Pilot Production And Pharmacodynamics Study Of Recombinant Human Microplasminogen

Posterior vitreous detachment(PVD)is a common complication of diabetic retinopathy and retinal vein occlusion(RVO).It also has a high morbidity in older and myopic people.Vitrectomy and reattachment of retina is an effective therapeutic approach for PVD,but the risk of vitrectomy must still be considered.Several clinical investigations demonstrated that intravitreous injection of plasmin before vitrectomy could reduce the risk of retinal bleeding or detachment.Retinal vein occlusion is the second commonest retinal vascular disorder after diabetic retinopathy and is one of the main causes of visual loss.At present,although systemic thrombolysis has been shown to be an effective treatment to some extent,but increased incidence of unacceptable hemorrhage put constraints on its extensive use.In addition,all of thrombolytic agents currently approved for clinical use are plasminogen activators(PAs)that must convert the inactive proenzyme plasminogen to plasmin, which cleaves blood fibrin clots.However,the dependence on a sufficient availability of plasminogen substrate represents a chilles' heel for PAs in situation when the clots are large and 'recruitment' of plasminogen from the circulation is limited.This study aims to develop microplasmin and applied it to the therapy and prophylaxis of eye diseases.Plasmin is a member of serinase superfamily.It can not only degrade fibrin for fibrinolysis but also can cleave several other extracellular proteins and play important role in inflammation,tissue restitution,ovulation,the insult and transferring of tumor cells.Human plasminogen is a kind of glycoprotein consisting of 791 amino acid residues and has a molecular weight of about 92 000 Dalton.In buffer at pH 11.0, plasmin can conditionally cleave the Arg⁵³⁰-Lys⁵³¹of plasminogen,resulting in the microplasmingen(μPlg),which consists of 261 amino acid residues.Microplasminogen is a truncated form of plasminogens that lacks N terminal peptides and all five kringle domains,but retains the serine proteinase domain.Microplasminogen can be activated by plasminogen activators to microplasmin,which has the fibrinolytic activity and is rarely inhibited byα₂-antiplasmin.A recombinant expression plasmid consisting of the DNA sequence of 246 amino acid residues between 546 and 791 of human plasminogen had been constructed using protein engineering and transformed into methanol inducible Pichia pastoris GS115.A strain with secreted expression at high level of rh-μPlg gene(clone pPIC9K -μPlg-GS115)had been selected.Base on the selected strain with high expression level, a rapid,effective and simple strategy for pilot production of rh-μPlg was established. Fermentation at high density(OD₆₀₀＞200)in a 7.5L fermenter was carried out and rh-μPlg was purified from the culture broth in a three step process;ultrafiltration,gel filtration,ion exchange chromatography.The final product had a purity of over 95%. Fermentation yielded approximately 200mg highly purified rh-μPlg per liter culture broth.The apparent Mr of rh-μPlg was 32 kDa under reducing conditions and its exact molecular weight was 27 877 Dalton examined by mass spectrum.Its isoelectric point was pH 7.5-7.8.After activated by urokinase,the material was fully active and the specific activity was 23.6 U/mg.Kcat/Km was127.87±0.01 mM^-1·s^-1as determined with the chromogenic peptide substrate S₂₃₉₀(NH₂-D-Val-Phe-Lys-p-nitroanilide), whereas the native plasmin had a specific activity of 18.2 U·mg^-1and a Kcat/Km of 94.15±0.03 mM^-1·s^-1under the same conditions.Recombinant human microplasminogen was incubated with tissue plasminogen activator with a 200;1 molar ratio at 37℃for 40 minutes to generate active microplasmin(μPlm),and then the incubation solution was injected into the rabbit vitreous cavity.Scanning electron microscopy(SEM),gross specimen examination, B-mode ultrasonography and optical coherence tomography(OCT)were performed 1 day and 7 day respectively to detect whether complete PVD had been produced.The determination with B-ultrasonograph showed that intravitreous bolus of 0.5 U,1.0 U and 1.5 U recombinantμPlm can induce complete PVD in 25%,75%and 82.5%rabbit eyes respectively at 1day.Whereas,intravitreal administration of 1.0 U plasmin can induce complete PVD in 66.7%rabbit eyes.The light-adapted amplitudes of both a-wave and b-wave of electroretinography(ERG)were reduced 1 day after treatments at all concentrations and recovered gradually at 3 day and 7 day.At 7 days after injection,a-and b-wave amplitudes did not differ significantly from the controls.The implicit times of a- and b-wave did not differ significantly from that of controls at 1,3,or 7 days.Light microscopy demonstrated normal retinal histologic findings in microplasmin- and plasmin- injected eyes.A similar result had been obtained from the research performed on SD rats. Immunofluoresent microscopy showed that in control rat eyes,most of the fibronectin (FN)is located at the anchoring points of internal limiting membrane(ILM);in contrast, laminin(LN)distributes not only at anchoring points,but also in internal limiting lamina (ILL)consisting of lamina densa and lamina lucida.In complete PVD induced by microplasmin,the LN and FN at the fusing points were completely degraded.But LN in the ILL was still preserved.Thus,rh-uPlm induced complete PVD by degrading the FN and LN in the anchoring points of vitreoretinal junction.Recombinant human microplasmin could also be effective in the therapy of retinal vein occlusion(RVO).The SD rat model of retinal vein occlusion was established by applying photodynamic thrombosis using photosensitizer Rose Bengal.Both fundus photographs and fluorescein fundus angiograms demonstrated the blocked venous flow 1 hour after laser irradiation.The formation of thrombi at the occlusion site and the disordered construction in the peripheral side to the irradiation site were observed 1 day after treatment through histopathologic studies.Transmission electron micrographs showed the autolysis of bipolar cells and ganglion cells.Then,intravitreal 0.04 U microplasmin was administered in the rats with RVO.According to the fluorescein angiographs,the recanalization happened in 75%eyes at 7 days,which was significantly higher than that of the rt-PA group and the control.Fundus photographs and histopathologic studies manifest the absorption of vitreous hemorrhage and regression of retinal edema.The pharmacodynamics studies above indicate a good perspective of recombinant microplasmin in the therapy of many eye diseases.