These agents can be classified by their mechanism of action as follows.141) Anticoagulants (such as heparin and coumarin), which prevent the formation of thrombi and the development of thrombi Nav1.7-IN-3 by interfering with the process of blood coagulation. Intro == Several providers are used to treat thromboembolic diseases. These agents can be classified by their mechanism of action as follows.141) Anticoagulants (such as heparin and coumarin), which prevent the formation of thrombi and the development of thrombi by interfering with the process of blood coagulation. 2) Antiplatelet medicines (such as aspirin, dipyridamole and hirudin), which inhibit the adhesion, aggregation and launch of platelets to prevent the formation of thrombi. These medicines are usually used in the treatment of cardiovascular diseases, cerebral ischemia and peripheral embolism. 3) Thrombolytic providers (such as streptokinase, urokinase and tissue-type plasminogen activator), which convert zymogen Nav1.7-IN-3 plasminogen into an active form of serine proteinase plasmin, and then initiate or accelerate the process of fibrinolysis and degrade the fibrin network of thrombi and blood clots. However, when apoplexy happens and is not treated in time, the thrombus would become structured over time. Organization of the thrombus by cells of the surrounding vessel cells was shown by Virchow and later on confirmed by Aschoff.5,6The matrix surrounding these cells was shown to be rich in collagen fibers.7The collagen of an organized thrombus then interferes with the effect of thrombolysis. Streptokinase and fibrinolysin have been demonstrated to have no effect on the solubility of native and denatured collagen, which could become degraded by collagenase.8Collagenase is a type of proteolytic enzyme that can hydrolyze collagen at appropriate pH and temp. Consequently, using collagenase would be a good strategy to dissolve an structured thrombus. Collagenase may achieve active focusing on by binding to a monoclonal antibody or antibody fragment, such as third-generation thrombolytic providers which have a special affinity to structured thrombus. The immunizing conjugation of collagenase can be targeted to a thrombus and dissolve the thrombus efficiently, and it also can reduce the side-effects of collagenase. A drug and a monoclonal antibody can be crosslinked by chemical reactions. Then, the biological activity of the drug and antibody of conjugation should be sustained, and the toxicity of conjugation does not increase. In the past few years, the method of crosslinking a drug and a monoclonal antibody having a carrier has been widely used. An intermediate is definitely 1st from a drug combined with some compounds or polymers or vehicle, then the intermediate binds having a monoclonal antibody Fn1 to form an immune-conjugate. The usual crosslinking reactions include direct oxidation,9the dextran T-40 method,10the triggered ester method,11the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDCI) method,12,13the cyanogens bromide method,14the combined anhydride method,15and so on. A reasonable crosslinking method should meet the following requirements: high yield of cured product, homogeneous composition, sustained activity, simple operation, good reproducibility, easy separation and purification. == Materials and methods == == Materials == Collagenase was purchased from your Shanghai Institute of Pharmaceutical Market (Shanghai, China). Collagen, EDCI and anti-collagen monoclonal antibody (McAb) were purchased from Sigma (USA). Ninhydrin was purchased from Shanghai SSS Reagent (China). Bovine serum albumin (BSA) was purchased from Shanghai Bio-Life Technology and Technology Co., Ltd (China). Fluorescein isothiocyanate (FITC) was purchased from Amresco (USA). Polyethylene glycol (PEG) 6000 was purchased from Bazhou Chemical Plant Manufacturing plant of Tianjin Crystal Clock Manufacturing plant (China). Rabbits, of body weight of 2.0 to 2.5 kg, were provided by the West China Laboratory Animal Center of Sichuan University. All experiments were authorized by the Institutional Animal Care and Use Committee of Sichuan University or college. == Methods == == Activity assay methods == == Collagenase assay == Ninhydrin-based assay:160.5 mL of solution was mixed with 0.5 mL of phosphate-buffered saline (PBS) (0.05 mol/L, pH 7.4) in test tubes. Nav1.7-IN-3 Then 0.5 mL of collagenase of different concentrations and 0.5 mL of CaCl2(0.05 mol/L) were separately added and the tubes were shaken evenly. The above mixtures were incubated at 37C for 30 minutes, mixed with 0.5 mL of freshly prepared ninhydrin reagent and heated for 20 minutes inside a boiling water.