Cinnamaldehyde reduction of platelet aggregation and thrombosis in rodents

INTRODUCTION: Cinnamaldehyde (CA) has been reported to inhibit in vitro aggregation in human and rabbit platelets; however, little is known about the antithrombotic activities of CA in vivo. MATERIALS AND METHODS: We tested the effects of CA on collagen- or thrombin-induced aggregation of rat platelets in vitro. Hemorrhage and coagulation times of mice treated with CA by the tail-cutting or slide method were measured. We also tested the life-saving effects of CA on experimental models of thrombosis in mice and rats. The anti-platelet effects of CA were examined in rats. RESULTS: CA inhibited collagen- and thrombin-induced platelet aggregation in vitro in a concentration-dependent manner. In mice, CA administration (250, 500 mg/kg orally and 50, 100 mg/kg i.p.) markedly prolonged hemorrhage and coagulation times and effectively reduced the mortality rate of collagen-epinephrine-induced acute pulmonary thromboembolism. In an arteriovenous shunt thrombosis rat model, the CA administration (250, 500 mg/kg orally and 50, 100 mg/kg i.p.) for 10 days dose-dependently decreased thrombus weight. Administration of CA also significantly inhibited collagen-induced platelet aggregation in the rat platelet-rich plasma (PRP). CONCLUSIONS: The results demonstrate that CA may be a promising antithrombotic agent, and its antithrombotic activity may be due to anti-platelet aggregation activity in vitro and in vivo.     

Experimental arterial thrombosis regulated by androgen and its receptor via modulation of platelet activation

OBJECTIVES: The aim of our study is to elucidate whether experimental arterial thrombosis is regulated by physiological doses of androgen and its receptor via modulation of platelet activation. METHODS: Surgical castration was performed in male rats and ferric chloride (FeCl(3)), as a stimulator, induced the experimental arterial thrombosis. Testosterone was measured directly by chemiluminescent immunoassay on the Bayer ADVIA Centaur analyzer. Dihydrotestosterone (DHT) was determined by ELISA using a commercially available kit. A platelet aggregometer was used to assess aggregation, and a platelet adherometer was used to measure adhesion. The contents of TXB(2) and 6-Keto-PGF(1alpha) were assayed by radio-immunoassay using commercially available kits. RESULTS: Our data showed that DHT replaced restored circulating DHT of castrated rats to physiological levels, without being altered by treatment with flutamide. Castration caused significant increases in the thrombus area and weight in castrated rats as compared with control group. In PRP diluted with autologous PPP, ADP-induced platelet aggregation rate was only 9.10%. However, in PRP diluted with Tyrode's buffer, 1 microM ADP-induced platelet aggregation rate rose to 63.65%. In PRP diluted with Tyrode's buffer, and pretreated with DHT (1 nM, 2 nM), ADP-induced platelet aggregation was significantly lowered again. Platelet aggregation in PRP diluted with autologous PPP was enhanced in castrated rats as compared with sham-operated rats, and DHT (2 nM) replacement suppressed platelet aggregation in castrated PRP to the level similar to that of sham-operated rats. However, presence of flutamide (3 microM) significantly increased platelet aggregation in PRP diluted with autologous PPP or Tyrode's buffer. DHT (2 nM) replacement significantly inhibited the ADP-induced platelet adhesion. However, presence of flutamide (3 microM) increased ADP-induced platelet adhesion again. DHT replacement obviously reduced the ratio of TXB(2) to 6-keto-PGF(1alpha) in castrated rats. However, administration of flutamide and DHT to castrated rats caused an increase in the ratio of TxB(2) to 6-keto-PGF1alpha. CONCLUSION: Inhibition of experimental arterial thrombosis by androgen at physiological doses and its receptor is mediated via modulation of platelet activation.     

Thromboxane and prostacyclin biosynthesis in patients with acute spontaneous intracerebral hemorrhage

OBJECTIVE: Elevated levels of 11-dehydrothromboxane B2 (11-dehydro-TXB2) excreted in urine have been observed in acute ischemic stroke. This marker of platelet activation has not been investigated in patients with acute spontaneous intracerebral hemorrhage (ICH). METHODS: We examined 43 patients with spontaneous ICH and 23 controls. Urinary excretion rates of 11-dehydro-TXB2, 2,3-dinor-thromboxane B2 (2,3 dinor-TXB2) and 2,3-dinor-6-ketoprostaglandin F(1alpha) (2,3-dinor-PGF(1alpha)) during the first week and at 3 months after ICH were compared between patients who had or had not used aspirin and controls. RESULTS: On admission, ICH patients without aspirin use had significantly higher urinary levels of 11-dehydro-TXB2 (p<0.001), 2,3-dinor-TXB2 (p<0.001) and 2,3-dinor-PGF(1alpha) (p=0.019) than controls. Aspirin users had significantly lower urinary levels of these metabolites than nonusers. The metabolite levels of aspirin users on admission did not significantly differ from those of controls. The differences between aspirin users and nonusers leveled off during the following 3-5 days, however, as the blocking effect of aspirin on the production of TXA2 and PGI2 ceased. Three months after ICH, the metabolite excretion levels in all the patients were similar to those in nonusers of aspirin on admission. On admission, aspirin users had longer bleeding times (p=0.032) than nonusers, but aspirin use did not associate with impaired recovery or hematoma enlargement. CONCLUSIONS: Urinary excretion levels of 11-dehydro-TXB2, 2,3-dinor-TXB2 and 2,3-dinor-PGF1alpha were higher in patients with acute ICH than in controls. The levels in aspirin users were equally low as in controls but rose to the levels of the other patients within a few days. The metabolite levels remained high 3 months after ICH in all patients. Prior use of aspirin did not seem to cause hematoma enlargement.     

Role of metalloproteinases in platelet function

Platelets contain and release matrix metalloproteinases (MMPs), their inhibitors (TIMPs) and disintegrin metalloproteinases (ADAMs) including MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP (MMP-14), ADAM-10, ADAM-17, ADAMTS-13, TIMP-1, TIMP-2 and TIMP-4. These proteins exert several effects regulating platelet functions such as agonist-stimulated platelet adhesion and aggregation, tumour cell-induced platelet aggregation and platelet-leukocyte aggregation. In this review, mechanisms of MMPs, TIMPs and ADAMs on platelets are discussed.     

Ginsenoside Rg1 inhibits platelet activation and arterial thrombosis

Introduction

Derived from the root of Panax ginseng C.A.Mey, Panax notoginsenosides (PNS) is a widely used herbal medicine to treat atherothrombotic diseases in Asian medicine. Ginsenoside Rg1 is one of the main compounds responsible for the pharmaceutical actions of PNS. As platelets play pivotal roles in atherothrombogenesis, we therefore studied the effect of Rg1 on platelet activation and its underlying mechanisms.

Materials and Methods

Human platelets are obtained from healthy subjects. Platelet activation and the inhibition of Rg1 were assessed by Born aggregometer, flow cytmetry, flow chamber and western blot. The in vivo thrombosis model was induced by 10% FeCl₃ on mesenteric arterioles of wild type B57/b6 mice.

Results

Rg1 significantly inhibited platelet aggregation induced by thrombin, ADP, collagen and U46619, e.g., aggregation rate stimulated by 0.1 U mL^- 1 thrombin was decreased 46% by Rg1. Rg1 also reduced thrombin (0.1 U mL^- 1)-enhanced fibrinogen binding and P-selectin expression of single platelet by 81% and 66%, respectively. Rg1 affected αIIbβ3-mediated outside-in signaling as demonstrated by diminished platelet spreading on immobilized fibrinogen. Rg1 also decreased the rate of clot retraction in platelet rich plasma. Furthermore, Rg1 decreased platelet adhesion on collagen surface under a shear rate correlated to the arterial flow (1000 s^- 1) by approximately 70%. Western blot showed that Rg1 potently inhibited ERK phosphrylation. The in vitro findings were further evaluated in the mouse model of in vivo arterial thrombosis, and Rg1 was found to prolong the mesenteric arterial occlusion time (34.9 ± 4.1 min without and 64.3 ± 4.9 min with Rg1; p < 0.01).

Conclusions

Rg1 inhibits platelet activation via the inhibition of ERK pathway, and attenuates arterial thrombus formation in vivo.     

A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation

Introduction

Prasugrel, a P2Y₁₂ adenosine diphosphate (ADP) receptor antagonist effectively inhibits ADP-mediated platelet activation and aggregation, and may be useful in reducing vaso-occlusive crises in sickle cell disease (SCD). In this study, we assess the effect of prasugrel on biomarkers of platelet activation and coagulation in patients with SCD.

Materials and Methods

Twelve adult patients with SCD and 13 healthy subjects were examined before and after 12 ± 2 days of 5.0 or 7.5 mg/day oral prasugrel. Assessed cellular biomarkers included monocyte- and neutrophil-platelet aggregates, activated glycoprotein IIb-IIIa (GPIIbIIIa), P-selectin, CD40 ligand (CD40L), tissue factor (TF) expression on circulating platelets and on monocyte-platelet aggregates, and platelet-erythrocyte aggregates. Soluble biomarkers included CD40L, prothrombin fragment 1.2 (F1.2), thromboxane B₂ (TXB₂), P-selectin, and TF.

Results

Patients with SCD had increased platelet baseline activation compared to healthy subjects, as measured by percentages of monocyte-platelet aggregates, neutrophil-platelet aggregates, and platelets expressing CD40L. Likewise, baseline levels of soluble F1.2 and TXB₂ were elevated in patients with SCD compared to healthy subjects. After 12 days of prasugrel, patients with SCD had a significant reduction in platelet-monocyte aggregates that was not observed in healthy subjects. Following prasugrel administration, those with SCD maintained higher levels of monocyte-platelet aggregates and soluble F1.2, but had lower levels of platelet-erythrocyte aggregates and soluble TF compared to healthy subjects.

Conclusions

These results provide evidence for chronic platelet activation in the SCD steady state, activation that was in part attenuated by prasugrel, thereby suggesting that ADP may mediate platelet activation in SCD.     

I(4), a new synthetic sulfonylurea compound, inhibits the action of TXA(2)in vivo and in vitro on platelets and aorta vascular smooth muscle

Introduction

1-[4-[2-(4-Bromobenzene-sulfonamino)ethyl]phenylsulfonyl]-3-(trans-4-methylcy-clohexyl)urea(I₄, CAS865483-06-3); a totally synthetic new sulfonylurea compound, combining the hypoglycemic active structure of Glimepiride (CAS 93479-97-1) and anti-TXA₂ receptor (TP) active structure of BM-531(CAS 284464-46-6), was designed and synthesized. Its effects on TXA₂ synthesis and TP have not been reported yet.

Aim

To study the inhibitory effects of I₄ and its mechanisms of action on TXA₂ and TP.

Methods

Platelet aggregation studies were performed on human platelet, rat whole blood platelet and rabbit platelet, platelets aggregation was induced by TP agonist U-46619(stable analog of TXA₂, CAS 56985-40-1). Plasma TXB₂ and 6-keto-prostaglandin F_1α (6-keto-PGF_1α) were used as markers to determine the effect of I₄ on thromboxane synthesis. Fluo-3-AM was used to measure the cytosolic Ca^2 + concentrations ([Ca^2 +]_i) in rabbit platelet. Aorta rings with and without endothelium were prepared and aorta contraction was induced by U-46619. A model of type 2 diabetes mellitus was established by intraperitoneal injection of low dose of streptozocin to rats fed a high-calorie diet. Both normal rats and type 2 diabetic rats were used to assay the inhibitory effect of I₄ on platelet aggregation induced by U-46619.

Results

I₄ exhibited a higher inhibitory potency than Glimepiride on U-46619 induced platelet aggregation in vitro and in vivo. I₄ increased the ratio of plasma PGI₂/TXA₂ and decreased [Ca^2 +]_i release from platelet internal stores. In addition, I₄ presented a vasorelaxant activity on isolated rat aorta contraction induced by U-46619.Oral administration of I₄ (1 ~ 10 mg/kg) markedly and dose-dependently inhibited platelet aggregation in both normal rats and type 2 diabetic rats.

Conclusion

I₄ significantly inhibited platelet aggregation induced by U-46619 in vitro and in vivo, and rat aorta contraction. It probably acts by partly blocking TXA₂ action, decreasing the platelet intracellular Ca^2 +, and increasing the PGI₂/TXA₂ ratio.     

The lowest VE/VCO2 ratio best identifies chronic thromboembolic pulmonary hypertension

Introduction

The natural history of acute pulmonary embolism (PE) under treatment is about a gradual resolution of the thrombi, and uncommonly, the development of chronic thromboembolic pulmonary hypertension (CTEPH). We hypothesized that ventilatory efficiency parameters during cardiopulmonary exercise testing (CPET) may be able to monitor the process and predict CTEPH.

Methods

15 patients rehabilitated from acute PE (total resolution of thrombi), 44 patients with chronic PE (with residual thrombi), 66 patients with CTEPH, and 36 sedentary healthy controls performed incremental CPET.

Results

The lowest VE/VCO₂ was higher in CTEPH patients than that in chronic PE and rehabilitated patients (43.4 L/min vs 29.9 L/min vs 27.1 L/min, p < 0.005). The VE/VCO₂ slope (48.4 L/min/L/min vs 29.9 L/min/L/min vs 28.0 L/min/L/min, p < 0.005) and oxygen uptake efficiency plateau (OUEP) (37.1 L/min vs 27.0 L/min vs 25.2 L/min, p < 0.005) had the similar changes. In logistic regression analysis, the lowest VE/VCO₂ ≥ 34.35 L/min was the best predictor of CTEPH (OR 159.0, 95% CI 36.0-702.3, p < 0.001). The lowest VE/VCO₂ was higher in chronic PE patients compared with the controls (29.9 L/min vs 26.5 L/min, p < 0.05), but there was no difference between the rehabilitated patients and the controls. In multiple linear regression analysis, the percentage of vascular obstruction by ventilation-perfusion lung scanning (PVO) was the most significant independent predictor for indices of ventilatory efficiency in chronic PE and rehabilitated patients.

Conclusions

CTEPH is associated with weakened ventilatory efficiency. The lowest VE/VCO2 ratio has the best capability to predict CTEPH. Ventilatory inefficiency improves along with recovery of acute PE.     

A comparative study of antithrombotic and antiplatelet activities of different fucoidans from Laminaria japonica

Introduction

Fucoidans extracted from brown algae represent an intriguing group of natural fucose-enriched sulfated polysaccharide, with excellent anticoagulant, antimetastatic, antiangiogenic and anti-inflammatory activities. In the present study, we compared antithrombotic activities of four fucoidan fractions with different molecular weight and sulfated ester content from Laminaria japonica in an electrical induced arterial thrombosis and their potential mechanism underlying such activity.

Results and Conclusions

In vivo middle molecular weight (MMW) fucoidan fractions with molecular weight about 28000 and 35000 exhibited better antithrombotic activity in electrical induced arterial thrombosis than low molecular weight (LMW) fucoidan LF1 and LF2 (Mw 7600 and 3900). Inhibition of arterial thrombosis occurred at dose of 0.1-0.25 mg/kg for MMW fucoidans, accompanied with moderate anticoagulant activity and significant decrease of whole blood viscosity and hematocrit. The antithrombotic effects of MMW Fucoidans might be related with promotion of TFPI content and decrease of TXB2 content, without affecting platelet aggregation and 6-keto-PGF1α content in vivo. In contrast, LMW fucoidans showed a correlation among anticoagulant, antiplatelet and antithrombotic effects in vivo. Antithrombotic action of LF1 and LF2 required high dose of 2.5-10 mg/kg, concomitantly with anticoagulant activity and specific inhibition of platelet aggregation in vivo. Their antithrombotic effect might be related to their promotion of TFPI and 6-keto-PGF1α, down regulation of TXB2, without affecting hemorheology. These findings suggested that fucoidan fractions with different molecular weight acted on the antithrombotic action by different mechanism. By comparison, highly sulfated fucoidan LF2 with molecular weight of 3900 seemed to be a more suitable choice of antithrombotic drug for its antithrombotic activity accompanied with specific inhibitory activity on platelet aggregation, low anticoagulant activity and low hemorrhagic risk in vivo.     

MC-002 exhibits positive effects against platelets aggregation and endothelial dysfunction through thromboxane A2 inhibition

Introduction

Thromboxane A₂ (TXA₂) induces platelet aggregation and vasoconstriction, and agents that inhibit TXA₂ production or interaction with receptors may exert potential application in stroke therapy.

Aim

To illustrate the platelet aggregation antagonistic and endothelial protective effect of (E) - 3 - (3 - methoxy - 4 - ((3, 5, 6 - trimethylpyrazin - 2 - yl) methoxy) phenyl) sodium acrylate (MC-002) through TXA₂ inhibition and underline mechanisms.

Materials and methods

Platelets aggregation and thoracic aorta ring contraction of rabbits were induced by U46619. Human umbilical vein endothelial cells (HUVECs) were further applied to explore the protective effect of MC-002 on endothelium when exposed to tumor necrosis factor - α (TNF-α). MTT method was used to assess cell damage, and ELISA analysis was exerted to estimate nitrogen monoxide (NO), endothelin-1 (ET-1), thromboxane B₂ (TXB₂) and 6-keto-prostaglandin F1α (6-keto-PGF1α) releasing. Fluorescence spectrophotometry was conducted to determine intracellular calcium concentration ([Ca^2 +]_i), and western blotting method was applied to evaluate the protein expressions of intracellular adhesion molecule-1 (ICAM-1), P-selectin and nuclear factor-kappa B (NF-κB).

Results and conclusions

TXA₂ analog U46619 mediated obvious platelet aggregation and vasoconstriction. MC-002 inhibited platelet aggregation through administration in vivo and incubation with platelet in vitro, and relaxed aorta ring in endothelium dependent manner. MC-002 alleviated cell damage, [Ca^2 +]_i overload, ET-1 overexcretion and TXB₂ activation, but improved NO availability reduction in HUVECs treated with TNF-α. Furthermore, MC-002 downregulated ICAM-1, P-selectin and NF-κB overexpression induced by TNF-α. In conclusion, MC-002 exerted antiplatelet aggregation effect through TXA₂ inhibition and relieved inflammatory injury of endothelial cells through NF-κB signal pathway.     

Increased basal platelet activity, plasma adiponectin levels, and diabetes mellitus are associated with poor platelet responsiveness to in vitro effect of aspirin

INTRODUCTION: Aspirin is one of the most effective antiplatelet agents and is now commonly used to prevent vascular events. In some patients, however, recurrent vascular events have been demonstrated despite aspirin therapy. Our objective was to characterize individuals showing poor response to in vitro effect of aspirin, using PFA-100. METHODS: One hundred sixty-eight healthy male subjects were analyzed. We assessed platelet function tests, including PFA-100, whole blood aggregation, and optical platelet aggregation. Also measured were hemostatic and other parameters including von Willebrand factor (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), soluble vascular adhesion molecule-1 (sVCAM-1), high sensitive C-reactive protein (hs-CRP), and adiponectin. Poor responders were defined as having a collagen/epinephrine-induced closure time (CEPI-CT) under 250 s with PFA-100 when incubated with 10 microM aspirin, whereas good responders were defined as having a CEPI-CT of more than 250 s. RESULTS AND CONCLUSIONS: PFA-100 tests revealed that 40 subjects (24%) were poor responders (PR) and 128 (76%) were good responders (GR). Poor responsiveness was significantly associated with (1) higher basal platelet activities in PFA-100, as well as in whole blood aggregation and aggregometer;(2) increased level of adiponectin (8.8+/-4.1 micro g/mL [PR] vs 7.3+/-2.9 micro g/mL [GR], p=0.010);and (3) the presence of diabetes mellitus (17.5% [PR] vs 4.7% [GR], p=0.009). Importantly, whereas 24% of the subjects showed insufficient inhibition in PFA-100 when incubated with 10 microM aspirin, almost all subjects showed maximum inhibition with 30 microM aspirin. These observations suggest that higher doses of aspirin might overcome aspirin resistance.     

Inefficient exercise gas exchange identifies pulmonary hypertension in chronic thromboembolic obstruction following pulmonary embolism

Introduction

Persistent obstruction in the pulmonary artery following acute pulmonary embolism (PE) can give rise to both chronic thromboembolic pulmonary hypertension (CTEPH) and chronic thromboembolic disease without PH (CTED). We hypothesised that cardiopulmonary exercise testing (CPET) may be able to differentiate patients with CTEPH and CTED following unresolved PE which may help guide patient assessment.

Materials and Methods

Fifteen patients with CTEPH and 15 with CTED all diagnosed after PE underwent CT pulmonary angiography, CPET and resting right heart catheterisation. Exercise variables were compared between patients with CTEPH, CTED and 10 sedentary controls and analysed as predictors of a CTEPH diagnosis. Proximal thrombotic burden in CTEPH and CTED was quantified using CT criteria.

Results

Physiological dead space (Vd/Vt) (34.5 ± 11.4 vs 50.8 ± 6.6 %, p < 0.001) and alveolar-arterial oxygen gradient (29 ± 16 vs 46 ± 12 mmHg, p < 0.001) at peak exercise strongly differentiated CTED and CTEPH groups respectively. Resting ventilatory efficiency also differed from control subjects. In both univariate and multivariate analyses, peak exercise Vd/Vt predicted a diagnosis of CTEPH (ROC AUC > 0.88, 0.67 - 0.97) despite a similar degree of proximal thrombotic obstruction to the CTED group (67.5, 55 - 70% and 72.5, 60 - 80% respectively, p = 0.08).

Conclusions

Gas exchange at peak exercise differentiates CTED and CTEPH after PE that can present with no apparent relation to the degree of proximal thrombotic burden. A potential role for CPET exists in guiding further clinical investigations in this setting.     

Propofol and erythropoietin antioxidant properties in rat brain injured tissue

So far, several treatment modalities have been attempted to brain protection in cases such as brain trauma, stroke or brain hemorrhage. However, a treatment method that the effect begins immediately and definitely helpful has not been discovered yet. In this study, we aimed to compare the effects of propofol and erythropoietin (Epo) on brain injury caused by oxidative stress and antioxidant properties of these agents after closed head injury (CHI) in rats. For this study, female Wistar Albino rats were divided into five groups: non-traumatic control group, trauma performed group CHI, trauma with propofol (100 mg/kg) intraperitoneally (i.p.), trauma with Epo (5000 U/kg) i.p. and trauma with propofol and Epo performed study groups. Twenty-four hours after CHI, rats were sacrificed and the brains were removed. Superoxide dismutase (SOD), catalase (CAT), xanthine oxidase (XO), nitric oxide (NO), and malondialdehyde (MDA) levels were measured in brain tissue. MDA and NO levels were decreased significantly in Groups Epo, Propofol and Epo+Propofol than Group CHI (p<0.01). XO activity was significantly lower in Group Epo than Group CHI (p<0.05). Epo and propofol decreased oxidative stress by decreasing MDA and NO level in brain tissue after CHI. However, combination of Epo and propofol has no significant beneficial advantage than Epo or propofol alone.     

Role of vascular endothelial growth factor in adult hippocampal neurogenesis: implications for the pathophysiology and treatment of depression

It is now well established that the adult brain has the capacity to generate new neurons throughout life. Although the functional significance of adult neurogenesis still remains to be established, increasing evidence has implicated compromised hippocampal neurogenesis as a possible contributor in the development of major depressive disorder. Antidepressants increase hippocampal neurogenesis and there is evidence in rodent models that the therapeutic efficacy of these agents is attributable, in part, to this neurogenic effect. As such, considerable interest has been directed at identifying molecular signals, including neurotrophic factors and related signaling pathways that are associated with antidepressant action and could operate as key modulators in the regulation of neurogenesis in the adult hippocampus. One interesting candidate is vascular endothelial growth factor (VEGF), which is known to possess strong neurogenic effects. In this review, we will discuss the involvement of VEGF signaling in the etiology and treatment of depression.     

The anti-thrombotic effect of hydrogen sulfide is partly mediated by an upregulation of nitric oxide synthases

Introduction

Hydrogen sulfide (H₂S) known as a gasotransmitter is increasingly recognized for its anti-adhesive, anti-inflammatory and vasoactive properties. Due to these properties, we analysed anti-thrombotic effects of H₂S and the participation of the nitric oxide synthase (NOS)-pathway.

Materials and Methods

In individual venules of the ear of hairless SKH1-hr mice, thrombus formation was induced using a phototoxic light/dye-injury model and intravital fluorescence microscopy. Animals were treated intravenously with the H₂S donor Na₂S or NaCl as control. In a second setting, the NOS inhibitor L-NAME was applied intraperitoneally as a bolus 12 h prior to Na₂S treatment and thrombus induction. Blood and ear tissue were sampled after microscopy for assessment of plasma concentrations of soluble (s)P-selectin, sE-selectin, sVCAM-1 and sICAM-1 and expression of endothelial (e)NOS and inducible (i)NOS, respectively.

Results

When mice were treated with Na₂S, venular thrombus formation was significantly delayed versus that in animals of the NaCl-treated control group. While plasma levels of pro-thrombotic adhesion molecules were not affected by Na₂S, immunohistochemistry of the vessel walls showed a significant up-regulation of eNOS and iNOS expression within the Na₂S-treated group. The delay of thrombus formation in the Na₂S-group was partly but significantly reverted by application of L-NAME.

Conclusions

The anti-thrombotic efficacy of H₂S involves the NOS-pathway and may be of preventive and therapeutic value for clinical disorders with increased risk of thrombotic events.     

Nitric oxide production and blood corpuscle dynamics in response to the endocrine status of female rats

Introduction

Menopause is associated with marked changes in the endocrine profile, and increases the risk of vascular disease. However, the effect of hormones on the vascular system is still unclear. Therefore, the aim of this study was to examine the effects of endocrine status in female rats on nitric oxide (NO) production, inflammatory reactions and thrombus organization potency in the mesenteric microcirculation.

Materials and Methods

Female Wistar rats were divided into four groups: proestrus, metestrus, ovariectomized (OVX) and OVX plus estradiol treatment (OVX + E₂). NO was imaged using an NO-sensitive dye. The leukocyte and platelet velocities relative to the erythrocyte velocity (V_W/V_RC and V_P/V_RE, respectively) and thrombi sizes created by laser radiation were measured as thrombogenesis indices.

Results

Changes in endocrine status did not affect vascular function in the arterioles. However, in venules, NO production, V_W/V_RC and V_P/V_RE were decreased in the OVX group compared with the proestrus and metestrus states. Thrombus size was significantly greater in the OVX group than in the proestrus and metestrus states. Administration of E₂ for 2 weeks restored NO production, V_W/V_RC and V_P/V_RE to control levels.

Conclusions

Changes in endocrine status did not affect arterioles. In contrast, in venules, reduced estrogen levels led to a decrease in NO production, thereby increasing thrombogenesis. Estrogen replacement restored NO production and leukocyte and platelet velocities, reducing thrombus formation relative to OVX. Although it is unclear how E₂ reduces thrombus formation, our results indicate that leukocyte and platelet adhesion to the endothelium is a target for E₂ via NO.     

The role of inflammation in regulating platelet production and function: Toll-like receptors in platelets and megakaryocytes

Platelets have been extensively studied as hemostatic regulators, stopping uncontrolled flow of blood from an injured vessel and allowing for repair. However, multiple studies have shown that platelets can interact with bacterial proteins, particularly seen during sepsis and inflammation. Immune cells recognize pathogens through Toll-like Receptors (TLRs). These same receptors allow platelets to recognize bacterial proteins and regulate platelet immunity and function. This review examines the TLRs expressed on platelets and megakaryocytes and how these receptors affect the function of these cells. Through TLRs, platelets go beyond hemostatic regulation and play a pivotal role in inflammation and infection.     

Release reaction of brain-derived neurotrophic factor (BDNF) through PAR1 activation and its two distinct pools in human platelets

Brain-derived neurotrophic factor (BDNF) is a cytokine that plays important roles in the survival, development, and plasticity of neurons. BDNF is also expressed in peripheral tissues and cells. In this article, we report the BDNF release reaction through thrombin stimulation and its localization in human platelets.Platelets from healthy volunteers were subjected to PAR1-AP or PAR4-AP stimulation. Release of BDNF was measured by ELISA. Localization of BDNF in resting and thrombin-activated platelets was examined by immunoelectron microscopy and sucrose gradient ultracentrifugation following western blotting.BDNF was released dose-dependently with PAR1-AP concentrations with drastic release at low PAR1-AP concentrations and gently release at high PAR1-AP concentrations. Maximum BDNF release was approximately 37% at 132 μM PAR1-AP. In contrast, 3.8% BDNF was released with 1.13 mM PAR4-AP stimulation. In immunoelectron microscopy and sucrose gradient ultracentrifugation analyses, BDNF was detected not only in α-granules but also cytoplasm in of the resting platelets, and it was distributed in the swollen open canalicular system fused to α-granules at 1 min and disappeared at 5 min after stimulation by thrombin. However, BDNF in cytoplasm remained throughout platelet activation.In conclusions, we demonstrate that BDNF is released from platelets through predominately PAR1 regulation. Furthermore, we identified two pools of BDNF in the α-granules and cytoplasm of human platelets, and only BDNF in α-granules is released through platelet activation.     

Evaluation of the effects of vitamin A supplementation on adult rat substantia nigra and striatum redox and bioenergetic states: Mitochondrial impairment,increased 3-nitrotyrosine and α-synuclein,but decreased D2 receptor contents

Vitamin A at moderate to high doses is applied in the treatment of some life threatening pathological conditions, for instance cancers. Additionally, vitamin A at low concentrations is a known antioxidant molecule. However, by increasing vitamin A (or its derivatives) concentrations, there is an increase in the levels of oxidative stress markers in several experimental models. Furthermore, it was reported that vitamin A therapy at high doses might induce cognitive decline among the patients, which may become anxious or depressive, for example, depending on vitamin A levels intake. We have previously reported increased levels of oxidative stress markers in rat substantia nigra and striatum. However, the mechanism by which this vitamin altered the redox environment in such rat brain regions remains to be elucidated. In the herein presented work, we have investigated the effects of vitamin A supplementation at clinical doses (1000–9000 IU/kg day^− 1) for 28 days on rat substantia nigra and striatum mitochondrial electron transfer chain (METC) activity, which may produce superoxide anion radical (O₂^−•) when impaired. Additionally, the levels of non-enzymatic antioxidant defenses were evaluated, as well as 3-nitrotyrosine, α- and β-synucleins and TNF-α levels through ELISA assay. We observed impaired METC in both rat brain regions. Moreover, we found increased O₂^−• production and nitrotyrosine content in the nigrostriatal axis of vitamin A-treated rats, suggesting that the use of vitamin A at therapeutic doses may be rethought due to this toxic effects found here.