The Antithrombotic Agent Pterostilbene Interferes with Integrin αIIbß3-Mediated Inside-Out and Outside-In Signals in Human Platelets.

Platelets play a crucial role in the physiology of primary hemostasis and pathological processes such as arterial thrombosis; thus, developing a therapeutic target that prevents platelet activation can reduce arterial thrombosis. Pterostilbene (PTE) has remarkable pharmacological activities, including anticancer and neuroprotection. Few studies have reported the effects of pterostilbene on platelet activation. Thus, we examined the inhibitory mechanisms of pterostilbene in human platelets and its role in vascular thrombosis prevention in mice. At low concentrations (2-8 µM), pterostilbene strongly inhibited collagen-induced platelet aggregation. Furthermore, pterostilbene markedly diminished Lyn, Fyn, and Syk phosphorylation and hydroxyl radical formation stimulated by collagen. Moreover, PTE directly hindered integrin αIIbß3 activation through interfering with PAC-1 binding stimulated by collagen. In addition, pterostilbene affected integrin αIIbß3-mediated outside-in signaling, such as integrin ß3, Src, and FAK phosphorylation, and reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Furthermore, pterostilbene substantially prolonged the occlusion time of thrombotic platelet plug formation in mice. This study demonstrated that pterostilbene exhibits a strong activity against platelet activation through the inhibition of integrin αIIbß3-mediated inside-out and outside-in signaling, suggesting that pterostilbene can serve as a therapeutic agent for thromboembolic disorders.

Ni-induced TGF-ß signaling promotes VEGF-a secretion via integrin ß3 upregulation.

Nickel compounds are associated with lung and skin cancer incidence increase and accumulation of nickel in the body contributes to carcinogenesis. Upregulation of certain integrins in the primary tumor is associated with cancer metastasis and poor prognosis. However, the molecular mechanisms of nickel-induced cancer metastasis are still unclear. The purpose of the present study was to investigate the effects of nickel chloride (NiCl2 ) on the progression of cancer during metastasis. The results of showed that NiCl2 induces the expression of integrin ß3 mRNA and protein in a dose- and time-dependent manner. Inhibition of integrin αvß3 activation by ITGB3 ligand mimetics and GR144053, as well as downregulation of ITGB3 by lentiviral shRNA gene silencing, diminished NiCl2 -induced secretion of vascular endothelial growth factor-a (VEGF-a). Furthermore, pretreatment with type I TGF-ß receptor inhibitor, SB525334, suppressed the expression of ITGB3 at cell surface and secretion of VEGF-a in NiCl2 -treated cells. In conclusion, NiCl2 induces the expression of ITGB3 through TGF-ß signaling activation, followed by increasing VEGF-a secretion, revealing a novel role for ITGB3 in nickel compound-induced cancer metastasis and tumor angiogenesis.

Blockade of integrin ß3 signals to reverse the stem-like phenotype and drug resistance in melanoma.

PURPOSE: Cancer cells with stem-like phenotype are frequently proliferative and show high resistance to chemotherapeutic agents. Specific cell markers to identify the cancer stem cells and reverse the drugs resistance are urgent needs in clinic cancer treatment. METHODS: To identify the potential role of integrin ß3 in melanoma stem cells. Flow cytometry and immunofluorescence were performed to detect the expression levels of integrin ß3 and integrin ß3 related signal molecules. qRT-PCR and western blotting were used to detect the signaling pathways induced by integrin ß3. Colony formation analysis and melanoma-bearing mice treatment by chemotherapeutic agents and integrin ß3 inhibitors were used to detect the curative effects. RESULTS: We proved that integrin ß3 could serve as a marker of stem-like cancer cells in melanoma, along with the acquired chemotherapeutic drugs resistance. Furthermore, we observed that the membrane-proximal complex of integrin ß3 with KRAS and Galectin-3 on the surface of melanoma cancer cells could recruit the RalB, resulting in the activation of TBK1. The phosphorylated TBK1 facilitates the activation of NF-κB signaling pathway, leading to the stem-like phenotype and drug resistance development in melanoma. Herein, the combination of cilengitide, an integrin ß3 inhibitor, and chemotherapeutic agents were capable of suppressing the tumor growth and reversing the drug resistance induced by integrin ß3. CONCLUSION: These findings identified integrin ß3 as a driver of melanoma stem-like cells with drug resistance and revealed an innovative strategy in clinic melanoma treatment.

Glycoprotein IIb/IIIa Inhibitor Bridging and Subsequent Endovascular Therapy in Vertebrobasilar Occlusion in 120 Patients.

PURPOSE: The treatment mode in acute vertebrobasilar occlusion (VBO) remains uncertain. We analyzed efficacy and safety of intravenous glycoprotein IIb/IIIa inhibitor (IV GPI) plus subsequent intra-arterial thrombolysis with or without additional endovascular mechanical therapy (percutaneous transluminal angioplasty/stenting or thrombus aspiration) and sought treatment factors that predict good clinical outcome. METHODS: We retrospectively analyzed 120 cases of patients with angiographically proven acute VBO. Multivariate logistic regression was used to identify independent predictors for clinical outcome and included level of consciousness, age, sex, time to angiography, GPI agent, admission mode, occlusion type, recanalization success, and endovascular treatment mode. Clinical follow-up was dichotomized in no to moderate disability (modified Rankin scale (mRS) 0-3) vs. severe disability or death (mRS 4-6). RESULTS: Median National Institutes of Health stroke scale (NIHSS) score on admission was 32, and mean NIHSS score was 24. A total of 49 patients (41 %) developed no to moderate disability (mRS 0-3), and 39 patients (33 %) died. Thrombolysis in myocardial infarction 2/3 recanalization success was achieved in 97 patients (80.8 %). Symptomatic intracerebral hemorrhages occurred in 11 patients (9 %). Mild impairment of consciousness (p < 0.001) and embolic occlusion type (p = 0.01) were significant predictors of favorable outcome. Clinical outcome in recanalized patients was better, but not statistically significant (p = 0.055). CONCLUSIONS: Our results indicate that combined therapy with IV GPI and subsequent endovascular therapy may be a valid treatment strategy in acute VBO. With this treatment approach, a preserved vigilance before treatment and an embolic occlusion type are associated with no to moderate disability.

Matrix rigidity regulates the transition of tumor cells to a bone-destructive phenotype through integrin ß3 and TGF-ß receptor type II.

Cancer patients frequently develop skeletal metastases that significantly impact quality of life. Since bone metastases remain incurable, a clearer understanding of molecular mechanisms regulating skeletal metastases is required to develop new therapeutics that block establishment of tumors in bone. While many studies have suggested that the microenvironment contributes to bone metastases, the factors mediating tumors to progress from a quiescent to a bone-destructive state remain unclear. In this study, we hypothesized that the "soil" of the bone microenvironment, specifically the rigid mineralized extracellular matrix, stimulates the transition of the tumor cells to a bone-destructive phenotype. To test this hypothesis, we synthesized 2D polyurethane (PUR) films with elastic moduli ranging from the basement membrane (70 MPa) to cortical bone (3800 MPa) and measured expression of genes associated with mechanotransduction and bone metastases. We found that expression of Integrin ß3 (Iß3), as well as tumor-produced factors associated with bone destruction (Gli2 and parathyroid hormone related protein (PTHrP)), significantly increased with matrix rigidity, and that blocking Iß3 reduced Gli2 and PTHrP expression. To identify the mechanism by which Iß3 regulates Gli2 and PTHrP (both are also known to be regulated by TGF-ß), we performed Förster resonance energy transfer (FRET) and immunoprecipitation, which indicated that Iß3 co-localized with TGF-ß Receptor Type II (TGF-ß RII) on rigid but not compliant films. Finally, transplantation of tumor cells expressing Iß3 shRNA into the tibiae of athymic nude mice significantly reduced PTHrP and Gli2 expression, as well as bone destruction, suggesting a crucial role for tumor-produced Iß3 in disease progression. This study demonstrates that the rigid mineralized bone matrix can alter gene expression and bone destruction in an Iß3/TGF-ß-dependent manner, and suggests that Iß3 inhibitors are a potential therapeutic approach for blocking tumor transition to a bone destructive phenotype.

Mice overexpressing integrin αv in fibroblasts exhibit dermal thinning of the skin.

BACKGROUND: Integrins, especially αv integrin (ITGAV), are thought to play central roles in tissue fibrosis and the pathogenesis of scleroderma. So far, skin phenotype of tissue-specific transgenic mice of ITGAV have not been investigated. OBJECTIVE: To investigate the role of ITGAV in the skin fibrosis, we engineered transgenic mice that overexpress ITGAV in the fibroblasts under the control of the COL1A2 enhancer promoter. METHODS: Protein or RNA expression was evaluated by real-time PCR, immunohistochemistry, immunoblotting and immunoprecipitation. RESULTS: Dermal thickness and Masson's trichrome staining were decreased in ITGAV transgenic (Tg) mice compared with wild-type (WT) mice. Protein and mRNA levels of COL1A2, COL3A1, CTGF and integrin ß3 were down-regulated in the skin of Tg mice. In addition, the cell proliferation of cultured dermal fibroblasts obtained from Tg mice skin was decreased compared to those of WT mice. FAK phosphorylation was reduced in fibroblasts cultured from Tg mice skin in comparison to WT mice fibroblasts. Integrin ß3 siRNA inhibited FAK phosphorylation levels, while FAK inhibitor reduced the expression of collagens and CTGF in mice dermal fibroblasts. CONCLUSIONS: The down-regulation of collagen or CTGF by decreased integrin ß3 and FAK phosphorylation may cause the dermal thinning in Tg mice. Lower CTGF may also result in reduced growth of Tg mice fibroblasts. Our hypothesis is that the balance between α and ß chain of integrins positively or negatively control collagen expression and dermal thickness. This study gave a new insight in the treatment of tissue fibrosis and scleroderma by balancing integrin expression.

Temporal changes in integrin-mediated cardiomyocyte adhesion secondary to chronic cardiac volume overload in rats.

Previous studies have established integrins as cell surface receptors that mediate cardiomyocyte-extracellular matrix (ECM) attachments. This study sought to determine the contributions of the myocardial ß1- and ß3-integrin subunits to ventricular dilatation and coronary flow regulation using a blood-perfused isolated heart preparation. Furthermore, cardiomyocyte adhesion to collagen types I and IV, fibronectin, and laminin with and without a ß1-integrin subunit neutralizing antibody was assessed during the course of remodeling secondary to a sustained cardiac volume overload, including the onset of heart failure. Isolated cardiomyocytes were obtained during the initial, compensated, and decompensated phases of remodeling resulting from an aortocaval fistula created in 8-wk-old male Sprague-Dawley rats. Blocking the ß1-integrin subunit in isolated normal hearts produced ventricular dilatation, whereas this was not the case when the ß3-subunit was blocked. Substantial reductions in cardiomyocyte adhesion coincided with the previously documented development of ventricular dilatation and decreased contractility postfistula, with the ß1-integrin contribution to adhesion ranging from 28% to 73% over the course of remodeling being essentially substrate independent. In contrast, both integrin subunits were found to be involved in regulating coronary vascular resistance. It is concluded that marked reductions in integrin-mediated cardiomyocyte adhesion to the ECM play a significant role in the progression of adverse myocardial remodeling that leads to heart failure. Furthermore, although both the ß1- and ß3-integrin subunits were involved in regulating coronary vascular resistance, only inhibition of ß1-integrin-mediated adhesion resulted in ventricular dilatation of the normal heart.

Mechanisms of aspirin resistance.

Aspirin is integral to the secondary prevention of cardiovascular disease and acts to impair the development of platelet-mediated atherothromboembolic events by irreversible inhibition of platelet cyclooxygenase-1 (COX-1). Inhibition of this enzyme prevents the synthesis of the potent pro-aggregatory prostanoid thromboxane A2. A large number of patients continue to experience atherothromboembolic events despite aspirin therapy, so-called 'aspirin treatment failure', and this is multifactorial in aetiology. Approximately 10% however do not respond appropriately to aspirin in a phenomenon known as 'aspirin resistance', which is defined by various laboratory techniques. In this review we discuss the reasons for aspirin resistance in a systematic manner, starting from prescription of the drug and ending at the level of the platelet. Poor medication adherence has been shown to be a cause of apparent aspirin resistance, and may in fact be the largest contributory factor. Also important is high platelet turnover due to underlying inflammatory processes, such as atherosclerosis and its complications, leading to faster regeneration of platelets, and hence of COX-1, at a rate that diminishes the efficacy of once daily dosing. Recent developments include the identification of platelet glycoprotein IIIa as a potential biomarker (as well as possible underlying mechanism) for aspirin resistance and the discovery of an anion efflux pump that expels intracellular aspirin from platelets. The absolute as well as relative contributions of such factors to the phenomenon of aspirin resistance are the subject of continuing research.

Genome-wide expression profiling of human lymphoblastoid cell lines implicates integrin beta-3 in the mode of action of antidepressants.

Selective serotonin reuptake inhibitors (SSRIs) are the first-line treatment for major depression. However, the link between inhibition of serotonin reuptake and remission from depression remains controversial: in spite of the rapid onset of serotonin reuptake inhibition, remission from depression takes several weeks, presumably reflecting synaptogenesis/neurogenesis and neuronal rewiring. We compared genome-wide expression profiles of human lymphoblastoid cell lines from unrelated individuals following treatment with 1 µM paroxetine for 21 days with untreated control cells and examined which genes and microRNAs (miRNAs) showed the most profound and consistent expression changes. ITGB3, coding for integrin beta-3, showed the most consistent altered expression (1.92-fold increase, P=7.5 × 10(-8)) following chronic paroxetine exposure. Using genome-wide miRNA arrays, we observed a corresponding decrease in the expression of two miRNAs, miR-221 and miR-222, both predicted to target ITGB3. ITGB3 is crucial for the activity of the serotonin transporter (SERT), the drug target of SSRIs. Moreover, it is presumably required for the neuronal guidance activity of CHL1, whose expression was formerly identified as a tentative SSRI response biomarker. Further genes whose expression was significantly modulated by chronic paroxetine are also implicated in neurogenesis. Surprisingly, the expression of SERT or serotonin receptors was not modified. Our findings implicate ITGB3 in the mode of action of SSRI antidepressants and provide a novel link between CHL1 and the SERT. Our observations suggest that SSRIs may relieve depression primarily by promoting neuronal synaptogenesis/neurogenesis rather than by modulating serotonin neurotransmission per se.

What is the optimum adjunctive reperfusion strategy for primary percutaneous coronary intervention?

Acute ST-segment elevation myocardial infarction (STEMI) is a dynamic, thrombus-driven event. As understanding of its pathophysiology has improved, the central role of platelets in initiation and orchestration of this process has become clear. Key components of STEMI include formation of occlusive thrombus, mediation and ultimately amplification of the local vascular inflammatory response resulting in increased vasoreactivity, oedema formation, and microvascular obstruction. Activation, degranulation, and aggregation of platelets are the platforms from which these components develop. Therefore, prompt, potent, and predictable antithrombotic therapy is needed to optimise clinical outcomes after primary percutaneous coronary intervention. We review present pharmacological and mechanical adjunctive therapies for reperfusion and ask what is the optimum combination when primary percutaneous coronary intervention is used as the mode of revascularisation in patients with STEMI.

Early glycoprotein IIb-IIIa inhibitors in primary angioplasty-abciximab long-term results (EGYPT-ALT) cooperation: individual patient's data meta-analysis.

BACKGROUND: Even although time to treatment has been shown to be a determinant of mortality in primary angioplasty, the potential benefits are still unclear from early pharmacological reperfusion by glycoprotein (Gp) IIb-IIIa inhibitors. Therefore, the aim of this meta-analysis was to combine individual data from all randomized trials conducted on upstream as compared with late peri-procedural abciximab administration in primary angioplasty. METHODS: The literature was scanned using formal searches of electronic databases (MEDLINE and EMBASE) from January 1990 to December 2010. All randomized trials on upstream abciximab administration in primary angioplasty were examined. No language restrictions were enforced. RESULTS: We included a total of seven randomized trials enrolling 722 patients, who were randomized to early (n = 357, 49.4%) or late (n = 365, 50.6%) peri-procedural abciximab administration. No difference in baseline characteristics was observed between the two groups. Follow-up data were collected at a median (25th-75th percentiles) of 1095 days (720-1967). Early abciximab was associated with a significant reduction in mortality (primary endpoint) [20% vs. 24.6%; hazard ratio (HR) 95% confidence interval (CI) = 0.65 (0.42-0.98) P = 0.02, P(het) = 0.6]. Furthermore, early abciximab administration was associated with a significant improvement in pre-procedural thrombolysis in myocardial infarction (TIMI) 3 flow (21.6% vs. 10.1%, P < 0.0001), post-procedural TIMI 3 flow (90% vs. 84.8%, P = 0.04), an improvement in myocardial perfusion as evaluated by post-procedural myocardial blush grade (MBG) 3 (52.0% vs. 43.2%, P = 0.03) and ST-segment resolution (58.4% vs. 43.5%, P < 0.0001) and significantly less distal embolization (10.1% vs. 16.2%, P = 0.02). No difference was observed in terms of major bleeding complications between early and late abciximab administration (3.3% vs. 2.3%, P = 0.4). CONCLUSIONS: This meta-analysis shows that early upstream administration of abciximab in patients undergoing primary angioplasty for ST-segment elevation myocardial infarction (STEMI) is associated with significant benefits in terms of pre-procedural epicardial re-canalization and ST-segment resolution, which translates in to significant mortality benefits at long-term follow-up.

Glabridin inhibits migration, invasion, and angiogenesis of human non-small cell lung cancer A549 cells by inhibiting the FAK/rho signaling pathway.

This study reports the antimigration, anti-invasive effect of glabridin, a flavonoid obtained from licorice, in human non-small cell lung cancer A549 cells. Glabridin exhibited effective inhibition of cell metastasis by decreasing cancer cell migration and invasion of A549 cells. In addition, glabridin also decreased A549-mediated angiogenesis. Further investigation revealed that glabridin's inhibition of cancer angiogenesis was also evident in a nude mice model. Blockade of A549 cells migration was associated with an increase of ανß3 integrin proteosome degradation. Glabridin also decreased the active forms of FAK and Src, and enhanced levels of inactivated phosphorylated Src (Tyr 527), decreasing the interaction of FAK and Src. Inhibition of the FAK/Src complex by glabridin also blocked Akt activation, resulting in reduced activation of RhoA and myosin light chain phosphorylation. This study demonstrates that glabridin may be a novel anticancer agent for the treatment of lung cancer in 3 different ways: inhibition of migration, invasion, and angiogenesis.

Diclofenac sodium inhibits NFkappaB transcription in osteoclasts.

A non-steroidal anti-inflammatory drug, diclofenac, acts efficiently against inflammation; however, down-regulation of diclofenac on bone remodeling has raised concerns. The inhibitory mechanisms of diclofenac are poorly understood. We hypothesized that diclofenac down-regulates osteoclast differentiation and activation via inhibition of the translocation of phosphorylated nuclear factor kappa B (NFkappaB). When osteoclasts prepared from mouse hematopoietic stem cells were treated with diclofenac, tartrateresistant acid phosphatase-positive multinucleated cells decreased in a concentration-dependent manner. Pit formation assay revealed the abolition of osteoclastic bone resorption; levels of cathepsin K transcripts, an osteoclastic resorption marker, were down-regulated time-dependently. Diclofenac induced the accumulation of the inhibitor of kappa B in cytosol, which led to suppression of the nuclear translocation of NFkappaB and phosphorylated NFkappaB. These results suggest that the novel mechanism of diclofenac for bone remodeling is associated with phosphorylated NFkappaB reduction, which regulates osteoclast differentiation and activation.

Integrin expression is altered after acute and chronic cocaine.

Cocaine addiction is associated with an increase in actin cycling and alterations in dendritic spines in the nucleus accumbens. Both actin polymerization and spine morphology are regulated in part by beta-(beta) integrins. Mice were administered acute or daily injections of cocaine or saline for 7 days. After 3 weeks of withdrawal, the level of beta-integrins in the postsynaptic density enriched subfraction from nucleus accumbens tissue was quantified by immunoblotting at 0, 30 or 120min following an a cocaine challenge injection. After chronic treatment and withdrawal the basal level of beta1-integrin was increased while beta3-integrin was unaltered. However, following a cocaine challenge in chronic cocaine, but not saline-treated animals, beta3-integrin was transiently up-regulated while beta1-integrin was transiently downregulated. These data demonstrate a bidirectional regulation of beta-integrins by chronic cocaine treatment that may contribute to cocaine-induced changes in actin cycling and dendrite morphology.

Effects of platelet glycoprotein IIb-IIIa number and glycoprotein IIIa Leu33Pro polymorphism on platelet aggregation and sensitivity to glycoprotein IIb-IIIa antagonists.

We investigated the influence of glycoprotein (GP) IIIa Leu33Pro polymorphism, platelet GP IIb-IIIa number, and plasma fibrinogen concentration on platelet aggregation and antiaggregatory action of GP IIb-IIIa antagonists. Healthy volunteers with GP IIIa Pro33(-) (Leu33Leu33, n = 20) and Pro33(+) (Leu33Pro33, n = 13, and Pro33Pro33, n = 2) genotypes were included into the study. GP IIIa Leu33Pro substitution was associated with the increase of the level and rate of platelet microaggregate formation induced by GP IIb-IIIa activating antibody CRC54 (100, 200, 400 microg/ml) against the epitope within 1-100 residues of GP IIIa N-terminal part (p from 0.001 to 0.047). No significant differences were detected between parameters of platelet aggregation induced by ADP (1.25, 2.5, 5.0, 20 microM) in GP IIIa Pro33(+) and Pro33(-) donors. GP IIb-IIIa antagonist Monafram (F(ab')(2) fragment of GP-IIb-IIIa blocking antibody CRC64) (1, 2, 3 microg/ml), but not eptifibatide (50, 100, 150 ng/ml) inhibited ADP-induced aggregation slightly less efficiently in GP IIIa Pro33(+) group (p < 0.05 at 1 and 2 microg/ml Monafram). GP IIb-IIIa number (evaluated as maximal binding of (125)I-labelled antibody CRC64) varied from 40.5 to 80.8 x 10(3) per platelet with no significant influence of GP IIIa genotype. Consistent correlations were revealed between GP IIb-IIIa quantity and the level and rate of ADP-induced aggregation (r from 0.353 to 0.583, p from <0.001 to 0.037) as well as resistance (level of residual aggregation) to both GP IIb-IIIa antagonists (r from 0.345 to 0.602, p from <0.001 to 0.042). ADP-induced aggregation was considerably increased and efficiency of GP IIb-IIIa antagonists decreased in donors with high in comparison with low GP IIb-IIIa quantity (>60 and 40-50 x 10(3) per platelet respectively, p < 0.01 for most tests). No correlations were observed between all tested parameters and plasma fibrinogen concentration. Our results indicate that inter-individual variability of platelet GP IIb-IIIa number significantly affects platelet aggregation and antiaggregatory effects of GP IIb-IIIa antagonists. Contribution of this factor is higher than that of GP IIIa Leu33Pro polymorphism and variations of fibrinogen concentration.

Effects of antiplatelet components of tomato extract on platelet function in vitro and ex vivo: a time-course cannulation study in healthy humans.

BACKGROUND: Natural antithrombotic agents that influence platelet function are of potential interest for primary prevention of cardiovascular disease. Previous reports showed that tomato extracts inhibit platelet aggregation in vitro, but little is known of the active components, their mode of action, or their efficacy in vivo. OBJECTIVE: The objectives of the study were to examine the antiplatelet activity of specific tomato components by in vitro experimentation and to establish their ex vivo efficacy in healthy humans. DESIGN: The mechanisms of action of antiplatelet components isolated from tomato extracts were examined in vitro. A 7-h time-course study was carried out in cannulated human subjects (n = 23) to determine the ex vivo efficacy of a supplement drink containing tomato extract and the onset and duration of antiplatelet effects. RESULTS: The inhibition of ADP-, collagen-, thrombin-, and arachidonate-mediated platelet aggregation by tomato extract components appears to be linked to the inhibition of glycoprotein IIb/IIIa and platelet secretory mechanisms. We found a significant inhibition of baseline platelet function, from 2.9 +/- 1.4% (optimal ADP concentrations; P = 0.03) to 20.0 +/- 4.9% (suboptimal ADP concentrations; P < 0.001), 3 h after supplementation with a dose of tomato extract equivalent to 6 tomatoes. The observed effects persisted for >12 h. Coagulation variables were not affected. CONCLUSIONS: The ingestion of tomato components with in vitro antiplatelet activity significantly affects ex vivo platelet function. The reported cardioprotective effects of tomatoes are potentially linked to a modulation of platelet function.

Ovarian stimulation with GnRH agonist, but not GnRH antagonist, partially restores the expression of endometrial integrin beta3 and leukaemia-inhibitory factor and improves uterine receptivity in mice.

BACKGROUND: The impact of different ovarian stimulation (OS) protocols on endometrial receptivity remains controversial. In this study, the effects of different OS on the expression of endometrial integrin beta3 subunit and leukaemia-inhibitory factor (LIF) during the implantation window and the implantation rate in mice were investigated. METHODS: Three OS protocols were used, involving either pregnant mare's serum gonadotrophin (PMSG) alone, PMSG plus GnRH agonist or PMSG plus GnRH antagonist. Uterus samples were collected at 48 h after OS or ovulation and were detected with immunohistochemistry, Western blot and RT-PCR analyses. Normal embryos at gestation day 4 were transferred into the uteri of mice in the control and OS groups. RESULTS: All OS groups showed a significant decrease in the expression of both the endometrial integrin beta3 subunit and LIF during the implantation window and the implantation rate. Among the three OS groups, GnRH agonist-treated mice showed a higher endometrial integrin beta3 subunit and LIF expression and a higher implantation rate. No significant difference was found in the measured indices between the GnRH antagonist and PMSG groups. CONCLUSIONS: OS may inhibit the expression of endometrial integrin beta3 subunit and LIF and impair endometrial receptivity in mice. OS with GnRH agonist, but not GnRH antagonist, may partially restore the endometrial physiological secretion and improve uterine receptivity.

Is platelet function as measured by Thrombelastograph monitoring in whole blood affected by platelet inhibitors?

Platelet inhibitors, especially the glycoprotein (GP) IIb/IIIa receptor antagonists, have demonstrated their effectiveness in reducing the acute ischemic complications of percutaneous coronary intervention (PCI) and in improving clinical outcomes in patients with acute coronary crisis. Three common platelet inhibitors observed in emergent cardiopulmonary bypass (CPB) for failed PCI are abciximab, eptifibatide, and tirofiban. An in vitro model was constructed in two parts to determine whether platelet aggregation inhibition induced by platelet inhibitors would be demonstrated by the Thrombelastograph (TEG) monitor when compared with baseline samples with no platelet inhibitor. In part A, 20 mL of fresh whole blood was divided into four groups: group I = baseline, group A = abcix-imab microg/mL, group E = eptifibatide ng/mL, and group T = tirofiban ng/mL. Platelet inhibitor concentrations in whole blood were derived starting with reported serum concentrations with escalation to achieve 80% platelet inhibition using the Medtronic hemoSTATUS and/or Lumi-aggregometer. A concentration range determined by our in vitro tests were chosen for each drug using concentrations achieving less than, equal to, or greater than 80% platelet inhibition. In part B, TEG analysis was then performed using baseline and concentrations for each drug derived in part A. Parameters measured were clot formation reaction time (R), coagulation time (K), maximum amplitude (MA) and alpha angle (A). Groups E1000 and E2000 extended R over control by 37% and 23%, respectively (p = 0.01 and 0.03). Groups E1000 and E2000 increased K times by 45% and 58% (p = .02 and .04). T160 samples prolonged K by 20% (p = 0.01). The angle or clot strength (A) was decreased in groups T160 and E1000 by 23% (+ 7.06 SD) and 18% (+ 11.23 SD), respectively (p = 0.001 and 0.01). The MA decrease was statistically significant in the T160, E1000 and E2000 by 9%, 6% and 13% respectively (p = 0.01). Samples treated with abciximab were comparable to control values for all parameters measured. Although statistical significance could be demonstrated with some parameters, sensitivity was only observed at increased doses and was not seen with all agents tested. In our in vitro model, the TEG monitor was unable to demonstrate clinically significant differences in platelet function and may not be reflective of platelet function in samples which have been treated with these GP IIb/IIIa inhibitors.

Mechanisms of poly-N-acetyl glucosamine polymer-mediated hemostasis: platelet interactions.

BACKGROUND: Investigations were performed to determine whether poly-N-acetyl glucosamine (p-GlcNAc) induces hemostasis by the activation of platelets. METHODS: Platelets were isolated from human blood, fixed in the presence poly-N-acetyl glucosamine fibers, and visualized with scanning electron microscopy. Platelet activation surface markers were measured by fluorescence multiphoton microscopy. Platelet aggregation in the presence of p-GlcNAc fibers and integrin receptor blockers was measured. RESULTS: Scanning electron microscopy indicated that contact of platelets with poly-N-acetyl glucosamine fibers resulted in platelet activation. Fluorescent microscopy showed that contact of platelets with the marine polymer increased intracellular levels of free calcium and resulted in surface exposure of platelet phosphatidylserine, P selectin, and the alphaIIbbeta3 integrin. Antibody inhibitors of the platelet alphaIIbbeta3 integrin inhibited p-GlcNAc to stimulate fibrin polymerization. CONCLUSION: Poly-N-acetyl glucosamine fiber material promotes hemostasis by the activation of platelets.