Dual antithrombotic therapy dose-dependently alters hemostatic plug structure and function.

BACKGROUND: Antithrombotic medications carry an inherent risk of bleeding, which may be exacerbated when anticoagulant and antiplatelet therapeutics are combined. Prior studies have shown different effects of antiplatelet vs anticoagulant drugs on the structure and function of hemostatic plugs in vivo. OBJECTIVES: We examined whether dual antithrombotic treatment consisting of combined antiplatelet and anticoagulant therapeutics alters hemostatic plug structure and function differently from treatment with either therapeutic alone. METHODS: Mice were treated with the P2Y12 antagonist clopidogrel and the factor Xa inhibitor rivaroxaban across a range of doses, either alone or in combination. The hemostatic response was assessed using a mouse jugular vein puncture injury model. Platelet accumulation and fibrin deposition were evaluated using quantitative multiphoton fluorescence microscopy, and bleeding times were recorded. RESULTS: Mice treated with clopidogrel alone exhibited a decrease in platelet accumulation at the site of injury, with prolonged bleeding times only at the highest doses of clopidogrel used. Mice treated with rivaroxaban alone instead showed a reduction in fibrin deposition with no impact on bleeding. Mice treated with both clopidogrel and rivaroxaban exhibited platelet and fibrin accumulation that was similar to that with either drug given alone; however, dual antithrombotic therapy resulted in impaired hemostasis at doses that had no impact on bleeding when given in isolation. CONCLUSION: Combined administration of antiplatelet and anticoagulant therapeutics exacerbates bleeding as compared to that with either drug alone, potentially via combined loss of both adenosine 5'-diphosphate- and thrombin-mediated platelet activation. These findings enhance our understanding of the bleeding risk associated with dual antithrombotic therapy.

Sparganii Rhizoma: A review of traditional clinical application, processing, phytochemistry, pharmacology, and toxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Sparganii Rhizoma (SR), a traditional Chinese medicine (TCM), is the rhizome of Sparganium stoloniferum Buch.-Ham. mainly distributed in East Asia. It has been used for eliminating blood stasis, promoting the flow of Qi, removing the retention of undigested food and relieving pain in China for hundreds of years. AIM OF THE REVIEW: This review summarizes comprehensive information in traditional clinical application, processing, phytochemistry, pharmacology, quality control and toxicity of SR, in exploring future scientific and therapeutic potentials. MATERIALS AND METHODS: Pertinent information was systematically collected from several electronic scientific databases (e.g., Web of Science, PubMed, China Knowledge Resource Integrated, Springer, Elsevier, ScienceDirect, and Google Scholar), PhD and MS dissertations, and classic Chinese medical books. RESULTS: SR is a gynecological drug which is often used to treat dysmenorrhea, mass in the abdomen, amenorrhea due to blood stasis, and abdominal distension in TCM. Two kinds of processed products of SR are included in Chinese Pharmacopoeia, which have better pharmacological effects than the crude herb. Approximately 180 compounds have been identified from SR, including phenylpropanoids, flavonoids, anthraquinones, organic acids, alkaloids, steroids, volatile oils, diarylheptanes, etc. The crude extracts and isolated components of SR have been reported to have anti-tumor, antithrombotic, estrogen antagonistic , anti-inflammatory, analgesic, antioxidant, anti organ fibrosis and other pharmacological activities. SR also has reproductive toxicity. CONCLUSIONS: As an important TCM, SR has been demonstrated by modern pharmacological researches to have significant bioactivities, especially on anti-tumor, antithrombotic, and estrogen antagonistic activities. These activities provide prospects for the development of new drugs and therapeutics for future applications. Nevertheless, quality control and evaluation, in-depth pharmacological mechanism, and toxicological effect of SR require further detailed research.

Improved Antithrombotic Activity and Diminished Bleeding Side Effect of a PEGylated αIIbß3 Antagonist, Disintegrin.

Polymer polyethylene glycol (PEG), or PEGylation of polypeptides improves protein drug stability by decrease degradation and reduces renal clearance. To produce a pharmaceutical disintegrin derivative, the N-terminal PEGylation technique was used to modify the disintegrin derivative [KGDRR]trimucrin for favorable safety and pharmacokinetic profiles and antithrombotic efficacy. We compared intact [KGDRR]trimucrin (RR) and PEGylated KGDRR (PEG-RR) by in vitro and in vivo systems for their antithrombotic activities. The activity of platelet aggregation inhibition and the bleeding tendency side effect were also investigated. PEG-RR exhibited optimal potency in inhibiting platelet aggregation of human/mouse platelet-rich plasma activated by collagen or ADP with a lower IC50 than the intact derivative RR. In the illumination-induced mesenteric venous thrombosis model, RR and PEG-RR efficaciously prevented occlusive thrombosis in a dose-dependent manner. In rotational thromboelastometry assay, there was no effect of PEG-RR in human whole blood coagulation even given at a higher concentration (30 µg/mL), while RR slightly prolonged clotting time. However, RR and PEG-RR were not associated with severe thrombocytopenia or bleeding in FcγRIIa-transgenic mice at equally efficacious antithrombotic dosages. We also found the in vivo half-life of PEGylation was longer than RR (RR: 15.65 h vs. PEG-RR: 20.45 h). In conclusion, injectable PEG-RR with prolonged half-life and decreased bleeding risk is a safer anti-thrombotic agent for long-acting treatment of thrombus diseases.

A Pharmacodynamic Study of CN-218, a Novel Antiplatelet and Antithrombotic Agent Primarily Targeting the P2Y12 Receptor.

PURPOSE: Drugs inhibiting the platelet P2Y12 receptor, such as clopidogrel and prasugrel, are potent antithrombotic agents and are widely used in cardiovascular disease. However, the adverse effects of these drugs have limited their clinical use. For example, clopidogrel resistance occurs in approximately one third of patients, while prasugrel increases the risk of major bleeding. Therefore, new generations of such drugs are of clinical interest. METHODS: In this study, the pharmacodynamics of a new P2Y12 antagonist, CN-218, was compared with that of clopidogrel and prasugrel in rats and mice. The differences between CN-218 and clopidogrel include deuteration of the 7-position methyl carboxylate and the introduction of cinnamate in the 2-position of thiophene. RESULTS: CN-218 had an antiaggregatory efficacy that was at least five times more potent than that of clopidogrel but not as potent as that of prasugrel. It had a significant impact on activated partial thromboplastin time (APTT), whereby the APTT of CN-218-treated rats was approximately 9 s longer than that of the vehicle- or clopidogrel-treated group, while it had no impact on prothrombin time (PT) in rats. CN-218 had a similar potent antithrombotic effect to that of prasugrel and clopidogrel and also reduced the risk of bleeding compared to prasugrel. CONCLUSION: CN-218 may be a promising antithrombotic agent, with potent antiplatelet and significant anticoagulant activity, as well as a lower risk of bleeding compared to clopidogrel and prasugrel.

Effect of acute exposure in swiss mice (Mus musculus) to a fibrinolytic protease produced by Mucor subtilissimus UCP 1262: An histomorphometric, genotoxic and cytological approach.

The fibrinolytic enzyme produced by Mucor subtilissimus UCP 1262 was obtained by solid fermentation and purified by ion exchange chromatography using DEAE-Sephadex A50. The enzyme toxicity was evaluated using mammalian cell lineages: HEK-293, J774.A1, Sarcoma-180 and PBMCs which appeared to be viable at a level of 80%. The biochemical parameters of the mice treated with an acute dose of enzyme (2000 mg/mL) identified alterations of AST and ALT and the histomorphometric analysis of the liver showed a loss of endothelial cells (P < 0.001). However, these changes are considered minimal to affirm that there was a significant degree of hepatotoxicity. The comet assay and the micronucleus test did not identify damage in the DNA of the erythrocytes of the animals treated. The protease did not degrade the Aα and Bß chains of human and bovine fibrinogens, thus indicating that it does not act as anticoagulant, but rather as a fibrinolytic agent. The assay performed to assess blood biocompatibility shows that at dose of 0.3-5 mg/mL the hemolytic grade is considered insignificant. Moreover, the enzyme did not prolong bleeding time in mice when dosed with 1 mg/kg. These results indicate that this enzyme produced is a potential competitor for developing novel antithrombotic drugs.

An αIIb ß3 antagonist prevents thrombosis without causing Fc receptor γ-chain IIa-mediated thrombocytopenia.

Essentials FcγRIIa-mediated thrombocytopenia is associated with drug-dependent antibodies (DDAbs). We investigated the correlation between αIIb ß3 binding epitopes and induction of DDAbs. An FcγRIIa-transgenic mouse model was used to evaluate thrombocytopenia among anti-thrombotics. An antithrombotic with binding motif toward αIIb ß-propeller domain has less bleeding tendency. SUMMARY: Background Thrombocytopenia, a common side effect of Arg-Gly-Asp-mimetic antiplatelet drugs, is associated with drug-dependent antibodies (DDAbs) that recognize conformation-altered integrin αIIb ß3 . Objective To explore the correlation between αIIb ß3 binding epitopes and induction of DDAb binding to conformation-altered αIIb ß3 , we examined whether two purified disintegrins, TMV-2 and TMV-7, with distinct binding motifs have different effects on induction of αIIb ß3 conformational change and platelet aggregation in the presence of AP2, an IgG1 inhibitory mAb raised against αIIb ß3 . Methods We investigated the possible mechanisms of intrinsic platelet activation of TMV-2 and TMV-7 in the presence of AP2 by examining the signal cascade, tail bleeding time and immune thrombocytopenia in Fc receptor γ-chain IIa (FcγRIIa) transgenic mice. Results TMV-7 has a binding motif that recognizes the αIIb ß-propeller domain of αIIb ß3 , unlike that of TMV-2. TMV-7 neither primed the platelets to bind ligand, nor caused a conformational change of αIIb ß3 as identified with the ligand-induced binding site mAb AP5. In contrast to eptifibatide and TMV-2, cotreatment of TMV-7 with AP2 did not induce FcγRIIa-mediated platelet aggregation and the downstream activation cascade. Both TMV-2 and TMV-7 efficaciously prevented occlusive thrombosis in vivo. Notably, both eptifibatide and TMV-2 caused severe thrombocytopenia mediated by FcγRIIa, prolonged tail bleeding time in vivo, and repressed human whole blood coagulation indexes, whereas TMV-7 did not impair hemostatic capacity. Conclusions TMV-7 shows antiplatelet and antithrombotic activities resulting from a mechanism different from that of all other tested αIIb ß3 antagonists, and may offer advantages as a therapeutic agent with a better safety profile.

Inhibition of tPA-induced hemorrhagic transformation involves adenosine A2b receptor activation after cerebral ischemia.

Tissue plasminogen activator (tPA) is administered after ischemic stroke to dissolve intravascular clots, but its use can lead to hemorrhagic transformation (HT). Therapeutic strategies to reduce hemorrhagic complications of tPA might be of benefit for stroke patients. Adenosine A2b receptor (A2bR) plays pivotal roles in regulating vascular protection in peripheral organs. This study explored whether A2bR agonist BAY 60-6583 reduces hemorrhage risk after tPA usage. Using a rat transient middle cerebral artery occlusion model, we showed that mRNA and protein expression of A2bR increased to a greater extent after ischemia-reperfusion than did expression of the other three adenosine receptors (A1, A2a, and A3). tPA administration reduced A2bR expression in ischemic brain microvessels. Post-treatment with BAY 60-6583 (1mg/kg) at the start of reperfusion reduced lesion volume in the absence or presence of tPA (10mg/kg) and attenuated brain swelling, blood-brain barrier disruption, and tPA-exacerbated HT at 24h. Additionally, BAY 60-6583 mitigated sensorimotor deficits in the presence of tPA. BAY 60-6583 inhibited tPA-enhanced matrix metalloprotease-9 activation, probably through elevation of tissue inhibitor of matrix metalloproteinases-1 expression, and thereby reduced degradation of tight junction proteins. These effects would likely protect cerebrovascular integrity. A2bR agonists as an adjuvant to tPA could be a promising strategy for decreasing the risk of HT during treatment for ischemic stroke.

ADPase CD39 Fused to Glycoprotein VI-Fc Boosts Local Antithrombotic Effects at Vascular Lesions.

BACKGROUND: GPVI (Glycoprotein VI) is the essential platelet collagen receptor in atherothrombosis. Dimeric GPVI-Fc (Revacept) binds to GPVI binding sites on plaque collagen. As expected, it did not increase bleeding in clinical studies. GPVI-Fc is a potent inhibitor of atherosclerotic plaque-induced platelet aggregation at high shear flow, but its inhibition at low shear flow is limited. We sought to increase the platelet inhibitory potential by fusing GPVI-Fc to the ectonucleotidase CD39 (fusion protein GPVI-CD39), which inhibits local ADP accumulation at vascular plaques, and thus to create a lesion-directed dual antiplatelet therapy that is expected to lack systemic bleeding risks. METHODS AND RESULTS: GPVI-CD39 effectively stimulated local ADP degradation and, compared with GPVI-Fc alone, led to significantly increased inhibition of ADP-, collagen-, and human plaque-induced platelet aggregation in Multiplate aggregometry and plaque-induced platelet thrombus formation under arterial flow conditions. GPVI-CD39 did not increase bleeding time in an in vitro assay simulating primary hemostasis. In a mouse model of ferric chloride-induced arterial thrombosis, GPVI-CD39 effectively delayed vascular thrombosis but did not increase tail bleeding time in vivo. CONCLUSIONS: GPVI-CD39 is a novel approach to increase local antithrombotic activity at sites of atherosclerotic plaque rupture or injury. It enhances GPVI-Fc-mediated platelet inhibition and presents a potentially effective and safe molecule for the treatment of acute atherothrombotic events, with a favorable risk-benefit ratio.

Trowaglerix Venom Polypeptides As a Novel Antithrombotic Agent by Targeting Immunoglobulin-Like Domains of Glycoprotein VI in Platelet.

OBJECTIVE: Currently prescribed antiplatelet drugs have 1 common side effect-an increased risk of hemorrhage and thrombocytopenia. On the contrary, bleeding defects associated with glycoprotein VI (GPVI) expression deficiency are usually slightly prolonged bleeding times. However, GPVI antagonists are lacking in clinic. APPROACH AND RESULTS: Using reverse-phase high-performance liquid chromatography and sequencing, we revealed the partial sequence of trowaglerix α subunit, a potent specific GPVI-targeting snaclec (snake venom C-type lectin protein). Hexapeptide (Troα6 [trowaglerix a chain hexapeptide, CKWMNV]) and decapeptide (Troα10) derived from trowaglerix specifically inhibited collagen-induced platelet aggregation through blocking platelet GPVI receptor. Computational peptide design helped to design a series of Troα6/Troα10 peptides. Protein docking studies on these decapeptides and GPVI suggest that Troα10 was bound at the lower surface of D1 domain and outer surface of D2 domain, which was at the different place of the collagen-binding site and the scFv (single-chain variable fragment) D2-binding site. The newly discovered site was confirmed by inhibitory effects of polyclonal antibodies on collagen-induced platelet aggregation. This indicates that D2 domain of GPVI is a novel and important binding epitope on GPVI-mediated platelet aggregation. Troα6/Troα10 displayed prominent inhibitory effect of thrombus formation in fluorescein sodium-induced platelet thrombus formation of mesenteric venules and ferric chloride-induced carotid artery injury thrombosis model without prolonging the in vivo bleeding time. CONCLUSIONS: We develop a novel antithrombotic peptides derived from trowaglerix that acts through GPVI antagonism with greater safety-no severe bleeding. The binding epitope of polypeptides on GPVI is novel and important. These hexa/decapeptides have therapeutic potential for developing ideal small-mass GPVI antagonists for arterial thrombogenic diseases.

Shear-sensitive nanocapsule drug release for site-specific inhibition of occlusive thrombus formation.

Essentials Vessel stenosis due to large thrombus formation increases local shear 1-2 orders of magnitude. High shear at stenotic sites was exploited to trigger eptifibatide release from nanocapsules. Local delivery of eptifibatide prevented vessel occlusion without increased tail bleeding times. Local nanocapsule delivery of eptifibatide may be safer than systemic antiplatelet therapies. SUMMARY: Background Myocardial infarction and stroke remain the leading causes of mortality and morbidity. The major limitation of current antiplatelet therapy is that the effective concentrations are limited because of bleeding complications. Targeted delivery of antiplatelet drug to sites of thrombosis would overcome these limitations. Objectives Here, we have exploited a key biomechanical feature specific to thrombosis, i.e. significantly increased blood shear stress resulting from a reduction in the lumen of the vessel, to achieve site-directed delivery of the clinically used antiplatelet agent eptifibatide by using shear-sensitive phosphatidylcholine (PC)-based nanocapsules. Methods PC-based nanocapsules (2.8 × 1012 ) with high-dose encapsulated eptifibatide were introduced into microfluidic blood perfusion assays and into in vivo models of thrombosis and tail bleeding. Results Shear-triggered nanocapsule delivery of eptifibatide inhibited in vitro thrombus formation selectively under stenotic and high shear flow conditions above a shear rate of 1000 s-1 while leaving thrombus formation under physiologic shear rates unaffected. Thrombosis was effectively prevented in in vivo models of vessel wall damage. Importantly, mice infused with shear-sensitive antiplatelet nanocapsules did not show prolonged bleeding times. Conclusions Targeted delivery of eptifibatide by shear-sensitive nanocapsules offers site-specific antiplatelet potential, and may form a basis for developing more potent and safer antiplatelet drugs.

Tissue-type plasminogen activator-binding RNA aptamers inhibiting low-density lipoprotein receptor family-mediated internalisation.

Recombinant tissue-type plasminogen activator (tPA, trade name Alteplase), currently the only drug approved by the US Food and Drug Administration and the European Medicines Agency for the treatment of cerebral ischaemic stroke, has been implicated in a number of adverse effects reportedly mediated by interactions with the low-density lipoprotein (LDL) family receptors, including neuronal cell death and an increased risk of cerebral haemorrhage. The tissue-type plasminogen activator is the principal initiator of thrombolysis in human physiology, an effect that is mediated directly via localised activation of the plasmin zymogen plasminogen at the surface of fibrin clots in the vascular lumen. Here, we sought to identify a ligand to tPA capable of inhibiting the relevant LDL family receptors without interfering with the fibrinolytic activity of tPA. Systematic evolution of ligands by exponential enrichment (SELEX) was employed to isolate tPA-binding RNA aptamers, which were characterised in biochemical assays of tPA association to low density lipoprotein receptor-related protein-1 (LRP-1, an LDL receptor family member); tPA-mediated in vitro and ex vivo clot lysis; and tPA-mediated plasminogen activation in the absence and presence of a stimulating soluble fibrin fragment. Two aptamers, K18 and K32, had minimal effects on clot lysis, but were able to efficiently inhibit tPA-LRP-1 association and LDL receptor family-mediated endocytosis in human vascular endothelial cells and astrocytes. These observations suggest that coadministration alongside tPA may be a viable strategy to improve the safety of thrombolytic treatment of cerebral ischaemic stroke by restricting tPA activity to the vascular lumen.

TGF-ß1 prevents blood-brain barrier damage and hemorrhagic transformation after thrombolysis in rats.

Transforming growth factor-beta1 (TGF-ß1) is well known to promote extracellular matrix accumulation. Recent studies demonstrated that TGF-ß1 protects against blood-brain barrier (BBB) disruption in the condition of inflammatory pain and stroke. In the present study, we investigated whether TGF-ß1 can maintain BBB integrity and prevent hemorrhagic transformation (HT) after recombinant tissue plasminogen activator (rt-PA) treatment in a rat model of thromboembolic middle cerebral artery occlusion (MCAO). Three hours after MCAO, rats were given saline, rt-PA alone or rt-PA combined with TGF-ß1 intravenously. Animals were sacrificed 24h after surgery. HT was calculated as hemorrhagic score. Evans blue dye extravasation was measured for BBB disruption. Basement membrane damage was observed by electron microscopy and quantified by collagen IV and laminin immunostaining. Gelatin zymography was used to measure the activities of matrix metalloproteinase (MMP)-2 and MMP-9. Western blot was performed for the expressions of MMP-2, MMP-9 and plasminogen activator inhibitor type-1 (PAI-1). Rats treated with rt-PA showed elevations in basement membrane damage, BBB disruption and HT. These phenomena were reduced in rats treated by TGF-ß1. We also showed that TGF-ß1 inhibited rt-PA mediated induction of MMP-2 and MMP-9. Meanwhile, TGF-ß1 upregulated PAI-1 expression which was reduced by rt-PA. Taken together, these results suggest that TGF-ß1 can reduce rt-PA induced basement membrane degradation, BBB disruption and HT. One possible mechanism is associated with the elevation of PAI-1. Suppression of MMP-2 and MMP-9 elevated by rt-PA may be another mechanism contributing to the protective effects of TGF-ß1.

Characterization of a novel function-blocking antibody targeted against the platelet P2Y1 receptor.

OBJECTIVE: Platelet hyperactivity is associated with vascular disease and contributes to the genesis of thrombotic disorders. ADP plays an important role in platelet activation and activates platelets through 2 G-protein-coupled receptors, the Gq-coupled P2Y1 receptor (P2Y1R), and the Gi-coupled P2Y12 receptor. Although the involvement of the P2Y1R in thrombogenesis is well established, there are no antagonists that are currently available for clinical use. APPROACH AND RESULTS: Our goal is to determine whether a novel antibody targeting the ligand-binding domain, ie, second extracellular loop (EL2) of the P2Y1R (EL2Ab) could inhibit platelet function and protect against thrombogenesis. Our results revealed that the EL2Ab does indeed inhibit ADP-induced platelet aggregation, in a dose-dependent manner. Furthermore, EL2Ab was found to inhibit integrin GPIIb-IIIa activation, dense and α granule secretion, and phosphatidylserine exposure. These inhibitory effects translated into protection against thrombus formation, as evident by a prolonged time for occlusion in a FeCl3-induced thrombosis model, but this was accompanied by a prolonged tail bleeding time. We also observed a dose-dependent displacement of the radiolabeled P2Y1R antagonist [(3)H]MRS2500 from its ligand-binding site by EL2Ab. CONCLUSIONS: Collectively, our findings demonstrate that EL2Ab binds to and exhibits P2Y1R-dependent function-blocking activity in the context of platelets. These results add further evidence for a role of the P2Y1R in thrombosis and validate the concept that targeting it is a relevant alternative or complement to current antiplatelet strategies.

SAR216471, an alternative to the use of currently available P2Y12 receptor inhibitors?

P2Y12 antagonism is a key therapeutic strategy in the management and prevention of arterial thrombosis. The objective of this study was to characterize the pharmacodynamic (PD) and pharmacokinetic (PK) properties of SAR216471, a novel P2Y12 receptor antagonist. SAR216471 blocks the binding of 2MeSADP to P2Y12 receptors in vitro (IC50=17 nM). This inhibition was shown to be reversible. It potently antagonized ADP-induced platelet aggregation in human and rat platelet-rich plasma (IC50=108 and 62 nM, respectively). It also inhibited platelet aggregation when blood was exposed to collagen or thromboxane A2. Its high selectivity was demonstrated against a large panel of receptors, enzymes, and ion channels. Despite its moderate bioavailability in rats, oral administration of SAR216471 resulted in a fast, potent, and sustained inhibition of platelet aggregation where the extent and duration of platelet inhibition were directly proportional to its circulating plasma levels. Pre-clinical study of SAR216471 in a rat shunt thrombosis model demonstrated a dose-dependent antithrombotic activity after oral administration (ED50=6.7 mg/kg). By comparison, ED50 values for clopidogrel, prasugrel and ticagrelor were 6.3, 0.35 and 2.6 mg/kg, respectively. Finally, the anti-hemostatic effect of SAR216471 and its competitors was investigated in a rat tail bleeding model, revealing a favorable safety profile of SAR216471. Together, these findings have established a reliable antiplatelet profile of SAR216471, and support its potential use in clinical practice as an alternative to currently available P2Y12 receptor antagonists.

Simple benchmark for complex dose finding studies.

While a general goal of early phase clinical studies is to identify an acceptable dose for further investigation, modern dose finding studies and designs are highly specific to individual clinical settings. In addition, as outcome-adaptive dose finding methods often involve complex algorithms, it is crucial to have diagnostic tools to evaluate the plausibility of a method's simulated performance and the adequacy of the algorithm. In this article, we propose a simple technique that provides an upper limit, or a benchmark, of accuracy for dose finding methods for a given design objective. The proposed benchmark is nonparametric optimal in the sense of O'Quigley et al. (2002, Biostatistics 3, 51-56), and is demonstrated by examples to be a practical accuracy upper bound for model-based dose finding methods. We illustrate the implementation of the technique in the context of phase I trials that consider multiple toxicities and phase I/II trials where dosing decisions are based on both toxicity and efficacy, and apply the benchmark to several clinical examples considered in the literature. By comparing the operating characteristics of a dose finding method to that of the benchmark, we can form quick initial assessments of whether the method is adequately calibrated and evaluate its sensitivity to the dose-outcome relationships.

Intracerebral transplantation of bone marrow stromal cells ameliorates tissue plasminogen activator-induced brain damage after cerebral ischemia in mice detected by in vivo and ex vivo optical imaging.

Detection and protection of the neurovascular unit (NVU) are essential for treatment of acute stroke patients, especially the use of tissue plasminogen activator (tPA). In the present study, we conducted in vivo and ex vivo optical imaging for detecting activation of matrix metalloproteinases (MMPs) and evaluated the protective effect of intracerebral transplantation of bone marrow stromal cells (BMSCs) obtained from green fluorescent protein (GFP) transgenic (Tg) mice on the NVU in tPA-mediated brain damage after transient middle cerebral artery occlusion (tMCAO) in mice. Compared with the tMCAO group, the tMCAO plus BMSC group showed significant reductions of in vivo and ex vivo fluorescent signals for MMPs at 48 hr after tMCAO, with a partial colocalization of BMSC-GFP signals. Intracerebrally transplanted BMSCs ameliorated MMP-9 activation by immunohistochemistry and Western blot with differentiation into microglial and astroglial cells. Double-immunofluorescence study revealed improved NVU disruption in the tMCAO plus BMSC group. The present study suggests that intracerebral BMSC transplantation reduced MMP activation and subsequent NVU disruption caused by tPA after tMCAO and that this MMP activation and BMSC effect were detectable with in vivo and ex vivo optical imaging.

Ent-16ß,17-dihydroxy-kauran-19-oic acid, a kaurane diterpene acid from Siegesbeckia pubescens, presents antiplatelet and antithrombotic effects in rats.

The antiplatelet and antithrombotic effects of ent-16ß,17-dihydroxy-kauran-19-oic acid (DDKA) isolated from Siegesbeckia pubescens were investigated with different methods both in vitro and in vivo. We tested the antithrombotic activity of DDKA in arterio-venous shunt model. The effects of DDKA on adenosine diphosphate (ADP)-, Thrombin-, Arachidonic acid-induced rat platelets aggregation were tested in vitro. We also assessed its bleeding side effect by measuring coagulation parameters after intravenous administration for 5 days and investigated the potential mechanisms underlying such activities. In vivo, DDKA significantly reduced thrombus weight in the model of arterio-venous shunt. Meanwhile, DDKA increased plasma cAMP level determined by radioimmunoassay in the same model. Notably, DDKA prolonged PT and APTT in rats after intravenous administration DDKA for successive 5 days. In vitro, pretreatment with DDKA on washed rat platelets significantly inhibited various agonists stimulated platelet aggregation and caused an increase in cAMP level in platelets activated by ADP. These findings support our hypothesis that DDKA possesses antiplatelet and antithrombotic activities. The mechanisms underlying such activities may involve the anticoagulatory effect and cAMP induction.

Effectiveness of dabigatran etexilate for thromboprophylaxis of mechanical heart valves.

OBJECTIVE: Warfarin reduces risk of stroke in patients with mechanical heart valves but increases risk of hemorrhage and is difficult to use. Dabigatran etexilate, a new oral direct thrombin inhibitor, is safe and effective in reducing risk of stroke among patients with atrial fibrillation. No data exist in the setting of mechanical heart valves. We tested the hypothesis that dabigatran etexilate is as effective as heparin for thromboprophylaxis of mechanical valves in a porcine heterotopic aortic valve model. METHODS: Thirty swine underwent implantation of modified bileaflet mechanical valved conduit bypassing the ligated, native descending thoracic aorta. Animals randomly received no anticoagulation (n = 10), enoxaparin 2 mg/kg subcutaneously twice daily (n = 10), or dabigatran etexilate 20 mg/kg orally twice daily. Primary end point was amount of valve thrombus at 30 days. Secondary end points included quantitative measurement of platelet deposition on valve prosthesis, thromboelastography, and hemorrhagic and embolic events. RESULTS: At 30 days, we observed 638 ± 895 mg thrombus in no anticoagulation group, 121 ± 128 mg in enoxaparin group, and 19 ± 31 mg in dabigatran etexilate group (P = .01 enoxaparin vs dabigatran etexilate). Fewer platelets were deposited on valves in dabigatran etexilate group (2.7 × 10(8)) than in enoxaparin group (1.8 × 10(9), P = .03). No major or occult hemorrhagic or embolic events were observed. By thromboelastographic analysis, dabigatran etexilate produced less prolongation of K value (P = .01) and less decreases in angle (P = .01) and maximum amplitude (P = .001) than enoxaparin. CONCLUSIONS: Dabigatran etexilate is as effective as enoxaparin for short-term thromboprophylaxis of mechanical valves. It prevents valve thrombus and platelet deposition at 30 days without increased adverse events. These promising results serve as a foundation for prospective clinical trials with dabigatran etexilate as an alternative to warfarin in patients with bileaflet mechanical aortic valves.

A disposable system for rapid purification of autologous plasmin as an adjunct to vitrectomy - performance and safety profile.

BACKGROUND: The generation of an atraumatic posterior vitreous detachment (PVD), a common goal in vitreoretinal surgery, is a challenge particularly in children and young trauma patients. Plasmin has been proposed as a surgical adjunct to enzymatically generate a PVD. This study assesses the performance and safety of a new system for rapid purification of plasmin as an adjunct to vitrectomy. METHODS: Plasminogen was isolated from human plasma by affinity chromatography using a disposable rapid purification kit, and activated to plasmin with streptokinase. Activities were assessed spectrophotometrically. For safety studies, 38 rabbits received intravitreal injections of one of the following compounds in 0.1 ml respectively: 4.7, 12.7 and 24 IU plasmin, 15 mg dextran, 4,100 U streptokinase, 500 µg ε-aminocaproic acid, 0.1 M potassium phosphate or balanced salt solution (BSS). Thirty min after injection, a two-port vitrectomy was performed. Rabbits were followed clinically and with bright flash electroretinography (ERG) for up to 9 months. The eyes were investigated by light and transmission electron microscopy. RESULTS: The specific plasmin activity obtained from blood of healthy volunteers averaged 42.3 ± 6.6 IU/ml (range 21.6 IU/ml to 54.5 IU/ml). The identity and purity of the enzyme was confirmed by several methods. Clinically, a mild to moderate inflammatory response was seen in most eyes on day 1, but had disappeared by day 7. ERG showed moderate depressions of a- and b-wave amplitudes on day 2, particularly in the potassium phosphate (a: -29.16 ± 4.56, b: -21.23 ± 6.31), 4.7 (a: -34.38 ± 6.64, b: -26.66 ± 6.06) and 24 IU (a: -38.25 ± 4.05, b: -23.38 ± 4.29) plasmin groups, but also in the BSS- (a: -11.19 ± 21.78, b: -11.41 ± 15.47) and dextran- (a: -17.86 ± 14.18, b: -6.67 ± 18.14) treated eyes. ERG changes recovered during follow-up. One rabbit each from the 12.7 and the 24 IU plasmin groups showed a minimal discoloration of one medullary ray after 9 months. Histology did not reveal morphologic signs of toxicity. CONCLUSION: The isolation system generated plasmin with a high degree of purity. A failure-mode analysis did not reveal significant risks of toxicity. A single preparation can provide a maximum dose of 10.9 IU/200 µl, the likely target clinical dose being 1.88 IU. Plasmin doses of at least 12.7 IU appear be safe when injected into rabbit eyes, followed by vitrectomy.

A novel finding of a low-molecular-weight compound, SMTP-7, having thrombolytic and anti-inflammatory effects in cerebral infarction of mice.

Tissue plasminogen activator (t-PA) has a short therapeutic time window for administration (3 h) and carries a risk of promoting intracerebral hemorrhage. The aim of the present study was to investigate a therapeutic time window and frequency of hemorrhagic region by treatment with Stachybotrys microspora triprenyl phenol-7 (SMTP-7). Thrombotic occlusion was induced by transfer of acetic acid-induced thrombus at the right common carotid artery into the brain of mice. Infarction area, neurological score, edema percentage, and regional cerebral blood flow (CBF) were determined as the index of the efficacy of SMTP-7. In order to evaluate the mechanism of SMTP-7, plasmin activities and the expressions of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), and IL-6 mRNA were examined. SMTP-7 (0.1, 1, 10 mg/kg) dose dependently reduced infarction area, neurological score, and edema percentage. Additionally, its therapeutic time window was longer than that of t-PA, a high-molecular-weight compound. In addition, little hemorrhagic region was induced by treatment with SMTP-7. SMTP-7 showed plasmin activity in vivo and caused a decreased CBF to recover. Furthermore, the expressions of inflammatory cytokine mRNA (IL-1beta, TNF-alpha, IL-6) were increased by t-PA treatment 3 h after ischemia but were not induced by SMTP-7 treatment. These results indicate that SMTP-7 shows potential thrombolytic and anti-inflammatory effects as well as a wide therapeutic time window and little hemorrhagic region compared with that of t-PA. Therefore, this novel low-molecular-weight compound may represent a novel approach for the treatment of cerebral infarction.