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

AIMS: Hydrogen (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 METHODS: After 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 FINDINGS: HRS 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. SIGNIFICANCE: These 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.

Plasma Phospholipid Transfer Protein Promotes Platelet Aggregation.

It remains unclear whether plasma phospholipid transfer protein (PLTP) is involved in hyper-coagulation or hypo-coagulation. This study investigated the direct effect of PLTP on platelet aggregation and the underlying mechanism. Washed platelets from humans or mice and mouse platelet-rich plasma and human recombinant PLTP were isolated. PLTP is present in human platelets. We assessed adenosine diphosphate (ADP)-, collagen- and thrombin-induced platelet aggregation, phosphatidylserine externalization and photothrombosis-induced cerebral infarction in mice. PLTP over-expression increased platelet aggregation, while PLTP deficiency had the opposing reaction. Human recombinant PLTP increased both mouse and human platelet aggregation in a dose-dependent manner. Phosphatidylserine externalization provides a water/lipid surface for the interaction of coagulation factors, which accelerates thrombosis. Compared with wild-type controls, platelets from PLTP transgenic mice had significantly more phosphatidylserine on the exterior surface of the plasma membrane, whereas platelets from PLTP-deficient mice had significantly less phosphatidylserine on the surface, thus PLTP influences fibrinogen binding on the plasma membrane. Moreover, recombinant PLTP together with ADP significantly increased phosphatidylserine exposure on the plasma membrane of PLTP-deficient platelets, thereby increasing fibrinogen binding. PLTP over-expression significantly accelerated the incidence of photothrombosis-induced infarction in mice, whereas PLTP deficiency significantly reduced the frequency of infarction. We concluded that PLTP promotes phosphatidylserine externalization at the plasma membrane of platelets and accelerates ADP- or collagen-induced platelet aggregation. This effect plays an important role in the initiation of thrombin generation and platelet aggregation under sheer stress conditions. Thus, PLTP is involved in hyper-coagulation. Therefore, PLTP inhibition could be a novel approach for countering thrombosis.

Characteristic enhancement of blood pressure V-shaped waves in sinoaortic-denervated rats in a conscious and quiet state.

A hemodynamic feature of chronic sinoaortic-denervated (SAD) rats is the increase in blood pressure variability (BPV) without significant changes in the average level of blood pressure (BP). The current study was designed to investigate the changes in BP V-shaped waves (V waves) in SAD rats. Sprague-Dawley (SD) rats were divided into 2 groups: SAD rats and sham-operated rats (n=13). Hemodynamics measurements were obtained in conscious, freely moving rats, four weeks after sinoaortic denervation or sham operation. V wave indices were evaluated in rats in both conscious and quiet states. Additionally, normal and high BPV was simulated by the production of V waves with different amplitudes. The results showed that the V wave amplitude was dramatically increased, with a significantly prolonged duration and reduced frequency in SAD rats. V wave BPV in SAD rats was significantly increased, though BP remained unchanged. The twenty-four hour BPV in all rats was positively correlated with amplitude, duration time and V wave BPV and negatively correlated with frequency. The systolic BP spectral powers in the low frequency range (0.38-0.45 Hz) were significantly reduced in the V waves of SAD rats. Moreover, there was a remarkable increase in mean BPV and a normal mean BP after simulating high BPV in SAD rats. These results suggest that enhancement of V waves might be a waveform character of BP in SAD rats in both the conscious and quiet states. These types of V waves appear to be related to a depression of sympathetic regulation of BP induced by sinoaortic denervation.

Salvia Miltiorrhiza Bge.f.alba Ameliorates the Progression of Monocrotaline-Induced Pulmonary Hypertension by Protecting Endothelial Injury in Rats.

Pulmonary hypertension (PH) is a life-threatening disease that is characterized by elevated pulmonary blood pressure, abnormally thickened pulmonary arteries, and right ventricular hypertrophy. Monocrotaline (MCT) has been used to generate an experimental model of PH in rats, with PH initiated from injuries of lung vascular endothelium. Salvia Miltiorrhiza Bge.f.alba is a widely used traditional herb in China, known to exert protective effects on vascular endothelial cell injury in animal experiments. However, the role of Salvia Miltiorrhiza Bge.f.alba in PH remains unclear. Thus, we investigated the effects of the aqueous extract of Salvia Miltiorrhiza Bge.f.alba (AESM) on MCT-induced PH and explored the pertinent mechanism. PH was induced in rats by a single subcutaneous injection of MCT (60 mg/kg body weight). Low or high dose (4.6 g/kg or 14 g/kg body weight) of AESM was then administered orally for 21 days to PH rats. Hemodynamic study showed that AESM reduced mean pulmonary artery pressure and improved right ventricle function. Lung pathological analysis revealed that AESM reduced wall thickness and lumen stenosis of pulmonary vessels. Also AESM ameliorated right ventricular hypertrophy. Measurement of biochemical parameters indicated that AESM decreased endothelin-1 and thromboxane A2 in plasma and increased nitrogen monoxide and prostacyclin in the plasma and reduced the increase of transforming growth factor ß1 in lung tissue. Our results suggest that AESM may ameliorate the progression of MCT-induced PH in rats, at least in part by its protective effect on endothelial injury. Therefore, Salvia Miltiorrhiza Bge.f.alba could be useful in the treatment of PH.

Cardioprotective Effect of Hydrogen-rich Saline on Isoproterenol-induced Myocardial Infarction in Rats.

BACKGROUND: Infusion with hydrogen gas-saturated saline has recently been reported to exert antioxidant and anti-inflammatory activity that may protect against organ damage induced by oxidative stress. Therefore because oxidative stress plays a significant role in the pathophysiology of myocardial infarction (MI), the aim of our study was to investigate whether hydrogen-rich saline has cardioprotective effects against isoproterenol-induced MI in rats. METHODS: An acute MI model was induced in male Wistar rats by subcutaneous injection of isoproterenol. Different doses of hydrogen-rich saline (5, 7.5, and 10 mL/kg body weight i.p.) or Vitamin C (250 mg/kg body weight i.g.) were administered to the rats. Oxidative stress indices including levels of myocardial marker enzymes, inflammatory cytokines, membrane-bound myocardial enzymes and histopathological changes were measured. RESULTS: Compared with those in isoproterenol-MI group, hydrogen-rich saline decreased malondialdehyde and 8-hydroxy-desoxyguanosine concentrations, enhanced superoxide dismutase and Na(+)-K(+)-ATPase activity, lowered Ca(2+)-ATPase activity and decreased interleukin-6 and tumour necrosis factor-α levels in the serum and/or cardiac tissue of rats. Hydrogen-rich saline pretreatment also diminished infarct size, improved left heart function, and ameliorated pathological changes of the left heart. CONCLUSION: From these results, hydrogen-rich saline exerts cardiovascular protective effects against isoproterenol-induced MI at least in part via interactions which evoke antioxidant and anti-inflammatory activities.

Increased active von Willebrand factor during disease development in the aging diabetic patient population.

Type 2 diabetes is known to cause endothelial activation resulting in the secretion of von Willebrand factor (VWF). We have shown that levels of VWF in a glycoprotein Ib-binding conformation are increased in specific clinical settings. The aim of the current study is to investigate whether active VWF levels increase during aging and the development of diabetes within the population of patients suffering from type 2 diabetes. Patients and controls were divided into two groups based on age: older and younger than 60 years of age. VWF antigen, VWF propeptide, VWF activation factor and total active VWF were measured. Patients older than 60 years of age had increased levels of total active VWF, VWF activation factor and VWF propeptide compared to younger patients and controls. All measured VWF parameters were associated with age in diabetic patients. Total active VWF and VWF propeptide correlated with the period of being diagnosed with diabetes. Regression analyses showed that especially the VWF activation factor was strongly associated with diabetes in patients older than 60 years of age. In conclusion, we found that the conformation of VWF could be involved in the disease process of diabetes and that the VWF in a glycoprotein Ib-binding conformation could play a role as risk marker during the development of diabetes in combination with an increase in age. Our study shows that the active quality of VWF was more important than the quantity.

Protective effects of hydrogen-rich saline on monocrotaline-induced pulmonary hypertension in a rat model.

BACKGROUND: Hydrogen-rich saline has been reported to have antioxidant and anti-inflammatory effects and effectively protect against organ damage. Oxidative stress and inflammation contribute to the pathogenesis and/or development of pulmonary hypertension. In this study, we investigated the effects of hydrogen-rich saline on the prevention of pulmonary hypertension induced by monocrotaline in a rat model. METHODS: In male Sprague-Dawley rats, pulmonary hypertension was induced by subcutaneous administration of monocrotaline at a concentration of 6 mg/100 g body weight. Hydrogen-rich saline (5 ml/kg) or saline was administered intraperitoneally once daily for 2 or 3 weeks. Severity of pulmonary hypertension was assessed by hemodynamic index and histologic analysis. Malondialdehyde and 8-hydroxy-desoxyguanosine level, and superoxide dismutase activity were measured in the lung tissue and serum. Levels of pro-inflammatory cytokines (tumor necrosis factor-α, interleukin-6) in serum were determined with enzyme-linked immunosorbent assay. RESULTS: Hydrogen-rich saline treatment improved hemodynamics and reversed right ventricular hypertrophy. It also decreased malondialdehyde and 8-hydroxy-desoxyguanosine levels, and increased superoxide dismutase activity in the lung tissue and serum, accompanied by a decrease in pro-inflammatory cytokines. CONCLUSIONS: These results suggest that hydrogen-rich saline ameliorates the progression of pulmonary hypertension induced by monocrotaline in rats, which may be associated with its antioxidant and anti-inflammatory effects.

Salvianolic acid B inhibits platelet adhesion under conditions of flow by a mechanism involving the collagen receptor alpha2beta1.

Salvianolic acid B (SAB) is a component of Danshen, a herb widely used in Chinese medicine, and was previously shown to exert a number of biological activities including inhibition of platelet function, but the exact mechanisms involved are unclear. SAB dose-dependently inhibited platelet deposition from flowing, anticoagulated whole blood to immobilized collagen at both venous and arterial shear rate, whereas platelet deposition to immobilized fibrinogen was not affected. The inhibitory effect of SAB on platelet adhesion to collagen was independent of alphaIIbbeta3, since SAB still inhibited platelet deposition in the presence of a alphaIIbbeta3-blocking peptide. SAB inhibited static platelet adhesion to a synthetic peptide specific for the collagen receptor alpha2beta1, whereas platelet adhesion to a glycoprotein VI-specific peptide was not affected. SAB inhibited binding of an antibody against alpha2beta1 to platelets as studied by flow cytometry, and inhibited the interaction of soluble alpha2beta1 to immobilized collagen in a solid phase binding assay. These combined results indicate that SAB inhibits platelet adhesion to immobilized collagen by interfering with the collagen receptor alpha2beta1.

The influence of the pulsatility of the blood flow on the extent of platelet adhesion.

A new improved flow system was developed to study the influence of blood flow pulsatility on platelet adhesion on adhesive proteins and bio-medical materials. The pulsatility was introduced by changing the shear rate every 15 s in blood that was aspirated through a perfusion chamber by a syringe pump. The advantage of this new system is that it avoids system related platelet activation. At steady low shear rate (300/s) after 5 min a collagen type III surface was covered for 24.2+/-3.8% with platelets. At steady high shear rate (1300/s) platelet coverage to collagen was 48.8+/-6.8%. When pulsatility was introduced by changing the shear rate was every 15 s form 300/s to 1300/s and vice-versa, platelet coverage after 5 min was increased to 60.4+/-4.0% (p<0.001). After 5 min perfusion samples were taken from the perfusate and the extent of platelet activation was measured. The significant difference in surface expression of P-selectin on platelets is only seen when comparing pulse flow with control (no flow). We concluded that a significant increase in platelet activation during blood pulsatile flow compared with steady flow, which results in an increased platelet adhesion to collagen.

Elevated plasma tissue-type plasminogen activator (t-PA) and soluble thrombomodulin in patients suffering from severe acute respiratory syndrome (SARS) as a possible index for prognosis and treatment strategy.

OBJECTIVE: To detect the presence of endothelial injury in patients with severe acute respiratory syndrome (SARS) via enhanced levels of tissue-type plasminogen activator (t-PA) and soluble thrombomodulin (sTM). METHODS: Case patients were from Xuanwu Hospital (Capital University of Medical Sciences, Beijing, China), and all of them met clinical criteria for SARS. Healthy controls were some of the hospital employees. Endothelial injury bio-markers tPA and sTM were detected by commercial ELISA-methods. RESULTS: Classic plasma markers of endothelial injury, tPA and sTM significantly elevated in SARS patients in comparison to controls [t-PA: 1.48 +/- 0.16 nmol/L versus 0.25 +/- 0.03 nmol/L (P<0.0001), and sTM: 0.26 +/- 0.06 nmol/L versus 0.14 +/- 0.02 nmol/L (P<0.05)]. The only patient who died had extremely high levels of these endothelial injury markers (t-PA: 2.77 nmol/L and sTM: 1.01 nmol/L). The likelihood ratio analysis indicated the excellent discriminating power for SARS at the optimal cut-point of 0.49 nmol/L for tPA and 0.20 nmol/L for sTM, respectively. Significant numerical correlations were found among these endothelial injury markers in SARS patients. The numerical coefficient of correlation Pearson r between t-PA and sTM was 0.5867 (P<0.05). CONCLUSION: Increased plasma concentrations of tPA and sTM in patients with SARS suggest the possibility of endothelial injury. SARS patients might need anticoagulant therapy or fibrinolytic therapy in order to reverse intraalveolar coagulation, microthrombi formation, alveolar and interstitial fibrin deposition. It may not only provide a useful treatment and prognostic index but also allow a further understanding of the pathological condition of the disease.