Alpha IIb beta 3 integrin expression and function in subpopulations of murine tumors.

Subpopulations of B16 amelanotic melanoma (B16a) cells, isolated by centrifugal elutriation from enzymatically dispersed solid tumors, demonstrated different abilities to form lung colonies when injected intravenously. In contrast, no differences in experimental metastasis were observed among subpopulations obtained from Lewis lung (3LL) tumors. Lung colonization by B16a and 3LL subpopulations correlated positively with observed differences (B16a) or lack of differences (3LL) in tumor cell ability to induce aggregation of homologous platelets, to adhere to subendothelial matrix or fibronectin, and with the percentage of cells in the G2/M phase of the cell cycle. Both B16a and 3LL cells express alpha IIb beta 3 integrin receptors; however, differences in the receptor expression level were found only among B16a subpopulations. Comparison of the amount of alpha IIb beta 3 receptor expressed on cell surface with tumor cell ability to induce platelet aggregation (TCIPA) and to adhere to fibronectin or subendothelial matrix revealed a positive correlation. Pretreatment of tumor cells with alpha IIb beta 3-specific antibodies inhibited tumor cell matrix adhesion, TCIPA, and lung colony formation. We propose that alpha IIb beta 3 integrin receptor expression, tumor cell matrix adhesion, and tumor cell-induced platelet aggregation can be important parameters to indicate the metastatic potential of some tumor cells and that the alpha IIb beta 3 is a multifunctional receptor involved in both tumor cell-matrix and tumor cell-platelet interactions. Further, the correlation among cell cycle phase, metastatic ability, and receptor expression suggests that metastatic propensity may be transiently expressed and/or increased in some tumor cell subpopulations.     

Molecular mechanisms of platelet activation and aggregation induced by breast cancer cells

Tumor cell-induced platelet aggregation represents a critical process both for successful metastatic spread of the tumor and for the development of thrombotic complications in cancer patients. To get further insights into this process, we investigated and compared the molecular mechanisms of platelet aggregation induced by two different breast cancer cell lines (MDA-MB-231 and MCF7) and a colorectal cancer cell line (Caco-2). All the three types of cancer cells were able to induce comparable platelet aggregation, which, however, was observed exclusively in the presence of CaCl₂ and autologous plasma. Aggregation was supported both by fibrinogen binding to integrin αIIbβ3 as well as by fibrin formation, and was completely prevented by the serine protease inhibitor PPACK. Platelet aggregation was preceded by generation of low amounts of thrombin, possibly through tumor cells-expressed tissue factor, and was supported by platelet activation, as revealed by stimulation of phospholipase C, intracellular Ca²⁺ increase and activation of Rap1b GTPase. Pharmacological inhibition of phospholipase C, but not of phosphatidylinositol 3-kinase or Src family kinases prevented tumor cell-induced platelet aggregation. Tumor cells also induced dense granule secretion, and the stimulation of the P2Y12 receptor by released ADP was found to be necessary for complete platelet aggregation. By contrast, prevention of thromboxane A₂ synthesis by aspirin did not alter the ability of all the cancer cell lines analyzed to induce platelet aggregation. These results indicate that tumor cell-induced platelet aggregation is not related to the type of the cancer cells or to their metastatic potential, and is triggered by platelet activation and secretion driven by the generation of small amount of thrombin from plasma and supported by the positive feedback signaling through secreted ADP.     

Role of tumor cell glycoproteins immunologically related to glycoproteins Ib and IIb/IIIa in tumor cell-platelet and tumor cell-matrix interactions

A panel of monoclonal and polyclonal antibodies raised against human platelet GpIb or the GpIIb/IIIa complex were used to detect immunologically related molecules on two cell lines derived from human solid tumors. Human cervical carcinoma (MS751) and human colon carcinoma (clone A) expressed molecules immunologically related to platelet GpIb and GpIIb/IIIa complex. These molecules were localized to their plasma membranes by immunofluorescence and immunocytochemistry. The immunologically related GpIb was evenly distributed on the tumor cell membrane with occasional areas of aggregates, whereas the immunologically related GpIIb/IIIa had a pronounced punctate distribution of aggregates in prefixed cells. When MS751 or clone A cells were pretreated with antibodies against platelet GpIb and/or the GpIIb/IIIa complex, their ability to induce platelet aggregation was significantly inhibited. In addition, when tumor cells were pretreated with antibodies against the platelet IIb/IIIa complex, adherence to fibronectin-coated plates was also significantly inhibited. These results suggest a role for these immunologically related tumor cell glycoproteins in tumor cell-host cell (i.e., platelet, endothelial cells) interactions, tumor cell interactions with components of the subendothelial matrix, and subsequent tumor metastasis.     

Pharmacological evaluation of the novel thromboxane modulator BM-567 (II/II). Effects of BM-567 on osteogenic sarcoma-cell-induced platelet aggregation

Evidence exists that a large number of tumor cells such as osteosarcoma cells stimulate platelet aggregation, which can be an early step in the metastatic processes of these tumors. Thromboxane A(2) (TXA(2)) is released during platelet aggregation, and it has been suggested that this release may be pathogenic for tumor metastasis for several reasons:Some tumors release large amounts of TXA(2) compared to normal tissue.TXA(2) potentiates tumor growth in culture and increases metastasis in animals.TXA(2) is a potent stimulant of platelet aggregation and causes vascular injuries that may promote implantation of tumor cell-platelet aggregates.If TXA(2) participates in tumor metastasis, it may be hypothesized that TXA(2) inhibitors should decrease tumor metastasis. So, we have evaluated the effects of the original TXA(2) synthase inhibitor and TXA(2) receptor antagonist BM-567 on platelet aggregation induced by osteosarcoma cells using MG-63 tumor cells. Results obtained showed that this drug inhibited both MG-63 tumor-cell-induced platelet aggregation and platelet TXA(2) release following the tumor cell stimulation with IC(50) values of 3.04x10(-7) and 2.51x10(-8)M, respectively.     

Involvement of the snake toxin receptor CLEC-2, in podoplanin-mediated platelet activation, by cancer cells

Podoplanin (aggrus), a transmembrane sialoglycoprotein, is involved in tumor cell-induced platelet aggregation, tumor metastasis, and lymphatic vessel formation. However, the mechanism by which podoplanin induces these cellular processes including its receptor has not been elucidated to date. Podoplanin induced platelet aggregation with a long lag phase, which is dependent upon Src and phospholipase Cgamma2 activation. However, it does not bind to glycoprotein VI. This mode of platelet activation was reminiscent of the snake toxin rhodocytin, the receptor of which has been identified by us as a novel platelet activation receptor, C-type lectin-like receptor 2 (CLEC-2) (Suzuki-Inoue, K., Fuller, G. L., Garcia, A., Eble, J. A., Pohlmann, S., Inoue, O., Gartner, T. K., Hughan, S. C., Pearce, A. C., Laing, G. D., Theakston, R. D., Schweighoffer, E., Zitzmann, N., Morita, T., Tybulewicz, V. L., Ozaki, Y., and Watson, S. P. (2006) Blood 107, 542-549). Therefore, we sought to evaluate whether CLEC-2 serves as a physiological counterpart for podoplanin. Association between CLEC-2 and podoplanin was confirmed by flow cytometry. Furthermore, their association was dependent on sialic acid on O-glycans of podoplanin. Recombinant CLEC-2 inhibited platelet aggregation induced by podoplanin-expressing tumor cells or lymphatic endothelial cells, suggesting that CLEC-2 is responsible for platelet aggregation induced by endogenously expressed podoplanin on the cell surfaces. These findings suggest that CLEC-2 is a physiological target protein of podoplanin and imply that it is involved in podoplanin-induced platelet aggregation, tumor metastasis, and other cellular responses related to podoplanin.     

Two-color multiparametric method for flow cytometric DNA analysis of carcinomas using staining for cytokeratin and leukocyte-common antigen

Solid tumors contain heterogenous cell populations, resulting in flow cytometric (FCM) DNA quantitations of a mixture of tumor and host cells. Such mixed populations can result in dilution of the tumor cells by the host cells, in difficulty defining the diploid reference mean and in histogram peak overlap, precluding cell-cycle analysis. In this study, epithelial (tumor) cells and contaminating host cells in 100 consecutively accessioned human mammary and colorectal carcinomas were segregated in a multiparametric two-color FCM DNA analysis of intact, ethanol-fixed cells. These two carcinomas and bladder carcinomas contain a cytoskeleton of simple epithelium that is selectively stained with an FITC-labeled monoclonal antibody (MAb) to cytokeratin (CK: CAM 5.2-FITC). This MAb detects the CK 8, CK 18 and CK 19 consistently present in all layers of normal and neoplastic urothelium, colonic epithelium and mammary epithelium. Gating on CK in these tumors enables the nonstaining leukocytes, stromal fibroblasts and endothelial cells to be excluded from DNA analysis. A separate aliquot of each tumor evaluated was labeled with an MAb to leukocyte-common antigen (LCA-FITC) to serve as a patient-specific intrinsic diploid reference standard. Both the CK-labeled and LCA-labeled cells were then dual labeled for DNA with propidium iodide. This method (1) correctly identified the intrinsic diploid (LCA-positive) channel, allowing an accurate definition of normal cell DNA content for calculation of the DNA index; and (2) resulted in an increased sensitivity in the identification of both diploid and abnormal hyperdiploid tumor cell populations. It also (3) limited DNA cell cycle analysis to urothelial, colonic and mammary epithelial cells, the majority of which were neoplastic in carefully selected tumor samples. In addition, this method (4) clarified near-tetraploid populations that overlap the normal nonepithelial G2M region by diminishing the normal G2M peak and accentuating the aneuploid tetraploid G0G1 peak and (5) deconvoluted overlapping histograms composed of normal host and diploid-range or aneuploid tumor cells by gating on tissue-specific markers. This exclusion of host cells in both classes of tumors resulted in more accurate cell-cycle calculations in the former and allowed calculation of the S-phase fractions in the latter.     

Molecular identification of Aggrus/T1alpha as a platelet aggregation-inducing factor expressed in colorectal tumors

Platelets play an important role in hemostasis, thrombosis, and antimicrobial host defense and are also involved in the induction of inflammation, tissue repair, and tumor metastasis. We have previously characterized the platelet aggregation-inducing sialoglycoprotein (Aggrus/gp44) overexpressed on the surface of tumor cells. Because a platelet aggregation-neutralizing 8F11 monoclonal antibody that could specifically recognize Aggrus suppressed tumor-induced platelet aggregation, we have previously purified Aggrus by 8F11-affinity chromatography and found that purified Aggrus possessed the ability to induce aggregation of platelets. Here we show that Aggrus is identical to the T1alpha/gp38P/OTS-8 antigen, the function of which in tumors is unknown. Expression of mouse Aggrus and its human homologue (also known as T1alpha-2/gp36) induced platelet aggregation without requiring plasma components. Using the 8F11 antibody, we identified the highly conserved platelet aggregation-stimulating domain with putative O-glycosylated threonine residues as the critical determinant for exhibiting platelet aggregation-inducing capabilities. We compared the expression level of human aggrus mRNA using an array containing 160 cDNA pair samples derived from multiple human tumorigenic and corresponding normal tissues from individual patients. We found that expression level of aggrus was enhanced in most colorectal tumor patients. To confirm the protein expression, we generated anti-human Aggrus polyclonal antibodies. Immunohistochemical analysis revealed that Aggrus expression was frequently up-regulated in colorectal tumors. These results suggest that Aggrus/T1alpha is a newly identified, platelet aggregation-inducing factor expressed in colorectal tumors.     

Functional analyses of the cancer stem cell-like properties of human endometrial tumor initiating cells

Recent data suggest that rare stem cell populations with the capacity to self renew and drive tumor formation are a feature of solid tumors. Several investigators have identified putative stem cells from solid tumors and cancer cell lines following isolation of a side population (SP) defined by dye exclusion. We investigated this parameter in our efforts to identify an endometrial cancer (EnCa) stem cell population. Multiple EnCa cell lines were assessed and verapamil sensitive SP and non-SP cells were isolated from two human EnCa cell lines. The functional significance of the SP and non-SP derived from AN3CA was evaluated in vitro and in vivo. SP cells proliferated at a significantly slower rate than the non-SP fraction, and a larger proportion of the SP cells were in G1 phase of the cell cycle as compared to the non-SP fraction. The SP fraction was more resistant to the chemotherapeutic agent paclitaxel. The SP comprised ~0.02% of the initial AN3CA cell population and this proportion of SP cells was maintained within the larger heterogeneous population following repeated passages of purified SP cells. These findings suggest that SP cells derived from the AN3CA cell line have the stem cell properties of low proliferative activity, chemoresistance, and self-renewal. We also tested relative tumor formation activity of the SP and non-SP fractions. Only the SP fraction was tumorigenic. Additionally, we identified SP fractions in primary EnCa. Together these results are consistent with the hypothesis that EnCa contain a subpopulation of tumor initiating cells with stem like properties.     

Bioactive polar lipids from Chroococcidiopsis sp. (Cyanobacteria)

Many studies indicate that various bioactive metabolites subsist in cyanobacteria. Glycolipids of cyanobacteria are reported as molecules that exert specific bioactivities. In this study, total lipids of Chroococcidiopsissp., a coccoid cyanobacterium isolated from a Greek cave, were separated into neutral and polar-lipids and the latter were further fractionated by high-performance liquid chromatography (HPLC). Each polar lipid fraction was tested in vitro for its ability to inhibit platelet-activating factor (PAF)- and thrombin-induced washed rabbit platelet aggregation and/or to cause platelet aggregation. The structures of the most active fractions were elucidated by biological assays and identified by electrospray mass spectrometry. One fraction was a potent inhibitor of PAF-induced platelet aggregation. Structural studies of this fraction indicated the existence of phospho-glyco analog of ceramide. Another fraction that was a potent inhibitor of PAF- as well as of thrombin-induced platelet aggregation was structurally elucidated as a phospho-acetylated glyco-analog of diglyceride. The fraction that induced platelet aggregation was identified as a phospho-acetylated-glyco analog of ceramide. These novel bioactive polar lipids in cyanobacteria in regard to the structure and biological activity may contribute to the allergic character of cyanobacteria.     

Electrophoretic Heterogeneity of Pigmented Hamster Melanoma Cells

Pigmented hamster melanoma tumors growing in situ contain two subpopulations of melanoma cells that have different electrophoretic mobilities (EPM). A mild neuraminidase treatment, which removes sialic acid residues from the cell surface glycoproteins, reduces the EPM of both groups of melanoma cells yielding an electrophoretically uniform population. This shows that the differences in the EPM between the subpopulations of pigmented melanoma cells stem from the different content of sialic acid residues on the cell surface. The relationship between the different EPM melanoma cell subpopulations was, therefore, examined during tumor growth, development, and formation of metastases. The relative content of cells having high electrophoretic mobility, the “fast moving” cells, increases as the tumors grow larger. However, tumors of the same diameter contain nearly the same fraction of “fast moving” cells despite their age. The proportion of the “fast moving” cells is significantly higher in the central part than in the outermost layer of pigmented melanoma tumors. These data suggest that the development of “fast moving” cells is promoted by some size-dependent changes in the intratumor environment. In vivo selection of melanoma cells for their ability to colonize lungs renders tumors that reveal elevated metastatic potential and contain a significantly higher fraction of cells possessing high electrophoretic mobility than the parent tumor. Moreover, the metastatic nodules contain a remarkably elevated fraction of the “fast moving” cells. The reported correlation between the “fast moving” cell fraction and the metastatic potential suggests that the relative content of cells having high electrophoretic mobility may determine the metastaticity of pigmented hamster melanoma.     

Adenosine diphosphate-induced aggregation of human platelets in flow through tubes. II. Effect of shear rate, donor sex, and ADP concentration.

The effect of shear rate on the adenosine diphosphate-induced aggregation of human platelets in Poiseuille flow was studied using the method described in part I (Bell, D.N., S. Spain, and H.L. Goldsmith. 1989. Biophys. J. 56:817-828). The rate and extent of aggregation in citrated platelet-rich plasma were measured over a range of mean transit time from 0.2 to 8.6 s and mean tube shear rate, G, from 41.9 to 1,920 s-1. At 0.2 microM ADP, changes in the single platelet concentration with time suggest that more than one type of platelet-platelet bond mediates platelet aggregation at physiological shear rates. At low G, a high initial rate of aggregation reflects the formation of a weak bond of high affinity, the strength of which diminishes with time. Here, the fraction of collisions yielding stable doublets, the collision efficiency, reached a maximum of 26%. The collision efficiency decreased with increasing G and was accompanied by a progressive delay in the onset of aggregation. However, the gradual expression of a more shear rate-resistant bond at high shear rates and long mean transit times produced a subsequent increase in collision efficiency and a corresponding increase in the rate of aggregation. Although the collision efficiencies here were less than 1%, the high collision frequencies were able to sustain a high rate of aggregation. At 0.2 microM ADP, aggregate size generally decreased with increasing G. At 1.0 microM ADP, aggregate size was still limited at high shear rates even though the rate of single platelet aggregation was much higher than at 0.2 microM ADP. Platelet aggregation was greater for female than for male donors, an effect related to differences in the hematocrit of donors before preparing platelet-rich plasma.     

Nuclear non-histone protein content of G0/G1 cells as related to growth fraction in mouse mammary tumors.

In eight mouse mammary tumors with varying growth fractions DNA and non-histone nuclear protein (NHNP) were determined by absorption cytophotometry of Feulgen-Naphthol Yellow S stained, isolated cells. It was found that: 1. The mean NHNP content of cells with postmitotic DNA content (G0 + G1) increased with increasing growth fraction. 2. The mean NHNP content of S and G2 cells in the eight tumors did not vary significantly with growth fraction. 3. The frequency distributions of NHNP in G0/G1 cells were unimodal and right-skewed. The results are interpreted as follows: A) G0 cells differ from G1 cells by their lower content of NHNP. B). If it is assumed that the G0 and G1 compartments are arranged in series, the cells in the transition from G0 to late G1 may account for the unimodality and skewedness of the NHNP frequency distributions of postmitotic cells.     

New variants of B16 mouse melanoma: differentiation and metastatic properties

A system of tumor transplantation has been developed to select metastatic variants of B16 in mutants of the C57BL/6J black strain of mice. The effects of transplantation into nonagouti a/a and mutant recipients on the production of melanin and on the metastatic potential of tumors were investigated. Transplantation of the pigmented B16 melanoma from a nonagouti black a/a host to a yellow mutant Ay/a recipient resulted in an achromic and metastatic variant melanoma, designated YB16. The amelanotic phenotype occurred consistently after more than ten passages through yellow mice and simultaneously with an increase in the incidence of pulmonary metastases. When YB16 was transplanted back to the nonagouti black a/a host, a second variant, MB16, characterized by its variable pigmentation, was obtained. Pigmented and/or entirely achromic tumors were observed. MB16 was dramatically more metastatic than B16 and YB16 when injected s.c. or i.v. Metastases in the lungs were pigmented and/or achromic. The properties of tumor cells derived from artificially induced metastases were investigated after s.c. and i.v. injections. Whereas the metastatic cells expressed a potent ability to generate metastases when injected s.c., no differences in the incidence of metastases, as compared to the metastatic potential of cells of parental origin, were observed after i.v. injection. In the MB16 variant, there appeared to be an inverse relationship between differentiation (production of melanins) and malignancy. Our results demonstrate that differentiation and metastatic behaviour are dependent on specific mutations in the host environment which generate a pool of tumor cells from which highly metastatic variants can be selected.     

Costimulation of Gi- and G12/G13-mediated signaling pathways induces integrin alpha IIbbeta 3 activation in platelets

Platelet activation is a complex process induced by a variety of stimuli, which act in concert to ensure the rapid formation of a platelet plug at places of vascular injury. We show here that fibrillar collagen, which initiates platelet activation at the damaged vessel wall, activates only a small fraction of platelets in suspension directly, whereas the majority of platelets becomes activated by mediators released from collagen-activated platelets. In Galpha(q)-deficient platelets that do not respond with activation of integrin alpha(IIb)beta(3) to a variety of mediators like thromboxane A2 (TXA2), thrombin, or ADP, collagen at high concentrations was able to induce aggregation, an effect that could be blocked by antagonists of the TXA2 or P2Y12 receptors. The activation of TXA2 or P2Y12 receptors alone, which in Galpha(q)-deficient platelets couple to G12/G13 and Gi, respectively, did not induce platelet integrin activation or aggregation. However, concomitant activation of both receptors resulted in irreversible integrin alpha(IIb)beta3-mediated aggregation of Galpha(q)-deficient platelets. Thus, the activation of G12/G13- and Gi-mediated signaling pathways is sufficient to induce integrin alpha(IIb)beta3 activation. Although G(q)-mediated signaling plays an important role in platelet activation, it is not strictly required for the activation of integrin alpha(IIb)beta3. This indicates that the efficient induction of platelet aggregation through G-protein-coupled receptors is an integrated response mediated by various converging G-protein-mediated signaling pathways involving G(q) and G(i) as well as G12/G13.     

EL-4 tumor cell-induced human and rabbit platelet aggregations

EL-4 tumor cells were assayed in vitro for their ability to aggregate two kinds of platelets. An inhibition study showed that the EL-4 tumor cell can induce platelet aggregation by at least two different mechanisms. One, mediated by thrombin, was dominant with rabbit platelets because hirudin, which specifically inhibits thrombin, considerably suppressed the rabbit platelet aggregation induced by EL-4 tumor cells. In contrast, EL-4 cells induced the aggregation of human platelets even in citrated PRP. It is the apyrase-sensitive pathway that is believed to work in human platelets. The human platelet responses to EL-4 tumor cells clearly differed from those of rabbit platelets in terms of inhibition by hirudin and apyrase and of reactivity in citrated PRP. Both phospholipase A2 and dibutyryl cAMP strongly inhibited EL-4 tumor cell-induced platelet aggregation in both rabbit and human platelets. These two compounds may block a vital step in platelet aggregation that is elicited by the EL-4 tumor cells. Our results show that human platelet response to tumor cells is not necessarily deducible from experimental data obtained with animal platelets.     

Effects of a stable prostacyclin analogue on platelet activity and on host immunocompetence in mice.

The stable prostacyclin (PGI2) analogue, iloprost, is a potent inhibitor of both tumor cell-induced platelet aggregation and of experimental metastasis in mice. To explore possible mechanisms of antimetastatic effect of iloprost, we measured the effect of this drug on both platelet aggregation and immunocompetence in the mouse. Iloprost (4 x 10(-8) M) inhibited platelet aggregation as induced by a mixture of collagen and epinephrine for at least 180 minutes of incubation, and completely reversed platelet aggregation when added during the second wave of aggregation. In addition, aggregation of platelets obtained from iloprost-treated mice (0.2 mg/kg) was completely inhibited for at least 90 minutes of incubation. Moreover, iloprost pretreatment in vivo counteracted tumor cell-induced thrombocytopenia. Thus, mouse platelets were equally sensitive to the inhibitory effect of iloprost on aggregation as platelets of other species including humans. Effects of iloprost on parameters of host immunocompetence that may influence tumor growth and metastasis formation were also evaluated. Iloprost treatment increased significantly macrophage cytostasis to tumor cells, natural killer (NK) lytic activity of spleen cells and T-cell mediated cytotoxicity ex vivo. These results suggested that the antimetastatic effect of iloprost in the mouse may be attributable to multiple mechanisms including inhibition of platelet aggregation and stimulation of certain host immune functions.     

Interaction of tumor cells and immune system in the metastatic process

The metastatic process is rather complicated and relatively inefficient. Millions of tumor cells are constantly shedding from the primary tumor into the blood stream, but very few of them are able to form metastatic tumors in the different organs or tissues of the host. It is widely accepted that metastatic cells have to possess a complex array of various properties that allow them to complete the metastatic cascade. The realization of the metastatic potential largely depends on the ability of tumor cells to evade host defense mechanisms. The potential role of specific and nonspecific immune mechanisms in the control of metastatic spread and growth is the subject of the present review. A better understanding of the mechanisms of antimetastatic defense is of prime importance for development of efficient immunotherapeutic methods for the treatment and eradication of disseminated tumor metastases.     

Effects of a stable prostacyclin analogue on platelet activity and on host immunocompetence in mice

The stable prostacyclin (PGI₂) analogue, iloprost, is a potent inhibitor of both tumor cell-induced platelet aggregation and of experimental metastasis in mice. To explore possible mechanisms of antimetastatic effect of iloprost, we measured the effect of this drug on both platelet aggregation and immunocompetence in the mouse. Iloprost (4×10⁻⁸M) inhibited platelet aggregation as induced by a mixture of collagen and epinephrine for at least 180 minutes of incubation, and completely reversed platelet aggregation when added during the second wave of aggregation. In addition, aggregation of platelets obtained from iloprost-treated mice (0.2 mg/kg) was completely inhibited for at least 90 minutes of incubation. Moreover, iloprost pretreatment counteracted tumor cell-induced thrombocytopenia. Thus, mouse platelets were equally sensitive to the inhibitory effect of iloprost on aggregation as platelets of other species including humans. Effects of iloprost on parameters of host immunocompetence that may influence tumor growth and metastasis formation were also evaluated. Iloprost treatment increased significantly macrophage cytostasis to tumor cells, natural killer (NK) lytic activity of spleen cells and T-cell mediated cytotoxicity . These results suggested that the antimetastatic effect of iloprost in the mouse may be attributable to multiple mechanisms including inhibition of platelet aggregation and stimulation of certain host immune functions.     

Glutathione-dependent regulation of platelet aggregation with neutrophils and tumor cells

It is shown that in the presence of reduced glutathione at low concentrations (1-5 microM) the extent of platelet aggregation with neutrophils increases and the lag period of platelet aggregation induced by tumor cells decreases. At the same time in the presence of reduced glutathione at high concentration (3 mM) the extent of platelet aggregation with neutrophils decreases, and the lag period of platelet aggregation induced by tumor cells increases. It is established that glutathione-dependent regulation of the intercellular contact formation between platelets and neutrophils depends on the ratio of glutathione oxidized and reduced forms: at fixed total glutathione concentration of 5 microM, increase of glutathione redox potential from -175 mV to 0 mV led to reduction in platelet aggregation with neutrophils. Thus, it is shown for the first time, that GSH has priming effect on the platelet aggregation with neutrophils and tumor cells, which may contribute to the regulation of inflammatory diseases and cancer.