THROMBOSIS RESEARCH 62; g-22,1991 0049-3848/91 $3.00 + .OO Printed in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
PLATELET PROAGGREGATING ACTIVITY OF THREE LYNPHOMA-DERIVED HUMAN CELL LINES.
Pulcinelli F.M., Manzari G., Faggioni A., La Mancusa R., Martinico E., Torrisi M.R., Zompetta C., and Gazzaniga P.P. University La Sapienza, Department of Experimental Medicine, Rome - Italy (Received
in revised form 20.12.1990
by Editor MB.
ABSTRACT vitro differential platelet in demonstrate a We -__-----Burkitt's lymphoma activity in three proaggregating derived human B cell lines, i.e. Daudi, Raji and P3H-Rl. indicated the ultrastructural findings Functional and cells to induce a marked secondary ability of Daudi while the Raji cells irreversible platelet aggregation, only induced a primary-type reversible platelet response; no evidence of proaggregating activity has been obtained Luminometric assays indicated that for P3H-Rl cells. but not P3H-Rl, cells with and Raji, contact of Daudi platelet rich plasma (PRP) or platelet poor plasma (PPP) was followed by ADP release, in the range of 2,2-3,5 FM and 0,4-0,6 PM respectively for Daudi and Raji cells. preincubation of PRP with apyrase Daudi cells After induced a reversible platelet response similar to that obtained the Raji cells: the with use of then, irreversible complete platelet response induced by Daudi cells was to be related to ADP release from degranulating platelets. Experiments in gel-filtered platelet systems showed that the plasma co-factor inducing ADP release from Daudi and Raji cells was not fibrinogen. Specific inhibition of platelet thrombin receptors, as well as of cycloxygenase and lipoxygenase pathways, did not modify the proaggregating activity of Daudi and Raji cells. Work is in progress to characterize the plasma factor interacting with Daudi and Raji, but not P3H-Rl cells, and the differences between the three cell lines which support this differential interaction. Key
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INTRODUCTION ----------aSince the initial observations (1) of tumor cell-induced in -several lines of evidence suggested vitro platelet aggregation, ----that the -------in vitro tumor cell-platelet interaction could reflect the possible involvement of platelet-associated events in tumor metastasis in vivo. Although extensively investigated, the -------mechanisms by which suspensions of, or materials derived from, both animal and human different tumor cell lines induce platelet activation are still largely obscure. The available data seem to indicate that different cellular and molecular mechanisms may be involved in the proaggregating activity of different tumor cell lines, including (a) release of adenosine-diphosphate from tumor cells (2, 3, 4); (b) generation of thrombin by tumor cells (5, 6, 7); (c) release from tumor cell membranes of microvesicles, acting by a thrombin-dependent or ADP-dependent mechanism (2); (d) generation and release from tumor cell membranes of a sialolipoprotein proaggregating material (PAM) (8, 9, 10); (e) surface interactions between GpIb or GpIIb/IIIa-like glycoproteins shared by tumor cells 'and platelets (11, 12, 13, 14, 15, 16, 17, 18, 19) and secondary involvement of platelet cytoskeleton proteins (20); (f) release of different types of proteinases from tumor cells (21, 22); (g) release from tumor cells of procoagulant unidentified yet thromboplastin-like materials (23). Also unclear are the intraplatelet mechanisms dealing with the amplification of the tumor cell-induced platelet response, probably involving both cycloxygenase (24, 25, 26, 27, 28) and lipoxygenase 28) pathways, as well as a modulation of (lh; intraplatelet Ca levels (29). In this paper we demonstrate that three Burkitt's lymphoma-derived, human B cell lines, i.e. Daudi, Raji and show marked differences in the --_--_-_ in vitro proaggregating PBH-Rl, activity on human platelets, and that different properties of the three cell types are probably involved in this differential tumor cell-platelet interaction.
MATERIALS AND METHODS -_-_-___-_-_-_-_-_-_Platelet EreEarations. Samples of human platelet-rich plasma ---------- -------(PRP) were obtained from healthy drug-free students of the Rome University Medical School. Venous blood was collected in polypropylene tubes with trisodium citrate (3.8%; 1:9 v/v) separated by centrifugation without stasis. PRP was immediately at 150 x g for 10 min. Platelet poor plasma (PPP) was obtained by centrifugation of samples of P5RP at 2.200 x g for 10 min. PRP was always adjusted to 3.5 x 10 platelets/p1 with autologous PPP
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(Platelet Counter PLlOO Sysmex TOA). Gel filtered Elatelets (GFP) were obtained by a gel filtration ----------------a--procedure derived from Tangen and Berman (30). 20 x 2.5 cm columns were polystyrene packed with Ultrogel A2 LKB in After prolonged calcium-free Tyrode buffer, pH 7.35 and 294 m0sm. washing with the same buffer supplemented with 0.1% glucose and 0.1% bovine serum albumin, 10 ml of PRP were added to a column. Elution was performed by use of the same buffer at a pressure of 40 cm H 0 and a rate of 6 drops/min, at 20°C. After first were discharged, we obtained a virtual1 20-25 ml o i: eluate 5 platelet suspension protein-free (Lowry method) (about 2 x 10 platelets/pi). Our GFPs aggregated in response to thrombin (>0.05 U/ml), arachidonic acid (>50 PM), and ristocetin (al.5 mg/ml) also in absence of added and to ADP (>0,8 PM), epinephrine fibrinogen, (> 5 PM) and collagen (24 pg/ml) after addition 2,"f human (final fibrinogen (final concentration 1.5 and Ca mg/ml) concentration 25 pg/ml). Cell lines and cultures. Daudi, Raji and P3H-Rl cells are three _-__-_--_--_---_-__---EBV genome positive Burkitt's lymphoma cell lines. The cell lines were provided by Dr. Dharam Ablashi, NCI, Bethesda, Maryland, USA. The cells were grown in 95% air, 5% CO in RPM1 1640 medium, 2 supplemented with 10% fetal calf serum and antibiotics. Tumor cells were harvested in the absence of proteases, washed three times with phosphate buffer solution (PBS)(centrifugation at 300 x g for 10 min) and resuspende in PBS in absence of added 4 calcium, at a concentration of 2 x 10 cells/ml. Cell viability was assayed by Trypan Blue exclusion procedure: cell suspensions were incubated (1:l v/v) with a 0.4% solution of Trypan Blue for 10 min at 20°C, and observed by light microscopy. In some experiments supernatants of sonicated tumor cells were used: the cells were sonicated by use of Mullard MSE cell disintegrator (60 set, 20 Kc/set at 4OC), then centrifuged at 14.550 x g (Microcentrifuge Biofuge A Heraeus). Aggregometric assays. Aggregometric assays were performed by use -- -----------of a 840 Elvi Logos dual sample aggregometer, in polystyrene tubes, at 37OC and 1000 rpm stirring. To 400 ~1 of PRP were adde+ 100 /Jl of Daudi, or Raji or P3H-Rl cell suspension (2 x 10 cells/ml). As a control, the same PRP (400 ~1 plus PBS 100 ~1) was stimulated by ADP (from 0.8 to 2.0 FM), or epinephrine (from 1 to 10 FM) or collagen (final concentration 4 pg/ml). In the experiments performed in supernatants from sonicated cells, increasing volumes of supernatant (50-100-150 ~1) were added to 350 ~1 of PRP; the final volume was adjusted to 500 ~1 with PBS. Electron microscoEx. Samples of PRP plus tumor cells were fixed ----------------in different steps of the tumor cell/platelet interaction, by direct addition of cold (4OC) 2% glutaraldehyde in PBS 0.1 M, pH 7.3 (380 mOsm), and then postfixed for 2 h at 4OC in a solution
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7.3 (380 mOsm), and then postfixed for 2 h at 4OC in a solution of osmium tetroxide 1% in Verona1 acetate buffer (pH 7.4). After dehydratation in acetone and 2 passages in toluol, the samples were embedded in EPON 812. Ultrathin sections were examined after staining with uranyl acetate and lead hydroxide. Adenine nucleotide release. ATP release following tumor _-_-___--_-_-_-_-_---------cell-platelet interaction was analyzed by use of LKB luminometer 1251. To 450 ~1 of PRP, 50 rl of a solution containing luciferin (80 rg/ml) and luciferase (8800 U/ml) were added, and ATP luminescence was measured after addition7of 100 ~1 of Daudi, Raji or PBH-Rl cell suspension in PBS (2x10 cells/ml). In parallel experiments the ATP release from Daudi, Raji or P3H-Rl cells was measured in identical experimental conditions, using PPP or normal human serum instead of PRP (with PBS instead of PPP as a control). In both cases, the increase in luminescence obtained in parallel assays by addition of a pyruvate kinase (0.75 U/ml)/phospho-enolpyruvate (0.8 mM) system, which completely converts ADP into ATP, indicated the amount of_ADP released. Experiments with Apxrase: in some experiments PRP or tumor cell ----------_----a ---supernatants were preincubated with an ADP scavenging system Sigma V grade, approximately 3.4 units/mg of protein, (Apyrase, final concentration 130 pg/ml, for 5 min at 37OC). Drug treatments. To inhibit the cycloxygenase pathway, either PRP --- ----------or tumor preincubated (10 min, at 37OC) with cells were indomethacin (Sigma Chemicals, St. Louis, final MO): concentrations were 28 ,uM for PRP and 14 ,uM for tumor cells, respectively. To inhibit the lipoxygenase pathway, PRP or tumor cells were preincubated min, at 37OC) with nor(10 of dihydroguaiaretic acid (NDGA, Sigma) in a final concentration 100 PM. In other experiments to inhibit platelet aggregation dependent upon both the cycleand lipoxygenase metabolites, 1-phenyl-3-pyrazolidone (BW 755C, Wellcome, Beckenham, Kent, UK) concentrations ranging from 1 mM to 5 mM was used in final (incubation for 10 min, at 37OC). To block the thrombin-activated D-phenylalanyl-L-L-prolyl-L-arginine platelet aggregation, La Jolla, CA) was used chloromethyl-ketone, (PPACK, Calbiochem, ranging from 6.3 PM to 18.9 in final concentrations rM (incubation for 10 min, at 37OC). To analyze the role of binding the platelet proteins in of and other adhesive fibrinogen aggregation induced by Daudi and Raji cells, PRP was preincubated for 2 min at 37OC with Arg-Gly-Asp-Ser tetrapeptide (RDGS)(Sigma) in final concentrations ranging from 23 to 230 PM, corresponding to lo-100 pg/ml).
RESULTS ------Proaggregating effect of tumor cell suspensions. When suspensions ----------- ---- ______--_-_-_-______----of intact Daudi or Raji cells were added to human PRPs, typical
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aggregation patterns were consistently observed. Daudi cell suspensions induced a marked irreversible platelet aggregation (maximum decrease of optical density 48 + n=20), 12%; characterized by the appearance of a monophasic or diphasic wave, whereas Raji suspensions always induced a cell monophasic reversible aggregation wave (maximum decrease of optical density 17 + 6%; n=15). Intact P3H-Rl cell suspensions as well as normal suspensions of peripheral blood human B lymphocytes, failed to cause platelet aggregation (in all experiments, maximum decrease of optical density