Aggregation Of Human Blood Platelets Research Articles

Synthesis, characterization and thrombolytic activity evaluation of novel pyrazolo[3,4-b]pyridine-4-carbohydrazide derivatives

Thrombosis, a leading cause of numerous fatalities, particularly in developed nations, accounts for thousands of deaths. The contemporary lifestyle inevitably leads to occurrences of venous thrombosis, including deep vein thrombosis and thromboembolism. This article presents a groundbreaking study where a novel series of N′-(arylmethylidene)-6-(p-methoxyphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-b] pyridine-4-carbohydrazide derivatives were efficiently synthesized and screened for thrombolytic activities. Compounds 4a, 4c, 4d, and 4h demonstrated the best antiplatelet activities when using different agonists such as arachidonic acid and collagen. These compounds showed >50 % activity by using agonist collagen and inhibited >50 % platelet aggregation in whole human blood. Compounds 4c and 4d showed the greatest inhibitory effect of 97.98 % and 98.97 %, respectively as compared to other synthesized compounds and aspirin. For platelet aggregation using arachidonic acid, all the synthesized compounds 4a, 4c, 4d and 4h shown >93 % inhibition than that of aspirin. In computational studies, ligands 4c(ap) and 4d(ap) exhibited potent interaction with the COX-1 active site, suggesting inhibitory potential against thrombolytic activities. Ligand 4c(ap) scored 8896 and ACE of -418.31 kcal/mol, while 4d(ap) scored 8742 and ACE of -417.43 kcal/mol. Similarly, ligands 4c(ap) and 4h(ap) displayed outstanding docking scores at the COX-2 active site, with 4c(ap) scoring 8574 (ACE:301.49 kcal/mol) and 4h(ap) scoring 8424 (ACE:276.16 kcal/mol). Synthesized compounds particularly 4c and 4d have the potential to be used for clinical trials and could be beneficial for the designing and development of novel therapeutics for cardiovascular and thromboembolic ailments.

Extracts of tamarillo, horned melon, and raspberry, but not extract of pear, inhibit human blood platelet aggregation: Investigating the underlying factors for their differential mechanisms

In addition to their roles in thrombosis, hyperactive platelets contribute to atherosclerosis.Since anti-platelet drugs are not recommended for the vulnerable population with hyperactive platelets to reduce the risk of cardiovascular disease, a search is on to find alternative dietary antiplatelets that can be safely used.We here describe that in a dose-dependent manner, sugar-free extracts of tamarillo, horned melon (kiwano), and raspberryinhibited ADP-induced platelet aggregation in platelet-rich plasma, whereas pear extract had no effects.Furthermore, analysisof untargeted metabolites revealed the presence of platelet inhibitory components such as benzoic acid, caffeic acid, and gallic acid in the sugar-free extracts of tamarillo, raspberry, and kiwano but absent in pear extract.All these three fruit extracts inhibited the platelet production of TxB2and the release of platelet factor 4. Our work suggeststhat these fruitsinhibitplatelet aggregation partly due to anti-platelet compounds such asbenzoic, caffeic, and gallic acids.

138 Escherichia coli-induced platelet aggregation in human whole blood is partly dependent on complement C3b

138 Escherichia coli-induced platelet aggregation in human whole blood is partly dependent on complement C3b

Complement C3b contributes to Escherichia coli-induced platelet aggregation in human whole blood.

Platelets have essential functions as first responders in the immune response to pathogens. Activation and aggregation of platelets in bacterial infections can lead to life-threatening conditions such as arterial thromboembolism or sepsis-associated coagulopathy. In this study, we investigated the role of complement in Escherichia coli (E. coli)-induced platelet aggregation in human whole blood, using Multiplate® aggregometry, flow cytometry, and confocal microscopy. We found that compstatin, which inhibits the cleavage of complement component C3 to its components C3a and C3b, reduced the E. coli-induced platelet aggregation by 42%-76% (p = 0.0417). This C3-dependent aggregation was not C3a-mediated as neither inhibition of C3a using a blocking antibody or a C3a receptor antagonist, nor the addition of purified C3a had any effects. In contrast, a C3b-blocking antibody significantly reduced the E. coli-induced platelet aggregation by 67% (p = 0.0133). We could not detect opsonized C3b on platelets, indicating that the effect of C3 was not dependent on C3b-fragment deposition on platelets. Indeed, inhibition of glycoprotein IIb/IIIa (GPIIb/IIIa) and complement receptor 1 (CR1) showed that these receptors were involved in platelet aggregation. Furthermore, aggregation was more pronounced in hirudin whole blood than in hirudin platelet-rich plasma, indicating that E. coli-induced platelet aggregation involved other blood cells. In conclusion, the E. coli-induced platelet aggregation in human whole blood is partly C3b-dependent, and GPIIb/IIIa and CR1 are also involved in this process.

Can Isoquinoline Alkaloids Affect Platelet Aggregation in Whole Human Blood?

Isoquinoline alkaloids have multiple biological activities, which might be associated with positive pharmacological effects as well as negative adverse reactions. As bleeding was suggested to be a side effect of the isoquinoline alkaloid berberine, we decided to ascertain if different isoquinoline alkaloids could influence hemocoagulation through the inhibition of either platelet aggregation or blood coagulation. Initially, a total of 14 compounds were screened for antiplatelet activity in whole human blood by impedance aggregometry. Eight of them demonstrated an antiplatelet effect against arachidonic acid-induced aggregation. Papaverine and bulbocapnine were the most potent compounds with biologically relevant IC50 values of 26.9 ± 12.2 μM and 30.7 ± 5.4 μM, respectively. Further testing with the same approach confirmed their antiplatelet effects by employing the most physiologically relevant inducer of platelet aggregation, collagen, and demonstrated that bulbocapnine acted at the level of thromboxane receptors. None of the alkaloids tested had an effect on blood coagulation measured by a mechanical coagulometer. In conclusion, the observed antiplatelet effects of isoquinoline alkaloids were found mostly at quite high concentrations, which means that their clinical impact is most likely low. Bulbocapnine was an exception. It proved to be a promising antiplatelet molecule, which may have biologically relevant effects.

In vitro and in vivo effects of carrot on human blood platelet aggregation

SummaryCarrots are a good source of essential nutrients and bioactive phytochemicals including carotenoids, phenolic compounds and polyacetylenes such as falcarinol. In this study, we investigated the inhibitory effect of a carrot extract on blood platelet aggregation in vitro and the effect of raw diced or juiced carrots on platelet aggregation in a human study. Carrot extract significantly reduced the platelet aggregation in vitro in blood obtained from healthy donors. In a randomised crossover human study, healthy volunteers consumed either 100 g of freshly diced carrots or 100 mL of freshly juiced carrots (with pulp). There was no significant effect on blood platelet aggregation in the participants at 2, 5, 8 and 24 h after the carrot meal due to wide interindividual variations. Further research is required to understand diet, genetics and lifestyle factors that may impact the physiological effects of falcarinol on platelet aggregation and blood flow.

Study of the Effect of Nanotechnology Drugs (mcs-b) on the Aggregation of Human Blood Platelets

This scientific article is devoted to the study of the effect of magnetite nanoparticles (MCS-B brand) on the functional platelets activity of human. 0.9% NaCl was used as a control. The results of the study showed that the use of NaCl saline solution shifts the colloidal suspension equilibrium of platelets towards a significant (P<0.001) increase in the rate and index of their aggregation. For the first time, the effect of 0.9% NaCl on function of platelets makes us reconsider the concept of safety of infusion solutions in patients with initial signs of platelet disorders hemostasis. On the contrary, the use of MCS-B nanoparticles significantly revealed (P<0.05) an increase in the stability of colloidal suspension of platelets. This is an important pathogenetic factor which affects the occurrence of correction of hemostasis in conditions of blood clotting disorders

Effects of functionalized silver nanoparticles on aggregation of human blood platelets.

PurposeWe studied the effects of silver nanoparticles (AgNPs) on human blood platelet function. We hypothesized that AgNPs, a known antimicrobial agent, can be used as blood-compatible, “ideal material’’ in medical devices or as a drug delivery system. Therefore, the aim of the current study was to investigate if functionalized AgNPs affect platelet function and platelets as well as endothelial cell viability in vitro.Methods AgNPs, functionalized with reduced glutathione (GSH), polyethylene glycol (PEG) and lipoic acid (LA) were synthesized. Quartz crystal microbalance with dissipation was used to measure the effect of AgNPs on platelet aggregation. Platelet aggregation was measured by changes in frequency and dissipation, and the presence of platelets on the sensor surface was confirmed and imaged by phase contrast microscopy. Flow cytometry was used to detect surface abundance of platelet receptors. Lactate dehydrogenase test was used to assess the potential cytotoxicity of AgNPs on human blood platelets, endothelial cells, and fibroblasts. Commercially available ELISA tests were used to measure the levels of thromboxane B2 and metalloproteinases (MMP-1, MMP-2) released by platelets as markers of platelet activation.Results2 nm AgNPs-GSH, 3.7 nm AgNPs-PEG both at 50 and 100 µg/mL, and 2.5 nm AgNPs-LA at 100 µg/mL reduced platelet aggregation, inhibited collagen-mediated increase in total P-selectin and GPIIb/IIIa, TXB2 formation, MMP-1, and MMP-2 release. The tested AgNPs concentrations were not cytotoxic as they did not affect, platelet, endothelial cell, or fibroblast viability.ConclusionAll tested functionalized AgNPs inhibited platelet aggregation at nontoxic concentrations. Therefore, functionalized AgNPs can be used as an antiplatelet agent or in design and manufacturing of blood-facing medical devices, such as vascular grafts, stents, heart valves, and catheters.

Study of the Effect of Nanotechnology Drugs (MСS-B) on the Aggregation of Human Blood Platelets

Study of the Effect of Nanotechnology Drugs (MСS-B) on the Aggregation of Human Blood Platelets

12‐LOX Oxylipins Derived from Omega‐3 Supplementation with Docosahexanoic Acid Potently Regulate Human Blood Platelets

Cardiovascular disease (CVD) is the leading cause of mortality in the United States in both men and women. Thrombotic‐associated ischemic injuries due to excessive platelet activation are largely responsible for the high level of death associated with CVD. Therapies that seek to pharmacologically inhibit platelet activation, aggregation, or clot stabilization, have the potential to reduce the high mortality rates associated with CVD but are also associated with an increased risk of bleeding. The oxygenated products of polyunsaturated fatty acids—oxylipins—have been shown to be critical regulators of platelet function. Previously, it has been shown that Docosahexanoic Acid (DHA) has antiplatelet activity; therefore, we hypothesize that DHA regulates platelet function at least in part through its 12‐lipoxygenase (12‐LOX) metabolites, 11‐ and 14‐ hydroxydocosahexaenoic acid (11‐ and 14‐HDHA). Our study sought to investigate the role that 12‐LOX derived products of DHA, 11‐ and 14‐ HDHA, have on human blood platelet aggregation and activation. Platelet aggregation, granule secretion, and integrin αIIbβ3 activation were measured following agonist stimulation in the presence of DHA, 11‐ or 14‐ HDHA. Both 11‐ and 14‐ HDHA were observed to dose‐dependently attenuate aggregation in response to sub‐maximal concentrations of either thrombin or collagen. Additionally, both 11‐ and 14‐ HDHA were observed to attenuate intracellular signaling as demonstrated by a decrease in granule secretion in platelets treated with DHA compared to vehicle control treated platelets. To further the study, the effects of DHA supplementation in vivo were investigated in wild‐type mice as well as mice lacking 12‐LOX. DHA was found to play an important role in regulation of platelet function in vivo. Future studies will help to elucidate the mechanism by which DHA and its metabolites inhibit platelet function and play a role in supplementation‐dependent regulation of blood clotting. Once completed this will be the first study to clearly define a role for DHA and its 12‐LOX metabolites in the regulation of platelet aggregation and give important insight into the benefits and risks of fish oil supplementation on the cardiovascular system.Support or Funding InformationThis work was funded in part through the National Institutes of Health grants R01 GM105671 (MH), R01 HL114405 (MH), R01 MD007880 (MH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The isoflavonoid tectorigenin has better antiplatelet potential than acetylsalicylic acid

BackgroundOne reason for the lower incidence of cardiovascular diseases in Asian countries may be the high intake of isoflavonoids and their antiplatelet effects may be an important factor. To date, there is limited comparison of a range of isoflavonoids and knowledge of their effects at different levels of platelet aggregation. PurposeTo screen the antiplatelet effects of a number of isoflavonoids on the arachidonic acid based aggregation pathway and investigate how the antiplatelet activity might occur. MethodsThe antiplatelet effects were first screened in whole human blood where platelet aggregation was induced by arachidonic acid. Further analysis was targeted at search of the mechanism of action. ResultsThirteen of the eighteen tested isoflavonoids had significant inhibitory effect on platelet aggregation in whole human blood. Genistein had the same potency as clinically used acetylsalicylic acid (ASA) while tectorigenin was clearly stronger than ASA. Further analyses showed that the effect of tectorigenin was not based on inhibition of cyclooxygenase-1 in contrast to ASA or thromboxane synthase but by competitive antagonism at thromboxane receptors. ConclusionTectorigenin is a more potent antiplatelet compound than ASA and thus an interesting substance for further testing.

Label-free detection of aggregated platelets in blood by machine-learning-aided optofluidic time-stretch microscopy.

According to WHO, about 10 million new cases of thrombotic disorders are diagnosed worldwide every year. Thrombotic disorders, including atherothrombosis (the leading cause of death in the US and Europe), are induced by occlusion of blood vessels, due to the formation of blood clots in which aggregated platelets play an important role. The presence of aggregated platelets in blood may be related to atherothrombosis (especially acute myocardial infarction) and is, hence, useful as a potential biomarker for the disease. However, conventional high-throughput blood analysers fail to accurately identify aggregated platelets in blood. Here we present an in vitro on-chip assay for label-free, single-cell image-based detection of aggregated platelets in human blood. This assay builds on a combination of optofluidic time-stretch microscopy on a microfluidic chip operating at a high throughput of 10 000 blood cells per second with machine learning, enabling morphology-based identification and enumeration of aggregated platelets in a short period of time. By performing cell classification with machine learning, we differentiate aggregated platelets from single platelets and white blood cells with a high specificity and sensitivity of 96.6% for both. Our results indicate that the assay is potentially promising as predictive diagnosis and therapeutic monitoring of thrombotic disorders in clinical settings.

Cardiovascular Protective Effects Of Standardized Extracts And Isolated Compounds Of Selected Medicinal Plants From The Tropical Rainforest

Compounds with diverse chemical structures including phenols, xanthones, prenylflavonoids, benzophenones, alkaloids and triterpenoids, isolated from several Malaysian plants, were investigated to discover leads for development of new cardiovascular protective agents. The compounds were tested for their ability to inhibit platelet aggregation in human whole blood using an electrical impedance method and their ability to inhibit human LDL oxidation using thiobarbituric acid reactive substances assay. Standardized extracts of several plants were evaluated for their cardiovascular protective effects against isoproterenol-induced myocardial infarction (MI) in rats and effects on serum lipid profiles of rats fed with high cholesterol diet (HCD) and their platelet-mediated antithrombotic effects. Among the compounds tested, xanthones from Guttiferae species showed strong inhibitory activities against platelet aggregation and LDL peroxidation. 2-(3-Methylbut-2-enyl)-1,3,5,-trihydroxyxanthone and 2-(3-methylbut-2-enyl)-1,3,5,6-tetrahydroxyxanthone showed strong inhibition on platelet aggregation induced by arachidonic acid (AA), macluraxanthone and 1, 7 dihydroxyxanthone showed significant and selective inhibitory activity against ADP-induced aggregation while rubraxanthone showed inhibitory activity against aggregation caused by all three inducers [1]. Recently we reported the isolation of a new xanthone, 1, 3, 6-trihydroxy-5-methoxy-7-(3′-methyl-2′-oxo-but-3′-enyl) xanthone and 1, 3, 5, 7-tetrahydroxyxanthone from Garcinia cantleyana var. cantleyana, with strong ability to inhibit LDL oxidation [2]. The standardized extracts of Zingiber officinale, Curcuma domestica and Labisia pumila var alata significantly (p < 0.05) reduced the total cholesterol, triglycerides, and LDL levels and helped in improving high density lipoprotein (HDL) and oxidase enzymes levels on HCD rats in a dose-dependent way. The extracts also exhibited protective effects on biochemical and histopathological alterations of the cardiac muscle cells in Isoproterenol-induced MI rats [3, 4].

Biological and biochemical characterization of venom from the broad-banded copperhead (Agkistrodon contortrix laticinctus): isolation of two new dimeric disintegrins.

Disintegrins represent a family of effective cell-cell and cell-matrix inhibitors by binding to integrin receptors. Integrins are heterodimeric, transmembrane receptors that are the bridges for these cell interactions. Disintegrins have been shown to have many therapeutic implications for the treatment of strokes, heart attacks, and cancer. Two novel heterodimeric disintegrins were isolated from the venom of the broad-banded copperhead (Agkistrodon contortrix laticinctus). Crude venom separated by cation-exchange chromatography resulted in several fractions possessing hemorrhagic, fibrinolytic, gelatinase, and platelet activities. Venom fractions 2-3 and 17-19 showed fibrinolytic activity. Fractions 2-6, 8-11, and 16-21 had hemorrhagic activity. Gelatinase activity was found in fractions 3, 11, and 19. The isolation of laticinstatins 1 and 2 was accomplished by fractionating crude venom using reverse phase chromatography. Data from both SDS-PAGE and N-terminal sequencing determined that laticinstatins 1 and 2 were heterodimeric disintegrins, and both were assayed for their ability to inhibit platelet aggregation in human whole blood. Future functional evaluation of snake venom disintegrins shows considerable promise for elucidating the biochemical mechanisms of integrin-ligand interactions that will allow the development of adequate medications for hemostatic pathologies such as thrombosis, stroke, and cerebral and cardiac accidents. In this study, we are presenting the first report of the purification, and partial characterization of two new dimeric disintegrins isolated from the venom of broad-banded copperhead snakes.

Vipegitide: a folded peptidomimetic partial antagonist of α2β1 integrin with antiplatelet aggregation activity.

Linear peptides containing the sequence WKTSRTSHY were used as lead compounds to synthesize a novel peptidomimetic antagonist of α2β1 integrin, with platelet aggregation-inhibiting activity, named Vipegitide. Vipegitide is a 13-amino acid, folded peptidomimetic molecule, containing two α-aminoisobutyric acid residues at positions 6 and 8 and not stable in human serum. Substitution of glycine and tryptophan residues at positions 1 and 2, respectively, with a unit of two polyethylene glycol (PEG) molecules yielded peptidomimetic Vipegitide-PEG2, stable in human serum for over 3 hours. Vipegitide and Vipegitide-PEG2 showed high potency (7×10−10 M and 1.5×10−10 M, respectively) and intermediate efficacy (40% and 35%, respectively) as well as selectivity toward α2 integrin in inhibition of adhesion of α1/α2 integrin overexpressing cells toward respective collagens. Interaction of both peptidomimetics with extracellular active domain of α2 integrin was confirmed in cell-free binding assay with recombinant α2 A-domain. Integrin α2β1 receptor is found on the platelet membrane and triggers collagen-induced platelet aggregation. Vipegitide and Vipegitide-PEG2 inhibited α2β1 integrin-mediated adhesion of human and murine platelets under the flow condition, by 50%. They efficiently blocked adenosine diphosphate- and collagen I-induced platelet aggregation in platelet rich plasma and whole human blood. Higher potency of Vipegitide than Vipegitide-PEG2 is consistent with results of computer modeling of the molecules in water. These peptidomimetic molecules were acutely tolerated in mice upon intravenous bolus injection of 50 mg/kg. These results underline the potency of Vipegitide and Vipegitide-PEG2 molecules as platelet aggregation-inhibiting drug lead compounds in antithrombotic therapy.

Biological properties of a DHA-containing structured phospholipid (AceDoPC) to target the brain

1-acetyl,2-docosahexaenoyl-glycerophosphocholine (AceDoPC) has been made to prevent docosahexaenoyl (DHA) to move to the sn-1 position as it rapidly does when present in 1-lyso,2-docosahexaenoyl-GPC (lysoPC-DHA), an efficient DHA transporter to the brain. When incubated with human blood, AceDoPC behaves closer to lysoPC-DHA than PC-DHA in terms of binding to plasma albumin and lipoproteins, and DHA incorporation into platelets and red cells. In addition, AceDoPC prevents more efficiently the deleterious effects of the experimental stroke in rats than does unesterified DHA. Also, AceDoPC inhibits platelet-activating factor-induced human blood platelet aggregation. Overall, AceDoPC might act as an efficient DHA transporter to the brain, and as a neuro-protective agent by itself.

Blocking the EP3 receptor for PGE2 with DG-041 decreases thrombosis without impairing haemostatic competence

Haemostasis interrupts bleeding from disrupted blood vessels by activating platelet aggregation and coagulation. A similar mechanism termed thrombosis generates obstructive thrombi inside diseased arteries. As a consequence of this similarity, current anti-thrombotic agents increase the risk of bleeding. Atherosclerotic plaques produce significant amounts of prostaglandin E2 (PGE2), which activates its receptor EP3 on platelets and aggravates atherothrombosis. We investigated whether blocking EP3 could dissociate atherothrombosis from haemostasis. Inhibiting in vivo the receptor EP3 for PGE2 with the blocking agent DG-041 reduced murine thrombosis triggered by local delivery of arachidonic acid or ferric chloride on healthy arteries. Importantly, it also reduced thrombosis triggered by scratching murine atherosclerotic plaques. PGE2 was not produced at the bleeding site after tail clipping. Consistently, blocking EP3 did not alter murine tail, liver, or cerebral haemostasis. Furthermore, blocking EP3 reduced murine pulmonary embolism and intensified platelet inhibition by clopidogrel leaving tail bleeding times unchanged. Human atherosclerotic plaques produced PGE2, which facilitated platelet aggregation in human blood and rescued the function of P2Y12-blocked platelets. Finally, in healthy patients, DG-041 reduced platelet aggregation, but did not significantly alter the cutaneous bleeding time at doses up to eight times the dose that inhibited the facilitating effect of PGE2 on platelets. In mice, blocking EP3 inhibited atherothrombosis without affecting haemostasis and intensified efficiency of conventional anti-platelet treatment without aggravating the bleeding risk. In patients, blocking EP3 should improve the prevention of cardiovascular diseases, which is currently limited by the risk of bleeding.

Inhibitory effects of kiwifruit extract on human platelet aggregation and plasma angiotensin-converting enzyme activity

Previous human studies suggest that supplementation with kiwifruits lowers several cardiovascular risk factors such as platelet hyperactivity, blood pressure and plasma lipids. The cardiovascular health benefit of fruit and vegetables is usually attributed to the complex mixture of phytochemicals therein; however, kiwifruit’s cardioprotective factors are not well studied. In this study, we investigated the effects of kiwifruit extract on human blood platelet aggregation and plasma angiotensin-converting enzyme (ACE) activity. A sugar-free, heat-stable aqueous extract with molecular mass less than 1000 Da was prepared from kiwifruits. Typically, 100 g kiwifruits produced 66.3 ± 5.8 mg (1.2 ± 0.1 mg CE) of sugar-free kiwifruit extract (KFE). KFE inhibited both human platelet aggregation and plasma ACE activity in a dose-dependent manner. KFE inhibited platelet aggregation in response to ADP, collagen and arachidonic acid, and inhibitory action was mediated in part by reducing TxA2 synthesis. The IC50 for ADP-induced platelet aggregation was 1.6 ± 0.2 mg/ml (29.0 ± 3.0 μg CE/ml), whereas IC50 for serum ACE was 0.6 ± 0.1 mg/ml (11.0 ± 1.2 μg CE/ml). Consuming 500 mg of KFE (9.0 mg CE) in 10 g margarine inhibited ex vivo platelet aggregation by 12.7%, 2 h after consumption by healthy volunteers (n = 9). All these data indicate that kiwifruit contains very potent antiplatelet and anti-ACE components. Consuming kiwifruits might be beneficial as both preventive and therapeutic regime in cardiovascular disease.

Regulation of human platelet aggregation by genetically modified pig endothelial cells and thrombin inhibition

Coagulation disorders remain barriers to successful pig-to-primate organ xenotransplantation. In vitro, we investigated the impact of pig genetic modifications on human platelet aggregation in response to pig aortic endothelial cells (pAEC). In comparison to human (h)AEC and wild-type (WT) pAEC, the expression of human complement- (CD46, CD55) or coagulation (thrombomodulin [TBM], endothelial protein C receptor [EPCR]) -regulatory proteins on pAEC from WT or α1,3-galactosyltransferase gene-knockout (GTKO) pigs was studied by flow cytometry. Using platelet-aggregometry, human whole blood platelet aggregation was evaluated after co-incubation with various AEC. Further, the inhibitory effect on aggregation of heparin, low molecular weight heparin, and hirudin was assessed. Heparin, low molecular weight heparin and hirudin almost completely prevented platelet aggregation induced by WT pAEC. The level of expression of human CD46, CD55, TBM and EPCR on pAEC was comparable to that on hAEC. Platelet aggregation induced by all genetically modified pAEC was significantly less (P < 0.05) than that by WT pAEC (which was 54%). GTKO/CD46/TBM pAEC induced the least platelet aggregation (27%)-a reduction of almost 50%-but this remained significantly greater (P < 0.01) than aggregation induced by hAEC (4%). There was significant positive correlation between reduction of aggregation and TBM or EPCR expression on pAEC (r = 0.89 and r = 0.86, respectively; P < 0.05). Platelet aggregation induced by GTKO/CD46/TBM pAEC in the presence of hirudin (1 IU/ml) was comparable to platelet aggregation induced by hAEC. Genetic modification of pAEC is associated with significant reduction of human platelet aggregation in vitro. With concomitant thrombin inhibition, platelet aggregation was comparable to that stimulated by hAEC.

Low-Density Lipoprotein (LDL) Antioxidants and Antiplatelet Compounds from Selected Malaysian Plants as Leads for the Development of Cardiovascular Protective Agents

Oxidative modification of low-density lipoprotein (LDL) plays a significant role in the onset and acceleration of atherosclerotic process and the development of cardiovascular diseases. The chronic antioxidant, hypolipidemic, antithrombotic and antiplatelet activities of many phytochemicals have important roles in prevention of lipoprotein oxidation and atherosclerotic lesion development. Phytochemicals of diverse chemical structures, including phenolic compounds, xanthones, prenylflavonoids, benzophenones, alkaloids and triterpenoids isolated from Malaysian plants were investigated for their ability to inhibit arachidonic acid (AA), collagen and adenosine diphosphate (ADP) induced platelet aggregation in human whole blood using an electrical impedance method. The compounds were also investigated for their ability to inhibit human LDL oxidation using thiobarbituric acid reactive substances (TBARS) assay. Among the compounds tested, 2-(3-methylbut-2-enyl)1,3,5,-trihydroxyxanthone, 2-(3-methylbut-2-enyl)-1,3,5,6-tetrahydroxyxanthone, rubraxanthone and 3,5,3′,5′-tetrahydroxy-4methoxybenzophenone isolated from Guttiferae species showed strong inhibition on platelet aggregation. Curcumin, cardamonin, pinocembrine, 5,6-dehydrokawain and 3-deacetylcrotepoxide were the strong antiplatelet compounds identified from Zingiberaceae species. Of the alkaloids tested, (-)-N-acetylnornuciferine, (-)-N-acetylanonaine and 1-(N-acetyl-Nmethylamino)ethyl-3,4,6-trimethoxy-7-hydroxyphenanthrene from Armodendron elegans showed strong inhibition on platelet aggregation caused by all three inducers. 3,5,3′,5′-Tetrahydroxy-4-methoxybenzophenone and 1, 7-dihydroxyxanthone showed strong inhibitory activity on LDL oxidation. Curcumin, demethoxycurcumin and bisdemethoxycurcumin exhibited stronger activity than the reference, probucol, as LDL antioxidants. Xanthorrhizol, zerumbone and ar-turmerone from the oils of Curcuma species showed relatively strong antioxidant activity. 3,5,3′,5′-Tetrahydroxy-4-methoxybenzophenone and 1, 7dihydroxyxanthone also showed strong inhibitory activity on LDL oxidation. Of the triterpenoids tested, garcihombronane B from Garcinia cantleyana showed moderate activity against LDL oxidation. The compounds are new classes of natural products that can be used as leads for development of new cardiovascular protective agents.