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.

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.

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.

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.

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.

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.

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.

Inhibition of plasminogen activation by apo(a): role of carboxyl-terminal lysines and identification of inhibitory domains in apo(a).

Apo(a), the distinguishing protein component of lipoprotein(a) [Lp(a)], exhibits sequence similarity to plasminogen and can inhibit binding of plasminogen to cell surfaces. Plasmin generated on the surface of vascular cells plays a role in cell migration and proliferation, two of the fibroproliferative inflammatory events that underlie atherosclerosis. The ability of apo(a) to inhibit pericellular plasminogen activation on vascular cells was therefore evaluated. Two isoforms of apo(a), 12K and 17K, were found to significantly decrease tissue-type plasminogen activator-mediated plasminogen activation on human umbilical vein endothelial cells (HUVECs) and THP-1 monocytes and macrophages. Lp(a) purified from human plasma decreased plasminogen activation on THP-1 monocytes and HUVECs but not on THP-1 macrophages. Removal of kringle V or the strong lysine binding site in kringle IV10 completely abolished the inhibitory effect of apo(a). Treatment with carboxypeptidase B to assess the roles of carboxyl-terminal lysines in cellular receptors leads in most cases to decreases in plasminogen activation as well as plasminogen and apo(a) binding; however, inhibition of plasminogen activation by apo(a) was unaffected. Our findings directly demonstrate that apo(a) inhibits pericellular plasminogen activation in all three cell types, although binding of apo(a) to cell-surface receptors containing carboxyl-terminal lysines does not appear to play a major role in the inhibition mechanism.

Staphylokinase promotes the establishment of Staphylococcus aureus skin infections while decreasing disease severity.

Skin infections are frequently caused by Staphylococcus aureus and can lead to a fatal sepsis. The microbial mechanisms controlling the initiation and progression from mild skin infection to a severe disseminated infection remain poorly understood. Using a combination of clinical data and in vitro and ex vivo assays, we show that staphylokinase, secreted by S. aureus, promoted the establishment of skin infections in humans and increased bacterial penetration through skin barriers by activating plasminogen. However, when infection was established, the interaction between staphylokinase and plasminogen did not promote systemic dissemination but induced the opening and draining of abscesses and decreased disease severity in neutropenic mice. Also, increased staphylokinase production was associated with noninvasive S. aureus infections in patients. Our results point out the dual roles of staphylokinase in S. aureus skin infections as promoting the establishment of infections while decreasing disease severity.

Toward the neurovascular unit. A journey in clinical translation: 2012 Thomas Willis Lecture.

The Nobel laureate Max Delbrück often said that it is the crossover between disciplines where advances are possible in science. This certainly has been true for our understanding of the vascular biology of the central nervous system in the setting of ischemic stroke. The ability to cross the boundaries of hemostasis, neurology, hematology, and neuroscience has facilitated our research direction to define the relation of the microvasculature to neuron function. Work begun with the clinical scientific exploration of the contributions of arterial thrombosis to the acute injury processes initiated by focal cerebral ischemia has led to an increased understanding of the effects of ischemia on microvessel integrity.

Flow-dependent channel formation in clots by an erythrocyte-bound fibrinolytic agent.

Studies in animal models have shown that plasminogen activators bound to erythrocytes (RBC-PA) have an extended lifetime in the circulation and are safer than free PAs. RBC-PAs incorporate into nascent thrombi, which are focally lysed from within, an attractive thromboprophylactic option. In static systems, RBC-PAs cleave surrounding fibrin fibers, forming pores larger than the cells themselves, and move around the pore edges, enlarging them until eventual clot dissolution. We hypothesized that under flow in blood vessels, RBC-PAs form functional patent channels before clot dissolution. Here we used perfusion chambers to study clot lysis by RBC-PAs under static versus arterial and venous flow conditions. We found that flow decelerates bulk clot lysis but quickly generates patent channels filled with passing RBCs, via pore enlargement and merging in the direction of flow. Formation of such channels by RBC-PAs may help rescue ischemic tissue before bulk dissolution of potentially occlusive clots.

Expression, purification and characterization of recombinant plasminogen activator from Gloydius brevicaudus venom in Escherichia coli.

The plasminogen activator (PA) in snake venom, a serine protease, can convert plasminogen to active plasmin, indirectly causing the degradation of fibrin. It is difficult to purify sufficient snake venom PA (SV-PA) for clinical applications due to the low SV-PA content in venom. The gene encoding PA was obtained from the venom gland of Gloydius brevicaudus using RT-PCR with primers designed according to the N-terminal amino acids of G. brevicaudus venom PA (GBV-PA), was cloned into the prokaryotic expression vector pET-42a, and recombinant GBV-PA (rGBV-PA) was expressed via Isopropyl-ß-d-1-Thiogalactopyranoside (IPTG) induction. Like human tissue PA, the purified renatured rGBV-PA could significantly reduce the rabbit plasma euglobulin lysis time (ELT) and prevent thrombus formation in the inferior vena cava of rats. Within the dosage range, the dosage and effects were positively correlated.

Serglycin is a major proteoglycan in polarized human endothelial cells and is implicated in the secretion of the chemokine GROalpha/CXCL1.

Proteoglycan (PG) expression was studied in primary human umbilical vein endothelial cells (HUVEC). RT-PCR analyses showed that the expression of the PG serglycin core protein was much higher than that of the extracellular matrix PG decorin and the cell surface PG syndecan-1. PG biosynthesis was further studied by biosynthetic [(35)S]sulfate labeling of polarized HUVEC. Interestingly, a major part of (35)S-PGs was secreted to the apical medium. A large portion of these PGs was trypsin-resistant, a typical feature of serglycin. The trypsin-resistant PGs were mainly of the chondroitin/dermatan sulfate type but also contained a minor heparan sulfate component. Secreted serglycin was identified by immunoprecipitation as a PG with a core protein of ∼30 kDa. Serglycin was furthermore shown to be present in perinuclear regions and in two distinct types of vesicles throughout the cytoplasm using immunocytochemistry. To search for possible serglycin partner molecules, HUVEC were stained for the chemokine growth-related oncogene α (GROα/CXCL1). Co-localization with serglycin could be demonstrated, although not in all vesicles. Serglycin did not show overt co-localization with tissue-type plasminogen activator-positive vesicles. When PG biosynthesis was abrogated using benzyl-ß-D-xyloside, serglycin secretion was decreased, and the number of vesicles with co-localized serglycin and GROα was reduced. The level of GROα in the apical medium was also reduced after xyloside treatment. Together, these findings indicate that serglycin is a major PG in human endothelial cells, mainly secreted to the apical medium and implicated in chemokine secretion.

Plasminogen activation by lactic acid bacteria.

Plasminogen was incubated with lactic acid bacteria and the plasmin activity in the mixture was measured. Three of 15 strains tested revealed significant plasminogen activation ability. Lactococcus lactis subsp. lactis biovar diacetylactis NIAI C59 showed the highest activity. The strain activated not only human plasminogen but also bovine plasminogen. The activity demonstrated a high level of thermal stability within a range of pH 3.0-9.0. The plasminogen activator activity in strain C59 increased after 15 h of cultivation, and reached a plateau after 21 h. A remarkable amount of activity was transferred to the solution when C59 cells were incubated in buffer solutions at pH 9.0 and above.

Effect of thyroxine on fibrinolytic system in rat.

Thyroid hormones have many effects on the cardiovascular system. Thyroid dysfunction accelerates atherosclerosis not only through conventional risk factors (dyslipidemia) but they also show a very close relationship with hemodynamic parameters. Thyroxine is determinant of the several components of fibrinolytic system even though the exact relationship is far from clear. Present study was carried out to determine the effect of thyroxine on fibrinolytic parameters such as plasminogen activators (PA) in rat heart, levels of PA and plasminogen activator inhibitor (PAI), glucose in plasma and serum lipid profile. Rats were injected with 50 ug eltroxine/100 gm(-1) body weight intraperitoneally for one week. Compared with controls, thyroxine treatment increased PA activity significantly in rat heart. No changes were seen in PA, PAI and glucose in plasma of two groups of rats. A significant decrease in total cholesterol and LDL-cholesterol levels was seen in serum of treated group resulting in the decrease of LDL/HDL and Total cholesterol/HDL-cholesterol ratios. These results suggest that thyroxine treatment may have considerable clinical significance. It raised PA activity in heart as well as reduced cholesterol content in blood. It is possible that thyroxine treatment may confer a beneficial effect on cardiovascular risk.

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.