Bleeding Time In Mice Research Articles

Blood Coagulation and Platelet Function Are Impaired By the ARP2 p.R258G Variant in Mice

Introduction: Venous thromboembolism (VTE) displays significant heritability, yet the genes explaining this heritability are not completely characterized. We previously identified that tissue factor pathway inhibitor heterozygosity ( Tfpi+/−) on the homozygous F5L background ( F5L/L) produces a lethal perinatal thrombotic phenotype. Using a sensitized ENU mutagenesis screen, we discovered an autosomal dominant missense mutation in the Actr2 gene (p.R258G, Actr2G) that suppresses thrombosis and restores survival to F5L/LTfpi+/− mice. Inheritance of the Actr2Gmutation is associated with nearly complete suppression of the lethal F5L/LTfpi+/- phenotype. Actr2 codes for the ARP2 protein, an essential component of the ARP2/3 complex, which mediates actin cytoskeletal rearrangements during platelet activation. We hypothesized that the Actr2G variant affects blood coagulation and platelet function to reduce thrombosis. Methods: To investigate potential effects of the Actr2G mutation on the coagulation cascade, prothrombin time (PT), activated partial thromboplastin time (aPTT), and plasma-dependent thrombin generation assays (TGA) were used to evaluate clot formation. A mouse tail clip assay was used to measure bleeding time and quantify blood loss. Finally, platelets were assessed for functional defects using platelet spreading and aggregometry assays. Results: Actr2 +/Gplasma had significantly prolonged PT and aPTT compared to wild type controls (N>17, p<0.03). There was also a significant reduction in Actr2+/G thrombin peak height compared to wildtypes (N=10; p=0.03). Despite the delayed time to clot formation, Actr2+/G mice did not exhibit a bleeding phenotype and showed no significant differences in blood loss from tail clip assays. Activated Actr2+/G platelets exhibited reduced surface area and adhesion (N=5; p<0.02), as well as significant defects in collagen induced whole blood aggregation (N=10; p<0.02). Discussion: The results of the PT, aPTT and TGA indicate reduced thrombin generation potential, suggesting a defect in the common pathway of coagulation. This hypocoaguability may be due to a decrease in circulating procoagulant factors, or an increase in endogenous thrombin inhibitors. Both the significantly decreased aggregation and defective platelet spreading in the Actr2+/G mice indicate that Actr2+/G platelets are less responsive to agonists, a protective mechanism that contributes to overall thrombosis suppression. Conclusion: In summary, our studies demonstrate the pleiotropic effects that the Actr2G mutation exerts in suppressing thrombosis. The combination of altered plasma coagulation and platelet reactivity likely contribute to suppression of the F5L/LTfpi+/− phenotype. Defects in these actin-mediated processes precipitated by Actr2+/G are the likely root of the F5L/LTfpi+/- phenotype rescue, demonstrating the power of this gene in maintaining hemostatic balance. However, the combination of these thrombosis suppressing phenotypes is not significant enough to prolong the bleeding time in mice with the Actr2G mutation. Thus, our results suggest that targeting actin proteins such as ARP2 for novel antithrombotic drug therapies may have clinical applicability with limited effects on bleeding.

Chemical constituents from Callicarpa kwangtungensis and their hemostatic activities

This study aims to identify the chemical constituents from Callicarpa kwangtungensis and determine their activities. MCI, ODS, and Sephadex LH-20 chromatography and semi-preparative HPLC were employed to separate the chemical constituents. A total of 15 compounds were separated, and their structures were identified on the basis of spectroscopic analysis and comparison with the data in relevant literature. Specifically, the 15 compounds were 3-O-α-L-rhamnopyranosyl-6-O-β-D-apiofuranosyl-4-O-E-caffeoyl-D-glucopyranoside(1), 3,6-O-α-L-dirhamnopyranosyl-4-O-E-caffeoyl-D-glucopyranoside(2), β-OH-forsythoside B(3), β-OH-poliumoside(4),(+)-lyoniresinol-3α-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside(5),(+)-lyoniresinol-3α-O-β-D-glucopyranoside(6),(-)-lyoniresinol-3α-O-β-D-glucopyranoside(7), kelampayoside A(8), descaffeoylpoliumoside(9), acteoside(10), alyssonoside(11), poliumoside(12), isacteoside(13), acetyl forsythoside B(14), and forsythoside B(15). Compounds 1 and 2 were novel, and the NMR data of compounds 3 and 4 were reported here for the first time. Furthermore, the hemostatic activities of the extract and abundant ingredients(compounds 12 and 15) of C. kwangtungensis were determined with Yunnan Baiyao as the positive control and normal saline as the negative control. The extract and compounds 12 and 15 significantly shortened the tail tip bleeding time in mice.

Garcinol acts as a novel integrin αIIbβ3 inhibitor in human platelets

AimsPlatelet activation plays a central role in arterial thrombosis. Platelets are activated by adhesive proteins (i.e., collagen) or soluble agonists (i.e., thrombin), the respective receptor-specific signaling cause inside–out signaling, leading to the binding of fibrinogen to integrin αIIbβ3. This binding triggers outside–in signaling, resulting in platelet aggregation. Garcinol, a polyisoprenylated benzophenone, is extracted from the fruit rind of Garcinia indica. Although garcinol exhibits considerable bioactivities, few studies have investigated the effect of garcinol on platelet activation. Main methodsAggregometry, immunoblotting, flow cytometer, confocal microscopic analysis, fibrin clot retraction, animal studies such as fluorescein-induced platelet plug formation in mesenteric microvessels, acute pulmonary thromboembolism, and tail bleeding time were performed in this study. Key findingsThis study indicates that garcinol inhibited platelet aggregation stimulated by collagen, thrombin, arachidonic acid, and U46619. Garcinol reduced integrin αIIbβ3 inside–out signaling, including ATP release; cytosolic Ca2+ mobilization; P-selectin expression; and Syk, PLCγ2/PKC, PI3K/Akt/GSK3β, MAPKs, and NF-κB activation stimulated by collagen. Garcinol directly inhibited integrin αIIbβ3 activation by interfering with FITC–PAC-1 and FITC-triflavin by collagen. Additionally, garcinol affected integrin αIIbβ3-mediated outside–in signaling, such as decreasing platelet adhesion and the single-platelet spreading area; suppressing integrin β3, Src, FAK, and Syk phosphorylation on immobilized fibrinogen; and inhibiting thrombin-stimulated fibrin clot retraction. Garcinol substantially reduced mortality caused by pulmonary thromboembolism and prolonged the occlusion time of thrombotic platelet plug formation without extending bleeding time in mice. SignificanceThis study identified that garcinol, a novel antithrombotic agent, acts as a naturally occurring integrin αIIbβ3 inhibitor.

Abstract 297: FHL2 Deficiency Facilitates Tissue Plasminogen Activator-mediated Thrombolysis Without Further Prolonging Bleeding Time

Introduction: A recent study showed a relationship between Four-and-a-half-LIM-domains 2 (FHL2) and venous thromboembolism. Here, we investigated FHL2 deficiency-mediated effects on arterial thrombus formation, tissue plasminogen activator (tPA)-mediated thrombolysis, and bleeding time. Methods: First, arterial thrombosis was induced in twelve-week-old male FHL2 +/+ mice (n=17) and FHL2 -/- mice (n=17) by applying FeCl 3 on the left common carotid artery (CCA) for 10 minutes. To measure thrombus volume at 30 minutes (n=10/group) or 3 hours (n=7/group) after the thrombus induction, microCT imaging was performed after intravenous injection of gold nanoparticles (2mg/kg in 200μL). Second, 32 mice (n=16/group) were treated with intravenous tPA (25 mg/kg) at 30 minutes (n=10/group) or 3 hours (n=6/group) after thrombosis, and microCT imaging was performed at baseline and post-tPA 0.5, 1, 3, and 24 hour time-points. Third, tail-bleeding test was performed with (n=6 and 8, respectively) or without (n=8/group) tPA treatment. Results: Thrombus volume at 30 minutes and 3 hours did not differ significantly between the groups. However, post-tPA 3-hour thrombus volume was significantly lower in FHL2 -/- mice than in FHL2 +/+ mice (71% vs. 46% reduction from the baseline, respectively; p=0.03), when the thrombolytic therapy was initiated at 30 minutes after thrombosis. There were no significant inter-group differences in thrombus volumes when tPA treatment was delayed (ie., started at 3 hours after thrombosis). Lastly, bleeding time did not show significant inter-group differences, regardless of tPA therapy (all p>0.05). Conclusions: FHL2 deficiency does not reduce baseline arterial thrombus volume but facilitates tPA-mediated thrombolysis without prolonging bleeding time in mice.

Platelet-Mimicking Nanosponges for Functional Reversal of Antiplatelet Agents.

During long-term antiplatelet agents (APAs) administration, patients with thrombotic diseases take a fairly high risk of life-threatening bleeding, especially when in need of urgent surgery. Rapid functional reversal of APAs remains an issue yet to be efficiently resolved by far due to the lack of any specific reversal agent in the clinic, which greatly restricts the use of APAs. Although platelet transfusion has become a common practice to counteract the function of APAs, personalized dosage of platelet transfusion is difficult to be judged by assessing the trade-off between its effectiveness and additional thrombotic risk. Here we reported a platelet-mimicking perfluorocarbon-based nanosponge, which potently reversed the antiplatelet effect of APAs by competitively binding with APAs. Platelet-mimicking perfluorocarbon-based nanosponges showed high binding affinity comparable to fresh platelets in vitro with first-line APAs, ticagrelor and tirofiban, and efficiently reversed their function in both tail bleeding and postischemic-reperfusion models. Moreover, the deficiency of platelet intrinsic thrombotic activity diminished the risk of thrombogenesis. In summary, our results demonstrated the safety and effectiveness of the platelet-mimicking nanosponge in ameliorating the bleeding risk of different APAs, which offers a promising strategy for the management of bleeding complications induced by antiplatelet therapy.

Synchronous evaluation of continuous dynamic dissolution and absorption of Compound Danshen Dropping Pills based on cellular bioelectrical effect

The real-time cell-based assay(RTCA) was used to establish the bioelectrical sensing model of Compound Danshen Dripping Pills with rat cardiomyocytes(H9 c2). The time/dose-dependent cell response profiles(TCRPs) of in vitro dissolution and absorption of the pills were determined to establish the continuous dynamic dissolution and absorption kinetic models. Thereby, the cell index(CI)-based dissolution and absorption kinetic curves and kinetic models of Compound Danshen Dripping Pills were obtained. The optimal dissolution kinetic model was Weibull model. The similarity factors f_2 of dissolution curves were greater than 50 and the correlation coefficients of absorption curves were larger than 0.95. With the experiment about the efficacy on mice, percentages of the bleeding time of mice administrated with Compound Danshen Dripping Pills were calculated, and there was a correlation among dissolution, absorption, and efficacy curves(r > 0.9). RTCA is applicable to the study of the dissolution and absorption kinetics of solid compound Chinese medicine preparations. Thus, it is an innovative and feasible method to evaluate the quality and batch consistency of compound Chinese medicine preparations.

Naringenin Inhibits Platelet Activation and Arterial Thrombosis Through Inhibition of Phosphoinositide 3-Kinase and Cyclic Nucleotide Signaling.

Citrus flavanoids intake can reduce the risk of cardiovascular diseases. Naringenin, a natural predominant flavonoid abundant in citrus fruits, possesses protective effects against atherothrombotic diseases. As platelet activation plays central roles in atherothrombogenesis, we studied the effects of naringenin on platelet activation, signaling, thrombosis and hemostasis. Naringenin dose-dependently inhibited agonist-induced platelet aggregation in vitro, and exhibited more-potent efficacy on ADP-induced platelet aggregation. It also suppressed platelet aggregation stimulated by ADP ex vivo. Naringenin inhibited ADP-induced platelet α-granule secretion, fibrinogen binding, intracellular calcium mobilization and platelet adhesion on collagen-coated surface. Naringenin also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in integrin signaling. Mechanism studies indicated that naringenin suppressed PI3K-mediated signaling and phosphodiesterase activity in platelets, in addition to increasing cGMP levels and VASP phosphorylation at Ser239. Furthermore, naringenin-induced VASP phosphorylation and inhibition of platelet aggregation were reversed by a PKA inhibitor treatment. Interestingly, naringenin inhibited thrombus formation in the (FeCl3)-induced rat carotid arterial thrombus model, but not cause a prolonged bleeding time in mice. This study suggests that naringenin may represent a potential antiplatelet agent targeting PI3K and cyclic nucleotide signaling, with a low bleeding risk.

Pimpinellin Inhibits Collagen-induced Platelet Aggregation and Activation Through Inhibiting Granule Secretion and PI3K/Akt Pathway.

Pimpinellin is a coumarin-like compound extracted from the root of Toddalia asiatica. Its effects on platelet function has not been investigated. This study found that pimpinellin pretreatment effectively inhibited collagen-induced platelet aggregation, but did not alter ADP- and thrombin-induced aggregation. Platelets pretreated with pimpinellin showed reduced α granule (CD62) level and secretion of dense granule (ATP release). Pimpinellin-treated platelets also exhibited decreased clot reaction and TxB2 production. Pimpinellin pretreatment suppressed adhesion and spreading of human platelets on the fibrinogen coated surface. Analysis of tail bleeding time of mice administered with pimpinellin (40 mg/kg) revealed that pimpinellin did not change tail bleeding time significantly, number of blood cells, and APTT and PT levels. Pimpinellin inhibited collagen-induced ex vivo aggregation of mice platelets. Immunoblotting results showed that pimpinellin suppressed collagen-induced phosphorylation of PI3K-Akt-Gsk3β and PKC/MAPK in platelets.

Reduction of NF-κB Signals in Platelets and Prolongation of Platelet Plug Formation against High Shear Flow in Whole Blood on Human Subject by Columbianadin

Myocardial infarction and cerebral ischemic stroke during the process of arterial thrombosis are prominently causes of death worldwide. Platelets are anucleated cells and play a critical factor in these diseases. Columbianadin (CBN), a coumarin derivative from plants, inhibits effective platelet activation. In this study, platelet function analysis revealed that the closure time of the platelet plug in human whole blood significantly prolonged by CBN, whereas CBN did not pointedly prolong the bleeding time in mice. BAY11-7082 (an inhibitor of IκB kinase) and MG-132 (an inhibitor of proteasome) inhibited collagen-stimulated platelet aggregation and ATP-release in human platelets, BAY11-7082 exhibited a higher potency than MG-132. Moreover, CBN markedly reduced NF-κB activation (e.g., IκBα and p65 phosphorylation) and reversed IκBα degradation in activated platelets. We investigated intercellular signaling events between mitogen-activated protein kinases and NF-κB, and found that BAY11-7082 abolished JNK1/2 and ERK1/2 phosphorylation. Interestingly, SP600125 (an inhibitor of JNK) but not PD98059 (an inhibitor of ERK) had no effect in NF-κB activation in activated platelets. Moreover, CBN but not BAY11-7082 significantly reduced hydroxyl radical (HO●) formation in platelets. Therefore, we propose that CBN inhibits NF-κB activation in human platelets and could present a potent clinical treatment for thromboembolic diseases.

Demonstration of an action pathway in mouse platelets leading to prolongation of bleeding time by fluoxetine

Fluoxetine is one of SSRIs commonly used as first-line antidepressants. It also induces adverse effects, including bleeding events. This study clarified the bleeding effect of fluoxetine and explored the action cascade of this drug leading to a longer bleeding time. A total of 48 male adult mice were evenly distributed into four groups and given fluoxetine in saline at 0, 4, 8, or 16 mg/kg, for 14 days. On day 15, tail bleeding time of 6 mice/group was measured, and their blood samples were collected for analyses of relevant platelet functions. The remained mice were allowed to survive for another 14 days without fluoxetine, and subjected to the same analyses on day 29. A significant effect of fluoxetine was reveled on bleeding time (F (3,20) = 16.842, P < 0.01) and intraplatelet serotonin (F (3,20) = 90.967, P < 0.01). Moreover, fluoxetine effectively inhibited platelet aggregation (F(3, 20) = 30.247, P < 0.01), decreased amount of GPIbα (F(3, 20) = 23.855, P < 0.01), suppressed GPIIb/IIIa activation (F(3, 20) = 89.441, P < 0.01), and lowered P-selectin (F(3, 20) = 7.960, P < 0.01) on platelet surface. Negative correlations existed between bleeding time and the aforementioned four indices, whereas correlations between intraplatelet serotonin and the same indices were positive. All changes returned to same levels as Control group after fluoxetine withdrawal. These data suggest an action pathway of fluoxetine starting at binding to serotonin transporter, followed by decreased intraplatelet serotonin, increased GPIbα shedding, suppressed GPIIb/IIIa activation, and inhibited α-granule release, and concluding with prolonged bleeding time in mice.

A novel di-peptide Met-Glu from collagen hydrolysates inhibits platelet aggregation and thrombus formation via regulation of Gq-mediated signaling.

Increasing evidence has shown that collagen peptides had various biological activities. In this study, a novel antiplatelet peptide Met-Glu (ME) was separated and identified from silver carp skin by YMC ODS-A C18 separation and ESI-MS/MS analysis. Peptide ME inhibited platelet aggregation and secretion of platelet granules induced by ADP, thrombin and collagen, and significantly attenuated ferric chloride-induced thrombus formation in rats. It did not prolong the bleeding time in mice even at the dose of 300μmol/kg body weight that showed potent anti-thrombosis effects. Additionally, peptide ME targeted at Gq-protein to downregulate the phosphorylation of PLCβ, an important upstream effector of PI3K/Akt and Erk/MAPK signaling to inhibit intracellular calcium ion mobilization. These results suggest that peptide ME inhibited thrombosis in vivo and inhibited Gq-mediated signaling in platelets, indicating the possibility that ME could potentially be developed as a novel therapeutic agent in the prevention and treatment of thrombotic diseases. PRACTICAL APPLICATIONS: Cardiovascular diseases (CVDs) are the leading cause of mortality and morbidity worldwide. The proximal cause of CVDs is intravascular thrombosis formation, which mostly results from platelet activation, aggregation, and granules secretion. Traditional drugs in the prevention of thrombotic disease, such as aspirin and clopidogrel, are still limited for their side effects, especially bleeding complications. Collagen is a natural source for bioactive peptides and our previous study has shown that collagen peptides could inhibit platelet aggregation in vitro. Understanding the mechanism of collagen peptides on regulation of platelet activation and their in vivo anti-thrombosis activities were important for the development of novel-specific medical food in the prevention of thrombotic diseases.

VITPOR AI, A Coagulation Factor XIIa Inhibitor from Porphyra yezoensis: In Vivo Mode of Action and Assessment of Platelet Function Analysis

Thrombosis represents as the prime contributor to the burden of diseases, worldwide. Conventional anticoagulants for thrombosis therapy have a common bleeding side effect. Bioactive peptides are studied to be an effective alternative for currently available therapeutic drugs. In this study, VITPOR AI peptide, a previously reported coagulation FXIIa inhibitor from Nori (Porphyra yezoensis), was assessed for its inhibitory activity against FXIIa and its in vivo mode of action. In vivo efficacy as well as the antithrombotic property of the peptide was evaluated in mice model by ex vivo activated Partial Thromboplastin Time assay, tail transection model and whole blood clotting time. The enzyme kinetics was studied using chromogenic substrate assay. The kinetic behaviour of VITPOR AI showed that the peptide is a competitive inhibitor of FXIIa. Peptide showed significant inhibition of platelet adhesion and aggregation. VITPOR AI exhibited significant antithrombotic activity. Furthermore, ex vivo activated Partial Thromboplastin Time assay revealed that VITPOR AI exhibited potent anticoagulant activity in vivo. Tail bleeding assay revealed that the peptide did not prolong bleeding time in mice even at a higher dose of 5 mg/kg. Cytotoxicity studies of the peptide against human blood leukocytes indicated the safety of the peptide. VITPOR AI could be prospected as a potent anticoagulant with Factor XIIa inhibition, antiplatelet aggregation and antithrombotic activity. It was also studied to have no bleeding side effect.

RhoA Targeting Suppresses Cold-Induced Platelet Lesion and Allows Refrigerated Storage of Functional Human Platelets to Fourteen Days

Platelet transfusion is required for the support therapy of patients with hematological disorders and cancer. Current practice of donor-derived platelet storage at room temperature (RT) associates with an inherent risk of microbial contamination and a limit of 5-7 day shelf life. Refrigerated platelets are hemostatically superior than RT platelets but their survival in circulation is severely reduced. Storage in cold temperature induces loss of galactosylation/sialylation of platelet GPIb with exposure of b-N-acetyl glucosamine of N-linked glycans, which clusters on the platelet plasma membrane. Upon transfusion, these clusters are recognized by macrophages and hepatocytes resulting in lectin-mediated platelet clearance. We hypothesized that the long-term refrigerated storage lesion depends on the mislocalization of membrane-bound glycosyl- and syalyl-transferases in lipid rafts and endocytotic intermediates of cold-stored platelets. By using a combination of genetic and pharmacological approaches, we investigated whether the three major members of the Rho GTPase family, RHOA, RAC1 and/or CDC42, which are molecular switches regulating actomyosin dynamics and signaling in platelets. We found that cooling of platelets induces a sustained activation of RhoA and Rac1, but not Cdc42 in mouse and human platelets. Inducible genetic deletion of platelet RhoA in mice prevents clearance of cold-stored platelets upon transfusion in wild-type congenic animals (WT: 2±0.5 hrs x fraction of infusate; RhoAΔ/Δ: 8± 0.7 hrs x fraction of infusate; p≤0.05). When human platelets were stored in either plasma or PAS-IIIM (PAS-E) for up to 14 days, the inclusion in the storage medium of a rationally designed RHOA inhibitor G04 can prevent the phagocytosis of 14-day refrigerated platelets by activated macrophages in vitro (RT-stored, vehicle-treated platelets: 20%±4; cold stored, vehicle-treated 70±5%; cold-treated, G04(10μM)- treated: 25±3%; p≤0.001) or in vivo clearance as assessed by the area under the curve of surviving human platelets (hrs x fraction of infusate) transfused into clodronate-treated, sub-lethally irradiated non-obese diabetic, γc-/- (NSG) mice (RT-stored, vehicle-treated platelets: 16±1.0; cold-stored, vehicle-treated: 6±0.5; cold-stored, G04(10μM)-treated: 15±0.6; p≤0.001). This inhibition is reversible by either a wash or a 3-fold dilution that resulted in a reversion of the inhibitory effect on aggregation and in vivo correction of the bleeding time of mice pre-treated with aspirin (RT-stored, vehicle-treated: 45±4 sec.; cold-stored, vehicle-treated: 150±10 sec.; cold-stored, G04(10μM)-treated: 76±3.8 sec.; p≤0.001). Mechanistically, RHOA inhibition prevents the cold-induced cytoskeleton/shape change and spreading on fibrinogen through the prevention of the formation of lipid rafts enriched in glycosyl- and syalyl-transferase activities and the endocytosis of vacuoles enriched in GPIbα. Addition of the lipid raft disruptor b-cyclodextrin, but not of the RHOA downstream effector ROCK inhibitor Fasudil, phenocopies the effect of G04 on the prevention of cold-induced platelet damage. Thus, RHOA is the key mediator of the platelet storage lesion through a ROCK independent mechanism and its reversible inhibition allows the functional maintenance of cold-stored platelets in both survival and hemostatic properties. Our pre-clinical data support the concept that a platelet additive solution containing a low-affinity RHOA inhibitor is useful in preventing platelet storage lesion while fully maintaining their hemostatic function after 14 days of storage. A rationally designed cold storage regimen is highly feasible, which could resolve the platelet clearance problem and meet an urgent need in transfusion medicine for the support therapy of patients with thrombocytopenia or thrombocytopathy. Disclosures Cancelas: Cellphire: Research Funding; Velico: Consultancy, Research Funding; Hemanext: Consultancy, Research Funding; Fresenius-Kabi: Research Funding; Cerus Co.: Research Funding; TerumoBCT: Consultancy, Research Funding; Macopharma Inc: Research Funding; Cytosorbents: Research Funding.

Antiplatelet Effectivity between Aspirin with Honey on Cardiovascular Disease Based on Bleeding Time Taken on Mice.

BACKGROUND:The prevalence of cardiovascular disease (CVD) and its mortality continues to increase. Various studies have shown aspirin can reduce CVD mortality but has adverse side effects. Research on a comparison between aspirin and honey has not been done, but both have antiplatelet effects.AIM:This study is aimed to prove the antiplatelet effects on honey and compare the antiplatelet effects of aspirin with honey based on the bleeding time in mice.METHODS:This study is a true experimental design with a post-test only control group using 32 male mice, Double Ditsch Webster, ± 3 months old, the weight of 20-30 g, divided into 4 groups. Consisting of a negative control group (placebo), aspirin and honey. The suspension has given orally for 12 days using the probe. The research was conducted at the Laboratory of Pharmacology Department of Pharmacology and Therapeutics Faculty of Medicine, the University of North Sumatra in September until December 2015. The data collected was bleeding time in mice. Data analysed by Shapiro Wilk test, Kruskal Wallis and Mann Whitney.RESULTS:The mean bleeding time was a placebo (102.88 seconds), aspirin (369.38 seconds) and honey (304.63 seconds). Mann Whitney test showed significant results in the aspirin and honey groups against the control group (placebo) with p = 0.001. There were no significant differences in the aspirin group against honey (p = 0.172). Honey has an antiplatelet effect in mice. The mean bleeding time in mice given honey is longer or closer to the mean bleeding time in the aspirin group.CONCLUSION:The results could be used as a basis for further research to determine its use in humans with cardiovascular disease.

Inhibitory effects of hydrogen on in vitro platelet activation and in vivo prevention of thrombosis formation

AimsHydrogen (H2) has antioxidant effects. The pharmacologic function of H2 in platelets is not yet clear. Therefore, in this study we sought to investigate the inhibitory effects of H2 on in vitro platelet activation and in vivo prevention of thrombus formation. Main methodsAfter platelets were incubated with H2-rich saline (HRS), platelet adhesion in whole human blood was assessed in fibrinogen-coated perfusion chambers, while rat platelet aggregation induced by ADP, collagen and H2O2 was detected through light transmission aggregometry. The level of P-selectin, thromboxane B2, nitric oxide (NO), malondialdehyde, reactive oxygen species (ROS), cGMP, extracellular signal-regulated kinases 1 and 2 (p-ERK1/2), and fibrinogen binding to platelets were evaluated in vitro. Besides, the in vivo effects were examined in arterio-venous shunt thrombosis, FeCl3-induced artery thrombus formation, and tail bleeding time in mice and rats. Key findingsHRS prolonged tail bleeding time in mice and rats, decreased thrombus weight and prolonged the time to occlusion in rat and mouse thrombosis models in vivo and inhibited platelet adhesion as well as aggregation in vitro. Additionally, HRS decreased P-selectin expression, release of thromboxane B2, ROS, and fibrinogen binding, but enhanced NO levels in H2O2-exposed platelets. HRS also decreased malondialdehyde levels in plasma of the rat arterial thrombosis or H2O2-exposed platelet model. Moreover, HRS increased cGMP level, decreased p-ERK1/2 (diminished with KT5823) in the platelets stimulated by H2O2. SignificanceThese results suggest that H2 has antithrombotic effects, which may be due to its antioxidant property and subsequent inhibition of platelet activation via NO/cGMP/PKG/ERK pathway.

Short-Acting Anti-VWF (von Willebrand Factor) Aptamer Improves the Recovery, Survival, and Hemostatic Functions of Refrigerated Platelets.

Refrigeration-induced binding of VWF (von Willebrand factor) to platelets contributes to the rapid clearance of refrigerated platelets. In this study, we investigate whether inhibiting VWF binding by a DNA-based aptamer ameliorates the clearance of refrigerated platelets without significantly impeding hemostatic functions. Approach and Results: Platelets were refrigerated with or without aptamer ARC1779 for 48 hours. VWF binding, the effective lifetime of ARC1779, platelet post-transfusion recovery and survival, and the hemostatic function were measured. ARC1779 treatment during refrigeration inhibited the platelet-VWF interaction. ARC1779-treated refrigerated murine platelets exhibited increased post-transfusion recovery and survival than untreated ones (recovery of ARC1779-treated platelets: 76.7±5.5%; untreated: 63.7±0.8%; P<0.01. Half-life: 31.4±2.36 hours versus 28.1±0.86 hours; P<0.05). A similar increase was observed for refrigerated human platelets (recovery: 49.4±4.4% versus 36.8±2.1%, P<0.01; half-life: 9.2±1.5 hours versus 8.7±0.9 hours, ns). The effective lifetime of ARC1779 in mice was 2 hours. Additionally, ARC1779 improved the long-term (2 hours after transfusion) hemostatic function of refrigerated platelets (tail bleeding time of mice transfused with ARC1779-treated refrigerated platelets: 160±65 seconds; untreated: 373±96 seconds; P<0.01). The addition of an ARC1779 antidote before transfusion improved the immediate (15 minutes after transfusion) hemostatic function (bleeding time of treated platelets: 149±21 seconds; untreated: 320±36 seconds; P<0.01). ARC1779 improves the post-transfusion recovery of refrigerated platelets and preserves the long-term hemostatic function of refrigerated platelets. These results suggest that a short-acting inhibitor of the platelet-VWF interaction may be a potential therapeutic option to improve refrigeration of platelets for transfusion treatment.

In Vitro Effects on Thrombin of Paris Saponins and In Vivo Hemostatic Activity Evaluation of Paris fargesii var. brevipetala.

The resource shortage of Rhizoma Paridis has never been effectively addressed, and the industry continues to search for alternative resources. The in vitro effects on thrombin of Paris saponins and in vivo hemostatic activity of Paris fargesii var. brevipetala (PF) were evaluated in this study. PF is considered to be an alternative source of Rhizoma Paridis (RP). The in vitro incubation experiment was designed to investigate the effects on thrombin activity of Paris saponin H (PS H) and saponin extract in PF. The bleeding time of mouse tail snipping was used to evaluate the in vivo hemostatic effects of Paris saponins. Also, in vivo changes in four blood coagulation parameters in rats after oral administration of different groups of Paris saponins were compared. The effects of Paris saponins on liver function and blood lipid parameters were examined in order to avoid drug-induced liver injury. Activity studies of thrombin after ultra-filtration centrifugation showed that Paris saponins were able to enhance thrombin activity. Ultra performance liquid chromatography mass spectrometry (UPLC-MS) analysis results of the substrates led us to speculate that there is a specific binding between Paris saponins and thrombin. PS H and Paris saponins in PF significantly shortened the bleeding time in mice. One pathway by which Paris saponins enhance in vivo blood coagulation is by increasing fibrinogen (FIB), among the four blood coagulation parameters in rats. At the same time, the effects on liver and blood lipid parameters were insignificant. P. fargesii var. brevipetala can be developed as an alternative medicinal source of Rhizoma Paridis.

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 Bβ 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.

Tannic Acid Inhibits Protein Disulfide Isomerase, Platelet Activation and Thrombus Formation

Tannic acid (TA) was a polyphenol that harbors anti-oxidant capacity. A recent report implied that surface coating with TA might blunt thrombosis via altering the structure of fibrinogen. However, the effect of TA on platelet function and in vivo thrombus formation has not been reported. In this study, we showed that TA inhibits PDI activity and attenuates platelet activation.To explore the effects of TA on platelet aggregation, gel-filtered human platelets from healthy human donors were pretreated with TA (10/30/50 μM) or vehicle (0.9% sodium chloride) before being stimulated by various agonists. Turbidity analyses on a Chronolog aggregometer showed that TA dose-dependently inhibited platelet aggregation induced by thrombin, SFLLRN, GYQGQV, collagen, CRP, U46619, and ristocetin. Next, we employed flow cytometry (FACS) to determine the role of TA in platelet activation, including α-granule secretion and integrin activation. Pretreatment of platelets with TA led to significant reductions in surface P-selectin expression and soluble fibrinogen binding, supporting the inhibition of diverse platelet activation pathways. Supportively, platelet spreading on immobilized fibrinogen was significantly suppressed by TA treatment. In addition, cell viability assay with Almar blue agent showed no detrimental impact of TA on the survival of platelets.To ask whether the antiplatelet role of TA might be translated into an antithrombotic efficacy, we tested the effect of TA in both ex vivo and in vivo thrombosis models. Calcein-labeled human whole blood was perfused through microfluidic channels coated with collagen, and adherent platelets were visualized under a fluorescent microscopy. However, treatment with TA suppressed the number of adherent platelets under flow conditions. Moreover, in laser-induced mouse cremaster muscle arteries, administration of TA (5mg/kg) significantly reduced the size of forming thrombi compared with the vehicle. Verification of bleeding risk using tail truncation assay indicated no prolongation of bleeding time in mice receiving TA. Thus, TA shows an antiplatelet effect and may also attenuate thrombus formation.To gain a mechanistic insight to the role of TA in platelet function, we performed a molecular docking screen of the structure of TA and platelet surface proteins using the Autodock Vina software, which displayed the binding of TA with protein disulfide isomerase at the enzymatic active center. We then measured the impact of TA on PDI reductase activity with the dieosin glutathione disulfide assay in vitro (di-GSSG), showing that TA significantly inhibited PDI activity in a concentration-dependent manner. The results were verified in platelets using the 3-(N-Maleimidylpropionyl) biocytin (MPB) labeling, which showed that TA abrogated thrombin-stimulated free thiol formation on platelet surface. Supportively, FACS demonstrated that TA significantly suppressed the binding of fluorescent-labeled PDI to Mn2+-activated platelet integrin β3. Taken together, our findings demonstrated that TA inhibits PDI activity and may become a novel antithrombotic agent. DisclosuresNo relevant conflicts of interest to declare.

The endogenous antimicrobial cathelicidin LL37 induces platelet activation and augments thrombus formation

AbstractPlatelet-associated complications including thrombosis, thrombocytopenia, and hemorrhage are commonly observed during various inflammatory diseases such as sepsis, inflammatory bowel disease, and psoriasis. Despite the reported evidence on numerous mechanisms/molecules that may contribute to the dysfunction of platelets, the primary mechanisms that underpin platelet-associated complications during inflammatory diseases are not fully established. Here, we report the discovery of formyl peptide receptor 2, FPR2/ALX, in platelets and its primary role in the development of platelet-associated complications via ligation with its ligand, LL37. LL37 acts as a powerful endogenous antimicrobial peptide, but it also regulates innate immune responses. We demonstrate the impact of LL37 in the modulation of platelet reactivity, hemostasis, and thrombosis. LL37 activates a range of platelet functions, enhances thrombus formation, and shortens the tail bleeding time in mice. By utilizing a pharmacological inhibitor and Fpr2/3 (an ortholog of human FPR2/ALX)–deficient mice, the functional dependence of LL37 on FPR2/ALX was determined. Because the level of LL37 is increased in numerous inflammatory diseases, these results point toward a critical role for LL37 and FPR2/ALX in the development of platelet-related complications in such diseases. Hence, a better understanding of the clinical relevance of LL37 and FPR2/ALX in diverse pathophysiological settings will pave the way for the development of improved therapeutic strategies for a range of thromboinflammatory diseases.