Recombinant Human Prourokinase Research Articles

Rationale and design of ProUrokinase in Mild IsChemic strokE (PUMICE): a multicentre, prospective, randomised, open-label, blinded-endpoint controlled trial.

Recombinant human prourokinase (rhPro-UK) is a new generation of specific plasminogen activator, that is non-inferior to alteplase in acute ischemic stroke. We aimed to investigate the efficacy and safety of rhPro-UK compared with standard medical treatment in acute mild ischemic stroke within 4.5 hours of symptom onset. Prourokinase in mild ischemic stroke is a multicentre, prospective, randomised, open-label, blinded-endpoint controlled trial. Patients who had an acute ischemic stroke within 4.5 hours from symptom onset and with baseline National Institutes of Health Stroke Scale (NIHSS) score ≤ 5 will be recruited. Patients will be randomly assigned (1:1) to receive intravenous rhPro-UK (35 mg) or standard medical treatment. The follow-up duration will be 90 days. The calculated sample size is 1446. Primary efficacy outcome is an excellent functional outcome, defined as a modified Rankin Scale (mRS) score ≤ 1 at 90 days. Secondary efficacy outcomes include ordinal distribution of mRS at 90 days, mRS score ≤ 2 at 90 days, early neurological improvement at 24 hours (a decrease of NIHSS score ≥ 4 points compared with baseline or NIHSS score ≤ 1 point), Barthel index of 75-100 points at 90 days, quality of life at 90 days, and activities of daily living at 90 days. Safety outcomes are symptomatic intracranial haemorrhage within 36 hours, mortality at 90 days, moderate and severe systematic bleeding at 90 days, and adverse events/serious adverse events within 90 days. This large phase III randomised clinical trial will answer the question of whether thrombolysis is beneficial for acute mild ischemic stroke, and may provide evidence for rhPro-UK in patients had an acute mild ischemic stroke within 4.5 hours of symptom onset. NCT05507645.

Efficacy and Safety of a Pharmaco-Invasive Strategy Using Half-Dose Recombinant Human Prourokinase in Patients with ST-Segment Elevation Myocardial Infarction During Hospitalization.

This study investigated the efficacy and safety of pharmaco-invasive strategy with half-dose recombinant human prourokinase (PHDP) during hospitalization for patients with ST-segment elevation myocardial infarction (STEMI) to provide references for the treatment of STEMI. Patients with STEMI who fulfilled the inclusion and exclusion criteria and attended Chengde Central Hospital, Hebei Province, China, between September 3, 2019, and December 28, 2021, were included in this study. The experimental group received PHDP and the control group underwent primary percutaneous coronary intervention (PPCI). This study enrolled 150 patients with STEMI, 75 in the experimental group and 75 in the control group. Coronary angiography revealed successful thrombolysis in 64 (85.33%) patients. Compared with the control group, the experimental group had shorter first medical contact-reperfusion time (P < 0.001), less slow flow/no-reflow (P < 0.001), and a lower utilization rate of Tirofiban (P < 0.001). Validity endpoints: no statistically significant differences between the two groups. Safety endpoints: no statistically significant differences between bleeding and major adverse cardiovascular and cerebrovascular events (MACCEs), but the experimental group was more prone to arrhythmias (P = 0.040), particularly premature ventricular beats (PVB) (P = 0.008). In conclusion, the efficacy and safety of PHDP in the treatment of patients with STEMI were positive. Complete epicardial and myocardial reperfusion rates, risk for bleeding during hospitalization, and incidence of MACCEs were similar to those of the PPCI strategy. Although the PHDP group has a higher incidence of PVB, it does not increase the incidence of malignant arrhythmia. This study aimed to provide a new therapeutic strategy for the treatment of STEMI in hospitals without adequate PPCI resources condition.

Retracted: Comparison of Efficacy and Safety of Recombinant Human Prourokinase and Alteplase in the Treatment of STEMI and Analysis of Influencing Factors of Efficacy.

[This retracts the article DOI: 10.1155/2021/6702965.].

Efficacy and Safety of Recombinant Human Prourokinase in the Treatment of Acute Ischemic Stroke Within 4.5 Hours of Stroke Onset

Recombinant human prourokinase (rhPro-UK) is a thrombolytic agent that has shown promising findings in a phase 2 clinical trial in patients with acute ischemic stroke (AIS). To evaluate the efficacy and safety of rhPro-UK thrombolysis within 4.5 hours of symptom onset in patients with AIS. This randomized, alteplase-controlled, open-label, phase 3 clinical trial was conducted from May 2018 to May 2020 at 35 medical centers in China. A total of 684 patients were screened and 674 patients were enrolled. Included patients were aged 18 to 80 years with a diagnosis of AIS and received treatment within 4.5 hours of stroke onset. Data were analyzed from June to October 2020. Eligible patients were randomly assigned (1:1) to receive intravenous rhPro-UK or alteplase. The primary objective was to assess whether rhPro-UK was noninferior to alteplase. The noninferiority margin was a between-group difference of less than 10%. The primary outcome was a modified Rankin Scale score of 0 to 1 at 90 days. Among 663 patients in the modified intention-to-treat population (mean [SD] age, 61.00 [10.20] years; 161 females [24.3%]), there were 330 patients in the rhPro-UK group and 333 patients in the alteplase group. The median (IQR) baseline National Institutes of Health Stroke Scale score was 6.00 (5.00-9.00). There were 23 deaths, and 619 patients (93.4%) completed the 3-month follow-up. The primary outcome occurred in 215 patients (65.2%) in the rhPro-UK group and 214 patients (64.3%) in the alteplase group (risk difference, 0.89; 95.4% CI, -6.52 to 8.29). Symptomatic intracerebral hemorrhage occurred in 5 patients (1.5%) in the rhPro-UK group and 6 patients (1.8%) in the alteplase group (P > .99). Systemic bleeding within 90 days occurred more frequently in the alteplase group (141 patients [42.2%]) than the rhPro-UK group (85 patients [25.8%]) (P < .001). By 90 days, 5 thrombolysis-related deaths each had occurred in the rhPro-UK group (1.5%) and alteplase group (1.5%) (P > .99). This study found that intravenous rhPro-UK within 4.5 hours of AIS onset was noninferior to alteplase. The rhPro-UK group showed a similar rate of symptomatic ICH but fewer cases of systemic bleeding than the alteplase group. ClinicalTrials.gov Identifier: NCT03541668.

Safety and efficacy of intracoronary recombinant human prourokinase administration in patients with acute myocardial infarction and ST‑segment elevation: A meta‑analysis of randomized controlled trials.

Slow blood flow or no reflow following percutaneous coronary intervention (PCI) in patients with acute ST-segment elevation myocardial infarction (STEMI) typically leads to an adverse prognosis. However, it is controversial whether to use prourokinase (Pro-UK) during PCI in patients with acute STEMI. The present meta-analysis compared the efficacy and safety of intracoronary Pro-UK administration in patients with acute STEMI. Published randomized controlled trials (RCTs) were analyzed to compare Pro-UK with non-Pro-UK treatment in patients with acute STEMI. PubMed, Cochrane Library and China National Knowledge Infrastructure were searched and meta-analysis was performed using Review Manager 5.3 software. A total of 13 RCTs were selected and 1,797 patients were considered in the meta-analysis, including 897 patients who received Pro-UK intervention and 900 patients who were in the control group. No significant heterogeneity was identified across these selected studies. Pro-UK therapy significantly decreased the incidence of major adverse cardiac events [risk ratio (RR), 0.68; 95% CI, 0.56-0.82, P<0.0001], left ventricular end-diastolic diameter [standardized mean difference (SMD), -0.26; 95% CI, -0.40 - -0.12; P=0.0003], corrected thrombolysis in myocardial infarction (TIMI) frame count [SMD, -0.45; 95% CI, -0.62 - -0.28; P<0.00001] and cardiac troponin I [SMD, -0.31; 95% CI, -0.46 - -0.17; P<0.0001]. In addition, Pro-UK administration increased TIMI grade 3 flow (RR, 1.16; 95% CI, 1.07-1.25; P=0.0003), TIMI myocardial perfusion grade 3 (RR: 1.39, 95% CI: 1.12-1.74, P=0.004), ST-segment resolution (RR, 1.23; 95% CI, 1.10-1.36; P=0.0002) and left ventricular ejection fraction (SMD, 0.38; 95% CI, 0.27-0.49; P<0.00001). No significant difference was identified in bleeding (RR, 1.12; 95% CI, 0.85-1.47; P=0.41). The present meta-analysis determined that intracoronary Pro-UK administration is efficacious and safe to decrease slow blood flow or no reflow phenomena following PCI and improve the prognosis of patients with acute STEMI.

Dual coronary embolization associated with atrial fibrillation: a case report

Background: Thrombotic occlusion of the coronary artery, which succeeds the atherosclerotic plaquerupture or erosion, gives rise to a major portion of acute myocardial infarction (AMI) incidences.Nevertheless, coronary embolism is gaining increasing recognition as another important factor contributingto AMI.&#x0D; Case presentation: A 72-year-old woman with atrial fibrillation (AF) and diabetes mellitus histories,presented with chest pain radiating to the left arm and shoulder that began 6 hours prior to admission.Electrocardiogram revealed AF plus ST-segment elevation in the anterior leads.Intervention: Patient was first treated with anti-platelet agents (aspirin plus ticagrelor) and atorvastatin.Emergency coronary angiography depicted multi-site coronary embolization of the left circumflex artery(LCX) and the left anterior descending artery (LAD). Blood flow was not restored after intracoronaryinjection of 600 ug tirofiban. 40 mg recombinant human prourokinase was then administered via aspirationthrombectomy catheter.&#x0D; Outcome: Two weeks later, coronary angiography showed no residual obstructive lesion in the LCX andLAD with TIMI (thrombolysis in myocardial infarction) 3 flow.&#x0D; Conclusion: Primary percutaneous coronary intervention is the most effective measure. In the case offailed blood flow restoration, thrombolytic treatment in both intravenous and intracoronary route should beconsidered.

Comparison of Efficacy and Safety of Recombinant Human Prourokinase and Alteplase in the Treatment of STEMI and Analysis of Influencing Factors of Efficacy.

Objective To compare the efficacy and safety of recombinant human prourokinase (rhPro-UK) and alteplase for thrombolytic therapy in acute ST-segment elevation myocardial infarction (STEMI) and to analyze the related factors affecting efficacy. Methods From January 2017 to December 2019, 100 patients diagnosed with STEMI were selected and randomly divided into the control group (n = 50) and the observation group (n = 50). Based on conventional treatments, the control group was treated with alteplase, and the observation group was treated with rhPro-UK, and both were treated for 7 days. After treatment, the vascular recanalization, left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), and left ventricular ejection fraction (LVEF) were compared. The bleeding and major adverse cardiovascular events (MACE) were recorded in both groups. According to the patient's vascular recanalization, it was divided into two subgroups: recanalization group and occlusion group. Multiple logistic regression models were used to analyze the related factors that affect the efficacy. Results The recanalization rate of the observation group (96.00%) was higher than that of the control group (84.00%) (P < 0.05). After treatment, LVDs and LVEDD in both groups were lower than those before treatment, and LVEF was higher than that before treatment. The LVDs and LVEDD in the observation group were lower than those in the control group, and the LVEF was higher than that in the control group (P < 0.05). The incidence of bleeding in the observation group (2.00%) was lower than that in the control group (12.00%), and the incidence of MACE (4.00%) was lower than that in the control group (16.00%) (P < 0.05). Univariate analysis showed that age, smoking history, diabetes history, myocardial infarction history, infarct location, and intravenous thrombolysis time were related to the efficacy after treatment (P < 0.05). Multivariate logistic analysis showed that age, history of diabetes, vascular infarction site, and venous thrombolysis time were independent influencing factors after treatment (P < 0.05). Conclusion Both rhPro-UK and alteplase thrombolytic therapy can effectively recanalize blood vessels and improve the cardiac function of patients with STEMI. However, rhPro-UK has better effect than alteplase and is safer and worth promoting. The curative effect is related to age, diabetes history, vascular infarction site, and venous thrombolysis time.

Safety and efficacy of intracoronary thrombolytic therapy via a new infusion catheter in patients with ST-segment elevation myocardial infarction with large thrombus burden: a pilot study.

A large intracoronary thrombus burden is associated with adverse clinical results. The optimal management of this scenario remains unknown. We aimed to determine the efficacy and safety of a new rapid infusion catheter combined with low-dose intracoronary thrombolysis in patients with ST-segment elevation myocardial infarction (STEMI) with a large thrombus burden. This pilot study included 22 patients with STEMI with a large thrombus burden. A large thrombus burden was defined as a definite thrombus with the largest dimension of at least two vessel diameters [thrombolysis in myocardial infarction (TIMI) thrombus grades 4 and 5]. All patients received primary percutaneous coronary intervention guided by the presence of recurrent chest pain or clinical myocardial ischemia evidences. All patients regained myocardial perfusion immediately after the infusion catheter crossed the thrombus. Local fibrinolysis with low-dose recombinant human prourokinase was administered continuously via the infusion catheter for 30 min. Repeat coronary angiography revealed marked thrombus resolution, with an improvement in TIMI flow from 0.14 ± 0.35 at baseline to 2.82 ± 0.40. Only one patient with postlysis thrombus grades 4-5 was observed. No major bleeding events were observed. In patients with STEMI presenting with a large thrombus burden, all patients regained myocardial perfusion immediately after the infusion catheter crossed over the thrombus, and low doses of intracoronary thrombolysis could significantly reduce the thrombus burden and improve the coronary flow without major bleeding.

The intracoronary injection of recombinant human prourokinase in emergency interventional therapy for high thrombus load during ST-segment elevation myocardial infarction: a retrospective cohort study

Vessel Plus is an open acccess journal, which publishes articles related to vascular diseases, including acute respiratory distress syndrome, aneurysm, atherosclerosis, hypertension, stroke, peripheral vascular or pulmonary vascular diseases, etc.

Thrombolysis with rhPro-UK 3 to 6 hours after embolic stroke in rat

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 subjected to embolic middle cerebral artery occlusion. RhPro-UK and positive control drugs rt-PA,UK were administered 3 h, 4.5 h, 6 h after inducing thromboem-bolic stroke. Neurological deficit scoring (NDS) was evaluated at 6 h and 24 h after the treatment. The lesion volume in cerebral hemispheres was measured by MRI scanning machine after 6 h of thrombolysis, and the infarct volume was measured by TTC stain, together with hemorrhagic volume quantified by a spectrophotometric assay after 24 h of thrombolysis.Results: RhPro-UK 10, 20 × 104 U/kg significantly improved the NDS after cerebral thromboembolism in rats at 3 h, 4.5 h TTW, and at the 6 h TTW, the NDS was improved by 28.0% (P = 0.0690) and 29.2% (P = 0.0927) at 6 h and 24 h after rhPro-UK 20 ×104 U/kg administration, respectively. RhPro-UK 10, 20 × 104 U/kg significantly reduced the brain lesions measured by MRI at 3 h and 4.5 h TTW. RhPro-UK 10, 20 × 104 U/kg significantly reduced the cerebral infarction measured by TTC at 3 h, 4.5 h TTW. There was no increase in cerebral hemorrhage compared with untreated group after rhPro-UK administration.Conclusions: RhPro-UK had an obvious therapeutic effect on ischemic stroke caused by thrombosis, and could be started within 4.5 h TTW with less side effects of cerebral hemorrhage than that of UK.

Cardiotonic Pills Plus Recombinant Human Prourokinase Ameliorates Atherosclerotic Lesions in LDLR–/– Mice

AimThis study was to explore the protective effects of cardiotonic pills (CP) or/and recombinant human prourokinase (proUK)on the atherosclerosis and the potential underlying mechanism.Methods and ResultsAtherosclerosis was induced in LDLR–/– mice by high fat diet contained 20% lard and 0.5% cholesterol. Daily oral administration of CP (130 mg/kg) or/and intravenous injection of proUK (2.5 mg/kg, twice a week) began at 8 weeks after feeding with high fat diet and continued for 4 weeks. CP alone treatment markedly decreased plasma triglyceride, but did not ameliorate atherosclerosis plaque. No effect was observed for proUK alone on any endpoints tested. CP plus proUK induced a significantly reduction in the atherosclerotic lesions, along with decreased levels of total cholesterol, triglyceride in the plasma. CP plus proUK inhibited the elevated hepatic total cholesterol and triglyceride in high fat diet-fed LDLR–/– mice, up-regulating the expressions of ATP-binding cassette gene 5 and 8, and adipose triglyceride lipase. In the aorta, CP plus proUK inhibited the expression of scavenger receptor A and CD36 in LDLR–/– mice. In addition, we observed that systemic inflammation was inhibited, manifested downregulation of plasma macrophage inflammatory protein-1α and intercellular cell adhesion molecule-1.ConclusionCP plus proUK effectively attenuated atherosclerosis plaque in LDLR–/– mice, which is associated with normalizing the lipid metabolism in the liver and aorta, reducing phagocytosis of receptor-mediated modified-LDL uptake and inhibiting systemic inflammation.

Abstract 79: Preliminary Efficacy and Safety of Intravenous Recombinant Human Pro-urokinase (rhpro-UK) for Acute Ischemic Stroke:AMulticenter Phase IIa, Randomized, Open, Positive Controlled, Trial(rhpro-UK STROKEIIa)

Objective: To investigate preliminary efficacy and safety of intravenous recombinant human pro-urokinase (rhpro-UK) in patients with acute ischemic stroke (AIS). Methods: In the 23-center phase IIa, time window stratified, randomized, open, positive controlled, clinical trial, patients within 6 hours after the onset of AIS were stratified into two therapeutic time windows. Patients within 4.5 h were randomized to receive intravenous rhpro-UK 50mg, 35mg, or recombinant tissue plasminogen activator (rt-PA; 0.9 mg/kg, maximum 90 mg). Patients within 4.5-6 h were randomized to receive rhpro-UK 50mg or 35mg. Primary outcome was a modified Rankin scale (mRS) score ≤1at 90 days. Secondary outcome was the treatment response based on ≥ 4-point reduction from the baseline National Institutes of Health stroke scale score at 24 hours after treatment. Safety outcomes included symptomatic intracerebral hemorrhage (sICH), death due to any cause, and other serious adverse events. Results: We enrolled 190 patients (Figure 1A). Within 4.5 h, the proportion of patients with a mRS score ≤1 at 90 days did not differ significantly among groups (57.9%, 55.6% and 52.6%, P=0.92). Similarly, there was no statistical significance among groups in term of treatment response at 24 hours (P=0.85). In the 4.5-6 h time window, the rates of functional independence of rhpro-UK 50mg and 35mg were 59.0% and 69.2% (P=0.34). The proportion of patients with functional response to treatment were 28.2% and 33.3% (P=0.62) (Figure1B, 1C). sICH occurred in one patient in the 50mg rphro-UK group within 4.5 h within 30 days after the intervention. There was no significant difference among the five groups in death and rate of other serious adverse events. Conclusion: As compared with rt-PA, intravenous rhpro-UK at two dose within the 4.5 hours after onset of symptoms showed similar efficacy and safety profiles in patients withAIS. Similarly, these two dose groups did not differ in efficacy and safety between 4.5-6.0 hours.

GW29-e0636 The Study of the Effects of Intracoronary Recombinant Human Prourokinase or Tirofiban on the Coronary Flow during Primary PCI for the Acute Myocardial Infartion

GW29-e0636 The Study of the Effects of Intracoronary Recombinant Human Prourokinase or Tirofiban on the Coronary Flow during Primary PCI for the Acute Myocardial Infartion

Effect of recombinant human prourokinase on thrombolysis in a rabbit model of thromboembolic stroke.

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), 36.8% (P<0.001) and 55.0% (P<0.001), respectively together with patency rates of 10% (P>0.05), 40% (P<0.05) and 70% (P<0.001). Furthermore, α2-AP levels were reduced by 5.3% (P>0.05), 5.3% (P>0.05) and 18.1% (P<0.05). At the 4.5 h time point, the thrombolysis rate was 18.8% (P<0.05), 29.9% (P<0.01) and 49.0% (P<0.001) together with patency rates of 10% (P>0.05), 30% (P<0.05) and 50% (P<0.01), and α2-AP levels were reduced by 2.4% (P>0.05), 6.5% (P>0.05) and 17.8% (P<0.05). At the 6 h time point, the thrombolysis rate was 14.7% (P<0.05), 24.1%(P<0.01) and 35.7% (P<0.001) together with patency rates of 20% (P>0.05), 30% (P<0.05) and 40% (P<0.01), and α2-AP levels were reduced by 5.7% (P>0.05), 12.7% (P>0.05) and 22.2% (P<0.01). No significant differences (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.

Investigation of Pharmacodynamics, Pharmacology, and Toxicology of Domestic Human Prourokinase

The comparison of the effect of fibrinolytic activity of recombinant human prourokinase (rhpro-UK) and urokinase(UK) with rabbits indicated rhpro-UK and UK all decreased resolution time of globular proteins, and the affect of rhpro-UK on parameter of fibrinolysis of rabbits was less than UK. The solution of thrombin of rhpro-UK in vitro was increased with dosage of rhpro-UK, and when rhpro-UK was compared with isodose UK, the difference is significant. Both rhpro-UK and UK all was marked solution of thrombin from Chinese piglets, and it were not evident effect on fibrinogen, plasminogen, and a2-antiplasmin of the piglets. The affect of rhpro-UK on bleeding time, clotting time, bleeding amount of unit time was less than UK. It indicated that the side effect of rhpro-UK was distinguished less than UK. The investigation of pharmacology of ileums of mice, rats, dogs, and guinea pigs indicated that rhpro-UK was not marked affect on behavior, state, central nervous system, cardiovascular system, respiratory system, and digestive system of the tested animals. But tested dogs were bleeding at location of operation, and the blood of the animal was heparinized. The studis of toxicology of rhpro-UK indicated that rhpro-UK was median lethal dose of 97.5 mg/kg in mice. When The Beagle dogs was administed rhpro-UK of 2, 8, 28 mg/kg, the toxic reaction was not observed, except for temporal hyperemia of gum, extend of clotting time, increasing of total cholerythrin, total proteins, albumin of the dogs, the direct damage of organs for the medicine was not observed in examination of pathology in the dosages of 8 and 28 mg/kg, any toxic reaction was not observed in dosage of 2 mg/kg. The special toxic experiments proved that rhpro-UK was not of mutagenesis and teratogenesis.

Catalytic and fibrinolytic properties of recombinant urokinase plasminogen activator from E. coli, mammalian, and yeast cells

The enzymatic and fibrinolytic properties of glycosylated and nonglycosylated recombinant human pro-urokinase (pro-UK) produced in yeast Pichia pastoris were characterized and compared with those of Escherichia coli and mammalian cell-derived pro-UK. Among the five different forms of pro-UK, the yeast glycosylated pro-UK was activated by plasmin with the lowest catalytic efficiency ( k cat/ K m). The yeast glycosylated urokinase (UK) also had the highest K m in its activation of Glu-plasminogen, and had a substantially lower fibrinolytic activity than the other four forms. These findings suggest that the poly-mannose on Asn-302 of yeast glycosylated pro-UK interfered with its activation by plasmin and its binding interaction with plasminogen. By contrast to plasminogen, the activation of the small synthetic substrate, S2444, was comparable for all five forms of recombinant UK. It is concluded that the glycosyl residue on pro-UK/UK is functionally important and modulates its activatability and its catalytic efficiency against its natural substrate. Therefore, pro-UK from different expression systems cannot be assumed to have comparable fibrinolytic activities.

Glycosylation of prourokinase produced by Pichia pastoris impairs enzymatic activity but not secretion.

Both glycosylated and nonglycosylated forms of recombinant human prourokinase were produced to the level of 20 mg/L by yeast Pichia pastoris in BMMY medium after 2 days of culture. The expressed pro-UK was 98% secreted into the culture medium and easily purified by carboxymethyl cellulose chromatography. More than 99% of pro-UK in the culture medium was found in single-chain form. This was contradictory to a previous finding which found that glycosylation of pro-UK by yeast inhibited its secretion. The absence of glycosylation at Asn302 of pro-UK has no measurable effect on its secretion from the yeast cells. However, the nonglycosylated pro-UK was much less stable in the culture medium, probably due to proteolysis. Nonglycosylated pro-UK from yeast had a clot lysing activity comparable to that of Escherichia coli-derived or mammalian cell-derived recombinant pro-UK. By contrast, the glycosylated yeast pro-UK was less activatable by plasmin and had a lower enzymatic activity against plasminogen and a lower clot lysing activity than nonglycosylated pro-UK from yeast, while their amidolytic activity against S2444 was equivalent. It was concluded that glycosylation of pro-UK by yeast P. pastoris interferes with the catalytic site but not secretion of this protein.

Validation of a Method for Automated Nucleotide Sequence Analysis and Estimation of the Limits of Detection of Variant Sequences in a Heterogenic DNA Sample

The assessment of the genetic stability of an expression-system used for the production of recombinant drugs and the verification of the nucleotide sequence of expression plasmids makes it necessary to estimate a limit of detection for variant sequences. Therefore, a series of experiments was performed in which defined mixtures of two different plasmid-DNAs were subjected to nucleotide sequence analysis using an automatic sequencer (ABI 377) and PCR-based sequencing reactions. A plasmid, pGR201 (Fig. 1a), encoding recombinant non-glycosylated human prourokinase (Saruplase), was spiked in varying ratios (90:10; 80:20; 70:30; 60:40) with a related plasmid, pSJ41 (Fig. 1a), differing in a single base-substitution and a cluster of multiple mutations, respectively. Each of these mixtures was sequenced six times in a 50 bp-region covering the variant sequences in order to estimate a limit of detection for point mutations and clusters of multiple mutations. The results are of general interest since we analysed enough point-mutations as well as deletion-mutations to allow a statistical assessment. It was shown that a single point mutation resulting in a base-substitution will be detected by sequencing in one direction if at least 20 % of the plasmids within the DNA sample are mutated, while the sequencing method is sensitive enough to detect a cluster of multiple mutations when at least 10 % of the sequenced plasmid DNA molecules carry the altered sequence. In contrast to other publications the sequencing-procedure presented in this paper results in electropherograms with small background signals although only a very simple purification of the sequencing reaction products was used in order to present a method that is not time-consuming.

GroE assists refolding of recombinant human pro-urokinase.

GroE, one of the molecular chaperones, facilitates correct protein folding both in vitro and in vivo. The refolding of recombinant human pro-urokinase, a protein with a high content of disulfide bonds, was used as a model system to illustrate the mechanism of action of GroE. Aggregation of this protein predominates during its in vitro refolding, as indicated by a strong, concentration-dependent increase in light scattering. The addition of GroE and Mg-ATP significantly increases the yield of the active protein. GroE specifically inhibits the aggregation reaction that competes with correct folding, as shown by a strong decrease in the intensity of light scattering. GroEL rapidly binds to unfolded or partially folded pro-urokinase molecules and thus protects them from the aggregation reaction. Interaction with GroES and ATP hydrolysis are required for the release of the polypeptide chain from GroEL and further acquisition of the completely folded, native conformation.

Enhancement of in vitro renaturation of recombinant human pro-urokinase by ampicillin

Ampicillin was used in in vitro renaturation process of recombinant human prourokinase and it can obviously improve the yield of the renaturation, although not as well as arginine. When ampicillin was used together with arginine or lysine, it decreased the yield of the renaturation comparing with the yield when arginine or lysine used alone.