Cloning and purification of an anti-thrombotic, chimeric Staphylokinase in Pichia pastoris.

There has been an increasing prevalence of cardiovascular diseases such as myocardial infarction and stroke in modern societies because of multiple lifestyle related issues like sedentariness and obesity, alcohol consumption and many more "life-style"factors. The FDA-approved thrombolytics such as Tissue Plasminogen Activator, Streptokinase etc. are used to lyse the clots in thrombotic disorders such as myocardial infarction, stroke etc. but re-occlusion and bleeding that are co-incident to their clinical usage are not addressed. Hence, there is need to develop thrombolytics having properties like increased fibrin clot specificity and thrombin inhibition capability to prevent re-occlusion. In the present work, a fusion protein construct containing two components i.e. Staphylokinase (SAK) and Epidermal Growth Factor (EGF) 4, 5, 6-like domains of human thrombomodulin (THBD) was expressed in Pichia pastoris after genetic optimization. SAK isolated from Staphylococcus aureus is a fibrin-specific plasminogen activator while EGF 4, 5, 6-like domains are reported to be responsible for imparting thrombin inhibition to human thrombomodulin, and therefore, expected could help prevent re-occlusion in the novel construct - SAK_EGF, which is a 43 kDa protein. After expression, it was purified (approx. 13-fold) using two-step purification protocol involving ion-exchange followed by Gel Filtration Chromatography (GFC). The functional characterization including plasminogen activation and thrombin inhibition showed that both the fusion partners viz. SAK and 4,5,6 EGF-like domains retained their respective activities after fusion, confirming it to be a bio-active construct. Thus, this engineered protein could be clinically promising due to the combinatorial effect of fibrin-specific thrombus lysis and prevention of re-occulusion.

Bio-inspired liposomal thrombomodulin conjugate through bio-orthogonal chemistry.

We report the synthesis of bioinspired liposomal thrombomodulin (TM) conjugates by chemoselective and site-specific liposomal conjugation of recombinant TM at C-terminus. TM is an endothelial cell membrane protein that acts as a major cofactor in the protein C anticoagulant pathway. To closely mimic membrane protein structural features of TM, we proposed membrane-mimetic re-expression of recombinant TM onto liposome. A recombinant TM containing the EGF-like 456 domains and an azidohomoalanine at C-terminus was expressed in E. coli. Conjugation of the recombinant TM onto liposome via Staudinger ligation and copper-free click chemistry were investigated as an optimal platform for exploring membrane protein TM's activity, respectively. The bioinspired liposomal TM conjugates were confirmed with Western blotting and protein C activation activity. The recombinant TM-liposome conjugates showed a 2-fold higher k(cat)/K(m) value for protein C activation than that of the recombinant TM alone, which indicated that the lipid membrane has a beneficiary effect on the recombinant TM's activity. The reported liposomal protein conjugate approach provides a rational design strategy for both studying membrane protein TM's functions and generating a membrane protein TM-based anticoagulant agent.

Production of an active recombinant thrombomodulin derivative in transgenic tobacco plants and suspension cells.

Thrombomodulin is a membrane-bound protein that plays an active role in the blood coagulation system by binding thrombin and initiating the protein C anticoagulant pathway. Solulin is a recombinant soluble derivative of human thrombomodulin. It is used for the treatment of thrombotic disorders. To evaluate the production of this pharmaceutical protein in plants, expression vectors were generated using four different N-terminal signal peptides. Immunoblot analysis of transiently transformed tobacco leaves showed that intact Solulin could be detected using three of these signal peptides. Furthermore transgenic tobacco plants and BY2 cells producing Solulin were generated. Immunoblot experiments showed that Solulin accumulated to maximum levels of 115 and 27 microg g(-1) plant material in tobacco plants and BY2 cells, respectively. Activity tests performed on the culture supernatant of transformed BY2 cells showed that the secreted Solulin was functional. In contrast, thrombomodulin activity was not detected in total soluble protein extracts from BY2 cells, probably due to inhibitory effects of substances in the cell extract. N-terminal sequencing was carried out on partially purified Solulin from the BY2 culture supernatant. The sequence was identical to that of Solulin produced in Chinese hamster ovary cells, confirming correct processing of the N-terminal signal peptide. We have demonstrated that plants and plant cell cultures can be used as alternative systems for the production of an active recombinant thrombomodulin derivative.

Marcadores de lesión endotelial implicados en la trombosis y fibrinolisis en la diabetes mellitus / Markers of endothelial damage involved in thrombosis and fibrinolysis in diabetes mellitus

La diabetes mellitus es un factor de riesgo conocido de enfermedad vascular. Las primeras fases de esta afección vascular implican la disfunción del endotelio vascular. El estudio funcional del endotelio supone una intervención demasiado laboriosa y costosa para formar parte de la valoración regular del paciente de riesgo. En consecuencia, ha crecido el interés en los últimos años en la valoración de la lesión endotelial por medio de la determinación de diferentes marcadores solubles. Los más estudiados son aquellos marcadores de síntesis predominantemente endotelial implicados en los procesos de trombosis y fibrinolisis. En este artículo se revisan aquellos que se han relacionado con la lesión endotelial en la diabetes: trombomodulina, tissue factor pathway inhibitor, factor von Willebrand, activador tisular del plasminógeno e inhibidor del activador del plasminógeno-1 (AU)

Separation and characterization of the molecular species of thrombomodulin in the plasma of diabetic patients.

Thrombomodulin (TM) and its molecular species have been identified as markers of vascular endothelial cells (EC). In the present study, using high-performance liquid chromatography (HPLC) in 7 normal subjects, 5 chronic glomerulonephritis (GN) patients, and 25 diabetes mellitus (DM) patients, the TM molecular species separated from plasma showed seven heterogeneous fragments of 94, 74, 48, 36, 27, 14, and 12 kDa. Comparing the diabetic patients and healthy subjects, it was found that plasma TM generally, and its 74-kDa molecular species particularly, were increased in diabetic patients and the increase became more apparent as the disease progressed in severity. Comparing the patients with diabetic nephropathy and those with nephritis of the same degree of renal dysfunction, both groups had increased levels of TM, but the distribution of the molecular species differed; that is, the 74-kDa form increased in the diabetic patients and the 12-kDa species increased markedly in the nephritis patients. Glycation of the various TM molecular species increased more in the diabetic patients than in healthy subjects. There was a significantly positive correlation between the HbA(1c) and the 74-kDa TM molecular species in diabetic patients. These findings suggest that a fluctuation in the blood glucose level is significantly related to vascular EC damage in DM.

Caracterización de la trombomodulina, un anticoagulante natural / Characterization of thrombomodulin, a natural anticoagulant

Durante el desarrollo de enfermedades tromboateroscleróticas se producen daños en el endotelio vascular los que están relacionados con el incremento en sangre de determinadas sustancias, consideradas como marcadores. La Trombomodulina es uno de estos, sus elevados niveles plasmáticos pudieran ser un indicador del desarrollo de fallas funcionales en el endotelio vascular. En este trabajo, se presentan algunos aspectos que caracterizan a la Trombomodulina, lo que permite mantener informados a los profesionales relacionados con la temática. Se reporta a modo de síntesis la localización de dicha molécula, su aislamiento y purificación, modo de detección, características estructurales y funcionales. También se hace referencia a la obtención de derivados de la Trombomodulina y su aplicación en el tratamiento, así como la prevención de enfermedades tromboateroscleroticas(AU)

Protective effect of human urinary thrombomodulin on ischemia- reperfusion injury in the canine liver.

This study was performed to determine whether human urinary soluble thrombomodulin plays a role in liver ischemia-reperfusion injury. Liver ischemia was induced in two groups of dogs. Group 1 was exposed to 60 min ischemia, and group 2 was exposed to 60 min ischemia after preischemic administration of human urinary soluble thrombomodulin. In group 1, the thrombin-antithrombin complex and hyaluronic acid were significantly elevated after ischemia, compared with the preischemic values. While liver issue blood flow and the plasmin-alpha(2)-plasmin inhibitor complex significantly decreased, AST, ALT and m-AST dramatically increased after reperfusion. In group 2, the increase in the thrombin-antithrombin complex and hyaluronic acid was significantly suppressed, and AST, ALT and liver tissue blood flow significantly improved, compared with group 1. Histologically, in group 2, the hepatic tissue structure, including endothelial cells, was relatively intact. These findings suggest that administration of thrombomodulin inhibits endothelial cell injury and coagulopathy and offers protection from liver ischemia-reperfusion injury.

Soluble thrombomodulin purified from human urine exhibits a potent anticoagulant effect in vitro and in vivo.

We examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity > 5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (> 1 TMU /ml), APTT (> 5 TMU/ml), TT (> 5 TMU/ml) and PT (> 40 TMU/ ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

Purification and characterization of two forms of soluble thrombomodulin from human urine.

We have isolated and characterized two forms of soluble thrombomodulin from human urine. The purification procedure consisted of ultrafiltration, chromatography on DEAE-Sepharose, affinity chromatography on diisopropyl-phosphate-thrombin and/or monoclonal anti-thrombomodulin IgG affigel followed by reverse-phase HPLC. An active soluble form of thrombomodulin was purified 1600-fold from 34-l urine. The purified protein migrated as a doublet, with molecular mass 76/72 kDa under reducing conditions and 63/57 kDa under non-reducing conditions as determined by SDS/PAGE. Amino acid analysis of the 63/57-kDa soluble thrombomodulin confirmed sequence identity with human thrombomodulin and demonstrated N-terminal heterogeneity. Compared to membrane-type thrombomodulin, the purified 63/57-kDa soluble thrombomodulin was more active as a cofactor for protein-C activation. The second major thrombomodulin fragment urine is an inactive 35-kDa thrombomodulin polypeptide derived from the N-terminal extracellular region of thrombomodulin.