thromboxane-b2 and Thrombophilia

Over the last 30 years, a growing body of evidence has documented the role of hyperhomocysteinemia (HHcy) as an independent vascular risk factor. However, the mechanisms through which elevated circulating levels of homocysteine (Hcy) cause vascular injury and promote thrombosis remain elusive. Most findings have been achieved in in vitro studies employing exceedingly high concentrations of Hcy, whereas only a few studies have been carried out in vivo in humans. In homocystinuric patients, homozygotes for mutations of the gene coding for the cystathionine beta-synthase enzyme, abnormalities of coagulation variables reflecting a hypercoagulable state, have been reported. In vitro studies provide a biochemical background for such a state. In homocystinuric patients, an in vivo platelet activation has also been reported. The latter abnormality is not corrected by the bolus infusion of concentrations of hirudin, which determines a long-lasting impairment of the conversion of fibrinogen to fibrin by thrombin; in contrast, it appears at least in part lowered by the administration of the antioxidant drug probucol. During the autooxidation of Hcy in plasma, reactive oxygen species are generated. The latter initiate lipid peroxidation in cell membranes (potentially responsible for endothelial dysfunction) and in circulating lipoproteins. Oxidized low-density lipoproteins (LDL) may trigger platelet activation as well as some of the hemostatic abnormalities reported in such patients. Thus the oxidative stress induced by Hcy may be a key process in the pathogenesis of thrombosis in HHcy. Accumulation of adenosylhomocysteine in cells (a consequence of high circulating levels of homocysteine) inhibits methyltransferase enzymes, in turn preventing repair of aged or damaged cells. This mechanism has been recently documented in patients with renal failure and HHcy and provides an additional direction to be followed to understand the tendency to thrombosis in moderate HHcy.

Topics: Adolescent; Adult; Arteriosclerosis; Blood Coagulation; Cardiovascular Diseases; Cellular Senescence; Child; Endothelium, Vascular; Female; Genetic Predisposition to Disease; Homocysteine; Homocystinuria; Humans; Hyperhomocysteinemia; Lipid Peroxidation; Lipoproteins, LDL; Male; Methyltransferases; Oxidation-Reduction; Platelet Activation; Reactive Oxygen Species; Renal Insufficiency; Risk Factors; S-Adenosylhomocysteine; Thrombophilia; Thromboxane B2; Vitamin K

Adenosine diphosphate (ADP) is involved in shear-induced platelet activation, which may be important for platelet responses to stress. We therefore tested the hypothesis that ADP receptor antagonism by clopidogrel treatment would attenuate exercise-induced platelet activation.. Fifteen healthy volunteers performed exhaustive exercise without and with clopidogrel pretreatment (75 mg/day; 7 days) in a randomised crossover study. Filtragometry readings (reflecting platelet aggregability in vivo) and 11-dehydro-thromboxane B(2) (TxM) in plasma were determined before and after exercise. Platelet and leukocyte activity, platelet-platelet (PPA), and platelet-leukocyte aggregates (PLAs) in vivo and their responsiveness to agonist stimulation in vitro were assessed by flow cytometry. Clopidogrel treatment inhibited ADP-induced platelet P-selectin expression by 72% (54-85%). Exercise increased platelet aggregation (filtragometry and PPAs), elevated plasma TxM, increased single platelet P-selectin expression, elevated circulating PLAs, and enhanced ADP and thrombin-stimulated P-selectin expression. Clopidogrel prolonged filtragometry readings and attenuated agonist stimulated P-selectin expression at rest, but did not influence TxM in plasma or urine or attenuate platelet or leukocyte responses to exercise. Clopidogrel treatment did not influence plasma CD40L (ligand) at rest or after exercise.. Clopidogrel treatment attenuates platelet activity in vivo at rest, but exercise counteracts the platelet stabilizing effects of clopidogrel. The hypothesis that ADP is involved in stress-induced platelet activation was not supported.

Topics: Adenosine Diphosphate; Adult; Blood Platelets; Cell Adhesion; Clopidogrel; Cross-Over Studies; Exercise; Humans; Leukocytes; Male; P-Selectin; Platelet Aggregation; Platelet Aggregation Inhibitors; Thrombophilia; Thromboxane B2; Ticlopidine

Previous studies suggest that cyclooxygenase-2 (COX-2) might influence megakaryocyte (MK) maturation and platelet production in vitro. Using a gene deletion model, we analysed the effect of COX-2 deficiency on megakaryopoiesis and platelet function. COX-2-/- mice (10-12 weeks old) have hyper-responsive platelets as suggested by their enhanced aggregation, TXA2 biosynthesis, CD62P and CD41/CD61 expression, platelet-fibrinogen binding, and increased thromboembolic death after collagen/epinephrine injection compared to wild-type (WT). Moreover, increased platelet COX-1 expression and reticulated platelet fraction were observed in COX-2-/- mice while platelet count was similar to WT. MKs were significantly reduced in COX-2-/- bone marrows (BMs), with high nuclear/cytoplasmic ratios, low ploidy and poor expression of lineage markers of maturation (CD42d, CD49b). However, MKs were significantly increased in COX-2-/- spleens, with features of MK maturation markers which were not observed in MKs of WT spleens. Interestingly, the expression of COX-1, prostacyclin and PGE2 synthases and prostanoid pattern were modified in BMs and spleens of COX-2-/- mice. Moreover, COX-2 ablation reduced the percentage of CD49b+ cells, the platelet formation and the haematopoietic stem cells in bone marrow and increased their accumulation in the spleen. Splenectomy decreased peripheral platelet number, reverted their hyper-responsive phenotype and protected COX-2-/- mice from thromboembolism. Interestingly, fibrosis was observed in spleens of old COX-2-/- mice (28 weeks old). In conclusion, COX-2 deletion delays BM megakaryopoiesis promoting a compensatory splenic MK hyperplasia, with a release of hyper-responsive platelets and increased thrombogenicity in vivo. COX-2 seems to contribute to physiological MK maturation and pro-platelet formation.

Topics: Animals; Antigens, CD; Antigens, Differentiation; Blood Platelets; Bone Marrow; Crosses, Genetic; Cyclooxygenase 1; Cyclooxygenase 2; Hematopoietic Stem Cells; Hyperplasia; Megakaryocytes; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Platelet Count; Ploidies; Purpura, Thrombocytopenic, Idiopathic; Receptors, Thromboxane A2, Prostaglandin H2; Spleen; Splenectomy; Thromboembolism; Thrombophilia; Thrombopoiesis; Thromboxane B2

The inability of aspirin (acetylsalicylic acid [ASA]) to adequately suppress platelet function is associated with future risk of myocardial infarction, stroke, and cardiovascular death. Genetic variation is a proposed but unproved mechanism for insufficient ASA responsiveness.. We examined platelet ASA responsiveness in 1880 asymptomatic subjects (mean age, 44+/-13 years; 58% women) recruited from 309 white and 208 black families with premature coronary heart disease. Ex vivo platelet function was determined before and after ingestion of ASA (81 mg/d for 2 weeks) with the use of a panel of measures that assessed platelet activation in pathways directly and indirectly related to cyclooxygenase-1, the enzyme inhibited by ASA. The proportion of phenotypic variance related to CHD risk factor covariates was determined by multivariable regression. Heritability of phenotypes was determined with the use of variance components models unadjusted and adjusted for covariates. ASA inhibited arachidonic acid-induced aggregation and thromboxane B2 production by > or = 99% (P<0.0001). Inhibition of urinary thromboxane excretion and platelet activation in pathways indirectly related to cyclooxygenase-1 was less pronounced and more variable (inhibition of 0% to 100%). Measured covariates contributed modestly to variability in ASA response phenotypes (r2=0.001 to 0.133). Phenotypes indirectly related to cyclooxygenase-1 were strongly and consistently heritable across races (h2=0.266 to 0.762; P<0.01), but direct cyclooxygenase-1 phenotypes were not.. Heritable factors contribute prominently to variability in residual platelet function after ASA exposure. These data suggest a genetic basis for the adequacy of platelet suppression by ASA and potentially for differences in the clinical efficacy of ASA.

Topics: Adult; Arachidonic Acid; Aspirin; beta-Thromboglobulin; Black or African American; Blood Platelets; Cardiovascular Diseases; Coronary Disease; Cyclooxygenase 1; Cyclooxygenase Inhibitors; Drug Resistance; Dyslipidemias; Female; Genetic Heterogeneity; Genetic Predisposition to Disease; Genetic Variation; Humans; Hyperglycemia; Hypertension; Male; Membrane Proteins; Middle Aged; Phenotype; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Prospective Studies; Risk Factors; Sex Characteristics; Smoking; Thrombophilia; Thrombosis; Thromboxane B2; White People

Hemoglobin vesicles (HbV) are artificial oxygen carriers that encapsulate a concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane. The oxygen transporting ability of HbV in vivo has been demonstrated by the transfusion of HbV into hemorrhagic shock rodent models. However, the compatibility of HbV with human blood cells must be evaluated. Preincubation of platelets with concentrations of 20% or 40% HbV had no effect on the binding of PAC-1, a monoclonal antibody that detects activation-dependent conformational changes in alphaIIbbeta3 on platelets, or the surface expression of CD62P in whole blood. ADP-induced increases in PAC-1 binding were significantly enhanced by exposing the platelets to concentrations of either 20% or 40% HbV, whereas the ADP-induced increases in CD62P expression were not affected by HbV treatment at either concentration. Preincubation of platelet-rich plasma (PRP) with HbV minimally reduced the spontaneous release of TXB2 and RANTES, but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with ADP. Similarly, preincubation of PRP with HbV minimally reduced the spontaneous release of RANTES but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with collagen, although collagen-induced serotonin release tended to decrease with HbV pretreatment. These data suggest that the exposure of human platelets to high concentrations of HbV (up to 40%) in vitro did not cause platelet activation and did not adversely affect the formation and secretion of prothrombotic substances or proinflammatory substances triggered by platelet agonists, although one of the earliest events in ADP-induced platelet activation was slightly potentiated by HbV pretreatment at the doses tested. Taken together, these results imply that HbV, at concentrations of up to 40%, do not have any aberrant interactions with either unstimulated or agonist-induced platelets.

Topics: Biomarkers; Blood Platelets; Chemokine CCL5; Drug Delivery Systems; Hemoglobins; Humans; Inflammation; Liposomes; P-Selectin; Phospholipids; Platelet Activation; Platelet Glycoprotein GPIIb-IIIa Complex; Thrombophilia; Thromboxane B2

A higher than normal incidence of thromboembolic events has been observed in adult patients with beta-thalassaemia major (TM) and certain haemostatic anomalies found in these patients suggest the existence of a chronic hypercoagulable state. Thalassaemic red blood cells (RBC) were demonstrated to facilitate thrombin formation due to altered asymmetry of the membrane phospholipids with enhanced exposure of phosphatidylserine. Since RBC anomalies exist in thalassaemia from the first months of life, we studied markers of hypercoagulability and thrombophilia in 36 adult patients (range 19-38 years) and 26 children (range 2-18 years) with beta-TM. All the patients were in steady state and none had experienced clinical signs or symptoms of thrombosis. Highly elevated urinary levels of 11-dehydro-thromboxane B2 and significantly elevated plasma levels of thrombin-antithrombin III (TAT) complexes were observed to the same extent in TM children and adults. The levels of factor II were decreased while factors V, VII + X and plasminogen were within the normal range. The natural coagulation inhibitors, protein C (PC) and protein S (PS) were significantly decreased in all TM patients investigated, regardless of age, but the PS binding protein (C4bBP) and antithrombin III levels were normal. The frequency of other thrombophilic mutations was not increased. Thus, a chronic hypercoagulable state already exists in thalassaemia in childhood and may contribute to the cardiac and pulmonary anomalies and the thrombotic events which occur later.

Topics: Adolescent; Adult; beta-Thalassemia; Blood Coagulation Factors; Child; Child, Preschool; Complement Inactivator Proteins; Glycoproteins; Humans; Mutation; Plasminogen; Prostaglandins F, Synthetic; Protein C; Protein S; Receptors, Complement; Thrombophilia; Thromboxane B2