A novel single‐chain enzyme complex with chain reaction properties rapidly producing thromboxane A₂ and exhibiting powerful anti‐bleeding functions

Figures

• 

  Figure 1. Schematic presentation of COX‐1 co‐ordinated with downstream three enzymes to maintain the balance of haemostasis. 3D crystal structures of human COX‐1 (PDB ID: 3N8Z28), PGIS (PDB ID: 3B6H25) and mPGES‐1 (PDB ID: 5T3729) used were downloaded from PDB. 3D structural model of human TXAS was created by homology modelling using the crystal structure of PGIS as a template. The co‐ordination of the upstream COX‐1 and downstream TXAS, PGIS and mPGES‐1 on ER membrane was schematically presented. A model demonstrating the three‐step catalytic activities: AA converted to PGG2, then PGH2 and finally to biologically active anti‐bleeding mediator TXA2, and the bleeding mediators PGE2 and PGI2 were shown

• 

  Figure 2. Design of a novel SCHEC. COX‐1‐10aa‐TXAS was created by linking the C‐terminus of COX‐1 to N‐terminus of TXAS through a linker, which contains 10‐amino acid residues (−10aa). The complete structural model of the COX‐1‐10aa‐TXAS with triple catalytically biological activities to convert AA to PGG2, then PGH2 and a final anti‐bleeding TXA2 within a single polypeptide chain were demonstrated

• 

  Figure 3. Construction and subcloning of the cDNA of COX‐1‐10aa‐TXAS into a mammalian expression vector, pcDNA3.1(+). The starting plasmid of TXAS and pcDNA3.1(+)‐COX‐1‐10aa‐PGIS vector was prepared previously.13 Full cDNA sequence of human TXAS was prepared by PCR after inactivated the BamHI site of the TXAS plasmid via site‐directed mutagenesis. On the other hand, the PGIS cDNA fragment was removed from the cDNA of COX‐1‐10aa‐PGIS, which was inserted in a pcDNA 3.1(+) vector. Next, the isolated TXAS cDNA was linked to the cDNA of the COX‐1‐10aa and then ligated to form a complete expression plasmid containing COX‐1‐10aa‐TXAS cDNA

• 

  Figure 4. A, Verification of the cDNA of COX‐1‐10‐TXS inserted in pCDNA3.1(+) vector. Restriction enzyme digestion was performed to verify the successful insert of the sequence of cDNA of COX‐1‐10aa‐TXAS in the pcDNA3.1(+) vector. The restriction enzyme BamHI was used and cut twice at the two recognition sites and got two DNA fragments: one was the pcDNA3.1(+) vector with COX‐1‐10aa, and the other one is the TXAS. The sizes of the vector plus COX‐1‐10aa should be equal to 7.3 kb (5.4 kb of vector +1.9 kb of COX‐1‐10aa), and the size of TXAS cDNA should be equal to 1.6 kb. Finally, the subcloned cDNA sequencing of COX‐1‐10aa‐TXAS in the plasmid was further verified by DNA sequencing the inserted region of 10aa‐TXAS (data not showed). B, Western blot analysis to verify the expression of SCHEC in HEK293 cells. The HKE293 cells were transfected with the pcDNA3.1(+) vector contained cDNA of COX‐1‐10aa‐TXAS and then screened for stable expression using G418 up to 60 d. Approximately 20 µg of the proteins of HEK293 cells was subjected to Western blot analysis using 10% PAGE gel and anti–COX‐1 antibody. The results were shown on (B) panel: left, markers; lane 1, negative control, HEK293 cells…

• 

  Figure 5. Immunofluorescent imaging of the expressed COX‐1‐10aa‐TXAs on HEK293 cells. The cells were cultured in the cover slides and transfected with cDNA: pcDNA3.1(+) empty vector (negative control [top]) or pcDNA3.1(+)‐COX‐1‐10aa‐TXAS (bottom). The cells were permeabilized by saponin and then incubated with mouse anti‐COX‐1 antibody. The bound primary antibodies were stained by rhodamine‐labelled goat antimouse IgG. The slides were imaged under a fluorescent microscope. Blue fluorescent DNA dye, DAPI, was used to image the nuclei of the cells

• 

  Figure 6. Schematic presentation for determination of the triple‐catalytic activities of the expressed COX‐1‐10aa‐TXAS using the highly sensitive and specific isotope‐labelled exogenous AA as a substrate. A, 3 µmol/L of [14C]‐AA was added to the HEK293 cells. After incubation for 15 min, the metabolized compounds from the [14C]‐AA were extracted out by buffer A (35% acetonitrile in 0.1% acetic acid) and then subjected to HPLC analysis using C‐18 column (4.5 × 250 mm) started with buffer A and then with a linear gradient of buffer B (100% acetonitrile) for 45 min. The eluents were directly connected to a scintillation analyzer to pick up the [14C]‐labelled compounds catalysed by the COX‐1‐10aa‐TXAS. B, [14C]‐mediators and [14C]‐products by the triple‐catalytic enzyme reactions of COX‐1‐10aa‐TXAS

• 

  Figure 7. Determination of the activities of the HEK293 cells stably expressing COX‐1‐10aa‐TXAS and comparison with that of co‐expressing individual COX‐1 and TXAS. HEK293 cells were transfected with COX‐1‐10aa‐TXAS cDNA (B), or cotransfected with the individual COX‐1 cDNA and TXAS cDNA (A). After G418 screening for 2 mo, the cells were stored in the liquid nitrogen. After re‐cultured the cells, a serial of tests of the enzymatic activities of the cells were designed for P1, P4 and P8 cells. The cells firstly reaching 100% confluency were marked as P1 cells; 1 week later, after three passages, the cells with 100% confluency were marked as P4 cells; and another week for the P8 cells. The procedures described in Figure 6 were used to determine the [14C]‐products enzymatically metabolized from the added [14C]‐AA by the HEK293 cells expressing COX‐1‐10aa‐TXAS (B), or co‐expressed individual COX‐1 and TXAS (B). The peaks of [14C]‐TXB2 and its derivatives between 10 and 20 min of the retention time, representing the biologically active TXA2, were indicated with arrows labelled with [14C]‐TXB2. Metabolized [14C]‐AA with retention time (after 30 min) was also indicated in each assay. HEK293 cells…

• 

  Figure 8. Determination of the biological activities of TXA2 produced by COX‐1‐10aa‐TXAS on promoting platelet aggregation. Human platelet‐rich plasma (PRP) obtained from the blood bank was used for the platelet aggregation tests. A, HEK293 cells expressing SCHEC (open triangles) or the HEK293 cells transfected with vector (HEK control, open circles) were incubated with PRP for 2 min. The aggregative activity was monitored by the addition of 10 µL AA (3 μmol/L of final concentration) using platelet aggregometer. The fibres (aggregated platelets) formed in the presence (B, right) or absence of SCHEC (B, left) were shown in (B). C, PRP was pre‐treated with 10 mmol/L aspirin for 20 min. The excessive aspirin was removed by centrifugation. The platelets were re‐suspended in clean platelet‐poor plasma (PPP) and then incubated with HEK293 cells expressing COX‐1‐10aa‐TXAS (open circles) or HEK control cells (open triangles) for 2 min. The platelet aggregation was monitored by addition of AA as described above. D, PRP was diluted by PPP (ration 1:4, 5 X) to mimic the platelet‐deficient conditions. The platelet aggregation was monitored in the presence (open circles) or absence (open triangles) of…

• 

  Figure 9. Examination of the anti‐bleeding effect of the expressed COX‐1‐10aa‐TXAs on bleeding site using tail‐cut arterial bleeding assay for normal mice in vivo. A, A 0.5 cm tip of mouse tail was cut by scissors to create an arterial bleeding site. The artery and vein displayed on the cutting site were shown in (B). Immediately, the bleeding tail was placed into the solution containing isolated microsomes of the HEK293 cells with (C, right) or without (C, left) expressing COX‐1‐10aa‐TXAS. The blood was blotted on a clean filter paper every 10 s until no detectable bleeding (A). The average and standard deviation of the bleeding time for each group (n = 6) were plotted in (C)

• 

  Figure 10. Examination of the anti‐bleeding effect of the expressed COX‐1‐10aa‐TXAs on bleeding site using tail‐cut arterial bleeding assay for transgenic mice with the bleeding tendency in vivo. A, The transgenic mice with bleeding tendency were created by injection of the cDNA of COX‐1‐10aa‐PGIS into the embryos of the mice (13). The arterial bleeding conditions were created by tail‐cut approach and then treated by the isolated microsomes of the HEK293 cells with (B, open bar) and without (B, closed bar) expressing COX‐1‐10aa‐TXAS. The averages and standard deviations of the bleeding times for each group (n = 6) were plotted (B)