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Journal overview. Special Issues. Academic Editor: Chih-Ching Huang. Received 14 Sep Revised 30 Nov Accepted 31 Dec Published 20 Mar Abstract Thrombin plays a vital role in blood coagulation, which is a key process involved in thrombosis by promoting platelet aggregation and converting fibrinogen to form the fibrin clot.
Introduction Thrombosis persists as a leading cause of death and incapacity worldwide [ 1 ]. Figure 1. The chemical structures of ten investigated phenolic compounds. Figure 2. Figure 3. Electrophoregrams of baicalein and acetone in running buffers containing different concentrations of thrombin. Table 1. Interactions of ten investigated compounds with thrombin evaluated by ACE. Figure 4. The 2D interaction diagram of baicalein with residues of thrombin.
Table 2. The docking results and residue interactions of the complexes of argatroban and six flavonoids with thrombin. Figure 5. Comparison of 3D structures of six flavonoids docked with the thrombin catalytic site. References J. Yau, K. Singh, Y. Hou et al. Tanaka-Azevedo, K. Morais-Zani, R. Torquato, and A. DiCera, Q. Dang, and Y.
Bijak, R. Ziewiecki, J. Saluk et al. Weitz and M. V—V, Dabbous, F. Sakr, and D. Stangier and A. Stangier, K. Rathgen, H. Staehle, D. Gansser, and W. Nutescu, N. Shapiro, A. Chevalier, and A. S2, Kong, H. Chen, Y. Wang, L. Meng, and J. Marqui de Almeida, T. Domingos et al. Liu, H. Ma, N. Yang et al. Wang, Q. Zhang, C. Li et al. Vuignier, J. Schappler, J.
Veuthey, P. Carrupt, and S. Mozafari, S. Balasupramaniam, L. The immobilization of thrombin on a CM5 chip was performed according to the BIA applications handbook A total of RU of immobilized proteins was obtained.
Argatroban and hydroxy alizarin were set as positive control and negative control, respectively. Solvent-correction procedures were included to compensate for any DMSO-related bulk refractive index variations. Reference flow cell without immobilized thrombin served as a non-specific binding control. Biacore traces were baseline subtracted and the signal was presented in sensorgrams and measured in RU.
Empirically in the BIAcore technology, 1 ng of analyte bound at the surface gives a response of RU 2 , The response units were measured before the end of injection. To further evaluate the anti-thrombin activities of BBR, inhibition of thrombin-induced platelet aggregation was measured using the turbidimetric method Male rabbits weight 2. A t is the platelet aggregation percentage of the test sample, and A 0 is the platelet aggregation percentage of the control sample.
HEK cells were seeded at 5. Different concentrations of BBR or 1. How to cite this article: Wang, X. Identification of berberine as a direct thrombin inhibitor from traditional Chinese medicine through structural, functional and binding studies. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Brummel, K. Thrombin functions during tissue factor-induced blood coagulation.
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N-benzoylpyrazoles are novel small-molecule inhibitors of human neutrophil elastase. Arooj, M. A lower nonsignificant effect was observed with 5nM CgA, pointing to a U-shaped dose-response curve. These results suggest that CgA, at concentrations close to its physiologic levels, can exert inhibitory effects on angiogenesis. In addition, CgA did not affect endothelial cell viability not shown and did not induce apoptosis supplemental Figure 2D.
Thus, the inhibitory effect on angiogenesis was probably related to an effect on endothelial cell migration and invasion, than on cell viability and proliferation. To characterize the biologic activity of fragments lacking the C-terminal region and to identify the structural determinants of the anti-angiogenic activity of CgA we then analyzed the activity of deletion mutants of CgA, that is, recombinant CgA, , and These fragments were designed taking into account the position of dibasic sites in the C-terminal region of CgA, which are potential cleavage sites of CgA.
Similar to CgA, a bell-shaped dose-response curve was observed with this fragment, maximal activity being obtained with 5nM CgA Notably, similar to CgA, CgA could partially inhibit VEGF-induced endothelial cell migration, but not cell proliferation, and did not cause cell apoptosis supplemental Figure 3. These results suggest that 1 an anti-angiogenic site is contained in the C-terminal region of CgA, 2 large N-terminal fragments lacking the C-terminal region do not inhibit angiogenesis, 3 small fragments corresponding to the C-terminal region can inhibit angiogenesis 5-fold less efficiently than full-length CgA, and 4 deletion of RRG from these fragments further reduces their anti-angiogenic activity.
The observation that CgA is approximately 5-fold less active than CgA in the RAR assay could have several explanations: one possibility is that the CgA peptide is more flexible, from a structural point of view, than the cognate sequence in full-length CgA.
Alternatively, other residues present in CgA might form accessory sites that contribute to the activity of its C-terminal region. The C-terminal domain of CgA is crucial for its anti-angiogenic activity. The role of the N-terminal region of CgA and fragments in angiogenesis was then investigated. CgA was markedly more potent, as significant inhibition of bFGF-induced microvessel sprouting occurred with 0. Notably, also in this case a U-shaped dose-response curve was observed, maximal activity occurring at 1nM concentration.
These results and the observation that CgA, , , and are inactive at this concentration, suggest that cleavage of QK77 bond is crucial for the full activation of an anti-angiogenic site located in the N-terminal region. To confirm that free CgA is a potent inhibitor of angiogenesis also in vivo, we treated mice bearing WEHI fibrosarcomas with low doses of this fragment 0. Immunofluorescence staining of tumor tissue sections with an anti-CD31 antibody an endothelial cell marker showed reduced vascularization in CgA—treated mice Figure 4 B.
Of note, significant delay in tumor growth also occurred Figure 4 B bottom panel , probably as a consequence of reduced angiogenesis. N-terminal fragments of CgA inhibit angiogenesis in vitro and in vivo. Vessel density and tumor volumes are shown. These and the above results suggest that CgA contains, in addition to a bioactive site located in the C-terminal region, also a latent anti-angiogenic site located in the N-terminal region, which can be activated by cleavage of the QK77 bond.
Considering that N-terminal fragments cleaved after Q76 are present in circulation at 0. Because this assay cannot detect fragments corresponding to the N-terminal region Figure 1 D , this result implies that no N-terminal processing occurred during the assay. In conclusion, these results suggest that full-length CgA is endowed of anti-angiogenic activity and that proteolytic cleavage is not necessary for activity. Angiogenesis can be promoted by thrombin, a proteolytic enzyme that is activated during blood coagulation.
Other fractions pool 2 and pool 3 were undetectable with both assays, suggesting that they corresponded to small C-terminal fragments. Accordingly, mass spectrometry analysis of thrombin-treated CgA showed the presence of fragments corresponding to cleavage after residue R, R, and R Figure 5 D. These results indicate that CgA is a substrate of thrombin and that degradation occurred in the C-terminal region Figure 5 E.
Thrombin cleaves the C-terminal region of CgA. B Gel filtration chromatography of thrombin-digested CgA. Fractions corresponding to the main peaks were collected and pooled pools 1, 2, and 3. The corresponding fragments and their expected molecular weight Exp are also shown. E Primary sequence of human CgA. Dibasic sites are indicated in bold. Thrombin is present in the blood as an inactive proenzyme that is activated during clot formation.
To assess whether the amount of active thrombin formed during blood coagulation is sufficient to cleave CgA, we spiked murine blood samples with 10nM recombinant CgA and monitored the presence of N and C-terminal fragments in plasma and serum samples ie, prepared with or without heparin by ELISA.
As expected, the recovery of full-length CgA was lower in serum compared with plasma Figure 6 A. These data are consistent with C-terminal fragmentation. Notably, this effect was inhibited by hirudin, a selective thrombin inhibitor Figure 6 A right , suggesting to thrombin was the proteolytic enzyme responsible for CgA cleavage during blood coagulation. No formation of CgA was observed data not shown , suggesting that clot formation promotes C-terminal, but not N-terminal, processing of CgA.
In conclusion, these results suggest that the amount of thrombin generated during clot formation is sufficient for CgA cleavage. No degradation was observed when CgA was added to plasma or serum samples after clot formation data not shown.
The C-terminal region of CgA is cleaved by thrombin in blood during fibrin clot formation. To assess whether also natural CgA can be processed during blood coagulation, we then analyzed the CgA levels in plasma and serum samples obtained from the same donor.
These data confirm the hypothesis that the C-terminal region of natural circulating CgA is cleaved during clot formation. The effect of thrombin on the anti-angiogenic activity of CgA was then investigated.
To this aim we digested CgA with thrombin immobilized on agarose beads, and after thrombin removal, we analyzed the product by RAR assay. Interestingly, the number of microvessels sprouting from aorta rings was higher than in controls, pointing to a proangiogenic effect. Notably, the pool 1 fraction obtained by gel filtration chromatography of the digestion mixture Figure 5 B; corresponding to fragments lacking the C-terminal region was sufficient to stimulate vessel formation Figure 7 A.
These results suggest that the biologic activity of thrombin-digested CgA is dominated by the proangiogenic effect of fragments lacking the C-terminal region. As bFGF is a potent proangiogenic factor, this finding may explain the proangiogenic activity of CgA Thrombin abrogates the anti-angiogenic activity of full-length CgA and generates proangiogenic fragments.
Bars represent the number of capillary-like structures emerging from the aorta rings, treated as indicated, expressed as percentage of the untreated control. The number of aorta rings tested is indicated n. The results show that the blood of healthy subjects contain subnanomolar levels of various CgA-related molecules, including full-length CgA, fragments lacking the C-terminal RRG residues, fragments lacking the entire C-terminal region, and fragments lacking both central and C-terminal regions CgA These findings raise the question as to whether these circulating polypeptides, all containing the N-terminal domain, have a role in the homeostatic regulation of angiogenesis.
Thus, full-length CgA might work as a blood-born anti-angiogenic factor in physiologic conditions. The observation that a fragment lacking the C-terminal region CgA was inactive at physiologic levels and that a peptide corresponding to CgA was sufficient to exert anti-angiogenic effects in the RAR assay although 5-fold less efficiently than full-length CgA suggests that the functional site of CgA is located in the C-terminal region and not in the N-terminal domain as originally hypothesized.
However, the finding that 0. Considering that biologically relevant levels of both CgA and CgA are present in circulation in healthy subjects, these findings suggest that these molecules, but not CgA and large fragments lacking the C-terminal region, contribute to the homeostatic inhibition of angiogenesis in normal conditions. The results also show that deletion of residues RRG from CgA drastically reduced fold its anti-angiogenic activity.
Thus, large fragments lacking the C-terminal RRG residues, which are also present in normal plasma, are unlikely to contribute in a significant manner to the homeostatic regulation of angiogenesis. What are the mechanisms underlying the inhibitory activities of CgA and its N-terminal fragments? The results of the present study suggest that full-length CgA, at 1nM physiologic level, does not affect endothelial cell viability and proliferation, whereas it can inhibit VEGF-induced cell migration.
Interestingly, in previous studies we showed that CgA can affect the expression of proteins involved in the regulation of cell cytoskeleton rearrangement such as phosphorylated cofilin , a process critical for endothelial cell migration.
Which proteases are responsible for CgA cleavage in normal conditions? The results of immunoassays performed on plasma and serum samples spiked with known amounts of recombinant CgA show that full-length CgA is stable even after prolonged incubations. Thus, it is unlikely that the proteases responsible for N and C-terminal cleavage of CgA in normal conditions are present in circulation.
Probably, these proteases are located in or close to the secretory cells. Notably, it was previously shown that plasminogen activation and plasmin formation, a serine protease capable of generating CgA and other CgA fragments, is present on the surface of chromaffin cells.
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