Do Y2K glitches deserve liability caps?
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Chronology as Topic↗
Biomedical subjects
K Fickenscher
Publications and source records attributed to K Fickenscher.
Coagulation factor XIIIa undergoes a conformational change evoked by glutamine substrate. Studies on kinetics of inhibition and binding of XIIIA by a cross-reacting antifibrinogen antibody.
Coagulation factor XIIIa, plasma transglutaminase (endo-gamma-glutamine:epsilon-lysine transferase EC 2.3.2.13) catalyzes isopeptide bond formation between glutamine and lysine residues and rapidly cross-links fibrin clots. A monoclonal antibody (5A2) directed to a fibrinogen Aalpha-chain segment 529-539 was previously observed from analysis of end-stage plasma clots to block fibrin alpha-chain cross-linking. This prompted the study of its effect on nonfibrinogen substrates, with the prospect that 5A2 was inhibiting XIIIa directly. It inhibited XIIIa-catalyzed incorporation of the amine donor substrate dansylcadaverine into the glutamine acceptor dimethylcasein in an uncompetitive manner with respect to dimethylcasein utilization and competitively with respect to dansylcadaverine. Uncompetitive inhibition was also observed with the synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetitive inhibition arises from preferential interaction of the inhibitor with the enzyme-substrate complex but is also found to inhibit gamma-chain cross-linking. The conjunction of the uncompetitive and competitive modes of inhibition indicates in theory that this bireactant system involves an ordered reaction in which docking of the glutamine substrate precedes the amine exchange. The presence of substrate enhanced binding of 5A2 to XIIIa, an interaction deemed to occur through a C-terminal segment of the XIIIa A-chain (643-658, GSDMTVTVQFTNPLKE), 55% of which comprises sequences occurring in the fibrinogen epitope Aalpha-(529-540) (GSESGIFTNTKE). Removal of the C-terminal domain from XIIIa abolishes the inhibitory effect of 5A2 on activity. Crystallographic studies on recombinant XIIIa place the segment 643-658 in the region of the groove through which glutamine substrates access the active site and have predicted that for catalysis, a conformational change may accompany glutamine-substrate binding. The uncompetitive inhibition and the substrate-dependent binding of 5A2 provide evidence for the conformational change.
Amino Acid Sequence↗
Lessons from the trenches in physician integration.
Beyond the theoretical basis for integration, three core considerations stand out as the primary reasons for pursuing integration from a physician's perspective. In the authors' experience, the ability to make a case for physician integration stands or falls based on the ability of the integrated delivery system to address these considerations: Gain greater access to capital; develop human resources with talents in managed care and the full spectrum of care services; and sustain an information infrastructure. This article explores the lessons learned in pursuing physician integration.
Community Networks↗
Coagulation assay with improved specificity to factor V mutants insensitive to activated protein C.
The prevalence of a new hereditary defect in the protein C anticoagulant pathway, the factor V-Leiden, has been reported to range between 20% to 60% in familial thrombophilia. In addition to differences in patient groups, these very divergent numbers might also be due to the detection method applied. In most studies a modified APTT was used, where activated protein C (APC) is added simultaneously with the start of the clotting reaction. However, this method is also influenced by other factors like protein S, factor VIII or lupus anticoagulants. Furthermore, heparin or oral anticoagulant therapy might interfere. We tried to develop a coagulation assay dependent only on those mutant forms of factor V stable against proteolytic attack by APC. For this purpose, samples were first diluted with a factor V deficient plasma (f.V-dp). Then, coagulation was initiated either on the intrinsic pathway (APTT) or on the extrinsic pathway (PT) or, by directly activating factor X (RVVT). Additionally, APC was added, which prolongation of the clotting time. Deficiencies in protein S or the presence of factor V-Leiden resulted in a less pronounced clotting time prolongation. Titration of protein S-deficient plasma samples with f.V-dp diminished this effect. In contrast, in samples with factor V-Leiden the difference to the clotting time obtained with normal plasma even increased in the order APTT>>RVVT>PT. In the APTT-based method high concentrations of factor VIII shortened the clotting times, thus mimicking a factor V-Leiden defect. This could be compensated for up to 4 U/ml factor VIII by using a f.V-dp containing factor VIII at physiological concentration. Neither unfractionated nor LMW-heparin (up to 2 U/ml) interfered with the determination. In a brief investigation on 16 plasma samples from patients under oral anticoagulation 5 (30%) showed a similar behaviour as observed with normal plasma from factor V-Leiden carriers. These results let us suggest that by simply mixing the patient sample with a factor V-deficient plasma factor V-Leiden might be detected also in patients under oral anticoagulant therapy. Inherited disorders of protein C or protein S are well known as thrombotic risk factors (1). The recent investigations by Dahlbäck et al. (2) led to the discovery of a new hereditary defect in the protein C anticoagulant pathway: the factor V-Leiden (3). This mutation renders activated factor V stable against proteolytic attack by activated protein C (APC). The reports on the prevalence of this mutation in thrombophilic patients show a considerable variation between 21% (4) and 64% (5).(ABSTRACT TRUNCATED AT 400 WORDS)
Administration, Oral↗
The PCAT--a simple screening assay for assessing the functionality of the protein C anticoagulant pathway.
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Anticoagulants↗
Evaluation of an automated chromogenic substrate assay for the rapid determination of hirudin in plasma.
A fully mechanized chromogenic substrate assay method for the rapid and specific determination of recombinant hirudin (r-hirudin) in citrated plasma on clinical chemistry analyzers (Hitachi 911 and Cobas Mira) is described. In a first step, 12 microliters sample volume is mixed with the chromogenic substrate. Due to the almost immediate action of hirudin the inhibitory reaction and the cleavage of the substrate is started simultaneously when bovine thrombin is added in excess. This excludes interferences by antithrombin III or heparin cofactor II. The change in absorbance/min is recorded at 405 nm. The measuring range is about 0.2-4.0 mg/l r-hirudin on both analyzers. Precision is characterized by intraassay coefficients of variation between 0.63% and 2.78% on the Hitachi 911 and 1.51% and 7.84% on the Cobas Mira, respectively and interassay coefficients of variation of 3.57% to 9.15% (Hitachi 911) and 3.72% to 12.99% (Cobas Mira) for the same r-hirudin plasma concentrations. The described determination of r-hirudin correlates well with an enzyme linked immunosorbent assay method for r-hirudin (Hitachi 911: r = 0.964, y = 0.978x + 0.038, n = 323; Cobas Mira: r = 0.964, y = 0.959x-0.003, n = 323).
Animals↗
Determination of factor XIII activity by a new photometric assay in plasma and platelets of healthy blood donors.
Crosslinking of fibrin monomers by activated factor XIII (F XIIIa) is a final event in blood coagulation. So the fibrin clot gains mechanical stability and resistance to plasmin degradation which is thought to be essential for normal blood clotting and wound healing (1-3). In addition, changes in plasma F XIII activity were found in several state of disease such as collagenoses, inflammatory bowel diseases, leukemias, subarachnoidal bleeding and delayed fracture healing (4-12). Because approximately 50% of the potential F XIII activity in plasma are present in platelets (1), the additional determination of F XIII activity in platelets is of clinical interest especially concerning platelet transfusions that may exert an additional benefit due to simultaneous substitution of platelet-bound F XIII. The latter differs from plasma F XIII as a dimer containing only a-chains (a2) compared to the plasmatic tetramer carrying additional b-chains (a2b2). We applied a recently described photometric assay suitable for the routine laboratory after adaption to an autoanalyser to determine F XIII activity in plasma and platelets of 64 healthy blood donors.
Adolescent↗
The Republican task force health reform proposal: loyal opposition or bipartisan collaboration?
In the November-December issue of Physician Executive, Drs. Fickenscher and Kindig explored the major elements of the Clinton health reform initiative, the Health Security Act of 1993. Although the Clinton proposal represents one of the major health reform proposals presently before Congress, it is by no means the only proposal. Over the next several issues, this column will provide an overview of other major proposals pending before Congress that will receive serious consideration in the coming months.
Health Care Reform↗
The rest of the spectrum: alternatives to managed competition.
In the November-December 1993 and January 1994 issues of Physician Executive, Kevin Fickenscher, MD, and David A. Kindig, MD, PhD, described the Clinton health reform plan and the Senate Republican Task Force proposals. At either end of the political spectrum are other proposals that are options to the managed competition model. This entry in the column is the last in a series that outlines the major proposals pending before Congress. It and the others are intended to highlight the major elements of the proposals, not their details. "A Matter of Policy" is jointly edited by Drs. Fickenscher and Kindig of the College's Forum on Health Policy.
Health Care Reform↗
Elements and strategies of an effective provider integration strategy.
Regardless of the outcome of the debate in our nation's capitol, a health care revolution is sweeping the nation. In fact, if the debate lasts much longer, policy makers will be playing catch-up and responding to policies already in place in the trenches. Everywhere we turn as health care leaders, there is evidence of major change on the horizon. Reimbursement methodologies are undergoing radical alteration, traditionally stable institutions are being challenged, new organizational models are evolving, the types and roles of providers best suited to provide care are being questioned, and consumer expectations are being heightened. One of the basic strategies that is receiving attention throughout the country as a response to all this change relates to the development of integrated delivery organizations (IDO), integrated delivery systems (IDS), or integrated delivery networks (lDN). This article discusses these emerging systems in terms of health care reform, describes the rationale for their creation, and provides some strategies for their successful development.
Group Practice↗
The National Health Care Reform Debate.
Because of increasing costs and decreasing access, most policymakers agree with the public perception that the health care system in the United States is in need of change. This chapter summarizes the various proposals under consideration, which ranges from a single-payer model similar to the system in Canada to the less radical concept of managed competition.
Journal Article↗
The computer and medicine in the year 2002.
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Clinical Medicine↗
A photometric assay for blood coagulation factor XIII.
An assay for a direct photometric determination of F XIII in untreated and undiluted plasma was developed. In a one-step procedure F XIII is activated by thrombin and Ca2+ and cross-links glycine-ethylester to a specific glutamine containing peptide substrate. The released ammonia is incorporated into alpha-ketoglutarate by glutamate dehydrogenase, and the NADH consumption of this reaction is measured photometrically at 340 nm. NADH-consumption is directly proportional to the F XIII activity. Fibrin polymerization and the corresponding turbidity is avoided by the use of a fibrin aggregation inhibitor. The procedure is rapid and simple and enables to measure within the range of 0 to 150% F XIII. It can be performed with automated analyzers as well as with common photometric equipment. The normal range of F XIII activity in 167 healthy donors was determined to be 70 to 140%.
Amino Acid Sequence↗
Anticoagulant properties of placenta protein 4 (annexin V).
A placenta protein, originally termed PP4, was found to inhibit the aPTT in a concentration-dependent manner. PP4 which turned out to be identical with a vascular anticoagulant of the annexin type, inhibits the blood clotting process by binding of the essential lipids in a reaction which is dependent on calcium ions. Also in the presence of calcium PP4 combines with platelet membranes neutralizing their procoagulant effect. By fluorescence-microscopy binding of PP4 to stimulated macrophages is shown. The antithrombotic effect of PP4 is demonstrated by means of thrombelastography of human blood. Coagulation triggered by the addition of thromboplastin/lipid-mixtures is extinguished by PP4.
Anticoagulants↗
Chloroplast glucose-6-phosphate dehydrogenase: Km shift upon light modulation and reduction.
Illumination of intact chloroplasts and treatment of chloroplast stroma with dithiothreitol (DTT) both inactivate glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) to less than 10% apparent activity when assayed under standard conditions. Illumination of intact protoplasts and incubation of leaf extract with DTT inactivate about 25-35% of the total G6PDH activity. In the leaf extract, however, further loss of activity is observed if NADP is absent. Light- and DTT-inactivated chloroplast G6PDH can be reactivated by oxidation with sodium tetrathionate or the thiol oxidant diamide. Chloroplast G6PDH is as sensitive toward reductive enzyme modulation in a stromal extract as are other light/dark modulated enzymes, e.g., NADP-malate dehydrogenase. Also, glutathione, provided it is kept reduced, is sufficient to cause inactivation. Light- and DTT-induced inactivation are shown to be due to a Km shift with respect to glucose-6-phosphate (G6P) from 1 to 35 and 43 mM, respectively, and with respect to NADP from 10 to 50 microM without any significant change of the Vmax. NADPH competitively (NADP) inhibits the enzyme (Ki = 8 microM). Reactivation by oxidation can be explained by an enhanced affinity of the oxidized enzyme toward G6P and NADP. The pH optimum of the reduced enzyme is more in the alkaline region (pH 9-9.5) as compared to that of the oxidized form (pH 8.0). The presence of 30 mM phosphate causes a shift of 0.5 to 1.0 pH unit into the alkaline region for both forms.
Chloroplasts↗
An improved heparin assay which is insensitive to platelet factor 4.
A new reagent for the determination of heparin in plasma has been developed. In the assay heparin which was bound to platelet factor 4 is also measured. That is why samples, which have to be assayed for heparin with this reagent, do not need any special pretreatment like fast and cooled processing in order to prevent release of platelet factor 4 from platelets. Heparin can be assayed in samples anticoagulated with citrate which are used routinely for the determination of other coagulation parameters like PT or aPTT. Freezing prior to the assay is possible and does not influence the result. The assay is based on the inactivation of factor Xa by antithrombin III which is catalysed by heparin or smaller fragments of it. It can therefore be used for the determination of heparins of low molecular weight, too. The sample is first mixed with AT III in order to compensate for a potentially decreased level in the probe. Then the F Xa reagent is added, which releases bound heparin from plasma proteins like platelet factor 4 by an added polysulfated dextran simultaneously to the onset of the inhibitory reaction towards F Xa. Free and secondarily released heparin are then available for determination. After a defined period of time a substrate for F Xa is added and the remaining activity is measured in a photometer. An incubation time of 1 min or 3 min is used for the normal range of 0.1 to 1 U/ml or the low dose range from 0.01 to 0.3 U/ml heparin, respectively.
Antithrombin III↗
Limited proteolysis of inactive tetrameric chloroplast NADP-malate dehydrogenase produces active dimers.
Carboxy-terminal amino acids of NADP-dependent malate dehydrogenase (EC 1.1.1.82) from pea chloroplasts were removed by treatment with carboxypeptidase Y. This results in the activation of the inactive oxidized enzyme, while activation by light in vivo is thought to occur via reduction of an intrasubunit disulfide bridge. After proteolytic activation the oxidized enzyme had a specific activity of 100 U/mg protein, which is 50% of the maximal activity of the control enzyme in the reduced state. When the truncated enzyme was reduced with dithiothreitol (DTT), the specific activity was further increased to 1200 U/mg. While the native enzyme is composed of four identical subunits of 38,900 Da, the truncated malate dehydrogenase forms dimers composed of two subunits of 38,000 Da. No further change of molecular mass or activity was noticed subsequent to prolonged incubation of native NADP-malate dehydrogenase with carboxypeptidase Y for several days. When the enzyme is denatured by 2 M guanidine-HCl, the proteolytic activation proceeds more rapidly, but only transiently. The truncated enzyme is less accessible to activation by reduced thioredoxin, but the stimulation of activity by DTT alone is more rapid than that of the native enzyme. These results indicate that only a small carboxy-terminal peptide of native NADP-malate dehydrogenase from pea chloroplasts is accessible to proteolytic degradation and that this peptide is involved in the regulation of activity, tetramer formation, and thioredoxin binding. While the pH optimum for catalytic activity of the intact reduced enzyme is at pH 8.0-8.5, it is shifted to more acidic values upon proteolysis of NADP-malate dehydrogenase. At pH values below 8 the reduced truncated enzyme exhibits substrate inhibition by oxaloacetate.
Amino Acids↗
Amino-terminal sequence of spinach chloroplast fructose-1,6-bisphosphatase.
The sequence of the NH2-terminal 25-amino acid residues of purified spinach chloroplast fructose-1,6-bisphosphatase was determined by automated Edman degradation. The amino acid sequence is as follows: Ala-Ala-Val-Gly-Glu-Ala-Ala-Thr-Gln-Thr-Lys-Ala- Arg-Thr-Arg-Ser-Lys-Tyr-Glu-Ile-Glu-Thr-Leu-Thr-Gly. A comparison of this sequence with the corresponding region of pig kidney and yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatases shows that the sequence of residues 1-19 of the chloroplast enzyme has no homology with the other fructose-1,6-bisphosphatases, but homology is evident after residue 20. The dissimilar sequence contains a region (residues (8-17) rich in basic and hydroxylated amino acids, a structure which is typical of presequences of mitochondrial and chloroplast proteins. Since chloroplast fructose-1,6-bisphosphatase is nuclear in origin, these results suggest that the chloroplast targeting region may have been retained within the amino acid sequence of the mature protein.
Amino Acid Sequence↗