scholarly journals Expression of Active Recombinant Human Tissue-Type Plasminogen Activator by Using In Vivo Polyhydroxybutyrate Granule Display

2010 ◽  
Vol 76 (21) ◽  
pp. 7226-7230 ◽  
Author(s):  
Yanping Geng ◽  
Shengjun Wang ◽  
Qingsheng Qi
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Human Tissue ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Display ◽  
Sodium Dodecyl ◽  
Tissue Type ◽  
Affinity Tag ◽  
Tissue Plasminogen

ABSTRACT Recombinant human tissue plasminogen activator (rPA) is a truncated version of tissue plasminogen activator (tPA), which contains nine disulfide bonds and is prone to forming inactive inclusion bodies when expressed in bacteria. To obtain functional rPA expression, we displayed the rPA on the surface of polyhydroxybutyrate (PHB) granules using phasin as the affinity tag. rPA was fused to the N terminus of the phasin protein with a thrombin cleavage site as the linker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis showed that rPA fusion was successfully displayed on the surface of PHB granules. An activity assay indicated that the rPA fusion is active. The in vivo surface display strategy for functional rPA expression in Escherichia coli is distinct for its efficient folding and easier purification and may be expanded to the expression of other eukaryotic proteins with complex conformation.

scholarly journals Catabolism of human tissue plasminogen activator in mice

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 539-544 ◽  
Author(s):  
HE Fuchs ◽  
H Jr Berger ◽  
SV Pizzo
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Active Site ◽  
Human Tissue ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Organ Distribution ◽  
Tissue Plasminogen ◽  
Iodine 125 ◽  
Distribution Studies

The catabolism of human tissue plasminogen activator (t-PA) was studied in mice. The clearance of t-PA labeled with iodine 125 was rapid (t1/2). The clearance of phenylmethylsulfonyl-125I-t-PA, which is active site-inhibited, was identical to the active enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the vast majority of 125I-t-PA injected into the circulation was present as free enzyme and not in a complex with inhibitors. The clearance of 125I-t-PA was unaltered by large molar excesses of several ligands of known clearance specificities, including macroalbumin, asialoorosomucoid, and diisopropylphosphorylthrombin and was also not altered in the presence of a 1,000-fold molar excess of unlabeled t-PA. Organ distribution studies demonstrated that the early rapid clearance of 125I-t-PA occurred in hepatocytes, followed by a later renal phase of clearance. The clearance of 125I-urokinase (UK) also was studied and was very similar in all aspects to the clearance of 125I-t-PA. These results suggest that both t-PA and UK are cleared from the circulation by unique nonsaturable processes localized in the liver that are independent of the proteinase active site.

scholarly journals Catabolism of human tissue plasminogen activator in mice

Blood ◽  
1985 ◽  
Vol 65 (3) ◽  
pp. 539-544 ◽  
Author(s):  
HE Fuchs ◽  
H Jr Berger ◽  
SV Pizzo
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Active Site ◽  
Human Tissue ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Organ Distribution ◽  
Tissue Plasminogen ◽  
Iodine 125 ◽  
Distribution Studies

Abstract The catabolism of human tissue plasminogen activator (t-PA) was studied in mice. The clearance of t-PA labeled with iodine 125 was rapid (t1/2). The clearance of phenylmethylsulfonyl-125I-t-PA, which is active site-inhibited, was identical to the active enzyme. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the vast majority of 125I-t-PA injected into the circulation was present as free enzyme and not in a complex with inhibitors. The clearance of 125I-t-PA was unaltered by large molar excesses of several ligands of known clearance specificities, including macroalbumin, asialoorosomucoid, and diisopropylphosphorylthrombin and was also not altered in the presence of a 1,000-fold molar excess of unlabeled t-PA. Organ distribution studies demonstrated that the early rapid clearance of 125I-t-PA occurred in hepatocytes, followed by a later renal phase of clearance. The clearance of 125I-urokinase (UK) also was studied and was very similar in all aspects to the clearance of 125I-t-PA. These results suggest that both t-PA and UK are cleared from the circulation by unique nonsaturable processes localized in the liver that are independent of the proteinase active site.

scholarly journals Tissue plasminogen activator release in vivo in response to vasoactive agents

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 835-839 ◽  
Author(s):  
D Smith ◽  
M Gilbert ◽  
WG Owen
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Tissue Type ◽  
Clot Lysis ◽  
Vasoactive Agents ◽  
Lysine Vasopressin ◽  
Type Plasminogen Activator ◽  
Transient Rise ◽  
Tissue Plasminogen

Release of tissue plasminogen activator into the circulation of rats in response to intravascular injections of vasoactive agents is studied by using a sensitive and specific clot lysis assay. Intra-arterial bradykinin elicits a rapid and transient rise in circulating plasminogen activator, which is maximum within one minute and is cleared within four to eight minutes. The plasminogen activator is fibrin dependent and is neutralized by an antiserum to human tissue- type plasminogen activator. Bradykinin is 1,000-fold more potent than the other agonists tested, which include histamine, norepinephrine, epinephrine, eledoisin-related peptide, arginine-vasopressin, lysine- vasopressin, desmopressin acetate, carbachol, and acetylcholine. Potency of bradykinin is related to its amino acid sequence. Sequential infusions of bradykinin produce a tachyphylactoid response that could be overcome by increasing the dose of the sequential bradykinin challenge. It is concluded that the characteristics of the responses to bradykinin and other agents in vivo differ significantly from those observed in isolated tissue preparations.

scholarly journals Tissue plasminogen activator release in vivo in response to vasoactive agents

Blood ◽  
1985 ◽  
Vol 66 (4) ◽  
pp. 835-839 ◽  
Author(s):  
D Smith ◽  
M Gilbert ◽  
WG Owen
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Tissue Type ◽  
Clot Lysis ◽  
Vasoactive Agents ◽  
Lysine Vasopressin ◽  
Type Plasminogen Activator ◽  
Transient Rise ◽  
Tissue Plasminogen

Abstract Release of tissue plasminogen activator into the circulation of rats in response to intravascular injections of vasoactive agents is studied by using a sensitive and specific clot lysis assay. Intra-arterial bradykinin elicits a rapid and transient rise in circulating plasminogen activator, which is maximum within one minute and is cleared within four to eight minutes. The plasminogen activator is fibrin dependent and is neutralized by an antiserum to human tissue- type plasminogen activator. Bradykinin is 1,000-fold more potent than the other agonists tested, which include histamine, norepinephrine, epinephrine, eledoisin-related peptide, arginine-vasopressin, lysine- vasopressin, desmopressin acetate, carbachol, and acetylcholine. Potency of bradykinin is related to its amino acid sequence. Sequential infusions of bradykinin produce a tachyphylactoid response that could be overcome by increasing the dose of the sequential bradykinin challenge. It is concluded that the characteristics of the responses to bradykinin and other agents in vivo differ significantly from those observed in isolated tissue preparations.

A Modified Human Tissue Plasminogen Activator with Extended Half–Life In Vivo

1987 ◽  
Vol 5 (9) ◽  
pp. 953-958 ◽  
Author(s):  
David Lau ◽  
Gregory Kuzma ◽  
Cha-Mer Wei ◽  
David J. Livingston ◽  
Nancy Hsiung
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Human Tissue ◽  
Half Life ◽  
Tissue Plasminogen

An Enzyme Linked Immunosorbent Assay for Determination of Tissue Plasminogen Activator Applied to Patients with Thromboembolic Disease

1983 ◽  
Vol 50 (03) ◽  
pp. 740-744 ◽  
Author(s):  
Nils Bergsdorf ◽  
Torbjörn Nilsson ◽  
Per Wallén
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Specific Activity ◽  
Thromboembolic Disease ◽  
Tissue Type ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type Plasminogen Activator ◽  
Tissue Plasminogen ◽  
Normal Human

SummaryUtilizing the immunoglobulin fraction from a goat antiserum against human uterine tissue plasminogen activator, an enzyme- linked immunoassay for tissue-type plasminogen activator in human plasma has been developed. With the new method, the concentration of t-PA in normal human acidified plasma is found to be 4.0 ± 1.8 (SD) ng/ml. It increases to 12 ng/ml after a tomiquet test, and to 14 ng/ml after strenous physical exercise. In a group of patients with idiopathic thromboembolic disease, the resting t-PA concentration was 5 ng/ml and the post-occlusion value 16 ng/ml. Furthermore, the patients also exhibited a normal post-occlusion rise in the concentration of plasmin-α2-antiplasmin complex. However, in 37% of the post-occlusion patient plasmas, virtually no increase in t-PA could be detected by a specific activity assay. The results indicate that the reason for a defective post-occlusion fibrinolytic activity in a majority of cases may be the presence of increased concentrations of a fast-acting specific t-PA inhibitor.

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