An active-site titrant for human tissue-type plasminogen activator.
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ABSTRACT:
SUBMITTER: Smith RA
PROVIDER: S-EPMC1147305 | biostudies-other | 1986 Oct
REPOSITORIES: biostudies-other
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Project description: Not available
| S-EPMC6418500 | biostudies-literature
Project description:Ischemia of the heart, brain, and limbs is a leading cause of morbidity and mortality worldwide. Treatment with tissue type plasminogen activator (tPA) can dissolve blood clots and can ameliorate the clinical outcome in ischemic diseases. But the underlying mechanism by which tPA improves ischemic tissue regeneration is not well understood. Bone marrow (BM)-derived myeloid cells facilitate angiogenesis during tissue regeneration. Here, we report that a serpin-resistant form of tPA by activating the extracellular proteases matrix metalloproteinase-9 and plasmin expands the myeloid cell pool and mobilizes CD45(+)CD11b(+) proangiogenic, myeloid cells, a process dependent on vascular endothelial growth factor-A (VEGF-A) and Kit ligand signaling. tPA improves the incorporation of CD11b(+) cells into ischemic tissues and increases expression of neoangiogenesis-related genes, including VEGF-A. Remarkably, transplantation of BM-derived tPA-mobilized CD11b(+) cells and VEGFR-1(+) cells, but not carrier-mobilized cells or CD11b(-) cells, accelerates neovascularization and ischemic tissue regeneration. Inhibition of VEGF signaling suppresses tPA-induced neovascularization in a model of hind limb ischemia. Thus, tPA mobilizes CD11b(+) cells from the BM and increases systemic and local (cellular) VEGF-A, which can locally promote angiogenesis during ischemic recovery. tPA might be useful to induce therapeutic revascularization in the growing field of regenerative medicine.
| S-EPMC2879105 | biostudies-literature
Project description:TNK-tPA products from the innovator and follow-on manufacturers were characterized and compared. All tryptic peptides including N-terminal, C-terminal, and mutated peptides as well as the disulfide-linked peptides were identified, with the demonstration of the same primary sequence and disulfide linkages between the innovator and follow-on products. The three N-linked and one O-linked fucose glycosylation sites were identified. The two N-linked fucose sites (N103 and N448) and one O-linked fucose site (T61) were fully glycosylated in both innovator and follow-on products. The other N-linked site (N184) was partially glycosylated and exhibited a approximately 2.5-fold difference between the innovator (60% occupancy) and follow-on (25% occupancy) products. Since the glycosylation occupancy at this site is known to affect biological activity in the clot lysis assay, this observed difference could cause a concern as to their bioequivalence. The cleavage site for the conversion of the zymogen form to active enzyme was also identified between R275 and I276, with a cleavage of 40% for the innovator and 10% for the follow-on products. Both the glycosylation occupancy (%) and the chain cleavage (%) were determined by two independent approaches, starting from either the peptide or intact protein separation, with consistent results by both methods. Subtle differences of modifications such as deamidation and oxidation between the innovator and biosimilar products were shown at M207, M445, M490 and N58, N184. The observation of different extents of oxidation at M207 and deamidation at N184, which could influence the clot lysis activity, were also of potential concern in drug efficacy between the follow-on and innovator products.
| S-EPMC3104925 | biostudies-literature
Project description:To address a species difference in the responsiveness to human recombinant tissue-type plasminogen activator (rt-PA) between rats and humans, tPA transgenic (Tg) rats were generated and characterized. In the rats, transcriptional regulation of tPA was designed under the control of the endogenous tPA promoter. There were no significant differences in hematological parameters between the tPA Tg and non Tg rats. Plasma tPA concentration was significantly increased and serum free PAI-1 was significantly decreased in the tPA Tg rats. Significant overexpression of tPA mRNA in five major organs was also confirmed in the tPA Tg rats. In contrast, the extent of tPA mRNA induction by pathophysiological stimuli (focal cerebral ischemia) was comparable in the two strains. Earlier increase in the plasma D-Dimer level was observed in the tPA Tg rats in a model of thromboembolism compared with the non Tg rats. On the other hand, there was no statistically significant prolongation of bleeding time in a rat model of bleeding between the two strains. rt-PA showed dose-related blood flow restoration in a rat model of thromboembolic stroke in the tPA Tg rats from a dose (1 mg/kg, i.v.) similar to clinical doses for human stroke patients. In conclusion, tPA Tg rats, in which tPA is overexpressed and endogenous fibrinolytic activity is enhanced without hemostatic abnormality, were generated. tPA Tg rats would be beneficial for the pharmacological and the toxicological evaluation of rt-PA and other various fibrinolytic enhancers.
| S-EPMC5756269 | biostudies-literature
Project description:Thrombosis is a leading cause of death worldwide [1]. Recombinant tissue-type plasminogen activator (tPA) is the FDA-approved thrombolytic drug for ischemic strokes, myocardial infarction and pulmonary embolism. tPA is a multi-domain serine protease of the trypsin-family [2] and catalyses the critical step in fibrinolysis [3], converting the zymogen plasminogen to the active serine protease plasmin, which degrades the fibrin network of thrombi and blood clots. tPA is rapidly inactivated by endogenous plasminogen activators inhibitor-1 (PAI-1) [4] (Fig. 1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort [5]. Tenecteplase (TNK-tPA) is a newer generation of tPA variant showing slower inhibition by PAI-1 [6]. Extensive studies to understand the molecular interactions between tPA and PAI-1 have been carried out [7], [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], however, the precise details at atomic resolution remain unknown. We report the crystal structure of tPA·PAI-1 complex here. The methods required to achieve these data include: (1) recombinant expression and purification of a PAI-1 variant (14-1B) containing four mutations (N150H, K154T, Q319L, and M354I), and a tPA serine protease domain (tPA-SPD) variant with three mutations (C122A, N173Q, and S195A, in the chymotrypsin numbering) [19]; (2) formation of a tPA-SPD·PAI-1 Michaëlis complex in vitro [19]; and (3) solving the three-dimensional structure for this complex by X-ray crystallography [deposited in the PDB database as 5BRR]. The data explain the specificity of PAI-1 for tPA and uPA [19], [20], and provide structural basis to design newer generation of PAI-1-resistant tPA variants as thrombolytic agents [19].
| S-EPMC4731420 | biostudies-literature
Project description:The neuronal serine protease tissue-type Plasminogen Activator (tPA) is an important player of the neuronal survival and of the synaptic plasticity. Thus, a better understanding the mechanisms regulating the neuronal trafficking of tPA is required to further understand how tPA can influence brain functions. Using confocal imaging including living cells and high-resolution cell imaging combined with an innovating labeling of tPA, we demonstrate that the neuronal tPA is contained in endosomal vesicles positives for Rabs and in exosomal vesicles positives for synaptobrevin-2 (VAMP2) in dendrites and axons. tPA-containing vesicles differ in their dynamics with the dendritic tPA containing-vesicles less mobile than the axonal tPA-containing vesicles, these laters displaying mainly a retrograde trafficking. Interestingly spontaneous exocytosis of tPA containing-vesicles occurs largely in dendrites.
| S-EPMC6491899 | biostudies-literature
Project description:The kinetics of inhibition of tissue-type plasminogen activator (t-PA) by the fast-acting plasminogen activator inhibitor-1 (PAI-1) was investigated in homogeneous (plasma) and heterogeneous (solid-phase fibrin) systems by using radioisotopic and spectrophotometric analysis. It is demonstrated that fibrin-bound t-PA is protected from inhibition by PAI-1, whereas t-PA in soluble phase is rapidly inhibited (K1 = 10(7) M-1.s-1) even in the presence of 2 microM-plasminogen. The inhibitor interferes with the binding of t-PA to fibrin in a competitive manner. As a consequence the Kd of t-PA for fibrin (1.2 +/- 0.4 nM) increases and the maximal velocity of plasminogen activation by fibrin-bound t-PA is not modified. From the plot of the apparent Kd versus the concentration of PAI-1 a Ki value of 1.3 +/- 0.3 nM was calculated. The quasi-similar values for the dissociation constants between fibrin and t-PA (Kd) and between PAI-1 and t-PA (Ki), as well as the competitive type of inhibition observed, indicate that the fibrinolytic activity of human plasma may be the result of an equilibrium distribution of t-PA between both the amount of fibrin generated and the concentration of circulating inhibitor.
| S-EPMC1135393 | biostudies-other
Project description:Elevated activity of plasminogen activator inhibitor-1 (PAI-1) and decreased tissue plasminogen activator (tPA) activity are considered to be important risk factors for type 2 diabetes mellitus (T2DM) and metabolic syndrome (MetS). The aim of this study was to investigate the association of the PAI-1 4G/5G and tPA Alu-repeat I/D polymorphisms with T2DM in Malaysian subjects. Serum insulin, coronary risk panel, plasma glucose, and PAI-1 4G/5G and tPA Alu-repeat I/D polymorphisms were studied in 303 T2DM subjects (227 with MetS and 76 without MetS) and 131 normal subjects without diabetes and MetS. Statistical analysis showed that the dominant and additive models of PAI-1 4G/5G polymorphism showed a weak association with T2DM without MetS (OR = 2.35, P = 0.045; OR = 1.67, P = 0.058). On the other hand, the recessive model of the tPA Alu-repeat I/D polymorphism showed an association with T2DM with MetS (OR = 3.32, P = 0.013) whereas the dominant and additive models of the tPA Alu-repeat I/D polymorphism were not associated with T2DM either with or without MetS.
| S-EPMC3336151 | biostudies-literature
Project description:Incubation of cultured human endothelial cells with 5 mM-dibutyryl cyclic AMP led to an approx. 2-fold increase in tissue-type plasminogen-activator (t-PA) production over a 24 h incubation period. The stimulating effect of dibutyryl cyclic AMP could be explained by the slow liberation of butyrate, as the effect could be reproduced by addition of free butyrate to the medium, but not by addition of 8-bromo cyclic AMP or forskolin, agents known to raise intracellular cyclic AMP levels. With butyrate, an accelerated accumulation of t-PA antigen in the conditioned medium (CM) was observed after a lag period of about 6 h. Increasing amounts of butyrate caused an increasingly stimulatory effect, reaching a plateau at 5 mM-butyrate. The relative enhancement of t-PA production in the presence of 5 mM-butyrate varied among different endothelial cell cultures from 6- to 25-fold in 24 h CM. Such an increase in t-PA production was observed with both arterial and venous endothelial cells. The butyrate-induced increases in t-PA production were accompanied by increased t-PA mRNA levels. Analysis of radiolabelled CM and cell extracts by SDS/polyacrylamide-gel electrophoresis indicated that the potent action of butyrate is probably restricted to a small number of proteins. The accumulation of plasminogen activator inhibitor type 1 (PAI-1) in CM from butyrate-treated cells varied only moderately. In our study of the relationship between structure and stimulatory activity, we found that a straight-chain C4 monocarboxylate structure with a methyl group at one end and a carboxy moiety at the other seems to be required for the optimal induction of t-PA in cultured endothelial cells.
| S-EPMC1148455 | biostudies-other
Project description:Human tissue plasminogen activator (tPA) belongs to the serine protease family. It converts plasminogen into plasmin and is used clinically to treat thrombosis. Human tPA is composed of 527 amino acids residues and contains 17 disulfide bonds. Escherichia coli has been used only rarely for the efficient production of recombinant tPA. However, the functional expression of full-length tPA that contains multiple disulfide bonds on an industrial scale remains challenging. Here, we describe the soluble expression and characterization of full-length tPA by auto-induction in E. coli.We achieved optimal levels of gene expression, minimized negative effects related to the production of heterologous proteins, and optimized cytoplasmic yields. Three different E. coli strains, BL21 (DE3), Rosetta, and Origami 2, could express tPA using an auto-induction mechanism. In addition, similar yields of recombinant protein were produced at temperatures of 33, 35, and 37°C. The E. coli strain origami 2 could increase disulfide bond formation in cytoplasmic tPA and produce purified soluble recombinant protein (~0.9 mg/l medium). The full-length tPA was monomeric in solution, and fibrin plate assays confirmed that the recombinant tPA displayed serine protease activity.This is the first report that describes the heterologous expression of correctly folded active full-length tPA. This could provide valuable information for using prokaryotic auto-induction expression systems to produce tPA at industrial and pharmaceutical levels without in vitro refolding during the production step.
| S-EPMC4379951 | biostudies-literature