Fibronectin binding to von Willebrand factor occurs via the A1 domain.

BACKGROUND: Collagen interactions with von Willebrand factor (VWF) perform an important role in initiation of hemostasis. OBJECTIVES: We hypothesized that in addition to collagen, other extracellular matrix (ECM) proteins such as fibronectin can bind VWF. METHODS: Fibronectin-VWF interactions were measured by ELISA using both plasma-derived and recombinant VWF-containing variants in specific domains. Inhibition was measured by antibody competition using antibodies directed against both VWF and fibronectin. Binding affinities were measured by the Octet Biosensor for fibronectin and collagen IV. RESULTS: Fibronectin was able to bind both plasma-derived and recombinant wild-type VWF. This interaction was inhibited by both anti-VWF antibodies and collagen types III and IV. Several VWF A1 domain variants in the region of the collagen IV binding site also demonstrated absent fibronectin binding, as did variants with defects in high-molecular-weight multimers. Binding affinity testing showed fibronectin has a strong affinity for VWF, in a range similar to that of collagen IV. Fibronectin binds VWF via a restricted region of the A1 domain. This interaction requires high-molecular-weight multimers and is similar to that seen with vascular collagens. CONCLUSIONS: Therefore, VWF would appear to be the common factor linking platelet adhesion to various ECM proteins and facilitating hemostasis under conditions of ECM exposure.

Systemic Metabolite Changes in Wild-type C57BL/6 Mice Fed Black Raspberries.

INTRODUCTION: Freeze-dried black raspberries (BRBs) elicit chemopreventive effects against colorectal cancer in humans and in rodents. The objective of this study was to investigate potential BRB-caused metabolite changes using wild-type (WT) C57BL/6 mice. METHODS AND RESULTS: WT mice were fed either control diet or control diet supplemented with 5% BRBs for 8 wk. A nontargeted metabolomic analysis was conducted on colonic mucosa, liver, and fecal specimens collected from both diet groups. BRBs significantly changed the levels of 41 colonic mucosa metabolites, 40 liver metabolites, and 34 fecal metabolites compared to control diet-fed mice. BRBs reduced 34 lipid metabolites in colonic mucosa and increased levels of amino acids in liver. One metabolite, 3-[3-(sulfooxy) phenyl] propanoic acid, might be a useful biomarker of BRB consumption. In addition, BRB powder was found to contain 30-fold higher levels of linolenate compared to control diets. Consistently, multiple omega-3 polyunsaturated fatty acids (ω-3 PUFAs), including stearidonate, docosapentaenoate (ω-3 DPA), eicosapentaenoate (EPA), and docosahexaenoate (DHA), were significantly elevated in livers of BRB-fed mice. CONCLUSION: The data from the current study suggest that BRBs produce systemic metabolite changes in multiple tissue matrices, supporting our hypothesis that BRBs may serve as both a chemopreventive agent and a beneficial dietary supplement.

Black raspberries suppress colonic adenoma development in ApcMin/+ mice: relation to metabolite profiles.

Freeze-dried black raspberries (BRBs) have demonstrated chemopreventive effects in a dietary intervention trial with human colorectal cancer patients. The aim of this study was to investigate BRB-caused metabolite changes using the Apc(Min/+) mouse as a model of human colorectal cancer. Wild-type (WT) mice were fed control diet, and Apc(Min/+) mice were fed either control diet or control diet supplemented with 5% BRBs for 8 weeks. Colonic and intestinal polyp size and number were measured. A non-targeted metabolomic analysis was conducted on colonic mucosa, liver and fecal specimens. Eight weeks of BRB treatment significantly decreased intestinal and colonic polyp number and size in Apc(Min/+) mice. The apc gene mutation significantly changed 52 metabolites in colonic mucosa associated with increased amino acid and decreased lipid metabolites, as well as 39 liver and 8 fecal metabolites. BRBs significantly reversed 23 apc-regulated metabolites, including 13 colonic mucosa, 8 liver and 2 fecal metabolites that were involved in amino acid, glutathione, lipid and nucleotide metabolism. Of these, changes in eight metabolites were linearly correlated with decreased colonic polyp number and size in BRB-treated Apc(Min/+) mice. Elevated levels of putrescine and linolenate in Apc(Min/+) mice were significantly decreased by BRBs. Ornithine decarboxylase expression, the key enzyme in putrescine generation, was fully suppressed by BRBs. These results suggest that BRBs produced beneficial effects against colonic adenoma development in Apc(Min/+) mice and modulated multiple metabolic pathways. The metabolite changes produced by BRBs might potentially reflect the BRB-mediated chemopreventive effects in colorectal cancer patients.

Beneficial Regulation of Metabolic Profiles by Black Raspberries in Human Colorectal Cancer Patients.

Dietary intervention of freeze-dried black raspberries (BRBs) in a group of human colorectal cancer patients has demonstrated beneficial effects, including proapoptosis, antiproliferation, and antiangiogenesis. The aim of this study was to investigate BRB-mediated metabolite changes from this same cohort of patients. Twenty-eight colorectal cancer patients were given 60 g BRB powder daily for 1 to 9 weeks. Urine and plasma specimens were collected before and after BRB intervention. A mass spectrometry-based nontargeted metabolomic analysis was conducted on each specimen. A total of more than 400 metabolites were annotated in each specimen. Of these 34 and 6 metabolites were significantly changed by BRBs in urine and plasma, respectively. Increased levels of 4-methylcatechol sulfate in both post-BRB urine and post-BRB plasma were significantly correlated with a higher level of apoptotic marker (TUNEL) in post-BRB tumors. One tricarboxylic acid (TCA) cycle metabolites, cis-aconitate, was increased in post-BRB urine. Furthermore, BRB-derived polyphenols were absorbed and metabolized to various benzoate species, which were significantly increased in post-BRB specimens. Increased benzoate levels were positively correlated with enhanced levels of amino acid metabolite. These results suggest that BRBs induce significant metabolic changes and affect energy generating pathways.This study supports the hypothesis that BRBs might be beneficial to colorectal cancer patients through the regulation of multiple metabolites.