The Role of CD36/GPIV in Platelet Biology.

CD36 (also known as platelet glycoprotein IV) is expressed by a variety of different cell entities, where it possesses functions as a signaling receptor, but additionally acts as a transporter for long-chain fatty acids. This dual function of CD36 has been investigated for its relevance in immune and nonimmune cells. Although CD36 was first identified on platelets, the understanding of the role of CD36 in platelet biology remained scarce for decades. In the past few years, several discoveries have shed a new light on the CD36 signaling activity in platelets. Notably, CD36 has been recognized as a sensor for oxidized low-density lipoproteins in the circulation that mitigates the threshold for platelet activation under conditions of dyslipidemia. Thus, platelet CD36 transduces atherogenic lipid stress into an increased risk for thrombosis, myocardial infarction, and stroke. The underlying pathways that are affected by CD36 are the inhibition of cyclic nucleotide signaling pathways and simultaneously the induction of activatory signaling events. Furthermore, thrombospondin-1 secreted by activated platelets binds to CD36 and furthers paracrine platelet activation. CD36 also serves as a binding hub for different coagulation factors and, thus, contributes to the plasmatic coagulation cascade. This review provides a comprehensive overview of the recent findings on platelet CD36 and presents CD36 as a relevant target for the prevention of thrombotic events for dyslipidemic individuals with an elevated risk for thrombosis.

Exostosin 1 Knockdown Induces Chemoresistance in MV3 Melanoma Cells by Upregulating JNK and MEK/ERK Signaling.

Heparan sulfate proteoglycans (HSPGs) possess various functions driving malignancy of tumors. However, their impact on tumor cell sensitivity to cytotoxic treatment is far less understood. Aiming to investigate this, we depleted HSPGs by downregulating Exostosin 1 (EXT1), a key enzyme in HS formation, or upregulating heparanase in human MV3 human melanoma cells, and investigated their response to cytotoxic drugs. Cytotoxicity of trametinib, doxorubicin, and mitoxantrone was detected by MTT assay. Insights into intracellular signaling was provided by kinome protein profiler array, and selected kinases were inhibited to investigate their impact on cell sensitization and migratory dynamics. EXT1 knockdown (EXT1kd) in MV3 cells affected the activity of doxorubicin and mitoxantrone, significantly increasing EC50 values two- or fourfold, respectively. Resistance formation was scarcely related to HSPG deficiency, suggested by enzymatic cleavage of HSPG in control cells. Notably, EXT1kd induced an upregulation of EGFR signaling via JNK and MEK/ERK, and hence blocking these kinases returned resistance to a sensitive level. JNK appeared as a key signal component, also inducing higher migratory activity of EXT1kd cells. Furthermore, EXT1kd upregulated thrombotic properties of MV3 cells, indicated by tissue factor and PAR-1 expression, functionally reflected by a stronger activation of platelet aggregation. EXT1 was confirmed to act as a tumor suppressor, shown here for the first time to affect chemosensitivity of melanoma cells.

The GPIb-IX complex on platelets: insight into its novel physiological functions affecting immune surveillance, hepatic thrombopoietin generation, platelet clearance and its relevance for cancer development and metastasis.

The glycoprotein (GP) Ib-IX complex is a platelet receptor that mediates the initial interaction with subendothelial von Willebrand factor (VWF) causing platelet arrest at sites of vascular injury even under conditions of high shear. GPIb-IX dysfunction or deficiency is the reason for the rare but severe Bernard-Soulier syndrome (BSS), a congenital bleeding disorder. Although knowledge on GPIb-IX structure, its basic functions, ligands, and intracellular signaling cascades have been well established, several advances in GPIb-IX biology have been made in the recent years. Thus, two mechanosensitive domains and a trigger sequence in GPIb were characterized and its role as a thrombin receptor was deciphered. Furthermore, it became clear that GPIb-IX is involved in the regulation of platelet production, clearance and thrombopoietin secretion. GPIb is deemed to contribute to liver cancer development and metastasis. This review recapitulates these novel findings highlighting GPIb-IX in its multiple functions as a key for immune regulation, host defense, and liver cancer development.

Tumour cell-activated platelets modulate the immunological activity of CD4+, CD8+, and NK cells, which is efficiently antagonized by heparin.

Platelets, key players in haemostasis, are progressively investigated with respect to their role in immunity and inflammation. Although the platelet support to haematogenous cancer cell metastasis has been the subject of multiple studies, their impact on anti-cancer immunity remains unaddressed. Here, we investigated the immunomodulatory potential of platelets upon their activation by MDA-MB-231 breast cancer cells in various in vitro approaches. We provide evidence that platelets as well as their tumour cell-induced releasates increased the ratio of regulatory T cells, shaping an immunosuppressive phenotype in isolated CD4+ cultures. The influence on CD8+ T cells was assessed by detecting the expression of activation markers CD25/CD69 and release of cytolytic and pro-inflammatory proteins. Notably, the platelet preparations differentially influenced CD8+ T cell activation, while platelets were found to inhibit the activation of CD8+ T cells, platelet releasates, in contrast, supported their activation. Furthermore, the NK cell cytolytic activity was attenuated by platelet releasates. Low molecular weight heparin (LMWH), the guideline-based anticoagulant for cancer-associated thrombotic events, is known to interfere with tumour cell-induced platelet activation. Thus, we aimed to investigate whether, unfractionated heparin, LMWH or novel synthetic heparin mimetics can also reverse the immunosuppressive platelet effects. The releasate-mediated alteration in immune cell activity was efficiently abrogated by heparin, while the synthetic heparin mimetics partly outperformed the commercial heparin derivatives. This is the first report on the effects of heparin on rebalancing immunosuppression in an oncological context emerging as a novel aspect in heparin anti-tumour activities.

Finite-time Lyapunov fluctuations and the upper bound of classical and quantum out-of-time-ordered expansion rate exponents.

This Letter demonstrates for chaotic maps [logistic, classical, and quantum standard maps (SMs)] that the exponential growth rate (Λ) of the out-of-time-ordered four-point correlator is equal to the classical Lyapunov exponent (λ) plus fluctuations (Δ^{(fluc)}) of the one-step finite-time Lyapunov exponents (FTLEs). Jensen's inequality provides the upper bound λ≤Λ for the considered systems. Equality is restored with Λ=λ+Δ^{(fluc)}, where Δ^{(fluc)} is quantified by k-higher-order cumulants of the (covariant) FTLEs. Exact expressions for Λ are derived and numerical results using k=20 furnish Δ^{(fluc)}∼ln(sqrt[2]) for all maps (large kicking intensities in the SMs).

Insulin-like Growth Factor Binding Protein-2 (IGFBP2) Is a Key Molecule in the MACC1-Mediated Platelet Communication and Metastasis of Colorectal Cancer Cells.

Tumor cell crosstalk with platelets and, subsequently, their activation are key steps in hematogenous tumor metastasis. MACC1 is an oncogene involved in molecular pathogenesis of colorectal cancer (CRC) and other solid tumor entities, mediating motility and metastasis, making MACC1 an accepted prognostic biomarker. However, the impact of MACC1 on platelet activation has not yet been addressed. Here, we investigated the activation of platelets by human CRC cells upon MACC1 modulation, indicated by platelet aggregation and granule release. These approaches led to the identification of insulin-like growth factor binding protein-2 (IGFBP2) as a functional downstream molecule of MACC1, affecting communication with platelets. This was confirmed by an shRNA-mediated IGFBP2 knockdown, while maintaining MACC1 activity. Although IGFBP2 displayed an attenuated platelet activation potential, obviously by scavenging IGF-I as a platelet costimulatory mediator, the MACC1/IGFBP2 axis did not affect the thrombin formation potential of the cells. Furthermore, the IGFBP2/MACC1-driven cell migration and invasiveness was further accelerated by platelets. The key role of IGFBP2 for the metastatic spread in vivo was confirmed in a xenograft mouse model. Data provide evidence for IGFBP2 as a downstream functional component of MACC1-driven metastasis, linking these two accepted oncogenic biomarkers for the first time in a platelet context.

Another structural correction for 1-oxo-1H-phenalene-2,3-dicarbonitriles: Synthesis of a potent BCL-2 inhibiting 7-phenoxy derivative.

Aromatic scaffolds are an important part of biologically active compounds and molecular probes used to study biochemical pathways and the involved targeted proteins of interest. 1-Oxo-1H-phenalene-2,3-dicarbonitrile-based compounds have been described as inhibitors of the BCL-2 family of proteins, and this core structure represents numerous possibilities for modifications that could lead to improved inhibitory potencies. Many studies demonstrated intriguing characteristics of these compounds in terms of reactivity and, interestingly, some contradictory literature reports appeared about reaction outcomes to synthesize them. Here, we initially provide a condensed overview of transformations performed on the phenalene scaffold, followed by the resynthesis of a 6-phenoxy-substituted derivative. We show that the initial determination of this particular structure was wrong and provide two-dimensional nuclear magnetic resonance (NMR) evidence to assign the structure properly. When preparing new derivatives using the same synthetic route, we observed 6- and 7-substituted regioisomers. After confirming their structures by NMR experiments, the ability of these compounds to inhibit BCL-2 was evaluated. The most potent 1-oxo-1H-phenalene-2,3-dicarbonitrile derivatives inhibited BCL-2 in the nanomolar range and showed double-digit micromolar cytotoxicity against four different cancer cell lines.

A Combined Activity of Thrombin and P-Selectin Is Essential for Platelet Activation by Pancreatic Cancer Cells.

Pancreatic cancer patients have an elevated risk of suffering from venous thrombosis. Among several risk factors that contribute to hypercoagulability of this malignancy, platelets possess a key role in the initiation of clot formation. Although single mechanisms of platelet activation are well-known in principle, combinations thereof and their potential synergy to mediate platelet activation is, in the case of pancreatic cancer, far from being clear. Applying an inhibitor screening approach using light transmission aggregometry, dense granule release, and thrombin formation assays, we provide evidence that a combination of tissue factor-induced thrombin formation by cancer cells and their platelet P-selectin binding is responsible for AsPC-1 and Capan-2 pancreatic cancer cell-mediated platelet activation. While the blockade of one of these pathways leads to a pronounced inhibition of platelet aggregation and dense granule release, the simultaneous blockade of both pathways is inevitable to prevent platelet aggregation completely and minimize ATP release. In contrast, MIA PaCa-2 pancreatic cancer cells express reduced levels of tissue factor and P-selectin ligands and thus turn out to be poor platelet activators. Consequently, a simultaneous blockade of thrombin and P-selectin binding seems to be a powerful approach, as mediated by heparin to crucially reduce the hypercoagulable state of pancreatic cancer patients.

Detection of Tumor Cell-Induced Platelet Aggregation and Granule Secretion.

Hematogenous metastatic spread of cancer is strongly dependent on and triggered by an intensive interplay of tumor cells with platelets. Immediately after entering the blood vascular system, tumor cells are surrounded by a platelet cloak, which protects them physically from shear stress and from attacks by the immune surveillance. Furthermore, tumor cell binding activates platelets, which in turn release growth factors and chemokines to recruit myeloid cells into the platelet/tumor cell microemboli, eventually create a permissive microenvironment in the early metastatic niche. Although the molecular mechanisms of tumor cells to activate platelets appear versatile being a matter of further research, interference with platelet activation turns out to be an attractive target to efficiently inhibit tumor metastasis. Some experimental assays are generally recognized to follow tumor cell-induced platelet activation (TCIPA), which provide an insight into the molecular mechanisms of TCIPA and allow searching for potential inhibitors. In this chapter, we describe the two most prominent experimental assays to follow TCIPA, namely platelet aggregation and platelet granule secretion, experimentally realized by dense granules´ ATP quantification. Although light transmission aggregometry and ATP detection from dense granule secretion are two age-old techniques, they are still highly relevant to provide reliable information concerning platelet activation status since all tumor cell-derived molecular triggers are covered and monitored in the experimental outcome.

Inhibition of Tumor-Host Cell Interactions Using Synthetic Heparin Mimetics.

Low-molecular-weight heparin (LMWH) is the guideline-based drug for antithrombotic treatment of cancer patients, while its direct antitumor effects are a matter of ongoing debate. Although therapeutically established for decades, LMWH has several drawbacks mainly associated with its origin from animal sources. Aiming to overcome these limitations, a library of synthetic heparin mimetic polymers consisting of homo- and copolymers of sulfonated and carboxylated noncarbohydrate monomers has recently been synthesized via reversible addition-fragmentation chain transfer polymerization. These heparin mimetics were investigated for their capacities to interfere with simulated steps of tumor cell metastasis. Among them, homo- and copolymers from sodium 4-styrenesulfonate (poly(SSS)) with acrylic acid (poly(SSS-co-AA)) with an MW between 5 and 50 kDa efficiently attenuated cancer cell-induced coagulation and thus platelet activation and degranulation similar to or even better than LMWH. Furthermore, independent of anticoagulant activities, these polymers affected other metastasis-relevant targets with impressive affinities. Hence, they blocked heparanase enzymatic activity outmatching commercial heparins or a glycosidic drug candidate. Furthermore, these polymers bind P-selectin and the integrin VLA-4 similar to or even better than heparin, indicated by a biosensor approach and thus efficiently blocked melanoma cell binding to endothelium under blood flow conditions. This is the first report on the prospects of synthetic heparin mimetics as promising nontoxic compounds in oncology to potentially substitute heparin as an anticoagulant and to better understand its role as an antimetastatic drug.

Insight into Cisplatin-Resistance Signaling of W1 Ovarian Cancer Cells Emerges mTOR and HSP27 as Targets for Sensitization Strategies.

The microenvironment possesses a strong impact on the tumor chemoresistance when cells bind to components of the extracellular matrix. Here we elucidate the signaling pathways of cisplatin resistance in W1 ovarian cancer cells binding to collagen type 1 (COL1) and signaling interference with constitutive cisplatin resistance in W1CR cells to discover the targets for sensitization. Proteome kinase arrays and Western blots were used to identify the signaling components, their impact on cisplatin resistance was evaluated by inhibitory or knockdown approaches. W1 cell binding to COL1 upregulates integrin-associated signals via FAK/PRAS40/mTOR, confirmed by ß1-integrin (ITGB1) knockdown. mTOR appears as key for resistance, its blockade reversed COL1 effects on W1 cell resistance completely. W1CR cells compensate ITGB1-knockdown by upregulation of discoidin domain receptor 1 (DDR1) as alternative COL1 sensor. COL1 binding via DDR1 activates the MAPK pathway, of which JNK1/2 appears critical for COL1-mediated resistance. JNK1/2 inhibition inverts COL1 effects in W1CR cells, whereas intrinsic cisplatin resistance remained unaffected. Remarkably, knockdown of HSP27, another downstream MAPK pathway component overcomes intrinsic resistance completely sensitizing W1CR cells to the level of W1 cells for cisplatin cytotoxicity. Our data confirm the independent regulation of matrix-induced and intrinsic chemoresistance in W1 ovarian cancer cells and offer novel targets for sensitization.

Targeting Discoidin Domain Receptor 1 (DDR1) Signaling and Its Crosstalk with ß1-integrin Emerges as a Key Factor for Breast Cancer Chemosensitization upon Collagen Type 1 Binding.

Collagen type 1 (COL1) is a ubiquitously existing extracellular matrix protein whose high density in breast tissue favors metastasis and chemoresistance. COL1-binding of MDA-MB-231 and MCF-7 breast cancer cells is mainly dependent on ß1-integrins (ITGB1). Here, we elucidate the signaling of chemoresistance in both cell lines and their ITGB1-knockdown mutants and elucidated MAPK pathway to be strongly upregulated upon COL1 binding. Notably, Discoidin Domain Receptor 1 (DDR1) was identified as another important COL1-sensor, which is permanently active but takes over the role of COL1-receptor maintaining MAPK activation in ITGB1-knockdown cells. Consequently, inhibition of DDR1 and ERK1/2 act synergistically, and sensitize the cells for cytostatic treatments using mitoxantrone, or doxorubicin, which was associated with an impaired ABCG2 drug efflux transporter activity. These data favor DDR1 as a promising target for cancer cell sensitization, most likely in combination with MAPK pathway inhibitors to circumvent COL1 induced transporter resistance axis. Since ITGB1-knockdown also induces upregulation of pEGFR in MDA-MB-231 cells, inhibitory approaches including EGFR inhibitors, such as gefitinib appear promising for pharmacological interference. These findings provide evidence for the highly dynamic adaptation of breast cancer cells in maintaining matrix binding to circumvent cytotoxicity and highlight DDR1 signaling as a target for sensitization approaches.

Glycosaminoglycans as Tools to Decipher the Platelet Tumor Cell Interaction: A Focus on P-Selectin.

Tumor cell-platelet interactions are regarded as an initial crucial step in hematogenous metastasis. Platelets protect tumor cells from immune surveillance in the blood, mediate vascular arrest, facilitate tumor extravasation, growth, and finally angiogenesis in the metastatic foci. Tumor cells aggregate platelets in the bloodstream by activation of the plasmatic coagulation cascade and by direct contact formation. Antimetastatic activities of unfractionated or low molecular weight heparin (UFH/LMWH) can undoubtedly be related to attenuated platelet activation, but molecular mechanisms and contribution of contact formation vs. coagulation remain to be elucidated. Using a set of non-anticoagulant heparin derivatives varying in size or degree of sulfation as compared with UFH, we provide insight into the relevance of contact formation for platelet activation. Light transmission aggregometry and ATP release assays confirmed that only those heparin derivatives with P-selectin blocking capacities were able to attenuate breast cancer cell-induced platelet activation, while pentasaccharide fondaparinux was without effects. Furthermore, a role of P-selectin in platelet activation and signaling could be confirmed by proteome profiler arrays detecting platelet kinases. In this study, we demonstrate that heparin blocks tumor cell-induced coagulation. Moreover, we identify platelet P-selectin, which obviously acts as molecular switch and controls aggregation and secretion of procoagulant platelets.

ß1-Integrin binding to collagen type 1 transmits breast cancer cells into chemoresistance by activating ABC efflux transporters.

Molecular interactions of tumor cells with the microenvironment are regarded as onset of chemotherapy resistance, referred to as cell adhesion mediated drug resistance (CAM-DR). Here we elucidate a mechanism of CAM-DR in breast cancer cells in vitro. We show that human MCF-7 and MDA-MB-231 breast cancer cells decrease their sensitivity towards cisplatin, doxorubicin, and mitoxantrone cytotoxicity upon binding to collagen type 1 (COL1) or fibronectin (FN). The intracellular concentrations of doxorubicin and mitoxantrone were decreased upon cell cultivation on COL1, while cellular cisplatin levels remained unaffected. Since doxorubicin and mitoxantrone are transporter substrates, this refers to ATP binding cassette (ABC) efflux transporter activities. The activation of the transporters BCRP, P-gp and MRP1 was shown by fluorescence assays to distinguish the individual input of these transporters to resistance in presence of COL1 and related to their expression levels by western blot. An ABC transporter inhibitor was able to re-sensitize COL1-treated cells for doxorubicin and mitoxantrone toxicity. Antibody-blocking of ß1-integrin (ITGB1) induced sensitization towards the indicated cytostatic drugs by attenuating the increased ABC efflux activity. This refers to a key role of ITGB1 for matrix binding and subsequent transporter activation. A downregulation of α2ß1 integrin following COL1 binding appears as clear indication for the relationship between ITGB1 and ABC transporters in regulating resistance formation, while knockdown of ITGB1 leads to a significant upregulation of all three transporters. Our data provide evidence for a role of CAM-DR in breast cancer via an ITGB1 - transporter axis and offer promising therapeutic targets for cancer sensitization.

The Impact of Integrin-Mediated Matrix Adhesion on Cisplatin Resistance of W1 Ovarian Cancer Cells.

BACKGROUND: Tumor cell binding to the microenvironment is regarded as the onset of therapeutic resistance, referred to as cell adhesion mediated drug resistance (CAM-DR). Here we elucidate whether CAM-DR occurs in ovarian cancer cells and contributes to still-existing cisplatin resistance. METHODS: Cultivation of W1 and cisplatin-resistant W1CR human ovarian cancer cells on collagen-type I (COL1) was followed by whole genome arrays, MTT assays focusing cisplatin cytotoxicity, and AAS detection of intracellular platinum levels. Expression of cisplatin transporters Ctr1 and MRP2 was analyzed. Mechanistic insight was provided by lentiviral ß1-integrin (ITGB1) knockdown, or inhibition of integrin-linked kinase (ILK). RESULTS: EC50 values of cisplatin cytotoxicity increased twofold when W1 and W1CR cells were cultivated on COL1, associated with significantly diminished intracellular platinum levels. Transporter deregulation could not be detected at mRNA levels but appears partially responsible at protein levels. The ITGB1 knockdown confirms that CAM-DR follows a COL1/ITGB1 signaling axis in W1 cells; thus, a blockade of ILK re-sensitized W1 cells on COL1 for cisplatin. In contrast, CAM-DR adds to cisplatin resistance in W1CR cells independent of ITGB1. CONCLUSIONS: CAM-DR appears relevant for ovarian cancer cells, adding to existing genetic resistance and thus emerges as a target for sensitization strategies.

Virulence-associated protein A from Rhodococcus equi is an intercompartmental pH-neutralising virulence factor.

Professional phagocytic cells such as macrophages are a central part of innate immune defence. They ingest microorganisms into membrane-bound compartments (phagosomes), which acidify and eventually fuse with lysosomes, exposing their contents to a microbicidal environment. Gram-positive Rhodococcus equi can cause pneumonia in young foals and in immunocompromised humans. The possession of a virulence plasmid allows them to subvert host defence mechanisms and to multiply in macrophages. Here, we show that the plasmid-encoded and secreted virulence-associated protein A (VapA) participates in exclusion of the proton-pumping vacuolar-ATPase complex from phagosomes and causes membrane permeabilisation, thus contributing to a pH-neutral phagosome lumen. Using fluorescence and electron microscopy, we show that VapA is also transferred from phagosomes to lysosomes where it permeabilises the limiting membranes for small ions such as protons. This permeabilisation process is different from that of known membrane pore formers as revealed by experiments with artificial lipid bilayers. We demonstrate that, at 24 hr of infection, virulent R. equi is contained in a vacuole, which is enriched in lysosome material, yet possesses a pH of 7.2 whereas phagosomes containing a vapA deletion mutant have a pH of 5.8 and those with virulence plasmid-less sister strains have a pH of 5.2. Experimentally neutralising the macrophage endocytic system allows avirulent R. equi to multiply. This observation is mirrored in the fact that virulent and avirulent R. equi multiply well in extracts of purified lysosomes at pH 7.2 but not at pH 5.1. Together these data indicate that the major function of VapA is to generate a pH-neutral and hence growth-promoting intracellular niche. VapA represents a new type of Gram-positive virulence factor by trafficking from one subcellular compartment to another, affecting membrane permeability, excluding proton-pumping ATPase, and consequently disarming host defences.

Mechanisms of Matrix-Induced Chemoresistance of Breast Cancer Cells-Deciphering Novel Potential Targets for a Cell Sensitization.

Tumor cell binding to microenvironment components such as collagen type 1 (COL1) attenuates the sensitivity to cytotoxic drugs like cisplatin (CDDP) or mitoxantrone (MX), referred to as cell adhesion mediated drug resistance (CAM-DR). CAM-DR is considered as the onset for resistance mutations, but underlying mechanisms remain elusive. To evaluate CAM-DR as target for sensitization strategies, we analyzed signaling pathways in human estrogen-positive MCF-7 and triple-negative MDA-MB-231 breast cancer cells by western blot, proteome profiler array and TOP-flash assay in presence of COL1. ß1-Integrins, known to bind COL1, appear as key for mediating COL1-related resistance in both cell lines that primarily follows FAK/PI3K/AKT pathway in MCF-7, and MAPK pathway in MDA-MB-231 cells. Notably, pCREB is highly elevated in both cell lines. Consequently, blocking these pathways sensitizes the cells evidently to CDDP and MX treatment. Wnt signaling is not relevant in this context. A ß1-integrin knockdown of MCF-7 cells (MCF-7-ß1-kd) reveals a signaling shift from FAK/PI3K/AKT to MAPK pathway, thus CREB emerges as a promising primary target for sensitization in MDA-MB-231, and secondary target in MCF-7 cells. Concluding, we provide evidence for importance of CAM-DR in breast cancer cells and identify intracellular signaling pathways as targets to sensitize cells for cytotoxicity treatment regimes.

The Low Molecular Weight Heparin Tinzaparin Attenuates Platelet Activation in Terms of Metastatic Niche Formation by Coagulation-Dependent and Independent Pathways.

An intimate interplay with platelets is an initial key issue for tumor cells in terms of hematogenous metastasis. Tumor cells activate platelets by different pathways and receive, upon forming a platelet cloak, protection from immune surveillance and support in metastatic niche creation. Therapeutic intervention with this early interaction is promising to antagonize the whole metastatic cascade. Here we aimed to investigate the capability of low molecular weight heparin (LMWH), unfractionated heparin (UFH), and a non-anticoagulant heparin derivative or FXa inhibitor fondaparinux to interfere with platelet activation by tumor cells. Coagulation-dependent and independent pathways of platelet activation by three tumor cell lines, and interference therewith were analyzed by fluorigenic thrombin formation assay, platelet aggregometry, ATP and VEGF release and endothelial tube formation assay. LMWH and UFH were found to repress various routes of platelet activation, reflected by attenuated endothelial tube formation. This confirms the duality of anti-coagulative and anti-adhesive properties of heparin. While non-anticoagulative heparin (RO-heparin) depressed platelets' ATP and VEGF release by contact inhibition sufficiently, fondaparinux just attenuated tissue factor mediated thrombin generation. Concluding, these data suggest that LMWH as a guideline-based drug for anticoagulative strategies in oncology is promising to provide additional benefit for interference with metastatic activities.

Unfractionated and Low Molecular Weight Heparin Reduce Platelet Induced Epithelial-Mesenchymal Transition in Pancreatic and Prostate Cancer Cells.

The interaction with platelets is of crucial importance for tumor cells passing through hematogenous metastasis. Platelets protect cancer cells from immune surveillance and exhibit many other prometastatic effects. Notably, platelets can change the epithelial tumor phenotype, a process termed epithelial-mesenchymal transition (EMT), which confers stem cell-like properties onto tumor cells associated with an increased motility and drug resistance. The aim of the study is to investigate the impact of heparin on the platelet induced EMT program in pancreatic and prostate tumor cells. Platelet activation and interaction with cancer cells were determined by static adhesion assays. Applying ELISAs, the platelet release of EMT inducing mediators was quantified. EMT marker protein expression by tumor cells was explored by western blot and qPCR. Our data show that different tumor cell entities have different platelet binding capacities and also that a weak interaction is sufficient to change tumor cell phenotype. Additionally, unfractionated heparin (UFH) as well as low molecular weight heparin (LMWH) reduced tumor cell platelet interaction. Subsequently, attenuated platelet-derived mediator release resulted in reduced EMT marker protein and transcription factor expression by the cancer cells and decreased cell migration. These data suggest that heparin reduces platelet induced EMT program and prevents the formation of cancer cells with stem cell-like properties. This additional mechanism argues for the use of heparin in oncological applications.