Clinical Pharmacokinetics and Pharmacodynamics of Desmoteplase.

Desmoteplase is a bat (Desmodus rotundus) saliva-derived fibrinolytic enzyme resembling a urokinase and tissue plasminogen activator. It is highly dependent on fibrin and has some neuroprotective attributes. Intravenous administration of desmoteplase is safe and well tolerated in healthy subjects. Plasma fibrinolytic activity is linearly related to its blood concentration, its terminal elimination half-life ranges from 3.8 to 4.92 h (50 vs. 90 µg/kg dose). Administration of desmoteplase leads to transitory derangement of fibrinogen, D-dimer, alpha2-antiplasmin, and plasmin and antiplasmin complex which normalize within 4-12 h. It does not alter a prothrombin test, international normalized ratio, activated partial thromboplastin time, and prothrombin fragment 1.2. Desmoteplase was tested in myocardial infarction and pulmonary embolism and showed promising results versus alteplase. In ischemic stroke trials, desmoteplase was linked to increased rates of symptomatic intracranial hemorrhages and case fatality. However, data from "The desmoteplase in Acute Ischemic Stroke" Trials, DIAS-3 and DIAS-J, suggest that the drug is well tolerated and its safety profile is comparable to placebo. Desmoteplase is theoretically a superior thrombolytic because of high fibrin specificity, no activation of beta-amyloid, and lack of neurotoxicity. It was associated with better outcomes in patients with significant stenosis or occlusion of a proximal precerebral vessels. However, DIAS-4 was stopped as it might have not reached its primary endpoint. Due to its promising properties, desmoteplase may be added into treatment of ischemic stroke with extension of the time window and special emphasis on patients presenting outside the 4.5-h thrombolysis window, with wake-up strokes and strokes of unknown onset.

Design and production of new chimeric reteplase with enhanced fibrin affinity: a theoretical and experimental study.

Plasminogen activators (PAs) are widely used for treatment of disorders caused by clot formation. Fibrin specific PAs are safe drugs from this group because of reducing the incidence of hemorrhage. The newer generation of PAs like tenecteplase, reteplase and desmoteplase were designed with the aim of achieving desirable properties such as improving specificity and affinity to fibrin and increasing half-life. Protein engineering and using of theoretical methods can help to rational and reliable design of new PAs with a set of favorable properties. In the present study, two new chimeric reteplase named M1-chr and M2-chr were designed with the aim of enhancing fibrin affinity also some potential properties include of increasing resistance to plasminogen activator inhibitor-1 and decreasing neurotoxicity. So, finger domain of desmoteplase was added to reteplase as a high fibrin specific domain. Some other point mutations were considering to achieve other mentioned properties. Three dimensional structure of wild-type reteplase and mutants were created by homology modeling and were evaluated by molecular dynamic simulation. Then, mutants docked to fibrin by HADDOCK web tools. Result of theoretical section verified the stability of mutants' structures. Also showed better interaction between M1-chr with fibrin than M2-chr. Wild-type and mutants were produced in bacterial expression system. Experimental assessment showed both mutants have appropriate enzymatic activity also 1.9-fold fibrin binding ability compared to wild-type. Therefore, this study offers new thrombolytic drugs with desirable properties specially enhanced fibrin affinity so they can represent a promising future in cost-effective production of favorable thrombolytic drugs.Communicated by Ramaswamy H. Sarma.

Corneal and scleral permeability of Desmoteplase in different species.

OBJECTIVE: Intraocular fibrin clots caused by severe uveitis can be a sight-threatening condition that needs to be resolved quickly and reliably. Intracameral injection of tissue-plasminogen activator (tPA) is commonly used to resolve intraocular fibrin. However, the drug does not reach fibrinolytic concentrations after topical application. Desmoteplase (DSPA) is a structurally similar but smaller fibrinolytic agent with a higher fibrin selectivity, a longer half-life, and better biocompatibility compared with tPA. This study was designed to evaluate the corneal and scleral permeability of DSPA in rabbits, pigs, dogs, horses, and humans ex vivo. PROCEDURES: Corneal and scleral tissues (n = 5 per group) were inserted into Franz-type diffusion chambers and exposed to 1.4 mg/mL DSPA for 30 minutes. Drug concentrations on the receiver side were determined by liquid chromatography-tandem mass spectrometry. RESULTS: Concentrations of DSPA after corneal and scleral permeation through fresh tissues ranged from 0.0 to 16.3 µg/mL and 0.0 to 11.4 µg/mL (rabbits), 0.3 to 5.6 µg/mL and 3.1 to 9.2 µg/mL (dogs), 2.1 to 14.9 µg/mL and 4 to 8.7 µg/mL (horses), and 0.6 to 3 µg/mL and 2.9 to 18.1 µg/mL (pigs), respectively. A concentration of 0.07-12.9 µg/mL DSPA was detectable after diffusion through tissue culture preserved human donor bank corneas (Table 1). CONCLUSIONS: Desmoteplase has the ability to permeate both cornea and sclera ex vivo in all species tested. Implications of the ex vivo permeability of DSPA suggest that in vivo permeability may be possible, and if so, it could lead to a novel topical application for lysing fibrin.

Tissue plasminogen activator attenuates outflow facility reduction in mouse model of juvenile open angle glaucoma.

Tissue plasminogen activator (tPA) has been shown to prevent steroid-induced reduction in aqueous humor outflow facility via an upregulation in matrix metalloproteinase (Mmp) expression. The purpose of this study was to determine whether tPA can rescue outflow facility reduction in the Tg-MYOCY437H mouse model, which replicates human juvenile open angle glaucoma. Outflow facility was measured in Tg-MYOCY437H mice following: periocular steroid exposure and intraocular protein treatment with enzymatically active or enzymatically inactive tPA. Effects of tPA on outflow facility were compared to those of animals treated with topical sodium phenylbutarate (PBA), a modulator of endoplasmic reticulum stress. Gene expression of fibrinolytic pathway components (Plat, Plau, and Pai-1) and matrix metalloproteinases (Mmp-2, -9, and -13) was determined in angle ring tissues containing the trabecular meshwork. Tg-MYOCY437H mice did not display further outflow facility reduction following steroid exposure. Enzymatically active and enzymatically inactive tPA were equally effective in attenuating outflow facility reduction in Tg-MYOCY437H mice and caused enhanced expression of matrix metalloproteinases (Mmp-9 and Mmp-13). tPA was equally effective to topical PBA treatment in ameliorating outflow facility reduction in Tg-MYOCY437H mice. Both treatments were associated with an upregulation in Mmp-9 expression while tPA also upregulated Mmp-13 expression. tPA increases the expression of matrix metalloproteinases and may cause extracellular matrix remodeling at the trabecular meshwork, which results in reversal of outflow facility reduction in Tg-MYOCY437H mice.

Oriented Immobilization on Gold Nanoparticles of a Recombinant Therapeutic Zymogen.

Direct immobilization of functional proteins on gold nanoparticles (AuNPs) affects their structure and function. Changes may vary widely and range from strong inhibition to the enhancement of protein function. More often though the outcome of direct protein immobilization results in protein misfolding and the loss of protein activity. Additional complications arise when the protein being immobilized is a zymogen which requires and relies on additional protein-protein interactions to exert its function. Here we describe molecular design of a glutathione-S-transferase-Staphylokinase fusion protein (GST-SAK) and its conjugation to AuNPs. The multivalent AuNP-(GST-SAK)n complexes generated show plasminogen activation activity in vitro. The methods described are transferable and could be adapted for conjugation and functional analysis of other plasminogen activators, thrombolytic preparations or other functional enzymes.

Evaluation of D-dimer levels in aqueous humor of rabbit eyes with and without induced intraocular fibrin and fibrinolytic treatment.

OBJECTIVE: To analyze D-dimer concentrations in aqueous humor (AH) of rabbit eyes under physiological conditions, after induction of fibrin clots, and following fibrinolytic therapy. ANIMALS STUDIED: Prospective study measuring D-dimers in aqueous humor of rabbit eyes with induced fibrin clots (n = 44). PROCEDURES: Rabbits were purchased in two groups, which led to two temporally separated experimentation groups. Different treatment protocols were compared for their efficacy in fibrin reduction (slit-lamp examination, high-resolution ultrasound). AH was taken from left eyes before clot induction (baseline, day 1), 24 hours later after clot establishment/prior to drug administration (post-induction, day 2) and 48 hours after clot induction (post-treatment, day 3). An enzyme-linked immunosorbent assay (ELISA) was performed to measure intraocular D-dimer concentrations RESULTS: D-dimer concentrations were measurable in all samples. There were no differences in D-dimer levels across time points or treatments within the arrival groups. However, a significant difference in mean D-dimer levels was observed between the two arrival groups (group 1:3.1 µg/mL; group 2:6.1 µg/mL; P < .0001), which made a direct comparison of treatment groups impossible. Clinically, all eyes displayed fibrin clots in the anterior chamber and different treatment types led to significant differences in clot resolution (clot size reduction after intracameral treatment: 98%, topical treatment: 60%, no treatment: 40%). CONCLUSION: D-dimers were identified in all AH samples of rabbits with large variability between samples. D-dimer levels were neither predictive for differences in induced fibrin formation nor for drug efficacy.

Design of a thrombin inhibitory staphylokinase based plasminogen activator with anti-reocclusion potential.

In the quest of a therapeutically improved thrombolytic drug, we designed and expressed a Staphylokinase based, recombinant fusion protein with fibrin specific clot lysis and anti-thrombin activities derived from human thrombomodulin, a transmembrane glycoprotein with anticoagulant properties, known to form a complex with thrombin and then activating Protein C. The fusion construct was expressed in the yeast Pichia pastoris for cost effective and facile production. The purified construct had comparable plasminogen activation and clot lysis capability as compared to native SAK in that it showed plasmin dependent Plasminogen activation, but exhibited significantly lower fibrinogenolysis (an indicator of fibrin-specific action) even at much higher doses than SAK. In addition, its successful activation of the thrombin-mediated Protein C anticoagulant pathway was reflected in increased in vitro plasma clotting time, as well as inhibition of clot bound thrombin, which led to nearly 15-25% lesser re-formation of fibrin clots after initial successful clot lysis in a specially designed in vitro assay for re-occlusion. Thus, this novel chimeric protein could be of high therapeutic potential as a thrombolytic drug with enhanced fibrin specific clot dissolving properties along with lower bleeding and re-thrombosis complications- a dire need today, especially in the treatment of thrombotic strokes.

Targeted Delivery of Plasminogen Activators for Thrombolytic Therapy: An Integrative Evaluation.

In thrombolytic therapy, plasminogen activators (PAs) are still the only group of drug approved to induce thrombolysis, and therefore, critical for treatment of arterial thromboembolism, such as stroke, in the acute phase. Functionalized nanocomposites have attracted great attention in achieving target thrombolysis due to favorable characteristics associated with the size, surface properties and targeting effects. Many PA-conjugated nanocomposites have been prepared and characterized, and some of them has been demonstrated with therapeutic efficacy in animal models. To facilitate future translation, this paper reviews recent progress of this area, especially focus on how to achieve reproducible thrombolysis efficacy in vivo.

Structural studies of plasmin inhibition.

Plasminogen (Plg) is the zymogen form of the serine protease plasmin (Plm), and it plays a crucial role in fibrinolysis as well as wound healing, immunity, tissue remodeling and inflammation. Binding to the targets via the lysine-binding sites allows for Plg activation by plasminogen activators (PAs) present on the same target. Cellular uptake of fibrin degradation products leads to apoptosis, which represents one of the pathways for cross-talk between fibrinolysis and tissue remodeling. Therapeutic manipulation of Plm activity plays a vital role in the treatments of a range of diseases, whereas Plm inhibitors are used in trauma and surgeries as antifibrinolytic agents. Plm inhibitors are also used in conditions such as angioedema, menorrhagia and melasma. Here, we review the rationale for the further development of new Plm inhibitors, with a particular focus on the structural studies of the active site inhibitors of Plm. We compare the binding mode of different classes of inhibitors and comment on how it relates to their efficacy, as well as possible future developments.

Design of a DNA-Programmed Plasminogen Activator.

Although the functional specificity and catalytic versatility of enzymes have been exploited in numerous settings, controlling the spatial and temporal activity of enzymes remains challenging. Here we describe an approach for programming the function of streptokinase (SK), a protein that is clinically used as a blood "clot buster" therapeutic. We show that the fibrinolytic activity resulting from the binding of SK to the plasma proenzyme plasminogen (Pg) can be effectively regulated (turned "OFF" and "ON") by installing an intrasteric regulatory feature using a DNA-linked protease inhibitor modification. We describe the design rationale, synthetic approach, and functional characterization of two generations of intrasterically regulated SK-Pg constructs and demonstrate dose-dependent and sequence-specific temporal control in fibrinolytic activity in response to short predesignated DNA inputs. The studies described establish the feasibility of a new enzyme-programming approach and serves as a step toward advancing a new generation of programmable enzyme therapeutics.

Fibrin-specific and effective clot lysis requires both plasminogen activators and for them to be in a sequential rather than simultaneous combination.

Thrombolysis with tissue plasminogen activator (tPA) has been a disappointment and has now been replaced by an endovascular procedure whenever possible. Nevertheless, thrombolysis remains the only means by which circulation in a thrombosed artery can be restored rapidly. In contrast to tPA monotherapy, endogenous fibrinolysis uses both tPA and urokinase plasminogen activator (uPA), whose native form is a proenzyme, prouPA. This combination is remarkably effective as evidenced by the fibrin degradation product, D-dimer, which is invariably present in plasma. The two activators have complementary mechanisms of plasminogen activation and are synergistic in combination. Since tPA initiates fibrinolysis when released from the vessel wall and prouPA is in the blood, they induce fibrinolysis sequentially. It was postulated that this may be more effective and fibrin-specific. The hypothesis was tested in a model of clot lysis in plasma in which a clot was first exposed to tPA for 5 min, washed and incubated with prouPA. Lysis was compared with that of clots incubated with both activators simultaneously. The sequential combination was almost twice as effective and caused less fibrinogenolysis than the simultaneous combination (p < 0.0001) despite having significantly less tPA, as a result of the wash. A mechanism is described by which this phenomenon can be explained. The findings are believed to have significant therapeutic implications.

Rapid Systematic Review: Intra-Arterial Thrombectomy ("Clot Retrieval") for Selected Patients with Acute Ischemic Stroke.

BACKGROUND: Acute ischemic stroke (AIS) is a leading cause of morbidity and mortality. However, precisely defining the optimal treatment for individual patients early after AIS onset remains elusive. There has recently been a surge in published studies documenting the effectiveness of mechanical intra-arterial thrombectomy for treatment of a subset of patients with AIS. This therapy has been proposed and studied for the small (<1.2%) subgroup of patients with ischemic strokes who have "large vessel" strokes or strokes that fail to improve after the administration of tissue plasminogen activator (t-PA). The current rapid systematic review provides practicing emergency physicians updated information regarding mechanical thrombectomy as a treatment option for carefully selected AIS patients. METHODS: A PubMed literature search was conducted from January 1996 to June 2016 and limited to human clinical trials written in English with relevant keywords. High-quality randomized controlled studies identified then underwent a structured review. RESULTS: In total, 179 papers fulfilling the search criteria were screened and 8 appropriate articles were rigorously reviewed in detail and recommendations given on the effectiveness and indication of mechanical intra-arterial thrombectomy for the treatment of AIS. CONCLUSIONS: Mechanical intra-arterial thrombectomy reduces long-term disability in a properly selected subset of patients who have an AIS caused by large vessel occlusion. Many of these patients will have failed to improve after intravenous administration of t-PA, and mortality is not increased when combined with t-PA. Careful screening criteria should be in place to identify the limited subset of patients to whom this therapy is delivered to derive optimal treatment benefits.

Desmoteplase 3 to 9 Hours After Major Artery Occlusion Stroke: The DIAS-4 Trial (Efficacy and Safety Study of Desmoteplase to Treat Acute Ischemic Stroke).

BACKGROUND AND PURPOSE: The DIAS-3 trial (Efficacy and Safety Study of Desmoteplase to Treat Acute Ischemic Stroke [phase 3]) did not demonstrate a significant clinical benefit of desmoteplase administered 3 to 9 hours after stroke in patients with major artery occlusion. We present the results of the prematurely terminated DIAS-4 trial together with a post hoc pooled analysis of the concomitant DIAS-3, DIAS-4, and DIAS-J (Japan) trials to better understand the potential risks and benefits of intravenous desmoteplase for the treatment of ischemic stroke in an extended time window. METHODS: Ischemic stroke patients with occlusion/high-grade stenosis in major cerebral arteries were randomly assigned to intravenous treatment with desmoteplase (90 µg/kg) or placebo. The primary outcome was modified Rankin Scale (mRS) score of 0 to 2 at day 90. Safety assessments included mortality, symptomatic intracranial hemorrhage, and other serious adverse events. RESULTS: In DIAS-4, 52 of 124 (41.9%) desmoteplase-treated and 46 of 128 (35.9%) placebo-treated patients achieved an mRS score of 0 to 2 (odds ratio, 1.45; 95% confidence interval, 0.79; 2.64; P=0.23) with equal mortality, frequency of symptomatic intracranial hemorrhage, and other serious adverse events in both the treatment arms. In the pooled analysis, mRS score of 0 to 2 was achieved by 184 of 376 (48.9%) desmoteplase-treated versus 171 of 381 (44.9%) placebo-treated patients (odds ratio, 1.33; 95% confidence interval, 0.95; 1.85; P=0.096). Treatment with desmoteplase was safe and increased the recanalization rate (107/217 [49.3%] versus 85/222 [38.3%]; odds ratio, 1.59; 95% confidence interval, 1.08-2.35; P=0.019). Recanalization was associated with favorable outcomes (mRS 0-2) at day 90 in both the treatment arms. CONCLUSIONS: Late treatment with intravenous 90 µg/kg desmoteplase is safe, increases arterial recanalization, but does not significantly improve functional outcome at 3 months. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00856661.

Clot Formation in the Presence of Acetylsalicylic Acid Leads to Increased Lysis Rates Regardless of the Chosen Thrombolysis Strategy.

BACKGROUND: Patients with acute ischemic strokes frequently take an acetylsalicylic acid (ASA) premedication. We determined the impact of ASA on different thrombolysis strategies in vitro. METHODS: For two clot types made from platelet-rich plasma (one with and one without ASA) lysis rates were measured by weight loss after 1 h for five different groups: in control group A clots were solely placed in plasma; in groups B and C clots were treated with rt-PA (60 kU/ml), and in groups D and E clots were treated with desmoteplase (DSPA; 2 µg/ml). Ultrasound (2 MHz, 0.179 W/cm2) was included in groups C and E. The fibrin mesh structures of the clots were investigated by electron microscopy. RESULTS: For both clot types lysis rates increased significantly for all treatment strategies compared to their control group (each p < 0.001). The addition of ASA significantly increased the lysis rate in all 5 groups (each p < 0.001) and led to a ceiling effect concerning the treatment. A semiquantitative analysis of transmission electron micrographs revealed a decreased fibrin density for clots with ASA. For both clot types DSPA and ultrasound led to a significant dissolution of the fibrin mesh (both p = 0.029). CONCLUSIONS: In vitro ASA pretreatment leads to significantly increased lysis rates due to a weaker fibrin mesh in platelet-rich plasma clots.

In Vitro Examination of the Thrombolytic Efficacy of Desmoteplase and Therapeutic Ultrasound Compared with rt-PA.

The aim of the study described here was to evaluate the thrombolytic efficacy of combined treatment with the fibrin-selective plasminogen activator desmoteplase (DSPA) and therapeutic ultrasound (sonothrombolysis [STL]) compared with conventional rt-PA (recombinant tissue plasminogen activator) treatment in vitro. Lysis rates were determined by the weight loss of platelet-rich plasma (PRP) clots treated with rt-PA (60 kU/mL) or DSPA (2 µg/mL) combined with pulsed wave ultrasound (2 MHz, 0.179 W/cm(2)). To reveal the individual effects of medication and ultrasound, lysis rates were also determined for DSPA monotherapy and for combined treatment with rt-PA and ultrasound. Clots solely placed in plasma served as the control group. Lysis increased significantly with rt-PA (26.5 ± 7.8%) and DSPA (30.5 ± 6%) compared with the control group (18.2 ± 5.9%) (each p < 0.001). DSPA lysis was more effective than rt-PA lysis (without STL: p = 0.015, with STL: p = 0.01). Combined treatment with DSPA and 2-MHz STL significantly exceeded rt-PA lysis (32.8% vs. 26.5%, p < 0.001).

Structure-activity relationships of the plasminogen modulator SMTP with respect to the inhibition of soluble epoxide hydrolase.

A family of fungal metabolites, SMTP, is a small-molecule plasminogen modulator that enhances plasminogen activation, leading to thrombolysis. We recently demonstrated that SMTP-7 effectively treats ischemic stroke due to its thrombolytic activity as well as anti-inflammatory action, which is attributable to soluble epoxide hydrolase (sEH) inhibition. In this paper, we studied detailed structure-activity relationships of plasminogen modulation and sEH inhibition using 25 SMTP congeners including six newly synthesized ones. The results clearly demonstrate that the structure of the N-linked side chain of SMTP congeners markedly affect their activities toward plasminogen modulation and inhibitions of the two activities of sEH (C-terminal epoxide hydrolase and N-terminal phosphatase). A slight change in the N-linked side chain results in affording selectivity of SMTP congeners. Many congeners, which lacked plasminogen modulation activity, differently inhibited the two sEH activities depending on the structures of the N-linked side chain. Some congeners were active in plasminogen modulation and inhibition of both activities of sEH. These results help comprehensive understanding of ideal design of a drug useful for ischemic diseases that are associated with inflammation, such as stroke.

Safety and efficacy of desmoteplase given 3-9 h after ischaemic stroke in patients with occlusion or high-grade stenosis in major cerebral arteries (DIAS-3): a double-blind, randomised, placebo-controlled phase 3 trial.

BACKGROUND: Current treatment of ischaemic stroke with thrombolytic therapy is restricted to 3-4·5 h after symptom onset. We aimed to assess the safety and efficacy of desmoteplase, a fibrin-dependent plasminogen activator, given between 3 h and 9 h after symptom onset in patients with occlusion or high-grade stenosis in major cerebral arteries. METHODS: In a prospective, double-blind, multicentre, parallel-group, randomised trial, we enrolled patients from 77 hospitals in 17 countries who had ischaemic stroke and occlusion or high-grade stenosis in major cerebral arteries. We randomly assigned patients in a 1:1 ratio, using computer-generated randomisation lists with stratification for baseline National Institutes of Health Stroke Scale and age, to treatment with desmoteplase (90 µg/kg) given 3-9 h after symptom onset or to placebo. Patients, investigators, staff, and the funder were masked to treatment assignment. The primary outcome was a favourable modified Rankin Scale score (0-2) at day 90 in all treated patients who had at least one postbaseline measurement of the modified Rankin Scale. Safety was assessed in all randomly assigned patients who received study drugs. This trial is registered with ClinicalTrials.gov, number NCT00790920. FINDINGS: Between Feb 6, 2009, and Nov 27, 2013, we enrolled 492 patients and randomly assigned 247 to desmoteplase and 245 to placebo (236 in the desmoteplase group and 237 in the placebo group were included in the analysis of the primary endpoint). Median time from stroke onset to treatment was 6·9 h (IQR 5·7-8·0) for placebo and 7·0 h (6·0-7·9) for desmoteplase. Modified Rankin Scale score (0-2) at day 90 occurred in 121 (51%) patients given desmoteplase and 118 (50%) patients given placebo (adjusted odds ratio 1·20, 95% CI 0·79-1·81, p=0·40). 24 (10%) of 240 patients given desmoteplase died compared with 23 (10%) of 238 patients given placebo. Serious adverse events occurred in 64 (27%) of 240 patients receiving desmoteplase compared with 69 (29%) of 238 patients receiving placebo; frequency of symptomatic intracranial haemorrhage (six [3%] patients in the desmoteplase group vs five [2%] in the placebo group), symptomatic cerebral oedema (five [2%] vs four [2%]), and major haemorrhage (ten [4%] vs 15 [6%]) was much the same between treatment groups. INTERPRETATION: Treatment with desmoteplase did not cause safety concerns and did not improve functional outcome when given to patients who had ischaemic stroke and major cerebral artery occlusion beyond 3 h of symptom onset. FUNDING: H Lundbeck A/S.

Thrombolytic and Antiplatelet Effects of a Novel Plasminogen Activator from the Venom of Gloydius Brevicaudus Viper.

AIM: To investigate the thrombolytic and antiplatelet effects of a novel plasminogen activator from the venom of the Gloydius brevicaudus viper (GBV-PA) in vitro and in vivo. METHODS: Thrombolytic experiments were performed in rabbit models of ear vein thrombosis and carotid artery thrombosis and in dog model of acute cerebral infarction. Inhibition of thrombus formation was evaluated in rat inferior vena cava thrombosis model and ferric chloride-induced arterial thrombosis. In vitro, we assayed the antithrombotic effect of GBV-PA on rabbit blood clots, euglobulin lysis time (ELT) of rabbit plasma, and ADP-induced platelet aggregation. RESULTS: GBV-PA intravenous administration significantly reduced vascular recanalization times of rabbit ear veins thrombosis and thrombus weight of rabbit carotid artery thrombosis. The arterial recanalization rates were dose- and time-dependently improved after the administration of GBV-PA in canine acute cerebral infarction model. Thrombus length and weight were significantly reduced by GBV-PA both in rat inferior vena cava and ferric chloride-induced arterial thrombosis models. Thrombus formation in the blood of rabbits that were administered of GBV-PA was also inhibited. GBV-PA radically reduced plasma ELT of the rabbit's blood clots. ADP-induced platelet aggregation was inhibited by GBV-PA in a dose-dependent manner with a half-maximal inhibitory concentration of 19.9 µg/mL. CONCLUSION: This study demonstrates that GBV-PA is a thrombolytic and antiplatelet agent. It has significant antithrombotic effects on various in vitro and in vivo experimental models of thrombosis. The mechanisms that underline its antithrombotic effects were related to GBV-PA's capabilities of increasing fibrinolytic activity and inhibition of platelet aggregation.

Neutrophil extracellular traps in ischemia-reperfusion injury-induced myocardial no-reflow: therapeutic potential of DNase-based reperfusion strategy.

Emerging evidence suggests a potential role of neutrophil extracellular traps (NETs) in linking sterile inflammation and thrombosis. We hypothesized that NETs would be induced during myocardial ischemia-reperfusion (I/R), and NET-mediated microthrombosis may contribute to myocardial "no-reflow". Male Wistar rats were randomly divided into I/R control, DNase (DNase I, 20 µg/rat), recombinant tissue-type plasminogen activator (rt-PA, 420 µg/rat), DNase + rt-PA, and sham control groups after 45-min myocardial ischemia. In situ NET formation, the anatomic "no re-flow" area, and infarct size were evaluated immediately after 3 h of reperfusion. Long-term left ventricular (LV) functional and histological analyses were performed 45 days after operation. Compared with the I/R controls, the DNase + rt-PA group exhibited reduced NET density [8.38 ± 1.98 vs. 26.86 ± 3.07 (per 200 × field), P < 0.001] and "no-flow" area (15.22 ± 0.06 vs. 34.6 ± 0.05%, P < 0.05) in the ischemic region, as well as reduced infarct size (38.39 ± 0.05 vs. 71.00 ± 0.03%, P < 0.001). Additionally, compared with the I/R controls, DNase + rt-PA treatment significantly ameliorated I/R injury-induced LV remodeling (LV ejection fraction: 64.22 ± 3.37 vs. 33.81 ± 2.98%, P < 0.05; LV maximal slope of the LV systolic pressure increment: 3,785 ± 216 vs. 2,596 ± 299 mmHg/s, P < 0.05). The beneficial effect was not observed in rats treated with DNase I or rt-PA alone. Our study provides evidence for the existence of NETs in I/R-challenged myocardium and confirms the long-term benefit of a novel DNase-based reperfusion strategy (DNase I + rt-PA), which might be a promising option for the treatment of myocardial I/R injury and coronary no-reflow.