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%.

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)

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.

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.

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).

Effects of an expanded medical curriculum on the number of graduates practicing in a rural state.

In 1973 the University of North Dakota School of Medicine (UNDSM), following the national trend toward four-year medical programs, expanded its previous two-year medical school curriculum to include all four years of medical education. It was hoped that this change, along with a renewed emphasis on primary care-oriented residency training within the state, would encourage medical students to establish practices within the state. In 1985 the UNDSM's Center for Rural Health mailed questionnaires to the 2,230 living graduates of the UNDSM to document a variety of their personal and practice characteristics. Based on the responses to the 924 completed questionnaires, the authors found that (1) the students from rural North Dakota were more likely than were urban students to practice in rural areas of the state, as were the students with primary care specialty training; and (2) the alumni completing residencies in North Dakota following the curriculum expansion (1976-1985) were more than twice as likely to establish practices in North Dakota. It was concluded that recruiting medical students (preferably in-state "natives") from rural areas, training them in primary care specialty areas, and enabling them to remain in North Dakota for the duration of their medical training (including residency training) combined to exert a considerable "retaining" effect on the UNDSM alumni.

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.

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.

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.

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.

Elements of the American Health Security Act of 1993.

Over the past several decades, there has been a plethora of proposals that were developed in response to the ongoing debate on how best to solve the problems of the American health care delivery system. In the past decade, calls for modification of our health system have become even more resonant, as measures to control rising costs were unsuccessful and access to basic services was diminished for many Americans. The most recent addition to the list of proposals for modifying the health care system is the American Health Security Act of 1993, introduced by President Clinton in September 1993. This article will examine the position of the Clinton Administration on health reform and the core elements of the reform package.

Health reform for small population areas.

There has been criticism of the managed competition model in terms of its impact on rural areas. It is suggested that the approach simply won't work for providers in rural areas and that an adjustment will be necessary. The author, acknowledging the flaw, proposes changes that will make competition work better for all providers. This column is jointly edited by Kevin M. Fickenscher, MD, and David A. Kindig, MD, PhD, chair and member, respectively, of the College's Forum on National Health Policy. Dr. Fickenscher is participating in various advisory capacities on health care in the Clinton Administration, and Dr. Kindig is Senior Advisor to HHS Secretary Donna Shalala.

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.

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.

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-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.