FUSION OF PROTEOLYZED LOW-DENSITY-LIPOPROTEIN IN THE FLUID-PHASE - A NOVEL MECHANISM GENERATING ATHEROGENIC LIPOPROTEIN PARTICLES

During atherogenesis, lipid droplets appear in the extracellular space of the arterial intima. We previously observed generation of lipid dr oplets on the surface of exocytosed mast cell granules when granule ne utral proteases degraded the granule-bound LDL particles and the parti cles became unstable and fused [Kovanen, P. T., and Kokkonen, J. O. (1 991) J. Biol. Chem. 266, 4430-4436]. We have now extended our studies to the fluid phase and examined the effects of several proteases (tryp sin, cl-chymotrypsin, Pronase, plasmin, kallikrein, and thrombin) all known for their ability to cleave the apolipoprotein B-100 component ( apoB-100) of LDL. The fused LDL particles were separated from unfused particles by gel filtration or by density gradient ultracentrifugation . Proteolytic degradation of LDL with trypsin, alpha-chymotrypsin, or Pronase led to fragmentation of apoB-100 and release of the fragments from the LDL particles and triggered particle fusion. In contrast, pro teolytic degradation of LDL with plasmin, kallikrein, or thrombin, whi ch also led to fragmentation of apoB-100 but not to release of fragmen ts, did not trigger particle fusion. With advancing degradation of apo B-100, particles having progressively lower densities and larger sizes were generated. Thus, after incubation for 24 h with alpha-chymotryps in (apoB-100:alpha-chymotrypsin mass ratio 10:1) 40% of the apoB-100 w as degraded and about 30% of the LDL particles had fused and reached d iameters of up to 70 nm and densities ranging from 1.020 to < 1,005 g/ mL. When the proteolyzed LDL particles, both unfused and fused, were i ncubated with macrophages, only those particles that had undergone fus ion were ingested and converted into intracellular cholesteryl ester d roplets. Thus proteolysis of LDL with release of apoB-100 fragments re nders the particles sufficiently unstable to fuse and thus to become l iable to ingestion by macrophages. Since the fused LDL particles resem ble the extracellular lipid droplets in the atherosclerotic arterial i ntima and generate foam cells in vitro, these findings support the ide a that proteolytic fusion of LDL is an atherogenic process.