Effect of Neutral Proteases from Blood Leukocytes on Human Platelets

Summary

Two highly purified neutral proteases from human leukocytes i.e. elastase-like protease (ELP) and chymotrypsin-like protease (CLP) do not destroy human platelets since no difference was found in ⁵¹Cr liberation from control and enzyme-treated platelets. As with pancreatic chymotrypsin (α-CT) ELP does not induce the release of ³H-serotonin while CLP provokes ³H- serotonin secretion, in an enzyme concentration and time dependent fashion. The rate and degree of ³H-serotonin release by CLP is similar to that produced by thrombin. Incubation of platelets at 37° C for 30 min with α-CT or ELP renders them resistant to thrombin-releasing activity. Thrombin did not liberate any additional label from platelets which lost over 60% of serotonin during the preceding incubation with CLP. α-CT and ELP do not aggregate platelets either in the presence or absence of apyrase. CLP does aggregate platelets suspended in Tyrode buffer without apyrase but not in the presence of apyrase (100 mg/1). The action of α-CT, ELP and CLP on washed platelets induces a progressive prolongation of lag phase and a decrease in changes of light transmission during aggregation by thrombin. Similarly to α-CT-treated platelets, those subjected to CLP action aggregate in the presence of human fibrinogen.

It is concluded that: (1) neutral proteases possibly contribute to development of defects in platelet function in pathological states associated with liberation of leukocyte content into the circulation, (2) CLP similarly to a-CT, exposes fibrinogen receptors but in contrast to α-CT, CLP aggregates platelets and stimulates serotonin secretion.

Keywords

Neutral leukocytic proteases - Human blood platelets

• References

• 1 Rulot H. Intervention des leukocytes dans l’autolyse de la fibrine (Fibrinolyse de Dastre). Arch Int Physiol 1904; 1: 152
• 2 Opie EL. Experimental pleurisy. Resolution of a fibrinous exudate J. Exp Med 1907; 9: 391
• 3 Haveman K, Janoff A. Neutral proteases of human polymorpho-nuclear leukocytes. Urban & Schwarzenberg; Baltimore - Munich: 1978
• 4 Egbring R, Schmidt W, Fuchs G, Haveman K. Demonstration of granulocytic proteases in plasma of patients with acute leukemia and septicemia with coagulation defects. Blood 1977; 49: 219-231
• 5 Kopeć M, Bykowska K, Lopaciuk S, Jeleńska M, Kaczanowska J, Sopata I, Wojtecka E. Effects of neutral proteases from human leukocytes on structure and biological properties of human factor VIII. Thromb Haemostas 1980; 43: 211-217
• 6 Varadi K, Marossy K, Asboth G, Elödi P, Elödi S. Inactivation of human factor VIII by granulocyte proteases. Thromb Haemostas 1980; 43: 45-48
• 7 Henriksson P, Nilsson IM, Ohlson K, Stenberg P. Granulocyte elastase activation and degradation of factor XIII. Thromb Res 1980; 18: 343-351
• 8 Kopeć M, Kaczanowska J, Stachurska J, Tomczak Z, Wojtecka-Lukasik E, Bykowska K, Lopaciuk S. Effects of granulocytic neutral proteases on some clotting factors. In: Progress in Fibrinolysis and Thrombolysis. Davidson JF, Nilsson IM, Astaed B. (eds) Churchill Livingston; Edinburgh, London, Melbourne, New York: 1981. 5 75-77
• 9 Kopéc M, Wȩgrzynowicz Z, Ardelt W, Teisseyre E, Latallo ZS. Effect of elastolytic enzymes from human leukocyte granules and from pig pancreas on plasminogen fibrinogen and fibrin. In: Progress in Chemical Fibrinolysis and Thrombolysis. Davidson JF, Rowan RM, Samama MM, Desnoyers CP. (eds) Raven Press; New York: 1978. 3 417-425
• 10 Gramse M, Bingenheimer C, Schmidt W, Egbring R, Haveman K. Degradation products of fibrinogen by elastase-like neutral protease from human granulocytes. J Clin Invest 1978; 61: 1027-1033
• 11 McDonald JA, Kelley DG. Degradation of fibronectin by human leukocytic elastase. J Biol Chem 1980; 255: 8848-8858
• 12 Vartio C. Characterization of the binding domains in the fragments cleaved by cathepsin G from human plasma fibronectin. Eur J Biochem 1982; 133: 223-233
• 13 Brower MS, Harpel PC. Proteolytic cleavage and inactivation of α₂-plasmin inhibitor and CT-inactivator by human polymorphonuclear elastase. J Biol Chem 1982; 257: 9849-9854
• 14 Klingeman HC, Egbring R, Holst F, Gramsa M, Haveman K. Digestion of α₂-plasmin inhibitor by neutral proteases from human leukocytes. Thromb Res 1981; 24: 479-483
• 15 Hummel BC W. A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin. Can J Biochem Physiol 1959; 37: 1393-1399
• 16 Visser L, Blout ER. The use of p-nitrophenyl N-tert-butylocarbonyl-L-alanine as a substrate for elastase. Biochim Biophys Acta 1972; 268: 257-260
• 17 Mustard JF, Perry DW, Ardlie NG, Packham MA. Preparation of suspensions of washed platelets from humans. Br J Haematol 1972; 22: 193-204
• 18 Nygaard K. A direct method of counting platelets in oxalated plasma. Proc Mayo Clin 1933; 8: 365
• 19 Podolak-Dawidziak M, Konopka L, Pawelski S. Uwalnianie³H-hydroksytryptaminy z plytek pod wplywem surowicy chorych z maloplytkowosciami. Pol Arch Med Wew 1982; 68: 43
• 20 Greenberg JP, Packham MA, Gucciona MA, Harfenist EJ, Orr JL, Kinlough-Rathbone RL, Perry DW, Mustard JF. The effect of pretreatment of human platelets with chymotrypsin on their responses to human fibrinogen and aggregation agents. Blood 1979; 54: 753-765
• 21 Niewiarowski S, Budzyński AZ, Morinelli TA, Brudzynńki TM, Stewart GJ. Exposure of fibrinogen receptor on human platelets by proteolytic enzymes. J Biol Chem 1981; 256: 917-925
• 22 Davey MG, Lüscher EF. Actions of thrombin and other coagulant and proteolytic enzymes on blood platelets. Nature 1967; 216: 857-858
• 23 Martin BM, Reinman RD, Detwiler TC. Platelet stimulation by thrombin and other proteases. Biochemistry 1975; 14: 1308-1314
• 24 Podolsak B. Effect of thrombin, chymotrypsin and aggregated gamma-globulins on the proteins of the human platelet membrane. Thromb Haemostas 1977; 37: 396-406
• 25 Okamura T, Jamieson GA. Platelet glycocalicin. Orientation of glycoproteins of the human platelet surface J Biol Chem 1976; 251: 5944-5949
• 26 Starkey PM. Elastase and cathepsin G; the serine proteinases of human neutrophil leukocytes and spleen. In: Proteinases in mamma-lian cells and tissues. Barret AJ. (eds) North-Holland Publishing Company; Amsterdam, New York, Oxford: 1977: 57-89
• 27 Plow EF. Leukocyte elastase release during blood coagulation. A potential mechanism for activation of the alternative fibrinolytic pathway J Clin Invest 1982; 69: 564-572
• 28 Cardamore JM, Edson JR, McArthur JR, Jacob HS. Abnormalities of platelet function in myeloproliferative disorders. JAMA 1972; 221: 270-273
• 29 Gerrard JM, Stoddard SR, Shapiro RS, Coccia PR, Ramsay NK C, Nesbit ME, Rao GH R, Krivit W, White JG. Platelet storage pool deficiency and prostaglandin synthesis in chronic granulocytic leukemia. Br J Haematol 1978; 40: 597-609
• 30 Castaldi PA, Bemdt MC, Booth W, Gregony C, Bull H, Greakes M. Evidence for a platelet membrane defect in the myeloproliferative disorders. Thromb Res 1982; 27: 601-609
• 31 Pareti FI, Gugliotta L, Mannucci L, Guardi A, Mannucci PM. Biochemical and metabolic aspects of platelet dysfunction in chronic myeloproliferative disorders. Thromb Haemostas 1982; 47: 84-89
• 32 Ireland H, Lane DA, Wolff S, Foadi M. In vivo platelet release in myèloproliferative disorders. Thromb Haemostas 1982; 48: 41-45
• 33 Harper JH, Wohl H, Harper E. Platelet factor 4 an inhibitor of collagenase. Science 1978; 199: 991-992
• 34 Lonky AS A, Marsh J, Wohl H. Stimulation of human granulocyte elastase by platelet factor 4 and heparin. Biochem Biophys Res Commun 1978; 85: 1113-1118