YM-254890, which was isolated from the culture broth of Chromobacterium sp., inhibits ADP-induced platelet aggregation and has antithrombotic and thrombolytic effects. YM-254890 blocks G␣ q/11 -coupled ADP receptor P2Y1-mediated Ca 2؉ mobilization. Here we report that YM-254890 is a selective G␣ q/11 inhibitor. YM-254890 blocked Ca 2؉ mobilization mediated by several G␣ q/11 -coupled receptors but not by G␣ i -or G␣ 15 -coupled receptor, indicating that phospholipase C␤ activation and subsequent signaling molecules are not the target of YM-254890. YM-254890 completely prevented the serum response factor (SRF)-mediated gene transcription induced by G␣ q R183C, which is constitutively active in a receptor-dependent manner because of its reduced k cat of GTP hydrolysis. Conversely, YM-254890 had only a modest effect on the SRF-mediated gene transcription by G␣ q Q209L, which is GTPase-deficient (activated) G␣ q . These suggested that the acting point of YM-254890 is receptor-G␣ q interaction or the subsequent guanine nucleotide exchange step. The fact that YM-254890 (i) inhibited the SRF-mediated gene transcription by G␣ qi5 , which interacts with G␣ i -coupled receptor and possesses the effector function of G␣ q , and (ii) had no effect on the K d value of high affinity [ These data indicate that YM-254890 blocks the exchange of GDP for GTP in G␣ q/11 activation. This novel G␣ q/11 -selective inhibitor is a promising and powerful tool for studying G␣ q/11 protein activation, G␣ q/11 -coupled receptor signaling, and G␣ q/11 -mediated biological events. G protein-coupled receptors (GPCR)1 and heterotrimeric G proteins, consisting of G␣, ␤, and ␥, transduce extracellular stimuli, such as hormones, neurotransmitters, chemokines, and other local mediators, into appropriate intracellular responses (1, 2). The activation of G␣ proteins is related to conformational change by guanine nucleotide interaction. The GPCRs, activated by the agonist, induced exchanges of GDP for GTP on the coupled G␣ subunit. The resultant G␣-GTP complex dissociates from the G␤␥ subunit and activates its downstream effectors, which in turn regulate various functions such as gene transcription, mitogenesis, metabolism, muscle contractile state, and ion channel regulation. The GTPase activity of ␣-subunit turns off effector signals by hydrolyzing G␣-GTP to G␣-GDP, which re-associates with G␤␥.There are over 20 G␣ subunits classified into subfamilies by its sequence homology and the downstream signal. These include the major four families, G␣ q/11 , G␣ s , G␣ i/o , and G␣ 12/13 . Main effector molecules of G␣ q/11 , G␣ s , G␣ i/o , and G␣ 12/13 are thought to be phospholipase C (PLC), adenylyl cyclase (activation), adenylyl cyclase (inhibition), and small GTPase families, respectively (3). Transient intracellular Ca 2ϩ mobilization is led by PLC␤ activation via the G␣ q/11 , G␣ 15/16 , or G␤␥ subunit with G␣ i . PLC␤ hydrolyzes phosphatidylinositol bisphosphate in the plasma membrane, and the generated inositol 1,4,5-trisphosphate (IP 3 ) activates ...

A novel platelet aggregation inhibitor, YM-254890, was isolated from the culture broth of strain QS3666. This strain was isolated from a soil sample collected at Okutama, Tokyo, Japan, and was identified as Chromobacterium sp. by morphological and physiological criteria. YM-254890 was purified from the culture supernatant by solvent extraction, ODS and silica gel flash chromatography, followed by preparative HPLC. YM-254890 inhibited ADP-induced the P2Y1 receptor-signal transduction pathway.

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SummaryThe thrombotic risk associated with elevated plasma levels of clotting factor VIII (FVIII) was investigated in a mouse model of thrombophilia. After the intravenous injection of recombinant human FVIII and/or of purified FVIII-free human von Willebrand factor (vWF), a controlled mild injury was inflicted on the carotid artery of FVB mice by irradiation with filtered green light in combination with intravenous injection of the dye rose bengal. Formation of a platelet-rich thrombus was continuously monitored for 40 min via transillumination and the thrombus size was measured via image analysis. Administration of recombinant human FVIII at 40 g/kg led to initial FVIII plasma activities equivalent to 250% of normal human plasma FVIII activity and significantly enhanced thrombus size. Immunohistochemical staining illustrated the accumulation of FVIII within the thrombi. Human vWF, even at 10 mg/kg, had no effect on thrombus formation. The thrombotic tendency induced by FVIII was significantly inhibited by the administration of human vWF in a dose-dependent manner. Separate plasma measurements revealed that human FVIII has comparable affinities for human and murine vWF but that human vWF does not effectively bind murine platelets. The inhibition by human vWF of the thrombotic tendency induced by human FVIII could therefore be explained by a lack of accumulation of FVIII within the developing thrombus because of the reduced affinity of human vWF for murine platelets and the reduced occupancy of murine von Willebrand factor by human FVIII after injection of human vWF. These results show that vWF actively participates in FVIII accumulation in the arterial thrombus and provide experimental evidence for epidemiological findings that elevated plasma FVIII levels are associated with an increased thrombotic risk, also in arteries.

Abstract-The relationship between platelet and leukocyte activation, coagulation, and neointima development was investigated in noninjured murine blood vessels subjected to blood stasis. The left common carotid artery of C57BL/6J mice was ligated proximal to the bifurcation. Tissue-factor expression in luminal leukocytes progressively increased over 2 weeks. On day 3 after ligation, in addition to infiltrated granulocytes, platelet microthrombi and platelet-covered leukocytes as well as tissue-factor-positive fibrin deposits lined the endothelium. Maximal neointima formation in carotid artery cross sections of control mice equaled 28Ϯ3.7% (nϭ11) and 42Ϯ5.1% (nϭ8) of the internal elastic lamina cross-sectional area 1 and 2 weeks after ligation. In FVIII Ϫ/Ϫ mice, stenosis was significantly lower 1 (11Ϯ3.6%, nϭ8) and 2 (21Ϯ4.7%, nϭ7) weeks after ligation (both PϽ0.01 versus background-matched controls). In u-PA Ϫ/Ϫ mice, luminal stenosis was significantly higher 1 (38Ϯ7.0%, nϭ7) and 2 (77Ϯ5.6%, nϭ6) weeks after ligation (PϽ0.05 and PϽ0.01, respectively, versus matched controls). In ␣ 2 -AP Ϫ/Ϫ mice, stenosis was lower at 1 week (14Ϯ2.6%, nϭ7, PϽ0.01) but not at 2 weeks. Responses in tissue-type plasminogen activator or plasminogen activator inhibitor-1 gene-deficient mice equaled that in controls. Reducing plasma fibrinogen levels in controls with ancrod or inducing partial thrombocytopenia with busulfan resulted in significantly less neointima, but inflammation was inhibited only in busulfan-treated mice. We conclude that stasis induces platelet activation, leading to microthrombosis and plateletleukocyte conjugate formation, triggering inflammation and tissue-factor accumulation on the carotid artery endothelium. Delayed coagulation then results in formation of a fibrin matrix, which is used by smooth muscle cells to migrate into the lumen. Blood containing veins, ligated for 30 to 60 minutes, attract leukocytes on the endothelium, but although stasis induces leukocyte migration through the endothelial layer, no platelet adherence, aggregation, or thrombus formation can be detected at this stage. 2 Only when veins are ligated and left in situ for as long as 24 to 72 hours can fibrin and deposition of leukocytes, platelets, and erythrocytes be detected. 3 The existence of hypoxemia in those sites where venous thrombosis commonly originates in humans 4 has suggested that hypoxic endothelial damage is involved in the ultimate development of venous thrombosis.In addition to causing delayed thrombosis, blood flow reduction also enhances intimal lesion formation in vascular grafts and balloon-injured vessels, 5-7 implying that alterations in blood flow affect the proliferative response of smooth muscle cells. Kumar and Lindner 8 have developed a model in which blood flow in the common carotid artery of the mouse is arrested by carefully ligating the vessel near the bifurcation. In this model, a neointima develops proximal to the ligation site as a consequence of partial blood stasis, reduced shear stress, enhanced art...