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Lida Tang - One of the best experts on this subject based on the ideXlab platform.
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Thrombolysis with rhPro-UK 3 to 6 hours after embolic stroke in rat
Neurological Research, 2019Co-Authors: Wen-xia Ding, Xin-xin Li, Weiting Wang, Zhuanyou Zhao, Lida TangAbstract:ABSTRACTObjectives: To investigate the thrombolysis with recombinant human Prourokinase (rhPro-UK) on thromboembolic stroke in rats at different therapeutic time windows (TTW).Methods: Rats were su...
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effect of human recombinant Prourokinase rhpro uk on thromboembolic stroke in rats
European Journal of Pharmacology, 2018Co-Authors: Wen-xia Ding, Xin-xin Li, Weiting Wang, Zhuanyou Zhao, Lida TangAbstract:Abstract We evaluated the efficacy and safety of human recombinant Prourokinase ( rhpro-UK) on thromboembolic stroke in rats. 60 rats with thromboembolic stroke were divided into 6 groups (n = 10). The model group was given saline, the reagent groups were given rhpro-UK (5, 10, 20 × 10 4 U/kg), and positive control groups were given urokinase (UK) 10 × 10 4 U/kg and recombinant tissue plasminogen activator (rt-PA) 9 mg/kg through intravenous infusion at 1.5 h after embolism. And other 10 rats without occluded by autologous blood clots as the sham group were given saline. At 6 h after treatment, neurological deficit score and Magnetic Resonance Imaging(MRI) including T 1 WI and T 2 WI sequence scanning were measured. At 24 h after treatment, the brain was cut for 2,3,5-triphenyltetrazolium chloride (TTC) staining and aspectrophotometric assay to measure the infarct area and intracerebral hemorrhage after neurological deficit detection. rhpro-UK (5, 10, 20 × 10 4 U/kg) improved neurological disorder by 39.1 ± 19.7% (n = 10, P > 0.05), 65.2 ± 14.2% (n = 10, P 4 U/kg) and each positive groups at intracerebral hemorrhage (P > 0.05). Rhpro-UK improved the damaged neural function, decreased the extent of the disease and did not raise bleeding, had protective effects for cerebral ischemia in rats.
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effect of recombinant human Prourokinase on thrombolysis in a rabbit model of thromboembolic stroke
Biomedical Reports, 2017Co-Authors: Wen-xia Ding, Xin-xin Li, Weiting Wang, Zhuanyou Zhao, Xiangwei Xu, Lida TangAbstract:: The aim of the present study was to investigate the efficacy of recombinant human Prourokinase (rhPro-UK) on thromboembolic stroke in rabbits. A total of 210 rabbits were used in experiments. The 180 thromboembolic stroke rabbits were divided into three therapeutic time windows with six groups in each time window (n=10). The model group was administered saline, the reagent groups were administered rhPro-UK (2.5×, 5× and 10×104 U/kg), and the positive control groups were administered 5×104 urokinase (UK) U/kg and 4.5 mg/kg recombinant human tissue plasminogen activator via intravenous infusion at 3, 4.5 and 6 h after embolism. The remaining 30 rats (that had not undergone occlusion by autologous blood clots) served as a sham group and were administered saline. The radioactive intensity was detected using a medical gamma counter before and after the administration of the drug for 15, 30, 45, 60, 75, 90, 105 and 120 min. At 24 h after treatment, the brain samples were coronally sliced into 5 mm sections and hemorrhage was estimated used a semiquantitative method by counting the number of section faces with hemorrhaging. The plasma was collected for prothrombin time, activated partial thromboplastin time, fibrinogen and thrombin time tests using a solidification method with a blood coagulation factor analyzer. In addition, α2-antiplasmin (α2-AP) was evaluated using ELISA methods using a RT-6100 microplate reader. At the 3 h time point, the thrombolysis rate of rhPro-UK(2.5×, 5× and 10×104 U/kg) was 21.5% (P 0.05), 40% (P 0.05), 5.3% (P>0.05) and 18.1% (P 0.05), 30% (P 0.05), 6.5% (P>0.05) and 17.8% (P 0.05), 30% (P 0.05), 12.7% (P>0.05) and 22.2% (P 0.05) were identified between rhPro-UK (2.5×, 5× and 10×104 U/kg) and the model group regarding hemorrhage type, size and blood coagulation factors at the different time points. Thus, rhPro-UK promoted thrombolysis and recanalization (patency rate), with reduced risk of cerebral hemorrhage, and thus exerted protective effects on cerebral ischemia rabbits.
Victor Gurewich - One of the best experts on this subject based on the ideXlab platform.
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thrombolysis vs bleeding from hemostatic sites by a Prourokinase mutant compared with tissue plasminogen activator
Journal of Thrombosis and Haemostasis, 2006Co-Authors: Victor Gurewich, Paolo Sarmientos, R Pannell, A Simmonsbyrd, S F BadylakAbstract:Summary. Background: A single site mutant (M5) of Prourokinase (proUK) was developed to make proUK less vulnerable to spontaneous activation in plasma. This was a problem that seriously compromised proUK in clinical trials, as it precluded proUK-mediated fibrinolysis at therapeutic concentrations. Methods and results: After completing dose-finding studies, 12 anesthetized dogs with femoral artery thrombosis were given either M5 (2.0 mg kg−1) or tissue plasminogen activator (t-PA) (1.4 mg kg−1) by i.v. infusion over 60 min (20% administered as a bolus). Two pairs of standardized injuries were inflicted at which hemostasis was completed prior to drug administration. Blood loss was quantified by measuring the hemoglobin in blood absorbed from these sites. Thrombolysis was evaluated at 90 min and was comparably effective by both activators. Rethrombosis developed in one t-PA dog. The principal difference found was that blood loss was 10-fold higher with t-PA (mean ∼40 mL) than with M5 (mean ∼4 mL) (P = 0.026) and occurred at more multiple sites (mean 2.7 vs. 1.2). This effect was postulated to be related to differences in the mechanism of plasminogen activation by t-PA and M5 in which the latter is promoted by degraded rather than intact (hemostatic) fibrin. In addition, two-chain M5 was efficiently inactivated by plasma C1 inactivator, an exceptional property which helped contain its non-specific proteolytic effect. Conclusions: Intravascular thrombolysis by M5 was accompanied by significantly less bleeding from hemostatic sites than by t-PA. This was attributed to the proUK paradigm of fibrinolysis being retained at therapeutic concentrations by the mutation.
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the blockage of the high affinity lysine binding sites of plasminogen by eaca significantly inhibits Prourokinase induced plasminogen activation
Biochimica et Biophysica Acta, 2002Co-Authors: Yuhong Chen, Ping Wang, Jing Zhang, Victor Gurewich, Peixiang ZhangAbstract:Abstract Prourokinase-induced plasminogen activation is complex and involves three distinct reactions: (1) plasminogen activation by the intrinsic activity of Prourokinase; (2) Prourokinase activation by plasmin; (3) plasminogen activation by urokinase. To further understand some of the mechanisms involved, the effects of epsilon-aminocaproic acid (EACA), a lysine analogue, on these reactions were studied. At a low range of concentrations (10–50 μM), EACA significantly inhibited Prourokinase-induced (Glu-/Lys-) plasminogen activation, Prourokinase activation by Lys-plasmin, and (Glu-/Lys-) plasminogen activation by urokinase. However, no inhibition of plasminogen activation by Ala 158 -Prourokinase (a plasmin-resistant mutant) occurred. Therefore, the overall inhibition of EACA on Prourokinase-induced plasminogen activation was mainly due to inhibition of reactions 2 and 3, by blocking the high-affinity lysine binding interaction between plasmin and Prourokinase, as well as between plasminogen and urokinase. These findings were consistent with kinetic studies which suggested that binding of kringle 1–4 of plasmin to the N-terminal region of Prourokinase significantly promotes Prourokinase activation, and that binding of kringle 1–4 of plasminogen to the C-terminal lysine 158 of urokinase significantly promotes plasminogen activation. In conclusion, EACA was found to inhibit, rather than promote, Prourokinase-induced plasminogen activation due to its blocking of the high-affinity lysine binding sites on plasmin(ogen).
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Prourokinase mutant that induces highly effective clot lysis without interfering with hemostasis
Circulation Research, 2002Co-Authors: Peixiang Zhang, Yuhong Chen, Jing Zhang, Victor GurewichAbstract:Prourokinase (proUK) is a zymogenic plasminogen activator that at pharmacological doses is prone to nonspecific activation to urokinase. This has handicapped therapeutic exploitation of its fibrin-specific physiological properties. To attenuate this susceptibility without compromising specific activation of proUK on a fibrin clot, a Lys300→His mutation (M5) was developed. M5 had a lower intrinsic activity and, therefore, remained stable in plasma at a 4-fold higher concentration than did proUK. M5 had a higher 2-chain activity and induced more rapid plasminogen activation and fibrin-specific clot lysis in vitro. Sixteen dogs embolized with radiolabeled clots were infused with saline, proUK, tissue plasminogen activator, or M5. The lower intrinsic activity allowed a higher infusion rate with M5, which induced the most rapid and efficient clot lysis (50% clot lysis by ≈600 μg/kg M5 versus ≈1200 μg/kg proUK). In association with this, M5 caused neither a significant increase in the primary bleeding time nor secondary bleeding (total blood loss). By contrast, these measurements increased 4-fold and 5-fold, respectively, with proUK and >5-fold and 8-fold, respectively, with tissue plasminogen activator. Clot lysis by M5 and hemostasis were further evaluated in 6 rhesus monkeys. M5 again induced rapid clot lysis without a significant increase in the primary bleeding time, and secondary bleeding did not occur. In conclusion, a site-directed mutation designed to improve the stability of proUK in blood at therapeutic concentrations induced superior clot lysis in vitro and in vivo without causing significant interference with hemostasis.
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Lipoprotein(a): A kinetic study of its influence on fibrin-dependent plasminogen activation by Prourokinase or tissue plasminogen activator
Biochemistry, 1993Co-Authors: Peter C. Harpel, Ralph Pannell, Victor GurewichAbstract:Lipoprotein(a) [Lp(a)] has been postulated to inhibit fibrinolysis due to its structural homology to plasminogen. Indeed, it has been reported that Lp(a) competitively inhibits the promotion by fibrin of tissue plasminogen activator (t-PA)-catalyzed plasminogen activation. However, it has also been reported that this inhibition is uncompetitive. No studies have been published, to our knowledge, of the effect of Lp(a) on Prourokinase (pro-UK)-catalyzed plasminogen activation. Plasminogen activation by pro-UK or a plasmin-resistant mutant pro-UK was previously shown to be promoted by fibrin fragment E 2 , whereas that by t-PA is promoted by fragment D
Kenneth Ouriel - One of the best experts on this subject based on the ideXlab platform.
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Prourokinase versus Urokinase for Recanalization of Peripheral Occlusions, Safety and Efficacy: The PURPOSE Trial
Journal of Vascular and Interventional Radiology, 1999Co-Authors: Kenneth Ouriel, Krishna Kandarpa, Debra M Schuerr, Micki Hultquist, Gillian Hodkinson, Bruce WallinAbstract:Background The intraarterial administration of thrombolytic agents is associated with clinical benefits in patients with acute peripheral arterial occlusion, and urokinase has been the agent that has become the standard of care in the United States. Recombinant Prourokinase (r-ProUK) offers potential as a novel agent with improved fibrin specificity and, as such, may offer advantages as an attractive alternative to urokinase. Methods A randomized, double-blind, parallel, phase II, prospective multicenter trial was undertaken to compare three doses of intra-arterial, catheter-directed r-ProUK (2 mg, 4 mg, or 8 mg/hr for 8 hrs, then 0.5 mg/hr) versus one dose of tissue-culture urokinase (4,000 IU/min for 4 hrs, then 2,000 IU/min) for the treatment of acute lower extremity arterial occlusion of 14 days' duration or less ( n = 241). The primary endpoint was complete (>95%) lysis of the occluding thrombus after 8 hours of infusion. Results Increased clot lysis at 8 hours, decreased fibrinogen concentration, and an increased rate of hemorrhagic events were observed as the r-ProUK dose was increased from 2 mg/hr to 8 mg/ hr. Similarly, a decreased duration of study drug infusion was seen, decreasing from 16.7 ± 0.90 hours in the 2 mg/hr group to 12.7 ± 0.97 hours in the 8 mg/hr group. The results for the urokinase group decreased to a level between those observed for the 2 mg and 8 mg r-ProUK group with respect to clot lysis at 8 hours, fibrinogen decrement, and bleeding complications, approximating those observed in the 4 mg/hr r-ProUK group. These results were achieved with a relatively low rate of major bleeding events and no episodes of intracranial hemorrhage. Conclusions The 8 mg/hr dose of r-ProUK was associated with an increased rate of thrombolysis relative to the other treatment groups, associated with a slightly increased frequency of bleeding complications and decrements in fibrinogen concentration. Conversely, the 2 mg/hr r-ProUK dose was associated with a slightly slower rate of thrombolysis, but bleeding complications and fibrinogenolysis were diminished. r-ProUK is a novel thrombolytic agent with a dose-related safety and efficacy profile. As such, it offers potential as a useful tool in the treatment of peripheral vascular occlusion.
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Prourokinase versus urokinase for recanalization of peripheral occlusions safety and efficacy the purpose trial
Journal of Vascular and Interventional Radiology, 1999Co-Authors: Kenneth Ouriel, Krishna Kandarpa, Debra M Schuerr, Micki Hultquist, Gillian Hodkinson, Bruce WallinAbstract:BACKGROUND: The intraarterial administration of thrombolytic agents is associated with clinical benefits in patients with acute peripheral arterial occlusion, and urokinase has been the agent that has become the standard of care in the United States. Recombinant Prourokinase (r-ProUK) offers potential as a novel agent with improved fibrin specificity and, as such, may offer advantages as an attractive alternative to urokinase. METHODS: A randomized, double-blind, parallel, phase II, prospective multicenter trial was undertaken to compare three doses of intra-arterial, catheter-directed r-ProUK (2 mg, 4 mg, or 8 mg/hr for 8 hrs, then 0.5 mg/hr) versus one dose of tissue-culture urokinase (4,000 IU/min for 4 hrs, then 2,000 IU/min) for the treatment of acute lower extremity arterial occlusion of 14 days' duration or less (n = 241). The primary endpoint was complete (>95%) lysis of the occluding thrombus after 8 hours of infusion. RESULTS: Increased clot lysis at 8 hours, decreased fibrinogen concentration, and an increased rate of hemorrhagic events were observed as the r-ProUK dose was increased from 2 mg/hr to 8 mg/hr. Similarly, a decreased duration of study drug infusion was seen, decreasing from 16.7 +/- 0.90 hours in the 2 mg/hr group to 12.7 +/- 0.97 hours in the 8 mg/hr group. The results for the urokinase group decreased to a level between those observed for the 2 mg and 8 mg r-ProUK group with respect to clot lysis at 8 hours, fibrinogen decrement, and bleeding complications, approximating those observed in the 4 mg/hr r-ProUK group. These results were achieved with a relatively low rate of major bleeding events and no episodes of intracranial hemorrhage. CONCLUSIONS: The 8 mg/hr dose of r-ProUK was associated with an increased rate of thrombolysis relative to the other treatment groups, associated with a slightly increased frequency of bleeding complications and decrements in fibrinogen concentration. Conversely, the 2 mg/hr r-ProUK dose was associated with a slightly slower rate of thrombolysis, but bleeding complications and fibrinogenolysis were diminished. r-ProUK is a novel thrombolytic agent with a dose-related safety and efficacy profile. As such, it offers potential as a useful tool in the treatment of peripheral vascular occlusion.
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thrombolysis with Prourokinase versus urokinase an in vitro comparison
Journal of Vascular Surgery, 1996Co-Authors: Kenneth Ouriel, Richard M Green, Julianne Stoughton, Patrick Riggs, Cathy CiminoAbstract:Purpose: Despite advantages demonstrated in vitro, no single thrombolytic agent has been clearly shown to be superior to another in the clinical setting. Prourokinase has recently received attention as a new thrombolytic agent with higher fibrin specificity. The thrombolytic activity of Prourokinase, however, remains ill defined. The purpose of this study was to evaluate thrombolysis with Prourokinase in comparison to urokinase in vitro. Methods: We used an in vitro parallel channel perfusion model that simulates catheter-directed thrombolysis in the peripheral arterial system. Radiolabeled thrombi were subjected to 90 minutes of endhole catheter-directed infusion with either Prourokinase 5000 IU/ml, urokinase 5000 IU/ml; or 5% dextrose in water at 4 ml/hr. Results: Prourokinase and urokinase were found to be equivalent with respect to thrombolytic effect. Percent lysis was maximal at 90 minutes in both the urokinase and Prourokinase groups. Prourokinase and urokinase were found to be equally effective in restoring flow through thrombosed graft segments. Conclusions: Prourokinase appears to offer little benefit over urokinase with respect to thrombolytic activity in an in vitro model that closely resembles the clinical setting. If Prourokinase is to be accepted as an alternative to urokinase, advantages must relate to differences in fibrin specificity.
Keld Dano - One of the best experts on this subject based on the ideXlab platform.
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effect of purified soluble urokinase receptor on the plasminogen Prourokinase activation system
FEBS Letters, 1996Co-Authors: Niels Behrendt, Keld DanoAbstract:The extracellular proteolytic pathway mediated by the urokinase plasminogen activator (uPA) is a cascade system, initiated by activation of the zymogen, pro-uPA. Pro-uPA as well as uPA binds to the cellular uPA receptor (uPAR) which has a central function in cell-dependent acceleration of the cascade system. This role of uPAR is generally assumed to be a positioning effect since uPAR-expressing cells exclusively stimulate the activation of cell surface-bound plasminogen (Ellis et al. (1993) Methods Enzymol. 223, 223–233). However, it was recently reported that a recombinant, soluble uPAR (suPAR) was capable of accelerating plasminogen activation in solution (Higazi et al. (1995) J. Biol. Chem. 270, 17375–17380). In this work we show that suPAR as such has no accelerative role. In contrast, the progress of the activation reactions in a soluble system with pro-uPA and plasminogen was found to be attenuated by suPAR. This delay of the activation system was shown to include a partial inhibition of the plasmin-mediated activation of pro-uPA as well as of the uPA-mediated activation of plasminogen.
Stephan Wnendt - One of the best experts on this subject based on the ideXlab platform.
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amyloid β peptides stimulate tissue type plasminogen activator but not recombinant Prourokinase
Thrombosis Research, 1997Co-Authors: Stephan Wnendt, Ingrid Wetzels, Wolfgang A GunzlerAbstract:Tissue-type plasminogen activator (rt-PA) and Prourokinase (rscu-PA) have been tested with respect to the influence of amyloid β peptides on plasminogen activation which was monitored by cleavage of the chromogenic plasmin substrate S-2251. It was shown that rt-PA is stimulated by amyloid β peptides at concentrations of 10 μg/ml in contrast to Prourokinase, which does not alter its catalytic properties in presence of amyloid β peptides. The stimulation of rt-PA can be inhibited by tranexamic acid indicating a molecular mode of stimulation similar to the fibrin mediated stimulation of rt-PA.
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construction and structure activity relationships of chimeric Prourokinase derivatives with intrinsic thrombin inhibitory potential
Protein Engineering, 1996Co-Authors: Stephan Wnendt, Elke Janocha, Johannes Schneider, Gerd J SteffensAbstract:: The blood clotting enzyme thrombin plays a central role in the aetiology of occlusive disorders such as stroke and acute myocardial infarction. During fibrinolytic therapy with plasminogen activators, thrombin is neutralized by anticoagulative drugs. In order to combine plasminogen-activating and thrombin-inhibitory activities we constructed chimeric derivatives of recombinant single-chain, urokinase-type plasminogen activator (rscu-PA) which comprise the kringle and protease domain of rscu-PA fused via a linker sequence to a thrombin-inhibitory domain. The inhibitory domain contains a sequence element directed to the active site of thrombin and a sequence taken from either hirudin or the human thrombin receptor both binding to the fibrinogen recognition site of thrombin. Analysing different sets of point mutants showed that the linker between the protease domain and the active site-directed sequence is contributing significantly to the thrombin-inhibitory potential. Kinetic analysis of thrombin inhibition revealed that most of the chimeras tested competitively inhibit the thrombin-mediated cleavage of a peptide substrate in a concentration-dependent manner; however, in two examples the insertion of one glycine residue into the active site directed-sequence abolished the blockade of the active site. This supports the conclusion that the chimeras with high thrombin-inhibitory potential interact with the active site and the fibrinogen recognition site of thrombin.
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Construction and structure–activity relationships of chimeric Prourokinase derivatives with intrinsic thrombin-inhibitory potential
Protein Engineering, 1996Co-Authors: Stephan Wnendt, Elke Janocha, Johannes Schneider, Gerd J SteffensAbstract:The blood clotting enzyme thrombin plays a central role in the aetiology of occlusive disorders such as stroke and acute myocardial infarction. During fibrinolytic therapy with plasminogen activators, thrombin is neutralized by anticoagulative drugs. In order to combine plasminogenactivating and thrombin-innibitory activities we constructed chimeric derivatives of recombinant single-chain, urokinase-type plasminogen activator (rscu-PA) which comprise the kringle and protease domain of rscu-PA fused via a linker sequence to a thrombin-inhibitory domain. The inhibitory domain contains a sequence element directed to the active site of thrombin and a sequence taken from either hirudin or the human thrombin receptor both binding to the fibrinogen recognition site of thrombin. Analysing different sets of point mutants showed that the linker between the protease domain and the active sitedirected sequence is contributing significantly to the thrombin-inhibitory potential. Kinetic analysis of thrombin inhibition revealed that most of the chimeras tested competitively inhibit the thrombin-mediated cleavage of a peptide substrate in a concentration-dependent manner; however, in two examples the insertion of one glycine residue into the active site directed-sequence abolished the blockade of the active site. This supports the conclusion that the chimeras with high thrombin-inhibitory potential interact with the active site and the fibrinogen recognition site of thrombin.
