Neonatal exposure to sevoflurane impairs preference for social novelty in C57BL/6 female mice at early-adulthood.

Social interaction deficit is core symptom of children with autism, owing to interaction of genetic predisposition and environmental toxins. Sevoflurane could induce neurotoxicity in developing brain in rodent models. This study aims to investigate whether sevoflurane anesthesia in neonatal period could impair social behaviors in male and female mice. Twenty-eight male and thirty-one female mice were randomly assigned to receive 3.0% sevoflurane or 60% oxygen on postnatal day 6. They were tested for social interaction behaviors at one- and two-month-old. In addition, the cortex and hippocampus of neonatal mice undergoing sevoflurane anesthesia were harvested for immunoblotting analysis. As a result, both male and female mice undergoing sevoflurane anesthesia showed strong sociability and weak preference for social novelty at juvenile age. In addition, the male mice developed normal preference for social novelty at early-adulthood; However, the female mice remained weak preference for social novelty. Furthurmore, sevoflurane anesthesia could decrease the levels of PSD95 but not Neuroligin-1 in the hippocampus but not cortex of neonatal mice. In conclusion, sevoflurane anesthesia in neonatal period could disturb development of social memory and impair preference for social novelty in female mice at early-adulthood, with the potential mechanism of decreasing PSD95 expression in the hippocampus of C57BL/6 mice.

Exploring the antiplatelet activity of serotonin 5-HT2A receptor antagonists bearing 6-fluorobenzo[d]isoxazol-3-yl)propyl) motif- as potential therapeutic agents in the prevention of cardiovascular diseases.

Small drug-like molecules that can block the function of serotonin 5-HT2A receptors have garnered considerable attention due to their ability to inhibit platelet aggregation and the possible prevention of atherosclerotic lesions. Although clinical data provided compelling evidence for the efficacy of this approach in the prevention of various cardiovascular conditions, the chemical space of 5-HT2A receptor antagonists is limited to ketanserin and sarpogrelate. To expand the portfolio of novel chemical motifs with potential antiplatelet activity, we evaluated the antiplatelet activity of a series of 6-fluorobenzo[d]isoxazole derivatives that possess a high affinity for 5-HT2A receptor. Here we describe in vitro studies showing that 6-fluorobenzo[d]isoxazole derivatives exert promising antiplatelet activity in three various in vitro models of platelet aggregation, as well as limit serotonin-induced vasoconstriction. Compound AZ928 showed in vitro activity greater than the clinically approved drug sarpogrelate. In addition to promising antiplatelet activity, the novel series was characterized by a favorable safety profile. Our findings show that the novel series exerts promising antiplatelet efficacy while being deprived of potential side effects, such as hemolytic activity, which render these compounds as potential substances for further investigation in the field of cardiovascular research.

Safety and Functional Pharmacokinetic Profile of APAC, a Novel Intravascular Antiplatelet and Anticoagulant.

ABSTRACT: Vascular intervention-induced platelet and coagulation activation is often managed with a combination of antiplatelets and anticoagulants, with evident benefits, but with a risk of systemic bleeding. Antiplatelet and anticoagulant (APAC) is a dual antiplatelet and anticoagulant heparin bioconjugate, which targets vascular injury sites to act as a local antithrombotic. We assessed the nonclinical safety and exposure of intravenously infused APAC in rats and cynomolgus monkeys by using single-day and 14-day repeat dose toxicology and pharmacodynamic markers. Activated partial thromboplastin time (APTT) was used as a functional surrogate of anticoagulant exposure of APAC. Routine clinical in-life observations were followed by clinical pathology and necropsy. The no-observed-adverse-effect level (NOAEL) in rats for the single APAC dose was 20 mg/kg and for the repeated administration was 10 mg/kg/d. Monkeys tolerated a single APAC dose of 10 mg/kg, although the red blood cell count reduced 16%-19% correlating with tissue hemorrhage at vein puncture and affected muscle sites during handling of the animals. However, after 2-week recovery, all clinical signs were normal. The single dose NOAEL exceeded 3 mg/kg. The repeat administration of 3-6 mg/kg/d of APAC was tolerated, but some clinical signs were observed. The NOAEL for repeated dosing was 0.5 mg/kg/d. APAC prolonged APTT dose-dependently in both species, returning to baseline after 1.5 (<10 mg/kg) or essentially by 6 hours also under repetitive dosing. The toxicology profile supports the safety of an intravenous APAC dose of 0.5 mg/kg/d for possible clinical applications. APTT is an acceptable indicator of the immediate systemic anticoagulation effect of APAC.

Targeting Thymidine Phosphorylase With Tipiracil Hydrochloride Attenuates Thrombosis Without Increasing Risk of Bleeding in Mice.

OBJECTIVE: Current antiplatelet medications increase the risk of bleeding, which leads to a clear clinical need in developing novel mechanism-based antiplatelet drugs. TYMP (Thymidine phosphorylase), a cytoplasm protein that is highly expressed in platelets, facilitates multiple agonist-induced platelet activation, and enhances thrombosis. Tipiracil hydrochloride (TPI), a selective TYMP inhibitor, has been approved by the Food and Drug Administration for clinical use. We tested the hypothesis that TPI is a safe antithrombotic medication. Approach and Results: By coexpression of TYMP and Lyn, GST (glutathione S-transferase) tagged Lyn-SH3 domain or Lyn-SH2 domain, we showed the direct evidence that TYMP binds to Lyn through both SH3 and SH2 domains, and TPI diminished the binding. TYMP deficiency significantly inhibits thrombosis in vivo in both sexes. Pretreatment of platelets with TPI rapidly inhibited collagen- and ADP-induced platelet aggregation. Under either normal or hyperlipidemic conditions, treating wild-type mice with TPI via intraperitoneal injection, intravenous injection, or gavage feeding dramatically inhibited thrombosis without inducing significant bleeding. Even at high doses, TPI has a lower bleeding side effect compared with aspirin and clopidogrel. Intravenous delivery of TPI alone or combined with tissue plasminogen activator dramatically inhibited thrombosis. Dual administration of a very low dose of aspirin and TPI, which had no antithrombotic effects when used alone, significantly inhibited thrombosis without disturbing hemostasis. CONCLUSIONS: This study demonstrated that inhibition of TYMP, a cytoplasmic protein, attenuated multiple signaling pathways that mediate platelet activation, aggregation, and thrombosis. TPI can be used as a novel antithrombotic medication without the increase in risk of bleeding.

Towards the Therapeutic Use of Thrombospondin 1/CD47 Targeting TAX2 Peptide as an Antithrombotic Agent.

OBJECTIVE: TSP-1 (thrombospondin 1) is one of the most expressed proteins in platelet α-granules and plays an important role in the regulation of hemostasis and thrombosis. Interaction of released TSP-1 with CD47 membrane receptor has been shown to regulate major events leading to thrombus formation, such as, platelet adhesion to vascular endothelium, nitric oxide/cGMP (cyclic guanosine monophosphate) signaling, platelet activation as well as aggregation. Therefore, targeting TSP-1:CD47 axis may represent a promising antithrombotic strategy. Approach and Results: A CD47-derived cyclic peptide was engineered, namely TAX2, that targets TSP-1 and selectively prevents TSP-1:CD47 interaction. Here, we demonstrate for the first time that TAX2 peptide strongly decreases platelet aggregation and interaction with collagen under arterial shear conditions. TAX2 also delays time for complete thrombotic occlusion in 2 mouse models of arterial thrombosis following chemical injury, while Thbs1-/- mice recapitulate TAX2 effects. Importantly, TAX2 administration is not associated with increased bleeding risk or modification of hematologic parameters. CONCLUSIONS: Overall, this study sheds light on the major contribution of TSP-1:CD47 interaction in platelet activation and thrombus formation while putting forward TAX2 as an innovative antithrombotic agent with high added-value.

Traditional Chinese medicine practices used in COVID-19 (Sars-cov 2/Coronavirus-19) treatment in clinic and their effects on the cardiovascular system.

OBJECTIVE: The aim of this study was to evaluate the effectiveness of plants used in the formulations of traditional Chinese medicine (TCM), which were also used in clinical trials to treat patients with the novel coronavirus COVID-19, and to assess their effects on the cardiovascular system. METHODS: A literature review of PubMed, ResearchGate, ScienceDirect, the Cochrane Library, and TCM monographs was conducted and the effects of the plants on the cardiovascular system and the mechanisms of action in COVID-19 treatment were evaluated. RESULTS: The mechanism of action, cardiovascular effects, and possible toxicity of 10 plants frequently found in TCM formulations that were used in the clinical treatment of COVID-19 were examined. CONCLUSION: TCM formulations that had been originally developed for earlier viral diseases have been used in COVID-19 treatment. Despite the effectiveness seen in laboratory and animal studies with the most commonly used plants in these formulations, the clinical studies are currently insufficient according to standard operating procedures. More clinical studies are needed to understand the safe clinical use of traditional plants.

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.

Cistanches alleviates sevoflurane-induced cognitive dysfunction by regulating PPAR-γ-dependent antioxidant and anti-inflammatory in rats.

This study aimed to investigate the protective effects and underlying mechanisms of cistanche on sevoflurane-induced aged cognitive dysfunction rat model. Aged (24 months) male SD rats were randomly assigned to four groups: control group, sevoflurane group, control + cistanche and sevoflurane + cistanche group. Subsequently, inflammatory cytokine levels were measured by ELISA, and the cognitive dysfunction of rats was evaluated by water maze test, open-field test and the fear conditioning test. Three days following anaesthesia, the rats were killed and hippocampus was harvested for the analysis of relative biomolecules. The oxidative stress level was indicated as nitrite and MDA concentration, along with the SOD and CAT activity. Finally, PPAR-γ antagonist was used to explore the mechanism of cistanche in vivo. The results showed that after inhaling the sevoflurane, 24- but not 3-month-old male SD rats developed obvious cognitive impairments in the behaviour test 3 days after anaesthesia. Intraperitoneal injection of cistanche at the dose of 50 mg/kg for 3 consecutive days before anaesthesia alleviated the sevoflurane-induced elevation of neuroinflammation levels and significantly attenuated the hippocampus-dependent memory impairments in 24-month-old rats. Cistanche also reduced the oxidative stress by decreasing nitrite and MDA while increasing the SOD and CAT activity. Moreover, such treatment also inhibited the activation of microglia. In addition, we demonstrated that PPAR-γ inhibition conversely alleviated cistanche-induced protective effect. Taken together, we demonstrated that cistanche can exert antioxidant, anti-inflammatory, anti-apoptosis and anti-activation of microglia effects on the development of sevoflurane-induced cognitive dysfunction by activating PPAR-γ signalling.

The activation of high mobility group Box1 and toll-like receptor 4 is involved in clopidogrel-induced gastric injury through p38 MAPK.

Clopidogrel-induced gastric injury is an important clinical problem. However, the exact mechanism was still unclarified. This study aimed to investigate the role of high mobility group box protein 1 (HMGB1) and the toll-like receptor 4 (TLR4) pathway in normal human gastric epithelial (GES-1) cells under clopidogrel exposure. Morphological alterations of the gastric epithelial cells were observed under a microscope. A laser scanning confocal microscope was used to determine the distribution of HMGB1 and TLR4 in clopidogrel-induced injury. MTT was used to compare the viability of GES-1 cell among the pretreated Cli-095 (TLR4 inhibitor) group, pretreated ethyl pyruvate (HMGB1 inhibitor) group, clopidogrel group, and control group. Moreover, expression of the HMGB1, TLR4, tight junction (TJ) proteins occludin and the phosphorylation of the mitogen-activated protein kinases (MAPK) p38 were examined by western blot. We found the expression of HMGB1 and TLR4 in the cytoplasm increased after clopidogrel administration. Besides, inhibited TLR4, which is a receptor of HMGB1, prevented the injury and occludin reducing caused by clopidogrel challenge. Furthermore, blocking HMGB1 or TLR4 activity by ethyl pyruvate (HMGB1 inhibitor) or cli-095(TLR4 inhibitor) can partially diminish the activation of p38MAPK. Gastric mucosal damages observed by clopidogrel challenge are associated with HMGB1, TLR4, through p38MAPK signal pathway.

Pioglitazone prevents sevoflurane­induced neuroinflammation and cognitive decline in a rat model of chronic intermittent hypoxia by upregulating hippocampal PPAR­Î³.

Post­operative cognitive dysfunction is a common complication after anesthesia and surgery. Sevoflurane (SEV), a widely used inhalational anesthetic, can exaggerate neuroinflammation and cause cognitive dysfunction under chronic intermittent hypoxia (CIH) conditions by downregulating hippocampal peroxisome proliferator­activated receptor­Î³ (PPAR­Î³). In the present study, it was examined whether treatment with PPAR­Î³ agonist pioglitazone (PIO) is beneficial in counteracting SEV­induced neuroinflammation and cognitive decline in a rat model of CIH. Rats were exposed to CIH for 4 weeks. After 2 weeks of CIH, these animals underwent either 2.6% SEV or control (CON) exposure for 4 h. PIO (60 mg/kg) or vehicle (VEH) was administered orally twice daily for 2 weeks, starting one day prior to SEV or CON exposure. Compared with CIH­CON+VEH rats, CIH­SEV+VEH rats exhibited significant cognitive decline as indicated by increased latency to locate the hidden platform and shorter dwell­time in the goal quadrant in the Morris Water Maze task. Molecular studies revealed that CIH­SEV+VEH rats had increased proinflammatory cytokine expression and microglial activation in the hippocampus, which were associated with decreased PPAR­Î³ activity. Notably, SEV­induced cognitive decline and increases in proinflammatory cytokine expression and microglial activation were prevented by PIO, which increased hippocampal PPAR­Î³ activity. PIO also increased hippocampal PPAR­Î³ activity in CIH­CON rats but did not alter proinflammatory cytokine expression and microglial activation as well as cognitive function. Additionally, expression of hippocampal PPAR­α and PPAR­ß, two other PPAR isotypes, were comparable among the groups. These data suggest that PIO prevents SEV­induced exaggeration of neuroinflammation and cognitive decline under CIH conditions by upregulating hippocampal PPAR­Î³. PIO may have the potential to prevent anesthetic SEV­induced cognitive decline in surgical patients with obstructive sleep apnea.

Optimizing Platelet GPVI Inhibition versus Haemostatic Impairment by the Btk Inhibitors Ibrutinib, Acalabrutinib, ONO/GS-4059, BGB-3111 and Evobrutinib.

Ibrutinib and acalabrutinib are approved for B cell malignancies and novel Bruton's tyrosine kinase (Btk) inhibitors undergo clinical testing also in B cell-driven autoimmune disorders. Btk in platelets mediates platelet activation via glycoprotein (GP) VI, which is crucial for atherosclerotic plaque-induced platelet thrombus formation. This can be selectively inhibited by Btk inhibitors. Since patients on second-generation Btk inhibitors apparently show less bleeding than patients on ibrutinib, we compared the effects of ibrutinib and four novel irreversible Btk inhibitors on GPVI-dependent platelet aggregation in blood and in vitro bleeding time. Low concentrations of collagen which induced the same low degree of GPVI-mediated platelet aggregation as atherosclerotic plaque material were applied. IC50 values for collagen (0.2-0.5 µg/mL)-induced platelet aggregation after 15-minute pre-incubation were: ibrutinib 0.12 µM, BGB-3111 0.51 µM, acalabrutinib 1.21 µM, ONO/GS-4059 1.20 µM and evobrutinib 5.84 µM. Peak venous plasma concentrations of ibrutinib (0.5 µM), acalabrutinib (2 µM) and ONO/GS-4059 (2 µM) measured after anti-proliferative dosage inhibited collagen-induced platelet aggregation, but did not increase PFA-200 closure time on collagen/epinephrine. Closure times were moderately increased by 2- to 2.5-fold higher concentrations of these inhibitors, but not by BGB-3111 (1 µM) and evobrutinib (10 µM). Prolonging platelet drug exposure to 60 minutes lowered IC50 values of any Btk inhibitor for GPVI-mediated aggregation by several fold, and 5- to 10-fold below anti-proliferative therapeutic drug plasma levels. In conclusion, low blood concentrations of ibrutinib and the novel Btk inhibitors suffice for GPVI selective platelet inhibition relevant for atherothrombosis but do not impair primary haemostasis.

Sevoflurane impairs learning and memory of the developing brain through post-transcriptional inhibition of CCNA2 via microRNA-19-3p.

The molecular mechanisms underlying sevoflurane (SEVO)-induced impairment of learning and memory remain unclear. Specifically, a role of microRNAs (miRNAs) in the control of the neuron proliferation in the developing brain exposed to SEVO has not been reported previously. Here, we studied the effects of SEVO exposure on the neural cell proliferation, and on the learning and memory of neonatal rats. We found that SEVO exposure significantly decreased neuron cell proliferation, reduced BDNF levels in brain, and impaired learning and memory of neonatal rats in Morris water maze test and Plus-Maze discriminative avoidance task (PM-DAT), likely through downregulation of CCNA2 protein. Next, we used bioinformatic tools to predict CCNA2-binding microRNAs (miRNAs), and found that miR-19-3p was upregulated in neurons exposed to SEVO. Moreover, miR-19-3p functionally inhibited the protein translation of CCNA2 in a human neural cell line, HCN-2. Furthermore, intracranial injection of adeno-associated virus carrying antisense of miR-19-3p under a CMV promoter into the neonatal rats significantly alleviated SEVO exposure-induced impairment of neuron cell proliferation, as well as the learning and memory of the rats. Together, our data suggest that SEVO-induced upregulation of miR-19-3p post-transcriptionally inhibits CCNA2, which contributes to the SEVO-associated impairment of learning and memory of the neonatal rats.

Sevoflurane induces neurotoxicity in the developing rat hippocampus by upregulating connexin 43 via the JNK/c-Jun/AP-1 pathway.

As one of the most popular anesthetics, sevoflurane is widely used in pediatric anesthesia. Unfortunately, an increasing number of studies have demonstrated that sevoflurane has potential neurotoxic effects on the developing brain and cognition, even in adolescence. Connexin 43 (Cx43) has been documented to contribute to cognitive dysfunction. The present study hypothesized that Cx43 may participate in sevoflurane-induced neuroinjury and investigated the underlying mechanisms in young Sprague Dawley (SD) rats. Seven-day-old SD rats (P7) were exposed to 3% sevoflurane for 4 h. The levels of Cx43,mitogen-activated protein kinase (MAPK) signaling pathway components(including total and phosphorylated p38, extracellular signal-regulated kinase (ERK), and c-Jun n-terminal kinase (JNK) and activator protein 1(AP-1) transcription factors (including total and phosphorylated c-Fos, and c-Jun) were assessed by Western blot analysis. Neuronal apoptosis was detected using immunohistochemistry (IHC). The Morris water maze (MWM) was performed to evaluate cognitive function from P28 to P33. The results showed that anesthesia with 3% sevoflurane for 4 h increased Cx43 levels in the rat hippocampus from 6 h to 3 d, and compared with sevoflurane exposure in the control group rats, exposure in P7 SD rats also increased the ratios of phosphorylated JNK to JNK and, phosphorylated c-Jun to c-Jun in the hippocampus from 6 h to 3 d. All these effects could be alleviated by pretreatment with the JNK inhibitor SP600125 (10 mg/kg). Neuroapoptosis was similarly increased from 6 h to 1 d after inhaled sevoflurane exposure. Finally, the MWM indicated that sevoflurane could increase the escape latency and, decrease the number of platform crossings from P28 to P33. Overall, our findings suggested that sevoflurane increased Cx43 expression and induced to apoptosis by activating the JNK/c-Jun signaling pathway in the hippocampus of P7 rats. This finding may reveal a new strategy for preventing sevoflurane-induced neuronal dysfunction.

Evaluation of recombinant factor VIIa, tranexamic acid and desmopressin to reduce prasugrel-related bleeding: A randomised, placebo-controlled study in a rabbit model.

BACKGROUND: Prasugrel is a thienopyridine that inhibits platelet aggregation more rapidly and effectively than clopidogrel, with an increased bleeding risk. OBJECTIVE: The current study aimed to evaluate the efficacy of three nonspecific haemostatic drugs - recombinant activated factor VII (rFVIIa), tranexamic acid and desmopressin (DDAVP) - to limit blood loss after administration of prasugrel in a rabbit model of bleeding while also evaluating any prothrombotic effects. DESIGN: Randomised, placebo-controlled study. SETTING: Faculty of Medicine, University of Geneva, Switzerland, in 2013. ANIMALS: Anaesthetised and artificially ventilated rabbits (n=56). INTERVENTIONS: Animals were randomly allocated to one of five groups: control (placebo-placebo), prasugrel-placebo, rFVIIa (prasugrel-rFVIIa 150 µg kg), tranexamic acid (prasugrel-tranexamic acid 20 mg kg) or DDAVP (prasugrel-DDAVP 1 µg kg). Two hours after an oral prasugrel loading dose (4 mg kg), a stenosis and an injury were inflicted on the carotid artery to induce cyclic flow reductions (CFRs) due to thrombosis. Haemostatic drugs were administered during the ensuing observation period. MAIN OUTCOME MEASURES: Standardised hepatosplenic sections were performed to evaluate the primary endpoint of blood loss, monitored for 15 min. Ear-immersion bleeding time and incidence of CFRs were secondary endpoints. RESULTS: Prasugrel decreased ADP-induced platelet aggregation (light transmission method) from 66 ±â€Š4% (mean ±â€ŠSD) to 41 ±â€Š7% (P < 0.001) and doubled blood loss: 10.7 g (10.1 to12.7) [median (interquartile range)] vs. 20.0 g (17.0 to 24.4), P = 0.003 in the control and prasugrel-placebo groups, respectively. rFVIIa, tranexamic acid and DDAVP reduced neither hepatosplenic blood loss [19.7 g (14.0 to 27.6), 25.2 g (22.6 to 28.7) and 22.9 g (16.8 to 28.8), respectively] nor bleeding time compared with placebo. Regarding safety, rVIIa induced three or more CFRs in 5/12 rabbits, vs. 0/12 in the prasugrel-placebo group (P = 0.037), whereas tranexamic acid and DDAVP did not increase them. CONCLUSION: The three studied haemostatic drugs rFVIIa, tranexamic acid and DDAVP failed to reduce prasugrel-related bleeding in this model. rFVIIa-treated rabbits were more prone to arterial thrombotic events. TRIAL REGISTRATION: NA.

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.

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.

Recombinant GPVI-Fc added to single or dual antiplatelet therapy in vitro prevents plaque-induced platelet thrombus formation.

The efficiency of current dual antiplatelet therapy might be further improved by its combination with a glycoprotein (GP) VI-targeting strategy without increasing bleeding. GPVI-Fc, a recombinant dimeric fusion protein binding to plaque collagen and concealing binding sites for platelet GPVI, acts as a lesion-focused antiplatelet drug, and does not increase bleeding in vivo. We investigated, whether GPVI-Fc added in vitro on top of acetylsalicylic acid (ASA), the P2Y12 antagonist ticagrelor, and the fibrinogen receptor antagonist abciximab alone or in combination would increase inhibition of platelet activation by atherosclerotic plaque. Under static conditions, GPVI-Fc inhibited plaque-induced platelet aggregation by 53 %, and increased platelet inhibition by ASA (51 %) and ticagrelor (64 %) to 66 % and 80 %, respectively. Under arterial flow, GPVI-Fc inhibited plaque-induced platelet aggregation by 57 %, and significantly increased platelet inhibition by ASA (28 %) and ticagrelor (47 %) to about 81 % each. The triple combination of GPVI-Fc, ASA and ticagrelor achieved almost complete inhibition of plaque-induced platelet aggregation (93 %). GPVI-Fc alone or in combination with ASA or ticagrelor did not increase closure time measured by the platelet function analyzer (PFA)-200. GPVI-Fc added on top of abciximab, a clinically used anti-fibrinogen receptor antibody which blocks platelet aggregation, strongly inhibited total (81 %) and stable (89 %) platelet adhesion. We conclude that GPVI-Fc added on top of single or dual antiplatelet therapy with ASA and/or a P2Y12 antagonist is likely to improve anti-atherothrombotic protection without increasing bleeding risk. In contrast, the strong inhibition of platelet adhesion by GPVI-Fc in combination with GPIIb/IIIa inhibitors could be harmful.

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.