Enhancement of Tissue Factor Dependent Procoagulant Activity and Factor Xa Generation by Alpha²-Macroglobulin.

Alpha₂-macroglobulin (α₂M) is a broad-spectrum proteinase inhibitor (M~718,000) which is unique in its ability to inhibit proteinases from all four mechanistic classes. α₂M inhibits coagulation proteinases, such as thrombin and factor Xa, and fibrinolytic proteinases, such as plasmin. α₂M is also a major carrier of growth factors and may regulate growth factor activity. The primary receptors for α₂M are the low density lipoprotein receptor related protein (LRP, K_D~5 nM) and a higher affinity receptor recently identified as the 78 kDa glucose regulated protein (Grp78, K_D~50 pM). In its native conformation α₂M is present in plasma at very high concentrations (up to 5 μM). Upon binding to serum proteinases or small primary amines α₂M is converted to the receptor-recognized form (α₂M*) and then binds to LRP. LRP-associated α₂M-proteinase complexes are rapidly internalized and degraded by phagocytic cells such as macrophages. Previous work in our laboratory has demonstrated that Grp78 binds to tissue factor (TF) and inhibits its functional activity. We now demonstrate that α₂M* enhances TF-dependent procoagulant activity and factor Xa generation and this function is dependent on α₂M* binding to LRP, but not Grp78, since receptor-associated protein (RAP) blocks the response. The effects of α₂M on TF-dependent procoagulant activity are most pronounced in RAW 264.7 macrophage-like cells, but also observed in THP-1 monocytic cells. We are performing additional studies, using both α₂M mutants and LRP-deficient cells, to further elucidate this novel mechanism by which α₂M* may regulate hemostasis.