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Intracellular Ca2+ rise in human platelets by polymorphonuclear (PMN) leukocyte-derived cathepsin G
S86
ABSTRACTS OF 12TH INTNAT’L CONGRESS
Vol. 65, Suppl. 1
Cl 67 INTRACELLULAR Cal* RISE IN HUMAN PLATELETS BY POLYMORPHONUCLEAR (PMN) LEUKOCYTE-DERIVED CATHEPSIN G M. Molino, M. Di Lallo, G. de Gaetano and C. Cerletti lstituto di Ricerche Farmacol. Mario Negri, Consorzio Negri Sud, S. Maria lmbaro (CH), 1 Cathepsin G. a serine protease released by PMN azurophibc granules upon stimulation, activates piatelets, increasing intraplatelet Ca2+ levels ([Ca2+],) in a concentration- dependent manner (50-200 nM). The [Cal+], rises elicited by low (50-80 nM) cathepsin G concentrations in fura-2-loaded platelets showed a biphasic mode, with a first small peak followed by a greater and more prolonged Ca*+ mobilization. Higher (100-200 r&l) cathepsin G concentrations induced monophasic [Cal+], increase. Neither aspirin, or nordihydroguaiaretic acid, nor ketanserin affected platelet activation by cathepsin G, while the ADP scavenger system CP/CPK significantly reduced Cal+ mobilization by 100 nM cathepsin G. Niz+ (4 mM), a divalent cation channel inhibitor, reduced cathepsin G-induced fluorescence rise by more than 90%. This effect was reversed by either reducing Nil’ or increasing cathepsin G concentration. Four mM EGTA totally abolished Cal+ transients. Evidence of a rapid and sustained divalent cation channel opening in the platelet membrane was obtained by adding Mn2+ to the platelet suspension 30 set or 3 min after cathepsin G. No accumulation of InsP, could be detected when platelets were stimulated with cathepsia G. Preincubation of platelets with staurosporine, an inhibitor of protein kinase C, enhances both [Cal+], rises and quenching by Mn” of fluorescence signal induced by cathepsin G. All these data indicate that cathepsm G induces [Ca”], increase mainly through an influx across the plasma membrane and that Cal+ channels may be under the control of protein kinase C.
Cl68 TRANCELLULAR METABOLISM OF ARACHIDONIC ACID (AA): INCREASED PLATELET THROMBOXANE (TX) GENERATION IN THE PRESENCE OF ACTIVATED POLYMORPHONUCLEAR LEUKOCYTES (PMN) N. Maugeri, V. Evangelista, P Piccardoni, A. Celardo, G. Dell’Elba, G. de Gaetano and C. Cerletti lstituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, Santa Maria lmbaro (CH), 1 Human PMN activated by n-formyl-methionyl-leucyl-phenylalanine (fMLP), in the presence of cytochalasin B, are able to induce activation of coincubated autologous platelets “via” cathepsin G released from the azurophilic granules. However TxB2 production in this system cannot be completely explained by cathepsin G-stimulated platelet arachidonate metabolism. Indeed the amount of TxB2 found in supematants of platelet/PMN suspensions challenged with IPM tMLP was 2 to 4 fold higher than that measured when platelets were stimulated by supematants from fMLP-activated PMN. We analyzed the possibility that PMN-induced TxBZ production in this system be the result of transcellular metabolism of AA between fMLP-activated PMN and cathepsin G-stimulated platelets. 3H-AA-labelled PMN were used to test if a transfer of AA or metabolite(s) occur from PMN to platelets. The results show that: I) )H-TxB2 is synthesized when )H-AA-labelled PMN are activated mixed to uulabelled platelets; 2) platelet cyclooxygenase inhibition completely prevents ‘H-TxB2 synthesis; 3) inhibition of cathepsin G-induced platelet activation by the antiprotease eglin C blocked the formation of 3H-TxB2. These data demonstrate that in this experimental system platelets utilize PMN-derived unmetabolized AA to synthesize TxB2.
ABSTRACTS OF 12TH INTNAT’L CONGRESS
Vol. 65, Suppl. 1
Cl 67 INTRACELLULAR Cal* RISE IN HUMAN PLATELETS BY POLYMORPHONUCLEAR (PMN) LEUKOCYTE-DERIVED CATHEPSIN G M. Molino, M. Di Lallo, G. de Gaetano and C. Cerletti lstituto di Ricerche Farmacol. Mario Negri, Consorzio Negri Sud, S. Maria lmbaro (CH), 1 Cathepsin G. a serine protease released by PMN azurophibc granules upon stimulation, activates piatelets, increasing intraplatelet Ca2+ levels ([Ca2+],) in a concentration- dependent manner (50-200 nM). The [Cal+], rises elicited by low (50-80 nM) cathepsin G concentrations in fura-2-loaded platelets showed a biphasic mode, with a first small peak followed by a greater and more prolonged Ca*+ mobilization. Higher (100-200 r&l) cathepsin G concentrations induced monophasic [Cal+], increase. Neither aspirin, or nordihydroguaiaretic acid, nor ketanserin affected platelet activation by cathepsin G, while the ADP scavenger system CP/CPK significantly reduced Cal+ mobilization by 100 nM cathepsin G. Niz+ (4 mM), a divalent cation channel inhibitor, reduced cathepsin G-induced fluorescence rise by more than 90%. This effect was reversed by either reducing Nil’ or increasing cathepsin G concentration. Four mM EGTA totally abolished Cal+ transients. Evidence of a rapid and sustained divalent cation channel opening in the platelet membrane was obtained by adding Mn2+ to the platelet suspension 30 set or 3 min after cathepsin G. No accumulation of InsP, could be detected when platelets were stimulated with cathepsia G. Preincubation of platelets with staurosporine, an inhibitor of protein kinase C, enhances both [Cal+], rises and quenching by Mn” of fluorescence signal induced by cathepsin G. All these data indicate that cathepsm G induces [Ca”], increase mainly through an influx across the plasma membrane and that Cal+ channels may be under the control of protein kinase C.
Cl68 TRANCELLULAR METABOLISM OF ARACHIDONIC ACID (AA): INCREASED PLATELET THROMBOXANE (TX) GENERATION IN THE PRESENCE OF ACTIVATED POLYMORPHONUCLEAR LEUKOCYTES (PMN) N. Maugeri, V. Evangelista, P Piccardoni, A. Celardo, G. Dell’Elba, G. de Gaetano and C. Cerletti lstituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, Santa Maria lmbaro (CH), 1 Human PMN activated by n-formyl-methionyl-leucyl-phenylalanine (fMLP), in the presence of cytochalasin B, are able to induce activation of coincubated autologous platelets “via” cathepsin G released from the azurophilic granules. However TxB2 production in this system cannot be completely explained by cathepsin G-stimulated platelet arachidonate metabolism. Indeed the amount of TxB2 found in supematants of platelet/PMN suspensions challenged with IPM tMLP was 2 to 4 fold higher than that measured when platelets were stimulated by supematants from fMLP-activated PMN. We analyzed the possibility that PMN-induced TxBZ production in this system be the result of transcellular metabolism of AA between fMLP-activated PMN and cathepsin G-stimulated platelets. 3H-AA-labelled PMN were used to test if a transfer of AA or metabolite(s) occur from PMN to platelets. The results show that: I) )H-TxB2 is synthesized when )H-AA-labelled PMN are activated mixed to uulabelled platelets; 2) platelet cyclooxygenase inhibition completely prevents ‘H-TxB2 synthesis; 3) inhibition of cathepsin G-induced platelet activation by the antiprotease eglin C blocked the formation of 3H-TxB2. These data demonstrate that in this experimental system platelets utilize PMN-derived unmetabolized AA to synthesize TxB2.