Platelet Thromboxane Synthesis Research Articles

Functional features of platelets at the beginning of ontogenesis in purebred calves of the Yaroslavl breed

After birth, calves develop an increased ability of platelets to aggregate. At the same time, during the newborn phase, the level of inactive platelets in the blood of the Yaroslavl breed calves slightly decreases. The total intravascular activity of platelets in their blood during the study increased by 10.4%. The number of various platelet aggregates suspended in the blood of calves during the observation showed an upward trend. This was caused by an increase in their platelet thromboxane synthesis, an increase in the number of mobile adenosine phosphates in platelet granules and an increase in actin and myosin levels in them. As a result of the study, it becomes clear that for the Yaroslavl breed calves during the first 10 days of life, an increase in the severity of platelet aggregation and secretion is characteristic. Apparently, this helps to create optimal conditions for them to realize hemocirculation and metabolism, and therefore for the successful growth of animals of this breed.

Physiological Properties of Platelets in Newborn Calves of the Kholmogory Breed

The platelet activity in newborn calves of the Kholmogory breed was assessed. The studies were conducted on 39 animals obtained from healthy cows of 2–3 calving. Calves were examined five times in 2 days during the observation period. A tendency towards an increase in the activity of the aggregation process of blood platelets with all tested inducers was revealed during the neonatal phase. It was noted that the number of discoid platelets in the calf blood tended to decrease during the observation period. In these conditions, the level of activated platelets increased by 12.9% in total and the content of freely circulating aggregates of small, medium, and large sizes increased. These changes were provided in the calves by a slightly increased synthesis of thromboxane in blood platelets, which was due to the increased activity of platelet cyclooxygenase and thromboxane synthetase. In addition, a large role in the increase in platelet activity was played by growth in the quantitative content of adenosine phosphates in platelet granules and some increase in their secretion. The level of actin and myosin in calf discoid platelets during the neonatal phase increased by 6.4 and 12.6%, respectively. This was accompanied by a tendency towards growth in the amount of actin and myosin in platelets during their aggregation in response to a weak and strong inducer by 6.4 and 10.1%, respectively. The calves of the Kholmogory breed are characterized by a slightly increased activity of the hemostatic properties of platelets during the neonatal period, which eliminates their risk of bleeding under optimal conditions for microcirculation. The increasing intravascular platelet activity forms the conditions for optimal anabolic processes in tissues of animals in the neonatal period.

Platelet function activity in black-motley calves during the dairy phase

Platelet activity in cattle can change under the influence of many factors. Their assessment in the second phase of their early ontogenesis is of great interest, taking into account the breed of calves. The purpose of the work is to evaluate platelet activity in black-and-white breed dairy calves. The study was conducted on 41 calf of black-motley breed, which was obtained from healthy cows as a result of 2–3 pregnancies. The calves were examined on the 11th, 15th, 20th, 25th and 30th day of ontogenesis. The study used biochemical, hematological and statistical methods. In animals, an increase in platelet aggregation with all tested inductors was detected during the milk feeding phase. The number of discoid platelets in the blood of calves observed during the second phase of early ontogenesis decreased by 10.5 %. Moreover, the total number of active platelets increased by 24.0 %. The levels of small, as well as medium and large aggregates of platelets present in the blood also increased during the milk feeding phase by 28.6 and 27.3 %, respectively. This was achieved in the observed calves by an increase of 9.6 % in the synthesis of thromboxane in platelets due to an increase in the activity of cyclooxygenase in them by 9.4 % and thromboxane synthetase by 9.3 %. This was also influenced by the increase in the platelet content of adenosine phosphates and the increase in their secretion. The levels of actin and myosin in inactive calf platelets increased during the milk feeding phase by 9.7 and 13.2 %, respectively. In animals, an increase in the additional generation of actin and myosin was revealed during platelet aggregation by 11.1 and 9.8 %, respectively. It is clear that calves of the black-motley breed of dairy food are characterized by a certain increase in the activity of platelet aggregation and secretion. This provides them with a high degree of preservation of blood volume in case of damage to blood vessels. The growth of intravascular platelet activity in these calves also contributes to the creation of the necessary conditions to minimize blood loss and ensure homeostasis.

Effects of Aspirin and Prednisone on Platelet Function and Thromboxane Synthesis in Healthy Dogs.

Glucocorticoid administration is a risk factor for thromboembolism in hypercoagulable dogs, and it is unknown if aspirin counteracts glucocorticoid-induced hypercoagulability. The objective was to determine the effects of sustained aspirin and prednisone administration on platelet function and thromboxane synthesis. Our hypothesis was that aspirin would consistently inhibit platelet function and thromboxane synthesis when administered with or without prednisone. In 24 healthy dogs, platelet aggregometry and urine 11-dehydro-thromboxane-B2 (11-dTXB2)-to-creatinine ratios were measured on days 0, 14, and 28. Dogs were administered placebos, aspirin (2 mg/kg/d), prednisone (2 mg/kg/d), or prednisone/aspirin combination therapy PO for 28 days in a randomized double-blinded study. Aspirin response was based on a >25% reduction in platelet aggregation compared to pre-treatment values. Results were compared using mixed model, split-plot repeated measures ANOVAs. P < 0.05 was considered significant. AUC differed significantly by time [F(2,40) = 10.2, P < 0.001] but not treatment or treatment-by-time. On day 14, 2 dogs were aspirin responders (aspirin, 1; placebo, 1). On day 28, 3 dogs were aspirin responders (aspirin, 2; prednisone/aspirin, 1). Urine 11-dTXB2-to-creatinine ratios differed significantly by group [F(3,20) = 3.9, P = 0.024] and time [F(2,40) = 8.7, P < 0.001), but not treatment-by-time. Post-hoc analysis revealed significant differences between aspirin and placebo groups (P=0.008), aspirin and prednisone/aspirin groups (P = 0.030), and placebo and prednisone groups (P = 0.030). In healthy dogs, sustained aspirin, prednisone, and combination therapy do not inhibit platelet aggregation, and when used as individual therapies, aspirin and prednisone decreased thromboxane synthesis. Additional studies using varied platelet function methodologies in hypercoagulable dogs are necessary.

Physiological parameters of thrombocytes in piglets during phase of plant nutrition early ontogeny

A very important mechanism for maintaining homeostasis in the animal body is platelet hemostasis. The level of his activity very strongly determines the rheology of blood in the capillaries and due to this the state of metabolism in the tissues. There is reason to believe that the growth and development of piglets depends on the level of functional activity of platelets. For this reason, studies of the age-related dynamics of platelet activity in piglets at the end of early ontogenesis are of great importance. In the study, it was found that the piglets of plant nutrition showed an increase in the aggregation capacity of platelets (aggregation of their platelets was accelerated by ADP from 34.10.12 s to 27.60.12 s, with collagen from 24.70.08 s to 17.50.10 s, with thrombin from 36.50.08 s to 28.40.12 s). The main reason for this may be an intensification of the work of receptor and postreceptor mechanisms of platelets. This was characteristic of piglets during the phase of plant nutrition of early ontogenesis to the same extent with respect to strong and weak inducers of platelet aggregation. The increase in thromboxane synthesis in platelets (from 48.50.05% to 59.80.09%) was largely due to an increase in the activity of cyclooxygenase in them (from 78,40.06% to 89.80.12%) and thromboxane synthetase (from 69.20.07% to 81.50.07%) and activation of ADP secretion from them (from 46.00.13% to 56.90.15%). The increase in hemostatic activity of platelets in piglets during the phase of plant nutrition of early ontogeny should be considered an important regulator of their microcirculation and metabolism in tissues under existing conditions of existence.

Physiological Features of Platelets in Milk and Vegetable Nutrition Piglets

A very important mechanism for maintaining homeostasis in the animal body is platelet hemostasis. The level of his activity very strongly determines the rheology of blood in the capillaries and due to this the state of metabolism in the tissues. There is reason to believe that the growth and development of piglets depends on the level of functional activity of platelets. For this reason, studies of the age-related dynamics of platelet activity in piglets during early ontogeny are of great importance. In the study, it was established that piglets of milk and vegetal nutrition showed an increase in the adhesive, aggregation and secretory capacity of platelets. The main reason for this may be an intensification of the work of receptor and postreceptor mechanisms of platelets. This was characteristic of piglets during dairy and plant nutrition in the same degree with respect to strong and weak inducers of platelet aggregation. At the base of the revealed increase in the activity of platelets in piglets throughout the course of the observation, the increase in the synthesis of thromboxane in platelets was largely due to the increase in the activity of cyclooxygenase and thromboxane synthetase in them and activation of the secretion of ADP from them. The increase in hemostatic activity of platelets in piglets during the phase of milk and plant nutrition should be considered an important regulator of their microcirculation and metabolism in tissues under changing conditions of existence.

Dietary supplements and drugs for preeclampsia prophylaxis

Pre-eclampsia (PE) is a major cause of mortality and morbidity during pregnancy. Many different strategies to prevent PE have been investigated in randomized controlled trials, but none have been found to be widely effective and this is not surprising given the complexities in pathogenesis. Low-dose aspirin, diminishing platelet thromboxane synthesis, seems to prevent or delay PE. A meta-analysis of individual patient data from 32,217 women (PARIS collaborative group) asses that low-dose aspirin during pregnancy is associated with moderate reduction in the relative risk of PE (RR 0.90 95%CI 0.84–0.97), of birth before 34 weeks’ gestation (RR 0.90 95%CI 0.83–0.98), and of having a pregnancy with a serious adverse outcome (RR 0.90 95%CI 0.85–0.96). This effect seems to be more significant in women at high risk of developing PE. Regarding diet and nutrients, there does not appear to be any clear benefit for prevention of PE from routine dietary supplementation. There is high quality evidence that vitamin C and vitamin E are not effective for prevention or treatment of PE. Actually a meta-analyses found that supplementation with these antioxidants is associated with a slightly increased risk of gestational hypertension and premature rupture of membranes. Also there is not enough high-quality evidence to show that dietary magnesium or omega-3 fatty acids supplementation during pregnancy is beneficial. Calcium supplementation instead appears to reduce the risk of PE and the rare occurrence of the composite outcome ’death or serious morbidity’ specially in women with a low baseline calcium intake. Some studies assess that preeclamptic women may be deficient in nitric oxide, which mediates vasodilatation and inhibits platelet aggregation. For this reason NO donors, such as isosorbide dinitrate, glyceryl trinitrate and S-Nitroglutathione, were used in some trials as treatment in preeclamptic women. Despite the reduction in blood pressure and in uterine artery pulsatility index, these substances were rarely used in clinical practice because of adverse effects such as headache or preoxynitrite production. Also l -arginine, the substrate for synthesis of nitric oxide, has been investigated as a mean of preventing PE. Even though a dysfunction of the l -arginine-NO pathway that seems to be present in preeclamptic women, l -arginine load appears to be associated with increased nitric oxide (NO) production, hypotension and reduction of PE/eclampsia rate. Also in obese women there is a dysfunction of the l -arginine-NO pathway that causes a decrease of endothelial-dependent vasodilatation. In these women a l -arginine supplement could be helpful. Conclusion Aspirin proven to be of some help provided dose and timing are adequate. Impact on PE is limited, and its use is better in high-risk. Calcium supply could be helpful in population with low dietary intake. l -Arginine supplementation is worth to receive the same attention aspirin had also considering that biological activity seems wider. Actual evidences are more robust than the ones that allows wasting time and money for Vitamins C and E.

Effects of Firocoxib, Flunixin Meglumine, and Phenylbutazone on Platelet Function and Thromboxane Synthesis in Healthy Horses.

Determine the effects of nonsteroidal anti-inflammatory drugs (NSAID) on platelet function and thromboxane synthesis immediately after drug administration and following 5 days of NSAID administration in healthy horses. Randomized cross-over study. Healthy adult horses (n=9; 6 geldings and 3 mares). Horses received either flunixin meglumine (1.1 mg/kg IV every 12 hours), phenylbutazone (2.2 mg/kg IV every 12 hours), or firocoxib (loading dose of 0.27 mg/kg IV on day 1, then 0.09 mg/kg IV every 24 hours for 4 days) for a total of 5 days. Blood samples were collected prior to drug administration (day 0), 1 hour after initial NSAID administration (day 1), and then 1 hour post-NSAID administration on day 5. Platelet function was assessed using turbidimetric aggregometry and a platelet function analyzer. Serum thromboxane B2 concentrations were determined by commercial ELISA kit. A minimum 14 day washout period occurred between trials. At 1 hour and 5 days postadministration of firocoxib, flunixin meglumine, or phenylbutazone, there was no significant effect on platelet aggregation or function using turbidimetric aggregometry or a platelet function analyzer. There was, however, a significant decrease in thromboxane synthesis at 1 hour and 5 days postadministration of flunixin meglumine and phenylbutazone that was not seen with firocoxib. Preoperative administration of flunixin meglumine, phenylbutazone, or firocoxib should not inhibit platelet function based on our model. The clinical implications of decreased thromboxane B2 synthesis following flunixin meglumine and phenylbutazone administration are undetermined.

Escherichia coli induces platelet aggregation in an FcγRIIa‐dependent manner

The discovery of pathogen-recognition receptors such as Toll-like receptors on platelets has led to the emergence of the concept of platelets as important components of the host response to infection. Escherichia coli (E. coli)-mediated sepsis is a serious illness characterized by the occurrence of thrombocytopenia. Whereas there has been a wealth of research on platelet activation by Gram-positive bacteria, little is known about the mechanisms associated with Gram-negative bacteria-induced platelet activation with Gram-negative bacteria. To determine the mechanisms by which Gram-negative E. coli induces platelet aggregation. Induction of platelet aggregation with E. coli strain O157:H7 was tested in platelet-rich plasma (PRP), washed platelets, and serum depleted of complement factors. Platelet inhibitors (against αII b β3 , glycoprotein Ibα and FcγRIIa) were used. Platelet thromboxane synthesis was analyzed after E. coli stimulation. Cell binding assays were used to assess the ability of E. coli to support platelet adhesion. Trypsinization was used to determine the role of E. coli surface proteins. E. coli-induced aggregation in PRP was donor-dependent. E. coli O157:H7 induced aggregation with a lag time of 6.9 ± 1.3 min in an αII b β3 -dependent and FcγRIIa-dependent manner. Furthermore, this interaction was enhanced by the presence of complement, and was dependent on thromboxane synthesis. These results show E. coli to be a potent inducer of platelet aggregation.

Abstract 12357: Enzyme Kinetic Model Reveals Novel Characteristics of Pharmacodynamic Interaction of NSAIDs and Aspirin at COX-1, Which Lead to High on Aspirin Platelet Reactivity (HAPR)

Introduction: Non-aspirin NSAIDs attenuate the antiplatelet action of aspirin leading to High on Aspirin Platelet Reactivity (HAPR). However, detailed features of this interaction are still unresolved. Both competitive as well as non-competitive interaction at the cyclooxygenase-1 (COX-1) enzyme have been proposed. Hypothesis: We examined whether competitive enzyme kinetics describe COX-1 inhibition by aspirin and HAPR and if they are compatible with results obtained experimentally. Methods: We have developed an enzyme kinetic model of the aspirin/NSAID interaction at COX-1 using mathematical modelling and have varied enzyme kinetic parameters to predict the occurrence and the extent of HAPR. Subsequently, the predictions obtained were experimentally verified with selected NSAIDs using light transmission aggregometry and immunoassay for platelet thromboxane (TX) synthesis. Results: Enzyme kinetic modelling predicted that only low- and medium-affinity NSAIDs (Ki ≥1 μM), but not high-affinity NSAIDs, interfere with COX-1 inhibition by aspirin. Furthermore, the interaction was predicted to be most extensive at high concentrations of arachidonic acid (1mM). Experimental results confirmed the model by showing that low or medium-affinity COX-1 inhibitors piroxicam, dipyrone and celecoxib (each n=5-10) largely prevented the inhibition of aggregation and platelet TX synthesis in vitro by 30 μmol/L aspirin whereas the high-affinity COX-1 inhibitor SC560 (n=6) did not interfere with aspirin at all. In accordance to the mathematical model, aspirin/NSAID interaction was more pronounced in presence of high (1mmol/L) versus low (0.3mmol/L) concentration of arachidonic acid. Hence, the kinetic model was validated experimentally. Conclusion: Both enzyme kinetic modelling and experimental results suggest competitive antagonism of NSAID and aspirin at COX-1 as a cause for HAPR, possibly resulting in cardiovascular events. Critical parameters that influence this interaction are drug concentrations and NSAID affinity. Interestingly, the concentration of arachidonic acid also influences the NSAID/aspirin interaction, suggesting that “fatty acid tone” and lipid metabolism may influence this unfavourable drug/drug interaction.

The Cause for Heart Disease and Strokes

There has been no solution to heart disease to date. I obtained discarded veins from bypass (CABG) surgeries and subjected them to phospholipid analysis. We also obtained arterial cells from human umbilical cords and cultured them with a decreasing concentration of either cholesterol or oxidized cholesterol. Patients undergoing CABG surgery and aging swine had significantly higher levels of sphingomyelin in their arterial cells than arterial cells from human umbilical cords. Oxidized low-density lipoprotein (OxLDL) and oxysterols further contribute to atherosclerosis by increasing the synthesis of thromboxane in platelets, a clotting factor. When we incubated arterial cells with cholesterol that had not been oxidized, even at twelve times the concentration of the oxidized cholesterols we used, there was no effect on sphingomyelin content, this shows that cholesterol itself is not the reason for heart disease, and has to be oxidized in order to cause harm. My study indicated that atherosclerosis is due to a diet that contains a high level of oxysterols. Normal levels of oxysterols in the plasma will not increase sphingomyelin levels. Removing oxidized fat from the diet should be considered as a therapeutic measure for atherosclerosis. Ancel Keys, who some consider the father of the cholesterol-heart disease hypothesis said in 1997: "There's no connection whatsoever between the cholesterol in food and cholesterol in the blood."

New analogues of 13-hydroxyocatdecadienoic acid and 12-hydroxyeicosatetraenoic acid block human blood platelet aggregation and cyclooxygenase-1 activity

BackgroundThromboxane A2 is derived from arachidonic acid through the action of cyclooxygenases and thromboxane synthase. It is mainly formed in blood platelets upon activation and plays an important role in aggregation. Aspirin is effective in reducing the incidence of complications following acute coronary syndrome and stroke. The anti-thrombotic effect of aspirin is obtained through the irreversible inhibition of cyclooxygenases. Analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid were shown previously to modulate platelet activation and to block thromboxane receptors.Results and discussionWe synthesized 10 compounds based on the structures of analogues of 12-hydroxyeicosatetraenoic acid and 13-hydroxyocatdecadienoic acid and evaluated their effect on platelet aggregation triggered by arachidonic acid. The structure activity relationship was evaluated. Five compounds showed a significant inhibition of platelet aggregation and highlighted the importance of the lipidic hydrophobic hydrocarbon chain and the phenol group. Their IC50 ranged from 7.5 ± 0.8 to 14.2 ± 5.7 μM (Mean ± S.E.M.). All five compounds decreased platelet aggregation and thromboxane synthesis in response to collagen whereas no modification of platelet aggregation in response to thromboxane receptor agonist, U46619, was observed. Using COS-7 cells overexpressing human cyclooxygenase-1, we showed that these compounds are specific inhibitors of cyclooxygenase-1 with IC50 ranging from 1.3 to 12 μM. Docking observation of human recombinant cyclooxygenase-1 supported a role of the phenol group in the fitting of cyclooxygenase-1, most likely related to hydrogen bonding with the Tyr 355 of cyclooxygenase-1.ConclusionsIn conclusion, the compounds we synthesized at first based on the structures of analogues of 12 lipoxygenase metabolites showed a role of the phenol group in the anti-platelet and anti-cyclooxygenase-1 activities. These compounds mediate their effects via blockade of cyclooxygenase-1.

High Glucose Inhibits the Aspirin-Induced Activation of the Nitric Oxide/cGMP/cGMP-Dependent Protein Kinase Pathway and Does Not Affect the Aspirin-Induced Inhibition of Thromboxane Synthesis in Human Platelets

Since hyperglycemia is involved in the “aspirin resistance” occurring in diabetes, we aimed at evaluating whether high glucose interferes with the aspirin-induced inhibition of thromboxane synthesis and/or activation of the nitric oxide (NO)/cGMP/cGMP-dependent protein kinase (PKG) pathway in platelets. For this purpose, in platelets from 60 healthy volunteers incubated for 60 min with 5–25 mmol/L d-glucose or iso-osmolar mannitol, we evaluated the influence of a 30-min incubation with lysine acetylsalicylate (L-ASA; 1–300 μmol/L) on 1) platelet function under shear stress; 2) aggregation induced by sodium arachidonate or ADP; 3) agonist-induced thromboxane production; and 4) NO production, cGMP synthesis, and PKG-induced vasodilator-stimulated phosphoprotein phosphorylation. Experiments were repeated in the presence of the antioxidant agent amifostine. We observed that platelet exposure to 25 mmol/L d-glucose, but not to iso-osmolar mannitol, 1) reduced the ability of L-ASA to inhibit platelet responses to agonists; 2) did not modify the L-ASA–induced inhibition of thromboxane synthesis; and 3) prevented the L-ASA–induced activation of the NO/cGMP/PKG pathway. Preincubation with amifostine reversed the high-glucose effects. Thus, high glucose acutely reduces the antiaggregating effect of aspirin, does not modify the aspirin-induced inhibition of thromboxane synthesis, and inhibits the aspirin-induced activation of the NO/cGMP/PKG pathway. These results identify a mechanism by which high glucose interferes with the aspirin action.

A role for adhesion and degranulation‐promoting adapter protein in collagen‐induced platelet activation mediated via integrin α2β1

Collagen-induced platelet activation is a key step in the development of arterial thrombosis via its interaction with the receptors glycoprotein (GP)VI and integrin α(2) β(1) . Adhesion and degranulation-promoting adapter protein (ADAP) regulates α(IIb) β(3) in platelets and α(L) β(2) in T cells, and is phosphorylated in GPVI-deficient platelets activated by collagen. To determine whether ADAP plays a role in collagen-induced platelet activation and in the regulation and function of α(2) β(1). Using ADAP(-/-) mice and synthetic collagen peptides, we investigated the role of ADAP in platelet aggregation, adhesion, spreading, thromboxane synthesis, and tyrosine phosphorylation. Platelet aggregation and phosphorylation of phospholipase Cγ2 induced by collagen were attenuated in ADAP(-/-) platelets. However, aggregation and signaling induced by collagen-related peptide (CRP), a GPVI-selective agonist, were largely unaffected. Platelet adhesion to CRP was also unaffected by ADAP deficiency. Adhesion to the α(2) β(1) -selective ligand GFOGER and to a peptide (III-04), which supports adhesion that is dependent on both GPVI and α(2) β(1), was reduced in ADAP(-/-) platelets. An impedance-based label-free detection technique, which measures adhesion and spreading of platelets, indicated that, in the absence of ADAP, spreading on GFOGER was also reduced. This was confirmed with non-fluorescent differential-interference contrast microscopy, which revealed reduced filpodia formation in ADAP(-/-) platelets adherent to GFOGER. This indicates that ADAP plays a role in mediating platelet activation via the collagen-binding integrin α(2) β(1). In addition, we found that ADAP(-/-) mice, which are mildly thrombocytopenic, have enlarged spleens as compared with wild-type animals. This may reflect increased removal of platelets from the circulation.

Mean Platelet Volume and Integrin Alleles Strongly Correlate with Increased Levels of Integrins αIIbβ3 and α2β1: Clinical Implications for Platelet Reactivity in Acute Coronary Syndome

Abstract 32 Background- Differences in the level of integrins expressed on the surface membranes of platelets can have a significant effect on platelet reactivity and adverse outcomes in a variety of thrombotic and hematologic dyscrasias. Among normal individuals, the basal level of platelet integrin aIIbb3 varies at least two-fold, while that of the integrin a2b1 exhibits a greater range of variation. The total number of integrin molecules and other receptors on platelets is influenced to a large extent by the total surface area of plasma membrane and consequently the mean platelet volume (MPV), which itself has a very strong genetic component. We sought to determine the relative contribution of mean platelet volume (MPV) and integrin gene alleles to expression levels of these integrins in patients with acute coronary syndrome (ACS) and normal subjects. Methods and Results- Monoclonal antibodies AP2 and 8C12 (or 12F1) were used in flow cytometry to measure platelet aIIbb3 and a2b1, respectively, in whole blood from 341 patients with ACS and 128 normal subjects. Comparisons were made with MPV, platelet count (PC) and in the normal subjects ITGA2 rs1126643 and ITGB3 rs5918 alleles. In both ACS patients and controls, there was a strong direct correlation between MPV and aIIbb3 level (p < 0.001). In control subjects, MPV and aIIbb3 level did not correlate with ITGB3 rs5918 alleles. For a2b1, MPV contributed modestly in either patients or controls, while ITGA2 rs1126643 alleles exerted the greatest effect. Moreover, we observed an inverse correlation between MPV and the rs1126643 minor allele. Conclusions- Our results indicate that MPV is the major effector of platelet membrane levels of aIIbb3, in both ACS patients and normal subjects, while ITGA2 rs1126643 contributes most strongly to a2b1 levels. Hyperactivity of larger platelets results from increased levels of key platelet receptors, notably integrin aIIbb3, in support of the contention that MPV is an accurate marker of risk for adverse outcome in ACS. This is consistent with published findings that MPV, the most common metric of platelet size, correlates very well with platelet reactivity, such that larger platelets have greater prothombotic potential, and elevated MPV is associated with increased platelet aggregation, thromboxane synthesis, b-thromboglobulin release, and expression of adhesion molecules. The most intriguing finding of our study is the inverse association of the ITGA2 rs1126643 minor allele T with MPV. The rs1126643 minor allele likely exerts this effect via the documented influence of a2b1 on megakaryocyte (MK) maturation. Disclosures:No relevant conflicts of interest to declare.

Ligand Switching in Cell-Permeable Peptides: Manipulation of the α-Integrin Signature Motif

A synthetic cell-permeable peptide corresponding to the highly conserved alpha-integrin signature motif, Palmityl-K(989)VGFFKR(995) (Pal-FF), induces integrin activation and aggregation in human platelets. Systematic replacement of the F(992)-F(993) with amino acids of greater or lesser hydrophobicity to create Pal-KVGxxKR peptides demonstrate that hydrophobic amino acids (isoleucine, phenylalanine, tyrosine, tryptophan) are essential for agonist potency. In marked contrast, substitution with small and/or hydrophilic amino acids (glycine, alanine, serine) causes a switch in the biological activity resulting in inhibition of platelet aggregation, adhesion, ADP secretion, and thromboxane synthesis. These substituted, hydrophilic peptides are not true pharmacological antagonists, as they actively induce a phosphotyrosine signaling cascade in platelets. Singly substituted peptides (Pal-AF and Pal-FA) cause preferential retention of pro- or anti-thrombotic properties, respectively. Because the alpha-integrin signature motif is an established docking site for a number of diverse cytoplasmic proteins, we conclude that eliminating critical protein-protein interactions mediated through the hydrophobic amino acids, especially F(993), favors an anti-thrombotic pathway in platelets. Agents derived from the inhibitory peptides described in this study may represent a new therapeutic strategy for anti-platelet or anti-integrin drug development.

Virgin olive oil polyphenol hydroxytyrosol acetate inhibits in vitro platelet aggregation in human whole blood: comparison with hydroxytyrosol and acetylsalicylic acid

Hydroxytyrosol acetate (HT-AC) is a polyphenol present in virgin olive oil (VOO) at a proportion similar to hydroxytyrosol (HT) (160-479 micromol/kg oil). The present study was designed to measure the in vitro platelet antiaggregating activity of HT-AC in human whole blood, and compare this effect with that of HT and acetylsalicylic acid (ASA). The experiments were designed according to the standard procedure to investigate the activity of ASA. HT-AC and HT inhibited platelet aggregation induced by ADP, collagen or arachidonic acid in both whole blood and platelet-rich plasma (PRP). ASA and HT-AC had a greater effect in whole blood than in PRP when ADP or collagen was used as inducer. ASA and HT-AC had a greater effect in PRP+leucocytes than in PRP alone. All three compounds inhibited platelet thromboxane B2 and leucocyte 6-keto-prostaglandin F1alpha (6-keto-PF1 alpha) production. The thromboxane/6-keto-PGF1alpha inhibition ratio (as an indirect index of the prostanoid equilibrium) was 10.8 (SE 1) for HT-AC, 1.0 (SE 0.1) for HT and 3.3 (SE 0.2) for ASA. All three compounds stimulated nitric oxide production, although HT was a weaker effect. In our experiments only concentrations higher than 500 microm (HT) or 1 mm (HT-AC and ASA) inhibited 3-nitrotyrosine production. All three compounds inhibited the production of TNFalpha by leucocytes, with no significant differences between them. In quantitative terms HT-AC showed a greater antiplatelet aggregating activity than HT and a similar activity to that of ASA. This effect involved a decrease in platelet thromboxane synthesis and an increase in leucocyte nitric oxide production.

COX-2 Inhibitors and Cardiovascular Risk

Placebo-controlled trials of nonsteroidal antiinflammatory drugs (NSAIDs) selective for COX-2 have revealed an enhanced risk for cardiovascular events. COX-2 inhibitors (coxibs) selectively reduce vascular prostacyclin synthesis without disrupting COX-1-derived thromboxane synthesis in platelets. Removal of prostacyclin's capacity to restrain all known endogenous compounds contributing to platelet activation and vasoconstriction is a well-recognized mechanism for coxib action in the cardiovascular system which can pre-dispose to thrombosis, hypertension and atherosclerosis. Novel mouse models of selective COX-2 inhibition and disruption of microsomal prostaglandin E synthase-1 have been exploited to reveal the relative importance of prostacyclin and prostaglandin E2 in cardiovascular homeostasis. This review discusses the background to our current understanding of coxibs and provides further information relating to recent mechanistic insights into how COX-2 inhibition promotes cardiovascular risk.

Group B streptococcus isolates from septic patients and healthy carriers differentially activate platelet signaling cascades

Infection with group B streptococcus (GBS) is the most common cause of early onset neonatal sepsis in many countries, leading to neonatal morbidity and mortality. There is much evidence for a direct involvement of platelets in the pathogenesis of inflammation and sepsis. Several bacteria are known to directly interact with platelets leading to activation and aggregation, a phenomenon also observed with GBS. Here, we demonstrate that GBS rapidly bound to platelets; however, only strains isolated from septic patients bound fibrinogen on their surface and induced platelet thromboxane synthesis, platelet aggregation, and P-selectin (CD62P) expression. In contrast, GBS strains isolated from healthy newborns or healthy pregnant women induced only shape change, but not platelet thromboxane synthesis, platelet aggregation, or CD62P expression. All GBS strains investigated were able to activate FcgammaRIIA receptor signaling pathways including phospholipase C gamma2 (PLCgamma2), as well as calcium/calmodulin-dependent myosin kinase II (CaMKII) and phosphorylation of myosin light chain (MLC). In contrast, protein kinase C (PKC) was exclusively activated by GBS strains isolated from septic patients, and p38 mitogen activated protein kinase (p38 MAP kinase) was preferentially activated by septic GBS strains. Furthermore, stress signaling kinase SEK1/MKK4 and focal adhesion kinase (FAK) were activated by all tested GBS strains in a FcgammaRIIA-independent way. This study demonstrates that septic, but not colonizing, GBS strains bind fibrinogen on their surface, and that septic GBS strains influence platelet function not only via the FcgammaRIIA receptor, but also via pathways distinct from IgG-mediated signalling. These mechanisms lead to platelet aggregation and secretion, thereby possibly modulating the pathophysiologic course of GBS infections.

Aspirin Resistance: Detection, Mechanisms and Clinical Implications

Aspirin, the most widely used antiplatelet agent, irreversibly acetylates the enzyme cyclooxygenase 1 (COX-1), thereby inhibiting platelet thromboxane synthesis and subsequent platelet aggregation. Although aspirin has been demonstrated to reduce the odds of serious atherothrombotic events and death in high-risk patients by 25%, subsets of patients fail to respond to therapy and continue to suffer atherothrombotic events. This aspirin treatment failure may be due to sub-optimal bioavailability (e.g. because of non-compliance or under-dosing) or may be a consequence of the as yet poorly understood phenomenon of aspirin resistance. In this review, we summarize the current laboratory methods used to identify aspirin-resistant patients, outline the cellular mechanisms that may contribute to aspirin resistance, and discuss the clinical implications of this important issue.