Stimulated Platelet Aggregation Research Articles

Qualification and Application of a Liquid Chromatography-Tandem Mass Spectrometric Method for the Simultaneous Quantification of Triflusal and its Active Metabolite Desacetyl Triflusal in Human Plasma Using Solid Phase Extraction Technique

Background: Triflusal and Desacetyl Triflusal prevents the formation of blood clots by inhibiting the enzyme cyclooxygenase, and reducing production of thromboxane A2 which is a stimulator of platelet aggregation and thereby reduces the risk of strokes, heart attacks or other serious circulatory problems. Introduction: A rapid, sensitive, specific, accurate, isocratic reversed-phase liquid chromatography coupled with triple quadrapole mass spectrometry method in its tandem operation mode was developed and validated for the simultaneous quantification of Triflusal and Desacetyl Triflusal, in human plasma. Method: Salicylic Acid and Hydrochlorothiazide are used as internal standards. The analytes were extracted from human plasma samples by solid-phase extraction technique using a Strata-X 33 µm polymeric sorbent. The reconstituted samples were chromatographed on an Oyster ODS3 100 x 4.6mm, 5µ column using a mobile phase composed of acetonitrile 40:60 (v/v) - 2mM ammonium acetate solutions (pH 3.25 ± 0.3) at a flow rate of 3.2 mL/min. The detection of precursor and product ions of analytes and their internal standards was performed by multiple reaction monitoring mode via a turbo ion spray interface in negative mode by MDS SCIEX API-3000. Result: The calibration curves obtained were linear over the concentration range of 0.100 to 35.086 µg/mL for Triflusal, 1.00 to 120.06 µg/mL for Desacetyl Triflusal with a correlation coefficient r ≥ 0.9952. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. The intra and inter batch precision was ≤ 12.7 for Triflusal and Desacetyl Triflusal with accuracy (%RE) between -6.6 and 6.9. Stability testing showed that all analytes were stable in plasma at 25 °C for 17.83 h, seven freeze–thaw cycles, and at 70°C for 53.65 days. The analytical method was successfully applied to an in vivo study evaluating the pharmacokinetics of Triflusal and Desacetyl Triflusal following single dose oral administration in 24 healthy volunteers in an open label, twoperiod, two sequence, randomized, crossover protocol under fasting conditions. Conclusion: Based on the 90% confidence interval of the individual ratios for Cmax and AUC0-inf, it was concluded that the method is efficient with a very short running time of 3.2min, sufficiently sensitive and suitable for pharmacokinetic studies. Keywords: Liquid chromatography, mass spectrometry, analysis, triflusal, desacetyl triflusal, solid phase extraction, pharmacokinetics, bioavailability.

Mildly elevated unconjugated bilirubin is associated with reduced platelet activation-related thrombogenesis and inflammation in Gilbert’s syndrome

Gilbert’s syndrome (GS) is associated with a mild unconjugated hyperbilirubinemia, increased circulating antioxidant capacity, and reduced cardiovascular disease (CVD) risk. The current study investigated whether mildly elevated circulating unconjugated bilirubin (UCB) is negatively associated with multiple thrombotic risk factors including platelet activity, hemostatic function, and inflammation in individuals with GS. Blood samples were collected from matched GS and control subjects (14 per group). Activation-dependent platelet surface marker expression of PAC-1 (binds to GPIIb/IIIa surface receptors on activated platelets) and CD62P (marker for P-selectin released from activated degranulated platelets) was assessed in adenosine diphosphate (ADP)-stimulated platelets using flow cytometry. Exogenous agonists, ADP, collagen, and arachidonic acid (AA), were used to stimulate platelet aggregation. A statistically significant decrease in the expression of P-selectin (P = 0.030) on activated platelets was observed in GS subjects. Collagen and AA-induced platelet aggregation were significantly (P = 0.018; P = 0.032 for respective agonists) reduced in GS versus control group. Elevated UCB (P = 0.001) and high density lipoprotein (P = 0.033) in addition to reduced low density lipoprotein (P = 0.024) and high sensitive C-reactive protein (P = 0.043) were also observed in GS when compared to the control group. Reduced P-selectin expression suggests decreased platelet activation-dependent degranulation, while reduced platelet aggregation by AA and collagen indicates a quantitative decrease in platelet aggregation consequently targeting the cyclooxygenase-1 and GP VI pathways, respectively. These findings are the first to demonstrate that the activation of platelets is mildly inhibited in individuals with GS, an effect that might contribute to protection from platelet hyperactivation-induced thrombosis and thus cardiovascular mortality in individuals with benign hyperbilirubinemia.

Prospective Evaluation of Genetic Variation in Platelet Endothelial Aggregation Receptor 1 Reveals Aspirin-Dependent Effects on Platelet Aggregation Pathways.

Genetic variation in the platelet endothelial aggregation receptor 1 (PEAR1) gene, most notably rs12041331, is implicated in altered on‐aspirin platelet aggregation and increased cardiovascular event risk. We prospectively tested the effects of aspirin administration at commonly prescribed doses (81, 162, and 324 mg/day) on agonist‐induced platelet aggregation by rs12041331 genotype in 67 healthy individuals. Prior to aspirin administration, rs12041331 minor allele carriers had significantly reduced adenosine diphosphate (ADP)‐induced platelet aggregation compared with noncarriers (P = 0.03) but was not associated with other platelet pathways. In contrast, rs12041331 was significantly associated with on‐aspirin platelet aggregation when collagen and epinephrine were used to stimulate platelet aggregation (P < 0.05 for all associations), but not ADP. The influence of PEAR1 rs12041331 on platelet aggregation is pathway‐specific and is altered by aspirin at therapeutic doses, but not in a dose‐dependent manner. Additional studies are needed to determine the impact of PEAR1 on cardiovascular events in aspirin‐treated patients.

Platelet function one and three months after coronary bypass surgery in relation to once or twice daily dosing of acetylsalicylic acid

IntroductionCurrent guidelines recommend acetylsalicylic acid (ASA) treatment after coronary artery bypass grafting (CABG) to reduce thrombotic vein graft occlusion. The optimal dosage of ASA is not known. Materials and methodsForty-two patients undergoing elective CABG were randomized to receive either ASA 75mg or 160mg once daily (OD) or 75mg twice daily (BID) after the operation. Platelet function testing was performed before, and one and three months after the operation. ResultsWhite blood cell counts increased during the initial postoperative days whereas platelet counts were initially slightly reduced after the operation but increased after one month without any major changes of mean platelet volumes. Serum thromboxane B2 was more effectively suppressed at one and three months after the operation with ASA 75mg BID or 160mg OD than with 75mg OD (p<0.001). ASA 75mg BID and 160mg OD were equally effective. Adenosine diphosphate stimulated platelet aggregation in whole blood (Multiplate®) was increased one and three months after the operation, and this was counteracted by ASA 75mg BID but not by 75 or 160mg OD. Arachidonic acid-induced aggregation was more effectively inhibited by 75mg BID or 160mg OD compared to 75mg OD at three months. ConclusionsLess effective inhibition of platelet activation was obtained with ASA 75mg OD than with ASA 160mg OD or 75mg BID up to three months after CABG. Especially the latter dose is of interest for further studies of efficacy and clinical outcomes after CABG.

Platelet function in baboons and humans — A comparative study of whole blood using impedance platelet aggregometry (Multiplate®)

BackgroundPlatelets play a pivotal role in coagulation, inflammation and wound healing. Suitable animal models that have the potential to mimic human platelet function are limited. The objective of the current study was to compare platelet aggregation response in the whole blood of baboons and humans using impedance aggregometry. MethodsBlood was drawn from 24 anesthetised male baboons and 25 healthy volunteers. The platelet aggregation response was determined by impedance aggregometry (Multiplate®). Platelets in the hirudinised whole blood samples were stimulated with four different activators: adenosine diphosphate (ADP), collagen (COL), thrombin receptor activating peptide-6 (TR1AP), and activation of PAR-4 thrombin receptor subtype (TR4AP) at standard concentrations. Higher than standard concentrations were tested in a subgroup of the animals. ResultsThe cell counts showed no differences between baboons and humans. The platelet aggregation response was significantly lower in baboons compared to humans when stimulated with the platelet agonists ADP (p<0.0001), COL (p=0.021) and TR4AP (p<0.0001). TR1AP did not stimulate platelet aggregation in the baboon blood. Doubling the concentration of ADP and of TR4AP significantly increased the AUC compared to the standard concentration. In contrast, increased COL levels did not further increase the AUC. ConclusionThe current study revealed that testing the platelet function in baboon blood by impedance aggregometry is feasible with ADP, COL and TR4AP, but not with TR1AP. Compared to humans, the aggregation response is lower in baboons. Considering the limitations in accordance to these results, baboons might represent a potential species for further platelet research.

A Real-Time Monitoring System to Assess the Platelet Aggregatory Capacity of Components of a Tissue-Engineered Blood Vessel Wall.

Native blood vessels contain both an antiaggregatory intimal layer, which prevents platelet activation in the intact vessel, and a proaggregatory medial layer, which stimulates platelet aggregation upon vascular damage. Yet, current techniques for assessing the functional properties of tissue-engineered blood vessels may not be able to assess the relative effectiveness of both these pro- and antiaggregatory properties of the vessel construct. In this study, we present a novel technique for quantitatively assessing the pro- and antiaggregatory properties of different three-dimensional blood vessel constructs made using a layered fabrication method. This technique utilizes real-time measurements of cytosolic Ca2+ signaling to assess platelet activation in fluorescently labeled human platelet suspensions using fluorescence spectrofluorimetry, while also permitting examination of thrombus formation upon the surface of the construct using fluorescent imaging of DiOC6-labeled platelets. Experiments using this method demonstrated that type I collagen hydrogels, commonly used as scaffolds for vascular tissue engineering, were unable to support significant platelet activation, while type I and III neo-collagen secreted from human coronary artery smooth muscle cells cultured within these hydrogels as the medial layer were able to support thrombus formation. The incorporation of an intimal layer consisting of human umbilical vein endothelial cells on top of the medial layer inhibited platelet activation and aggregation. These data demonstrate that the methodology presented here is able to quantitatively compare the capacity of different constructs to trigger or prevent platelet activation. As such, this technique may provide a useful tool for standardizing the assessment of the functional properties of tissue-engineered blood vessel constructs developed using different culturing techniques.

Plasma L5 levels are elevated in ischemic stroke patients and enhance platelet aggregation

AbstractL5, the most electronegative and atherogenic subfraction of low-density lipoprotein (LDL), induces platelet activation. We hypothesized that plasma L5 levels are increased in acute ischemic stroke patients and examined whether lectin-like oxidized LDL receptor-1 (LOX-1), the receptor for L5 on endothelial cells and platelets, plays a critical role in stroke. Because amyloid β (Aβ) stimulates platelet aggregation, we studied whether L5 and Aβ function synergistically to induce prothrombotic pathways leading to stroke. Levels of plasma L5, serum Aβ, and platelet LOX-1 expression were significantly higher in acute ischemic stroke patients than in controls without metabolic syndrome (P < .01). In mice subjected to focal cerebral ischemia, L5 treatment resulted in larger infarction volumes than did phosphate-buffered saline treatment. Deficiency or neutralizing of LOX-1 reduced infarct volume up to threefold after focal cerebral ischemia in mice, illustrating the importance of LOX-1 in stroke injury. In human platelets, L5 but not L1 (the least electronegative LDL subfraction) induced Aβ release via IκB kinase 2 (IKK2). Furthermore, L5+Aβ synergistically induced glycoprotein IIb/IIIa receptor activation; phosphorylation of IKK2, IκBα, p65, and c-Jun N-terminal kinase 1; and platelet aggregation. These effects were blocked by inhibiting IKK2, LOX-1, or nuclear factor–κB (NF-κB). Injecting L5+Aβ shortened tail-bleeding time by 50% (n = 12; P < .05 vs L1-injected mice), which was prevented by the IKK2 inhibitor. Our findings suggest that, through LOX-1, atherogenic L5 potentiates Aβ-mediated platelet activation, platelet aggregation, and hemostasis via IKK2/NF-κB signaling. L5 elevation may be a risk factor for cerebral atherothrombosis, and downregulating LOX-1 and inhibiting IKK2 may be novel antithrombotic strategies.

Abstract 17547: Syncope and Inferior ST Elevations: A Diagnosis Within the Heart

A 53 year old woman with a history of diabetes and hypertension presented with syncope, hypotension, and electrocardiography demonstrating inferior ST elevations. The week prior to admission she treated an episode of left knee pain with an admixture of Chinese herbs. Two days prior to presentation she noted the onset of fevers, myalgias and dizziness culminating in syncope the day of admission. Initial electrocardiogram demonstrated complete heart block and junctional escape with inferior ST elevations. Cardiac catheterization demonstrated no epicardial coronary disease. Laboratory studies revealed acute kidney injury, elevated cardiac biomarkers, and a normal white blood cell count with normal differential. Upon transfer to our hospital, the patient was hypotensive requiring temporary ventricular pacing and vasopressor support. Echocardiogram demonstrated severe systolic dysfunction with LVEF of 31%. On arrival, she was noted to have episodes of monomorphic ventricular tachycardia. Etiologies of fulminant myocarditis considered included viral (lymphocytic), giant cell myocarditis, and sarcoid a well as acute eosinophilic myocarditis given recent ingestion of novel herbal mixture. Given the potential for steroid-responsive etiologies, endomyocardial biopsy was performed with institution of mechanical support ( intra-aortic balloon pump) and administration of parenteral steroids. Inotropic and mechanical support were weaned within 72 hours of administration of steroids and she demonstrated normalization of electrical conduction. Biopsy demonstrated acute necrotizing eosinophilic myocarditis (ANEM). Cardiac MRI demonstrated acute myocarditis, subacute inferior infarct, and no LV thrombus. ANEM represents an often fatal, fulminant form of eosinophilic heart disease and can present without systemic eosinophilia. Triggers can include antecedent viral illness and drug hypersensitivity (including herbal mixtures). Eosinophilic proteins can stimulate platelet aggregation and coronary vasoconstriction. Considered mechanisms of inferior infarct included LV thrombus with coronary embolism. The patient was treated with steroids and anticoagulation with recovery of baseline cardiopulmonary function.

Modification of platelet proteins by 4-hydroxynonenal: Potential Mechanisms for inhibition of aggregation and metabolism

Platelet aggregation is an essential response to tissue injury and is associated with activation of pro-oxidant enzymes, such as cyclooxygenase, and is also a highly energetic process. The two central energetic pathways in the cell, glycolysis and mitochondrial oxidative phosphorylation, are susceptible to damage by reactive lipid species. Interestingly, how platelet metabolism is affected by the oxidative stress associated with aggregation is largely unexplored. To address this issue, we examined the response of human platelets to 4-hydroxynonenal (4-HNE), a reactive lipid species which is generated during thrombus formation and during oxidative stress. Elevated plasma 4-HNE has been associated with renal failure, septic shock and cardiopulmonary bypass surgery. In this study, we found that 4-HNE decreased thrombin stimulated platelet aggregation by approximately 60%. The metabolomics analysis demonstrated that underlying our previous observation of a stimulation of platelet energetics by thrombin glycolysis and TCA (Tricarboxylic acid) metabolites were increased. Next, we assessed the effect of both 4-HNE and alkyne HNE (A-HNE) on bioenergetics and targeted metabolomics, and found a stimulatory effect on glycolysis, associated with inhibition of bioenergetic parameters. In the presence of HNE and thrombin glycolysis was further stimulated but the levels of the TCA metabolites were markedly suppressed. Identification of proteins modified by A-HNE followed by click chemistry and mass spectrometry revealed essential targets in platelet activation including proteins involved in metabolism, adhesion, cytoskeletal reorganization, aggregation, vesicular transport, protein folding, antioxidant proteins, and small GTPases. In summary, the biological effects of 4-HNE can be more effectively explained in platelets by the integrated effects of the modification of an electrophile responsive proteome rather than the isolated effects of candidate proteins.

Role of protein kinase CK2 in the dynamic interaction of platelets, leukocytes and endothelial cells during thrombus formation

IntroductionThrombus formation is a complex process, which is characterized by the dynamic interaction of platelets, leukocytes and endothelial cells. The activation of these cells is strictly mediated by different phospho-regulated signaling pathways. Recently, it has been reported that inhibition of protein kinase CK2 affects platelet function by suppressing phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K) signaling. Based on this finding, we herein analyzed whether CK2 acts as a crucial regulator of thrombus formation. Materials and methodsWe examined the effect of CK2 inhibition on platelet activation and aggregation, the formation of platelet-leukocyte aggregates (PLA), the endothelial expression of von Willebrand factor (vWF), intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, and the subcellular localization of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and phospho-p65 in human dermal microvascular endothelial cells (HDMEC). Dorsal skinfold chambers were prepared in BALB/c mice to analyze in vivo the effect of CK2 inhibition on photochemically induced thrombus formation using intravital fluorescence microscopy. ResultsCK2 inhibition by CX-4945 suppressed adenosin diphosphate (ADP)- and proteinase-activated receptor-1-peptide (PAR-1-AP)-stimulated platelet aggregation, which was associated with down-regulation of P-selectin, GPIIb/IIIa and a reduced formation of PLA. Expression and secretion of vWF was diminished in CX-4945-treated HDMEC. Moreover, CK2 inhibition attenuated the endothelial expression of VCAM-1, whereas the expression of ICAM-1 was not affected. Finally, CX-4945-treated mice exhibited a significantly delayed photochemically induced thrombus formation when compared to vehicle-treated controls. ConclusionThese results indicate that CK2 is a pleiotropic regulator of thrombus formation, affecting multiple interactions of platelets, leukocytes and endothelial cells.

Cocaine, Marijuana, Hypertension and Cardiovascular Effects

Cocaine is used by more than 14 million people worldwide, about 0.3 percent of the global population age is 15 to 64 years. After alcohol, cocaine is the most common cause of acute drug-related emergency department visits in the United States. Cocaine consumption is more frequently associated with acute cardiovascular illness. Cocaine stimulates α1, α2, β1 and β2 adrenergic receptors through increased levels of norepinephrine and a lesser extent epinephrine. The cardiovascular effects of cocaine are thought to be similar and regardless to the route of consumption. An acute coronary syndrome is the most common cardiac problem including myocardial ischemia and infarction even in young persons without atherosclerosis, aortic dissection and rupture, arrhythmias, ventricular tachycardia and fibrillation, asystole and finally sudden death. Other cardiovascular effects that caused by cocaine include coronary artery aneurysm, palpitation, sinus tachycardia, increased systemic vascular resistance and hypertension crisis, left ventricular hypertrophy, myocarditis, cardiomyopathy, myocardial fibrosis, bundle branch block, heart block, supraventricular arrhythmia, accelerated atherosclerosis, hypotension, bradycardia and infective endocarditis among intravenous users.Cocaine by three mechanisms cause ischemia: 1. increased myocardial oxygen demand, 2. decreased coronary blood flow due to coronary artery vasoconstriction and spasm and 3. Coronary artery thrombosis via activation of platelets, stimulation of platelet aggregation and potentiation of thromboxane production.

Inhibitory Effects of Rice Bran Water Extract Fermented Lactobacillus plantarum due to cAMP-dependent Phosphorylation of VASP (Ser157) on human Platelet Aggregation

In this study, we investigated the effect of rice bran water extract fermented with Lactobacillus plantarum KCCM-12116 (RBLp) on ADP (20 μM)-, collagen (10 μg/mL)-, and thrombin (0.2 U/mL)-stimulated platelet aggregation. RBLp dose-dependently inhibited ADP-, collagen-, and thrombin-induced platelet aggregation, with IC50 values of 501.1, 637.2, and > 2,000 μg/mL, respectively. The platelet aggregation induced by ADP plus RBLp (750 μg/mL) was increased by the adenylate cyclase inhibitor, SQ22536, and the cAMP-dependent protein kinase (A-kinase) inhibitor, Rp-8-Br-cAMPS. Treatment with RBLp increased the phosphorylation of VASP (Ser 157 ), an A-kinase substrate, which was also inhibited by SQ22536 and Rp-8-Br-cAMPS. It is thought that the RBLp-induced increases in cAMP contributed to the phosphorylation of VASP (Ser 157 ), which in turn resulted in an inhibition of ADP-induced platelet aggregation, thereby indicating that RBLp has an antiplatelet effect via cAMP-dependent phosphorylation of VASP (Ser 157 ). Thus, RBLp may have therapeutic potential for the treatment (or prevention) of platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

Inhibitory Mechanisms of CME-1, a Novel Polysaccharide from the Mycelia of Cordyceps sinensis, in Platelet Activation

CME-1 is a polysaccharide purified from the mycelia of medicinal mushroom Cordyceps sinensis, its molecular weight was determined to be 27.6 kDa by using nuclear magnetic resonance and gas chromatography-mass spectrometry. The initiation of arterial thromboses is relevant to various cardiovascular diseases (CVDs) and is believed to involve platelet activation. Our recent study exhibited that CME-1 has potent antiplatelet activity via the activation of adenylate cyclase/cyclic AMP ex vivo and in vivo. The aggregometry, and immunoblotting were used in this study. In this study, the mechanisms of CME-1 in platelet activation is further investigated and found that CME-1 inhibited platelet aggregation as well as the ATP-release reaction, relative intracellular [Ca(+2)] mobilization, and the phosphorylation of phospholipase C (PLC)γ2 and protein kinase C (PKC) stimulated by collagen. CME-1 has no effects on inhibiting either convulxin, an agonist of glycoprotein VI, or aggretin, an agonist of integrin α2β1 stimulated platelet aggregation. Moreover, this compound markedly diminished thrombin and arachidonic acid (AA) induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 2, c-Jun N-terminal kinase 1, and Akt. Treatment with SQ22536, an inhibitor of adenylate cyclase, markedly diminished the CME-1-mediated increasing of cyclic AMP level and reversed prostaglandin E1- or CME-1-mediated inhibition of platelet aggregation and p38 MAPK and Akt phosphorylation stimulated by thrombin or AA. Furthermore, phosphodiesterase activity of human platelets was not altered by CME-1. The crucial finding of this study is that the antiplatelet activity of CME-1 may initially inhibit the PLCγ2-PKC-p47 cascade, and inhibit PI3-kinase/Akt and MAPK phosphorylation through adenylate cyclase/ cyclic AMP activation, then inhibit intracellular [Ca(+2)] mobilization, and, ultimately, inhibit platelet activation. The novel role of CME-1 in antiplatelet activity indicates that this compound exhibits high therapeutic potential for treating or preventing CVDs.

Whole Blood Stored at 4°C for 7 Days Is Equivalent to Fresh Whole Blood for Resuscitation of Severe Polytrauma

Background: Currently, whole blood is rarely used in trauma resuscitation due, in part, to the widely held belief that refrigeration will reduce the hemostatic efficacy of stored platelets. Recently, however, Pidcoke et al. (Transfusion 2013, 53:137s) showed that hemostatic function of human whole blood was well preserved when stored at 4°C for up to 21 days. The hemostatic and resuscitative efficacy of cold-stored whole blood has not been tested in a coagulopathic animal polytrauma model.Hypothesis: We hypothesized that blood stored for 7 days at 4°C is equivalent to fresh whole blood with regard to hemostatic and coagulation function in resuscitation of severe trauma.Method: Sprague-Dawley rats (300-400g) were anesthetized with Isoflurane. Polytrauma was induced by damaging the small intestines, the left and medial liver lobes, the right leg skeletal muscle, and by fracturing the right femur. The rats were then bled to a mean arterial pressure of 40mmHg and held there until 40% of the blood volume was removed. Hemorrhage was usually completed between 30-60 min. Resuscitation was started at 1hr and included the following groups: Lactated Ringer’s (LR), fresh whole blood (FWB) or FWB stored at 4°C for 7 days (sFWB). The resuscitation volume was 20% of blood volume and represents the approximate volume used in prehospital care of trauma patients in both civilian and military settings. The experiment was terminated at 2hrs. Blood samples were taken before (time 0) and 2hrs after trauma (1hr after resuscitation) to assess hemostatic function. Prothombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen were measured on ST-4 (Stago). Platelet aggregation was measured with Multiplate (Diapharma) after stimulation with ADP, collagen or thrombin (PAR4) and expressed as area under the curve per 1000 platelets. Clotting function was assessed using ROTEM (Tem International).Results: Resuscitation with FWB and sFWB led to recovery of mean arterial blood pressure to levels similar to baseline (FWB 92±2.4 to 86±3, sFWB 93±3 to 91±4). Resuscitation with LR led to a significantly lower arterial pressure (96±3 to 61±3.8mmHg, p<0.05). Plasma lactate levels were significantly elevated in all groups. However, plasma lactate was lower after resuscitation with FWB and sFWB (0.52±0.06 to 1.22±0.08, and 0.5±0.05 to 1.28±0.12mM, respectively), as compared to LR (0.47±0.05 to 2.36±0.24mM). Several coagulation parameters changed significantly after resuscitation (PT, aPTT, fibrinogen, mean clotting firmness, clotting time and alpha angle). However, there was no difference in the change of any of these parameters between animals treated with FWB or sFWB. Because platelets make up most of the clot strength, we assessed the ability of agonists (ADP, thrombin agonist, collagen) to stimulate platelet aggregation. The degree of aggregation after resuscitation with all fluids was significantly decreased to stimulation with collagen (15 to 26%). However there was no significant difference in the aggregation changes between the FWB or sFWB groups. Resuscitation with any of the fluids had no effect on ADP or PAR4 stimulation of aggregation.Conclusion: These data strongly suggest that FWB and FWB stored for 7days at 4° are equivalent for treating severe polytrauma and hemorrhage when considering recovery of arterial pressure and plasma lactate, changes in clotting function and changes in platelet aggregation as endpoints necessary for recovery of the patient. This project was funded by the US Army Medical Research and Materiel Command. DisclosuresNo relevant conflicts of interest to declare.

Inhibitory Effects of Water Extract from Rice Bran Due to cAMP-dependent Phosphorylation of VASP (Ser157) on ADP-induced Platelet Aggregation

In this study, we investigated the effect of water extract from rice bran (RB) on ADP (20 μM)-stimulated platelet aggregation. RB dose-dependently inhibited ADP-induced platelet aggregation, and its IC50 value was 224.0 μg/mL, which was increased by adenylate cyclase inhibitor SQ22536 and cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS. RB elevated the phosphorylation of VASP (Ser 157 ) which was also inhibited by SQ22536 and Rp-8-Br-cAMPS. It is thought that RB-elevated cAMP contributed to the phosphorylation of VASP (Ser 157 ) to inhibit ADP-induced platelet aggregation. Therefore, we demonstrate that RB has an antiplatelet effect via cAMP-dependent phosphorylation of VASP (Ser 157 ), and RB may have preventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

(-)-Epigallocatechin gallate selectively inhibits adenosine diphosphate‑stimulated human platelet activation: suppression of heat shock protein 27 phosphorylation via p38 mitogen‑activated protein kinase.

(‑)‑Epigallocatechin gallate (EGCG) is a major component of green tea. It has been demonstrated that EGCG has an antithrombotic effect by inhibiting platelet aggregation. However, the detailed mechanisms underlying the effects of EGCG remain to be elucidated. The present study examined the effects of EGCG on human platelet activation by various stimulators and the exact underlying mechanisms. EGCG suppressed adenosine diphosphate (ADP)‑stimulated platelet aggregation dose dependently between 30 and 70 µM. By contrast, EGCG failed to affect platelet aggregation stimulated by collagen, U46619 (a TP agonist) or ristocetin (an activator of GPIb/IX/V). EGCG attenuated the ADP‑induced phosphorylation of p38 mitogen‑activated protein (MAP) kinase and heat shock protein 27 (HSP27). The ADP‑stimulated release of platelet‑derived growth factor (PDGF)‑AB and the soluble CD40 (sCD40) ligand was inhibited by EGCG. These findings suggest that EGCG selectively inhibits ADP‑stimulated human platelet activation and that EGCG reduces the release of PDGF‑AB and the sCD40 ligand due to suppressing HSP27 phosphorylation via p38 MAP kinase.

Cordycepin-enriched WIB801C from Cordyceps militaris inhibits ADP-induced [Ca(2+)] i mobilization and fibrinogen binding via phosphorylation of IP 3R and VASP.

In this study, we investigated the effect of cordycepin-enriched (CE)-WIB801C from Cordyceps militaris on ADP (20µM)-stimulated platelet aggregation. CE-WIB801C dose-dependently inhibited ADP-induced platelet aggregation, and its IC50 value was 18.5μg/mL. CE-WIB801C decreased TXA2 production, but did not inhibit the activities of COX-1 and thromboxane synthase (TXAS) in ADP-activated platelets, which suggests that the inhibition of TXA2 production by CE-WIB801C is not resulted from the direct inhibition of COX-1 and TXAS. CE-WIB801C inhibited ATP release and [Ca(2+)]i mobilization, and increased cAMP level and IP3RI (Ser(1756)) phosphorylation in ADP-activated platelets. cAMP-dependent protein kinase (A-kinase) inhibitor Rp-8-Br-cAMPS increased CE-WIB801C-inhibited [Ca(2+)]i mobilization, and strongly inhibited CE-WIB801C-increased IP3RI (Ser(1756)) phosphorylation. CE-WIB801C elevated the phosphorylation of VASP (Ser(157)), an A-kinase substrate, but inhibited fibrinogen binding to αIIb/β3. These results suggest that CE-WIB801C-elevated cAMP involved in IP3RI (Ser(1756)) phosphorylation to inhibit [Ca(2+)]i mobilization and, VASP (Ser(157)) phosphorylation to inhibit αIIb/β3 activation. Therefore, in this study, we demonstrate that CE-WIB801C may have apreventive or therapeutic potential for platelet aggregation-mediated diseases, such as thrombosis, myocardial infarction, atherosclerosis, and ischemic cerebrovascular disease.

Anti-platelet effect of ginkgolide a from Ginkgo biloba

Effects of ginkgolide A (GA) from Ginkgo biloba leaves in collagen (10 μg/mL)-stimulated platelet aggregation were investigated. Zymographic analysis confirmed that pro-matrix metalloproteinase-9 (MMP-9) (92 kDa) was activated by GA to form an activated MMP-9 (86-kDa) on gelatinolytic activities. GA concentration-dependently inhibited platelet aggregation, intracellular Ca2+ mobilization, and thromboxane A2 (TXA2) formation by inhibiting the cyclooxygenase-1 (COX-1) activity in collagen-stimulated platelets. In addition, GA increased the formation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which have an anti-platelet function in both resting and collagen-stimulated platelets. On the other hand, GA did not prolong prothrombin time (PT) and activated partial thromboplastin time (aPTT) associated with the extrinsic and intrinsic coagulation pathways on human plasma, respectively. Therefore, we suggest that the inhibitory effect of GA on platelet aggregation might involve the following pathway. GA may increase the MMP-9 activity and intracellular cAMP and cGMP production, inhibit intracellular Ca2+ mobilization, and decrease TXA2 production by down-regulating the COX-1, thereby leading to inhibition of platelet aggregation without cytotoxicity. These results strongly indicate that GA is a potent inhibitor of collagen-stimulated platelet aggregation. It may play an important role as a negative regulator during platelet activation.

Chromium picolinate inhibits cholesterol-induced stimulation of platelet aggregation in hypercholesterolemic rats

Hypercholesterolemia indirectly increases the risk of myocardial infarction by enhancing platelet aggregation. Chromium has been shown to lower plasma lipids. This study was designed to investigate whether chromium inhibits platelet aggregation under hypercholesterolemic conditions. Albino rats were divided into four groups: control rats fed with a normolipemic diet (NLD group), chromium-supplemented rats fed with NLD (NLD + Cr group), rats fed with a high-fat diet (HF group), and chromium-supplemented rats fed with HF (HF + Cr group). After 10 weeks, blood was collected to determine adenosine diphosphate and collagen-induced platelet aggregation and plasma levels of total cholesterol, triglycerides, high-density lipoprotein cholesterol, apolipoprotein A1, apolipoprotein B, and thromboxane B2. Low-density lipoprotein cholesterol was calculated by Friedewald formula. High-fat diet animals displayed significant elevation of plasma lipids and platelet aggregation which was normalized to control levels by chromium supplementation. Chromium supplementation in normolipemic (NLD + Cr) rats did not produce significant changes in either plasma lipids or platelet activity. Chromium supplementation to hypercholesterolemic rats improves the lipid profile and returns platelet hyperaggregability to control levels. This normalization is mostly due to a reduction in plasma cholesterol level.

PP-300 The Effect of Heparine on in vitro Stimulated Platelet Aggregation by Elective Percutaneous Coronary Intervention Patients

PP-300 The Effect of Heparine on in vitro Stimulated Platelet Aggregation by Elective Percutaneous Coronary Intervention Patients