Dffierent Effects of Human Neutrophil Elastase on Platelet Glycoproteins IIb and IIIa of Resting and Stimulated Platelets^[*]

Summary

The effect of human neutrophil elastase (HNE) on the structure and receptor activity of platelet glycoprotein IIb/IIIa complex was studied. Resting platelets, which bound only traces of ¹²⁵I-fibrinogen in the absence of ADR were found to be barely susceptible to HNE. As shown by immunoblotting experiments, treatment of such platelets with HNE (14 μg/ml) did not provoke a detectable cleavage of GPIIb but resulted in a partial digestion of GPIIIa and appearance of 110 kDa fragment. Such proteolytic modification of the GPIIb/IIIa complex was accompanied by a slight increase in the binding of fibrinogen to blood platelets in the absence of ADP. Treatment of partially activated platelets (spontaneous activation during washing procedure) with HNE caused a progressive loss of GPIIb and degradation of GPIIIa to 110 kDa and 60 kDa fragments. These spontaneously stimulated platelets had initially a high number of fibrinogen binding sites exposed, corresponding to approximately 50% of receptor capacity observed in platelets activated by the optimal concentration of ADP. Digestion of GPIIb/IIIa by HNE of such platelets markedly increased the exposure of fibrinogen receptors. Thus, the stimulation of platelets increases significantly the susceptibility of the GPIIb/IIIa complex to proteolysis by HNE. However, such modification of the GPIIb/IIIa does not destroy its function as a receptor for fibrinogen either on the resting or activated platelets.

Dedicated to Professor M. Verstraete on the occasion of his 65th birthday.

• References

• 1 Brower MS, Levin RI, Garry K. Human neutrophil elastase modulates platelet function by limited proteolysis of membrane glycoproteins. J Clin Invest 1985; 75: 657-666
• 2 Bykowska K, Kaczanowska J, Karpowicz M, Lopaciuk S, Kopec M. Alterations of blood platelet function induced by neutral proteases from human leukocytes. Thromb Res 1985; 38: 535-546
• 3 Bykowska K, Kaczanowska J, Karpowicz M, Stachurska J, Kopec M. Effect of neutral proteases from blood leukocytes on human platelets. Thromb Haemostas 1983; 50: 768-772
• 4 Selak MA, Chignard M, Smith JB. Cathepsin G is a strong platelet agonist released by neutrophils. Biochem J 1988; 251: 293-299
• 5 Taki M, Miura T, Inagaki M, Tomita Y, Saito N, Okada Y, Tsuda Y, Meguro T, Yamada K. Influence of granulocyte elastase-like proteinase (ELP) on platelet functions. Thromb Res 1986; 41: 837-846
• 6 Wicki AN, Glemetson KJ. Structure and function of platelet membrane glycoproteins Ib and V. Effects of leukocyte elastase and other proteases on platelet response to von Willebrand factor and thrombin. Eur J Biochem 1985; 153: 1-11
• 7 Kornecki E, Ehrlich YH, Egbring R, Gramse M, Seitz R, Eckardt A, Lukasiewicz H, Niewiarowski S. Granulocyte-platelet interactions and platelet fibrinogen exposure. Am J Physiol 1988; 255: H651-H658
• 8 Kornecki E, Ehrlich YH, De Mars DD, Lenox RH. Exposure of fibrinogen receptors in human platelets by surface proteolysis with elastase. J Clin Invest 1986; 77: 750-756
• 9 Baugh RJ, Travis J. Human leukocyte granule elastase: rapid isolation and characterization. Biochemistry 1976; 15: 836-841
• 10 Kruze D, Wojtecka E. Activation of leukocyte collagenase proenzyme by rheumatoid synovial fluid. Biochim Biophys Acta 1972; 285: 436-446
• 11 Thorsen LI, Brosstad F, Gogstad G, Sletten K, Solum NO. Binding of 125I-labelled fibrin(ogen) fragments to platelets and to immuno-precipitated glycoprotein Ilb-IIIa complex. Thromb Res 1986; 42: 645-659
• 12 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685
• 13 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc Natl Acad Sri USA 1979; 76: 4350-4354
• 14 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275
• 15 Miller GL. Protein determination for large numbers of samples. Anal Chem 1959; 31: 964
• 16 Ciemiewski CS, Kowalska MA, Krajewski T, Janiak A. Binding of fibrinogen molecules to pig platelets and their membranes. Biochim Biophys Acta 1982; 714: 543-548
• 17 Scatchard G. The attraction of proteins for small molecules and ions. Ann NY Acad Sri 1949; 51: 660-662
• 18 Klotz IM. Numbers of receptor sites from Scatchard graphs: facts and fantasies. Science 1982; 217: 1247-1249
• 19 Sanibaldi AR, Lowe GD O, Barbenel JC, Forbes CD. Further studies on the role of red blood cells in spontaneous platelet aggregation. Thromb Res 1985; 38: 225-232
• 20 Breddin K, Ziemen M, Bauer O, Herrmann W, Schaudinn L, Schlosser U, Winterhagen A, Krzywanek HJ. Time and temperature dependent changes of ADP- and collagen-induced and “spontaneous” aggregation. Thromb Res 1980; 19: 621-638
• 21 Phillips DR, Charo IF, Parise LV, Fitzgerald LA. The platelet membrane glycoprotein IIb-IIIa complex. Blood 1988; 71: 831-843
• 22 Niiya K, Hodson E, Bader R, Byers-Ward V, Koziol JA, Plow EF, Rugger ZM. Increased surface expression of the membrane glycoprotein Ilb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation. Blood 1987; 70: 475-483
• 23 Niewiarowski S, Budzynski A, Morinelli TA, Brudzynski TM, Stewart GJ. Exposure of fibrinogen receptor on human platelets by proteolytic enzymes. J Biol Chem 1981; 256: 917-925
• 24 Hato T, Ikeda K, Yasukawa M, Watanabe A, Kobayashi Y. Exposure of platelet fibrinogen receptors by a monoclonal antibody to CD 9 antigen. Blood 1988; 72: 224-229
• 25 Woods VL, Wolff LE, Keller DM. Resting platelets contain a substantial centrally located pool of glycoprotein IIb-IIIa complex which may be accessible to some but not other extracellular proteins. J Biol Chem 1986; 261: 15242-15251
• 26 Shulman S, Wiezner R, Troll W, Karpatkin S. Reevaluation of the presence of the major antigen Ca++ complex in Bemard-Soulier syndrome platelets. Thromb Res 1983; 30: 61-69
• 27 Coller BS. A new murine monoclonal antibody reports an activation-dependent change in the conformation and/or microenvironment of the platelet glycoprotein IIb/IIIa complex. J Clin Invest 1985; 76: 101-108
• 28 Coller BS. Activation affects access to the platelet receptor for adhesive glycoproteins. J Cell Biol 1986; 103: 451-456
• 29 Peerschke EI B, Coller BS. A murine monoclonal antibody that blocks fibrinogen binding to normal platelets also inhibits fibrinogen interactions with chymotrypsin-treated platelets. Blood 1984; 64: 59-63
• 30 Komecki E, Tuszynski GP, Niewiarowski S. Inhibition of fibrinogen receptor-mediated platelet aggregation by heterologous anti-human platelet membrane antibody. Significance of an Mr = 66,000 protein derived from glycoprotein Ilia. J Biol Chem 1983; 258: 9349-9356
• 31 Niewiarowski S, Norton KJ, Eckardt A, Lukasiewicz H, Holt JC, Komecki E. Structural and functional characterization of major platelet membrane components derived by limited proteolysis of glycoprotein Ilia. Biochim Biophys Acta 1989; 983: 91-99
• 32 McGregor JL, Clezardin P, James E, Mcgregor L, Dechavanne M, Clemetson KJ. Identification and characterization of fragments of major glycoproteins from platelet membrane after chymotrypsin treatment. Eur J Biochem 1985; 148: 97-106