Allergic inflammation induces a persistent mechanistic switch in thromboxane-mediated airway constriction in the mouse

Actions of thromboxane (TXA(2)) to alter airway resistance were first identified over 25 years ago. However, the mechanism underlying this physiological response has remained largely undefined. Here we address this question using a novel panel of mice in which expression of the thromboxane receptor (TP) has been genetically manipulated. We show that the response of the airways to TXA(2) is complex: it depends on expression of other G protein-coupled receptors but also on the physiological context of the signal. In the healthy airway, TXA(2)-mediated airway constriction depends on expression of TP receptors by smooth muscle cells. In contrast, in the inflamed lung, the direct actions of TXA(2) on smooth muscle cell TP receptors no longer contribute to bronchoconstriction. Instead, in allergic lung disease, TXA(2)-mediated airway constriction depends on neuronal TP receptors. Furthermore, this mechanistic switch persists long after resolution of pulmonary inflammation. Our findings demonstrate the powerful ability of lung inflammation to modify pathways leading to airway constriction, resulting in persistent changes in mechanisms of airway reactivity to key bronchoconstrictors. Such alterations are likely to shape the pathogenesis of asthmatic lung disease.     

BM-520, an original TXA2 modulator, inhibits the action of thromboxane A2 and 8-iso-prostaglandin F2alphain vitro and in vivo on human and rodent platelets, and aortic vascular smooth muscles from rodents

Thromboxane A(2) (TXA(2)) and 8-iso-PGF(2alpha) are two prostanoid agonists of the thromboxane A(2) receptor (TP), whose activation has been involved in platelet aggregation and atherosclerosis. Agents able to counteract the actions of these agonists are of great interest in the treatment and prevention of cardiovascular events. Here, we investigated in vitro and in vivo the pharmacological profile of BM-520, a new TP antagonist. In our experiments, this compound showed a great binding affinity for human washed platelets TP receptors, and prevented human platelet activation and aggregation induced by U-46619, arachidonic acid and 8-iso-PGF(2alpha). The TP receptor antagonist property of BM-520 was confirmed by its relaxing effect on rat aorta smooth muscle preparations precontracted with U-46619 and 8-iso-PGF(2alpha). Further, its TP antagonism was also demonstrated in vivo in guinea pig after a single intravenous injection (10 mg kg(-1)). We conclude that this novel TP antagonist could be a promising therapeutic tool in pathologies such as atherosclerosis where an increased production of TXA(2) and 8-iso-PGF(2alpha), as well as TP activation are well-established pathogenic events.     

Thromboxane A2 induces airway constriction through an M3 muscarinic acetylcholine receptor-dependent mechanism

Thromboxane A2 (TXA2) is a potent lipid mediator released by platelets and inflammatory cells and is capable of inducing vasoconstriction and bronchoconstriction. In the airways, it has been postulated that TXA2 causes airway constriction by direct activation of thromboxane prostanoid (TP) receptors on airway smooth muscle cells. Here we demonstrate that although TXA2 can mediate a dramatic increase in airway smooth muscle constriction and lung resistance, this response is largely dependent on vagal innervation of the airways and is highly sensitive to muscarinic acetylcholine receptor (mAChR) antagonists. Further analyses employing pharmacological and genetic strategies demonstrate that TP-dependent changes in lung resistance and airway smooth muscle tension require expression of the M2 mAChR subtype. These results raise the possibility that some of the beneficial actions of anticholinergic agents used in the treatment of asthma and chronic obstructive pulmonary disease result from limiting physiological changes mediated through the TP receptor. Furthermore, these findings demonstrate a unique pathway for TP regulation of homeostatic mechanisms in the airway and suggest a paradigm for the role of TXA2 in other organ systems.     

New therapeutic approaches to combat arterial thrombosis: better drugs for old targets, novel targets, and future prospects

Cardiovascular disease and stroke are predominant causes of death in developed countries. Rupture of atherosclerotic plaque in an artery wall and the ensuing thrombotic events are the triggers for acute ischemic injury in these diseases. Platelet activation and aggregation play key roles in this process of atherothrombosis. Anti-platelet drugs thus provide the primary therapeutic strategy to combat these diseases. Dual therapy with aspirin and clopidogrel is the current standard of care for most patients, but it has significant limitations. This provides an impetus for developing new anti-platelet drugs. One new drug has received FDA approval recently; prasugrel targets the platelet P2Y(12) receptor, just like clopidogrel. Several other new drugs are showing great promise in clinical trials and appear to be nearing approval. Some of these drugs have traditional targets on the platelets; others, such as vorapaxar, terutroban, and sarpogrelate, generate more excitement as they are directed against novel targets.     

Coupling of thromboxane A2 receptor isoforms to Galpha13: effects on ligand binding and signalling.

Previous subtyping of thromboxane A2 (TXA2) receptors in platelets and vascular smooth muscle cells was based on pharmacological criteria. Two distinct carboxy-terminal splice variants for TXA2 receptors exist and they couple to several different G protein alpha subunits including Galpha13, but it has not been established whether either or both isoforms interact with and signal through it. We sought to determine: (1) which TXA2 receptor isoforms exist in vascular smooth muscle, (2) if Galpha13 is present in vascular smooth muscle and (3) if Galpha13 interacts with either or both of the two TXA2 receptor isoforms as determined by changes in ligand binding properties and generation of intracellular signals. Both TXA2 receptor isoforms and Galpha13 were found in vascular smooth muscle cells. Both the alpha and beta isoforms of the TXA2 receptors were transiently transfected with or without Galpha13 into COS-7 (radioligand binding assays) or CHO cells (agonist induced Na+/H+ exchange). Co-expression of each receptor isoform with Galpha13 significantly (P<0.05) increased the affinity of each receptor for the two agonists, I-BOP and ONO11113, and decreased the affinity of the receptor for the antagonists, SQ29,548 and L657,925. I-BOP stimulated Na+/H+ exchange in vascular smooth muscle cells. Co-expression of Galpha13 with each TXA2 receptor isoform in CHO cells resulted in a significant (P<0.04) agonist induced increase in Na+/H+ exchange compared to cells not transfected with Galpha13. The results support the possibility that the previous classification of TXA2 receptor subtypes based on pharmacological criteria reflect unique interactions with specific G protein alpha subunits.     

The value of using polymorphisms in anti-platelet therapy

Objectives and Backgrounds

Cardiovascular events occure as a result of various risk factors, such as uric acid (UA), inflammation, hormones and other materials that induce C- reactive protein (CRP) expression. These factors lead to complement activation, and endothelial damages. Damaged endothelial cells release heparan sulfate which inhibits tissue factor activity and von Willed brand factor (VWVF) and causes aggregation. Finally this cascade of events cause platelets aggregation and leads to heart ischemia and cardiovascular events.

Discussion

Anti-platelet therapy is an interesting premise. Anti-platelet resistance patients and bleeding as a result of using ticagrelor and prasugrel should be considered in this treatment methods. Anti-platelet drugs such as clopidogrel are prescribed in cardiovascular events. Platelets have VWF receptors and P2Y12 receptors on their surface, and thus, targeting these receptors can be useful in treatment. The active metabolites of clopidogrel bind to P2Y12R and inhibit ADP binding; thus, clopidogrel inhibits aggregation by interfering in several events as a result of the inhibition of ADP attachment to P2Y12R of the platelet. However, the polymorphisms of P2Y12 and other genes mentioned in Table 1 showed treatment resistance in anti-platelet therapy, highlighting that these SNPs can be helpful in anti-platelet therapy.

Conclusion

The knowledge of these SNPs may decrease the number of unwanted effects that endanger patients with cardiovascular diseases and avoids ineffective anti-platelet therapy in several patients. Clopidogrel, ticagrelor, prasugrel, and aspirin and CYP2C19 and their SNPs are very important subjects in anti-platelet therapy. To present the importance of using pharmacogenetics in anti-platelet therapy, we discuss here the association between these drugs and the SNPs for therapeutic resistance.

     

Inhibition of tumor necrosis factor alpha-mediated NFkappaB activation and leukocyte adhesion,with enhanced endothelial apoptosis,by G protein-linked receptor (TP) ligands

Tumor necrosis factor (TNF) alpha is a critical mediator of inflammation; however, TNFalpha is rarely released alone and the "cross-talk" between different classes of inflammatory mediators is largely unexplored. Thromboxane A(2) (TXA(2)) is released during I/R injury and exerts its effects via a G protein-linked receptor (TP). In this study, we found that TXA(2) mimetics stimulate leukocyte adhesion molecule (LAM) expression on endothelium via TPbeta. The potential interaction between TXA(2) and TNFalpha in altering endothelial survival and LAM expression was examined. IBOP, a TXA(2) mimetic, attenuated TNFalpha-induced LAM expression in vitro, in a concentration-dependent manner, by preventing TNFalpha-enhanced gene expression, and also reduced TNFalpha-induced leukocyte adhesion to endothelium both in vitro and in vivo. IBOP abrogated TNFalpha-induced NFkappaB activation in endothelial cells, as determined by reduced IkappaB phosphorylation and NFkappaB nuclear translocation, by inhibiting the assembly of signaling intermediates with the intracellular domain of TNF receptors 1 and 2 in response to TNFalpha. This inhibition resulted from the Galpha(q)-mediated enhancement of STAT1 activation and was reversed by anti-STAT1 antisense oligonucleotides. TNFalpha-mediated TNFR1-FADD association and caspase 8 activation were not inhibited by IBOP co-stimulation, however, resulting in a 2.6-fold increase in endothelial cell apoptosis. By stimulating the vessel wall and inducing endothelial cell apoptosis, TXA(2), in combination with TNFalpha, may hamper the angiogenic response during inflammation or ischemia, thus reducing revascularization and tissue viability.     

血栓素A2与糖尿病心血管并发症

心血管疾病是现今导致病人发病和死亡的首要因素,很多因素在血管性疾病发病发展中起着重要作用,血栓形成是参与脑中风及急性冠状动脉综合症的首要因素。血栓素A2（TXA2）是一种强血小板活化因子,在糖尿病患者体内的合成显著增加,并通过作用于血栓素受体诱导血小板聚集,血管收缩,血栓形成参与糖尿病心血管并发症的发生发展。因此,以TXA2为靶点开发抗血栓类药物对心血管疾病起着预防及治疗作用。本文对TXA2介导的糖尿病血管并发症的发病机制,及以此为靶点开发的抗血栓药物进行综述,为糖尿病心血管并发症的治疗及新型低副作用抗血栓药物的研发提供新的靶点。     

Pharmacological evaluation of the novel thromboxane modulator BM-567 (I/II). Effects of BM-567 on platelet function

The aim of this work was to evaluate the effects of BM-567 (N-pentyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on both thromboxane A(2) (TXA(2)) receptors (TP) and thromboxane synthase of human platelets. The drug affinity for TP receptors of human washed platelets has been determined. In this test, BM-567 showed a high affinity (IC(50): 1.1+/-0.1nM) for the TP receptors in comparison with BM-531 (IC(50): 7.8+/-0.7nM) and sulotroban (IC(50): 931+/-85nM), two TXA(2) antagonists. We also demonstrated that BM-567 prevented platelet aggregation induced by arachidonic acid (AA) (600 microM) (ED(100): 0.20+/-0.10 microM), U-46619, a stable TXA(2) agonist (1 microM) (ED(50): 0.30+/-0.04 microM) and collagen (1microgram ml(-1)) (% of inhibition: 44.3+/-4.3% at 10 microM) and inhibited the second wave of ADP (2microM). Moreover, when BM-567 was incubated in whole blood from healthy donors, the closure time measured by the Platelet Function analyzer (PFA-100((R))) was significantly prolonged (closure time: 215+/-21s) by using collagen/epinephrine cartridges. Finally, at the concentration of 1 microM, BM-567 completely reduced the TXB(2) production from human platelets stimulated with AA (600 microM). These results indicate that BM-567 is a novel combined TXA(2) receptor antagonist and thromboxane synthase inhibitor characterized by a powerful antiplatelet potency.     

Characterization of endothelial thromboxane receptors in rabbit aorta

An increased synthesis of thromboxane (TX) A(2) is associated with a number of cardiovascular diseases including atherosclerosis, unstable angina and hypertension. We previously identified a subgroup of NZW rabbits in which isolated arteries failed to contract to the TX agonists, U46619 or I-BOP. In vascular smooth muscle membranes, there was a significant decrease in TX receptors, termed TP. These rabbits are referred to as vTP- and those with the TP receptor are called vTP+. Because TP receptors are expressed in some types of endothelial cells, the present study was designed to determine whether functional TP receptors are present in endothelial cells cultured from aortas of vTP+ and vTP- rabbits. Radioligand binding studies were performed with (125)I-BOP. Aortic endothelial cells from vTP+ rabbits exhibited specific and saturable binding. In contrast, in endothelial preparations from vTP- rabbit aortas, no measurable binding to (125)I-BOP was detected. Using an anti-TP receptor antibody, we compared the amount of receptor expressed in endothelial cell lysates obtained from vTP+ and vTP- rabbits. Consistent with the results observed radioligand binding assays, the expression of TP receptor protein was decreased in vTP- compared to vTP+ endothelial cells. An in vitro wound healing assay was used on confluent monolayers of endothelial cells. In the untreated vTP+ cells, the area of the scratch was completely closed by 30 h. In the vTP+ cells treated with U46619 (3 microM), the rate of closure of the scratch area was reduced with approximately 12% of the scratch area remaining at 30 h. Pretreatment with the TP receptor antagonist, SQ 29548 (10 microM) prevented the inhibitory effect of U46619. The rate of closure of the scratch in the vTP- was not altered by U46619. In a separate study, U46619 (3 microM) increased the release of 6-keto PGF(1alpha), the stable metabolite of prostacyclin, in vTP+ but not vTP- endothelial cells. Pretreatment with SQ29548 (10 microM) or the cyclooxygenase inhibitor, indomethacin (10 microM) blocked the increase in vTP+ endothelial cells. In vascular reactivity studies in aortas from vTP+ rabbits, removal of the endothelium enhanced the vasoconstrictor response to U46619 indicating that activation of endothelial TP receptors may modulate vascular tone via the release of the vasodilator, prostacyclin. The results of this study suggest an important role for endothelial TP receptors in modulating vascular function.     

辛伐他汀对糖尿病大鼠阿司匹林抵抗的影响及其机制

目的：观察辛伐他汀对糖尿病大鼠阿司匹林抵抗的作用及机制。方法：选取雄性8周龄Wistar大鼠96只,随机分成糖尿病组和正常组（n=48）。糖尿病组大鼠腹腔一次性注射1%链脲佐菌素（STZ） （65 mg/kg,溶于0.l mmol/L、pH 4.5的柠檬酸缓冲液）来诱发糖尿病模型,正常组大鼠注射相同剂量的柠檬酸缓冲液。血糖大于16.8 mmol/L且同时具备多饮、多尿、体重减轻的糖尿病症状的大鼠认为糖尿病造模成功。糖尿病和正常大鼠分别随机分为糖尿病对照组（DM）、糖尿病阿司匹林组（DM-ASA）、糖尿病辛伐他汀组（DM-SIM）、糖尿病阿司匹林联合辛伐他汀组（DM-ASA+SIM）和正常对照组（NC）、正常阿司匹林组（NC-ASA）、正常辛伐他汀组（NC-SIM）、正常阿司匹林联合辛伐他汀组（NC-ASA+SIM）（n=12）。阿司匹林组、辛伐他汀组及阿司匹林联合辛伐他汀组分别给予10 mg/kg阿司匹林、2 mg/kg辛伐他汀、10 mg/kg阿司匹林加2 mg/kg辛伐他汀溶于PBS灌胃,对照组给予等量PBS灌胃。连续灌胃8周后,评价血小板聚集功能和血小板活化指标,检测大鼠血清一氧化氮（NO）、内皮素（ET）、前列环素（PGI2）、脂联素（APN）、血栓素B2（TXB2）水平和血清超氧化物歧化酶（SOD）、丙二醛（MDA）水平;进行大鼠胸主动脉离体血管张力实验评价内皮功能;检测大鼠胸主动脉血红素氧合酶-1（HO-1）、血红素氧合酶-2（HO-2）、内皮型NO合酶（eNOS）、磷酸化-内皮型NO合酶（p-eNOS）、抗凋亡蛋白（Bcl-2）、环加氧酶-2 （COX-2）等蛋白的表达水平。结果：与对照组大鼠相比,糖尿病大鼠其血小板活化及聚集值增高,且模型组呈现阿司匹林的反应性减低现象（P<0.05）;在糖尿病大鼠,阿司匹林联合辛伐他汀较单用阿司匹林组显著降低血小板活化及聚集值（P<0.05）,其通过上调HO-1、eNOS、p-eNOS、Bcl-2等蛋白的表达水平及升高血清APN水平,下调COX-2蛋白表达水平,通过改善内皮功能,调节TXA2/PGI2水平、增加NO水平,发挥其抗血小板作用。结论：糖尿病大鼠呈现阿司匹林反应性减低,辛伐他汀具有潜在的改善血小板对阿司匹林的反应性的作用。     

Controversies and future perspectives of antiplatelet therapy in secondary stroke prevention

Antiplatelet agents are a cornerstone in the treatment of acute arterial thrombotic events and in the prevention of thrombus formation. However, existing antiplatelet agents (mainly aspirin, the combination of aspirin and dipyridamole and clopidogrel) reduce the risk of vascular events only by about one quarter compared with placebo. As a consequence, more efficacious antiplatelet therapies with a reduced bleeding risk are needed. We give an overview of several new antiplatelet agents that are currently investigated in secondary stroke prevention: adenosine 5′-diphosphonate receptor antagonists, cilostazol, sarpogrelate, terutroban and SCH 530348. There are unique features in secondary stroke prevention that have to be taken into account: ischaemic stroke is a heterogeneous disease caused by multiple aetiologies and the blood–brain barrier is disturbed after stroke which may result in a higher intracerebral bleeding risk. Several small randomized trials indicated that the combination of aspirin and clopidogrel might be superior to antiplatelet monotherapy in the acute and early post-ischaemic phase. There is an ongoing debate about antiplatelet resistance. Decreasing response to aspirin is correlated independently with an increased risk of cardiovascular events. However, there is still no evidence from randomized trials linking aspirin resistance and recurrent ischaemic events. Similarly, randomized trials have not demonstrated a clinical significantly decreased antiplatelet effect by the concomitant use of clopidogrel and proton pump inhibitors. Nevertheless, a routine use of this drug combination is not recommended.     

Increased spontaneous tone in renal arteries of spontaneously hypertensive rats

The spontaneous tone of vascular smooth muscle is augmented in hypertension. The present study examined the role of nitric oxide (NO), cyclooxygenase (COX), thromboxane A(2)/prostanoid (TP) and PGE(2)/prostanoid (EP-1) receptors, reactive oxygen species, and large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels in the regulation of spontaneous tone in renal arteries of young and mature Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Rings of arteries, with and without endothelium, were suspended in a myograph for isometric force recording. Spontaneous tone (increase above initial tension) was observed only in arteries of mature SHR and was greater in arteries without endothelium. N(omega)-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NO synthases) induced larger contractions in arteries of SHR than WKY. Indomethacin (a COX inhibitor), SC-19220 (an EP-1 receptor antagonist), and terutroban (a TP receptor antagonist) reduced the L-NAME-evoked contractions. Tiron (a superoxide anion scavenger), catalase (an enzyme that degrades H(2)O(2)), and deferoxamine (a hydroxyl radical scavenger) augmented the L-NAME-induced contractions in arteries of mature SHR. Charybdotoxin (a BK(Ca) channel blocker) caused contractions in arteries of mature SHR without endothelium and in arteries with endothelium incubated with L-NAME. A decreased protein level of endothelial NO synthase, an increased release of prostacyclin, and an increased expression of EP-1 receptors were observed in arteries of mature SHR. The present study suggests that spontaneous tone is precipitated by age and hypertension. The reduced production of NO, leading to decreased activation of BK(Ca) channels, may leave the actions of endogenous vasoconstrictors unopposed. COX products that activate EP-1 and TP receptors are involved in the development of spontaneous tone.     

Identification of an interaction between the TPalpha and TPbeta isoforms of the human thromboxane A2 receptor with protein kinase C-related kinase (PRK) 1: implications for prostate cancer

In humans, thromboxane (TX) A(2) signals through the TPα and TPβ isoforms of the TXA(2) receptor or TP. Here, the RhoA effector protein kinase C-related kinase (PRK) 1 was identified as an interactant of both TPα and ΤPβ involving common and unique sequences within their respective C-terminal (C)-tail domains and the kinase domain of PRK1 (PRK1(640-942)). Although the interaction with PRK1 is constitutive, agonist activation of TPα/TPβ did not regulate the complex per se but enhanced PRK1 activation leading to phosphorylation of its general substrate histone H1 in vitro. Altered PRK1 and TP expression and signaling are increasingly implicated in certain neoplasms, particularly in androgen-associated prostate carcinomas. Agonist activation of TPα/TPβ led to phosphorylation of histone H3 at Thr(11) (H3 Thr(11)), a previously recognized specific marker of androgen-induced chromatin remodeling, in the prostate LNCaP and PC-3 cell lines but not in primary vascular smooth muscle or endothelial cells. Moreover, this effect was augmented by dihydrotestosterone in androgen-responsive LNCaP but not in nonresponsive PC-3 cells. Furthermore, PRK1 was confirmed to constitutively interact with TPα/TPβ in both LNCaP and PC-3 cells, and targeted disruption of PRK1 impaired TPα/TPβ-mediated H3 Thr(11) phosphorylation in, and cell migration of, both prostate cell types. Collectively, considering the role of TXA(2) as a potent mediator of RhoA signaling, the identification of PRK1 as a bona fide interactant of TPα/TPβ, and leading to H3 Thr(11) phosphorylation to regulate cell migration, has broad functional significance such as within the vasculature and in neoplasms in which both PRK1 and the TPs are increasingly implicated.     

Lipid phosphate phosphatases regulate lysophosphatidic acid production and signaling in platelets: studies using chemical inhibitors of lipid phosphate phosphatase activity

Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature. Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in the blood and atherosclerotic plaques. LPA receptors on platelets, leukocytes, endothelial cells, and smooth muscle cells regulate growth, differentiation, survival, motility, and contractile activity. Definition of the opposing pathways of synthesis and degradation that control extracellular LPA levels is critical to understanding how LPA bioactivity is regulated. We show that intact platelets and platelet membranes actively dephosphorylate LPA and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1). Localization of LPP1 to the platelet surface is increased by exposure to LPA. A novel receptor-inactive sn-3-substituted difluoromethylenephosphonate analog of phosphatidic acid that is a potent competitive inhibitor of LPP1 activity potentiates platelet aggregation and shape change responses to LPA and amplifies LPA production by agonist-stimulated platelets. Our results identify LPP1 as a pivotal regulator of LPA signaling in the cardiovascular system. These findings are consistent with genetic and cell biological evidence implicating LPPs as negative regulators of lysophospholipid signaling and suggest that the mechanisms involve both attenuation of lysophospholipid actions at cell surface receptors and opposition of lysophospholipid production.     

Identification of Galpha13 as one of the G-proteins that couple to human platelet thromboxane A2 receptors.

Previous studies have shown that ligand or immunoaffinity chromatography can be used to purify the human platelet thromboxane A2 (TXA2) receptor-Galphaq complex. The same principle of co-elution was used to identify another G-protein associated with platelet TXA2 receptors. It was found that in addition to Galphaq, purification of TXA2 receptors by ligand (SQ31,491)-affinity chromatography resulted in the co-purification of a member of the G12 family. Using an antipeptide antibody specific for the human G13 alpha-subunit, this G-protein was identified as Galpha13. In separate experiments, it was found that the TXA2 receptor agonist U46619 stimulated [35S]guanosine 5'-O-(3-thiotriphosphate) incorporation into G13 alpha-subunit. Further evidence for functional coupling of G13 to TXA2 receptors was provided in studies where solubilized platelet membranes were subjected to immunoaffinity chromatography using an antibody raised against native TXA2 receptor protein. It was found that U46619 induced a significant decrease in Galphaq and Galpha13 association with the receptor protein. These results indicate that both Galphaq and Galpha13 are functionally coupled to TXA2 receptors and dissociate upon agonist activation. Furthermore, this agonist effect was specifically blocked by pretreatment with the TXA2 receptor antagonist, BM13.505. Taken collectively, these data provide direct evidence that endogenous Galpha13 is a TXA2 receptor-coupled G-protein, as: 1) its alpha-subunit can be co-purified with the receptor protein using both ligand and immunoaffinity chromatography, 2) TXA2 receptor activation stimulates GTPgammaS binding to Galpha13, and 3) Galpha13 affinity for the TXA2 receptor can be modulated by agonist-receptor activation.     

Cardiovascular protection of magnolol: cell-type specificity and dose-related effects

Magnolia officinalis has been widely used in traditional Chinese medicine. Magnolol, an active component isolated from Magnolia officinalis, is known to be a cardiovascular protector since 1994. The multiplex mechanisms of magnolol on cardiovascular protection depends on cell types and dosages, and will be reviewed and discussed in this article. Magnolol under low and moderate dosage possesses the ability to protect heart from ischemic/reperfusion injury, reduces atherosclerotic change, protects endothelial cell against apoptosis and inhibits neutrophil-endothelial adhesion. The moderate to high concentration of magnolol mainly acts on smooth muscle cells and platelets. Magnolol induces apoptosis in vascular smooth muscle cells at moderate concentration and inhibits proliferation at moderate and high concentration. High concentration of magnolol also abrogates platelet activation, aggregation and thrombus formation. Magnolol also serves as an smooth muscle relaxant only upon the high concentration. Oral intake of magnolol to reach the therapeutic level for cardiovascular protection is applicable, thus makes magnolol an agent of great potential for preventing cardiovascular diseases in high-risk patients.     

Immunohistochemical studies on the distribution of cellular myosin II isoforms in brain and aorta.

The distribution of nonmuscle myosin isoforms in brain and aorta was studied by using polyclonal antibodies against two synthetic peptides selected from a region near the carboxyl terminus of bovine brain (peptide IIB) and human macrophage (peptide IIA) myosin. Immunoblots of brain homogenates and purified myosin showed two major bands stained by anti-peptide IIB (MIIB1 and MIIB2) and a minor band stained by anti-peptide IIA (MIIA2). Polyclonal anti-human platelet myosin antibodies did not react with MIIB isoforms. In cryosections from bovine, rat, and mouse brains, anti-peptide IIB stained most neuronal cells. In bovine cryosections, glial staining was also observed. In contrast, anti-peptide IIA and anti-platelet myosin antibodies primarily stained blood vessels. In bovine aorta, the anti-peptide antibodies recognized four bands, MIIB3, MIIB4, MIIA1, and MIIA2. Only MIIA2 was recognized by anti-human platelet myosin antibodies. In bovine aorta cryosections, anti-peptide IIB stained smooth muscle cells in tunica intima and tunica media but did not stain endothelial cells. Anti-peptide IIA stained smooth muscle cells in the tunica media, and endothelial cells of vaso vasorum but not of aorta. Only polyclonal anti-platelet myosin antibodies stained the endothelial cells of aorta tunica intima. These results indicate that multiple isoforms of cellular myosins exist in mammals, that these isoforms are expressed in a cell specific manner, and that the major myosin isoforms isolated from whole brain originate from neurons and, at least in bovine brain, from glia, but not from blood vessels.     

Primary platelet adhesion receptors

Thrombotic diseases such as heart attack and stroke remain a major health concern in the Western world despite existing anti-thrombotic drugs. Current studies are revealing structure-function relationships of primary platelet adhesion receptors mediating adhesion, activation and aggregation, and the molecular mechanisms underlying platelet thrombus formation. Platelet adhesion is relevant not only to thrombotic disease, but there is increasing evidence of a specific role for platelets in vascular processes such as inflammation and atherogenesis. This review focuses on recent advances in understanding the molecular basis for platelet thrombus formation, in particular the receptors, glycoprotein (GP)Ib-IX-V and GPVI, that initiate platelet adhesion and activation at high shear stress.