| Atherosclerosis is one of global diseases, which seriously threatens the health of our people and become a heavily burden of every country, the pathological changes of which always occurred in arteries including the big and middle arteries. Atherosclerosis can lead to ischemic cardiovascular disease. Several theories or hypotheses about atherosclerosis such as lipid infiltration, smooth muscle sell proliferation, thrombogenesis have been proposed during the past decades. The thrombogenesis theory emphasizes that the rising of platelet activating factor makes the aggregation or adhension of platelet on the endomembrane of blood vessel, promotes the cross-linking, aggregation and deposition of fibrin, the thrombolytic system has been inhibited and different cytokines were released, thus promoting the damage and proliferation of endothelial cell, LDL infiltration, monocyte aggregation, proliferation and migration of smooth muscle cell, fibroblast proliferation, vasoconstriction, and thrombopoiesis. It was revealed that the activation and adhesion of platelet and fibrin cross-linking play a role in the progression of atherosclerosis.Staphylokinase (SAK) is a protein of single chain polypeptide consisted of 136 amino acid residues, which is a fibrin-selective agent with an important potency as a thrombolytic. SAK can mediate efficiently the lysis of fibrint-rich clots with excellent fibrin specificity. Thus, its application to clinical treatment would not cause the adverse effects such as the activation of thrombolytic system, extensive thrombolysis and bleeding. SAK displays better effect than that of rt-PA in animal models. Being a heterogeneous protein, SAK has high antigenicity which restricts its repeated use. RGD is the motif that can bind the platelet glycoprotein GPⅡb /Ⅲa . The exogenous RGD can compete to bind GPⅡb /Ⅲa with fgn that inhibits the aggregation of the activated platelets; a segment of amino acid residues in the C terminal of hirudin is the smallest one with anticoagulation effect, which can bind with thrombin to inhibit the function of thrombin. The engineering E.coli strain harboring the mutant SAK with introduced RGD sequence and 12 amino acid residues at the C-ternminus and without antigenic epitopes was obtained in our early studies. This engineering strain was expressed and the expressed product was purified by molecular sieve and ion-exchange chromatographies. The obtained product was named RXP with a purity of 95%, and proved bioactive by cell assay. In the basis of the above-mentioned work, we first explored the function of RXP both in vivo and in vitro. It had been found that RXP possesses the characters of anti-platelet aggregation, inhibition of thrombin, degradation of fibrin, and low immunogenicity. The effect of RXP was evaluated in the artherosclerosis animal model (rabbits, rats and apoE-/-mice were all induced by feeding on high fat diet). It was confirmed that RXP can antagonize the progression of the atherosclerosis, preserve blood vessel. Finally, the signaling molecule was screened out, which can regulate fat metabolism and ameliorate inflammation status. It was certified that the RXP can inhibit the progression of atherosclerosis in many ways (anti-platelet, degradation of fibrin, anti-thrombin, regulation of lipid metablish, diminishing oxidative damage, and anti-inflammation). This study might provide the experimental foundation for RXP developing into the anti-atherosclerosis medicine.Evaluation of RXP activity and its safetyThe thrombolytic activity was determined by the method of haemolysis ring on fibrin clot plate, and the result confirmed that there was no significant statistical difference between wild SAK and RXP (4.35±0.8;3.5±1.4, p>0.05), The plasminogen activation rate was also examined, so was it. Both activation progresses were similar to each other. ELISA showed that both SAK and RGD-SAK can bind with fibrin, which showed no significant statistical difference. The efficiency of fibrin clot lysis was investigated on the ELISA plates. Data showed that the fibrinolytic efficiencies of the two proteins on the fibrin clot were not significantly different. To examine whether the activated platelet binding ability enhanced by RXP would lead to a faster clot lysis, the platelet-rich clot lysis experiments were performed, which partially simulated the physiological situation. The differences between SAK and RXP were observed. The platelet aggregation was testified by the anti-platelet aggregation assay in vitro. A significantly higher inhibitory effect of RXP on platelet aggregation was observed, compared with that of SAK. Thrombin inhibition assay also proved that the inhibition rate of the thrombin activity by RXP was 18% at the 5% concentration. The purified and sterilized preparations were devoid of common reaction of mice after intravenous bolus injection at a dose of 3 mg per mice in groups of 6 mice.The evaluation of the RXP's antigenicity and the effects of the RXP on macaque's coagulation/fibrinolysis system for long-term and repeated use10 macaque's was treated with RXP once two days at the same dose of 0.02mg/kg body weight for 2 weeks, this procedure was repeated after ten weeks interval. The observation was lasted for about three weeks from the end of second procedure. The whole process contained two procedures of administration, and lasted for about six months. The sera at 15 different time points of the whole process were obtained. The level of antibody against wtSAK and RXP were tested by using ELISA. The results suggested that the level of antibody against RXP was obviously lower than that of wtSAK. The neutralizing activity of antibody against wtSAK was obviously higher than that of RXP, which was displayed by fibrin plate assay. After being incubated with each antibody, only 3.8% thrombolysis activity was kept in wtSAK, but that of in RXP was kept over 90%. In the six months,some indexes were tested such as FIB,PLG,α2-PI and PT,APTT,TT. The results indicated that the injections of both two proteins would not cause a notable change of the index in safety aspect.Intervention of Atherosclerosis Progress by RXPThe low-antigenicity and high safety were tested and verified repeatedly on monkey by administrating the low dose of RXP. The function of RXP had been evaluated with rats, rabbits and the ApoE-/-gene knockout mice. The results certified that RXP can decrease the levels of cholesterol and low density lipoprotein (LDL) in the blood plasma, and some indications for the cardiovascular damages also decreased. The pathological examination confirmed that RXP can inhibit the plaque formation in the arterial wall. The rabbits assay proved its function, while the level of blood fat was not lowered. Evaluation on the action of RXP with the apoE knockout mice certified that the RXP contributes to prevent the progression of atherosclerosis by the following reasons: 1. it can lower the density of cholesterol and LDL; 2. it can ameliorate the inflammation in vivo; 3. it can elevate the anti-oxidative capability; 4. it can interrupt the adhension of the blood elements.Molecular Basis of Intervention of Atherosclerosis by RXPThe different gene expression between the two apoE gene knockout mice groups was screened by gene chip. It was found that the administration of RXP in earlier phases can contribute to intervention of atherosclerosis for several aspects. In the later phases, the RXP showed a little effect on the atherosclerosis. RXP can influence the progression of atherosclerosis by the mediation of fat metabolism, inhibition of inflammation and anti-oxidative stress. In this study, 2412 diverse genes have been obtained, including 233 genes for fat metabolism, 458 inflammatory genes, 28 regulation genes for blood vascular system. while, in the later phases, the total differential genes reached to 182, 6 genes for fat metabolism,13 inflammatory genes, 28 regulation genes for blood vascular system were found. Our results indicated that RXP suppressed the atherosclerosis by regulation of fat metabolism, inhibition of inflammation, and anti-oxidative stress.RXP can protect the cardiovascular system and regulate the function of anti-atherosclerosis, which displayed the perspective for exploration and development of new therapeutic medicines...
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