9-12 days in the circulation. Stabilization experiments with a functional immunoassay indicated that the activity of PAI-1 in both platelets and in isolated -granules was prolonged in comparison to the rapid inactivation of this molecule in their lysates (t 1 h). Although combined ligand blot/immunoblot analysis revealed that vitronectin was the major PAI-1 binding protein in platelets, vitronectin/PAI-1 complexes were not detected in -granules using a two-site immunoassay. Co-incubation of -granules with a number of agents that disrupt pH gradients (e.g. ionophores) had no effect on the stability of PAI-1 activity, whereas incubation of -granules with the calcium ionophore A23187 reduced the half-life of PAI-1 to the levels observed for PAI-1 in solution. Addition of calcium ions to intact -granules was an effective means of neutralizing the ionophore's effect on PAI-1 activity. Fractionation of -granule proteins on molecular sieving columns using conditions known to be present within storage granules (e.g. a high calcium concentration) revealed the presence of PAI-1 in fractions with a molecular mass of >10 daltons. Immunoabsorption of PAI-1 from these column fractions followed by negative staining revealed 25-nm diameter complexes of -granule proteins under the electron microscope. PAI-1 activity associated with these complexes was prolonged in the presence of calcium ions and these high M complexes were shown to be composed of a defined set of proteins that can be dissociated from PAI-1 by chelation of calcium ions. These data indicate that PAI-1 is stabilized by its packaging with other -granule proteins in a calcium-dependent manner.

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