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Biomedical subjects

K J Norton

Publications and source records attributed to K J Norton.

18 recordsLinked to original sources
PubMed1997

Collecting data on potentially harmful events: a method for monitoring incidents in general practice.

OBJECTIVE: The prediction and subsequent prevention of errors, which are an integral element of human behaviour, require an understanding of their cause. The incident monitoring technique was developed in the study of aviation errors in the Second World War and has been applied more recently in the field of anaesthetics. This pilot study represents one of the first attempts to apply the incident monitoring technique in the general practice environment. METHOD: A total of 297 GPs across Australia anonymously reported details of unintended events which harmed or could have harmed the patient. Reports were contemporaneously recorded on prepared forms which allowed a free text description of the incident, and structured responses for contributing and mitigating factors, immediate and long-term out-comes, additional costs etc. RESULTS AND DISCUSSION: The first 500 reports were analysed using both of qualitative and quantitative methods and a brief overview of results is presented. The methodological issues arising in the application of this technique to such a large, widely spread profession, in which episodes of care are not necessarily confined to a single consultation, are discussed. This study demonstrated that the incident monitoring technique can be successfully applied in general practice and that the resulting information can facilitate the identification of common factors contributing to such events and allow the development of preventive interventions.

Australia↗
PubMed1996

Tests and investigations. Indicators for better utilisation.

Diagnostic tests and investigations are standard tools for today's general practitioner. However, without reliable systems for the management of tests and test results, these valuable assets may become a liability to the GP, with the possibility of unforeseen adverse events arising at several points in the total investigative process. Exploring GPs' reports of such incidents may provide opportunities for improvements in patient care.

Communication↗
PubMed1993

Immunologic analysis of the cloned platelet thrombin receptor activation mechanism: evidence supporting receptor cleavage, release of the N-terminal peptide, and insertion of the tethered ligand into a protected environment.

The recently cloned functional thrombin receptor is thought to be activated by thrombin cleavage of the bond between R41 and S42, followed by the insertion of the new N-terminal region ("tethered ligand") into an unknown site in the receptor. Antibodies to peptides at or near the cleavage site have been reported to inhibit thrombin-induced platelet activation to varying extents, but the precise mechanism(s) of their inhibition is unknown. We have produced: (1) a polyclonal antibody in rabbits to a peptide containing amino acids 34 to 52 (anti-TR34-52); enzyme-linked immunosorbent assays (ELISA) indicate that anti-TR34-52 contains antibodies to regions on both sides of the thrombin cleavage site; (2) two murine monoclonal antibodies (MoAbs) to a peptide containing amino acids 29 to 68; one antibody reacts primarily with residues N-terminal to the thrombin cleavage site, and the other reacts primarily with residues C-terminal to the cleavage site; and (3) a polyclonal rabbit antibody to a peptide containing amino acids 83 to 94 (anti-TR83-94). Anti-TR34-52 binds to platelets as judged by flow cytometry, and pretreating platelets with a thrombin receptor peptide ligand does not lead to loss of antibody reactivity, suggesting that platelet activation does not initiate redistribution or internalization of surface thrombin receptors. In contrast, pretreating platelets with thrombin leads to complete loss of anti-TR34-52 binding. Similarly, the binding of both MoAbs to platelets is dramatically reduced by pretreatment with thrombin. However, the binding of anti-TR83-94 is not decreased by thrombin activation, confirming that the receptor is not internalized. Anti-TR34-52 profoundly inhibits low dose thrombin-induced platelet shape change and aggregation, but the inhibition can be overcome with higher thrombin doses. However, anti-TR34-52 does not inhibit platelet aggregation induced by tethered ligand peptides. The TR34-52 peptide is a thrombin substrate, with cleavage occurring at the R41-S42 bond as judged by high performance liquid chromatography (HPLC) and platelet aggregation analysis. Anti-TR34-52 prevented cleavage of the TR34-52 peptide, suggesting that the antibody prevents platelet activation, at least in part, by preventing cleavage of the thrombin receptor. These data, although indirect, provide additional support for a thrombin activation mechanism involving thrombin cleavage of the receptor; in addition, they provide new evidence indicating that receptor cleavage is followed by loss of the N-terminal peptide, and insertion of the tethered ligand into a protected domain.

Amino Acid Sequence↗
PubMed1993

The thrombin receptor extracellular domain contains sites crucial for peptide ligand-induced activation.

A thrombin receptor (TR) demonstrating a unique activation mechanism has recently been isolated from a megakaryocytic (Dami) cell line. To further study determinants of peptide ligand-mediated activation phenomenon, we have isolated, cloned, and stably expressed the identical receptor from a human umbilical vein endothelial cell (HUVEC) library. Chinese hamster ovary (CHO) cells expressing a functional TR (CHO-TR), platelets, and HUVECs were then used to specifically characterize alpha-thrombin- and peptide ligand-induced activation responses using two different antibodies: anti-TR34-52 directed against a 20-amino acid peptide spanning the thrombin cleavage site, and anti-TR1-160 generated against the NH2-terminal 160 amino acids of the TR expressed as a chimeric protein in Escherichia coli. Activation-dependent responses to both alpha-thrombin (10 nM) and peptide ligand (20 microM) were studied using fura 2-loaded cells and microspectrofluorimetry. Whereas preincubation of CHO-TR with anti-TR34-52 abolished only alpha-thrombin-induced [Ca2+]i transients, preincubation with anti-TR1-160 abrogated both alpha-thrombin- and peptide ligand-induced responses. This latter inhibitory effect was dose dependent and similar for both agonists, with an EC50 of approximately 90 micrograms/ml. Anti-TR1-160 similarly abolished peptide ligand-induced [Ca2+]i transients in platelets and HUVECs, whereas qualitatively different responses characterized by delayed but sustained elevations in [Ca2+]i transients were evident using alpha-thrombin. Platelet aggregation to low concentrations of both ligands was nearly abolished by anti-TR1-160, although some shape change remained; anti-TR34-52 only inhibited alpha-thrombin-induced aggregation. These data establish that a critical recognition sequence for peptide ligand-mediated receptor activation is contained on the NH2-terminal portion of the receptor, upstream from the first transmembrane domain. Furthermore, alpha-thrombin-induced activation of HUVECs and platelets may be partially mediated by an alternative mechanism(s) or receptor(s).

Animals↗
PubMed1992

Thromboerythrocytes. In vitro studies of a potential autologous, semi-artificial alternative to platelet transfusions.

In an attempt to overcome the limitations and drawbacks of using fresh platelets for transfusion therapy of thrombocytopenic patients, we have performed in vitro experiments on an autologous, semi-artificial alternative to platelet transfusions. Based on our previous studies of the interactions of unactivated and activated platelets with beads coated with peptides of various lengths, all of which contained the arginine-glycine-aspartic acid (RGD) cell recognition sequence, the peptide Ac-CGGRGDF-NH2 was chosen for covalent coupling to erythrocytes. A heterobifunctional crosslinking reagent (N-maleimido-6-aminocaproyl ester of 1-hydroxy-2-nitrobenzene-4-sulfonic acid) was used to crosslink via the peptide's free sulfhydryl group and the erythrocyte's surface amino groups. Approximately 0.5-1.5 x 10(6) peptide molecules bound per erythrocyte after 2 h of incubation, and most of the peptides appeared to crosslink to glycophorin A. The resulting cells, termed thromboerythrocytes, interacted selectively with activated platelets to form mixed aggregates. Studies with fluid phase RGD peptides and monoclonal antibodies indicated that the RGD peptides on the thromboerythrocytes interacted with the GPIIb/IIIa receptors on activated platelets. Thromboerythrocytes could also bind to platelets adherent to collagen. There was minimal erythrocyte hemolysis during the formation of thromboerythrocytes and studies of thromboerythrocyte osmotic fragility and cellular deformability showed no significant changes from control erythrocytes. Whereas there is a 20:1 ratio of erythrocytes to platelets in the circulation of normal individuals, the erythrocytes from as little as 50 ml of blood could be transformed into the equivalent of 2 U of platelets by numbers (equivalent to 18 U of platelets by mass), and reinfused into the same individual within several hours. These data encourage us to proceed to in vivo studies to assess the hemostatic efficacy of thromboerythrocytes in thrombocytopenic animals.

Amino Acid Sequence↗
PubMed1991

Platelet fibrinogen and vitronectin in Glanzmann thrombasthenia: evidence consistent with specific roles for glycoprotein IIb/IIIA and alpha v beta 3 integrins in platelet protein trafficking.

To assess the individual contributions of the platelet glycoprotein (GP) IIb/IIIa receptor and the alpha v beta 3 vitronectin receptor to platelet levels of fibrinogen and vitronectin, we analyzed the platelets from two groups of Glanzmann thrombasthenic patients: Iraqi-Jews, whose platelets lack both receptors, and Arab patients in Israel, whose platelets lack GPIIb/IIIa, but have normal or increased numbers of alpha v beta 3 vitronectin receptors. The platelets from both thrombasthenic groups had profound deficiencies of fibrinogen, but the defect in the Iraqi-Jewish patients' platelets appeared to be slightly more severe. This finding indicates that GPIIb/IIIa is the major determinant of platelet fibrinogen, presumably acting by receptor-mediated uptake, and that the alpha v beta 3 vitronectin receptor plays little or no role. Arab patients' platelets have normal amounts of platelet vitronectin, whereas Iraqi-Jewish patients' platelets have nearly five times as much vitronectin as control or Arab patients' platelets. To account for these data, we propose a working hypothesis in which vitronectin is synthesized in megakaryocytes and the alpha v beta 3 vitronectin receptor is involved in transport of the protein out of megakaryocytes and/or platelets. Collectively, these observations suggest that in addition to their recognized roles in cell adhesion and in the interaction of cells with extracellular proteins, integrin receptors may be important in protein trafficking into, and perhaps out of, platelets.

Blood Platelets↗
PubMed1989

Structural and functional characterization of major platelet membrane components derived by limited proteolysis of glycoprotein IIIa.

The authors isolated a product of proteolytic degradation of glycoprotein IIIa (GPIIIa) which is formed on the surface of human platelets during incubation with chymotrypsin and which was previously described as the 66 kDa platelet membrane component. This component migrated with an apparent Mr 62,400 in a non-reduced system of sodium dodecyl sulfate polyacrylamide gel electrophoresis. In a reduced system it yielded two major subunits migrating with apparent Mr 14,000-17,000 and 65,000. The low-molecular weight component began with the NH2-terminal sequence of GPIIIa (GPNICTTR...) and the larger component with residue 348 of GPIIIa (GKIRSKKA...) as deduced from a cDNA clone of this glycoprotein. The two subunits appeared to be linked by one or more S-S bridges supporting the contention that GPIIIa is a highly folded molecule on the platelet membrane. In contrast to GPIIIa, the '66 kDa component' did not bind to GRGDSPK-agarose, to fibrinogen-agarose nor to insolubilized monoclonal antibody recognizing the GPIIb/IIIa complex. The exposure of fibrinogen receptors during the course of incubation of platelets with chymotrypsin preceded the formation of the '66 kDa component' characterized in this study. An intermediate product of GPIIIa proteolysis migrating with an apparent Mr 120,000 in a non-reduced system and Mr 80,000 in a reduced system was identified as a precursor of the '66 kDa component'. The '120 kDa component' was not retained on GRGDSPK-agarose or on fibrinogen-agarose but it was retained on insolubilized antibody recognizing the GPIIb/IIIa complex. Incubation of platelets with porcine pancreatic elastase or human granulocytic elastase resulted in the formation of similar proteolytic degradation fragments.

Amino Acid Sequence↗
PubMed1989

High molecular weight kininogen inhibits fibrinogen binding to cytoadhesins of neutrophils and platelets.

Fibrinogen inhibited 125I-high molecular weight kininogen (HMWK) binding and displaced bound 125I-HMWK from neutrophils. Studies were performed to determine whether fibrinogen could bind to human neutrophils and to describe the HMWK-fibrinogen interaction on cellular surfaces. At 4 degrees C, the binding of 125I-fibrinogen to neutrophils reached a plateau by 30 min and did not decrease. At 23 and 37 degrees C, the amount of 125I-fibrinogen bound peaked by 4 min and then decreased over time because of proteolysis of fibrinogen by human neutrophil elastase (HNE). Zn++ (50 microM) was required for binding of 125I-fibrinogen to neutrophils at 4 degrees C and the addition of Ca++ (2 mM) increased the binding twofold. Excess unlabeled fibrinogen or HMWK completely inhibited binding of 125I-fibrinogen. Fibronectin degradation products (FNDP) partially inhibited binding, but prekallikrein and factor XII did not. The binding of 125I-fibrinogen at 4 degrees C was reversible with a 50-fold molar excess of fibrinogen or HMWK. Binding of 125I-fibrinogen, at a concentration range of 5-200 micrograms/ml of added radioligand, was saturable with an apparent Kd of 0.17 microM and 140,000 sites/cell. The binding of 125I-fibrinogen to neutrophils was not inhibited by the peptide RGDS derived from the alpha chain of fibrinogen or by the mAb 10E5 to the platelet glycoprotein IIb/IIIa heterodimer. Fibrinogen binding was inhibited by a gamma-chain peptide CYGHHLGGAKQAGDV and by mAb OKM1 but was not inhibited by OKM10, an mAb to a different domain of the adhesion glycoprotein Mac-1 (complement receptor type 3 [CR3]). HMWK binding to neutrophils was not inhibited by OKM1. These observations were consistent with a further finding that fibrinogen is a noncompetitive inhibitor of 125I-HMWK binding to neutrophils. Fibrinogen binding to ADP-stimulated platelets was increased twofold by Zn++ (50 microM) and was inhibited by HMWK. These studies indicate that fibrinogen specifically binds to the C3R receptor on the neutrophil surface through the carboxy terminal of the gamma-chain and that HMWK interferes with the binding of fibrinogen to integrins on both neutrophils and activated platelets.

Antibodies, Monoclonal↗
PubMed1989

The effects of a topical PGE2 analogue on global flap ischemia in rats.

The present study evaluated the ability of DHV-PGE2ME, a topically effective 16-vinyl prostaglandin E2 analogue, to improve the tolerance of skin flaps to a period of ischemia. DHV-PGE2ME and placebo were applied to bilateral island flaps on 70 anesthetized rats; then the vascular pedicle of each flap was clamped for 10 hours. Treated flaps evidenced significantly better reperfusion, as documented by quantification of fluorescein dye delivery at 90 minutes after clamp release, and they had significantly greater ultimate viability (p less than 0.05, by ANOVA). While less than 3 percent of untreated flaps survived, those treated with 1.75 and 17.5 microgram/cm2 of drug evidenced 76 and 86 percent survival, respectively. Treatment of a given flap did not affect its contralateral mate, since there was no evidence of a systemic effect. Especially since its effect can be limited to the site of application, DHV-PGE2ME should be valuable for the treatment of compromised perfusion in a variety of settings.

Administration, Topical↗
PubMed1988

Fluorescence assessment of skin perfusion after oral fluorescein.

This study evaluated oral ingestion as the route of fluorescein administration for fluorescence assessment of skin perfusion. In 10 anesthetized rats, dorsal pedicle flaps were raised to produce graded perfusion; then fluorescein (7.5 mg/kg) was administered via an oropharyngeal tube. Quantification of skin fluorescence, performed with the fiberoptic fluorometer 45 minutes after dye ingestion, delineated a significant difference between the perfusion of flap regions that remained viable and those that subsequently became dystrophic (p less than 0.05). The precision was equivalent to that previously reported after intravenous dye. In addition, the gradual delivery of dye after oral administration permitted monitoring of slope of uptake and time to peak. Each of these pharmacokinetic parameters delineated a significant difference between viable and nonviable sections. As measures of relative change in fluorescence, they are independent of skin color and thickness. This feature suggests that the oral route actually may improve the accuracy and reliability of the fluorescein test.

Administration, Oral↗
PubMed1987

Fluorometric quantification of low-dose fluorescein delivery to predict amputation site healing.

This retrospective study evaluated quantification of skin fluorescein delivery by fiberoptic fluorometry as a means of predicting the healing potential of an amputation site. Fluorometry uses a dual-channel fiberoptic light guide--one channel transmits blue light to excite the fluorescein in the skin under study, and the other transmits emitted fluorescence from the skin to a photomultiplier tube where it is measured. Ten minutes after intravenous administration of sodium fluorescein (4 to 8 mg/kg), fluorometric readings were obtained at more than 100 reading sites. In the 86 cases without preoperative cellulitis at the site of amputation, preoperative fluorometry clearly distinguished between healing and nonhealing sites. Healing sites averaged 79% of the fluorescence of a healthy reference area (dye fluorescence index [DFI] = 79), while failing sites averaged only 27% (p less than 0.01 by ANOVA). In all but one case where the DFI was greater than 42, the amputation healed. In all cases where the DFI was less than 38, the amputation failed. In general, uncertainty was limited to sites with values between these limits. The technique maintained its high accuracy in patients with diabetes and for distal amputations. However, it was not accurate at sites of active cellulitis (12 cases). There were no significant adverse effects from the slow injection of the low dose of fluorescein used for this technique. We conclude that fluorometry is an effective means of predicting healing in patients undergoing amputation.

Amputation, Surgical↗
PubMed1985

Comparative assessment of blood flow to canine island flaps.

Fluorometric quantification of dye delivery, laser Doppler flowmetry, and transcutaneous measurement of tissue oxygen level were evaluated in canine island flaps subjected to vascular occlusion. Each technique readily identified the clamping insult. Relative fluorescein delivery to the flap (compared with a reference area) decreased from 66% before clamping to almost zero during clamping. Laser Doppler readings rapidly declined from an average of 230 to an average of 31 mV. Oxygen readings rapidly declined from 57 to 1 mm Hg. Laser Doppler flowmetry and transcutaneous oxygen monitoring seem best suited for continuous monitoring of trends at a single site. Fluorometry is more precise and can be used to monitor multiple sites. One should weigh the benefits of this technique against the need for dye injection.

Animals↗
PubMed1985

Fluorometric prediction of successful amputation level in the ischemic limb.

The present study was undertaken to compare fluorometric documentation of fluorescein dye delivery with the standard means of determining the level at which an amputation should be performed in the dysvascular extremity. Thirty-nine patients underwent lower-extremity amputation at the level determined by the surgeon based upon physical examination, angiography, segmental pressure indices, and/or pulse volume recordings. In addition, fiberoptic fluorometry was performed preoperatively. After intravenous administration of sodium fluorescein (4-8 mg/kg), fluorometric readings were obtained by placing the fluorometer's light guide on 126 reading sites. Fluorometric findings were evaluated retrospectively, and therefore did not influence the surgeon's decision. Of the 39 amputations performed overall, only 26 healed. The accuracy of the standard criteria was lowest for the 20 below-ankle amputations, where only 12 cases healed. Alternatively, fluorometric indices separated healing from nonhealing sites in 36 of the 39 cases and in 18 of the 20 below-ankle amputations. Overall, healing sites averaged 94 percent of the fluorescence of the healthy reference area, while nonhealing sites averaged only 29 percent. We conclude that fluorometry should prove to be a valuable adjunct in the assessment of the dysvascular extremity. It uses a low dose of dye, is easy to perform, and is readily repeatable.

Amputation, Surgical↗
PubMed1985

Serial fluorometric documentation of fluorescein dye delivery.

Since its introduction in 1980, fiberoptic fluorometry has improved the precision and objectivity of the fluorescein test as a means to delineate nutritive blood flow and predict tissue viability. This study was undertaken primarily to determine if fiberoptic fluorometry could be repeated reliably at close time intervals. Four injections of fluorescein dye were administered at varying dose and time schedules to determine what factors influenced the repeatability of this technique. The results confirm the high accuracy of fluorometric indices as a means to predict the ultimate fate of a skin flap. A method of scanning the flap within 2 minutes after injection of 1.5 mg/kg of fluorescein dye correctly predicted the fate of almost 100% of flap sections. Repeated injections of 1.5, 3.0, and 6.0 mg/kg of dye at 15-minute intervals provided virtually identical predictions of flap viability. Thus fluorometry is readily repeatable and should be of value in assessing the state of tissue perfusion as well as changes in perfusion caused by pharmacologic manipulation or surgical intervention.

Animals↗
PubMed1984

Assessment of delay in bilateral thoracoabdominal island flaps in the rat.

This study demonstrates improved perfusion and viability following surgical delay in a bilateral paired flap model that minimizes the effects of systemic variables in the rat. Surgical delay was achieved in one of two paired thoracoabdominal island flaps by ligating and cutting the inferior epigastric (distal) pedicle three days before flap construction. This created a hypoperfused zone. Within three days of delay, perfusion of the delayed region, as documented by quantification of tissue fluorescein delivery, increased markedly and often exceeded nondelayed values. Construction of 2 X 11-cm island flaps based on the superior epigastric pedicle at this time was associated with an average of 1.1-cm necrosis, compared with 2.6 cm in nondelayed flaps.

Abdomen↗
PubMed1984

Vascular island skin-flap tolerance to warm ischemia: an analysis by perfusion fluorometry.

Fiberoptic perfusion fluorometry and assessment of ultimate viability were used to analyze the tolerance to warm ischemia of rat vascular island skin flaps. Both acute flaps and flaps raised 24 hours previously and then reraised were subjected to 0 (control), 6, 8, 10, and 12 hours of vascular pedicle clamping. Following clamp release, serial fluorometry documented the progressive delay in effective reflow resulting from extended periods of ischemia. Fluorometry, furthermore , suggested that flaps constructed 24 hours previously had an improved hemodynamic status with a significantly reduced period of poor reflow following clamp release. The improved hemodynamics were associated with increased viability, confirming the increased tolerance of 24-hour-old flaps to warm ischemia.

Animals↗