Minoru Okuma

Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor γ‐chain

We have recently shown that collagen activates platelets through a pathway dependent on the Fc receptor γ‐chain and the tyrosine kinase Syk. We report here that the Fc receptor γ‐chain and the candidate collagen receptor glycoprotein VI (GPVI) co‐associate. Furthermore, cross‐linking GPVI stimulates a similar pattern of tyrosine phosphorylation to that stimulated by collagen, including tyrosine phosphorylation of Fc receptor γ‐chain. These results support a model where GPVI couples collagen‐stimulation of platelets to phosphorylation of the Fc receptor γ‐chain leading to activation of Syk and phospholipase Cγ2.

Two thromboxane A2 receptor isoforms in human platelets: Opposite coupling to adenylyl cyclase with different sensitivity to Arg60 to Leu mutation

Thromboxane A2 (TXA2) receptor is a key molecule in hemostasis as its abnormality leads to bleeding disorders. Two isoforms of the human TXA2 receptor have been cloned; one from placenta and the other from endothelium, here referred to as TXR alpha and TXR beta, respectively. These isoforms differ only in their carboxyl-terminal tails. We report that both isoforms are present in human platelets. The two isoforms expressed in cultured cells show similar ligand binding characteristics and phospholipase C (PLC) activation but oppositely regulate adenylyl cyclase activity; TXR alpha activates adenylyl cyclase, while TXR beta inhibits it. The Arg60 to Leu mutant of TXR alpha, which has been shown to impair PLC activation (Hirata, T., A. Kakizuka, F. Ushikubi, I. Fuse, M. Okuma, and S. Narumiya. 1994. J. Clin. Invest. 94: 1662-1667), also impairs adenylyl cyclase stimulation, whereas that of TXR beta retains its activity to inhibit adenylyl cyclase. These findings suggest that the pathway linked to adenylyl cyclase inhibition might be involved in some of the TXA2-induced platelet responses such as shape change and phospholipase A2 activation which remain unaffected in the patients with this mutation.

Collagen-platelet interaction : Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen

OBJECTIVE Peptides consisting of a repeat Gly-Pro-Hyp sequence are potent platelet agonists. The aim of this study was: (1) to examine the specificity of this sequence for platelet activation; (2) to confirm its recognition by platelet glycoprotein VI; and (3) to assess with suitable peptides the relative importance of glycoprotein VI and integrin alpha 2 beta 1 in platelet activation by collagen. METHODS Peptides were synthesized by standard Fmoc chemistry and tested for their ability to support adhesion of human platelets and HT 1080 cells, induce platelet aggregation, bind integrin alpha 2 subunit A-domain and to cause tyrosine phosphorylation of platelet proteins. RESULTS (1) Peptides consisting of a repeat Gly-Pro-Pro, Gly-Pro-Ala or Gly-Pro-Arg sequence exhibited little if any platelet-reactivity. (2) The platelet-reactive peptide consisting of a repeating Gly-Pro-Hyp sequence failed to induce tyrosine phosphorylation in glycoprotein VI-deficient platelets. Platelet adhesion to this peptide was inhibited by intact anti-glycoprotein VI antibody and its Fab fragment. The latter inhibited aggregation by the peptide and fibres of both collagens I and III. (3) A peptide containing a 15-mer alpha 2 beta 1-binding sequence in a repeat Gly-Pro-Pro structure supported alpha 2 beta 1-mediated platelet and HT 1080 cell adhesion and bound alpha 2 A-domain, but failed to activate platelets or to induce tyrosine phosphorylation. Conversely, a peptide containing this sequence but with an essential Glu replaced by Ala and inserted in a repeat Gly-Pro-Hyp structure did not recognize alpha 2 beta 1, but was highly platelet activatory. CONCLUSIONS Platelet activation by collagen involves the highly-specific recognition of the Gly-Pro-Hyp sequence by platelet glycoprotein VI. Recognition of alpha 2 beta 1 is insufficient to cause activation. Interaction between collagen and glycoprotein VI is unique since Gly-Pro-Hyp is common in collagens but occurs rarely in other proteins, and glycoprotein VI may be expressed solely by platelets. This sequence could provide a basis for a highly-specific anti-thrombotic reagent to control thrombosis associated with plaque rupture.

Requirement of AP-1 for Ceramide-induced Apoptosis in Human Leukemia HL-60 Cells

Ceramide has emerged as a novel lipid mediator in cell proliferation, differentiation, and apoptosis. In this work, we demonstrate that the levels of c-jun mRNA, c-Jun protein, and DNA binding activity of a nuclear transcription factor AP-1 to 12-o-tetradecanoylphorbol 13-acetate responsive elements all increased following treatment with the cell-permeable ceramide, N-acetylsphingosine in human leukemia HL-60 cells. N-Acetylsphingosine (1-10 μM) increased the levels of c-jun mRNA in a dose-dependent manner, and maximal expression was achieved 1 h after treatment. Increase of c-jun expression treated with 5 μMN-acetyldihydrosphingosine, which could not induce apoptosis, was one third of that with 5 μMN-acetylsphingosine. Ceramide-induced growth inhibition and DNA fragmentation were both prevented by treatment with curcumin, 1,7-bis[4-hydroxy-3-methoxy-phenyl]-1,6-heptadiene-3,5-dione (an inhibitor of AP-1 activation), or antisense oligonucleotides for c-jun. These results suggest that the transcription factor AP-1 is critical for apoptosis in HL-60 cells and that an intracellular sphingolipid mediator, ceramide, modulates a signal transduction inducing apoptosis through AP-1 activation.

Ceramide-induced Translocation of Protein Kinase C-δ and -ϵ to the Cytosol IMPLICATIONS IN APOPTOSIS

Ceramide is now recognized as an intracellular lipid signal mediator, which induces various kinds of cell functions including apoptosis. Ceramide-induced apoptosis was reported to be blocked by 12-O-tetradecanoylphorbol 13-acetate, a protein kinase C (PKC) activator, but its mechanism remained unclear. Therefore, we investigated whether ceramide has any effects on PKC in the induction of apoptosis. We here report that N-acetylsphingosine (synthetic membrane-permeable ceramide) induced translocation of PKC-δ and -ε isozymes from the membrane to the cytosol within 5 min in human leukemia cell lines. Treatment with sphingomyelinase, tumor necrosis factor-α, or anti-Fas antibody, all of which can induce apoptosis by generating natural ceramide, similarly induced cytosolic translocation of PKC-δ and -ε. In Fas-resistant cells anti-Fas antibody did not induce cytosolic translocation of PKC-δ and -ε because of no generation of ceramide, whereas N-acetylsphingosine induced apoptosis with cytosolic translocation of PKC-δ and -ε. Furthermore, both 12-O-tetradecanoylphorbol 13-acetate and a nonspecific kinase inhibitor, staurosporine, prevented ceramide-induced apoptosis by inhibiting cytosolic translocation of PKC-δ and -ε. These data suggest that cytosolic translocation of PKC-δ and -ε plays an important role in ceramide-mediated apoptosis.

Arg60 to Leu mutation of the human thromboxane A2 receptor in a dominantly inherited bleeding disorder.

Recent advances in molecular genetics have revealed the mechanisms underlying a variety of inherited human disorders. Among them, mutations in G protein-coupled receptors have clearly demonstrated two types of abnormalities, namely loss of function and constitutive activation of the receptors. Thromboxane A2 (TXA2) receptor is a member of the family of G protein-coupled receptors and performs an essential role in hemostasis by interacting with TXA2 to induce platelet aggregation. Here we identify a single amino acid substitution (Arg60-->Leu) in the first cytoplasmic loop of the TXA2 receptor in a dominantly inherited bleeding disorder characterized by defective platelet response to TXA2. This mutation was found exclusively in affected members of two unrelated families with the disorder. The mutant receptor expressed in Chinese hamster ovary cells showed decreased agonist-induced second messenger formation despite its normal ligand binding affinities. These results suggest that the Arg60 to Leu mutation is responsible for the disorder. Moreover, dominant inheritance of the disorder suggests the possibility that the mutation produces a dominant negative TXA2 receptor.

Affinity labeling displays the stepwise activation of ICE-related proteases by Fas, staurosporine, and CrmA-sensitive caspase-8.

The activation of multiple interleukin-1β converting enzyme-related proteases (caspases) in apoptotic mammalian cells raises questions as to whether the multiple active caspases have distinct roles in apoptotic execution as well as how these proteases are organized in apoptotic signaling pathways. Here we used an affinity-labeling agent, YV(bio)KD-aomk, to investigate the caspases activated during apoptotic cell death. YV(bio)KD-aomk identified six distinct polypeptides corresponding to active caspases in Fas-stimulated Jurkat T cells. On staurosporine treatment, four polypeptides were detected. Competition experiments showed that the labeled caspases have distinct substrate preferences. Stepwise appearance of the labeled caspases in each cell death event was consistent with the view that the activated caspases are organized into protease cascades. Moreover, we found that stepwise activation of caspases similar to that induced by Fas ligation is triggered by exposing non-apoptotic Jurkat cell extracts to caspase-8 (MACH/FLICE/Mch5). Conversely, CrmA protein, a viral suppressor of Fas-induced apoptosis, inhibited the protease activity of caspase-8. Overall, these findings provide evidence that caspase-8, a CrmA-sensitive protease, is responsible for initiating the stepwise activation of multiple caspases in Fas-stimulated cells.

Thrombopoietin, c‐Mpl ligand, induces tyrosine phosphorylation of Tyk2, JAK2, and STAT3, and enhances agonists‐induced aggregation in platelets in vitro

We investigated in vitro effects of recombinant human thrombopoietin (TPO), or c‐Mpl ligand, on human platelets. TPO induced rapid dose‐dependent tyrosine phosphorylation of several proteins. We identified Janus tyrosine kinases, Tyk2 and JAK2, and a member of STAT (signal transducers and activators of transcription) family, STAT3, as the tyrosine‐phosphorylated proteins in response to TPO. TPO by itself did not cause platelet aggregation and shape change, but augmented ADP‐induced aggregation in a dose‐dependent manner. Acetylsalicylic acid inhibited the secondary aggregation enhanced by TPO, but not the TPO‐induced potentiation of the primary aggregation. TPO modulates platelet activation possibly through protein‐tyrosine phosphorylation.

Generation of prostacyclin-like substance and lipid peroxidation in vitamin E-deficient rats.

Endogenous generation of prostacyclin (PGI2)-like substance and lipid peroxidation were studied in the aorta of rats fed on vitamin E-deficient diet and/or vitamin E-supplemented one for 4 to 10 months after they were weaned at 4 weeks. PGI2-like substance was produced by the incubation of the aortic ring in pH 9.0 borate-buffered saline and was estimated by comparison of its antiaggregatory activity with that produced by known amounts of synthetic PGI2. Thiobarbituric acid-reacting substance (TBARS) was determined as an indicator of lipid peroxidation. The generation of PGI2-like substance was significantly reduced in rats fed on vitamin E-deficient diet for 8 and 10 months as compared with that in the animals fed on vitamin E-supplemented one for the same period (p less than 0.001). Mean concentration of TBARS in the aortae of rats fed on vitamin E-deficient diet for 10 months was significantly higher than that of the animals fed on vitamin E-supplemented diet for the same feeding period (p less than 0.001). These alterations in the aortae of rats fed on vitamin E-deficient diet were corrected by feeding them on vitamin E-supplemented diet for subsequent 2 months.

Vascular complications in living related liver transplantation detected with intraoperative and postoperative Doppler US

BACKGROUND/AIMS The purpose of this study was to clarify changes in the graft hemodynamics induced by vascular complications in living related liver transplantation. METHODS This study included 46 pediatric recipients who underwent partial liver transplantation from living related donors. The blood flow was evaluated in the portal system, the hepatic artery and the hepatic vein with serial intra- and post-operative Doppler ultrasound (US). RESULTS In 12 patients, intraoperative Doppler US showed a decrease in portal venous inflow (< 9 ml.min-1.kg-1) toward the liver graft and could act as a guide for ligation of collaterals in seven patients, portal re-construction in two, thrombectomy in one and relief of hepatic venous outflow obstruction in two for increasing the portal venous inflow. In five patients, intraoperative Doppler US showed poor arterial inflow, i.e. dampened arterial waveforms which involved both low pulsatility index (< 0.90) and low peak-systolic velocity (< 31 cm/s). In three of them, the waveform was more pulsatile after re-anastomosis or relief from stretching of the hepatic artery. The remaining two patients developed hepatic artery thrombosis. Most of the hepatic venous outflow obstruction (four of five patients) had flat waveforms, low flow velocity (< 10 cm/s) of the hepatic vein, and poor portal inflow (flow velocity < 14 cm/s). Postoperative Doppler US showed hepatic venous outflow obstruction in three patients, hepatic artery thrombosis in three (twice in one patient), portal vein stenosis in two and portal vein thrombosis in one. These complications were successfully managed with surgical procedures in three patients, transhepatic angioplasty in three and conservative treatments in four. Six patients died of non-vascular complications. CONCLUSIONS Serial intra- and post-operative Doppler US was a useful technique for making an early diagnosis of abnormal hemodynamics of the graft circulation. Furthermore, intraoperative Doppler US could assess reconstructed vessels objectively and would reduce the incidence of vascular complications following transplantation.