Biomedical subjects

M Handa

Publications and source records attributed to M Handa.

At least 109 records · Page 6Linked to original sources

PubMed1996

Crkl is constitutively tyrosine phosphorylated in platelets from chronic myelogenous leukemia patients and inducibly phosphorylated in normal platelets stimulated by thrombopoietin.

Platelet functions such as aggregation and clot retraction are often abnormal in chronic mylogenous leukemia (CML) patients. However, the molecular mechanisms of these altered functions are unknown. As expression of the p210bcr-abl oncogene product, a constitutively active tyrosine kinase, is known to have an essential role in the pathogenesis of CML and tyrosine phosphorylation is intimately involved in various aspects of platelet activation, we examined the pattern of protein tyrosine phosphorylation in platelets from 15 CML patients by immunoblotting with a monoclonal antiphosphotyrosine antibody (4G10). Before and after stimulation with thrombin, the only consistent difference between normal and CML platelets was the presence of a tyrosine phosphorylated protein with a relative molecular weight of 39 kD. This tyrosine phosphorylated protein was identified as crid, an SH2, SH3 containing adapter protein. Thus, as previously demonstrated for neutrophils from CML patients, tyrosine phosphorylation of p39crkl persists in mature platelets. No tyrosine phosphorylation of crid was detected following stimulation with thrombin in normal platelets. However, crkl became incorporated into the Triton X-100 insoluble residue following thrombin stimulation in a manner dependent on platelet aggregation. Further, we found that crkl is an endogenous substrate for calpain, a protease that may be involved in postaggregation signaling processes. This suggests that crkl may be involved in the reorganization of the cytoskeleton during normal platelet aggregation and its tyrosine phosphorylation in CML platelets may contribute to the abnormal platelet function in CML patients. Finally, we found that thrombopoietin induces tyrosine phosphorylation of crk1 in normal platelets and FDCP cells genetically engineered to express human c-Mpl. This suggests that crk1 can be phosphorylated by a kinase other than p210bcr-abl and that crk1 may have a role in signaling by thrombopoietin.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Affinity modulation of the platelet integrin alpha IIb beta 3 by alpha-chymotrypsin: a possible role for Na+/Ca2+ exchanger.

In the present study, we have investigated the mechanism of affinity modulation of alpha IIb beta 3 by chymotrypsin. We first confirmed that alpha-chymotrypsin could activate alpha IIb beta 3 (approximately 7,000 molecules per platelet) without major intracellular signaling. However, we unexpectedly found that high concentrations of amiloride dose-dependently inhibited 125I-fibrinogen binding to the chymotrypsin-treated platelets, as well as the platelet aggregation (IC50 [50% inhibitory concentration] for fibrinogen binding, 530 mumol/L). In contrast, amiloride did not inhibit alpha IIb beta 3 activation induced by anti-alpha IIb beta 3 monoclonal antibody PT25-2 or AP5. To identify the pathway involved, the effects of alteration of Na+ gradient in platelets were examined. Lowering Na+ gradient by replacing extracellular Na+ with tetramethylammonium (TMA) increased the number of activated alpha IIb beta 3 by twofold, as assessed by fibrinogen-binding assay. The incubation of platelets with ouabain, a Na+/K(+)-adenosine triphosphatase (ATPase) inhibitor, further augmented alpha IIb beta 3 activation. These data suggested that a likely candidate for the pathway was Na+/Ca2+ exchanger. At 140 mmol/L [Na+]o, 45Ca2+ influx to the chymotrypsin-treated platelets was twofold greater than that to non-treated platelets. Replacement of Na+ with TMA further increased the Ca2+ influx, and the increase was inhibited by amiloride dose-dependently. 3',4'-Dichlorobenzamil (DCB) and bepridil, relatively specific inhibitors of Na+/Ca2+ exchanger, also inhibited the chymotrypsin-induced alpha IIb beta 3 activation, and the IC50 values of these inhibitors for fibrinogen binding were 25 mumol/L and 52 mumol/L, respectively. Moreover, platelet aggregation induced by various physiologic agonists was inhibited by DCB or bepridil, while platelet agglutination by ristocetin was not. Our data newly suggest that Na+/Ca2+ exchanger operating in reverse mode may be directly involved in inside-out signaling that activates alpha IIb beta 3.

Amiloride↗

PubMed1996

p120c-cbl is present in human blood platelets and is differentially involved in signaling by thrombopoietin and thrombin.

To investigate the signaling processes induced by recombinant thrombopoietin, we used human platelets to recently show that thrombopoietin induces rapid tyrosine phosphorylation of Jak2, Tyk2, Shc, Stat3, Stat5, and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine-phosphorylated protein in platelets stimulated by thrombopoietin is approximately 120 kD, we examined the possibility that this could be p120c-cbl, a protein known to be involved in signaling by many growth factors. Specific antisera against p120c-cbl recognized the same 120-kD protein in lysates of Jurkat cells, which are known to express p120c-cbl, and platelets, indicating that platelets have p120c-cbl. Thrombopoietin induced rapid tyrosine phosphorylation of p120c-cbl in platelets. Thrombopoietin also induced tyrosine phosphorylation of p120c-cbl in FDCP cells genetically engineered to express the thrombopoietin receptor, c-Mpl. Interestingly, FDCP cells, expressing a truncated c-Mpl devoid of the box-2 domain, proliferate in response to thrombopoietin. However, no increase in tyrosine phosphorylation of p120c-cbl was observed upon treatment of these cells with thrombopoietin, indicating that in this system tyrosine phosphorylation of p120c-cbl may not be essential for cell proliferation. This suggests that tyrosine phosphorylation of p120c-cbl may be required for nonmitogenic responses induced by thrombopoietin in postmitotic cells such as platelets. On the other hand, p120c-cbl was not significantly tyrosine-phosphorylated upon treatment of platelets with thrombin. However, it became incorporated into the Triton X-100-insoluble, 10,000g-sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. p120c-cbl was constitutively associated with a 28-kD adapter protein, Grb2, that was also incorporated into the Triton X-100-insoluble, sedimentable residue dependent on aggregation. Further, we found that p120c-cbl is an endogenous substrate for calpain, a protease that may play a role in postaggregation signaling processes. Our data suggest that p120c-cbl may be involved in signal transduction following ligand binding to c-Mpl through its inducible tyrosine phosphorylation, and it may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.

Adaptor Proteins, Signal Transducing↗

PubMed1996

Thrombopoietin primes human platelet aggregation induced by shear stress and by multiple agonists.

Recombinant thrombopoietin has been reported to stimulate megakaryocytopoiesis and thrombopoiesis and it may be quite useful to treat patients with low platelet counts after chemotherapy. As little is known regarding the possible activation of platelets by thrombopoietin, we examined the effects of thrombopoietin on platelet aggregation induced by shear stress and various agonists in native plasma. Using hirudin as an anticoagulant, thrombopoietin (1 to 100 ng/mL) enhanced platelet aggregation induced by 2 micromol/L adenosine-diphosphate (ADP) in a dose dependent fashion. The enhancement was not affected by treatment of platelets with 1 mmol/L aspirin plus SQ-29548 (a thromboxane antagonist, 1 micromol/L) but was inhibited by a soluble form of the thrombopoietin receptor, suggesting that the enhancement was mediated by the specific receptors and does not require thromboxane production. Epinephrine (1 micromol/L), which does not induce platelet aggregation in hirudin platelet rich plasma (PRP), did so in the presence of thrombopoietin (10 ng/mL). Thrombopoietin (10 ng/mL) also enhanced or primed platelet aggregation induced by collagen (0.5 micron.mL),. thrombin, serotonin, and vasopressin. Thrombopoietin does not induce any rise in cytosolic ionized calcium concentration nor activation of protein kinase C, as estimated by phosphorylation of preckstrin, indicating that the priming effects of thrombopoietin does not require those processes. The ADP- or thrombin-induced rise in cytosolic ionized calcium concentration was not enhanced by thrombopoietin (100 ng/mL). Further, shear (ca. 90 dyn/cm2)-induced platelet aggregation was also potentiated by thrombopoietin. The priming effect on epinephrine-induced platelet aggregation in hirudin PRP was unique to thrombopoietin, with no effects seen using interleukin-6 (IL-6), IL-11, IL-3, erythropoietin, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, or c-kit ligand. These data indicate that monitoring of platelet functions may be necessary in the clinical trials of thrombopoietin.

Adenosine Diphosphate↗

PubMed1996

Establishment and characterization of transgenic mice expressing human platelet glycoprotein Ib alpha.

The platelet glycoprotein (GP) Ib/IX/V is a hetero-oligomeric receptor complex for von Willebrand factor (vWF) and mediates platelet adhesion and aggregation under high shear stress conditions. It is composed of alpha and beta chain of GP Ib, GP IX, AND and GP V. To establish transgenic mice carrying human GP Ib alpha, we injected into mouse zygotes a 6 kb DNA fragment containing human GP Ib alpha gene that included entire coding sequence and putative promoter region. One hundred and thirteen offsprings were screened, and only one was found to express human GP Ib alpha protein and has passed the human GP Ib alpha gene as well as the expression of the gene to next generation. The expression of human GP Ib alpha in transgenic mice was limited to platelets and megakaryocytes. Glycocalicin, a proteolytic fragment of human GP Ib alpha found in normal human plasma, was not detected in transgenic mouse plasma. Human vWF in the presence of ristocetin supported agglutination of transgenic mouse platelets, but not of control mouse platelets.

Animals↗

PubMed1996

Hemodynamic forces modulate the effects of cytokines on fibrinolytic activity of endothelial cells.

We investigated the effects of hemodynamic force on fibrinolytic activity of cultured human umbilical vein endothelial cells stimulated by cytokines, using a modified cone-plate viscometer in which well-controlled and -defined shear forces were generated. Treatment of the cells with interleukin (IL)-beta or tumor necrosis factor alpha (TNFalpha) under static conditions had no effect on tissue plasminogen activator (t-PA) secretion, while release of plasminogen activator inhibitor 1 (PAI-1) increased. When cells were exposed to increasing shear stress up to 24 dynes/cm2, levels of t-PA and t-PA/PAI-1 complex significantly increased relative to shear stress, while total PAI-1 and active PAI-1 secretion decreased gradually. In the presence of IL-1beta or TNFalpha, the increase in production of t-PA and the t-PA/PAI-1 complex was further augmented. Dot blot hybridization analysis of cultured cells in similar experimental conditions using t-PA and PAI-1 cDNA probes revealed no t-PA mRNA in 3 microg total RNA from static endothelial cells under resting or cytokine-stimulated conditions, but abundant t-PA mRNA was detected in cells subjected to a shear force of 18 dynes/cm2, and the increase was further augmented by addition of cytokines. In contrast, PAI-1 mRNA was detected in resting and cytokine-stimulated, nonsheared endothelial cells, but levels decreased after exposure to shear stress, even in the presence of cytokines. These results indicate a role for hemodynamic forces in regulating fibrinolytic activity with or without cytokine stimulation.

Cells, Cultured↗

PubMed1996

Purification and cloning of a soluble ATP-diphosphohydrolase (apyrase) from potato tubers (Solanum tuberosum).

A soluble ATP-diphosphohydrolase (apyrase, EC 3.6.1.5) has been purified from potato tubers. Solanum tuberosum, to a specific activity of 10,000 mumol P(i)/mg/min. The cDNA corresponding to the potato apyrase has been isolated and termed RROP1. The deduced amino acid sequence contains a putative signal sequence, two hydrophobic regions at the carboxy terminus, two potential Asn-linked glycosylation sites, and four regions in the amino-terminal half that we term ACR (apyrase conserved regions) 1-4 that are highly conserved in known apyrases and related enzymes; garden pea nucleoside triphosphatase, Toxoplasma gondii nucleoside triphosphate hydrolases, and Saccharomyces cerevisiae golgi guanosine diphosphatase. A yeast 71.9-kDa hypothetical protein on chromosome V, a Caenorhabditis elegans hypothetical 61.3-kDa protein on chromosome III, and human CD39, a lymphoid cell activation antigen, also share the conserved ACR regions, but their ability to hydrolyze nucleotides has not been assessed.

Amino Acid Sequence↗

PubMed1996

Thrombopoietin induces tyrosine phosphorylation of Stat3 and Stat5 in human blood platelets.

Thrombopoietin is known to be essential for megakaryocytopoiesis and thrombopoiesis. Recently, we and others have shown that thrombopoietin induces rapid tyrosine phosphorylation of Jak2 and other proteins in human platelets and BaF3 cells, genetically engineered to express c-Mpl, a receptor for thrombopoietin. The Jak family of tyrosine kinases are known to mediate some of the effects of cytokines or hematopoietic growth factors by recruitment and tyrosine phosphorylation of a variety of Stat (signal transducers and activators of transcription) proteins. Hence, we have investigated whether Stat proteins are present in platelets and, if so, whether they become tyrosine phosphorylated in response to thrombopoietin. We immunologically identified Stat1, Stat2, Stat3, and Stat5 in human platelet lysates. Thrombopoietin induced tyrosine phosphorylation of Stat3 and Stat5 in these cells. Thrombopoietin also induced tyrosine phosphorylation of Stat3 and Stat5 in FDCP-2 cells genetically engineered to constitutively express human c-Mpl. Thus, our data indicate that Stat3 and Stat5 may be involved in signal transduction after ligand binding to c-Mpl and that this event may have a role in megakaryopoiesis/thrombopoiesis or possibly a mature platelet function such as aggregation.

Animals↗

PubMed1996

Fusion of an ETS-family gene, EIAF, to EWS by t(17;22)(q12;q12) chromosome translocation in an undifferentiated sarcoma of infancy.

EIAF is a newly isolated ETS-family gene that is located on 17q21 and codes for the adenovirus EIA enhancer-binding protein. In our chromosome analysis of 18 of the Ewing family of tumors and undifferentiated sarcomas, we found t(17;22)(q12;q12) in an MIC2 antigen-positive undifferentiated sarcoma of infancy. On Southern blot analysis, EWS and EIAF cDNA probes hybridized to the same rearranged band, indicating that an EWS-EIAF fusion gene was formed in the tumor. Further Southern blot analysis using four EIAF cDNA probes of different sizes showed that the breakpoint lies in the region upstream to the ETS domain of the EIAF gene. EIAF may be the fourth ETS-family gene to be identified forming a fusion gene with EWS. We assume that the RNA binding domain of EWS may have been replaced by the DNA binding domain of EIAF in the EWS-EIAF fusion protein as in other fusion proteins previously characterized in Ewing sarcoma and other types of sarcomas.

Adenovirus E1A Proteins↗

PubMed1996

An increased NM23H1 copy number may be a poor prognostic factor independent of LOH on 1p in neuroblastomas.

In a study of 154 neuroblastomas, loss of heterozygosity (LOH) was observed on 1p (13%, 19/143), 11q (19%, 11/59), 14q (15%, 15/97), 17p (5%, 5/105) and 17q (17%, 9/52). We also found an increase in NM23H1 copy number in 14% (13/95) of neuroblastomas. All except one tumour with an increased copy number stained positive with anti-NM23H1 monoclonal antibody. Event-free survival (EFS) was significantly shorter in 19 patients with LOH on 1p than in 128 without (41% vs 77% 4 year EFS, P=0.0093), and in 13 patients with increased NM23H1 copy numbers than in 82 with normal copy numbers of the gene (61% vs 84% 4 year EFS, P=0.0103). LOH on 11q, 14q or 17q did not affect EFS. Most tumours with LOH on 1p, increased NM23H1 copy numbers or MYCN amplification occurred in patients aged 12 months or more, those with advanced stage disease, and those who showed near diploidy or pseudodiploidy. However, LOH on 1p was found in only 1 of the 13 tumours with increased NM23H1 copy numbers, and MYCN amplification of four copies occurred in only one other such tumour. These findings suggest that the increased NM23H1 copy number may be a predictor for poor prognosis, independent of LOH on 1p, and probably also of MYCN amplification.

Alleles↗

PubMed1996

Development of low potassium solution (EP4 solution) for long-term preservation of a lung transplant: evaluation in primate and murine lung transplant model.

The results of recent studies on long-term preservation using low potassium flush solution, which was originally produced in our department, are presented and discussed. In the primate model, a lung preserved for 24 h with EP4 solution retained sufficient function in single lung allotransplantation. In the murine experiments, we have evaluated the ion transport properties across alveolar epithelial cells in the transplanted lung using the tools of alveolar fluid clearance tests. The alveolar fluid clearance rate, which can be inhibited by 10(-3) M ouabain, is also well preserved for the first 24 h, indicating that the alveolar epithelial Na-K-ATPase is still functioning and that the 72 h preservation decreases those function of Na-K-ATPase in a time-dependent manner. In conclusion, because 24 h hypothermic preservation with EP4 solution did not show any obvious disadvantage on primate lung allografts and the cellular and molecular viability of the preserved lung flushed with EP4 solution could be retained for 24 h, this kind of solution with extracellular electrolyte composition can be applicable in the clinical settings.

Animals↗

PubMed1996

Preservation of alveolar epithelial fluid transport mechanisms in rewarmed human lung after severe hypothermia.

Although hypothermia abolishes alveolar fluid clearance in the in situ goat lung and in the ex vivo human lung, it is unknown whether alveolar fluid clearance resumes in lungs that are rewarmed after severe hypothermia. An isosmolar albumin solution was instilled into resected human lungs that were rewarmed to 37 degrees C after hypothermia (7 +/- 3 degrees C), and then alveolar fluid clearance was measured by the concentration of albumin in the alveolar fluid sample after 4 h. In control experiments in lungs that had not been cooled and rewarmed, alveolar fluid clearance was 11 +/- 2% over 4 h. In separate experiments, hypothermia completely abolished alveolar fluid clearance. However, alveolar fluid clearance resumed to a normal level of 12 +/- 1% over 4 h in the lungs that were rewarmed after hypothermia. Amiloride decreased alveolar fluid clearance by 47% in the rewarmed lungs. Terbutaline increased alveolar fluid clearance by nearly 300% in 2-h experiments in the rewarmed lungs (P < 0.05). The results of this study indicate that alveolar sodium-channel transport mechanisms are preserved in resected human lungs that are exposed to rewarming after hypothermia.

Aged↗

PubMed1996

Mitochondrial aldehyde dehydrogenase in diabetes associated with mitochondrial tRNA(Leu(UUR)) mutation at position 3243.

OBJECTIVE: To ascertain why alcohol is prone to manifest unpleasant effects in diabetes associated with mitochondrial tRNA(Leu(UUR) mutation at position 3243 (DM-Mt3243), we investigated the genotype of aldehyde dehydrogenase (ALDH) 2 and alcohol dehydrogenase 2 (ADH2) in DM-Mt3243. RESEARCH DESIGN AND METHODS: Nineteen unrelated patients with DM-Mt3243 were included in the study (12 men and 7 women). They were recruited from approximately 700 diabetic patients at three different institutes, without prior information of alcohol habit. ALDH2, ADH2, and 3243 mutation were genotyped by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) methods. There were 461 unrelated Japanese individuals and 170 non-3243 mutant NIDDM patients enrolled as control subjects. RESULTS: In the DM-Mt3243 group, 15 (79%) patients had inactive ALDH2 and 18 (95%) had atypical ADH2. The frequency of the inactive ALDH2 genotype was higher than that in the normal control subjects (P < 0.002) and that in the NIDDM control subjects (P < 0.003). However, the frequencies of ADH2 genotype in the DM-Mt3243 group, the normal control subjects, and the NIDDM control subjects were not different. CONCLUSIONS: Inactive ALDH2 genotype was frequently observed in DM-Mt3243. It suggests that DM-Mt3243 is associated with ALDH2 inactivity. We speculate the trait of acetaldehyde accumulation on ALDH2 inactivity may favor mitochondrial DNA abnormalities, thereby worsening ATP production and impairing insulin secretion. In addition, the interaction of ALDH1 and ALDH2 may alter the retinoid metabolism in the pancreas, thereby influencing insulin secretion and precipitating diabetes. Thus, this association of ALDH2 genotype with DM-Mt3243 provides insight into the etiology of diabetes in the mitochondrial diseases.

Adult↗

PubMed1996

[Reconstruction after wide resection of the chest wall: application of plates as rigid prosthetic materials].

Wide resection of the chest wall requires reconstruction with rigid prosthetic material to protect the thoracic organs and to avoid flail chest. We tried to use three kinds of plates for chest wall reconstruction in 4 cases: acrylic plate for 1, ultra-high-molecular-weight polyethylene (UHMWP) plate for 1 and methyl metacrylic resin (MMR) plate for 2. Marlex mesh was utilized to strengthen the rebuilt chest wall sandwiching rigid prosthesis in UHMWP plate and MMR plate. Except for somewhat local accumulation of serous fluid in two cases of acrylic plate and MMR plate, postoperative courses were uneventful in all cases. Acrylic plate which was used for patient with metastatic sternal tumor was easily damaged by heat. UHMWP plate which was used for patient with large benign sternal tumor was expensive and took a long time to make. On the other hand, MMR plates which were used for two patients with malignant lateral chest wall tumor were easy to handle on the spot and not expensive. MMR plate sandwiched by Marlex mesh seems to be more suitable and available for chest wall reconstruction.

Adolescent↗

PubMed1996

[Assessment of a new TNM classification produced by UICC for resected lung cancer with intrapulmonary satellite tumor(s)].

We examined the efficacy of a new classification produced by International Union Against Cancer (UICC) in 1993 for 61 patients who underwent resection of lung cancer with intrapulmonary satellite tumor(s) during 1952 to June 1991. Survival rate after resection was calculated according to the new classification. Survival rate of patients with stage I lung cancer was 67% at four years after surgery, that with stage IIIA was 33% and stage IIIB 5% at five years after surgery. There was a significant difference between the survival of stage IIIA and stage IIIB. Five year survival of patients with N0 or N1 lung cancer was 62%, which was significantly better than four year survival (3%) with N2, N3, or NX lung cancer. Five year survival for patients with squamous cell carcinoma was 61%, which was significantly better than that (9%) with adenocarcinoma. The new TNM classification produced by UICC is acceptable in investigating lung cancer with ipsilateral satellite tumor(s). It is necessary to investigate lung cancer with contralateral satellite tumor(s). Prognosis of patients with N0 or N1 lung cancer, especially squamous cell carcinoma, even if with intrapulmonary satellite tumor(s), was relatively good after resection. Such lung cancers may be synchronous multiple lung cancers from a therapeutic point of view.

Adult↗

PubMed1996

A novel regulatory epitope defined by a murine monoclonal antibody to the platelet GPIIb-IIIa complex (alpha IIb beta 3 integrin).

We characterized a murine monoclonal antibody, PT25-2 (IgG1), raised against washed human platelets. The antibody and its Fab fragments were both capable of inducing platelet aggregation in a fibrinogen-dependent manner and induced 125I-fibrinogen binding to unstimulated platelets (120,000 molecules/platelet at a 100 nM IgG concentration). The antibody immunoprecipitated the alpha IIb beta 3 complex from lysates of iodinated platelets but did not react with the respective subunits when complex formation was disrupted by treatment with 5 mM EDTA at 37 degrees C for 30 min. However, simply removing the extracellular divalent cation with EDTA had no effect on antibody binding indicating that the antibody's epitope depends upon a conformational structure maintained by alpha beta subunit association. Antibody binding to unstimulated, washed platelets yielded binding parameters (Kd = 40 nM, Bmax = 100,000 molecules/platelet), which were found to be virtually unchanged when binding was performed using thrombin or RGDS-peptide-stimulated platelets. Thus, the PT25-2 antibody defines a novel regulatory epitope expressed by the alpha IIb beta 3 integrin on unstimulated, quiescent platelets.

Animals↗

PubMed1995

Recombinant thrombopoietin induces rapid protein tyrosine phosphorylation of Janus kinase 2 and Shc in human blood platelets.

A cDNA for the thrombopoietin has been cloned by several groups. The recombinant thrombopoietin has been reported to stimulate the megakaryocytopoiesis and thrombopoiesis. Little is known regarding the molecular basis of its effects. To elucidate the molecular mechanism involved in signal transduction, we have investigated the effects of thrombopoietin on platelet tyrosine phosphorylation. We report here that thrombopoietin induced time- and dose-dependent tyrosine phosphorylation of several proteins including Janus kinase 2 (Jak2) and a 52-kD protein, Shc, in human blood platelets. Both Jak2 and Shc were tyrosine phosphorylated within 15 seconds after stimulation. The tyrosine phosphorylation of Jak2 was accompanied by increased kinase activity, whereas Shc tyrosine phosphorylation induced its association with a 25-kD protein, Grb2. Thus, our data suggest that Jak2, Shc, and Grb2 may be involved in signal transduction after ligand binding to c-mpl in human platelets.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Substitution of Val for Met at residue 239 of platelet glycoprotein Ib alpha in Japanese patients with platelet-type von Willebrand disease.

Genomic DNA was studied from four patients with platelet-type von Willebrand disease (vWD) from two Japanese families previously reported. The entire coding region of platelet glycoprotein (GP) Ib alpha, a component of the platelet receptor for von Willebrand factor (vWF), was examined by polymerase chain reaction (PCR) followed by direct DNA sequence analysis. A single point mutation was found in all patients resulting in substitution of Val (GTG) for Met (ATG) at residue 239 of GPIb alpha. All patients were heterozygous for the mutation, whereas none of the unaffected family members had an amino acid substitution at residue 239. Because the nucleotide substitution destroys an NIa III restriction site on GPIb alpha, PCR products were subjected to digestion with this enzyme; DNA fragments from both normal and mutant alleles were detected in all affected individuals. In allele-specific PCR, DNA was amplified from patients' genomic DNA using either adenine- or guanine-containing primers, whereas only adenine-containing primer successfully amplified DNA from normal individuals. Cloning of amplified DNA into bacteriophage M13mp19 and subsequent DNA sequence analysis confirmed the mutation in these families. The absence of the amino acid substitution at residue 239 of GPIb alpha in the normal individuals tested, together with the linkage of this substitution to the phenotypic expression of disease in these two families and in a family recently described suggest that this amino acid change is a molecular basis for platelet-type vWD, and the substitution may produce a quite similar phenotype to the one reported previously (Gly to Val at residue 233 of GPIb alpha).

Amino Acid Sequence↗