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Biomedical subjects

M Handa

Publications and source records attributed to M Handa.

At least 91 records · Page 5Linked to original sources
PubMed1997

Glycoprotein Ib-von Willebrand factor interactions activate tyrosine kinases in human platelets.

von Willebrand factor (vWF ) in the presence of botrocetin induces p72(syk) activation, assessed as its autophosphorylated level and in vitro kinase assays, the transient association of p72(syk) with p60(c-src), and the translocation of p60(c-src) and p54/58(lyn) to cytoskeletal fractions. Jararaca glycoprotein Ib-binding protein (GPIb-BP), which specifically binds to GPIb, abolished these phenomena, suggesting that they are mediated by the vWF-GPIb interaction. These tyrosine kinase-related events were not inhibited by GRGDS peptide (plus EGTA), indicating that GPIIb/IIIa is not involved in the observed responses. Shc, an adaptor protein, was also tyrosine phosphorylated by the botrocetin-vWF activation. When GPIb was immunoprecipitated with nonfunctional monoclonal antibodies (MoAbs) directed against GPIb, a kinase activity was found to associate with GPIb upon botrocetin-vWF activation. On the other hand, anti-GPIb MoAbs that inhibit the vWF-GPIb interaction did not coprecipitate a kinase activity. Because the recovery of GPIb did not differ significantly, it is suggested that the excessive presence of inhibitory anti-GPIb MoAb dissociated a kinase activity from GPIb. Phosphoamino acid analysis showed that the kinase activity was that of a tyrosine kinase. The identity of the tyrosine kinase and the mode of interaction with the cytoplasmic region of GPIb await to be determined. Our findings suggest that the tyrosine kinase associated with GPIb serves at a most proximal step in the signal transduction pathway involved in the vWF-GPIb-induced platelet activation, which leads to other tyrosine kinase-related intracellular signals.

Blood Platelets↗
PubMed1997

Expression and functional characterization of an abnormal platelet membrane glycoprotein Ib alpha (Met239 --> Val) reported in patients with platelet-type von Willebrand disease.

Platelet-type von Willebrand disease (vWD) is a congenital bleeding disorder characterized by heightened ristocetin-induced platelet aggregation caused by abnormally high affinity between the platelet membrane glycoprotein (GP) Ib/IX complex and von Willebrand factor (vWF). Two distinct point mutations, Gly233 to Val and Met239 to Val, have been reported in GPIb alpha. We have constructed a recombinant GPIb alpha fragment containing the latter mutation, Met239 to Val (M239V) and characterized the mutant molecule using two methods, ie, interaction between soluble vWF and immobilized M239V and inhibition of platelet aggregation by purified soluble M239V. Spontaneous binding (ie, binding without any inducers) was observed between 125I-vWF and immobilized M239V but not between 125I-vWF and immobilized wild-type (WT) GPIb alpha. The addition of low concentrations of ristocetin (0.2 mg/mL) induced specific 125I-vWF binding to immobilized M239V, but not to WT GPIb alpha. At high concentrations of ristocetin (1.2 mg/mL), both WT GPIb alpha and M239V specifically bound to 125I-vWF. Thus, M239V reproduced the unique functional abnormality of the GPIb/IX complex in platelet-type vWD. Moreover, the purified soluble M239V inhibited platelet aggregation induced by low concentration of ristocetin (0.3 mg/mL) in platelet-rich plasma from a patient having Met239 to Val mutation, whereas purified WT did not. These results provide direct evidences that the reported point mutation is the responsible molecular basis of this disorder.

Analysis of Variance↗
PubMed1997

Characterization of the novel murine monoclonal anti-von Willebrand factor (vWf) antibody GUR76-23 which inhibits vWf interaction with alpha IIb beta 3 but not alpha v beta 3 integrin.

von Willebrand factor (vWf) is known to interact with the two beta 3 integrins, alpha IIb beta 3 and alpha v beta 3, in an RGD-dependent manner. We characterized a novel murine monoclonal antibody to human vWf, GUR76-23, which recognized a site within the carboxy-terminal half of the molecule containing the RGD sequence. This antibody inhibited high shear-induced platelet aggregation and blocked adhesion of ADP plus epinephrine-stimulated platelets to vWf, indicating that it interferes with the interaction with alpha IIb beta 3. Unlike antibodies against the RGD site, however, the antibody was without effect on adhesion of cultured human umbilical vein endothelial cells to vWf, a phenomenon known to involve the interaction with alpha v beta 3. GUR76-23 binding was not displaced by anti-RGD antibodies. These results suggest that the adhesive interaction of vWf with these two beta 3 integrins may be differentially modulated by a site(s) other than the common RGD module.

Animals↗
PubMed1997

High shear stress attenuates agonist-induced, glycoprotein IIb/IIIa-mediated platelet aggregation when von Willebrand factor binding to glycoprotein Ib/IX is blocked.

High shear stress facilitates von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib/IX, causing activation of GPIIb/IIIa to induce platelet aggregation. Here we report that activated GPIIb/IIIa, even occupied by ligands, is not sufficient to mediate platelet aggregation under high shear stress conditions when vWF binding to GPIb/IX is blocked. Platelet rich plasma or washed platelet suspension supplemented with purified human fibrinogen at a concentration of 2 mg/mL were treated with an anti-vWF monoclonal antibody NMC-4 which blocks the binding of vWF to GPIb/IX. After addition of 10 mumol/L ADP, aggregation was continuously monitored under various shear stress conditions (0-108 dyne/cm2) using a cone-plate type aggregometer previously described (Ikeda Y et al J Clin Invest 1991; 87:1234). The extent of maximal aggregation of agonist-stimulated platelets in the presence of NMC-4 correlated inversely with the level of shear stress applied, with the virtual absence of aggregation at 108 dyne/cm2. Once aggregated by 10 mumol/L ADP under low shear stress (12 dyne/cm2), platelets could be disaggregated, in part, by the application of high shear stress (108 dyne/cm2), and reaggregated when shear stress was returned to 12 dyne/cm2. Flow cytometric analysis revealed that platelets stimulated with 10 mumol/L ADP at 108 dyne/cm2 bound fluorescein isothiocyanate (FITC)-labeled fibrinogen, although aggregation was absent in this experimental condition. These results demonstrate the dual effect of shear stress on platelet functions; a pro-aggregating activity that induces vWF-GPIb/IX interaction leading to platelet activation, and an anti-aggregating force to prevent the growth of platelet thrombi. It is suggested that the efficacy of vWF blockade is greater under high shear than low shear stress conditions, and that a selective inhibition of platelet functions can be possible.

Adenosine Diphosphate↗
PubMed1997

Thrombopoietin and thrombin induce tyrosine phosphorylation of Vav in human blood platelets.

Thrombopoietin has an essential role in megakaryopoiesis and thrombopoiesis. To investigate the signaling processes induced by thrombopoietin, we have employed human platelets and recently demonstrated that thrombopoietin induces rapid tyrosine phosphorylation of Jak-2, Tyk2, Shc, Stat3, Stat5, p120(c-cbl) and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine phosphorylated protein in platelets stimulated by thrombopoietin is approximately 85 to 95 kD, we examined the possibility that this could be Vav, a 95-kD proto-oncogene product. Specific antisera against Vav recognized the same 95 kD protein in lysates of Jurkat cells, which are known to express Vav, and platelets, indicating that platelets have Vav. Thrombopoietin induced rapid tyrosine phosphorylation of Vav in platelets without an elevation in cytosolic free calcium concentration or activation of protein kinase C. Vav was also tyrosine phosphorylated upon treatment of platelets with thrombin, collagen, or U46619, which activate phospholipase C, leading to an increased ionized calcium concentration and activation of protein kinase C. Ionomycin or phorbol 12-myristate 13-acetate (PMA) also induces tyrosine phosphorylation of Vav, suggesting that an increase in ionized calcium concentration or activation of protein kinase C may lead to phosphorylation of Vav. Thrombopoietin also induced tyrosine phosphorylation of Vav in FDCP-2 cells, genetically engineered to express human c-Mpl (FDCP-hMpl5). However, neither ionomycin nor PMA induced an increase in tyrosine phosphorylation of Vav in FDCP-hMpl5 cells, suggesting that the calcium and protein kinase C pathways of Vav phosphorylation may be unique to platelets. Further, Vav became incorporated into the Triton X-100 insoluble 10,000 g sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. Vav was constitutively associated with a 28-kD adapter protein, Grb2, which is also incorporated into the cytoskeleton in an aggregation-dependent fashion. Lastly, we found that Vav is cleaved when there is activation of calpain, a protease that may have a role in postaggregation signaling processes. Our data suggest that thrombopoietin and other agonists may induce tyrosine phosphorylation of Vav by different mechanisms and Vav may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.

Animals↗
PubMed1997

Increased alveolar fluid clearance following thoracotomy: report of a case and results of an analysis.

Alveolar fluid clearance was studied in the resected lung of a 58-year-old man who had undergone exploratory thoracotomy 9 days earlier. An isosmolar albumin solution was instilled into the distal air spaces, and the albumin and electrolyte concentrations were measured over 4 h. Alveolar sodium and fluid clearance had increased by nearly 200% from the control values in the resected lungs of patients without prior thoracotomy (n = 5), and histological examination showed that the number of alveolar type II epithelial cells was markedly elevated. These results suggest that an increase in the number of alveolar type II cells may accelerate alveolar fluid clearance under certain clinical conditions.

Aged↗
PubMed1997

Hypothermia inhibits the alveolar epithelial injury caused by hyposmotic albumin solution during preservation of the resected human lung.

This study was conducted to determine whether hypothermia inhibited alveolar epithelial injury in the resected human lung during preservation. Hyposmotic albumin solution, 248 mOsm/kg, was instilled into the alveolar spaces of resected human lungs which were inflated with an airway pressure of 7 cmH2O and stored at either 37 degrees C or 8 degrees C for 4 h. Alveolar fluid was aspirated and the influx of lactate dehydrogenase (LDH) and globulin into the alveolar spaces, as markers of alveolar epithelial injury, was measured. Ion transport and fluid clearance across the alveolar epithelium were calculated by the changes in electrolyte and albumin concentrations in the alveolar fluid, respectively. While the LDH levels and globulin concentrations increased significantly in the hyposmotic experiments at 37 degrees C, hypothermia inhibited these increases. Alveolar fluid clearance at 37 degrees C increased to 20% in the hyposmotic experiments compared with 12% in the control isosmotic experiments; however, sodium and chloride transport in the hyposmotic experiments was not significantly different from that in the isosmotic experiments. Thus, we conclude that hypothermia at 8 degrees C inhibits alveolar epithelial injury caused by the hyposmotic solution in resected human lungs. Moreover, alveolar ion and fluid clearance mechanisms were preserved across the injured alveolar epithelial cells.

Epithelium↗
PubMed1997

Re-expression of functional P-selectin molecules on the endothelial cell surface by repeated stimulation with thrombin.

P-selectin (GMP-140, PADGEM, CD62P) is a cell adhesion receptor which is believed to play an important role in inflammatory diseases by supporting leucocyte rolling. P-selectin is located on the granule membrane of Weibel-Palade bodies in resting endothelial cells and is expressed on the cell surface during cellular activation with various stimulators such as thrombin. Thereafter, P-selectin is internalized and sorted to the Golgi region and Weibel-Palade bodies again. However, whether P-selectin is re-expressed upon subsequent cellular stimulation has, to date, been unclear. To address this question, we measured the cellular content and surface expression of P-selectin, using indirect immunofluorescence and confocal laser cytometry. Surface expression of P-selectin reached a maximum < 2 min after thrombin stimulation and declined to basal levels after 180 min. Rechallenge with thrombin induced rapid surface re-expression of P-selectin, which was independent of de novo protein synthesis, since cycloheximide did not inhibit re-expression. Moreover, re-expressed P-selectin supported the adherence of HL60 promyelocytic cells. These results clearly demonstrated that functional P-selectin molecule was recycled after repeated stimulation with thrombin, raising the possibility that P-selectin is involved in chronic inflammation.

Endothelium, Vascular↗
PubMed1997

EWS-ERG fusion transcript produced by chromosomal insertion in a Ewing sarcoma.

The EWS gene is fused in Ewing sarcoma-like tumors by a chromosomal translocation to one of the four ETS-family genes: FLI1, ERG, ETV1, and E1AF. The orientation of EWS and FLI1 on chromosomes 22 and 11, respectively, is 5' centromeric and 3' telomeric, whereas that of ERG on chromosome 21 is the reverse. Although 10% of Ewing-family tumors express the EWS-ERG fusion transcript, there have been no reports on tumors with t(21;22)(q22;q12) identified by banding cytogenetics. We found the karyotype 50, XY, +8, +8, +12, +mar in all metaphase cells from a tumor. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis performed on the tumor and direct sequencing of the products identified the EWS-ERG fusion transcript. Subsequent two-color fluorescence in situ hybridization (FISH) analysis with EWS and ERG clones showed the fused signals on the der(21) chromosome, but no ERG signals on the chromosome 22 homologs. Thus, our RT-PCR and FISH analyses indicated that the chromosome 22 fragment containing the 5' portion of EWS had been inverted and inserted into chromosome 21 and had fused to the 3' portion of ERG. This subtle chromosome aberration could not be identified by routine cytogenetics. A chromosomal inversion/insertion has also been described in acute leukemia with the MLL-AF10 fusion gene, and this may be a common pathway for producing fusion of reverse-oriented genes in leukemias and solid tumors.

Bone Neoplasms↗
PubMed1997

[Effect of thoracoscopic procedure versus standard posterolateral/muscle-sparing thoracotomy on pulmonary lung function].

To understand the effects of thoracoscopic approach to thoracotomy on lung function, we investigated the changes in lung function after thoracoscopic operation (n = 7) and compared them to those after standard posterolateral (n = 5) or muscle-sparing thoracotomy (n = 6). In 18 patients with benign lung tumor in which surgical treatment resected in partial resection of the lung tissue, we measured TLC, VC, RV, FEV1.0, FEV1.0%, Peak Flow and DLco preoperatively and at 2, 4 weeks postoperatively. In the standard posterolateral or muscle-sparing thoracotomy groups, FVC, TLC, FEV1.0 and Peak Flow decreased to about 80% of preoperative value at 2 weeks. At 4 weeks after operation, these values showed to increase some what, but did not reach to the preoperative value. Whereas, the thoracoscopic procedure group, did not show the decrease in lung function through the time course. There was not significant different between standard posterolateral and muscle-sparing thoracotomy. We conclude that thoracoscopic approach is an useful technique to thoracotomy because of its minimizing after thoracotomy effect of lung function loss.

Adult↗
PubMed1997

[Video-assisted thoracoscopic surgery of bilateral dissemination of invasive thymoma: report of a case].

Eight years ago, A 29-year-old woman was admitted by complaining with facial edema and dyspnea. A chest x-ray film and computed tomography showed an anterior mediastinal tumor that invaded to the superior vena cava and the trachea. A bronchoscopic biopsy revealed that the tumor was a thymoma. We performed radiotherapy and chemotherapy because a surgical operation was not possible. The size of the tumor was slightly reduced after those therapies roentgenographically. We followed up the subsequent of history of the patient. Eight years later, July, 1995, chest computed tomography showed bilateral disseminations of invasive thymoma. The disseminated tumors were removed by two-stage video assisted thoracoscopic surgery (VATS), to preserve collateral veins so that collateral venous circulations were preserved. She discharged 20 days after second VATS uneventfully.

Adult↗
PubMed1997

[Prevention of posttransfusion graft-versus-host disease by leukocyte depletion filter].

Posttransfusion graft-versus-host disease (PTGVHD) is characterized by a fatal alloimmune reaction induced by donor lymphocytes contaminated in blood products. It is generally accepted that gamma irradiation of the blood products prior to transfusion is a most efficacious and reliable means to prevent this reaction. In contrast, leukocyte reduction by a third generation, high performance filter has been reported to fail the prevention of PTGVHD. With the filter function being considerably improved recently, leukocyte reduction may be an alternative for gamma irradiation to prevent PTGV-HD.

Adult↗
PubMed1997

[The efficacy of leukodepleted platelet transfusion].

The use of leukopoor blood cellular products is now considered as being a simple and efficacious method to prevent unfavorable transfusion complications such as nonhemolytic febrile transfusion reaction and HLA-alloimmunization which are induced by contaminated leukocytes. Several prospective studies demonstrated that leukocyte depletion by filtration has been found to be effective to prevent HLA alloimmunization and/or platelet transfusion refractoriness, although cost effectiveness of the filter use still remains unclear. Lueukodepletion is also efficient in preventing CMV infection in patients undergoing stem cell transplantation, and its efficacy is readily comparable to CMV seronegative blood products. With more efficient and reliable filters (4th generation filters) having been developed recently, the clinical application of leukodepletion filters in transfusion medicine may be considerably widened.

Cell Separation↗
PubMed1997

[Successful two-stage approach to treating excessive hemorrhage from pulmonary arterial stump in post-lobectomy bronchopleural fistula].

A 62-year-old man underwent right lower lobectomy for adenocarcinoma (pT2N0M0) and nine days later, a bronchopleural fistula with empyema was evident. Six weeks following the lobectomy, excessive hemorrhage from the site of chest drainage and hemoptysis were noted. The bleeding and empyema were controlled by a two-stage approach. Anterior transpericardial approach was first made through the median sternotomy to clamp the right main pulmonary artery and then postero-lateral thoracotomy was conducted for the bronchopleural fistula with empyema. The right bronchial stump was covered with a pedicled muscle flap and pseudomonas aeruginosa, always positive in drainage effusion, consequently disappeared. The patient was discharged with a closed bronchus 4 months following the operation.

Bronchial Fistula↗
PubMed1997

Expression of nucleolar protein p120 in human lung cancer: difference in histological types as a marker for proliferation.

The function of proliferation-associated nucleolar protein p120 is unclear. A recent report that a yeast protein, NOP2, 67% homologous to human p120, is up-regulated during the onset of growth and influences the morphology of the nucleolus supports the notion that this protein could serve as a marker for proliferation in neoplastic cells. Lung cancer is characteristic in that different histological types show different biological features. We attempted to evaluate the levels of p120 expression in resected human lung cancer tissues of different histological types and the relation of p120 expression and cell proliferation using human lung cancer cell lines. When 37 frozen specimens of human lung cancer and normal lung were stained with a p120 monoclonal antibody, the nucleoli of cancer cells were positively stained, whereas a few macrophages in normal lung revealed only weak staining. The labeling index of p120 in squamous cell carcinoma (67.7 +/- 12.4%) was significantly higher than that in adenocarcinoma (35.3 +/- 12.6%) or in large cell carcinoma (30.1 +/- 17.3%; P < 0.01). In six human lung cancer cell lines and one normal lung fibroblast cell line cultured in vitro, there was a significant correlation between S-phase fraction and p120 mRNA (r = 0.851, P < 0.02)/p120 protein (r = 0.869, P < 0.01) or between doubling time and p120 protein (r = -0.928, P < 0.01). In the context of the reports that indicate higher [3H]thymidine incorporation and shorter doubling time in the squamous cell carcinoma, these results indicate that p120 can be a marker for proliferation in human lung cancer cells in vivo and in vitro, and that it has an important function in the cell cycle of tumor proliferation.

Adenocarcinoma↗
PubMed1996

Synergistic effect of epinephrine and shearing on platelet activation.

Platelet activation mediated through the interaction between von Willebrand factor (vWF) and platelet glycoprotein (GP) Ib is known to occur under high shear rate. We have demonstrated that low concentration of epinephrine could reduce the threshold level of shear rate necessary to cause platelet activation with purified system devoid of the effect of plasma proteins other than vWF. Both the extent of platelet aggregation and [Ca2+]i were continuously measured with optically modified cone-and-plate viscometer. No aggregation with no change in [Ca2+]i occurred under shear rate less than 7,200 s-1 in the absence of exogeneously added epinephrine. Epinephrine enhanced platelet aggregation under moderate level of shear rate (7,200 s-1) in a dose dependent manner. Significant aggregation with rise in [Ca2+]i was demonstrated even under the low shear rate of 1,200 s-1 when exogeneously added low concentration of epinephrine (0.05 microM) which did not cause platelet activation by itself was present. Aggregation and rise in [Ca2+]i under low shear in the presence of epinephrine was abolished by monoclonal antibodies against A1 domain of vWF or GP Ib, like aggregation and the rise in [Ca2+]i occurred under high shear rate (10,800 s-1), alpha 2-receptor blockade yohimbine completely antagonized the enhancing effects of epinephrine. Our findings suggested that epinephrine and shearing synergistically activated platelets through vWF interaction to GP Ib, which might suggest the role of sympathetic stimulation for the onset of acute arterial thrombosis.

Blood Viscosity↗