Hsp47 Inhibitor Col003 Attenuates Collagen-Induced Platelet Activation and Cerebral Ischemic–Reperfusion Injury in Rats

Ischemic stroke is a major type of stroke worldwide currently without effective treatment, although antiplatelet therapy is an existing option for it. In previous studies, heat shock protein 47 (Hsp47) was found to be expressed on the surface of human and mice platelets and to strengthen the interaction between platelets and collagen. In recent years, Col003 was discovered to inhibit the interaction of Hsp47 with collagen. We evaluated whether the Hsp47 inhibitor Col003 is a promising therapeutic agent for ischemic stroke. Here, we first verified that Hsp47 is also expressed on the surface of rat platelets, and its inhibitor Col003 significantly inhibited thrombus formation in the FeCl3-induced rat carotid arterial thrombus model. Both Col003 and clopidogrel did not alter the bleeding time or coagulation parameters, while aspirin increased the tail-bleeding time (p < 0.05). The low cytotoxicity level of Col003 to rat platelets and human liver cells was similar to those of aspirin and clopidogrel. Col003 inhibited collagen-induced platelet aggregation, adhesion, [Ca2+]i mobilization, P-selectin expression, reactive oxygen species production and the downstream signal pathway of collagen receptors. The results of the middle cerebral artery occlusion model indicated that Col003 has a protective effect against cerebral ischemic–reperfusion injury in rats. The Hsp47 inhibitor Col003 exerted antiplatelet effect and protective effect against brain damage induced by ischemic stroke through the inhibition of glycoprotein VI (GPVI)and mitogen-activated protein kinase (MAPK) signaling events, which might yield a new antiplatelet agent and strategy to treat ischemic stroke.

Effects of Glycosaminoglycan from Urechis Unicinctus on the P2Y12 Receptor Signaling Pathway in Rat Platelets

This study aims to investigate the effects of glycosaminoglycan (GAG) from Urechis unicinctus on the P2Y12 receptor signaling pathway in rat platelets. The concentrations of cyclic adenosine monophosphate (cAMP), thromboxane B2 (TXB2) and glycoprotein IIb/IIIa (GPIIb/IIIa) in rat platelets were determined using enzyme-linked immunosorbent assay (ELISA) kits. The phosphorylation levels of protein kinase A (PKA) and vasodilator-stimulated phosphoprotein (VASP) in rat platelets were detected through Western blot. The expression of P2Y12 gene in rat platelets was analyzed via real-time fluorescence quantitative PCR and reverse-transcription PCR. It was observed that GAG significantly increased the cAMP content (plessthan 0.05, plessthan 0.01) and decreased the TXB2 and GPIIb/IIIa concentrations (plessthan 0.05,plessthan 0.01) in rat platelets. GAG significantly enhanced the phosphorylation levels of PKA and VASP in rat platelets plessthan 0.01) and had a synergistic effect with the P2Y12 receptor blocker. GAG significantly reduced the expression level of P2Y12 gene in rat platelets (plessthan 0.01). We speculated that GAG from U. unicinctus inhibited platelet aggregation in rats through the P2Y12 receptor signaling pathway.

Anti-platelet aggregation effects of extracts from Arbutus unedo leaves

It is well known that platelet hyperactivity is a risk factor for cardiovascular diseases such as atherosclerosis, stroke and myocardial infarction. This study aimed to examine the effects of extracts enriched in flavonoids obtained from Arbutus unedo leaves on platelet aggregation. Rat platelets were prepared and incubated in vitro with different doses of the tested extracts, and aggregation was trigged by physiological agonists. Platelet treatment with increasing concentrations (0.1 - 1 mg/ml) of diethyl ether extract (genins = free flavonoids) or ethyl acetate extract (hetrosidic flavonoids) inhibited platelet aggregation evoked by thrombin in a concentration-dependant manner. The IC50 values were 0.22 ± 0.03 and 0.36 ± 0.05 mg/ml for genins and heterosidic flavonoids respectively. Treatment with Arbutus unedo extracts also significantly reduced the initial rate of platelet aggregation. At 1 mg/ml, the rate inhibition was 97.8 ± 0.74 and 90.8 ± 1.55 % for genins and heterosidic flavonoids respectively. In addition, flavonoids significantly inhibited platelet aggregation induced by ADP, collagen or epinephrine. We conclude that Arbutus unedo extracts show antiaggregant effects due mainly to flavonoids. These results may partly explain the traditional use of Arbutus unedo leaves for the treatment of cardiovascular disorders such as hypertension.

Influence of glycosaminoglycan from Mactra veneriformison antiplatelet aggregation

In this study, influence of glycosaminoglycan from Mactra veneriformis on antiplatelet aggregation in rats was studied. The results showed that glycosaminoglycan (GAG) from Mactra veneriformis could reduce the platele aggregation and the platelet adhesion rates of rats in vivo and in vitro (P<0.05, P<0.01), attenuate calcium ion concentration in rat platelets (P<0.01), increase cAMP concentration in rat platelets (P< 0.05, P<0.01), diminish TXB2 concentrationin in rat plasma (P<0.01), elevate 6-keto-PGF1a concentration in rat plasma (P<0.05, P<0.01) . This study suggests that GAG has obviously inhibitory effects on ADP-induced platelet aggregation in rats, which might account for its anti-thrombitic activity.

Red Wine Inhibits Aggregation and Increases ATP-diphosphohydrolase (CD39) Activity of Rat Platelets in Vitro

Moderate consumption of red wine has been shown to exert a peculiar cardioprotective effect compared with other alcoholic beverages; inhibition of platelet aggregation seems to be one of the mechanisms underlying this beneficial effect. CD39/ATP-diphosphohydrolase is an integral membrane glycoprotein metabolizing ATP and ADP to AMP; in concert with CD73/ecto-5′-nucleotidase, it contributes to extracellular adenosine accumulation. CD39 is considered a key modulator of thrombus formation; it inhibits platelet aggregation by promoting ADP hydrolysis. There is evidence that red wine consumption increases CD39 activity in platelets from streptozotocin-induced diabetic rats. Here we show that two kinds of Aglianico red wines inhibit aggregation and increase ATP-and ADPase activity in rat platelets.