Concurrent signaling from Gαq- and Gαi-coupled pathways is essential for agonist-induced αvβ3 activation on human platelets

Summary.  The integrin αvβ3 mediates platelet adhesion to the matrix protein osteopontin and likely is the predominant integrin mediating platelet adhesion to the matrix protein vitronectin. To address the mechanism that regulates αvβ3 activity in platelets, we measured the effect of the P2Y₁ antagonist adenosine 3'-phosphate-5'-phosphate (A3P5P) and the P2Y₁₂ antagonist AR-C66096 on ADP-stimulated platelet adhesion to osteopontin and vitronectin. Each antagonist completely inhibited platelet adhesion, implying that concurrent stimulation of P2Y₁ and P2Y₁₂ was required to activate αvβ3. The reducing agent dithiothreitol and Mn²⁺ also induced platelet adhesion to osteopontin, but did so without stimulating platelet activation. Thus, these data suggest that ADP stimulation regulates αvβ3 activity by perturbing the conformation of its extracellular domain. The actin polymerization inhibitors cytochalasin D and latrunculin A also induced platelet adhesion to osteopontin and vitronectin. Thus, αvβ3 activity in resting platelets appears to be constrained by the platelet cytoskeleton. Moreover, the effect of these agents was inhibited by A3P5P and AR-C66096 at micromolar and subnanomolar concentrations, respectively, suggesting that subthreshold platelet stimulation by ADP was required. Our data suggest that signals from both Gα_q- and Gα_i-coupled receptors converge to release cytoskeletal constraints on αvβ3. We propose that the release of cytoskeletal constraints and a concurrent increase in affinity for ligands is responsible for αvβ3-mediated platelet adhesion.     

心肌缺血再灌注损伤时细胞核被膜钙泵活性特征的研究

目的　研究心肌缺血再灌注损伤时Ca2 + 、ATP、ADP和AMP对细胞核被膜钙泵 (Ca2 + ATPase)活性的调节。方法　采用大鼠心肌缺血再灌注模型 ,密度梯度分离纯化细胞核 ,定磷法测定Ca2 + ATPase活性。结果　与正常大鼠相比 ,缺血再灌注损伤动物血浆MDA和FFA显著升高 (P <0 0 1) ;细胞核Ca2 + ATPase活性下降 ,对Ca2 + 亲和力降低 ;ADP、AMP对核Ca2 + ATPase活性的影响明显减弱 ;ATP对Ca2 + ATPase的作用在 [ATP]小于 1 0mmol L时 ,与正常细胞Ca2 + AT Pase活性无显著差异 ,浓度增至 2 0mmol L时 ,活性低于正常细胞 ,ATP浓度继续增加 ,核Ca2 + ATPase活性快速增加。结论　心肌缺血再灌注损伤时Ca2 + 、ATP、ADP、AMP对心肌细胞核Ca2 + ATPase活性的影响发生了明显改变 ,其病理生理意义值得进一步探讨。     

Acidic ATP activates lymphocyte outwardly rectifying chloride channels via a novel pathway

Using whole-cell patch-clamp techniques we found that ATP activated an outwardly rectifying current in Daudi human B lymphoma cells under acidic conditions. The substitution of Cl^– for gluconate^– shifted the reversal potential, while Cl^– channel blockers, 4,4-diisothiocyanostibene-2,2-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC), blocked the current, indicating that ATP induces this current by activating the outwardly rectifying chloride channel (ORCC). The effect of ATP on ORCC was mimicked by ADP, but not by other P2 receptor agonists such as ATPS (a poorly hydrolyzable analog of ATP), 2,3-O-benzoyl-4-benzoyl-ATP (BzATP), and UTP. The ATP-induced ORCC current was completely blocked by 100 M suramin (a P2 receptor antagonist), and was partially blocked by 100 M pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid tetrasodium (PPADS), which is another P2 receptor antagonist. Neither inactivation of G proteins nor elimination of extracellular Ca²⁺ affected the ATP-induced current, indicating that G protein-coupled P2Y receptors and Ca²⁺-permeable P2X receptors are not involved. Based on the pharmacological profile and the fact that acidic conditions are required for ATP to activate the ORCC, we suggest that acidic ATP activates the lymphocyte ORCC via a novel pathway, which is not associated with any previously described purinergic receptors.     

Drugs affecting coagulation

The clotting cascade is a complex process and is an important survival mechanism. Major haemorrhage and thromboembolic events remain major causes of increased morbidity and mortality. Drugs affecting coagulation have primarily been utilized to treat or reduce the risk of thromboembolic events. However, the recent progress in the management of major trauma and treating coagulopathy has resulted in further research and development of drugs that improve clotting function. Knowledge of drugs used for both clinical circumstances is now required when working in anaesthesia or intensive care.     

心脑通对大鼠凝血功能的影响

目的　观察心脑通对大鼠凝血功能的影响。方法　心脑通给大鼠连续灌胃８ｄ 后,观察其对大鼠的出血时间、凝血酶时间、凝血酶原时间、部分凝血活酶时间、血小板聚集以及血浆中ＴＸＢ２ 与６ － 酮－ ＰＧＦ１α含量的影响。结果　心脑通０．４ｇ／ｋｇ、０．６ｇ／ｋｇ、０ ．８ｇ／ｋｇ 剂量组的出血时间、凝血酶时间、凝血酶原时间、部分凝血活酶时间均较生理盐水组明显延长（ Ｐ＜ ０．０５） ;且对ＡＤＰ诱导的血小板聚集有明显抑制作用;血浆中ＴＸＢ２ 与生理盐水组相比含量下降。结论　心脑通有良好的活血化瘀作用。     

Differential effects of zidovudine and zidovudine triphosphate on mitochondrial permeability transition and oxidative phosphorylation

1. The effects of zidovudine (ZDV) and zidovudine triphosphate (ZDV-3P) on Ca²⁺-induced mitochondrial permeability transition (MPT), respiratory control ratio (RCR) and ATP synthesis have been investigated on isolated rat liver mitochondria.
2. ZDV slightly but significantly decreased RCR and ATP synthesis but was ineffective in inhibiting MPT. In contrast, ZDV-3P did not alter RCR and ATP synthesis but strongly inhibited MPT (IC₅₀=3.0±0.9 μM).
3. The effect of ZDV-3P on mitochondrial swelling required a preincubation time. When incubated 10 min with mitochondria, ZDV-3P (8 μM) totally inhibited the rate of swelling.
4. ADP, ATP and atractyloside, which are agents known to interact with the mitochondrial adenine nucleotide carrier (ANC), antagonized the effect of ZDV-3P on mitochondrial swelling. Indeed, the IC₅₀ value of ZDV-3P increased from 3.0 to 17.4, 93.6 and 66.5 μM, in the presence of 20 μM, ADP, ATP or atractyloside, respectively.
5. ZDV-3P did not displace [³H]-ATP from its mitochondrial binding site(s) whereas ADP and atractyloside did, suggesting that ZDV-3P and [³H]-ATP do not share the same binding sites.
6. ZDV-3P did not affect either mitochondrial respiration or ATP synthesis but inhibited Ca²⁺-dependent mitochondrial swelling. It was concluded that mitochondrial toxic effects observed during the chronic administration of ZDV cannot be related to its active metabolite (ZDV-3P).

     

"In vitro" activity of urokinase on platelet function and on ADP degradation by vascular tissue

The effects of urokinase on ADP breakdown by vessel-wall, platelet aggregation and the related prostaglandin system "in vitro" were investigated. It is confirmed that urokinase does not induce platelet aggregation both in humans and rabbits "in vitro". Conversely, in high concentrations, urokinase inhibits ADP-induced platelet aggregation in human and rabbit platelet-rich plasma. No effects were observed on rabbit platelet thromboxane A2 release and on rat vascular prostacyclin production, both measured by radioimmunoassay of thromboxane B2 and 6-keto-F1 alpha prostaglandin, respectively. Moreover, the incubation of urokinase with vascular endothelium resulted in an increased disappearance rate.     

Pharmacodynamic properties of antiplatelet agents: current knowledge and future perspectives

Platelets play an important role in atherothrombotic disease. The currently available antiplatelet drugs target key steps of platelet activation including thromboxane A₂ synthesis, ADP-mediated signaling, and glycoprotein IIb/IIIa-mediated platelet aggregation. The improvement of our understanding on the pharmacokinetic and pharmacodynamic characteristics of these drugs enables the tailoring of the most appropriate anti-thrombotic therapy to the individual patient and risk situation in the daily clinical practice. However, current antiplatelet therapies are associated with increased bleeding risk. Thus, further research on platelet functions may give rise to numerous new antiplatelet agents with high anti-thrombotic efficiency and low adverse hemorrhagic side effects.     

Analysis of IgG subclass antibodies and expression of T-Cell receptor signaling molecules in anti-CD4 monoclonal antibody treated mice with autoimmune cardiomyopathy

T-cell immune abnormality in patients of dilated cardiomyopathy has been intensively studied over the past 10 years. In this study, we aim to focus on the molecular mechanism of T-cells in autoimmune cardiomyopathy mouse model by detecting the expression of three T-cell signaling molecules. Balb/C mice (n = 12) were immunized with the peptides derived from human ADP/ATP carrier on the 1st, 14th, 28th, 49th and 79th days, and half of them were also injected with anti-L3T4 McAb on the ? 1st, 0 and 1st days. The sham-immunized mice were taken as the controls (n = 6). The main result shows that the antibody response of IgG subclasses such as IgG1, IgG2b and IgG3 were definitely blocked except IgG2a in CD4⁺ cell-depleted Balb/C mice. In addition, the average mRNA expression of p56lck, p59fyn and zap-70 were all found to be dramatically higher in the mice immunized with only ADP/ATP carrier peptides than in the control-group. At meantime, reduced levels of the protein kinases p56lck, p59fyn and zap-70 were clearly observed in anti-CD4 McAb immunized group compared with DCM group. We propose that the proliferation of T-cells was significantly inhibited in anti-CD4 treated mice and CD4⁺ T-cells may play a critical role in ADP/ATP carrier caused mouse DCM.