The Arachidonic Acid Metabolism Mechanism Based on UPLC-MS/MS Metabolomics in Recurrent Spontaneous Abortion Rats.

Recurrent spontaneous abortion (RSA) remains a critical and challenging problem in reproduction. To discover novel biomarkers for RSA, ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) metabolomics approach was applied to detect RSA serum metabolic profiles and explore its possible pathogenesis and mechanism. The abortion rat model was established, and a metabolomics analysis was performed to evaluate the differentially expressed metabolites between the control and model groups. Immunohistochemistry (IHC), qRT-PCR, and Western blot further examined the expression of Arachidonic acid metabolism-related genes in uterus tissues. To identify arachidonic acid metabolism-related changes in RSA, ELISA's potential mechanisms were further confirmed in serum. Ninety-one metabolites were significantly different between the two groups, as indicated by a VIP ≥1, fold change ≥1. The metabolic pathways involving arachidonic acid metabolism pathway (P = 0.00044) are related to RSA. Verification by experimental showed that compared with the control rats, the expression of the COX-1, COX-2, PTGFR, and TBXA2R genes associated with the arachidonic acid metabolism pathway has significantly increased the uterus and serum of RSA rats (P < 0.05). Regulation of the arachidonic acid metabolism pathway might serve as a promising therapeutic strategy for relieving RSA women's symptoms.

Platelet Shp2 negatively regulates thrombus stability under high shear stress.

Essentials Shp2 negatively regulates thrombus stability under pathological shear rate. Shp2 suppresses TXA2 receptor-mediated platelet dense granule secretion. Through αIIbß3 outside-in signaling, Shp2 targets calmodulin-dependent activation of Akt. Shp2 may serve to prevent the formation of unwanted occlusive thrombi. SUMMARY: Background Perpetuation is the final phase of thrombus formation; however, its mechanisms and regulation are poorly understood. Objective To investigate the mechanism of Shp2 in platelet function and thrombosis. Methods and results We demonstrate that the platelet-expressed Src homology region 2 domain-containing protein tyrosine phosphatase Shp2 is a negative regulator of thrombus stability under high shear stress. In a ferric chloride-induced mesenteric arteriole thrombosis model, megakaryocyte/platelet-specific Shp2-deficient mice showed less thrombi shedding than wild-type mice, although their occlusion times were comparable. In accordance with this in vivo phenotype, a microfluidic whole-blood perfusion assay revealed that the thrombi formed on collagen surfaces by Shp2-deficient platelets were more stable under high shear rates than those produced by wild-type platelets. Whereas Shp2 deficiency did not alter platelet responsiveness towards thrombin, ADP and collagen stimulation, Shp2-deficient platelets showed increased dense granule secretion when stimulated by the thromboxane A2 analog U46619. Shp2 appears to act downstream of integrin αIIb ß3 outside-in signaling, inhibiting the phosphorylation of Akt (Ser473 and Thr308) and dense granule secretion. Calmodulin was also shown to bind both Shp2 and Akt, linking Shp2 to Akt activation. Conclusions Platelet Shp2 negatively regulates thrombus perpetuation under high shear stress. This signaling pathway may constitute an important mechanism for the prevention of unwanted occlusive thrombus formation, without dramatically interfering with hemostasis.

Investigation of a Thromboxane A2 Receptor-Based Vaccine for Managing Thrombogenesis.

BACKGROUND: Despite the well-established role for the thromboxane A2 receptor (TPR) in the development of thrombotic disorders, none of the antagonists developed to date has been approved for clinical use. To this end, we have previously shown that an antibody targeted against TPR's ligand-binding domain inhibits platelet activation and thrombus formation, without exerting any effects on hemostasis. Thus, the goal of the present studies is to design a novel TPR-based vaccine, demonstrate its ability to trigger an immune response, and characterize its antiplatelet and antithrombotic activity. METHODS AND RESULTS: We used a mouse keyhole limpet hemocyanin/peptide-based vaccination approach rationalized over the TPR ligand-binding domain (ie, the C-terminus of the second extracellular loop). The biological activity of this vaccine was assessed in the context of platelets and thrombotic diseases, and using a host of in vitro and in vivo platelet function experiments. Our results revealed that the TPR C-terminus of the second extracellular loop vaccine, in mice: (1) triggered an immune response, which resulted in the development of a C-terminus of the second extracellular loop antibody; (2) did not affect expression of major platelet integrins (eg, glycoprotein IIb-IIIa); (3) selectively inhibited TPR-mediated platelet aggregation, platelet-leukocyte aggregation, integrin glycoprotein IIb-IIIa activation, as well as dense and α granule release; (4) significantly prolonged thrombus formation; and (5) did so without impairing physiological hemostasis. CONCLUSIONS: Collectively, our findings shed light on TPR's structural biological features, and demonstrate that the C-terminus of the second extracellular loop domain may define a new therapeutic target and a TPR vaccine-based approach that should have therapeutic applications.

Aspirin in coronary artery disease: an appraisal of functions and limitations.

Aspirin (acetylsalicylic acid) is an antiplatelet drug used to treat and prevent coronary artery disease. Aspirin is used more frequently than any other drug in the world, however it does not inhibit platelet function equally well in all patients. The risk of platelet-dependent cardiovascular events is increased in patients, in whom aspirin inhibits platelet function suboptimally. Platelet inhibition with aspirin can be evaluated by use of modern whole blood platelet function tests. The overall aim of this dissertation was to identify and describe functions and limitations of aspirin. We used whole blood platelet aggregometry (Multiplate® Analyzer and VerifyNow® Aspirin) as the primary measure of platelet function. To identify biological mechanisms explaining our findings we also measured a number of biological markers, including markers of cyclooxygenase-1 activity, platelet activation, and platelet turnover. All participants (except healthy individuals in study 1) were treated with non-enteric coated aspirin 75 mg once daily during study participation and received no other drugs affecting platelet function. We used serum TXB2 measurements to verify that all patients were adherent to aspirin. In study 1, we investigated the association between platelet aggregometry results and platelet count, red blood cell count, and white blood cell count. The study population consisted of 417 aspirin-treated patients with stable coronary artery disease and 21 drug-naïve healthy individuals. We found consistent associations between aggregation and platelet count, red blood cell count, and white blood cell count. In particular, platelet count was an independent predictor of platelet aggregation, although generally associations were rather weak. In study 2, we focused on patients previously suffering definite stent thrombosis because these patients may be at a prothrombotic state. We compared levels of platelet aggregation and platelet turnover in 39 patients with previous definite stent thrombosis with levels in 78 patients with stable coronary artery disease. We found that patients with previous definite stent thrombosis displayed increased platelet aggregation and had a tendency towards increased platelet turnover. In study 3, we investigated if the antiplatelet effect of aspirin is sustained through the standard 24-hour dosing interval. We included 50 patients with previous definite stent thrombosis, 100 patients with stable coronary artery disease, and 50 healthy individuals. We found that platelet aggregation increased significantly through the 24-hour dosing interval, and so did cyclooxygenase-1 activity and platelet activation. The increase in platelet aggregation did not differ between groups, but patients with previous definite stent thrombosis had higher levels of platelet turnover indices and thrombopoietin. In study 4, we addressed an ongoing debate concerning potential interactions between antiplatelet drugs and proton pump inhibitors. In patients with coronary artery disease, we investigated if the antiplatelet effect of aspirin was reduced in 54 patients treated with aspirin and a proton pump inhibitor compared to 364 patients treated with aspirin only. We found increased levels of platelet aggregation and platelet activation in patients treated with aspirin and a proton pump inhibitor.

Platelet dysfunction associated with the novel Trp29Cys thromboxane A2 receptor variant.

BACKGROUND: Genetic variations that affect the structure of the thromboxane A2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are very rare in unselected populations. OBJECTIVES: To determine the functional consequences of the TP receptor Trp29Cys (W29C) substitution. PATIENTS/METHODS: We performed a detailed phenotypic analysis of an index case (P1) with reduced platelet aggregation and secretion responses to TP receptor pathway activators, and a heterozygous TP receptor W29C substitution. An analysis of the variant W29C TP receptor expressed in heterologous cells was performed. RESULTS: Total TP receptor expression in platelets from P1 was similar to that of controls, but there was reduced maximum binding and reduced affinity of binding to the TP receptor antagonist [(3) H]SQ29548. HEK293 cells transfected with W29C TP receptor cDNA showed similar total TP receptor expression to wild-type (WT) controls. However, the TP receptor agonist U46619 was less potent at inducing rises in cytosolic free Ca(2+) in HEK293 cells expressing the W29C TP receptor than in WT controls, indicating reduced receptor function. Immunofluorescence microscopy and cell surface ELISA showed intracellular retention and reduced cell surface expression of the W29C TP receptor in HEK293 cells. Consistent with the platelet phenotype, both maximum binding and the affinity of binding of [(3) H]SQ29548 to the W29C TP receptor were reduced compared to WT controls. CONCLUSION: These findings extend the phenotypic description of the very rare disorder TP receptor deficiency, and show that the W29C substitution reduces TP receptor function by reducing surface receptor expression and by disrupting ligand binding.

Soy isoflavones affect platelet thromboxane A2 receptor density but not plasma lipids in menopausal women.

OBJECTIVES: It has been suggested that isoflavones protect the cardiovascular system, in part by improving lipid profile. The purpose of the present research was to examine the effect of a 12-week soy isoflavone supplementation on lipoprotein status and platelet thromboxane A2 receptor density. METHODS: Twenty-nine healthy postmenopausal women were invited to take part in a randomised study to receive either 100 mg/day isoflavone supplement (n=15) or identical placebo capsules (n=14). Blood samples obtained at baseline and after 12 weeks were analysed for isoflavones, total cholesterol, high density lipoprotein cholesterol, triglycerides, glucose, insulin, estradiol, testosterone, gonadotrophins, sex hormone-binding globulin (SHBG) and platelet thromboxane A2 receptor density. Blood pressure measurements, body mass index, subcutaneous fat at entrance and at the end of treatment were also registered. Changes in variables between groups were compared by ANOVA for repeated measures. RESULTS: Blood pressure, body mass index, subcutaneous fat, insulin, serum lipoprotein, sex hormones and SHBG did not differ among groups. However, platelet thromboxane A2 receptor density declined significantly (from 181.9+/-30.9 to 115.2+/-16.2 fmol/10(8) platelets) in the experimental group, remaining mostly unchanged in the placebo group (176.3+/-27.3 to 170.4+/-28.2 fmol/10(8) platelets). The dissociation constant (Kd) values were unchanged. The change in platelet thromboxane A2 receptors correlated negatively with isoflavones serum concentration (r=-0.59, p<0.001). CONCLUSIONS: In this study we demonstrated that the beneficial effects of isoflavones in menopausal women could be more related to platelet function than to improving classical cardiovascular risk factors.

Platelet receptors for adenine nucleotides and thromboxane A2.

Adenosine diphosphate (ADP) and thromboxane A (2) (TXA (2)) are important physiological activators of platelets and exert their effects by acting on cell surface receptors. Platelet nucleotide receptors can be distinguished as three separate subtypes of the P2 receptor family. The P2X (1) receptor is a ligand-gated adenosine triphosphate (ATP) receptor that was originally mistaken for an ADP receptor. This calcium-influx-causing receptor mediates platelet shape change and plays an important role in thrombus formation in small arterioles. The P2Y (1) receptor, through activation of G (q) and phospholipase C, is required for ADP-induced platelet shape change, fibrinogen receptor activation, and TXA (2) generation. The G (i)-coupled P2Y (12) receptor plays an important role in platelet aggregation, potentiation of dense granule release, and TXA (2) generation. Both the P2Y receptors are crucial for in vivo thrombus formation. TXA (2) stimulates two subtypes of G protein-coupled TP receptor, TPalpha and TPbeta, but its effects in platelets are mediated predominantly through the alpha isoform. Although interference with the activation of G protein-coupled ADP or TP receptors results in increased bleeding times and protection from thromboembolism, TP receptor antagonists did not translate into effective antiplatelet drugs. Blockade of ADP receptor is a mode of newer classes of antithrombotic drugs in the coming era. This review focuses on the contribution of different nucleotide receptors and TP receptors to platelet function and their potential as antithrombotic agents.

Diversity of signaling underlying responses mediated by selective activation of the platelet thromboxane A2 (TXA2) receptor; pharmacological and kinetic studies in vitro.

Selective activation of the platelet TXA2 receptor is sufficient to mediate concurrent aggregation, deaggregation and shape change (SC) responses without activation of known Gi-coupled receptors (Platelets 2003; 14: 89). However, Gi-coupled receptor activation strongly influences the hemostasis response in vivo. This study investigated the modulatory effects of two signaling pathways related to Gi-coupled receptor activation, stimulation of phosphoinositide 3-kinases (PI3Ks) and inhibition of adenylyl cyclase (AC), on the aggregation, deaggregation and SC components of the platelet activation response. A novel turbidimetric approach was applied to separate these responses and to characterize their pharmacology and kinetics. The SC response was more sensitive to TXA2 receptor activation (lower EC50 value) but less sensitive to a TXA2 receptor antagonist (higher Kd value) than the net aggregation response. Epinephrine and sulprostone, agonists of the Gi-coupled alpha2A-adrenoceptor and EP3 receptor, respectively, amplified the SC, decelerated deaggregation and enhanced net aggregation responses. SQ22536 and 2',5'-dideoxyadenosine, inhibitors of AC activity, elicited smaller but qualitatively similar effects. The PI3K inhibitor wortmannin did not affect the SC response but accelerated deaggregation and inhibited net aggregation. These data are consistent with a differential modulation of the platelet SC response by each pathway associated with Gi-coupled receptor activation, while both pathways cooperatively enhance the net aggregation response by decelerating deaggregation. We propose that the TXA2 receptor mediated concurrent platelet aggregation and SC responses, that are differentially modulated by different signaling pathways, provide a model for studying the underlying cellular pharmacology of platelet physiology.