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• W2916508081 abstract "Blood platelets are required for normal wound healing, but they are also involved in thrombotic diseases, which are usually managed with anticoagulant drugs. Here, using genetic engineering, we coupled the disintegrin protein echistatin, which specifically binds to the platelet integrin αIIbβ3 receptor, to annexin V, which binds platelet membrane-associated phosphatidylserine (PS), to create the bifunctional antithrombotic molecule recombinant echistatin–annexin V fusion protein (r-EchAV). Lipid binding and plasma coagulation studies revealed that r-EchAV dose-dependently binds PS and delays plasma clotting time. Moreover, r-EchAV inhibited ADP-induced platelet aggregation in a dose-dependent manner and exhibited potent antiplatelet aggregation effects. r-EchAV significantly prolonged activated partial thromboplastin time, suggesting that it primarily affects the in vivo coagulation pathway. Flow cytometry results indicated that r-EchAV could effectively bind to the platelet αIIbβ3 receptor, indicating that r-EchAV retains echistatin’s receptor-recognition region. In vivo experiments in mice disclosed that r-EchAV significantly prolongs bleeding time, indicating a significant anticoagulant effect in vivo resulting from the joint binding of r-EchAV to both PS and the αIIbβ3 receptor. We also report optimization of the r-EchAV production steps and its purification for high purity and yield. Our findings indicate that r-EchAV retains the active structural regions of echistatin and annexin V and that the whole molecule exhibits multitarget-binding ability arising from the dual functions of echistatin and annexin V. Therefore, r-EchAV represents a new class of anticoagulant that specifically targets the anionic membrane-associated coagulation enzyme complexes at thrombogenesis sites and may be a potentially useful antithrombotic agent. Blood platelets are required for normal wound healing, but they are also involved in thrombotic diseases, which are usually managed with anticoagulant drugs. Here, using genetic engineering, we coupled the disintegrin protein echistatin, which specifically binds to the platelet integrin αIIbβ3 receptor, to annexin V, which binds platelet membrane-associated phosphatidylserine (PS), to create the bifunctional antithrombotic molecule recombinant echistatin–annexin V fusion protein (r-EchAV). Lipid binding and plasma coagulation studies revealed that r-EchAV dose-dependently binds PS and delays plasma clotting time. Moreover, r-EchAV inhibited ADP-induced platelet aggregation in a dose-dependent manner and exhibited potent antiplatelet aggregation effects. r-EchAV significantly prolonged activated partial thromboplastin time, suggesting that it primarily affects the in vivo coagulation pathway. Flow cytometry results indicated that r-EchAV could effectively bind to the platelet αIIbβ3 receptor, indicating that r-EchAV retains echistatin’s receptor-recognition region. In vivo experiments in mice disclosed that r-EchAV significantly prolongs bleeding time, indicating a significant anticoagulant effect in vivo resulting from the joint binding of r-EchAV to both PS and the αIIbβ3 receptor. We also report optimization of the r-EchAV production steps and its purification for high purity and yield. Our findings indicate that r-EchAV retains the active structural regions of echistatin and annexin V and that the whole molecule exhibits multitarget-binding ability arising from the dual functions of echistatin and annexin V. Therefore, r-EchAV represents a new class of anticoagulant that specifically targets the anionic membrane-associated coagulation enzyme complexes at thrombogenesis sites and may be a potentially useful antithrombotic agent. Platelets are irregular fragments formed by megakaryocytes and play an important role in hemostasis, thrombosis, and inflammation. When endothelial cells are damaged, platelets are activated and adhere to the collagen fibers exposed to the injured site. The factors affecting platelet adhesion include platelet membrane glycoprotein, collagen of endothelial tissue (collagen), and hemophilia factor (von Willebrand factor). Adherent platelets release ADP, thromboxane A2, 5-hydroxytryptamine, and other contents to promote platelet activation (1Vorchheimer D.A. Becker R. Platelets in atherothrombosis.Mayo Clin. Proc. 2006; 81 (16438480): 59-6810.4065/81.1.59Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 2Wolf P. The nature and significance of platelet products in human plasma.Br. J. Haematol. 1967; 13 (6025241): 269-288Crossref PubMed Scopus (1106) Google Scholar). At this point, multiple signaling pathways are initiated and interact with each other, and the internal signal mechanism is amplified to activate the platelet surface αIIbβ3 receptor. The damaged endothelial tissue releases coagulation factor III to activate the exogenous coagulation pathway, and the local platelets produce thrombin quickly, causing more platelet aggregation, forming a softer hemostatic embolus (3Heemskerk J.W. Bevers E.M. Lindhout T. Platelet activation and blood coagulation.Thromb. Haemost. 2002; 88 (12195687): 186-19310.1055/s-0037-1613209Crossref PubMed Scopus (444) Google Scholar). Platelets play an important role in normal wound healing and are also important causes of pathological thrombotic diseases. Thrombotic diseases are characterized by high morbidity, disability, and mortality and seriously endanger human life and health. Anticoagulant and antithrombotic drugs play an important role in the prevention and treatment of thrombotic diseases. To achieve high-efficiency antithrombosis, until recently most clinical guidelines recommended both anticoagulation and dual antiplatelet therapy (triple therapy) (4Verlinden N.J. Coons J.C. Iasella C.J. Kane-Gill S.L. Triple antithrombotic therapy with aspirin, P2Y12 inhibitor, and warfarin after percutaneous coronary intervention: an evaluation of prasugrel or ticagrelor versus clopidogrel.J. Cardiovasc. Pharmacol. Ther. 2017; 22 (28279076): 546-55110.1177/1074248417698042Crossref PubMed Scopus (27) Google Scholar, 5Cannon C.P. Bhatt D.L. Oldgren J. Lip G.Y.H. Ellis S.G. Kimura T. Maeng M. Merkely B. Zeymer U. Gropper S. Nordaby M. Kleine E. Harper R. Manassie J. Januzzi J.L. et al.Dual antithrombotic therapy with dabigatran after PCI in atrial fibrillation.N. Engl. J. Med. 2017; 377 (28844193): 1513-152410.1056/NEJMoa1708454Crossref PubMed Scopus (897) Google Scholar). Personalized detection, treatment, and evaluation are also the current research hot spots of antithrombotic therapeutic agents. To improve the efficacy of the drug while not increasing the amount of drug used and reducing drug use costs, we attempted to couple echistatin (ECH), 2The abbreviations used are: ECHechistatinANVannexin V or annexin A5aPTTactivated partial thromboplastin timeDAVdiannexinFIBfibrinogen assayIPTGisopropyl β-d-thiogalactopyranosidePCphosphatidylcholinePPPplatelet-poor plasmaPRPplatelet-rich plasmaPSphosphatidylserinePSPprescission proteasePTplasma clotting timeSPRsurface plasmon resonanceTTthrombin timeGSTGSH S-transferaseTEMEDN,N,N′,N′-tetramethylethylenediaminer-EchAVrecombinant echistatin–annexin V fusion proteinSECsize-exclusion chromatographyTDDtargeted drug deliveryFVafactor VaFXafactor XaKPIKunitz-type protease inhibitorsTFsoluble tissue factorHRPhorseradish peroxidase. which specifically binds to the platelet αIIbβ3 integrin receptor, with annexin V (ANV), which specifically binds to the platelet membrane PS molecule, by genetic engineering techniques to form an efficient bifunctional recombinant antithrombotic molecule r-EchAV. echistatin annexin V or annexin A5 activated partial thromboplastin time diannexin fibrinogen assay isopropyl β-d-thiogalactopyranoside phosphatidylcholine platelet-poor plasma platelet-rich plasma phosphatidylserine prescission protease plasma clotting time surface plasmon resonance thrombin time GSH S-transferase N,N,N′,N′-tetramethylethylenediamine recombinant echistatin–annexin V fusion protein size-exclusion chromatography targeted drug delivery factor Va factor Xa Kunitz-type protease inhibitor soluble tissue factor horseradish peroxidase. PS accounts for ∼2–10% of the total lipid content of cells (6Vance J.E. Steenbergen R. Metabolism and functions of phosphatidylserine.Prog. Lipid. Res. 2005; 44 (15979148): 207-23410.1016/j.plipres.2005.05.001Crossref PubMed Scopus (360) Google Scholar) and is often considered a “fingerprint” molecule for apoptotic cells (7Choo H.J. Kholmukhamedov A. Zhou C. Jobe S. Inner mitochondrial membrane disruption links apoptotic and agonist-initiated phosphatidylserine externalization in platelets.Arterioscler. Thromb. Vasc. Biol. 2017; 37 (28663253): 1503-151210.1161/ATVBAHA.117.309473Crossref PubMed Scopus (37) Google Scholar, 8Obydennyy S.I. Sveshnikova A.N. Ataullakhanov F.I. Panteleev M.A. Dynamics of calcium spiking, mitochondrial collapse and phosphatidylserine exposure in platelet subpopulations during activation.J. Thromb. Haemost. 2016; 14 (27343487): 1867-188110.1111/jth.13395Crossref PubMed Scopus (56) Google Scholar). It must be mentioned here that PS is also a fingerprint molecule of activated platelets (9Nagata S. Suzuki J. Segawa K. Fujii T. Exposure of phosphatidylserine on the cell surface.Cell Death Differ. 2016; 23 (26891692): 952-96110.1038/cdd.2016.7Crossref PubMed Scopus (232) Google Scholar, 10Zhao L. Wu X. Si Y. Yao Z. Dong Z. Novakovic V.A. Guo L. Tong D. Chen H. Bi Y. Kou J. Shi H. Tian Y. Hu S. Zhou J. Shi J. Increased blood cell phosphatidylserine exposure and circulating microparticles contribute to procoagulant activity after carotid artery stenting.J. Neurosurg. 2017; 127 (28009236): 1041-1054Crossref PubMed Scopus (10) Google Scholar). When the platelets change from a resting state to an activated state, the PS molecules originally located in the inner membrane of the cells are largely translocated to the outside of the cell membrane. The flipping of the PS will initiate or directly participate in physiological processes such as coagulation or phagocytosis. The PS-rich platelet surface catalyzes the onset of coagulation and ultimately leads to the formation of thrombin, which in turn stabilizes platelet-rich thrombotic tissue through fibrin formation (3Heemskerk J.W. Bevers E.M. Lindhout T. Platelet activation and blood coagulation.Thromb. Haemost. 2002; 88 (12195687): 186-19310.1055/s-0037-1613209Crossref PubMed Scopus (444) Google Scholar). ANV, a nonglycosylated single-chain protein discovered as a class of anticoagulant proteins of vascular tissue (11Reutelingsperger C.P. Hornstra G. Hemker H.C. Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord.Eur. J. Biochem. 1985; 151 (3896792): 625-62910.1111/j.1432-1033.1985.tb09150.xCrossref PubMed Scopus (159) Google Scholar), is a member of the annexin family of calcium-dependent phospholipid-binding proteins and binds with very high affinity to PS-containing phospholipid bilayers (12Raynal P. Pollard H.B. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- and phospholipid-binding proteins.Biochim. Biophys. Acta. 1994; 1197 (8155692): 63-9310.1016/0304-4157(94)90019-1Crossref PubMed Scopus (1027) Google Scholar13Huber R. Römisch J. Paques E.P. The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes.EMBO J. 1990; 9 (2147412): 3867-387410.1002/j.1460-2075.1990.tb07605.xCrossref PubMed Scopus (355) Google Scholar, 14Voges D. Berendes R. Burger A. Demange P. Baumeister W. Huber R. Three-dimensional structure of membrane-bound annexin V. A correlative electron microscopy-X-ray crystallography study.J. Mol. Biol. 1994; 238 (8158649): 199-21310.1006/jmbi.1994.1281Crossref PubMed Scopus (162) Google Scholar15Lewit-Bentley A. Morera S. Huber R. Bodo G. The effect of metal binding on the structure of annexin V and implications for membrane binding.Eur. J. Biochem. 1992; 210 (1446685): 73-7710.1111/j.1432-1033.1992.tb17392.xCrossref PubMed Scopus (54) Google Scholar). ANV also binds to human platelets with a Kd of 7 nm (16Thiagarajan P. Tait J.F. Binding of annexin V/placental anticoagulant protein I to platelets. Evidence for phosphatidylserine exposure in the procoagulant response of activated platelets.J. Biol. Chem. 1990; 265 (2145274): 17420-17423Abstract Full Text PDF PubMed Google Scholar, 17Thiagarajan P. Tait J.F. Collagen-induced exposure of anionic phospholipid in platelets and platelet-derived microparticles.J. Biol. Chem. 1991; 266 (1662206): 24302-24307Abstract Full Text PDF PubMed Google Scholar). Binding to quiescent platelets in vitro is minimal, although maximally stimulated platelets contain nearly 200,000 ANV-binding sites; this substantially exceeds the number of binding sites for antibodies to integrin αIIbβ3 (∼25,000 on activated platelets) (18Shattil S.J. Hoxie J.A. Cunningham M. Brass L.F. Changes in the platelet membrane glycoprotein IIb. IIIa complex during platelet activation.J. Biol. Chem. 1985; 260 (2411729): 11107-11114Abstract Full Text PDF PubMed Google Scholar). Anticoagulant effects can be achieved by competitively binding PS sites (19Shi J. Gilbert G.E. Lactadherin inhibits enzyme complexes of blood coagulation by competing for phospholipid-binding sites.Blood. 2003; 101 (12517809): 2628-263610.1182/blood-2002-07-1951Crossref PubMed Scopus (150) Google Scholar, 20Uchman B. Triplett D.A. Inhibition of binding of β2-glycoprotein 1 to phosphatidylserine by polymyxin B, a lupus-like anticoagulant.Clin. Appl. Thromb. Hemost. 2015; 21 (24275099): 584-58510.1177/1076029613512417Crossref PubMed Scopus (2) Google Scholar). ANV exerts antithrombotic activity by binding to PS, inhibiting the activation of serine proteases important in blood coagulation. ANV is a relatively small protein (Mr 36,000) that is easily produced in quantity by recombinant DNA methods. It has been shown that recombinant ANV has the same PS-binding ability as the native state of ANV present in human tissues (21Marder L.S. Lunardi J. Renard G. Rostirolla D.C. Petersen G.O. Nunes J.E. de Souza A.P. de O Dias A.C. Chies J.M. Basso L.A. Santos D.S. Bizarro C.V. Production of recombinant human annexin V by fed-batch cultivation.BMC Biotechnol. 2014; 14 (24766778): 33-4310.1186/1472-6750-14-33Crossref PubMed Scopus (8) Google Scholar, 22Maurer-Fogy I. Reutelingsperger C.P. Pieters J. Bodo G. Stratowa C. Hauptmann R. Cloning and expression of cDNA for human vascular anticoagulant, a Ca2+-dependent phospholipid-binding protein.Eur. J. Biochem. 1988; 174 (2455636): 585-59210.1111/j.1432-1033.1988.tb14139.xCrossref PubMed Scopus (82) Google Scholar23Liang P. Zhang X. Jing J. The recombinant expression and properties of human annexin V.J. Beijing Normal Univ. (Natur. Sci.). 2009; 45: 378-380Google Scholar), and as a human protein it would not be expected to induce an immune response, unlike murine monoclonal antibodies. It is also virtually absent from normal human plasma (24Flaherty M.J. West S. Heimark R.L. Fujikawa K. Tait J.F. Placental anticoagulant protein-I: measurement in extracellular fluids and cells of the hemostatic system.J. Lab. Clin. Med. 1990; 115 (2137157): 174-181PubMed Google Scholar) because it rapidly passes from the blood into the kidneys due to its relatively small size. How to couple different mechanisms to achieve a multieffect therapy is an important direction of antithrombotics or anticoagulant research and development. Therefore, rational coupling of different mechanisms of action to design fusion proteins has become the key to successful research. We constructed the ECH-linker–ANV recombinant fusion protein (r-EchAV) by genetic engineering technology. ECH can efficiently bind to the integrin receptor αIIbβ3 exposed on the surface of activated platelets, inhibiting platelet aggregation efficiently to achieve an antithrombotic effect; ANV binds a large number of PS molecules valgus to the surface of activated platelets in a calcium-dependent manner. We hope that the constructed r-EchAV can simultaneously couple the above two anticoagulant/antithrombotic mechanisms to become a bifunctional antithrombotic recombinant protein molecule. This paper describes the phospholipid binding and platelet integrin receptor binding characteristics of r-EchAV, and its anticoagulant effect in vivo was evaluated based on animal models. The preparation process and physicochemical characteristics of r-EchAV were also studied and elaborated. The DNA fragments containing the constructed ECH gene and the rh-ANV gene were further amplified and subjected to agarose gel electrophoresis, and the detection results are shown in Fig. 1A. These results showed that the DNA size met the expected requirements. Combined with the results of DNA sequencing, it can be confirmed that the base sequence of the DNA fragment of the ECH gene or the ANV gene is completely correct. The translated amino acid sequences of these synthetic genes match the published sequence of ANV and ECH listed in the GenBankTM database. The ECH-L and L-ANV genes were used as templates, and E3F and LAA-3 were used as primers for upstream and downstream, respectively; PCR was carried out to construct a 1226-bp DNA fragment containing the r-EchAV gene (as shown in the Fig. 1B). After DNA sequencing, the base sequence was confirmed to be completely correct and to conform to the expected base sequence requirement of the recombinant r-EchAV gene; its nucleotide sequence thereof is shown in Table 1B. The r-EchAV gene was finally cloned into the pGEX vector, and soluble expression was achieved in Escherichia coli. Furthermore, we explored and optimized the bacterial culture and important induction expression conditions of the recombinant r-EchAV fusion protein and placed a good foundation for successful follow-up preparation of the r-EchAV.Table 1Design, synthesis and identification of r-EchAV recombinant gene The optimal IPTG dosage in the induction of r-EchAV expression is shown in Fig. 2A. Our studies showed that under the conditions of different IPTG concentrations in the medium, significant amounts of expression products were induced, with a size of 70 kDa. The molecular weight of this induced expression product was in accordance with the expected theoretical value of GST–r-EchAV (70.1 kDa). However, we also found that when the final concentration of IPTG in the medium reached 100 μm, the expression level of the macromolecule fusion recombinant protein GST–r-EchAV also reached its highest value and no longer increased in response to increases in the IPTG concentration. At the same time, considering that an excessive IPTG concentration will promote the formation of inclusion bodies in E. coli and that a too high IPTG concentration can also exhibit strong toxicity against E. coli cells, we finally determined the optimal concentration of the cell inducer IPTG of r-EchAV expression to be 100 μm for subsequent large-scale bacterial culture, induction of expression, and preparation of recombinant proteins. The experimental identification results of optimal temperature control during r-EchAV–induced expression are shown in Fig. 2B. The IPTG-induced expression of recombinant E. coli was carried out under the control conditions of 37, 30, and 25 °C, respectively. Our studies have shown that under the above temperature conditions, r-EchAV expression products could be achieved efficiently and in large quantities; the target recombinant protein expression products were found in both the isolated supernatant and the bacterial inclusion bodies, and the yield of the expressed products was not significantly different. Considering that the formation of E. coli inclusion bodies is positively correlated with the temperature, reducing the temperature during induction will help to reduce the formation of bacterial inclusion bodies, so we finally controlled the temperature during the induction of r-EchAV at 25 °C and used it as the optimum temperature for the later large-scale E. coli culture and r-EchAV preparation. We also explored the optimal harvest time during the induction of r-EchAV expression, as shown in Fig. 2C. The results showed that the content of recombinant foreign gene expression products in the whole-cell protein of the bacteria increased gradually with the prolongation of induced expression control times at 0, 5–12, and 20 h. However, we found that when the harvest time was controlled between 11 and 12 h, the recombinant protein content reached its highest value. Further prolongation of the induced expression time decreased the content of the target protein. We speculate that because of the excessive prolongation of the induction time, a degradation tendency of the recombinant expression product is also obvious, resulting in a decrease in target protein content; another reason may be that too long of an induction time will result in decreased utilization of nutrients by E. coli cells, excessive accumulation of harmful products, and reduced ability to synthesize foreign proteins in the later stage. Based on the above results, we finally determined that the induction time of r-EchAV was controlled within 12 h. Based on the above studies, recombinant fusion proteins (GST–r-EchAV) formed by r-EchAV and GST were present in a soluble form in a large amount in the supernatant after disruption of the cells. Therefore, after the induction of expression was completed, the cells were collected by centrifugation and disrupted, and then the supernatant was collected for further purification and property studies. Most of the E. coli–expressed fusion proteins were found in the inclusion bodies. Fortunately, our expression product was present in a soluble form in E. coli. Therefore, the subsequent purification steps circumvent complex and cumbersome steps such as inclusion body extraction and protein de-renaturation. The results are shown in Fig. 2D. Our studies have shown that combined with GST column-affinity chromatography technology, GST–r-EchAV recombinant foreign protein was obtained with relatively good purification, and the preparation yield was also relatively high. The purity of the target protein GST–r-EchAV was above 92%, and the yield reached 19 mg/liter (as shown in Table 2).Table 2Approximate yield and purity of the total and target proteins after extraction and purification with affinity chromatography and r-EchAV after specific digestion and ultrafiltrationTotal proteinaThe extraction and purification start from 1 liter of bacteria cultured and induced following the method described in the text.GST–r-EchAVGSTr-EchAVbr-EchAV was prepared from GST–r-EchAV by protease digestion with scissor-protease.YieldPurityYieldPurityYieldPuritymgmg%mg%mg%275.2 ± 1336.1 ± 1.69614.7 ± 0.89919.8 ± 1.199a The extraction and purification start from 1 liter of bacteria cultured and induced following the method described in the text.b r-EchAV was prepared from GST–r-EchAV by protease digestion with scissor-protease. Open table in a new tab In the specific digestion process of the GST–r-EchAV recombinant protein, we explored different substrate/enzyme molecular molar ratios to achieve optimal protease digestion. As shown in Fig. 2E, the enzymatic cleavage effect of the PSP enzyme on GST–r-EchAV was not enhanced with the increase in the amount of protease. Conversely, we observed that when the molar ratio of the recombinant protein GST–r-EchAV to PSP enzyme was 800:1 and the enzyme digestion was 12 h, the enzymatic cleavage effect was very good. GST–r-EchAV (70 kDa) can be sufficiently cleaved to form the products r-EchAV (44 kDa) and GST (26 kDa). Therefore, in the later large-scale preparation process, the proportion of substrate/enzyme was controlled at around 800:1 (molar ratio). Using the anti-human ANV polyclonal antibody, we performed a Western blotting assay on the purified r-EchAV recombinant protein. The results are shown in Fig. 2G. Our studies have shown that r-EchAV exhibits a full ANV antibody-binding effect. This indicates that the r-EchAV recombinant fusion protein retains the intact ANV epitope structure. The experimental results of molecular sieve SEC-HPLC of r-EchAV are shown in Fig. 2H. Our studies have shown that the absorption peak of the r-EchAV protein was detected in the range of 8.150–9.093-min retention time and reached its peak at 8.513 min. The peak shape symmetry was good. This result is consistent with the peak time parameter of the molecular mass of the 44-kDa protein identified by SEC-HPLC. The absorption peak-to-peak area percentage of the target protein r-EchAV reached 92.852%. This fully demonstrates that the r-EchAV recombinant protein exists in a single form; the monomer form is stably present, and no polymer is formed in a solution state. This structural feature is different from that of ANV. ANV can form dimers or even polymers. This suggests that the addition of the ECH structure to the N-terminal of the ANV molecule hinders the intermolecular polymerization of ANV and changes the original polymerization conditions of the ANV molecule. The r-EchAV molecule is preferably present in solution in a stable form as a single molecule. The test results are shown in Fig. 3A. Our studies have shown that r-EchAV, like ANV molecules, exhibits different binding abilities to different phospholipids, i.e. it does not bind to PC, and the binding effect with PS increases with increasing sample protein concentration, showing a significant dose-dependent relationship. This indicates that the phospholipid-binding properties of r-EchAV are not significantly different from those of ANV. This also suggests that the ECH structure in the r-EchAV molecule does not block the interaction site of ANV with PS. The results are shown in Fig. 3B. As seen from this figure, when different doses of PS were immobilized in the wells of the ELISA plate, the binding of r-EchAV to PS showed a dose-dependent relationship, and at PS >2.5 μg, the protein and PS were gradually saturated. The results are shown in Fig. 3C. Our studies have shown that the binding activity of r-EchAV to PS increases with an increase in Ca2+ concentration when the Ca2+ concentration is between 0.5 and 10 mm. This finding indicates that the specific binding between the r-EchAV molecule and PS requires the presence of Ca2+. This characteristic is similar in nature to ANV. As a calcium ion-dependent phospholipid-binding protein, ANV can reversibly bind to negatively charged PS when Ca2+ is present. To determine the binding parameters of the interaction between r-EchAV and PS or PC, we performed surface plasmon resonance assays. The preparations of r-EchAV or ANV were allowed to flow over the chip in the presence of 2 mm Ca2+. Preliminary measurements show that r-EchAV and ANV have very similar phospholipid binding characteristics. The binding range between the two proteins and PS is approximately Kd = 1.83–2.15 nm, as shown in Fig. 3, D–F. At the same time, the binding of the two proteins to PC is much weaker than that of PS, and it is generally considered that there is no specific binding. Under the same conditions, BSA did not bind to PS or PC (data not shown). Based on the above findings (Fig. 3), it was shown that the activity of the ANV structure in the r-EchAV molecule was completely retained. The activated partial thromboplastin time (aPTT) assays are useful in in vitro global tests for assessing the activities of the classical extrinsic, intrinsic, and common pathways of coagulation and for monitoring anticoagulant therapy. At the same concentration, the aPTT value prolonged by the r-EchAV group was slightly higher than that of the ANV group; r-EchAV was about 1.5-fold more potent than ANV, and both were significantly higher than the normal reference range (as shown in Fig. 4A). In terms of the effects on PT (plasma clotting time) value, FIB (fibrinogen) value, and TT (thrombin time) value, neither of them caused statistically significant changes; and they were all within the normal clinical reference range. Furthermore, r-EchAV showed a dose-dependent effect on aPTT value (as shown in Fig. 4B). With the increase in the r-EchAV concentration, the aPTT value was significantly prolonged; and when the r-EchAV concentration was greater than 5 μmol/liter, the amplitude of aPTT changed more than the maximum within the normal reference range. However, when the concentration of r-EchAV increased, the PT value, FIB value, and TT value did not change much, and there was basically no statistically significant change. The aPTT measures the intrinsic pathway activity, such as coagulation factors XII, XI, IX, and X, and the reduction in these coagulation factors will cause prolongation of the aPTT value. Based on the above analysis results, we speculate that r-EchAV cannot fully activate factor X by binding to Ca2+ and phospholipid molecules, thereby affecting the endogenous coagulation pathway and thus affecting the common coagulation pathway. The results are shown in Fig. 5; the turbidity dynamics of different plasma samples were measured using a microplate reader. PS alone only weakly promotes plasma coagulation (Fig. 5A). This may be because PS binds to coagulant factors Xa and Va, which are involved in steps that are rather late in the coagulation cascade. As shown in Fig. 5B, when r-EchAV was added to plasma in a PS-coated well, the onset of coagulation was delayed; a concentration of 20 μm r-EchAV delayed the onset time of coagulation from 30 to 50 min. As the concentration of r-EchAV increased, the plasma clotting time was significantly prolonged. The delay effects of coagulation by r-EchAV were dose-dependent, but the effect was barely presented in a PC-coated well as compared with a PS-coated" @default.
• W2916508081 created "2019-03-02" @default.
• W2916508081 creator A5039905681 @default.
• W2916508081 creator A5048358778 @default.
• W2916508081 date "2019-04-01" @default.
• W2916508081 modified "2023-09-29" @default.
• W2916508081 title "An αIIbβ3- and phosphatidylserine (PS)-binding recombinant fusion protein promotes PS-dependent anticoagulation and integrin-dependent antithrombosis" @default.
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