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C Cerletti

Publications and source records attributed to C Cerletti.

At least 55 records · Page 3Linked to original sources
PubMed1993

Effects of leukocyte-derived cathepsin G on platelet membrane glycoprotein Ib-IX and IIb-IIIa complexes: a comparison with thrombin.

Cathepsin G is a serine, chymotrypsin-like protease released by activated polymorphonuclear leukocytes (PMN) that may act as a platelet agonist. The effect of this enzyme on platelet surface glycoproteins (Gp) Ib and IIb-IIIa was evaluated by means of a cytofluorimetric assay, using fluorescein isothiocyanate-labeled monoclonal antibodies (MoAbs) directed at the alpha chain of Gp Ib (SZ2), at Gp IX or at the complex Gp IIb-IIIa (P2), and the fibrinogen-receptor-specific MoAb PAC-1. In human washed platelets, cathepsin G increased the binding of P2 and PAC-1, decreased the binding of SZ2, but only slightly affected the binding of anti-Gp IX. SZ2 binding decrease was more rapid in cathepsin G- than in thrombin-stimulated platelets, whereas the increase of P2 and PAC-1 binding occurred to a comparable extent with either agonist. In paraformaldehyde (PFA)-fixed and energy-depleted platelets, no effect on either Gp Ib or Gp IIb-IIIa complex was observed with thrombin. At variance, cathepsin G was still able to reduce binding of SZ2, whereas increased binding of P2 or PAC-1 antibodies was not observed. Triton X-100 permeabilization of cathepsin G-treated, PFA-fixed platelets did not restore SZ2 binding at variance with thrombin. Moreover, platelet incubation with cathepsin G resulted in the loss of ristocetin-induced agglutination in the presence of the von Willebrand factor and in the appearance of Gp Ib-derived proteolytic products in supernatants. After dissociation by EDTA pretreatment of surface Gp IIb-IIIa complexes, cathepsin G still induced increased binding of P2. Aspirin and an adenosine diphosphate scavenger system had only a slight but not significant effect on changes in antibody binding induced by cathepsin G. All these data would indicate that cathepsin G, like thrombin, interacts with platelet-surface Gp, inducing the exposure of the intracellular pool of the Gp IIb-IIIa complex with concomitant expression of a functional fibrinogen receptor. Moreover, it induces a loss of antigenic sites on Gp Ib, but the mechanism involved, a proteolytic cleavage of Gp Ib, is substantially different from that of thrombin. These changes, induced by a product of activated PMN, might reduce the reactivity of platelets to the subendothelium, while increasing their ability to undergo aggregation and release reaction.

Adenosine Diphosphate↗
PubMed1993

Effects of glycosaminoglycans on platelet and leucocyte function: role of N-sulfation.

The effect of glycosaminoglycans (GAGs) such as sulodexide, low molecular mass dermatan sulfate, heparin and some derivatives with different degrees and types of sulfation was studied on cathepsin G- or thrombin-stimulated platelets and n-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated polymorphonuclear leucocytes (PMNs). All GAGs (0.01-20 micrograms/mL) inhibited both platelet aggregation induced by cathepsin G and its catalytic activity. Thrombin-induced platelet aggregation in contrast was only prevented by heparin, sulodexide and dermatan (2-100 micrograms/mL). All GAGs, except 2-O,N-desulfated heparin, inhibited beta-glucuronidase and lysozyme release, as well as beta-glucuronidase activity and PMN superoxide production by the peptide fMLP. The efficacy of GAGs was clearly dependent on the degree and type of sulfation since dermatan and N-desulfated heparins were comparatively less effective. The observation that heparin and other GAGs inhibit platelet activation induced by the PMN protease cathepsin G may help determine whether mechanisms of action other than anticoagulation are critical in the antithrombotic activity of heparin and related compounds.

Blood Platelets↗
PubMed1993

Cathepsin G-dependent platelet stimulation by activated polymorphonuclear leukocytes and its inhibition by antiproteinases: role of P-selectin-mediated cell-cell adhesion.

Human PMN stimulated by fMLP are able to activate coincubated, autologous platelets. Cathepsin G, a neutral serine protease stored in the azurophilic granules of PMN, is the major platelet activator in this system. We previously proposed that shear-induced close PMN-platelet contact creates the conditions for which cathepsin G activity on platelets is protected against antiproteinases. The aim of this study was to investigate the adhesive mechanisms, possibly creating between PMN and platelet membranes the microenvironment in which cathepsin G, discharged from stimulated PMN onto adherent platelets, is protected against antiproteinases. Microscopic examination showed that under conditions of high shear, 71.3% +/- 6.1% of PMN were associated to platelets forming small clumps. This percentage decreased to 10% +/- 2% and 13% +/- 4%, respectively, in the presence of an inhibitory antibody to P-selectin or 20 mmol/L mannose-1-phosphate and to 10.8% +/- 3.7% when cells were not stirred. Similarly, PMN pretreatment with neuraminidase abolished PMN binding to platelets. These results indicate that P-selectin mediates PMN-platelet adhesion occurring before PMN stimulation. Prevention of PMN-platelet contact significantly potentiated the inhibitory effect of alpha 1-protease inhibitor on subsequent cathepsin G-induced platelet serotonin release. Because anti-P-selectin antibody, mannose-1-phosphate, and neuraminidase treatment of PMN did not modify PMN-induced platelet activation in the absence of antiproteinases, it is suggested that P-selectin-mediated PMN-platelet adhesion results in the formation of a sequestered microenvironment between cell membranes, in which higher amounts of antiproteinases are required to prevent the activity of released cathepsin G. These data add a new functional role to P-selectin-mediated PMN-platelet adhesion that could be important in vivo because of the presence of antiproteinases in plasma.

Amino Acid Sequence↗
PubMed1993

Antiplatelet activity of 2-(6-carboxyhexyl)-3-n-hexylcyclohexylamine (IBI P-05006), a thromboxane receptor antagonist.

2-(6-Carboxyhexyl)-3-n-hexylcyclohexylamine (IBI P-05006) is a molecule with cytoprotective and antisecretory actions. Since its chemical structure resembles that of the prostanoid nucleus of thromboxane (Tx) A2, the possibility of antiplatelet activity was investigated. The aims of our study were to evaluate a possible inhibitory effect of IBI P-05006 on in vitro platelet aggregation induced by different stimuli and to characterize its mechanism of action. For this purpose, selected mechanisms of platelet function were evaluated. IBI P-05006 inhibited platelet aggregation induced by arachidonic acid or U46619, at concentrations ranging from 0.4 to 4 microM, dose dependently, without interfering with arachidonic acid metabolism. The possibility that the compound acts through adenylate cyclase stimulation was ruled out by measurement of cAMP levels, which were not increased. Receptor binding studies indicated competitive inhibition by IBI P-05006 of the binding of the ligand [3H]SQ29548 to the TxA2/(prostaglandin) PG-cyclic endoperoxides receptor with IC50 in the range of concentrations active on platelet aggregation. In conclusion, IBI P-05006 inhibits in vitro human platelet aggregation through a specific mechanism of competition for the TxA2/PG-cyclic endoperoxides receptor.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1993

Receptor-activated calcium influx in human monocytes exposed to monocyte chemotactic protein-1 and related cytokines.

Stimulation of human monocytes with monocyte chemotactic protein-1 (MCP-1) resulted in an increase of [Ca2+]i. The [Ca2+]i rise was dependent on external Ca2+, could be reconstituted by the addition of external Ca2+ and was blocked by Ni2+. Agonist-stimulated Ca2+ influx was demonstrated directly by the use of Mn2+: in the presence of extracellular Mn2+, MCP-1 and FMLP stimulated a dose-dependent quench in fluorescence of Fura-2-loaded monocytes. This quench was the result of stimulation of Mn2+ influx and was blocked by Ni2+ and by the Ca2+ channel inhibitor SC38249. Pretreatment of monocytes with verapamil and nifedipine or high depolarizing [K+] had no effect. MCP-1 did not induce production of inositol triphosphate nor turnover of phosphatidylinositol 4,5-biphosphate. Collectively, these results show that MCP-1 does not induce discharge of intracellular stores. This chemokine stimulates divalent cation entry (Ca2+ or Mn2+) by a mechanism independent of changes in [Ca2+]i, unrelated to voltage-dependent Ca2+ channels and presumably involving receptor-activated channels. In addition to MCP-1, also two other members of the chemokine Cys-Cys family, RANTES and MIP-1 alpha, stimulated [Ca2+]i increase. Exposure of human monocytes to either RANTES or MIP-1 alpha, had no effect on a subsequent stimulation by MCP-1. On the contrary, MCP-1 cross-desensitized monocytes for a subsequent stimulation with RANTES and MIP-1 alpha. These results suggest a certain level of receptor sharing among members of the Cys-Cys chemokine family.

Calcium↗
PubMed1993

Current concepts of platelet physiopathology.

The genesis of the modern concept of platelet physiopathology can be traced historically to many individuals and developments, including the pioneering work of Giulio Bizzozero over 100 years ago. Much knowledge has since been gained to the point that we can now define many of the elements of the platelet function in relation to the hemostatic process in terms of their molecular biology. This presentation will attempt to synthesize a simple, understandable, yet global perspective of platelet function and of its participation to the pathogenesis of arterial thrombosis. Focus will be placed on the crucial importance of cell-cell interaction. A symbolic representation of platelet plug formation using simple mathematical figures like the line, point, and triangle is helpful. The line represents the vascular system with intact endothelial cell linings. The irritation/disruption of vascular endothelium can be viewed as a point on the line representative of a localized event. From this initial (often small) stimulus, amplification occurs rapidly to produce an appropriate cellular and humoral response, represented by a triangle. Each component (vessel wall, platelets, leukocytes, coagulation and fibrinolysis systems) of this complicated response has biological characteristics which localize and amplify subsequent reactions to the site of primary injury until bleeding is arrested and the wound is healed or arterial thrombosis progresses to vascular occlusion.

Arteriosclerosis↗
PubMed1993

Cathepsin G--induced release of PAI-1 in the culture medium of endothelial cells: a new thrombogenic role for polymorphonuclear leukocytes?

Activated polymorphonuclear leukocytes (PMNs) may affect the integrity of blood vessels by endothelial cell injury. We investigated the effects of cathepsin G purified from human neutrophils on the fibrinolytic potential of cultured human umbilical vein endothelial cells (HUVECs). Cathepsin G (5 and 10 micrograms/ml) induced marked intercellular gap formation after 1 hour of treatment, whereas 1 microgram/ml did not, even after 6 hours incubation. In contrast, plasminogen activator inhibitor-1 (PAI-1) antigen levels, measured by a double antibody enzyme-linked immunosorbent assay, were significantly increased in culture media (CM) on cathepsin G (1 microgram/ml) treatment after 15 minutes (5.1 +/- 1.2 ng/ml vs 2.6 +/- 0.6 ng/ml for controls, p < 0.01) and 6 hours of incubation (69.6 +/- 17.5 ng/ml vs 40.0 +/- 9.0 ng/ml for controls, p < 0.01). Likewise, PAI activity, measured by reverse fibrin autography, increased on cell treatment with cathepsin G. Preincubation of cathepsin G with eglin C (10 micrograms/ml) almost completely abolished the increase in both PAI antigen and activity levels induced by cathepsin G. Cycloheximide, a protein synthesis inhibitor, did not block cathepsin G-induced PAI-1 release. PAI-1 mRNA levels were not affected by HUVEC treatment with cathepsin G (1 microgram/ml for 15 minutes), even after 24 hours. In the extracellular matrix (ECM) PAI-1 antigen levels decreased to 77% and 40% of controls, respectively, after 15 minutes and 6 hours of cathepsin G (1 micrograms/ml) treatment. Reverse fibrin autography also demonstrated a dose-dependent reduction of PAI activity in the ECM on 6 hours of cell treatment with 1 or 5 micrograms/ml cathepsin G. Moreover, ECM prepared from confluent HUVECs released PAI-1 in supernatants on 1 micrograms/ml cathepsin G incubation in a cell-free system. Tissue-type plasminogen activator (t-PA) activity was strongly depressed on cathepsin G treatment, both in CM from HUVECs or in a cell-free system. Finally, PAI-1 was also released from cathepsin G-stimulated platelets in a dose-dependent manner. In summary, our results support a potentially thrombogenic role of cathepsin G, which could impair the fibrinolytic potential of the endothelium. These data give a new insight into the mechanisms by which activated PMNs may promote thrombus formation. On the other hand, the decrease of PAI-1 in ECM could favor penetration and migration of inflammatory or tumor cells through the subendothelial layers.

Cathepsin G↗
PubMed1992

Intracellular Ca2+ rise in human platelets induced by polymorphonuclear-leucocyte-derived cathepsin G.

Cathepsin G, a serine protease released by polymorphonuclear-leucocyte azurophilic granules upon stimulation, activates human platelets, inducing an increase in intra-platelet Ca2+ concentration ([Ca2+]i) in a concentration-dependent manner (50-200 nM). The [Ca2+]i 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 Ca2+ transient. Higher (100-200 nM) cathepsin G concentrations induced a monophasic increase in intracellular Ca2+. Acetylsalicylic acid, nordihydroguaiaretic acid and ketanserin did not affect platelet activation by cathepsin G, whereas the ADP-scavenger system phosphocreatine/creatine kinase significantly decreased Ca2+ mobilization, platelet aggregation and 5-hydroxytryptamine secretion by cathepsin G. Preventing cathepsin G-induced platelet aggregation with the synthetic peptide RGDSP (Arg-Gly-Asp-Ser-Pro) did not significantly affect cathepsin G-induced Ca2+ transients. Ni2+ (4 mM), a bivalent-cation-channel inhibitor, decreased the cathepsin G-induced fluorescence rise by more than 90%. This effect was reversed by either decreasing Ni2+ or increasing cathepsin G concentration. Preventing Ca2+ influx across the plasma membrane with 4 mM-EGTA totally abolished Ca2+ transients. However, EGTA also strongly decreased catalytic activity of cathepsin G, which is essential for platelet activation. Evidence of a rapid and sustained bivalent-cation channel opening in the platelet membrane was obtained by adding Mn2+ to the platelet suspension 30 s or 3 min after cathepsin G. No accumulation of InsP3 could be detected when platelets were stimulated with cathepsin G. All these data indicate that cathepsin G induces a [Ca2+]i increase mainly through an influx across the plasma membrane. This massive Ca2+ entry is probably due to opening of receptor-operated channels and is amplified by endogenous ADP release.

Adult↗
PubMed1992

Transcellular metabolism of arachidonic acid: increased platelet thromboxane generation in the presence of activated polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (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, thromboxane (Tx) B2 production in this system cannot be completely explained by cathepsin G-stimulated platelet arachidonate metabolism. Indeed, the amount of TxB2 found in supernatants of platelet/PMN suspensions challenged with 1 mumol/L fMLP was twofold to fourfold higher than that measured when platelets were stimulated by supernatants from fMLP-activated PMN. In the present report, we analyzed the possibility that PMN-induced TxB2 production in this system is the result of transcellular metabolism of arachidonic acid (AA) between fMLP-activated PMN and cathepsin G-stimulated platelets. 3H-AA-labeled PMN were used to test if a transfer of AA or metabolite(s) occur from PMN to platelets. Our results showed that: (1) 3H-TxB2 and 3H-12-HHT are synthesized when 3H-AA-labeled PMN are activated mixed to unlabeled platelets; (2) total radioactivity released by fMLP-stimulated PMN is increased in the presence of platelets, whereas the membrane content of unesterified 3H-AA is reduced; (3) platelet cyclooxygenase inhibition completely prevents 3H-TxB2 synthesis; and (4) inhibition of cathepsin G-induced platelet activation with the antiprotease eglin C blocks the formation of 3H-TxB2. These data show that in the experimental system used, platelets use PMN-derived unmetabolized AA to synthesize TxB2.

Arachidonic Acids↗
PubMed1992

Inhibition by heparin of platelet activation induced by neutrophil-derived cathepsin G.

Heparin is the most widely used anticoagulant drug for prevention and treatment of thrombosis. However, inhibition of blood coagulation might not fully explain the antithrombotic activity of this drug. The present study shows that different heparin preparations (50 nM) completely prevent human platelet aggregation, serotonin release and thromboxane B2 production induced by purified neutrophil-derived cathepsin G (E.C. No. 3.4.21.20). This inhibitory effect was not related to the anticoagulant property of the compounds, since a heparin preparation with an inactivated active for antithrombin III was also effective. Heparins inhibited the protease activity of the enzyme over the same range of concentrations. Since the effect of cathepsin G on platelets requires an intact proteolytic active site, the inhibitory effect of the drugs on cathepsin G-induced platelet activation may be explained by a blockade of protease activity. Heparins were also shown to reduce platelet activation induced by cathepsin G released from activated polymorphonuclear leucocytes in mixed cell suspensions. As polymorphonuclear leucocytes might contribute to both arterial and venous thrombosis through platelet activation induced by the release of cathepsin G, this novel property of heparin could be used to optimize its antithrombotic efficacy.

Amino Acid Sequence↗
PubMed1992

Defibrotide inhibits platelet activation by cathepsin G released from stimulated polymorphonuclear leukocytes.

Defibrotide is a polydeoxyribonucleotide with antithrombotic effects in experimental animal models. Most of the actions of this drug have been observed in in vivo test models but no effects have been reported in in vitro systems. In this paper we demonstrate that defibrotide interferes with polymorphonuclear leukocyte-induced human platelet activation in vitro. This effect was not related to any direct interaction with polymorphonuclear leukocytes or platelets, but was due to the inhibition of cathepsin G, the main biochemical mediator of this cell-cell cooperation. Since cathepsin G not only induces platelet activation but also affects some endothelial cell functions, the anticathepsin G activity of defibrotide could help to explain the antithrombotic effect of this drug.

Catalysis↗
PubMed1992

Synthesis of some guanylhydrazones and imidazolinylhydrazones as thromboxane-synthase and platelet aggregation inhibitors.

The imidazolinylhydrazones of (3-pyridinyloxy)-acetaldehyde and of 6-[3-(2-formyl-pyridinyl)oxy]hexanoic acid were synthesized as cyclic analogues of the corresponding guanylhydrazones which were found to be selective inhibitors of human thromboxane-synthase. The benzene isosters were also prepared in order to define the importance of the ring nitrogen for the activity. Moreover, the guanyl- and imidazolinyl-hydrazones of two 6-[(3-pyridinyl)oxy]hexanoic acids showing in the 2 position an alkyl chain with an alpha, beta-unsaturated ketonic function were prepared. Imidazolinylhydrazones 7 and 18 are selective inhibitors of thromboxane-synthase, while the two guanylhydrazones 14 and 15 which do not affect prostanoid biosynthesis seemed to be antagonists at the thromboxane receptor.

Dinoprostone↗
PubMed1992

Modulation of fibrinolytic response to venous occlusion in humans by a combination of low-dose aspirin and n-3 polyunsaturated fatty acids.

Aspirin at high but not at low doses reduces the fibrinolytic response to venous occlusion. Inhibition of vascular prostacyclin synthesis could be involved in this effect. Fish oil supplementation may redirect prostanoid metabolism toward an overall "antithrombotic" condition but with controversial effects on prostacyclin formation. In this study we investigated the effect of low-dose aspirin together with n-3 polyunsaturated fatty acid (PUFA) supplementation on the fibrinolytic response to venous occlusion. Following a double-blind, randomized, crossover design, six healthy volunteers (three men and three women, 24-37 years old) were given for 29 days 5.3 g eicosapentaenoic and docosahexaenoic acids or a corresponding dose of n-6 PUFAs as control; aspirin (40 mg/day) was then added for an additional 14 days. A 2-month washout period was allowed before the crossover. Blood was collected before and after venous stasis on days 0, 29, and 43 of each test period. A combination of aspirin with n-3 PUFAs reduced the fibrinolytic response to venous occlusion in all subjects, the mean value of fibrinolytic activity after stasis being 240 +/- 40 mm2, a value significantly lower than at baseline (366 +/- 51 mm2, mean +/- SEM, p < 0.05). Similarly, the tissue-type plasminogen activator (t-PA) antigen level was lower in the aspirin + PUFA-treated group. Plasminogen activator inhibitor activity before stasis was enhanced by n-3 PUFA supplementation (from 7.5 +/- 2 to 14.8 +/- 3 IU/ml, p < 0.05), an effect not affected by aspirin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Effects of heparan-sulphate administration on clotting parameters and serum thromboxane B2 levels in cholesterol fed rabbits.

We evaluated the effects of heparan-sulphate administration on clotting times, thromboelastographic parameters and serum thromboxane B2 levels in hypercholesterolemic rabbits with aortic atherosclerotic lesions (sudanophilic areas). 24 New Zealand male rabbits were divided into three groups of 8 animals each. Group A and B were fed a rabbit chow diet containing 0.7% of cholesterol whereas Group C was fed a standard rabbit diet without cholesterol. Group A was treated by subcutaneous route with 6 mg/kg/day of heparan sulphate. At the beginning of the study and after 3 and 6 months of treatment, serum cholesterol and thromboxane B2 levels were tested. Furthermore, at the end of the experiment, we evaluated plasma fibrinogen, aPTT, PT and TT values. The administration of heparan-sulphate in cholesterol fed rabbits produced: a reduction of plasma fibrinogen levels, without modifying aPTT and TT; a protective effect vs the lengthening in PT values, likely induced by cholesterol rich diet; a reduction of plasma thrombophilic activities and of aortic atheromasic involvement induced by dietetic cholesterol intake. However, increased serum thromboxane B2 levels, likely through a proaggregant activity were observed. We suggest that heparan-sulphate administration, in cholesterol fed rabbits, has a favourable effect on clotting parameters, while contrasting effects were found on platelet activity.

Animals↗
PubMed1991

The signal transduction pathway involved in the migration induced by a monocyte chemotactic cytokine.

Recombinant monocyte-chemotactic and activating factor (rMCAF; alternative acronyms MCP-1, TDCF, human JE) induced migration of human monocytes across polycarbonate or nitrocellulose filters. Maximal induction of migration was observed at a concentration of 10 ng/ml (10(-9) M). Checkerboard analysis revealed that rMCAF elicited true gradient-dependent chemotactic migration, although a gradient independent chemokinetic effect was observed at low concentrations (1-5 ng/ml). rMCAF caused a rapid (less than 5 s) and transient (approximately 1.5 min) increase of free cytosolic Ca2+ ions, as assessed by the fura-2 probe. No Ca2+ increase was detected in neutrophils or lymphocytes stimulated by rMCAF. Studies conducted in the absence of extracellular Ca2+ or in the presence of Ni2+ (an inhibitor of Ca2+ influx) suggested that the increase of intracellular Ca2+ induced by rMCAF is dependent on the influx of extracellular Ca2+ through plasma membrane channels. Bordetella pertussis toxin inhibited the intracellular Ca2+ elevation and chemotaxis caused by rMCAF. The possible involvement of Ca(2+)-dependent protein kinases in rMCAF signaling pathway(s) was explored using inhibitors. Inhibitors of GMP-dependent kinase and myosin L chain kinase had no effect on rMCAF-induced monocyte migration. In contrast, protein kinase C/cAMP-dependent kinase inhibitors (such as, C-I, H-7, HA-1004, KT5720, and Staurosporine) markedly decreased rMCAF induced chemotaxis suggesting the involvement of a serine/threonine protein kinase, possibly protein kinase C, in rMCAF signaling pathway.

Calcium↗
PubMed1991

Endotoxic lipid A induces intracellular Ca2+ increase in human platelets.

The activation of protein kinase C by endotoxic lipid A was observed with both intact platelets and in a cell-free system [Romano & Hawiger (1990) J. Biol. Chem. 265, 1765-1770]. We have now studied the action of lipid A on intracellular Ca2+ concentration ([Ca2+]i). Lipid A induced a concentration-dependent rise in [Ca2+]i in human platelets loaded with fura-2, which reached a maximum at 37.1 +/- 3.8 s (tmax). Maximum [Ca2+]i levels, observed at 30 microM lipid A, were 432 +/- 60 nM. EGTA (2 mM) or NiCl2 (1 mM) each decreased the lipid A-dependent elevation of [Ca2+]i by 50-60% without significant modification of tmax, but shortening the time for 50% recovery (t50) from greater than 400 s to 113.1 +/- 29.1 s and 54 +/- 2.1 s, respectively. Quenching of the fura-2 signal was also observed in lipid A-stimulated platelets resuspended with MnCl2 (1 mM), suggesting that both mobilization and external influx of Ca2+ occur. Intracellular Ca2+ mobilization depended on release from Ins(1,4,5)P3-sensitive stores, since Ins(1,4,5)P3 accumulation was detected in lipid A-activated platelets. Staurosporine, an inhibitor of protein kinase C, blocked the [Ca2+]i rise generated by lipid A in platelets [concn. giving 50% inhibition (IC50) = 0.1 microM], prolonging the tmax. to 54.7 +/- 5.1 s, but decreasing the t50 to 157.5 +/- 31.8 s. Staurosporine also suppressed InsP3 accumulation (IC50 = 0.15 microM). These results suggest that platelet activation by lipid A involves an interaction between [Ca2+]i elevation and protein kinase C activation.

Blood Platelets↗