Recombinant Tissue Plasminogen Activator-conjugated Nanoparticles Effectively Targets Thrombolysis in a Rat Model of Middle Cerebral Artery Occlusion

The efficacy and safety of recombinant tissue plasminogen activator （rtPA） need to be improved due to its low bioavailability and requirement of large dose administration. The purpose of this study was to develop a fibrin-targeted nanoparticle （NP） drug delivery system for thrombosis combination therapy. We conjugated rtPA to poly（ethylene glycol）- poly（ε-caprolactone） （PEG-PCL） nanoparticles （rtPA-NP） and investigated its physicochemical characteristics such as particle size, zeta potential, enzyme activity of conjugated rtPA and its storage stability at 4℃. The thrombolytic activity of rtPA-NP was evaluated in vitro and in vivo as well as the half-life of rtPA-NP, the properties to fibrin targeting and its influences on systemic hemostasis in vivo. The results showed that rtPA-NP equivalent to 10% of a typical dose of rtPA could dissolve fibrin clots and were demonstrated to have a neuroprotective effect after focal cerebral ischemia as evidenced by decreased infarct volume and improved neurological deficit （P〈0.001）. RtPA-NP did not influence the in vivo hemostasis or coagulation system. The half-life of conjugated rtPA was shown to be approximately 18 times longer than that of free rtPA. These experiments suggested that rtPA-conjugated PEG-PCL nanoparticles might be a promising fibrin-targeted delivery system for a combination treatment of thrombosis.

Roles of tissue plasminogen activator and its inhibitor in proliferative diabetic retinopathy

AIMTo investigate the role of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) in proliferative diabetic retinopathy (PDR) and to discuss the correlations among t-PA, PAI and vascular endothelial growth factor (VEGF) expressions.

The pleiotropic effects of tissue plasminogen activator in the brain:implications for stroke recovery

Tissue plasminogen activator （tPA） use in the treatment of isch- emic stroke： tPA is a serine protease that catalyzes the breakdown of blood dots. Because of its thrombolytic properties, tPA is used to treat specific types of stroke, including ischemia, but is contra- indicated for treatment of hemorrhagic stroke or head trauma. Although a life saving and powerful ＇dot buster＇, tPA has a short therapeutic window. When administered outside of this prescribed timeframe, research suggests that tPA can produce neurotoxic ef- fects in the brain, due in part to activation of several signalling pro- cesses associated with cell apoptosis, degradation of the extracel- lular matrix, and increase in the permeability of the neurovascular unit （Yepes et al., 2009）. Concerted research has been dedicated to- ward understanding the mechanisms mediating the impact of tPA on the brain, using both in vivo and in vitro animal models.

Comment on roles of tissue plasminogen activator and its inhibitor in proliferative diabetic retinopathy

Dear Sir,We congratulate Wu et al for their study entitled＂Roles of tissue plasminogen activator and its inhibitor in proliferative diabetic retinopathy＂.The authors investigated the effects of tissue plasminogen activator（t-PA）and plasminogen activator inhibitor（PAI）in the pathogenesis of proliferative diabetic retinopathy（PDR）.The authors reported that t-PA and PAI are involved in the pathogenesis of PDR.

Therapeutic effect of recombinant tissue plasminogen activator on acute cerebral infarction at different times

BACKGROUND:The study aimed to compare the therapeutic effect of recombinant tissue plasminogen activator(rt-PA) on the onset of acute cerebral infarction(ACI) at different time points of the first 6 hours.METHODS:A retrospective analysis was conducted in 74 patients who received rt-PA thrombolysis treatment within 4.5 hours after ACI and another 15 patients who received rt-PA thrombolysis treatment between 4.5-6 hours after ACI.RESULTS:National Institute of Health Stroke Scale(NIHSS) scores were statistically decreased in both groups(P>0.05) at 24 hours and 7 days after ACI.There was no significant difference in modified ranking scores and mortality at 90 days after the treatment between the two groups(P>0.05).CONCLUSIONS:The therapeutic effect and mortality of rt-PA treatment in patients with ACI between 4.5-6 hours after the onset of the disease were similar to those in patients who received rtPA within 4.5 hours after the onset of this disease.Therefore,intravenous thrombolytic therapy for ACI within 4.5-6 hours after ACI was effective and safe.

Factors influencing clinical outcomes of acute ischemic stroke treated with intravenous recombinant tissue plasminogen activator

Background Thrombolysis with recombinant tissue plasminogen activator （rt-PA） has gained international recognition, clinical outcomes following this thrombolytic therapy varied from patient to patient. Factors affecting clinical outcomes have not been well understood yet, so this retrospective case-control study aimed to investigate factors that may influence clinical outcomes of acute ischemic stroke treated with intravenous rt-PA. Methods One hundred and one patients with acute ischemic stroke who received intravenous rt-PA thrombolysis within 4.5 hours from disease onset were included. Patients were divided into good or poor outcome group according to modified Rankin Scale （mRS） score, good outcome group： mRS score of 0-1; poor outcome group： mRS of 2-6. Stroke characteristics were compared between the two groups. Factors for stroke outcomes were analyzed via univariate analysis and Logistic regression. Results Of the 101 patients studied, patients in good outcome group （n=55） were significantly younger than patients in poor outcome group （n=46, （62.82±14.25） vs. （68.81±9.85） years, P=0.029）. Good outcome group had fewer patients with diabetic history （9.09% vs. 28.26%, P=0.012）, fewer patients with leukoaraiosis （7.27% vs. 28.26%, P=-0.005） and presented with lower blood glucose level （（5.72±1.76） vs. （6.72±1.32） mmol/L, P=0.012）, lower systolic blood pressure level （（135.45±19.36） vs. （148.78±19.39） mmHg, P=0.003）, lower baseline NIHSS score （12.02±5.26 vs. 15.78±4.98, P=0.002） and shorter onset-to-treatment time （OTT） （（2.38±1.21） vs. （2.57±1.03） hours, P=0.044） than poor outcome group. Logistic regression analysis showed that absence of diabetic history （odds ratio （OR） 0.968 （95% CI 0.941-0.996））, absence of leukoaraiosis （OR 0.835 （95% C/0.712-0.980））, lower baseline NIHSS score （OR 0.885 （95% Cl 0.793- 0.989））, lower pre-thrombolysis systolic blood pressure （OR 0.962 （95% CI 0.929-0.997））, and lower blood glucose level （OR 0.699 （95% Cl 0.491-0.994）） before thrombolysis were significantly associated with better outcome. Conclusion Patients with no history of diabetes, no leukoaraiosis, low blood glucose level, low systolic blood pressure level and low baseline NIHSS score before thrombolvsis have a better outcome.

Myocardial infarction following recombinant tissue plasminogen activator treatment for acute ischemic stroke： a dangerous complication

Thrombolysis with intravenous tissue plasminogen activator （t-PA） is currently an approved therapy for patients with acute ischemic stroke. Acute myocardial infarction （AMI） immediately following t-PA treatment for stroke is a rare but serious complication. A case of acute myocardial infarction （MI） following IV t-PA infusion for acute stroke was observed. This is a 52-year-old male with a known history of hypertension and chest pain, who subsequently developed MI four hours after IV t-PA was administered for acute ischemic stroke. The disruption of intra-cardiac thrombus and subsequent embolization to the coronary arteries may be an important mechanism. In addition, spontaneous recanalization of infarct-related arteries may be associated with greater myocardial salvage and better prognosis.

Study on the Mechanism of the Annexin -Mediated Co-Assembly of t-PA and Plasminogen

In order to further investigate the effect of annexinⅡ(Ann Ⅱ) on tissue plasminogen activator (t PA) dependent plasminogen (PLG) activation and its interactive mechanism, recombinant native Ann Ⅱ bound t PA, PLG and plasmin with high affinity was examined. The flow cytometric assay showed that the ann Ⅱexpression rate was higher in the human umbilical vein endothelial cell (HUVEC) (87 65 %) than in the HL 60 cells as controls (35.79 %). Two irrelevant proteins, bovine serum albumin (BSA) and equine IgG (EIG) had no effect on the production of plasmin. Ann Ⅱ mediated enhancement of t PA dependent PLG activation was inhibited by ε aminocaproic acid or by pretreatment of Ann Ⅱ with carboxypeptidase B with the inhibitive rate being 77.8 % and 77.0 %, respectively. It was revealed that the effect of Ann Ⅱon PLG activation was specific for t PA. Urokinase didn't bind to Ann Ⅱ, demonstrating the role of receptor related lysine residues on activation of PLG, showing that the Ann Ⅱ PLG interaction was dependent upon carboxyl terminal lysine residues. These findings suggest that annexin Ⅱ mediated co assembly of t PA and PLG may promote plasmin generation and play a key role in modulating fibrinolysis on the endothelial surface.

Relationship between tissue type plasminogen activator and coronary vulnerable plaque in patients with acute coronary syndrome： virtual histological study

Background The association between vulnerability of plaque assessed with intravascular ultrasound （IVUS） and plasma levels of fibrinolytic biomarkers was determined in patients with acute coronary syndrome （ACS）. However, few data are available on the relationship between the levels of tissue type plasminogen activator （t-PA） and virtual histological intravascular ultrasound （VH-IVUS） signs of plaque instability. Methods Eighty-nine patients with ACS were enrolled in the study. Blood was collected to measure t-PA levels by liquid phase bead flow cytometry. Eighty-nine nonbifurcate lesions （identified by coronary angiography and ECG） were investigated using IVUS before catheterization. IVUS radiofrequency data obtained with a 20 MHz catheter were analyzed with IVUS virtual histological software. The areas of plaque and media were calculated and lesions were classified into two groups： VH-IVUS derived thin cap fibroatheroma （VH-TCFA） and non-VH-TCFA plaque. Results Plasma t-PA level in the patients with TCFA was significantly lower than that with non-TCFA （（1489 ± 715） pg/ml vs （2163 ± 1004） pg/ml）. Decreased plasma levels of t-PA were associated with plaque vulnerability. Plasma levels of t-PA correlated negatively with plaque plus media and necrotic core in plaque in patients with ACS. Conclusions t-PA is an independent risk factor and a powerful predictor of vulnerable plaques. Decreased levels of t-PA may reflect instability of atherosclerotic plaques and might therefore serve as noninvasive determinants of those at high risk for consequent adverse events.

Effect of Zhutan Tongluo Tang on fibrinolytic activity following intracerebral hemorrhage in rats

The neuroprotective effect of Zhutan Tongluo Tang is associated with the activities of the fibrinolytic system. Thus the present study was designed to investigate therapeutic effects of Zhutan Tongluo Tang on intracerebral hemorrhage in rats, induced by injecting collagenase into one side of the caudate nucleus. Fibrinolytic indices including tissue plasminogen activator, plasminogen activator inhibitor-1 and D-Dimer were determined. The results obtained demonstrated that Zhutan Tongluo Tang treatment could alleviate the neural and behavioral impairments of intracerebral hemorrhaging rats. Increased frequencies of placing their forelimb correctly and decreased frequencies of turning left were observed. Enzyme linked immunosorbent assay showed that Zhutan Tongluo Tang could significantly elevate the concentration of plasma tissue plasminogen activator and D-Dimer, while depress plasminogen activator inhibitor-1. Immunohistochemistry demonstrated increased levels of catalase and glutathione in whole brain rat tissue following intracerebral hemorrhage. In addition, elevated expression of Bcl-2 in various hippocampal regions was seen. These findings describe the ability of Zhutan Tongluo Tang to activate the fibrinolytic system, and suggests that antioxidant and apoptosis inhibitory mechanisms may be playing a crucial role in protecting against collagenase-induced intracerebral hemorrhage.

Management of subretinal hemorrhage

Subretinal hemorrhage is a vision threatening complication of exudative age related macular degeneration(AMD) and polypoidal choroidal vasculopathy(PCV). Timely removal or displacement of subretinal hemorrhage from the central macula, ideally within 7 to 10 days after onset, is critical to allowing potential recovery of vision. Surgical techniques with the use of a bubble to displace the subretinal hemorrhage can now be performed with tissue plasminogen activator to lyze the blood and with or without vitrectomy.

Submacular hemorrhage:treatment update and remaining challenges

Submacular haemorrhage(SMH)is a sight threatening complication that can occur in exudative age related macular degeneration(AMD),but has been described to occur more frequently in eyes with polypoidal choroidal vasculopathy(PCV).Left untreated,SMH carries a grave visual prognosis.Thus,expedient diagnosis and effective management of this complication is of paramount importance.The treatment strategies for SMH include(I)displacement of blood from the fovea,usually by injection of an expansile gas;(II)pharmacologic clot lysis such as with recombinant tissue plasminogen activator(rtPA);and(III)treatment of the underlying choroidal neovascularization(CNV)or PCV,such as with anti-vascular endothelial growth factor(anti-VEGF)agents.These three strategies have been employed in isolation or in combination,some concurrently and others in stages.rtPA has demonstrable effect on the liquefaction of submacular clots but there are remaining uncertainties with regards to the dose,safety and the timing of initial and repeat treatments.Potential side effects of rtPA include retinal pigment epithelial toxicity,increased risk of breakthrough vitreous haemorrhage and systemic toxicity.In cases presenting early,pneumatic displacement alone with anti-VEGF may be sufficient.Anti-VEGF monotherapy is a viable treatment option particularly in patients with thinner SMH and those who are unable to posture post pneumatic displacement.

Coagulation and fibrinolytic activity in patients with acute cerebral infarction

Objective To measure the concentration of D-dimer (DD), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen (PLG) activity in plasma and cerebrospinal fluid in patients with acute cerebral infarction and to investigate their clinical significance.Methods The concentrations of D-dimer, t-PA, and PAI-1 in plasma and cerebrospinal fluid in patients were measured by enzyme-linked immunosorbent assay (ELISA). The PLG biological activity was detected using the chromophore method. The results were compared with those of the controls.Results The concentrations of D-dimer, t-PA and PAI-1 in cerebrospinal fluid and plasma in patients with acute cerebral infarction were much higher than those of normal subjects (P<0.01). Conversely, the level of PLG activity was significantly lower in the patients than in the controls (P<0.01).Conclusion Hypercoagulability and secondary hyperfibrinolysis exist in patients with acute cerebral infarction.

Factors Associated with Thrombolysis Outcome in Ischemic Stroke Patients with Atrial Fibrillation

The outcome of early intravenous thrombolysis for ischemic stroke in patients with atrial fibrillation（AF）is worse than that without thrombosis. How to increase the efficacy of intravenous thrombolysis for AF-related ischemic stroke remains largely unknown. In this study, we investigated factors that influence the effect of intravenous thrombolysis in these patients. Our results showed that thrombolysis was independently associated with a favorable outcome（P ／ 0.001） and did not influence the mortality of AF-related ischemic stroke, although it increased the risk of hemorrhage within 24 h after treatment. Risk factors for a poor outcome at admission were：heart failure（P = 0.045）; high systolic pressure（P = 0.039）; high blood glucose（P = 0.030）; and a high National Institutes of Health Stroke Scale（NIHSS） score（P ／ 0.001）. Moreover, high systolic pressure at admission（P = 0.007）, high blood glucose（P = 0.027）, and a high NIHSS score（P ／ 0.001） were independent risk factors for mortality at 3 months. Besides thrombolysis, a high NIHSS score（P = 0.006） and warfarin taken within 48 h before stroke onset（P = 0.032） were also independent risk factors for symptomatic hemorrhage within 24 h after treatment. Ischemic stroke patients with AF benefited from intravenous thrombolysis with recombinant tissue plasminogen activator within 4.5 h after stroke.

Protocoled thrombolytic therapy for frostbite improves phalangeal salvage rates

Background:Frostbite is a cold injury that has the potential to cause considerable morbidity and long-term disability.Despite the complexity of these patients,diagnostic and treatment practices lack standardization.Thrombolytic therapy has emerged as a promising treatment modality,demonstrating impressive digit salvage rates.We review our experience with thrombolytic therapy for severe upper extremity frostbite.Methods:Retrospective data on all frostbite patients evaluated at our institution from December 2017 to March 2018 was collected.A subgroup of patients with severe frostbite treated with intraarterial thrombolytic therapy(IATT)were analysed.Results:Of the 17 frostbite patients treated at our institution,14(82%)were male and the median age was 31(range:19–73).Substance misuse was involved in a majority of the cases(58.8%).Five(29.4%)patients with severe frostbite met inclusion criteria for IATT and the remaining patients were treated conservatively.Angiography demonstrated a 74.5%improvement in perfusion after tissue plasminogen activator thrombolysis.When comparing phalanges at risk on initial angiography to phalanges undergoing amputation,the phalangeal salvage rate was 83.3%and the digit salvage rate was 80%.Complications associated with IATT included groin hematoma,pseudoaneurysm and retroperitoneal hematoma.Conclusions:Thrombolytic therapy has the potential to greatly improve limb salvage and functional recovery after severe frostbite when treated at an institution that can offer comprehensive,protocoled thrombolytic therapy.A multi-center prospective study is warranted to elucidate the optimal treatment strategy in severe frostbite.