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ABSTRACT: Platelets are the primary players in both thrombosis and hemostasis. Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA)
are important signaling molecules in the regulation of platelet function, such as adhesion, aggregation, and secretion. Elevation
of intracellular cAMP, which induces the activation of PKA, results in the inhibition of platelet function. Thus, tight control
of the intracellular cAMP/PKA signaling pathway has great implications for platelet-dependent hemostasis and effective cardiovascular
therapy. In this review, we summarize the PKA substrates and their contributions to platelet function, especially the advancing
understanding of the cAMP/PKA-dependent signaling pathway in platelet physiology. In addition, we suggest the possibility
that cAMP/PKA is involved in the platelet procoagulant process and receptor ectodomain shedding.
Frontiers of Biology in China 04/2012; 4(1):7-14.
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ABSTRACT: The gelatin plasma substitute is often polydisperse and heterogenous, making it difficult to determine the elimination rate and half-life in the body. In this study, one method was developed based on quantitative determination of hydroxyproline derivatives. Two plasma substitutes were prepared by succinylation and genipin-crosslinking, respectively. After transfusion, the blood samples were hydrolyzed and derivatized, and then analyzed by HPLC. A two-phase exponential association equation was used for fitting the time-concentration curves. The results indicated that this method could be used for quantitative determination of gelatin in blood, and the pharmacokinetic parameters such as elimination rate and half-life.
Artificial Cells Blood Substitutes and Biotechnology (formerly known as Artificial Cells Blood Substitutes and Immobilization Bi 02/2011; 39(1):19-25. · 0.94 Impact Factor
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ABSTRACT: Serious thrombotic and hemorrhagic problems or even fatalities evoked by either microgravity or hypergravity occur commonly in the world. We recently reported that platelet functions are inhibited in microgravity environments and activated under high-G conditions, which reveals the pathogenesis for gravity change-related hemorrhagic and thrombotic diseases. However, the mechanisms of platelet functional variations under different gravity conditions remain unclear. In this study we show that the amount of filamin A coimmunoprecipitated with GPIbalpha was enhanced in platelets exposed to modeled microgravity and, in contrast, was reduced in 8 G-exposed platelets. Hypergravity induced actin filament formation and redistribution, whereas actin filaments were reduced in platelets treated with modeled microgravity. Furthermore, intracellular Ca2+ levels were elevated by hypergravity. Pretreatment of platelets with the cell-permeable Ca2+ chelator BAPTA-AM had no effect on cytoskeleton reorganization induced by hypergravity but significantly reduced platelet aggregation induced by ristocetin/hypergravity. Two anti-platelet agents, aspirin and tirofiban, effectively reversed the shortened tail bleeding time and reduced the death rate of mice exposed to hypergravity. Furthermore, the increased P-selectin surface expression was obviously reduced in platelets from mice treated with aspirin/hypergravity compared with those from mice treated with hypergravity alone. These data suggest that the actin cytoskeleton reorganization and intracellular Ca2+ level play key roles in the regulation of platelet functions in different gravitational environments. The results with anti-platelet agents not only further confirm the activation of platelets in vivo but also suggest a therapeutic potential for hypergravity-induced thrombotic diseases.
Journal of Applied Physiology 05/2010; 108(5):1241-9. · 3.75 Impact Factor
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ABSTRACT: Carboxylmethylated konjac glucomannan (CKGM) is a carboxylmethylated polymer of mannose and glucose that is derived from the plant Amorphophallus konjac cultivated in East Asia. The CKGM solution had a high volume-expanding efficacy and was evaluated as a plasma substitute in the present study. Ameliorative hemorrhagic shock rabbits were used as the model animals. The in vivo hemodynamic and hemorheologic properties, including blood pressure, blood viscosity, hematocrit, erythrocyte deformation index and erythrocyte aggregation index, were measured in animals treated in the CKGM solution. The in vitro colloid osmotic pressure (COP) of the CKGM solution was measured to estimate its plasma-expanding efficacy. These parameters of the CKGM-treated group were compared with groups exposed to four other treatments: human serum albumin (HSA), hydroxyethyl starch (HES), polygeline and normal saline. The CKGM solution showed an exceptionally higher COP than other therapy solutions. For example, the COP of 1% (weight in volume [w/v]) CKGM solution is comparable to those of 6% (w/v) HES solution and 5% (w/v) HSA solution. Accordingly, the CKGM solution can be transfused in a much lower dosage while maintaining its plasma-expanding efficacy. The CKGM-treated group showed an improved intravascular persistence and good hemodynamic and hemorheological properties. Biopsy analysis suggested no organ dysfunction in the group treated in CKGM solution. Moreover, the high plasma-expanding efficacy and inexpensive availability of the CKGM solution may facilitate its clinical application as a potential plasma substitute.
Glycobiology 04/2010; 20(8):950-8. · 3.58 Impact Factor
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ABSTRACT: Hemorrhage is a significant pathological feature of some fever or hyperthermia-related diseases, such as dengue fever and heatstroke. Although the mechanisms of hemorrhage in these diseases are thought to be complex, whether there is an association between hemorrhage and hyperthermia or fever remains unclear. Platelets play a central role in maintaining integrity of endothelium and biological hemostasis. To explore the effect of hyperthermia on platelet physiology, platelet-rich plasma or washed platelets were incubated at hypothermia (22 degrees C), normothermia (37 degrees C) or hyperthermia (40 and 42 degrees C) for 1 or 2 hours. ADP and alpha-thrombin induced platelet aggregations were obviously reduced in platelets incubated at hyperthermia. Hyperthermia induced apoptotic events in platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 dependent gelsolin cleavage and phosphatidylserine exposure. Furthermore, hyperthermia incurred platelet glycoprotein Ibalpha ectodomain shedding. Thus, these data suggest that hyperthermia induces platelet apoptosis and dysfunction. These findings have important implications for the pathogenesis of hemorrhage in fever or hyperthermia-related diseases, and also suggest that attention should be paid to platelet apoptosis under relatively high temperature conditions.
Platelets 02/2010; 21(3):229-37. · 1.85 Impact Factor
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ABSTRACT: Calmodulin (CaM) antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. In platelets, CaM has been found to bind directly to the cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet functions. However, it is still unknown whether CaM antagonists induce platelet apoptosis. Here we show that CaM antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), tamoxifen (TMX), and trifluoperazine (TFP) induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, and phosphatidylserine exposure. CaM antagonists did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP-, botrocetin-, and alpha-thrombin-induced platelet aggregation, platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonists. Furthermore, cytosolic Ca(2+) levels were obviously elevated by both W7 and TMX, and membrane-permeable Ca(2+) chelator BAPTA-AM significantly reduced apoptotic events in platelets induced by W7. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis. The elevation of the cytosolic Ca(2+) levels may be involved in the regulation of CaM antagonists-induced platelet apoptosis.
Thrombosis Research 02/2010; 125(4):340-50. · 2.44 Impact Factor
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ABSTRACT: Shear-induced platelet adhesion through the interaction of glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) exposed at the injured vessel wall or atherosclerotic plaque rupture is a prerequisite for the physiological hemostatic process or pathological thrombus formation in stenosed arteries. Here we show that shear-induced interaction of platelets with immobilized VWF results in GPIbalpha ectodomain shedding. Washed platelets were exposed to VWF-coated glass capillary or cone-and-plate viscometer at different shear rates, and GPIbalpha ectodomain was shed from platelets, while a small mass of GPIbalpha COOH-terminal peptide, approximately 17 kDa, was increased correspondingly. The extent of GPIbalpha shedding was enhanced with the concentration of immobilized VWF and the time duration of constant shear stress, whereas it was obviously reduced with the decreased number of adherent platelets. Pretreatment of platelets with membrane-permeable calpain inhibitors and metalloproteinase inhibitor abolished shear-induced GPIbalpha shedding. Furthermore, GPIbalpha shedding was obviously diminished by anti-integrin-alphaIIbbeta3 monoclonal antibody SZ21, phosphatidylinositol 3-kinase inhibitor wortmannin, and cell-permeable calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. These results indicate that shear-induced platelet-VWF interaction results in calpain and metalloproteinase-dependent GPIbalpha ectodomain shedding. These findings not only have a physiological implication in understanding the presence of glycocalicin in normal circulation, but also suggest a novel mechanism for the negative regulation of platelet function and the limitation of platelet thrombus infinite formation under pathophysiological flow conditions.
AJP Heart and Circulatory Physiology 10/2009; 297(6):H2128-35. · 3.71 Impact Factor
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ABSTRACT: Many serious thrombotic and haemorrhagic diseases or fatalities have been documented in human being exposed to microgravity or hypergravity environments, such as crewmen in space, roller coaster riders, and aircrew subjected to high-G training. Some possible related organs have been examined to explore the mechanisms underlying these gravity change-related diseases. However, the role of platelets which are the primary players in both thrombosis and haemostasis is unknown. Here we show that platelet aggregation induced by ristocetin or collagen and platelet adhesion to von Willebrand factor (VWF) were significantly decreased after platelets were exposed to simulated microgravity. Conversely, these platelet functions were increased after platelets were exposed to hypergravity. The tail bleeding time in vivo was significantly shortened in mice exposed to high-G force, whereas, was prolonged in hindlimb unloaded mice. Furthermore, three of 23 mice died after 15 minutes of -8 Gx stress. Platelet thrombi disseminated in the heart ventricle and blood vessels in the brain, lung, and heart from the dead mice. Finally, glycoprotein (GP) Ibalpha surface expression and its association with the cytoskeleton were significantly decreased in platelets exposed to simulated microgravity, and obviously increased in hypergravity-exposed platelets. These data indicate that the platelet functions are inhibited in microgravity environments, and activated under high-G conditions, suggesting a novel mechanism for gravity change-related haemorrhagic and thrombotic diseases. This mechanism has important implications for preventing and treating gravity change-related diseases, and also suggests that special attentions should be paid to human actions under different gravity conditions.
Thrombosis and Haemostasis 06/2009; 101(5):902-10. · 5.04 Impact Factor
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ABSTRACT: The interaction of platelet glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) exposed at the injured vessel wall initiates platelet adhesion and thrombus formation. Thus GPIbalpha ectodomain shedding has important implications for thrombosis and hemostasis. A disintegrin and metalloproteinase 17 (ADAM17) was identified recently to play an essential role in agonist induced GPIbalpha shedding. Here we show that prolonged inhibition of protein kinase A (PKA) results in metalloproteinase-dependent GPIbalpha shedding.
GPIbalpha was shed from platelets prolongedly incubated with PKA inhibitors in a dose-dependent manner. In platelets treated with PKA inhibitor H89, the level of GPIbalpha shedding was significantly higher than that in calcium ionophore or alpha-thrombin treated platelets, however, P-selectin surface expression was significantly lower. PKA inhibition mediated GPIbalpha shedding was reversed by PKA activator forskolin and partially inhibited by membrane-permeable calpain inhibitors. Furthermore, the metalloproteinase inhibitor GM6001 or EDTA completely inhibited H89 induced GPIbalpha shedding, indicating that it was metalloproteinase-dependent. Time course experiments revealed that the maximum GPIbalpha shedding occurred at 30 minutes after treatment with PKA inhibitor. Platelets prolongedly treated with PKA inhibitor exhibited significant decrease in botrocetin-induced aggregation and shear-induced adhesion on VWF.
These data show that prolonged inhibition of PKA results in metalloproteinase-dependent platelet GPIbalpha ectodomain shedding. This finding has physiological implications for hemostasis and limiting thrombus infinite formation after platelet activation, and it also suggests a novel strategy to develop new drugs for thrombotic diseases.
Thrombosis Research 02/2009; 124(1):101-9. · 2.44 Impact Factor
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ABSTRACT: Inulin, a plant-source polysaccharide, was cross-linked and evaluated as a new potential plasma expander in vitro and in vivo. The cross-linked inulin (CLI) in three different molecular sizes (54, 100 and 146 kDa) was characterized by gel filtration chromatography coupled with multi-angle laser light scattering. Their in vivo physiological properties, including blood pressure and organ function, were tested in a rabbit model. Compared to the other two CLI samples, the CLI2 with a molecular weight (Mw) of 100 kDa is advisable as a potential plasma expander for its strong expansion efficacy and no organ dysfunction. Moreover, CLI2 showed a high colloid osmotic pressure (COP) where the COP of 3% CLI2 is equal to that of 6% HES. Thus, much less CLI2 can be infused with the same effects as normal HES, and its potential side effects can be minimized.
Carbohydrate Polymers.
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ABSTRACT: IntroductionThe interaction of platelet glycoprotein (GP) Ibα with von Willebrand factor (VWF) exposed at the injured vessel wall initiates platelet adhesion and thrombus formation. Thus GPIbα ectodomain shedding has important implications for thrombosis and hemostasis. A disintegrin and metalloproteinase 17 (ADAM17) was identified recently to play an essential role in agonist induced GPIbα shedding. Here we show that prolonged inhibition of protein kinase A (PKA) results in metalloproteinase-dependent GPIbα shedding.Methods and ResultsGPIbα was shed from platelets prolongedly incubated with PKA inhibitors in a dose-dependent manner. In platelets treated with PKA inhibitor H89, the level of GPIbα shedding was significantly higher than that in calcium ionophore or α-thrombin treated platelets, however, P-selectin surface expression was significantly lower. PKA inhibition mediated GPIbα shedding was reversed by PKA activator forskolin and partially inhibited by membrane-permeable calpain inhibitors. Furthermore, the metalloproteinase inhibitor GM6001 or EDTA completely inhibited H89 induced GPIbα shedding, indicating that it was metalloproteinase-dependent. Time course experiments revealed that the maximum GPIbα shedding occurred at 30 minutes after treatment with PKA inhibitor. Platelets prolongedly treated with PKA inhibitor exhibited significant decrease in botrocetin-induced aggregation and shear-induced adhesion on VWF.ConclusionsThese data show that prolonged inhibition of PKA results in metalloproteinase-dependent platelet GPIbα ectodomain shedding. This finding has physiological implications for hemostasis and limiting thrombus infinite formation after platelet activation, and it also suggests a novel strategy to develop new drugs for thrombotic diseases.
Thrombosis Research.
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ABSTRACT: Hemorrhage is a significant pathological feature of some fever or hyperthermia-related diseases, such as dengue fever and heatstroke. Although the mechanisms of hemorrhage in these diseases are thought to be complex, whether there is an association between hemorrhage and hyperthermia or fever remains unclear. Platelets play a central role in maintaining integrity of endothelium and biological hemostasis. To explore the effect of hyperthermia on platelet physiology, platelet-rich plasma or washed platelets were incubated at hypothermia (22 C), normothermia (37 C) or hyperthermia (40 and 42 C) for 1 or 2 hours. ADP and -thrombin induced platelet aggregations were obviously reduced in platelets incubated at hyperthermia. Hyperthermia induced apoptotic events in platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 dependent gelsolin cleavage and phosphatidylserine exposure. Furthermore, hyperthermia incurred platelet glycoprotein Ib ectodomain shedding. Thus, these data suggest that hyperthermia induces platelet apoptosis and dysfunction. These findings have important implications for the pathogenesis of hemorrhage in fever or hyperthermia-related diseases, and also suggest that attention should be paid to platelet apoptosis under relatively high temperature conditions.
