Summary Direct fibrinolytics are proteolytic enzymes that degrade fibrin without requiring an intermediate step of plasminogen activation. This review summarizes the current information available for five such agents, namely, plasmin (the prototypical form), three derivatives of plasmin (mini-plasmin, micro-plasmin, and delta-plasmin), and alfimeprase, a recombinant variant of a snake venom alpha-fibrinogenase, fibrolase. Biochemical attributes of molecular size, fibrin binding and inhibitor neutralization are compared. Pre-clinical foundations that assess potential for thrombolytic efficacy in vitro and in animal models of vascular occlusion and for hemostatic safety in animal models of bleeding are detailed. Clinical potential has been assessed in patients with peripheral arterial and graft occlusion, acute ischemic stroke, access catheter and hemodialysis shunt occlusions. The direct fibrinolytic agents have impressive biochemical and pre-clinical foundation for ultimate clinical application. However, clinical trial results for micro-plasmin and alfimeprase have not measured up to their anticipated benefit. Plasmin has thus far shown encouraging hemostatic safety, but efficacy data await completion of clinical trials. Whether direct fibrinolytics will impart clinical superiority in major thrombotic disorders over currently utilized indirect fibrinolytics such as tissue plasminogen activator remains to be determined.

We investigated the in vitro fibrinolytic properties of microplasmin, the isolated proteinase domain of plasmin, and its thrombolytic efficacy in a coronary artery thrombosis model in dogs. The amidolytic and fibrinolytic activity of recombinant microplasmin was compared with natural human plasmin. The thrombolytic efficacy of microplasmin was studied in a canine model of copper coil induced coronary artery thrombosis. Animals were randomly assigned to one of six treatment regimens, each with five animals per cohort. Four treatment groups received an intravenous bolus of microplasmin followed by an intravenous infusion of microplasmin for 1 h (1 mg/kg + 1.5 mg/kg/h with or without abciximab or 2 mg/kg + 3 mg/kg/h). In two treatment groups, microplasmin was administered intracoronary. Bolus administration was followed by a 1-h infusion if coronary flow was incompletely restored after the initial bolus administration (1 mg/kg + 1.5 mg/kg/h or 2 mg/kg + 3 mg/kg/h, respectively). The thrombolytic efficacy was documented by repeated angiographies and the coronary perfusion was assessed with the Thrombolysis in Myocardial Infarction (TIMI) grading. No significant differences between plasmin and microplasmin were observed with respect to the catalytic efficiencies towards the synthetic chromogenic substrates S-2403 or S-2444. The concentration required for 50% lysis of purified fibrin clots in 3 h, was ~100 nM for microplasmin compared to 20 nM for natural plasmin. Intravenous bolus administration of microplasmin restored TIMI 3 coronary flow in 0/5, 0/5, 1/5 and 2/5, respectively, whereas intracoronary bolus administration restored TIMI 3 coronary flow in 1/5 and 4/5 (1 mg/kg and 2 mg/kg, respectively)(ANOVA P < 0.05). TIMI 3 coronary flow was obtained in 0/5, 2/5, 2/5 and 3/5, respectively, during subsequent intravenous administration and in 5/5 and 4/5 in case of intracoronary administration (ANOVA P < 0.05). When compared to natural plasmin, the catalytic efficiency of microplasmin towards chromogenic substrates was similar, but the fibrinolytic potency of microplasmin towards fibrin clots was lower. Intracoronay administration of microplasmin effectively lysed coronary thrombosis.

Fibrinolytic activity has been shown to be reduced in many vascular diseases, including hepatic veno-occlusive disease after stem cell transplantation, a microangiopathy characterized by sinusoidal endothelial cell injury. Defibrotide is a polydisperse oligonucleotide with antithrombotic, profibrinolytic, anti-ischemic, and antiadhesive properties. Numerous clinical studies have shown promising activity of defibrotide in the treatment and prevention of veno-occlusive disease, with minimal toxicity. In corollary laboratory studies, defibrotide has been shown to decrease plasminogen activator inhibitor-1, increase tissue plasminogen activator levels, and increase overall plasma fibrinolytic activity in patients. Plasmin, a potent and nonspecific serine protease, plays a pivotal role in fibrinolysis by virtue of its ability to effectively degrade fibrin clots. In this study, defibrotide increases the activity of plasmin in hydrolyzing its substrate in a dose-dependent and length-dependent manner. Similar concentration-dependent effects of defibrotide were observed when plasmin was generated by tissue plasminogen activator or urokinase activation of plasminogen. In contrast, defibrotide had no direct effect on the activation of plasminogen to plasmin. Defibrotide was also able to enhance the activity of plasmin in degrading fibrin clot formed from fibrinogen, plasminogen, and thrombin. This effect was also concentration-dependent and directly correlated with the enzymatic activity of plasmin. This study therefore demonstrates that defibrotide is capable of enhancing the activity of plasmin and so contributes to its fibrinolytic activity. Taken together, these results support the effect of defibrotide in restoring the fibrinolytic vascular phenotype, in microangiopathic conditions such as veno-occlusive disease.

Plasmin is the prototype of a distinct class of "direct-acting" fibrinolytic agents, with biochemical and physiological attributes that are favorable for catheter-delivered thrombolytic therapy. Our studies indicate that plasmin is superior to plasminogen activators for hemostatic safety and thrombolytic efficacy in experimental models, and that plasmin has potential to avoid the bleeding risk that accompanies therapy of deep vein thrombosis with currently-used thrombolytic agents.

Several randomized trials of catheter-directed thrombolysis versus operative revascularization in patients with acute lower extremity ischemia were performed in the mid-1990s. Although the outcomes of these trials were not definitive, they did provide insight into potential uses and techniques of catheter-directed thrombolysis and lytic agents. This article reviews the outcomes of these randomized trials and describes advances in thrombolytic techniques and technology, including percutaneous mechanical thrombectomy devices and innovative catheter designs that accelerate lysis and the development of direct-acting lytic agents.

INTRODUCTION: Hemodialysis grafts often fail because of stenosis at the venous anastomosis resulting in thrombosis. Percutaneous intervention involves thrombolysis with plasminogen activators, mechanical removal of thrombus, and angioplasty of the stenotic lesions. OBJECTIVES: This article describes a phase I trial using plasmin (human) TAL 05-00018, a direct-acting fibrinolytic agent, to evaluate safety and, secondarily, to establish effective thrombolytic dosing. PATIENTS/METHODS: Six cohorts of five patients with acute HD graft occlusion documented by fistulagrams were treated with increasing dosages of plasmin (1, 2, 4, 8, 12, and 24 mg) infused within the graft over 30 min via two criss-crossed pulse-spray catheters. The primary efficacy endpoint was at least 50% thrombolysis, as determined by fistulography. RESULTS: There was no significant change in plasma alpha-2 antiplasmin or fibrinogen concentration, major bleeding did not occur, and there were no deaths. Serious adverse events in four patients were not related to the study drug. There was a dose-response relationship for the primary efficacy endpoint, all five subjects receiving 24 mg achieving more than 75% lysis. CONCLUSION: This first phase I study of plasmin (human) TAL 05-00018, infused into thrombosed HD grafts, documents safety at all doses and an effective thrombolytic dosage of 24 mg indicating that further investigation into locally delivered plasmin is warranted.

Catheter-directed thrombolysis is an important treatment option for many patients with acute lower extremity ischemia due to arterial and/or graft thrombosis. A growing body of evidence demonstrates that thrombolytic therapy for acute limb ischemia reduces the need for amputation; however, bleeding complications are more likely with lytic therapy. New direct-acting fibrinolytic compounds are being developed that facilitate thrombolysis while reducing the risk of bleeding. Plasmin, the active enzyme produced by all plasminogen activators, works directly upon thrombus and is neutralized instantly in the systemic circulation. This report reviews the principles for effective catheter directed thrombolysis for acute arterial occlusions and describes the development and evaluation of the new thrombolytic, plasmin.

Background:  Current treatment of acute peripheral artery or bypass graft occlusion utilizes catheter-directed thrombolysis of a plasminogen activator (PA). Plasmin is a direct-acting thrombolytic with a striking safety advantage over PA in pre-clinical models. Objectives:  We report the first use of purified plasmin for acute lower extremity arterial or bypass graft thrombosis in a Phase I, dose-escalation study of catheter-delivered agent. Methods:  Eighty-three patients with non-embolic occlusion of infrainguinal native arteries or bypass grafts were enrolled (safety population) into seven sequential dose cohorts to receive 25-175 mg of plasmin by intra-thrombus infusion over 5 hours. Arteriograms were performed at baseline, 2 and 5 hours and subjects were monitored for 30 days for clinical outcomes and laboratory parameters of systemic fibrinolysis. Results:  Major bleeding occurred in 4 patients (4.8%), minor bleeding alone in 13 (15.7%), with no trend towards more bleeding at higher dosages of plasmin. There was a trend towards lower plasma concentrations of fibrinogen, α(2)-antiplasmin, and α(2)-macroglobulin with increasing doses of plasmin, but the nadir fibrinogen was >350 mg/dL at the highest plasmin dose. Individual nadir values were above 200 mg/dL in 82 of 83 subjects, and were not different in patients with or without bleeding. Thrombolysis (≥50%) occurred in 79% of subjects receiving 125-175 mg of plasmin, compared with 50% who received 25-100 mg. Conclusions:  Catheter-delivered plasmin is safely administered to patients with acute lower extremity arterial occlusion at dosages of 25 to 175 mg. © 2012 International Society on Thrombosis and Haemostasis.

Plasmin is a direct thrombolytic which has been shown to have a strikingly favorable benefit to risk profile in comparison with plasminogen activators, notably tissue plasminogen activator (t-PA). As heparin is known to increase the risk of hemorrhage when co-administered with a plasminogen activator, we asked whether adjunct antithrombotic agents such as aspirin and heparin would affect the safety of plasmin. Three groups of rabbits were administered plasmin at a dose (4 mg kg-1) designed to induce significant decreases in antiplasmin, fibrinogen and factor (F)VIII, to about 25, 40 and 40%, respectively, of baseline values, but not cause prolongation of the ear puncture bleeding time. In a blinded and randomized trial, the results show that an intravenous aspirin bolus plus heparin administered as a bolus followed by a maintenance continuous infusion did not significantly prolong the bleeding time during plasmin infusion. These data indicate that in the rabbit, concomitant use of aspirin plus heparin does not affect the safety of a therapeutic dose of plasmin.

The purpose of this study is to investigate the possible underlying pathogenesis of erectile dysfunction(ED) in young men with low risk of coronary heart disease and no well-known aetiology. To conduct this study, 122 patients with ED under the age of 40 were enrolled, along with 33 age-matched normal control subjects. The patients with ED had significantly higher levels of systolic blood pressure (SBP), total cholesterol and triglyceride, high sensitivity C-reactive protein (hs-CRP), greater carotid intima-media thickness (CIMT) and Framingham risk score (FRS) than the control group, though all of these values were within the respective normal range. Further, the brachial artery flow- mediated vasodilation (FMD) values were significantly lower in ED patients and correlated positively with the severity of ED (r = 0.714, p < 0.001). When these significant factors were studied in the multivariate logistic regression model, FMD, SBP, hs-CRP and FRS remained the statistical significance. The receiver-operating characteristic (ROC) analysis demonstrated that FMD had a high ability to predict ED in young male with low FRS [area under the curve (AUC) 0.921, p < 0.001]. The cutoff value of FMD <10.25% had sensitivity of 82.8% and specificity of 100% for diagnosis of ED. FRS and hs- CRP were also proven to be predictors of ED (AUC 0.812, p < 0.001; AUC 0.645, p = 0.011, respectively). The results of this study validated that subclinical endothelial dysfunction and low-grade inflammation may be the underlying pathogenesis of ED with no well-known aetiology. Young patients complaining of ED should be screened for cardiovascular risk factors and possible subclinical atherosclerosis. Measurement of FMD, hs-CRP and FRS can improve our ability to predict and treat ED, as well as subclinical cardiovascular disease early for young male.

Robust reversible watermarking (RRW) methods are popular in multimedia for protecting copyright, while preserving intactness of host images and providing robustness against unintentional attacks. However, conventional RRW methods are not readily applicable in practice. That is mainly because 1) they fail to offer satisfactory reversibility on large-scale image datasets; 2) they have limited robustness in extracting watermarks from the watermarked images destroyed by different unintentional attacks; and 3) some of them suffer from extremely poor invisibility for watermarked images. Therefore, it is necessary to have a framework to address these three problems, and further improve its performance. This paper presents a novel pragmatic framework, wavelet-domain statistical quantity histogram shifting and clustering (WSQH-SC). Compared with conventional methods, WSQH-SC ingeniously constructs new watermark embedding and extraction procedures by histogram shifting and clustering, which are important for improving robustness and reducing run-time complexity. Additionally, WSQH-SC includes the property inspired pixel adjustment (PIPA) to effectively handle overflow and underflow of pixels. This results in satisfactory reversibility and invisibility. Furthermore, to increase its practical applicability, WSQH-SC designs an enhanced pixel-wise masking (EPWM) to balance robustness and invisibility. We perform extensive experiments over natural, medical, and synthetic aperture radar (SAR) images to show the effectiveness of WSQH-SC by comparing with the histogram rotation (HR)-based and histogram distribution constrained (HDC) methods.

TAL1/SCL is a hematopoietic-specific oncogene and its activity is regulated by associated transcriptional co-activators and corepressors. Dysregulation of TAL1 activity has been associated with T-cell leukemogenesis. However, it remains unclear how the interactions between TAL1 and corepressors versus co-activators are properly regulated. Here, we reported that protein kinase A (PKA)-mediated phosphorylation regulates TAL1 interaction with the lysine-specific demethylase (LSD1) that removes methyl group from methylated Lys 4 on histone H3 tails. Phosphorylation of serine 172 in TAL1 specifically destabilizes the TAL1-LSD1 interaction leading to promoter H3K4 hypermethylation and activation of target genes that have been suppressed in normal and malignant hematopoiesis. Knockdown of TAL1 or LSD1 led to a derepression of the TAL1 target genes in T-cell acute lymphoblast leukemia (T-ALL) Jurkat cells, which is accompanied by elevating promoter H3K4 methylation. Similarly, treatment of PKA activator forskolin resulted in derepression of target genes by reducing its interaction with LSD1 while PKA inhibitor H89 represses them by suppressing H3K4 methylation levels. Consistent with the dual roles of TAL1 in transcription, TAL1-associated LSD1 is decreased while recruitment of hSET1 is increased at the TAL1 targets during erythroid differentiation. This process is accompanied by a dramatic increase in H3K4 methylation. Thus, our data revealed a novel interplay between PKA phosphorylation and TAL1-mediated epigenetic regulation that regulates hematopoietic transcription and differentiation programs during hematopoiesis and leukemogenesis.Oncogene advance online publication, 6 February 2012; doi:10.1038/onc.2012.8.

Purpose. To evaluate the safety and efficacy of the Possis rheolytic thrombectomy with or without indwelling catheter-directed pharmacolysis for the treatment of massive pulmonary embolus in patients presenting with right heart strain and/or a pulseless electrical activity (PEA). Materials and Methods. Retrospective review of patients undergoing pulmonary pharmacolysis was performed (07/2004-06/2009). Pre- and posttreatment Miller index scoring weres calculated and compared. Patients were evaluated for tPA doses, ICU stay, hospital stay, and survival by Kaplan-Meier analysis. Results. 11 patients with massive PE were found, with 10/11 presenting with a Miller score of >17 (range: 16-27, mean: 23.2). CTPA and/or echocardiographic evidence of right heart strain was found in 10/11 patients. 3 (27%) patients presented with a PEA event. Two (18%) patients had a contraindication to pharmacolysis and were treated with mechanical thrombectomy alone. The intraprocedural mortality was 9%(n = 1/11). Of the 10 patients who survived the initial treatment, 7 patients underwent standard mechanical thrombectomy initially, while 5 received power pulse spray mechanical thrombectomy. Eight of these 10 patients underwent adjunctive indwelling catheter-directed thrombolysis. The mean catheter-directed infusion duration was 18 hours (range of 12-26 hours). The average intraprocedural, infusion, and total doses of tPA were 7 mg, 19.7 mg, and 26.7 mg, respectively. There was a 91%(10/11) technical success rate. The failure was the single mortality. Average reduction in Miller score was 9.5 or 41%(P = 0.009), obstructive index of 6.4 or 47%(P = 0.03), and perfusion index of 2.7 or 28%(P = 0.05). Average ICU and hospital stay were 7.4 days (range 2-27 days) and 21.3 days (range 6-60 days), respectively. Intent to treat survival was 90% at 6, 12, and 18 months. Conclusion. Rheolytic thrombectomy with or without adjunctive catheter-directed thrombolysis provides a safe and effective method for treatment of acute PE in patients who present with right heart strain and/or a PEA event.

Increased activation of epidermal growth factor receptor (EGFR) family members such as HER2/Erbb2 can result in more aggressive disease, resistance to chemotherapy and reduced survival of head and neck squamous cell carcinoma (HNSCC) patients. In order to identify mechanisms through which these receptor tyrosine kinases accelerate tumor progression, the regulation of metalloprotease expression by EGFR family members was investigated in 11 squamous cell carcinoma (SCC) cell lines. HER2 expression was significantly correlated with ADAM12 (A Disintegrin And Metalloprotease 12) expression in these cell lines and was co-expressed in human head and neck cancers. Inhibition of HER2 or EGFR decreased ADAM12 transcripts whereas HER2 transfection upregulated ADAM12 expression. To understand the molecular mechanisms underlying HER2 regulation of ADAM12, we investigated the signaling pathways directing ADAM12 production in SCC cells. Inhibition of phosphatidyl inositol-3-kinase or mammalian target of rapamycin decreased ADAM12 transcripts in HER2-expressing SCC cells, whereas transfection with AKT increased ADAM12 mRNA. Experiments utilizing ADAM12 transfection or siRNA targeting of ADAM12 revealed that the protease increased both the migration and invasiveness of oral SCC cells. Surprisingly, ADAM12 also increased HER2 message, protein levels and activity through an Ets1-dependent mechanism. Collectively, these results reveal a novel positive activation loop between ADAM12 and HER2 that may contribute to HNSCC progression.Oncogene advance online publication, 10 October 2011; doi:10.1038/onc.2011.460.

Two issues have held the focus of thrombolysis research for over 50 years, namely, choosing between a plasminogen activator (PA) or plasmin as the best therapeutic agent and choosing between systemic or local administration. The original plasmin product of the 1950s was both ineffective and contaminated with PA, and catheter technology was not yet developed for routine clinical use. For decades, clinical practice has focused on PA and systemic administration, but today, PAs are often administered by catheter into thrombosed vessels, notably for peripheral arterial and graft occlusion and deep vein thrombosis, and increasingly for acute ischaemic stroke. Despite using catheter-delivered therapy, bleeding complications still occur, most severely expressed as symptomatic intracranial haemorrhage. New experimental data indicate that we should now reconsider plasmin as a viable, even preferable, thrombolytic agent. Plasmin requires catheter delivery to achieve thrombolysis, but this technical issue has been solved with modern technology and widespread presence of interventional suites. After local administration, plasmin will lyse thrombi; thereafter, any plasmin in the circulation will be rapidly neutralised. Pre-clinical studies confirm that plasmin has marked haemostatic safety advantage over t-PA. After more than 50 years, the field has come full circle, and plasmin as the thrombolytic agent and catheter use for local delivery of agent may represent a step forward in thrombolytic therapy.

All existing video coding standards developed so far deem video as a sequence of natural frames (formed in the XY plane), and treat spatial redundancy (redundancy along X and Y direction) and temporal redundancy (redundancy along T direction) differently and separately. In this paper, we investigate into a new compression (redundancy reduction) method for video in which the frames are allowed to be formed in a non-XY plane. We are to exploit fuller extent of video redundancy, and propose an adaptive optimal compression plane (OCP) determination process to be used as a preprocessing step prior to any standard video coding scheme. The essence of the scheme is to form the frames in the plane formed by two axes (among X, Y and T) corresponding to signal correlation evaluation, which enables better prediction (therefore better compression). In spite of the simplicity of the proposed method, it can be used for both lossless and lossy compression, and with and without inter-frame prediction. Extensive experimental results show that the new coding method improves the performance of the video coding, for a number of coding methods (inclusive of lossless and near-lossless Motion JPEG-LS, Motion JPEG, Motion JPG2K, H.264 intra-only profile and H.264) and videos with different visual content.

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