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Biomedical subjects

J Ware

Publications and source records attributed to J Ware.

At least 55 records · Page 3Linked to original sources
PubMed1995

Identification of three tyrosine residues of glycoprotein Ib alpha with distinct roles in von Willebrand factor and alpha-thrombin binding.

The interaction between von Willebrand factor (vWF) and the platelet membrane glycoprotein (GP) Ib-IX-V complex is essential for platelet adhesion at sites of vascular injury under high shear stress flow conditions. Moreover, GP Ib-IX-V may contribute to the mechanisms of platelet activation through its high affinity binding of alpha-thrombin. There are two distinct but partially overlapping regions of GP Ib alpha thought to be involved in interacting with vWF (residues 251-279) and alpha-thrombin (residues 271-284); they share three tyrosine residues (positions 276, 278, and 279) that have recently been shown to be sulfated (Dong, J., Li, C. Q., and Lopez, J.A. (1994) Biochemistry 33, 13946-13953). To define the functional role of these three residues, we have introduced selected mutations in a soluble recombinant GP Ib alpha fragment (corresponding to the sequence 1-302 of the mature protein) that binds vWF and alpha-thrombin with the same attributes as intact GP Ib-IX-V complex. Fragments containing a single Tyr-->Phe substitution either at position 276 or 278 or 279 exhibited normal interaction with vWF but markedly reduced or absent binding of alpha-thrombin. GP Ib alpha fragment with normal sequence but synthesized under sulfate-free conditions also failed to bind alpha-thrombin and, in addition, had markedly reduced interaction with vWF. The simultaneous substitution of three neighboring Asp residues with Asn at positions 272, 274, and 277, a multiple mutation that may impair Tyr sulfation, also resulted in loss of binding of both ligands. These results define distinct structural features of GP Ib alpha selectively involved in supporting the interaction with vWF or alpha-thrombin.

Animals↗
PubMed1994

Disease-avoidance model: factor analysis of common animal fears.

This paper reports the results of two studies investigating a disease-avoidance model of common animal fears. In Study 1, a factor analysis of a variety of self-rated animal fears revealed that these fears can be grouped into two theoretically meaningful categories which correspond to predatory animals and fear-relevant animals. Study 2 found a significant correlation between disgust sensitivity and fear to animals in the fear-relevant category, but not between disgust sensitivity and fear to animals in the predatory category. In addition, fear of animals in the fear-relevant category was significantly associated with scores on the obsessive washing sub-scale of the Maudsley Obsessive-Compulsive Inventory, but was not associated with obsessive behaviour in general.

Adolescent↗
PubMed1994

Casualty risk reduction from safety seat belts in a desert country.

A prospective study was carried out between December 1991 and October 1992 to assess the knowledge, attitudes and practices of hospitalized drivers regarding seat belt usage in United Arab Emirates (UAE). During this period, a total of 800 vehicle drivers were seen and treated in the Accident and Emergency Departments of two general hospitals. A total of 706 (88%) drivers gave consent for the study. The study showed that the rate of constant seat belt usage among drivers was 10.5%, and the rate of frequent seat belt usage was 5.8%. There was a statistically significant difference between seat belting and age groups (p = 0.0051). However, there were no significant statistical differences between seat belt usage versus non usage by sex, nationality and marital status. There were statistically significant differences between user and non-users of seat belts concerning their educational level and occupation. The results showed a reduction in the number of injuries due to usage of seat belts. Those patients who were not wearing seat belts were at risk twice as often as drivers who were restrained by belts. There was a statistically significant difference between the number of persons injured wearing and not wearing seat belts, (p = 0.032). It was observed that the majority of patients stated that seat belts are the best protective measure against all injuries (62.1%) and severe injuries (29.1%) of road traffic accidents. Also, there was strong support for the mandatory use of safety seat belts (56%). We may conclude that these data are encouraging, and would suggest general acceptance of seat belt legislation in the United Arab Emirates.

Accidents, Traffic↗
PubMed1993

Analysis of structure-function relationships in the platelet membrane glycoprotein Ib-binding domain of von Willebrand's factor by expression of deletion mutants.

We have used a series of Escherichia coli-expressed deletion mutants of the glycoprotein (GP) Ib-binding domain of von Willebrand factor (vWF) to study the structural basis of its function. In addition to the prototypic molecule (rvWF441-733), we constructed 11 mutants; seven had deletions of sequence on the amino- and/or carboxyl-terminal side of the Cys509-Cys695 intrachain disulfide loop, and four had limited deletions inside the loop. Other cysteine residues in addition to 509 and 695, when present in the corresponding native sequence, were mutated to glycine; all molecules were purified in the oxidized as well as reduced and alkylated state. The smallest species retaining the ability to interact with GP Ib in the absence of modulators was the oxidized rvWF508-696; the latter, as well as rvWF441-696, became inactive after reduction and alkylation. In contrast, all the other fragments with deletions outside of the loop, but extending at least to residue 700, showed better binding to platelets after reduction and alkylation than when the Cys509-Cys695 disulfide bond was oxidized. Any limited deletion of sequence inside the loop caused complete loss of GP Ib-binding function both in the absence or in the presence of botrocetin, and this persisted even after reduction and alkylation. In contrast, all mutants with intact sequence between residues 509 and 695 bound to GP Ib in the presence of botrocetin, regardless of whether the 2 cysteine residues were oxidized or reduced and alkylated. Ristocetin, unlike botrocetin, appeared to have no effect in modulating the binding of any of the expressed fragments to platelets. Our findings suggest that the GP Ib-binding domain of vWF contains multiple interaction sites, but integrity of the sequence 509-695 is important for function.

Amino Acid Sequence↗
PubMed1993

Expression of human platelet glycoprotein Ib alpha in transgenic mice.

Platelets are cytoplasmic fragments of megakaryocytes and, therefore, their membrane proteins cannot be manipulated by expression methods in culture. To overcome this limitation, we have expressed human glycoprotein (GP) Ib alpha in transgenic mouse megakaryocytes and found that it was present on the surface of platelets associated with the mouse GP Ib beta subunit. This finding demonstrates that assembly of the heterooligomeric GP Ib complex occurs through mechanisms conserved across species. In contrast, the receptor function of GP Ib exhibited restricted species specificity, since only the chimeric complex containing human GP Ib alpha bound human von Willebrand factor and supported platelet aggregation mediated by ristocetin. These studies demonstrate the transgenic engineering of a platelet adhesion receptor under control of the human GP Ib alpha promoter and illustrate a new approach to manipulate platelet receptors and study structure-function relationships in hemostasis and thrombosis.

Animals↗
PubMed1993

Disulfide bond requirements for assembly of the platelet glycoprotein Ib-binding domain of von Willebrand factor.

von Willebrand factor (vWF) is a multimeric glycoprotein of plasma and the subendothelial matrix that interacts with specific platelet receptors to establish platelet adhesion at a site of vascular injury. The vWF domain containing the platelet receptor glycoprotein Ib-binding site can be expressed in heterologous cells as a recombinant homodimeric fragment that mediates platelet-platelet interaction, analogous to multimeric vWF. The recombinant domain, r116, contains 7 Cys residues within its 290-residue monomeric subunit paired in an unidentified intra- and intermolecular disulfide bond arrangement. In this report we define the disulfide bond-dependent framework of r116 that provides the domain with its essential structural features that support dimer formation and the generation of a disulfide bond-dependent epitope. The results demonstrate that a triplet of Cys residues at positions 459, 462, and 464 are essential for efficient dimer formation. An intramolecular Cys509/Cys695 disulfide loop is required for generating a functional dimeric molecule, and monomeric molecules containing a Cys509/Cys695 intramolecular disulfide bond are unable to support ristocetin-mediated platelet aggregation. The disulfide arrangement in r116 is similar, if not identical, to the proposed arrangement within the corresponding region of plasma vWF, and these studies document the inherent Cys-dependent maturation of an isolated vWF domain.

Disulfides↗
PubMed1993

von Willebrand factor.

von Willebrand factor is a multimeric glycoprotein essential for the normal arrest of bleeding after tissue injury (hemostasis). The molecule is present in blood, both in plasma and inside platelets, as well as in endothelial cells and the subendothelial matrix of the vessel wall. Through multiple functional domains, von Willebrand factor mediates the attachment of platelets to exposed tissues, where discontinuity of the vascular endothelium occurs, and the subsequent platelet aggregation leading to the formation of platelet thrombi. The crucial role of von Willebrand factor in platelet function is particularly apparent when hemodynamic conditions create blood flow with high shear stress, as in capillaries under physiologic conditions or in stenosed and partially occluded arteries in disease states. The involvement of von Willebrand factor in the processes that lead to acute thrombosis has attracted considerable interest on the molecular and functional biology of the protein. Indeed, understanding the mechanisms and structural bases for von Willebrand factor function may result in new and effective approaches to anti-thrombotic intervention.

Amino Acid Sequence↗
PubMed1993

A role for von Willebrand factor proline residues 702-704 in ristocetin-mediated binding to platelet glycoprotein Ib.

Mutant domains of von Willebrand factor (vWF) were constructed to determine the effects of altering net charge, and presumably conformation, within a peptide sequence (residues 694-708) previously shown to be involved in the platelet receptor glycoprotein (GP) Ib binding function of vWF. Non-conservative substitutions replaced a triplet of proline residues (proline702-704) with either a triplet of arginine (positively-charged) or aspartic acid (negatively-charged) residues. After establishing stable CHO cell transformants, we observed the secretion of covalently-linked dimeric molecules analogous to a domain with native sequence. Functional assays using immunopurified molecules revealed that the ristocetin-dependent binding to GP Ib was abolished with both charge mutants. However, in the absence of disulfide-bond dependent conformation both mutant molecules and the molecule with native sequence interacted with GP Ib. The results demonstrate that vWF proline702-704 are important for the ristocetin-mediated interaction between vWF and GP Ib, but are not essential residues of the GP Ib binding site within vWF.

Amino Acid Sequence↗
PubMed1993

Expression of the phenotypic abnormality of platelet-type von Willebrand disease in a recombinant glycoprotein Ib alpha fragment.

The platelet GP Ib-IX receptor supports platelet adhesion and activation by binding to vWf in the exposed subendothelial matrix. An abnormal GP Ib-IX complex exists in platelet-type or pseudo-von Willebrand disease and has a characteristic increased affinity for soluble vWf resulting in impaired hemostatic function due to the removal of larger vWf multimers from the circulation. Genetic studies within an afflicted family have demonstrated that the disease is linked to a Gly233-->Val amino acid substitution within the alpha-subunit of the oligomeric GP Ib-IX complex (Miller, J.L., D. Cunningham, V.A. Lyle, and C. L. Finch. 1991. Proc. Natl. Acad. Sci. USA. 88:4761-4765). To evaluate the functional consequences of this mutation, we constructed a recombinant analogue of the alpha-subunit of GP Ib containing Val233. Experiments comparing molecules with either Gly233 or Val233 revealed that the Val substitution generates a molecule with increased affinity for vWf. The recombinant fragment reproduces the functional abnormality of the GP Ib-IX complex in platelet-type von Willebrand disease, thus establishing the molecular basis of the bleeding disorder within this family. Moreover, it becomes apparent that structural elements responsible for the regulation of hemostasis through modulation of vWf affinity for platelets reside within the alpha-subunit of the GP Ib-IX complex.

Amino Acid Sequence↗
PubMed1993

von Willebrand factor mutation enhancing interaction with platelets in patients with normal multimeric structure.

Variant von Willebrand disease designated as type I New York or type Malmö is characterized by enhanced ristocetin-induced platelet agglutination with normal von Willebrand factor multimeric distribution in plasma. We have studied four such patients belonging to three unrelated families and found in all of them a unique cytosine-to-thymine transition changing the codon for Pro503 (CCG) to Leu (CTG). In three patients the mutant allele also had a silent mutation in the codon for Ser500 (TCG-->TCA). Both nucleotide changes are present in the von Willebrand factor pseudogene; however, the characterization of distinctive markers where the gene and pseudogene differ, as well as the examination of amplified cDNA derived from platelet mRNA, confirmed that the abnormality occurs in the von Willebrand factor gene of the patients. Moreover, recombinant expression of the isolated glycoprotein Ib-binding domain of von Willebrand factor provided direct evidence that the Pro503-->Leu mutation is responsible for enhanced platelet reactivity to lower ristocetin concentrations. These results define a new structural element affecting the affinity of von Willebrand factor for glycoprotein Ib and establish the molecular basis of a variant form of von Willebrand disease.

Adult↗
PubMed1993

Point mutation in a leucine-rich repeat of platelet glycoprotein Ib alpha resulting in the Bernard-Soulier syndrome.

Leucine-rich repeats are a conserved structural motif, of yet undefined significance, found in a group of proteins from different species. Among these are the four components of the human platelet glycoprotein Ib-IX-V complex, a membrane receptor that performs an essential role in the thrombogenic function of platelets by interacting with the adhesive protein, von Willebrand factor. We have found that a single amino acid substitution (Ala156-->Val) within one of the six leucine-rich repeats in the alpha-subunit of glycoprotein Ib results in a variant form of the congenital bleeding disorder, Bernard-Soulier syndrome, characterized by giant dysfunctional platelets. Genetic studies of the propositus and his family members were complemented by immunological and functional analysis of expressed recombinant GP Ib alpha fragments to demonstrate that the observed mutation is the cause of defective von Willebrand factor binding. These studies define the molecular basis of the Bernard-Soulier syndrome within this family and demonstrate that structural integrity of a leucine-rich repeat is necessary for normal function of the glycoprotein Ib-IX-V receptor complex and, possibly, for normal platelet morphology.

Amino Acid Sequence↗
PubMed1992

Genetic and structural characterization of an amino acid dimorphism in glycoprotein Ib alpha involved in platelet transfusion refractoriness.

The platelet-specific alloantigen, Siba, located within the alpha-subunit of the glycoprotein (GP) Ib-IX membrane receptor, has been found to be involved in the pathogenesis of platelet transfusion refractoriness. We have identified the existence of a naturally occurring threonine/methionine dimorphism at position 145 of the GPIb alpha sequence, and determined that the Siba antigen corresponds to the molecule containing methionine145. The diallelic codons can be detected by restriction enzyme analysis of amplified genomic DNA fragments from the GPIb alpha gene. Evaluation of 61 healthy blood donors showed that the allele frequencies are 89% and 11% for the threonine145 and methionine145 codons, respectively. A positive correlation exists between platelet reactivity with the anti-Siba antibody and the presence of a methionine145-encoding allele. Moreover, recombinant expression of two soluble GPIb alpha fragments differing only at residue 145, provided definitive evidence that the human anti-Siba antibody reacts only with the molecule containing methionine145. These results explain the structural basis of the Siba human alloantigen system and define screening methodologies useful in transfusion medicine to match donor and recipient platelets accordingly.

Amino Acids↗
PubMed1992

Epitope mapping of human factor VIII inhibitor antibodies by site-directed mutagenesis of a factor VIII polypeptide.

Previous epitope mapping studies of human factor VIII (FVIII) inhibitor antibodies with heavy chain specificity localized epitopes to the amino-terminal half of the FVIII A2 domain. In this report we have used unidirectional deletion analysis and site-directed mutagenesis to identify a minimum length polypeptide and amino acid residues that contribute to the FVIII conformation recognized by these antibodies. Bacterial expression plasmids were exploited to demonstrate that a FVIII polypeptide of approximately 150 residues is required to generate a common heavy chain epitope(s). Another series of plasmids were constructed that synthesize: a FVIII polypeptide containing an internal deletion; four polypeptides with single residue substitutions; two polypeptides with triple residue changes; and a quadruple amino acid replacement within one polypeptide. The relative reactivities of the wild-type and mutant FVIII polypeptides were tested by immunoblotting, inhibitor neutralization assays and ELISA with a variety of human FVIII inhibitor auto- and alloantibodies. These techniques illustrate that the internal deletion mutant and one of the relatively conservative amino acid substitution triple mutants, mutant 389, resulted in significantly decreased immunoreactivity. The data identify FVIII Glu389,390,391 as three critical components of an epitope for human FVIII inhibitor antibodies and identify a major inhibitory epitope involved in the immune response to FVIII.

Amino Acid Sequence↗
PubMed1992

Measurements of hepatic malondialdehyde and conjugated diene concentrations in 24-hour endotoxemic rats.

The aim of our study was to analyze if intraperitoneally administered E-coli endotoxin (1-7 mg.kg-1) causes an increase in liver malondialdehyde (MDA) and conjugated dienes, 24 h postinjection. Blood glucose, white blood cell counts, liver edema and mortality were also measured. The results of our study show that there were no signs of increased MDA or diene production in the liver. To our surprise, blood leukocyte counts of the experimental groups were not statistically different from controls. In the endotoxemic animals there was a slight liver edema which, however, did not influence the measurements of the lipid peroxidation metabolites. Blood glucose levels were decreased in the endotoxemic animals. There was no mortality in our study, perhaps due to the fact that the rats were fed and not starved. We conclude that, in our study, a bolus dose of endotoxin i.p. does not cause any signs of increased liver lipid peroxidation 24 h postinjection. The liver seems, however, to be affected with slight edema and low blood glucose levels.

Animals↗
PubMed1992

Evidence for gene-environmental interactions in Utah families with hypertension, dyslipidaemia and early coronary heart disease.

1. Among 45,258 Utah families surveyed, about 4% have a strong aggregation of early coronary disease. In detailed clinical evaluation, about 21% of such high risk coronary families were found to have familial dyslipidaemic hypertension (FDH) and about 3% were found to have heterozygous familial hypercholesterolaemia (hFH). 2. Common and potentially modifiable environmental factors seem to play an important role in these high risk families. Non-genetic obesity promotes the expression of FDH. A high fat diet promotes the expression of FH. Cigarette smoking promotes earlier death in all coronary prone families. 3. Practical approaches are suggested for helping coronary prone pedigrees by applying our understanding of genetic and environmental factors that promote earlier coronary disease onset.

Adult↗
PubMed1992

Prevention of familial cardiovascular disease by screening for family history and lipids in youths.

We analyzed medical family history information from 51,053 families of high school students in Utah and Texas and cholesterol measurements from 853 youths and 1618 adults in Utah families with cardiovascular disease (CVD) to assess the utility of different approaches to risk-factor evaluation for youths. The major question addressed was in which youths should blood cholesterol be tested? Applying National Cholesterol Education Program recommendations suggested that 36% in Utah and 38% in Texas be tested. Heterozygous familial hypercholesterolemia (hFH) is the best documented and most serious cholesterol disorder readily diagnosed in youths. In Utah families ascertained for CVD in adults, blood cholesterol levels among youths were significantly bimodal with hFH present in 84% of youths in the upper cholesterol mode. Blood cholesterol levels in adults from the same families were less bimodal with hFH present in 38% of adults in the upper mode. More overlap existed between high and normal modes in adults than in youths. Data from this study suggest that family histories and cholesterol concentrations obtained from high school students may meet the needs of cholesterol screening, education, and follow-up in a controlled and cost-effective setting.

Adolescent↗