A new functional assay for human protein S activity using activated factor V as substrate.
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Adult↗
Biomedical subjects
D Meyer
Publications and source records attributed to D Meyer.
[von Willebrand disease].
Von Willebrand disease (vWD) is a frequent congenital bleeding disorder with an autosomal, usually dominant, mode of inheritance. Its clinical expression and mechanism (quantitative or qualitative abnormality of von Willebrand Factor) are extremely heterogeneous. The bleeding symptoms vary in intensity from one patient to the other (including in the same family) and also with time in a given patient. The role of vWF in platelet adhesion to the subendothelium and as a carrier of Factor VIII explains the prolongation of the bleeding time and of the APTT in the typical forms of vWD. These tests, however, may be only slightly abnormal or even normal in the numerous mild forms of the disease which are often diagnosed because of bleeding complications following surgery or delivery. The specific tests (assay of vWF antigen, assay of ristocetin-cofactor activity, assay of Factor VIII activity) usually lead to the diagnosis. The latter remains uncertain, however, in some cases where results are at the lower limit of normal. Once the diagnosis is established, highly specialized methods are required to determine the type of vWD. The therapy of vWD relies on the use of either virus inactivated plasma concentrates or DDAVP.
Bleeding Time↗
Production in Escherichia coli of a biologically active subfragment of von Willebrand factor corresponding to the platelet glycoprotein Ib, collagen and heparin binding domains.
A full-length cDNA for vWF has been cloned from a human lung cDNA library and a fragment of this cDNA has been modified to allow its expression in E. coli. This fragment, which corresponds to Val 449-Asn 730 of vWF and includes the GPIb-binding domain and binding sites for collagen and heparin, was subcloned into an expression vector containing an inducible lambda PL promoter. On induction, the expressed recombinant vWF subfragment migrated with a mol wt of around 38,000 after SDS-PAGE. It was identified as a vWF fragment by Western blotting using either a polyclonal or a monoclonal antibody which inhibits the binding of vWF to GPIb. Following solubilization in urea, the bacterial extract inhibited ristocetin-induced platelet aggregation and bound to ristocetin-treated platelets, to collagen and to heparin.
Binding Sites↗
[Thrombin-antithrombin III complexes as a measure of effective heparin treatment?].
To study the effect of heparin on thrombin formation in vivo, we measured thrombin-antithrombin III complexes (TAT) in 26 patients treated with heparin for acute coronary artery disease or deep vein thrombosis. There was a statistically significant correlation between activated partial thromboplastin time and heparin concentration, but none was found between these tests and TAT. Abnormal TAT values in 45% of the patients showed that therapeutic heparin concentration did not inhibit thrombin formation in vivo, or did so only partially.
Antithrombin III↗
Localization within the 106 N-terminal amino acids of von Willebrand factor (vWF) of the epitope corresponding to a monoclonal antibody which inhibits vWF binding to factor VIII.
We have used an in vitro transcription-translation system to localize the epitope corresponding to a monoclonal antibody (MAb 418) to vWF which specifically inhibits its binding to F. VIII. We have subcloned 1.5 Kb of vWF cDNA encoding for the N-terminal part of the vWF subunit which contains a binding site for F.VIII into the Bluescribe expression vector. After in vitro transcription and translation using the resulting construction (pBS-TG3522), a polypeptide of mol wt 60,000 (AA 1-495) was immunoprecipitated with a polyclonal antibody to vWF and with MAb 418. The MAb 418 epitope was further localized by reducing the size of the cDNA insert. This resulted in the production of two polypeptides of 18,000 (AA 1 to 142) and 13,000 (AA 1 to 106) which both retained reactivity with MAb 418. Thus the epitope corresponding to MAb 418 is localized to the first 106 AA of the mature vWF subunit.
Antibodies, Monoclonal↗
Oculomotor nerve palsy precipitating acute angle-closure glaucoma. A case report.
A case of acute angle-closure glaucoma precipitated by oculomotor nerve palsy in a patient with shallow anterior chambers is reported. The different ways in which a palsy of the oculomotor nerve can influence the intra-ocular pressure are discussed.
Acute Disease↗
Potential use of human tissues for toxicity research and testing.
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Cell Transformation, Neoplastic↗
Export of prepro-alpha-factor from Escherichia coli.
Yeast prepro-alpha-factor translocates posttranslationally into yeast microsomes in vitro. This process is strongly influenced by the extreme carboxyl-terminal region of the protein. These features contrast with the properties of most eucaryotic proteins which are translocated into the endoplasmic reticulum. We have extended these studies by introducing the gene for the wild-type and several mutant forms of prepro-alpha-factor into Escherichia coli. Prepro-alpha-factor is secreted into the periplasm and processed to pro-alpha-factor. Its translocation across the plasma membrane requires the membrane potential and the secY gene product. Deletion mutant analysis showed that features of the pro-segment were essential for secretion of prepro-alpha-factor in E. coli, while the carboxyl-terminal region, which is required in yeast, is dispensible in E. coli. Neither size nor the presence of a unique topogenic sequence was sufficient to explain the requirement for the pro-segment.
Chromosome Deletion↗
Expression of von Willebrand factor in porcine vessels: heterogeneity at the level of von Willebrand factor mRNA.
The expression of the von Willebrand factor (vWF) gene by cultured endothelial cells from the porcine pulmonary artery, aorta, and lung was compared at the levels of messenger (m)RNA and antigen. Steady-state levels of vWF mRNA were determined by dot-blot analysis using a partial human vWF cDNA as the hybridization probe; vWF mRNA from cultured aortic endothelial cells, and vWF antigen secreted into the culture supernatants were barely detectable. In contrast, vWF mRNA and antigen from the pulmonary artery endothelial cells were approximately eight to nine times that demonstrated by aortic cells. Levels of vWF mRNA and antigen in cultured lung cells were intermediate of those found in the pulmonary artery and aorta and correlated with the estimated number of cells demonstrated to be of endothelial origin in the mixed cell populations grown from the lung. Differences between the levels of vWF mRNA found in cultured cells from the pulmonary artery and those found in the aorta were maintained in cells processed directly from these vessels. Correlation between the levels of vWF mRNA and antigen in endothelial cells from different vessels of the pig suggests that the differential control of vWF synthesis is at the level of transcription. Furthermore, maintenance in cultured cells of the difference in transcription rates that were observed in vivo suggests that vWF gene expression is not exclusively regulated through environmental factors.
Animals↗
Phase II study with etoposide in previously untreated advanced breast cancer.
A phase II study was carried out to evaluate the efficacy and safety of etoposide used as first-line chemotherapy for patients with advanced breast carcinoma. A total of 20 patients received 230 mg/m2 i.v. etoposide per day for 3 days (total, 690 mg/m2 per course) every 4 weeks. A total of 95 courses were given. Observed responses included 3 partial remissions (PR) and 14 cases of stable disease (NC). The median duration of response was 6 (PR) and 5.6 months (NC). Contrary to the severe hematological toxicity in heavily pretreated patients described in previous studies, no substantial problems were observed in this trial. No dose reduction was necessary, and only once did leukopenia lead to a 1-week delay in therapy. An increase in platelets up to a maximum of 685,000/mm3 was seen in all patients, particularly in those with bone metastases. No relation to the quality of remission or pretreatment was seen. Nausea, vomiting, and fatique were mild and transient, but alopecia occurred in all cases. One patient developed nonfatal anaphylactic shock after etoposide treatment.
Adult↗
von Willebrand factor and platelet function.
vWF is an adhesive protein that binds to two distinct platelet glycoproteins, GP Ib and GP IIb-IIa complex. Its interaction with GP Ib is primarily responsible for platelet adhesion to the subendothelium. The current model is that vWF binds to collagen and/or another component of the subendothelium, after which a conformational change in the vWF molecule exposes the GP Ib binding site. This interaction may not only promote the initial attachment of platelets to the subendothelium but also play a role in thrombus formation through exposure of GP IIb-IIIa to which vWF and fibrinogen can bind. The second important function of vWF is to be a carrier for F. VIII, protecting it from degradation and playing a role in its activation by thrombin. Circulating vWF has a complex multimeric structure that ranges in Mrs from 0.5 to 20 x 10(6) Daltons. The basic subunit has a Mr of 270 kDa. Amino acid sequencing of vWF demonstrated that the basic subunit or mature vWF is made up of 2050 amino acids. Molecular cloning of the vWF cDNA revealed that the primary transcript consists of 8900 base pairs that encode for 2813 amino acids, including a 22 amino acid signal peptide and a propolypeptide of 741 amino acids, called vWF antigen II. Recent studies on the expression of recombinant vWF molecules indicate that the propolypeptide is involved in the multimerization of vWF. The domains on the vWF molecule involved in the interactions of vWF with GP Ib, GP IIb-IIIa, collagen, F. VIII and heparin have been localized to varying extents. It is anticipated that peptide analysis and recombinant DNA techniques, such as in vitro mutagenesis, will further define the structural requirements of these binding domains. vWF is synthesized in a cell-specific manner by endothelial cells and megakaryocytes. It undergoes a complex intracellular biosynthesis involving transcription of a 200 kb gene, splicing out more than 42 introns, translation of a 8900 bp mRNA, glycosylation, disulphide bond formation, sulphatation, multimerization and proteolytic cleavage. The molecule can be secreted in a constitutive or regulated manner upon perturbation of the endothelial cells with physiological and non-physiological secretagogues. The mechanisms that control the synthesis of vWF should be an exciting area of further research. vWD is probably the most common of all congenital disorders of haemostasis. It is an extremely heterogeneous syndrome involving quantitative or qualitative disorders of vWF.(ABSTRACT TRUNCATED AT 400 WORDS)
Binding Sites↗
[Clinical model concept of the evolution of malignant tumors: implication for hormone therapy of breast cancer].
An analysis is made of current concepts about heterogeneity, instability and autonomy of tumor cells which accounts for the clinical course of incurable, late-stage malignant disease which as such is characterized by a growing independence from growth control mechanisms of the host, by progressive metastatic spread and by unpredictable malignancy associated events. Increasing heterogeneity, instability and autonomy are best demonstrated by the development of resistance phenomena and are potentially reversible processes. This reversibility can be exploited using biological response modifiers. Understanding this triad also helps explain certain clinical phenomena such as qualitative differences in remissions and mixed responses, early vs. late stage disease, and the potential therapeutical hazards which can lead to further tumor heterogeneity. On the breast cancer example further clinical implications are illustrated, namely receptor heterogeneity, autocrine autonomy, and combination therapy with hormones. The concepts discussed are the basis for the development of non-toxic tumor therapies which are aimed less at tumor cell kill than at stabilization.
Breast Neoplasms↗
The use of bone marrow aspirations and lumbar punctures at the time of diagnosis of retinoblastoma.
Lumbar punctures (n = 115) and bone marrow aspirations (n = 114) were performed as part of the routine initial diagnostic evaluation of 115 children with retinoblastoma. Three spinal fluid examinations were positive for tumor cells, and bone marrow smears of three children demonstrated clumps of tumor cells. Five of the six positive studies were in patients with stage IV (extraglobar) disease. These results show that demonstrable CSF or bone marrow involvement is so infrequent an event at diagnosis in patients without symptoms, signs, or histologic evidence of tumor dissemination (stages I-II) as to support a recommendation that these studies need not be performed routinely in such patients. If, after enucleation, there is evidence of extraglobar extension, or if patients have symptoms or signs of CNS or systemic spread (stages III or IV), both procedures should be performed to accurately stage disease and provide baseline measurements of tumor involvement for monitoring of response to chemotherapy and/or irradiation. These results have importance in terms of justification of invasive work-up of most (greater than 85%) affected children, and cost containment.
Adolescent↗
New variant of von Willebrand disease with defective binding to factor VIII.
A new variant of von Willebrand disease (vWD) was identified by a new analytic method which characterizes the ability of plasma von Willebrand Factor (vWF) to bind to purified factor VIII (F.VIII). vWF was isolated from small amounts of plasma by immunoadsorption with a selected monoclonal antibody to vWF previously coated onto wells of microtitration plates. Plasma F.VIII was removed from immobilized vWF by washing with 0.4 mol/L CaCl2; purified F.VIII was then added to the well. The amount of bound F.VIII was estimated directly in the wells by a chromogenic assay and immobilized vWF was estimated by an immunologic a pool of normal plasma, ten control individuals, 13 with hemophilia A and five with type I vWD. In all cases, the dose-response curves were linear and the slopes of the regression lines were essentially the same. The method was then applied to investigate the binding of vWF to F.VIII in two vWD patients (sister and brother) who demonstrated significantly lower activity of F.VIII than of vWF. The first patient, with a long history of epistaxis, bruising, and hematomas, showed a slightly prolonged bleeding time (10 minutes); 15% VIII:C and 39% of vWF:Ag and vWFRCo. Her brother, who has a bleeding syndrome but no hematomas, showed similar data (bleeding time 9 minutes, 20% VIII:C, 53% vWF:Ag and vWFRCo). Similar levels of F.VIII were observed in the two propositi by four different methods (one- and two-stage clotting and chromogenic and immunologic assays). Sodium dodecyl sulfate (SDS) 1.4% agarose gel electrophoresis showed that all multimers of vWF were present in both patients. vWF binding to F.VIII was markedly decreased in the two propositi. The abnormal binding of vWF to F.VIII was not corrected during pregnancy or after infusion of 1-deamino (8-D-arginine) vasopressin despite an increase in vWF levels. The qualitative abnormality of vWF in both patients was associated with a subtle alteration of the multimeric structure by SDS 3% agarose gel electrophoresis in which the two central subbands of the quintuplet of individual oligomers were undetectable or poorly visible. SDS-polyacrylamide gel electrophoresis under reducing conditions demonstrated a single band of 275 Kd in the plasma of both patients, and there was no evidence of a second band corresponding to pro-vWF, the precursor of the mature vWF subunit, suggesting that proteolytic processing of vWF was normal.(ABSTRACT TRUNCATED AT 400 WORDS)
Blood Coagulation Tests↗
Action stat! Ethylene glycol poisoning.
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Emergencies↗
Family physicians' perceptions of the family conference.
Ninety-one of 127 graduates (72 percent) of one family practice residency returned a questionnaire in which they estimated the likelihood that their patients would want a physician-family conference for each of 21 clinical situations. For each situation the physicians also rated their own preferences regarding patients' interest in family conferences. Serious medical illnesses received the highest ratings for both sets of ratings. For all 21 situations, physicians' estimates of patients' responses were significantly lower than physicians' preferred response ratings. The physicians' estimates and preferences regarding patients' interest in family conferences were compared with actual patients' ratings obtained in a previous study. The patient ratings were significantly greater than the physicians' estimates of patient ratings for 14 of 21 situations; the physicians' preferences ratings were significantly higher than the actual patient ratings for 11 situations and lower for three situations. The mean number of actual family conferences conducted in the previous month was 2.6, and 66 percent of respondents had conducted at least one such conference during this time. These data indicate that physicians may be more interested in family conferences than their patients are, but that they may underestimate the degree to which patients do, in fact, want them. The implications of these data for teaching, practice, and research are discussed.
Adult↗
Patients' attitudes toward physician involvement in family conferences.
Patients' interest in family conferences was investigated using Doherty and Baird's concept of level of physician involvement with families. Patients entering two primary care clinics (N = 239) completed a questionnaire assessing their interest in physician level of involvement for each of six representative clinical situations: hospitalization for serious illness, new diagnosis of serious illness, depression, marital or relationship problems, stress-related symptoms, and frequent visits without improvement. Most patients indicated that they would want family conferences with their primary physician if a family member experienced hospitalization, new diagnosis of a serious illness, or depression. Slightly less than one half of the patients indicated that they would want family conferences for the remaining situations. Among those patients desiring family conferences, majorities responded that they would want their primary physician to provide all of Doherty and Baird's levels 2 through 5 (ongoing medical information and advice, feelings and support, systematic assessment and planned intervention, and family therapy), especially for hospitalization for serious illness and for depression. Most patients who indicated that they would want their physician to provide family therapy in the family conference also responded that they would want referral to a mental health professional for family therapy. The implications of these findings for clinical practice, residency training, and future research are discussed.
Adolescent↗
Abnormality of the N-terminal portion of von Willebrand factor in type IIA and IIC von Willebrand disease.
We have established a new analytical method which allows the characterization of von Willebrand factor (vWF) degradation fragments in minute amounts (10 microliters) of plasma, without the need for immunopurification of vWF. Plasma vWF was hydrolysed by S aureus V-8 protease (V-8 protease) and the cleaved fragments separated by SDS-agarose gel electrophoresis followed by staining with 125I-labeled polyclonal or monoclonal antibodies against vWF and autoradiography. Quantification of the amount of each product was estimated by counting the incorporated radioactivity following excision. V-8 protease limitedly hydrolysed vWF in normal as well as type I von Willebrand disease (vWD) plasma and produced two distinct fragments with identical electrophoretic and antigenic characteristics to those produced from purified vWF, i.e. a C-terminal SpII and a series of N-terminal SpIII fragments (SpIIIa, b and c). The method was applied to further characterize the molecular abnormalities of vWF in eighteen patients with type II vWD. In seven individuals with type IIA and five patients with type IIC, SpIII appeared significantly modified as compared to normal. In type IIA, there was a marked decrease or absence of SpIIIa and an increase of SpIIIb and c. In type IIC, SpIIIb was lacking. In three patients with type IIB and in three patients with type IID, there was no significant modification of SpIII. In all cases, SpII was apparently not modified. In conclusion, the molecular abnormality of vWF in type IIA and IIC vWD appears to reside in SpIII, the N-terminal portion of the vWF-subunit (residues 1 to 1,365).
Amino Acid Sequence↗