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Role of Platelet Membrane Glycoproteins Ib/IX and Llb/Llla, and of Platelet S-Granule Proteins in Platelet Aggregation Induced by Human Osteosarcoma Cells

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Role of Platelet Membrane Glycoproteins Ib/IX and Llb/Llla, and of Platelet S-Granule Proteins in Platelet Aggregation Induced by Human Osteosarcoma Cells Role of Platelet Membrane Glycoproteins Ib/IX and llb/llla, and of Platelet s-Granule Proteins in Platelet Aggregation Induced by Human Osteosarcoma Cells Philippe Clezardin, 2 Jeanne Drouin, Marie-Christine Morel-Kopp, Michel Hanss, Beate Kehrel, Claire-Marie Serre, C~cile Kaplan, and Pierre D. Delmas INSERM Research Unit 234, Pavilion F [P C., C-M. S., P. D. D.] and Pavilion E [M. H.], H@ital Edouard Herriot, Place d'Arsonval, 69437Lyon, France; Division of Hematology, Ottawa General Hospital, Ottawa, Ontario, Canada [J. D.]; Service lmmunologie Leuco-Plaquettaire, Institut National de TransJitsion Sanguine, Rue Alexandre-Cabanel, 75739 Paris, France [C. K., M-C. M-K.]; and ExperimenteUe Hiimostaseforschung, lnnere Medizin A, Universitiitskliniken Miinster, Germany [B. K.]. ABSTRACT cells to endothelial cells and subendothelial matrix largely depend on the interaction of tumor cells with platelets (1-3). Such an interaction We have previously shown that the platelet-aggregating activity of hu- of tumor cells with platelets involves a series of sequential events man MG-63 and HOS osteosarcoma cells depends at least in part upon (1, 4). Initially, a few platelets adhere to tumor cells which activate tumor cell surface-associated thrombospondin, and suggested that plate- let-osteosarcoma cell interactions could occur through interactions with them through expression of prothrombogenic activities, depending on specific platelet membrane receptors. In this study, the platelet-aggregat- the origin of the tumor cells. This results in the recruitment of addi- ing activity of MG-63 and HOS ceils was studied by using a variety of tional platelets, and formation of focal aggregates of activated, de- platelet disorders. Both osteosarcoma cdl lines induced a biphasic platelet granulated platelets attached to the tumor cell surface. Subsequently, aggregation response when added to normal platelet-rich plasma, while tumor cells respond to activated platelets with the appearance of the second phase of aggregation was absent when added to gray platelets cellular processes that interdigitate with platelet aggregates, suggest- (deficiency in a-granule proteins) and to aspirin-treated platelets. Platelets ing tumor cell cytoskeletal alterations. Several cell surface\adhesive from two unrelated patients with type I Gianzmann's thrombasthenia proteins have been implicated in linking platelets to tumor cE~ls. (deficiency in glycoprotein (GP) GPIIb/IIIa) did not aggregate at all with Among these adhesive proteins are fibronectin (5), von Willebrahd osteosarcoma ceils. Using giant platelets from three patients with Bernard- factor (5), fibrinogen (6), and thrombospondin (7, 8). During hemo- Soulier syndrome (deficiency in GPIb/IX), the aggregation response in- duced by MG-63 and HOS cells was monophasic and reversible when stasis, von Willebrand factor binds to platelet membrane glycoprotein compared to normal-sized platelets and to giant platelets from a patient complex GPIb/IX, 3 and the membrane glycoprotein complex GPIIb/ with May-Hegglin anomaly (no membrane GP defect). Because GPIb Ilia serves as a platelet receptor for fibrinogen, fibronectin, von Wil- serves as a receptor for von Willebrand factor during hemostasis, aggre- lebrand factor, and thrombospondin (for review see Ref. 9). Adhesive gation experiments were also conducted with the platelet-rich plasma of proteins are also secreted from a-granules upon platelet activation and two patients with a low plasma von Wiilebrand factor concentration (type help to stabilize platelet aggregates (10). The functions of these major I yon Willebrand's disease) before and after the infusion of deamino-D- platelet glycoproteins have been elucidated by the study of platelets arginine vasopressin. MG-63 and HOS cells induced biphasic platelet from patients with Bernard-Soulier syndrome, Glanzmann's throm- aggregation both before and after deamino-D-arginine vasopressin treat- basthenia, and gray platelet syndrome which lack GPIb/IX, GPIIb/Illa ment, while the ristocetin-dependent binding of von Willebrand factor to and a-granule proteins, respectively (9, 11). Immunoinhibition has platelets only occurred after deamino-D-arginine vasopressin treatment. Preincubation of normal platelet-rich plasma with monocional antibody been widely used in evaluating the effects of antibodies against plate- SZ-2 directed against the yon Wiilebrand binding domain of GPIb did not let GPIIb/IIIa on the platelet-aggregating activity of tumor cells (5, inhibit the platelet-aggregating activity of osteosareoma cells, whereas 12-15). Pretreatment of platelets with anti-GPIIb/IIIa monoclonal anti-GPIb antibody SZ.2 did inhibit ristocetin-induced platelet agglutina- antibodies inhibit both the binding of mouse CT26 and human HCT8 tion. In addition, anti-GPIX antibodies did not affect platelet-osteosar- colon carcinoma cells to platelets (5), and the platelet-aggregating coma cell interactions. In conclusion, our data demonstrate that the first activity of different tumor cell lines, including human HMV-I mela- phase of the platelet-aggregating activity of human osteosarcoma cells is noma cells and M7609 colon carcinoma cells (12-15). By contrast, the initiated by the interaction of these tumor cells with platelet membrane significance of the results obtained with anti-GPIb monoclonal anti- GPIIb/IIIa, whereas the second phase, even if plasma von Willebrand bodies remains unclear. Anti-GPIb monoclonal antibodies have no factor is deficient, involves platelet membrane GPIb and the participation effect on the binding of mouse CT26 and human HCT8 colon carci- of platelet a-granule proteins in membrane-mediated events, making ag- gregation irreversible. noma cells to platelets (5) and on the platelet-aggregating activity of M3Dau melanoma cells (14), while they inhibit the platelet-aggregat- ing activity of human HMV-I melanoma cells and M7609 colon INTRODUCTION carcinoma cells (15). We have previously shown that the platelet- Metastasis is a complex phenomenon that involves tumor cell dis- aggregating activity of human osteosarcoma cells depend at least in semination, and tumor cell interactions with host cells (platelets, endo- part upon tumor cell surface-associated thrombospondin, and sug- thelial cells) and subendothelial matrix (For review see Ref. 1). The gested that platelet-osteosarcoma cell interactions could occur through survival of circulating metastatic cells and the attachment of tumor interactions with specific platelet membrane receptors (8). However, it is not known whether platelet receptors such as GPIb/IX or GPIIb/IIIa Received 5/10/93; accepted 7/27/93. are involved during platelet aggregation induced by osteosarcoma The costs of publication of this article were defrayed in part by the payment of page cells, or if platelet-released a-granule proteins play a role in this charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. context. The present study investigates the role of platelet membrane 1 This study was supported by grants from INSERM/Medical Research Council (P. C., glycoproteins GPIb/IX and GPIIb/IIIa, and of a-granule proteins dur- J. D.), la FEdEration Nationale des Centres de Lutte contre le Cancer (P. C.), the Groupe- ing platelet aggregation induced by human osteosarcoma cells, using ment des Entreprises Franqaises pour la Lutte contre le Cancer (P. C.), the Deutsche Forschungs Gemeinschaft (Ke397/1-2) (B. K.), and the Ottawa General Hospital Medical Research Fund (J. D.). 2 To whom requests for reprints should be addressed, at INSERM Unit Research 234, 3 The abbreviations used are: GP, glycoprotein;DDAVP, deamino-o-arginine vasopres- Pavilion F, H6pital Edouard Herriot, 3 Place d'Arsonval, 69437 Lyon CEdex 03, France. sin. 4695 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1993 American Association for Cancer Research. PLATELET GPs IN OSTEOSARCOMA CELL-INDUCED PLATELET AGGREGATION (Immunotech, France). Mouse monoclonal antibodies P5 and P35 (clones FMC25 and GR-P, respectively) directed against platelet GPIX (CD42a), and mouse monoclonal antibody Gi27 directed against the/3 chain of platelet GPIb were obtained from the IVth and Vth International Workshop on Leucocyte Typing (18). Goat anti-mouse IgG conjugated with fluorescein isothiocyanate was obtained from Coulter Immunology. Platelet Aggregation. Platelet-rich plasma was obtained by centrifugation of freshly drawn blood of healthy donors and patients [collected in heparin (10 units/ml)] at room temperature for 20 min at 160 X g. The remaining blood was centrifuged at 1500 X g for 15 min to obtain platelet-poor plasma. Platelet-poor plasma was used to calibrate light transmission at 100%. The' platelet count in platelet-rich plasma was adjusted to 2 x 108 platelets/ml for each experiment. EDTA-treated osteosarcoma cells (106 cells/50 ~l) in Hanks' solution containing 0.2% (mass/volume) bovine serum albumin were either preincubated with an antibody and washed to remove unbound antibodies or directly added to the platelet-rich plasma. The aggregation pattern was then recorded in a Chrono-Log Dual Channel aggregometer at 37~ with continu- ous stirring. Electron Microscopy of Platelet-Osteosarcoma Cell Interaction. At the peak of the aggregation curve, platelet-osteosarcoma cell aggregates were allowed to settle at the bottom of Eppendorf tubes. Cell aggregates were washed twice in phosphate-buffered saline, and fixed with 2% (v/v) glutaral-
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