Études sur une protéase élastinolytique des plaquettes sanguines humaines

Abstract

Elastinolytic protease in human blood platelets Experiments are described devised in order to study the “liberation” of elastinolytic protease(s) from human blood platelet suspensions as well as the separation of these proteases from the acid-cathepsins, their partial purification and characterization. ADP, adrenaline, and collagen produced a significant activation of the elastinolytic activity of the platelet suspension as well as a partial “liberation” of this activity in the supernatant. Triton-X-100 and sonication produced a strong activation and a near 100% liberation of the elastinolytic activity. This suggested the existence of two different and independent mechanisms: “activation” and “liberation”. Triton-X-100 stabilised the elastinolytic activity sufficiently to render possible its partial purification. Ammonium sulfate at 40% saturation precipitated most of the acid-catheptic activity. This activity could be further purified by gel-filtration on Sephadex G 200. The partially purified acid-cathepsin fraction was not activated by mercaptoethanol and migrated with the mobility of γ-globulins. Its behaviour reminds that of the cathepsin C isolated from carrageenine granuloma extracts 8,9. About half of the elastinolytic activity remained in the 60% ammonium sulfate supernatant and was not further purified. Part of it (approx. 8%) precipitated at 60% ammonium sulfate and was further separated on Sephadex G-200 in two peaks; the first, excluded peak (approx. 90% of total proteins charged) having a specific activity of 21,400 counts/min/mg protein; the second, penetrating the column (approx. 5% of total protein, ⋍ 1.2 mg) having a specific activity of 144,000 counts/min/mg protein. The first fraction was still heterogenous, with a major band migrating with the albumins. Its pH optimum is near 8.0, it is strongly inhibited by Ca 2+ which precipitates it. The other agents of platelet aggregation (ADP, adrenaline, EDTA, 5-hydroxytryptamine) had only a weak activating or inhibiting action. These experiments confirm the presence of several proteolytic enzymes in human blood platelet preparations and show that the elastolytic protease is distinct from the major acid-catheptic enzymes. The elastolytic enzymes may be present in several protein fractions or they may have several forms differing in molecular weight. Further experiments should clarify this point.