CALCIUM-DEPENDENT STABILIZATION OF TYPE-I PLASMINOGEN-ACTIVATOR INHIBITOR WITHIN PLATELET ALPHA-GRANULES

Type 1 plasminogen activator inhibitor (PAI-1) is known to be synthesi zed in an active conformation but it is rapidly converted into an inac tive conformation (t(1/2) 1 h) upon incubation at 37 degrees C. This s tudy was initiated to investigate the mechanism that account for the p resence 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 p latelets and in isolated alpha-granules was prolonged in comparison to the rapid inactivation of this molecule in their lysates (t(1/2) 1 h) . Although combined ligand blot/immunoblot analysis revealed that vitr onectin was the major PAI-1 binding protein in platelets, vitronectin/ PAI-1 complexes were not detected in alpha-granules using a two-site i mmunoassay. Co-incubation of alpha-granules with a number of agents th at disrupt pH gradients (e.g. ionophores) had no effect on the stabili ty of PAI-1 activity, whereas incubation of alpha-granules with the ca lcium ionophore A23187 reduced the half-life of PAI-1 to the levels ob served for PAI-1 in solution. Addition of calcium ions to intact alpha -granules was an effective means of neutralizing the ionophore's effec t on PAI-1 activity. Fractionation of alpha-granule proteins on molecu lar sieving columns using conditions known to be present within storag e granules (e.g. a high calcium concentration) revealed the presence o f PAI-1 in fractions with a molecular mass of > 10(6) daltons. Immunoa bsorption of PAI-1 from these column fractions followed by negative st aining revealed 25-nm diameter complexes of alpha-granule proteins und er the electron microscope. PAI-1 activity associated with these compl exes was prolonged in the presence of calcium ions and these high M(r) 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 dat a indicate that PAI-1 is stabilized by its packaging with other alpha- granule proteins in a calcium-dependent manner.