Decreased Human Platelet Activation and Mouse Pulmonary Thrombosis by Rutaecarpine and Comparison of the Relative Effectiveness with BAY11-7082: Crucial Signals of p38-NF-κB

Platelets play a critical role in arterial thrombosis. Rutaecarpine (RUT) was purified from Tetradium ruticarpum, a well-known Chinese medicine. This study examined the relative activity of RUT with NF-κB inhibitors in human platelets. BAY11-7082 (an inhibitor of IκB kinase [IKK]), Ro106-9920 (an inhibitor of proteasomes), and RUT concentration-dependently (1–6 μM) inhibited platelet aggregation and P-selectin expression. RUT was found to have a similar effect to that of BAY11-7082; however, it exhibits more effectiveness than Ro106-9920. RUT suppresses the NF-κB pathway as it inhibits IKK, IκBα, and p65 phosphorylation and reverses IκBα degradation in activated platelets. This study also investigated the role of p38 and NF-κB in cell signaling events and found that SB203580 (an inhibitor of p38) markedly reduced p38, IKK, and p65 phosphorylation and reversed IκBα degradation as well as p65 activation in a confocal microscope, whereas BAY11-7082 had no effects in p38 phosphorylation. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay shows that RUT and BAY11-7082 did not exhibit free radical scavenging activity. In the in vivo study, compared with BAY11-7082, RUT more effectively reduced mortality in adenosine diphosphate (ADP)-induced acute pulmonary thromboembolism without affecting the bleeding time. In conclusion, a distinctive pathway of p38-mediated NF-κB activation may involve RUT-mediated antiplatelet activation, and RUT could act as a strong prophylactic or therapeutic drug for cardiovascular diseases.

Changes of starch and sucrose content and related gene expression during the growth and development of Lanzhou lily bulb

As the main forms of carbohydrates, starch and sucrose play a vital role in the balance and coordination of various carbohydrates. Lanzhou lily is the most popular edible lily in China, mainly distributed in the central region of Gansu. To clarify the relationship between carbohydrate metabolism and bulb development of Lanzhou lily, so as to provide a basis for the promotion of the growth and development in Lanzhou lily and its important economic value, we studied lily bulbs in the squaring stage, flowering stage, half withering stage and withering stage. The plant height, fresh weight of mother and daughter bulbs continued to increase during the whole growth period and fresh weight of stem and leaf began to decrease in the half withering stage. The content of starch, sucrose and total soluble sugar in the lily mother bulb accumulated mostly in the flowering, withering and half withering stages, respectively. Starch, sucrose and total soluble sugar accumulated in the daughter bulb with the highest concentration during the withering stage. In the transcription level, sucrose synthase (SuSy1) and sucrose invertase (INV2) expressed the highest in squaring stage, and the expression was significantly higher in the mother bulb than in the daughter bulb. In flowering stage, the expression levels of soluble starch synthase (SSS1), starch-branching enzyme (SBE) and adenosine diphosphate-glucose pyrophosphorylase (AGP1) genes were higher in the mother bulb than in the daughter bulb. Altogether, our results indicate that starch and sucrose are important for the bulb growth and development of Lanzhou lily.

Design, Synthesis, and Docking Studies of Arylhydrazone Compounds and Evaluation of Their Platelet Aggregation Inhibitory Effect and Cytotoxicity

In view of proven antiplatelet activity of hydrazone group containing compounds, two series of hydrazone derivatives were synthesized by coupling appropriate aldehydes with phenyl hydrazine and Isonicotinic acid in the presence of distilled water and a catalytic amount of glacial acetic acid. All synthesized compounds were screened for their antiplatelet activity against induced aggregation by adenosine diphosphate (ADP) and arachidonic acid (AA). The results indicate that compounds in arylhydrazone group had shown satisfactory activity. Among them, 1-(3-methoxybenzylidene)-2-phenylhydrazine (1c), 2-methoxy-4-(2-phenylhydrazono) methyl phenol (1g), and 2-((2-phenylhydrazono) methyl)-1H-pyrrole (1h) were found to be the most potent antiplatelet compounds with IC50 less than 39 μM. Furthermore, the cell toxicity assay, (MTT test) indicates their noncytotoxic in various cell lines. None of the synthesized N-isonicotinohydrazide derivatives in this study excreted sufficient antiplatelet activity.

ĐIỀU TRỊ DUY TRÌ BẰNG OLAPARIB Ở BỆNH NHÂN UNG THƯ BUỒNG TRỨNG TIẾN XA MỚI CHẨN ĐOÁN

Đặt vấn đề: Hầu hết phụ nữ ung thư buồng trứng (UTBT) giai đoạn tiến xa mới chẩn đoán, sau khi điều trị chuẩn bằng phẫu thuật và hóa trị liệu có chứa Platinum sẽ tái phát trong 3 năm đầu. Olaparib là thuốc ức chế enzym poly (adenosine diphosphate–ribose) polymerase, có hiệu quả tốt trong điều trị ung thư buồng trứng tái phát, nhưng lợi ích của Olaparib trong điều trị duy trì đối với các trường hợp mới được chẩn đoán chưa được chứng minh. Phương pháp: Thử nghiệm lâm sàng pha 3, đa trung tâm, ngẫu nhiên, mù đôi, nhằm đánh giá hiệu quả của Olaparib như một liệu pháp duy trì ở những bệnh nhân mới được chẩn đoán ung thư buồng trứng giai đoạn III – IV theo FIGO; gồm các thể: ung thư biểu mô thanh dịch hoặc ung thư dạng nội mạc tử cung, độ ác tính cao, ung thư phúc mạc nguyên phát, ung thư vòi tử cung (hoặc các dạng kết hợp), với đột biến ở gen BRCA1, BRCA2 hoặc cả hai; đã có đáp ứng lâm sàng hoàn toàn hoặc một phần sau khi hóa trị liệu bằng hóa trị có chứa Platinum. Các bệnh nhân được chỉ định ngẫu nhiên, theo tỷ lệ 2:1, được uống Olaparib (300mg hai lần mỗi ngày) hoặc giả dược. Tiêu chí chính của nghiên cứu là sống thêm bệnh không tiến triển (PFS). Kết quả: Trong số 391 bệnh nhân, sau chọn ngẫu nhiên có 260 người được chỉ định nhận Olaparib và 131 nhận giả dược. Tổng cộng 388 bệnh nhân có đột biến gen BRCA1/2 dạng di truyền (germline) và 2 bệnh nhân đột biến dạng mắc phải (somatic) được xác nhận bởi phòng xét nghiệm trung tâm. Sau khi theo dõi trung vị 41 tháng, nguy cơ bệnh tiến triển hoặc tử vong ở nhóm dùng Olaparib thấp hơn 70% so với giả dược; tỷ số nguy cơ (HR) bệnh tiến triển hoặc tử vong là 0,30; CI 95% [0,23 – 0,41], p < 0,001). Các biến cố ngoại ý phù hợp với độc tính đã biết của Olaparib. Kết luận: Điều trị duy trì bằng Olaparib giúp cải thiện đáng kể thời gian sống thêm bệnh không tiến triển ở phụ nữ ung thư buồng trứng giai đoạn tiến xa mới chẩn đoán, có đột biến gen BRCA1/2; làm giảm 70% nguy cơ tiến triển bệnh hoặc tử vong so với với giả dược.

Platelet SHARPIN regulates platelet adhesion and inflammatory responses through associations with αIIbβ3 and LUBAC

Platelets form hemostatic plugs to prevent blood loss and they modulate immunity and inflammation in several ways. A key event during hemostasis is activation of integrin αIIbβ3 through direct interactions of the β3 cytoplasmic tail with talin and kindlin-3. Recently, we showed that human platelets express the adapter molecule, SHARPIN, that can associate directly with the αIIb cytoplasmic tail and can separately promote NF-κB pathway activation as a member of the Met-1 linear ubiquitination activation complex (LUBAC). Here we investigated the role of SHARPIN in platelets after crossing Sharpin flox/flox (fl/fl) mice with PF4-Cre or GPIbα-Cre mice to selectively delete SHARPIN in platelets. SHARPIN-null platelets adhered to immobilized fibrinogen through αIIbβ3, and they spread more extensively than littermate control platelets in a manner dependent on feedback stimulation by platelet adenosine diphosphate (ADP) (P &lt; 0.01). SHARPIN-null platelets showed increased colocalization of αIIbβ3 with talin as assessed by super-resolution microscopy and increased binding of soluble fibrinogen in response to sub-maximal concentrations of ADP (P &lt; 0.05). However, mice with SHARPIN-null platelets showed compromised thrombus growth on collagen and slightly prolonged tail bleeding times. Platelets lacking SHARPIN also showed reduced NF-κB activation and linear ubiquitination of protein substrates upon challenge with classical platelet agonists. Furthermore, the loss of platelet SHARPIN resulted in significant reduction in inflammation in murine models of colitis and peritonitis (P &lt; 0.01). Thus, SHARPIN plays differential and context-dependent roles in platelets to regulate important inflammatory and integrin adhesive functions of these anucleate cells.

The principal mitochondrial K+ uniport is associated with respiratory complex I

Mitochondria generate ATP via coupling the negative electrochemical potential (proton motive force, Capital Greek (Deltap), consisting of a proton gradient (Capital Greek DeltapH+) and a membrane potential (Capital Greek Psim) across the respiratory chain, to phosphorylation of adenosine diphosphate nucleotide. In turn, DeltapH+ and Capital Greek Psim, are tightly balanced by the modulation of ionic uniporters and exchange-diffusion systems which preserve integrity of mitochondrial membranes and regulate ATP production. Here, we provide direct electrophysiological, pharmacological and genetic evidence that the main mitochondrial electrophoretic pathway for monovalent cations is associated with respiratory complex I, contrary to the long-held dogma that only H+ gradients are built across proteins of the mammalian electron transport chain. Here we propose a theoretical framework to describe how monovalent metal cations contribute to the buildup of H+ gradients and the proton motive force, extending the classical Mitchellian view on chemiosmosis and vectorial metabolism. Keywords: mitochondrial electrogenic transport, chemiosmotic theory, vectorial metabolism, whole-mitochondria electrophysiology.

Pharmacodynamic profiles of dual-pathway inhibition with or without clopidogrel vs dual antiplatelet therapy in patients with atherosclerotic disease

Aim: Inhibition of thrombin-mediated signaling processes using a vascular dose of rivaroxaban in adjunct to antiplatelet therapy, known as dual-pathway inhibition (DPI), reduces atherothrombotic events in patients with stable atherosclerotic disease. However, there are limited data on the pharmacodynamic (PD) effects of this strategy. Methods and Results: This investigation was conducted in selected cohorts of patients (n=40) with stable atherosclerotic disease enrolled within a larger prospective PD study who were treated with either aspirin plus clopidogrel (DAPT), aspirin plus rivaroxaban 2.5 mg/bid (DPI) or DAPT plus rivaroxaban 2.5 mg/bid. Multiple PD assays assessing of markers of thrombosis were used. PD endpoints included platelet-mediated global thrombogenicity measured by light transmittance aggregometry (LTA) following stimuli with CATF [collagen‐related peptide +adenosine diphosphate (ADP) +tissue factor (TF)], markers of P2Y12 reactivity, markers of platelet aggregation using LTA following several stimuli (arachidonic acid, ADP, collagen, TF, and TRAP), thrombin generation and thrombus formation. There was no difference in platelet-mediated global thrombogenicity between groups. Rivaroxaban significantly reduced thrombin generation and was associated with a trend towards reduced TF-induced platelet aggregation. Clopidogrel-based treatments reduced markers of P2Y12 signaling and TRAP‐induced platelet aggregation. There were no differences between groups on markers of cyclooxygenase‐1 mediated activity. Conclusions: Compared with DAPT, DPI does not result in any differences in platelet-mediated global thrombogenicity, but reduces thrombin generation. These PD observations support that modulating thrombin generation by means of factor Xa inhibition in adjunct to antiplatelet therapy provides effective antithrombotic effects, supporting the efficacy and safety findings of DPI observed in clinical

Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear

Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s−1). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A2 (TxA2), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen.

Current Treatments and New Possible Complementary Therapies for Epithelial Ovarian Cancer

Epithelial ovarian cancer (EOC) is one of the deadliest gynaecological malignancies. The late diagnosis is frequent due to the absence of specific symptomatology and the molecular complexity of the disease, which includes a high angiogenesis potential. The first-line treatment is based on optimal debulking surgery following chemotherapy with platinum/gemcitabine and taxane compounds. During the last years, anti-angiogenic therapy and poly adenosine diphosphate-ribose polymerases (PARP)-inhibitors were introduced in therapeutic schemes. Several studies have shown that these drugs increase the progression-free survival and overall survival of patients with ovarian cancer, but the identification of patients who have the greatest benefits is still under investigation. In the present review, we discuss about the molecular characteristics of the disease, the recent evidence of approved treatments and the new possible complementary approaches, focusing on drug repurposing, non-coding RNAs, and nanomedicine as a new method for drug delivery.

PKD-dependent PARP12-catalyzed mono-ADP-ribosylation of Golgin-97 is required for E-cadherin transport from Golgi to plasma membrane

Adenosine diphosphate (ADP)-ribosylation is a posttranslational modification involved in key regulatory events catalyzed by ADP-ribosyltransferases (ARTs). Substrate identification and localization of the mono-ADP-ribosyltransferase PARP12 at the trans-Golgi network (TGN) hinted at the involvement of ARTs in intracellular traffic. We find that Golgin-97, a TGN protein required for the formation and transport of a specific class of basolateral cargoes (e.g., E-cadherin and vesicular stomatitis virus G protein [VSVG]), is a PARP12 substrate. PARP12 targets an acidic cluster in the Golgin-97 coiled-coil domain essential for function. Its mutation or PARP12 depletion, delays E-cadherin and VSVG export and leads to a defect in carrier fission, hence in transport, with consequent accumulation of cargoes in a trans-Golgi/Rab11–positive intermediate compartment. In contrast, PARP12 does not control the Golgin-245–dependent traffic of cargoes such as tumor necrosis factor alpha (TNFα). Thus, the transport of different basolateral proteins to the plasma membrane is differentially regulated by Golgin-97 mono-ADP-ribosylation by PARP12. This identifies a selective regulatory mechanism acting on the transport of Golgin-97– vs. Golgin-245–dependent cargoes. Of note, PARP12 enzymatic activity, and consequently Golgin-97 mono-ADP-ribosylation, depends on the activation of protein kinase D (PKD) at the TGN during traffic. PARP12 is directly phosphorylated by PKD, and this is essential to stimulate PARP12 catalytic activity. PARP12 is therefore a component of the PKD-driven regulatory cascade that selectively controls a major branch of the basolateral transport pathway. We propose that through this mechanism, PARP12 contributes to the maintenance of E-cadherin–mediated cell polarity and cell–cell junctions.