A widely targeted metabolite modificomics strategy for modified metabolites identification in tomato.

The structural and functional diversity of plant metabolites is largely created via chemical modification of a basic backbone. However, metabolite modifications in plants have still not been thoroughly investigated by metabolomics approaches. In this study, a widely targeted metabolite modificomics (WTMM) strategy was developed based on ultra-high performance liquid chromatography-quadrupole-linear ion trap (UHPLC-Q-Trap) and UHPLC-Q-Exactive-Orbitrap (UHPLC-QE-Orbitrap), which greatly improved the detection sensitivity and the efficiency of identification of modified metabolites. A metabolite modificomics study was carried out using tomato as a model, and over 34,000 signals with MS2 information were obtained from approximately 232 neutral loss transitions. Unbiased metabolite profiling was also performed by utilizing high-resolution mass spectrometry data to annotate a total of 2,118 metabolites with 125 modification types; of these, 165 modified metabolites were identified in this study. Next, the WTMM database was used to assess diseased tomato tissues and 29 biomarkers were analyzed. In summary, the WTMM strategy is not only capable of large-scale detection and quantitative analysis of plant-modified metabolites in plants, but also can be used for plant biomarker development.

Population analysis reveals the roles of DNA methylation in tomato domestication and metabolic diversity.

DNA methylation is an important epigenetic marker, yet its diversity and consequences in tomato breeding at the population level are largely unknown. We performed whole-genome bisulfite sequencing (WGBS), RNA sequencing, and metabolic profiling on a population comprising wild tomatoes, landraces, and cultivars. A total of 8,375 differentially methylated regions (DMRs) were identified, with methylation levels progressively decreasing from domestication to improvement. We found that over 20% of DMRs overlapped with selective sweeps. Moreover, more than 80% of DMRs in tomato were not significantly associated with single-nucleotide polymorphisms (SNPs), and DMRs had strong linkages with adjacent SNPs. We additionally profiled 339 metabolites from 364 diverse accessions and further performed a metabolic association study based on SNPs and DMRs. We detected 971 and 711 large-effect loci via SNP and DMR markers, respectively. Combined with multi-omics, we identified 13 candidate genes and updated the polyphenol biosynthetic pathway. Our results showed that DNA methylation variants could complement SNP profiling of metabolite diversity. Our study thus provides a DNA methylome map across diverse accessions and suggests that DNA methylation variation can be the genetic basis of metabolic diversity in plants.

Integrated Metabolomics and Transcriptomics Analyses Reveal the Metabolic Differences and Molecular Basis of Nutritional Quality in Landraces and Cultivated Rice.

Rice (Oryza sativa L.) is one of the most globally important crops, nutritionally and economically. Therefore, analyzing the genetic basis of its nutritional quality is a paramount prerequisite for cultivating new varieties with increased nutritional health. To systematically compare the nutritional quality differences between landraces and cultivated rice, and to mine key genes that determine the specific nutritional traits of landraces, a seed metabolome database of 985 nutritional metabolites covering amino acids, flavonoids, anthocyanins, and vitamins by a widely targeted metabolomic approach with 114 rice varieties (35 landraces and 79 cultivars) was established. To further reveal the molecular mechanism of the metabolic differences in landrace and cultivated rice seeds, four cultivars and six landrace seeds were selected for transcriptome and metabolome analysis during germination, respectively. The integrated analysis compared the metabolic profiles and transcriptomes of different types of rice, identifying 358 differentially accumulated metabolites (DAMs) and 1982 differentially expressed genes (DEGs), establishing a metabolite-gene correlation network. A PCA revealed anthocyanins, flavonoids, and lipids as the central differential nutritional metabolites between landraces and cultivated rice. The metabolite-gene correlation network was used to screen out 20 candidate genes postulated to be involved in the structural modification of anthocyanins. Five glycosyltransferases were verified to catalyze the glycosylation of anthocyanins by in vitro enzyme activity experiments. At the same time, the different mechanisms of the anthocyanin synthesis pathway and structural diversity in landrace and cultivated rice were systematically analyzed, providing new insights for the improvement and utilization of the nutritional quality of rice landrace varieties.

Multi-Omics Analysis Reveals That SlERF.D6 Synergistically Regulates SGAs and Fruit Development.

Steroidal glycoalkaloids (SGAs) are cholesterol-derived molecules that contribute to the pathogen defense in tomato but are toxic and considered to be antinutritional compounds to humans. APETALA2/Ethylene Responsive Factor (AP2/ERF) family transcription factors (TFs) play an indispensable role in various biological processes, such as plant growth and development, fruit ripening, biotic and abiotic stresses responses, and SGA biosynthesis. In this study, we identified 176 AP2/ERF genes that were domesticated or improved SlAP2/ERF in the tomato variome (Solanum lycopersicum) within either domestication or improvement sweeps, respectively. According to the RNA-sequencing data, 93 of the ERF genes with high transcriptional level (Transcripts Per Million, TPM > 1) belong to six clusters. Weighted gene co-expression network analysis (WGCNA) and metabolite-based genome-wide association study (mGWAS) analyses revealed that the expression level of the Solyc04g071770 (SlERF.D6) gene in the cluster six gradually increased as the fruit matured. Transient transformation verified that the overexpression of SlERF.D6 significantly promoted fruit ripening and regulated the expression of multiple genes in the SGA synthesis pathway, thereby affecting the SGA content of the fruit. Virus-induced gene silencing (VIGS) showed that the silencing of SlERF.D6 delayed fruit ripening and influenced the content of SGAs. Our data provide new insights into AP2/ERF TFs in tomato, offer a candidate TF for fruit development and steroidal glycoalkaloids, and provide new resources for tomato breeding and improvement.

Integration of rhythmic metabolome and transcriptome provides insights into the transmission of rhythmic fluctuations and temporal diversity of metabolism in rice.

Various aspects of the organisms adapt to cyclically changing environmental conditions via transcriptional regulation. However, the role of rhythmicity in altering the global aspects of metabolism is poorly characterized. Here, we subjected four rice (Oryza sativa) varieties to a range of metabolic profiles and RNA-seq to investigate the temporal relationships of rhythm between transcription and metabolism. More than 40% of the rhythmic genes and a quarter of metabolites conservatively oscillated across four rice accessions. Compared with the metabolome, the transcriptome was more strongly regulated by rhythm; however, the rhythm of metabolites had an obvious opposite trend between day and night. Through association analysis, the time delay of rhythmic transmission from the transcript to the metabolite level was ∼4 h under long-day conditions, although the transmission was nearly synchronous for carbohydrate and nucleotide metabolism. The rhythmic accumulation of metabolites maintained highly coordinated temporal relationships in the metabolic network, whereas the correlation of some rhythmic metabolites, such as branched-chain amino acids (BCAAs), was significantly different intervariety. We further demonstrated that the cumulative diversity of BCAAs was due to the differential expression of branched-chain aminotransferase 2 at dawn. Our research reveals the flexible pattern of rice metabolic rhythm existing with conservation and diversity.

Comparative Metabolomics Reveals Two Metabolic Modules Affecting Seed Germination in Rice (Oryza sativa).

The process of seed germination is crucial not only for the completion of the plant life cycle but also for agricultural production and food chemistry; however, the underlying metabolic regulation mechanism involved in this process is still far from being clearly revealed. In this study, one indica variety (Zhenshan 97, with rapid germination) and one japonica variety (Nipponbare, with slow germination) in rice were used for in-depth analysis of the metabolome at different germination stages (0, 3, 6, 9, 12, 24, 36, and 48 h after imbibition, HAI) and exploration of key metabolites/metabolic pathways. In total, 380 annotated metabolites were analyzed by using a high-performance liquid chromatography (HPLC)-based targeted method combined with a nontargeted metabolic profiling method. By using bioinformatics and statistical methods, the dynamic changes in metabolites during germination in the two varieties were compared. Through correlation analysis, coefficient of variation analysis and differential accumulation analysis, 74 candidate metabolites that may be closely related to seed germination were finally screened. Among these candidates, 29 members belong to the ornithine-asparagine-polyamine module and the shikimic acid-tyrosine-tryptamine-phenylalanine-flavonoid module. As the core member of the second module, shikimic acid's function in the promotion of seed germination was confirmed by exogenous treatment. These results told that nitrogen flow and antioxidation/defense responses are potentially crucial for germinating seeds and seedlings. It deepens our understanding of the metabolic regulation mechanism of seed germination and points out the direction for our future research.

In Vivo Detection of Extracellular Adenosine Triphosphate in a Mouse Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) is traditionally characterized by primary and secondary injury phases, both contributing to pathological and morphological changes. The mechanisms of damage and chronic consequences of TBI remain to be fully elucidated, but synaptic homeostasis disturbances and impaired energy metabolism are proposed to be a major contributor. It has been proposed that an increase of extracellular (eATP) adenosine triphosphate (ATP) in the area immediately surrounding impact may play a pivotal role in this sequence of events. After tissue injury, rupture of cell membranes allows release of intracellular ATP into the extracellular space, triggering a cascade of toxic events and inflammation. ATP is a ubiquitous messenger; however, simple and reliable techniques to measure its concentration have proven elusive. Here, we integrate a sensitive bioluminescent eATP sensor known as pmeLUC, with a controlled cortical impact mouse model to monitor eATP changes in a living animal after injury. Using the pmeLUC probe, a rapid increase of eATP is observed proximal to the point of impact within minutes of the injury. This event is significantly attenuated when animals are pretreated with an ATP hydrolyzing agent (apyrase) before surgery, confirming the contribution of eATP. This new eATP reporter could be useful for understanding the role of eATP in the pathogenesis in TBI and may identify a window of opportunity for therapeutic intervention.

Human Recombinant Apyrase Therapy Protects Against Myocardial Ischemia/Reperfusion Injury and Preserves Left Ventricular Systolic Function in Rats, as Evaluated by 7T Cardiovascular Magnetic Resonance Imaging.

OBJECTIVE: The occurrence of intramyocardial hemorrhage (IMH) and microvascular obstruction (MVO) in myocardial infarction (MI), known as severe ischemia/reperfusion injury (IRI), has been associated with adverse remodeling. APT102, a soluble human recombinant ecto-nucleoside triphosphate diphosphohydrolase-1, can hydrolyze extracellular nucleotides to attenuate their prothrombotic and proinflammatory effects. The purpose of this study was to temporally evaluate the therapeutic effect of APT102 on IRI in rats and to elucidate the evolution of IRI in the acute stage using cardiovascular magnetic resonance imaging (CMRI). MATERIALS AND METHODS: Fifty-four rats with MI, induced by ligation of the origin of the left anterior descending coronary artery for 60 minutes, were randomly divided into the APT102 (n = 27) or control (n = 27) group. Intravenous infusion of APT102 (0.3 mg/kg) or placebo was administered 15 minutes before reperfusion, and then 24 hours, 48 hours, 72 hours, and on day 4 after reperfusion. CMRI was performed at 24 hours, 48 hours, 72 hours, and on day 5 post-reperfusion using a 7T system and the hearts were collected for histopathological examination. Cardiac function was quantified using cine imaging and IMH/edema using T2 mapping, and infarct/MVO using late gadolinium enhancement. RESULTS: The extent of infarction (p < 0.001), edema (p < 0.001), IMH (p = 0.013), and MVO (p = 0.049) was less severe in the APT102 group than in the control group. IMH size at 48 hours was significantly greater than that at 24 hours, 72 hours, and 5 days after reperfusion (all p < 0.001). The left ventricular ejection fraction (LVEF) was significantly greater in the APT102 group than in the control group (p = 0.006). There was a negative correlation between LVEF and IMH (r = -0.294, p = 0.010) and a positive correlation between IMH and MVO (r = 0.392, p < 0.001). CONCLUSION: APT102 can significantly alleviate damage to the ischemic myocardium and microvasculature. IMH size peaked at 48 hours post reperfusion and IMH is a downstream consequence of MVO. IMH may be a potential therapeutic target to prevent adverse remodeling in MI.

Modulation of CD39 and Exogenous APT102 Correct Immune Dysfunction in Experimental Colitis and Crohn's Disease.

BACKGROUND AND AIMS: CD39/ENTPD1 scavenges pro-inflammatory nucleotides, to ultimately generate immunosuppressive adenosine, which has a central role in immune homeostasis. Global deletion of Cd39 increases susceptibility to experimental colitis while single nucleotide polymorphisms within the human CD39 promoter, and aberrant patterns of expression during experimental hypoxia, predispose to Crohn's disease. We aimed to define the impact of transgenic human CD39 [hTG] overexpression in experimental colitis and to model therapeutic effects using the recombinant apyrase APT102 in vivo. We also determined the in vitro effects of APT102 on phenotypic and functional properties of regulatory T-lymphocytes derived from patients with Crohn's disease. METHODS: Colitis was induced by administration of dextran sulfate sodium in wild-type [WT] or hTG mice, and, in another model, by adoptive transfer of CD45RBhigh cells with or without WT or hTG regulatory T cells [Treg]. In additional experiments, mice were treated with APT102. The effects of APT102 on phenotype and function of Treg and type-1 regulatory T [Tr1] cells were also evaluated, after purification from peripheral blood and lamina propria of Crohn's disease patients [n = 38]. RESULTS: Overexpression of human CD39 attenuated experimental colitis and protected from the deleterious effects of systemic hypoxia, pharmacologically induced by deferoxamine. Administration of APT102 in vivo enhanced the beneficial effects of endogenous Cd39 boosted by the administration of the aryl hydrocarbon receptor [AhR] ligand unconjugated bilirubin [UCB]. Importantly, supplemental APT102 restored responsiveness to AhR stimulation by UCB in Treg and Tr1 cells, obtained from Crohn's disease patients. CONCLUSIONS: hCD39 overexpression ameliorated experimental colitis and prevented hypoxia-related damage in vivo. Exogenous administration of APT102 boosted AhR-mediated regulatory effects in vivo while enhancing Treg functions in Crohn's disease in vitro.

A non-contrast CMR index for assessing myocardial fibrosis.

PURPOSE: Safe, sensitive, and non-invasive imaging methods to assess the presence, extent, and turnover of myocardial fibrosis are needed for early stratification of risk in patients who might develop heart failure after myocardial infarction. We describe a non-contrast cardiac magnetic resonance (CMR) approach for sensitive detection of myocardial fibrosis using a canine model of myocardial infarction and reperfusion. METHODS: Seven dogs had coronary thrombotic occlusion of the left anterior descending coronary arteries followed by fibrinolytic reperfusion. CMR studies were performed at 7days after reperfusion. A CMR spin-locking T1ρ mapping sequence was used to acquire T1ρ dispersion data with spin-lock frequencies of 0 and 511Hz. A fibrosis index map was derived on a pixel-by-pixel basis. CMR native T1 mapping, first-pass myocardial perfusion imaging, and post-contrast late gadolinium enhancement imaging were also performed for assessing myocardial ischemia and fibrosis. Hearts were dissected after CMR for histopathological staining and two myocardial tissue segments from the septal regions of adjacent left ventricular slices were qualitatively assessed to grade the extent of myocardial fibrosis. RESULTS: Histopathology of 14 myocardial tissue segments from septal regions was graded as grade 1 (fibrosis area, <20% of a low power field, n=9), grade 2 (fibrosis area, 20-50% of field, n=4), or grade 3 (fibrosis area, >50% of field, n=1). A dramatic difference in fibrosis index (183%, P<0.001) was observed by CMR from grade 1 to 2, whereas differences were much smaller for T1ρ (9%, P=0.14), native T1 (5.5%, P=0.12), and perfusion (-21%, P=0.05). CONCLUSION: A non-contrast CMR index based on T1ρ dispersion contrast was shown in preliminary studies to detect and correlate with the extent of myocardial fibrosis identified histopathologically. A non-contrast approach may have important implications for managing cardiac patients with heart failure, particularly in the presence of impaired renal function.

Human recombinant apyrase therapy protects against canine pulmonary ischemia-reperfusion injury.

BACKGROUND: There is accumulating evidence that extracellular adenosine triphosphate (eATP) promotes many of the underlying mechanisms that exacerbate acute lung injury. However, much of these data are from inbred rodent models, indicating the need for further investigation in higher vertebrates to better establish clinical relevance. To this end we evaluated a human recombinant apyrase therapy in a canine warm pulmonary ischemia-reperfusion injury (IRI) model and measured eATP levels in human lung recipients with or without primary lung graft dysfunction (PGD). METHODS: Warm ischemia was induced for 90 minutes in the left lung of 14 mongrel dogs. Seven minutes after reperfusion, the apyrase APT102 (1 mg/kg, n = 7) or saline vehicle (n = 7) was injected into the pulmonary artery. Arterial blood gases were obtained every 30 minutes up to 180 minutes after reperfusion. Bronchioalveolar lavage fluid (BALF) was analyzed for eATP concentration, cellularity, and inflammatory mediator accumulation. Thirty bilateral human lung transplant recipients were graded for immediate early PGD and assessed for BALF eATP levels. RESULTS: APT102-treated dogs had progressively better lung function and less pulmonary edema during the 3-hour reperfusion period compared with vehicle-treated controls. Protection from IRI was observed, with lower BALF eATP levels, fewer airway leukocytes, and blunted inflammatory mediator expression. Human lung recipients with moderate to severe PGD had significantly higher eATP levels compared with recipients without this injury. CONCLUSIONS: Extracellular ATP accumulates in acutely injured canine and human lungs. Strategies that target eATP reduction may help protect lung recipients from IRI.

Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk.

In patients with acute myocardial infarction undergoing reperfusion therapy to restore blood flow through blocked arteries, simultaneous inhibition of platelet P2Y12 receptors with the current standard of care neither completely prevents recurrent thrombosis nor provides satisfactory protection against reperfusion injury. Additionally, these antiplatelet drugs increase the risk of bleeding. To devise a different strategy, we engineered and optimized the apyrase activity of human nucleoside triphosphate diphosphohydrolase-3 (CD39L3) to enhance scavenging of extracellular adenosine diphosphate, a predominant ligand of P2Y12 receptors. The resulting recombinant protein, APT102, exhibited greater than four times higher adenosine diphosphatase activity and a 50 times longer plasma half-life than did native apyrase. Treatment with APT102 before coronary fibrinolysis with intravenous recombinant human tissue-type plasminogen activator in conscious dogs completely prevented thrombotic reocclusion and significantly decreased infarction size by 81% without increasing bleeding time. In contrast, clopidogrel did not prevent coronary reocclusion and increased bleeding time. In a murine model of myocardial reperfusion injury caused by transient coronary artery occlusion, APT102 also decreased infarct size by 51%, whereas clopidogrel was not effective. These preclinical data suggest that APT102 should be tested for its ability to safely and effectively maximize the benefits of myocardial reperfusion therapy in patients with arterial thrombosis.

Combination treatment of r-tPA and an optimized human apyrase reduces mortality rate and hemorrhagic transformation 6h after ischemic stroke in aged female rats.

Recombinant tissue plasminogen activator (r-tPA) is the only FDA-approved drug treatment for ischemic stroke and must be used within 4.5h. Thrombolytic treatment with r-tPA has deleterious effects on the neurovascular unit that substantially increases the risk of intracerebral hemorrhage if administered too late. These therapeutic shortcomings necessitate additional investigation into agents that can extend the therapeutic window for safe use of thrombolytics. In this study, combination of r-tPA and APT102, a novel form of human apyrase/ADPase, was investigated in a clinically-relevant aged-female rat embolic ischemic stroke model. We propose that successfully extending the therapeutic window of r-tPA administration would represent a significant advance in the treatment of ischemic stroke due to a significant increase in the number of patients eligible for treatment. Results of our study showed significantly reduced mortality from 47% with r-tPA alone to 16% with co-administration of APT102 and r-tPA. Co-administration decreased cortical (47 ± 5% vs. 29 ± 5%), striatal (50 ± 2%, vs. 40 ± 3%) and total (48 ± 3%vs. 33 ± 4%) hemispheric infarct volume compared to r-tPA alone. APT102 improved neurological outcome (8.9±0.6, vs. 6.8 ± 0.8) and decreased hemoglobin extravasation in cortical tissue (1.9 ± 0.1mg/dl vs. 1.4 ± 0.1mg/dl) striatal tissue (2.1 ± 0.3mg/dl vs. 1.4 ± 0.1mg/dl) and whole brain tissue (2.0 ± 0.2mg/dl vs. 1.4 ± 0.1mg/dl). These data suggest that APT102 can safely extend the therapeutic window for r-tPA mediated reperfusion to 6h following experimental stroke without increased hemorrhagic transformation. APT102 offers to be a viable adjunct therapeutic option to increase the number of clinical patients eligible for thrombolytic treatment after ischemic stroke.

Improved insulin sensitivity by calorie restriction is associated with reduction of ERK and p70S6K activities in the liver of obese Zucker rats.

Calorie restriction (CR) improves obesity-related insulin resistance through undefined molecular mechanisms. Insulin receptor substrate (IRS)-1 serine/threonine kinases have been proposed to modulate insulin sensitivity through phosphorylation of IRS proteins. The aim of this study is to test the hypothesis that changes in the activity of IRS1 serine/threonine kinases may underlie the molecular mechanism of CR in improving insulin sensitivity. Obese and lean Zucker rats were subjected to 40% CR or allowed to feed ad libitum (AL) for 20 weeks; body weight and insulin sensitivity were monitored throughout this period. The activity of IRS1 serine/threonine kinases - including JNK, ERK, MTOR/p70(S6K) (RPS6KB1 as listed in the MGI Database), glycogen synthase kinase 3beta (GSK3B), AMPK (PRKAA1 as listed in the MGI Database), and protein kinase C (PRKCQ) in liver tissue extracts was measured by an in vitro kinase assay using various glutathione-S-transferase (GST)-IRS1 fragments as substrates, while phosphorylation of IRS1 and serine kinases was determined by western blotting using phosphospecific antibodies. CR in obese rats significantly reduced body weight and increased insulin sensitivity compared to AL controls. Serine kinase activity toward IRS1(S612) (corresponding to S616 in human IRS1) and IRS1(S632/635) (corresponding to S636/639 in human IRS1) was increased in obese rats compared to lean littermates, and was markedly decreased following CR. Concomitantly, obesity increased and CR decreased the activity of hepatic ERK and p70(S6K) against IRS1. The close association between the activity of hepatic ERK and p70(S6K) with insulin resistance suggests an important role for ERK and p70(S6K) in the development of insulin resistance, presumably via phosphorylation of IRS proteins.

Apyrase treatment prevents ischemia-reperfusion injury in rat lung isografts.

OBJECTIVE: Endothelial cells express the ectoenzyme ectonucleoside adenosine triphosphate diphosphohydrolase, an apyrase that inhibits vascular inflammation by catalyzing the hydrolysis of adenosine triphosphate and adenosine diphosphate. However, ectonucleoside adenosine triphosphate diphosphohydrolase expression is rapidly lost following oxidative stress, leading to the potential for adenosine triphosphate and related purigenic nucleotides to exacerbate acute solid organ inflammation and injury. We asked if administration of a soluble recombinant apyrase APT102 attenuates lung graft injury in a cold ischemia reperfusion model of rat syngeneic orthotopic lung transplantation. METHODS: Male Fisher 344 donor lungs were cold preserved in a low-potassium dextrose solution in the presence or absence of APT102 for 18 hours prior to transplantation into syngeneic male Fisher 344 recipients. Seven minutes after reperfusion, lung transplant recipients received either a bolus of APT102 or vehicle (saline solution). Four hours after reperfusion, APT102- and saline solution-treated groups were evaluated for lung graft function and inflammation. RESULTS: APT102 significantly reduced lung graft extracellular pools of adenosine triphosphate and adenosine diphosphate, improved oxygenation, and protected against pulmonary edema. Apyrase treatment was associated with attenuated neutrophil graft sequestration and less evidence of tissue inflammation as assessed by myeloperoxidase activity, expression of proinflammatory mediators, and numbers of apoptotic endothelial cells. CONCLUSIONS: Administration of a soluble recombinant apyrase promotes lung function and limits the tissue damage induced by prolonged cold storage, indicating that extracellular purigenic nucleotides play a key role in promoting ischemia-reperfusion injury following lung transplantation.

APT102, a novel adpase, cooperates with aspirin to disrupt bone metastasis in mice.

Platelets contribute to the development of metastasis, the most common cause of mortality in cancer patients, but the precise role that anti-platelet drugs play in cancer treatment is not defined. Metastatic tumor cells can produce platelet alphaIIb beta3 activators, such as ADP and thromboxane A(2) (TXA(2)). Inhibitors of platelet beta3 integrins decrease bone metastases in mice but are associated with significant bleeding. We examined the role of a novel soluble apyrase/ADPase, APT102, and an inhibitor of TXA(2) synthesis, acetylsalicylic acid (aspirin or ASA), in mouse models of experimental bone metastases. We found that treatment with ASA and APT102 in combination (ASA + APT102), but not either drug alone, significantly decreased breast cancer and melanoma bone metastases in mice with fewer bleeding complications than observed with alphaIIb beta3 inhibition. ASA + APT102 diminished tumor cell induced platelet aggregation but did not directly alter tumor cell viability. Notably, APT102 + ASA treatment did not affect initial tumor cell distribution and similar results were observed in beta3-/- mice. These results show that treatment with ASA + APT102 decreases bone metastases without significant bleeding complications. Anti-platelet drugs such as ASA + APT102 could be valuable experimental tools for studying the role of platelet activation in metastasis as well as a therapeutic option for the prevention of bone metastases.

Platelet aggregation-induced by caco-2 cells: regulation by matrix metalloproteinase-2 and adenosine diphosphate.

Formation of tumor cell-platelet aggregates facilitates hematogenous metastases. However, molecular mechanisms implicated in tumor cell-induced platelet aggregation (TCIPA) in colon cancer are unclear. To investigate mechanisms of TCIPA induced by colon adenocarcinoma cells in vitro, human Caco-2 cells were used to study their interactions with platelets using aggregometry, zymography, phase-contrast microscopy, and flow cytometry. Caco-2-induced platelet aggregation in a concentration-dependent manner. This aggregation resulted in the release of matrix metalloproteinase (MMP)-2, as measured by zymography. In addition, flow cytometry showed a significant up-regulation of activated GpIIb/IIIa, total GpIIb/IIIa, GpIb, and P-selectin receptors on platelets. Inhibition of MMP-2 by phenantroline and degradation of ADP by APT102, respectively, resulted in inhibition of TCIPA. Furthermore, both phenantroline and APT102 significantly down-regulated the surface abundance of platelet receptors. Caco-2 cells aggregate platelets, at least in part, via releasing MMP-2 and ADP. Modulation of MMP-2 and ADP actions could have therapeutic value in colonic cancer.