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Biomedical subjects

D A Lane

Publications and source records attributed to D A Lane.

At least 163 records · Page 9Linked to original sources
PubMed1986

Direct analysis of plasma fibrinogen-derived fibrinopeptides by high performance liquid chromatography: investigation of A alpha-chain N-terminal heterogeneity.

Fibrinopeptide A (FPA), released from the fibrinogen A alpha-chain by thrombin, can be resolved by high-performance liquid chromatography (HPLC) into three forms, the intact peptide (A), a modified peptide phosphorylated at the serine in position 3 (AP), and an N-terminally degraded form (AY). A new method has been developed, using HPLC, that allows direct measurement of the proportions of AP, A, and AY released by thrombin from fibrinogen in plasma samples of 200 ul or less. The method was used to examine variations in the proportions of AP and AY expressed as a % of total FPA in a number of patient and control groups. The mean percentages of AP and AY of plasma fibrinogen were found to be 21.7 and 14.2%, respectively, in normal laboratory controls. In older, apparently normal, individuals these figures were 27.0 and 15.5%, respectively. Cord plasma exhibited very high AP and slightly reduced AY levels (41.6 and 12.4%, respectively) compared with normal adults. Patients with liver failure had low AP levels and high AY levels (11.6 and 21.1%, respectively). Patients recovering from major surgery or acute thrombotic stroke showed an acute-phase rise in fibrinogen level that was accompanied by an increase in AP and variable reduction in AY. Incubation of heparinized whole blood for 8 days in vitro demonstrated a gradual decrease in the proportion of AP and increase in AY of plasma fibrinogen. These results provide some support for the idea that an increased "aging" of fibrinogen in the circulation may result in a decrease in the AP content of fibrinogen accompanied by a more variable increase in AY.

Aged↗
PubMed1986

Direct analysis of plasma fibrinogen-derived fibrinopeptides by high-performance liquid chromatography.

The fibrinopeptides released from fibrinogen by thrombin can be conveniently quantitated by high-performance liquid chromatography (HPLC). This work describes a method that enables direct analysis of fibrinopeptides derived from fibrinogen contained in small amounts of plasma or whole blood without the necessity of fibrinogen purification. Aliquots of plasma or whole blood were treated with thrombin, diluted in buffer at pH 6.0, boiled and centrifuged. After filtration to 0.22 micron the supernatant was analysed directly by HPLC using a 3 microns C18 analytical column fitted with a pre-column. All the known normal fibrinopeptide peaks and their degradation products were clearly resolved from other plasma-derived peptides and the release pattern was identical to that observed with purified fibrinogen. Some samples contained interfering endogenous peptides which were removed by rapid gel-filtration of 200 microliters of plasma before thrombin treatment. The method was quantitative and the peak heights of fibrinopeptide B (FPB) could be used to accurately measure plasma fibrinogen concentrations directly in samples equivalent to 20 microliters of plasma. These fibrin determinations were unaffected by the type of anticoagulant used were performed on blood samples of both human and animal origin collected and/or stored under a variety of conditions. Variations of the method were specifically developed for different applications requiring quantitative fibrinopeptide analysis.

Animals↗
PubMed1986

The anticoagulant effect of heparinoid Org 10172 during haemodialysis: an objective assessment.

The heparinoid of natural origin Org 10172 has anti-factor Xa activity but minimal anti-thrombin activity, and little effect upon broad spectrum assays such as the KCCT in vitro. Its anticoagulant effects have been compared to those of commercial heparin in 7 patients undergoing haemodialysis for chronic renal failure. Commercial heparin was administered in a dose (5,000 iu bolus + 1,500 iu/hour continuous iv infusion) previously shown to inhibit fibrin formation during haemodialysis. This produced mean anti-factor Xa levels in plasma between 0.7-1.0 iu/ml and largely suppressed fibrin formation for 5 h dialysis measured as mean FPA levels in plasma. Administration of Org 10172 as a bolus of 1,350 anti-factor Xa u or 2,000-2,400 anti-factor Xa u produced plasma anti-factor Xa levels of less than 0.5 u/ml and allowed fibrin clot and FPA generation during dialysis. Org 10172 administered as a bolus dose of 4,000-4,800 anti-factor Xa u produced mean anti-factor Xa levels of greater than 0.5 u/ml, allowed dialysis of 6 patients for 5 h and appreciably suppressed FPA generation during dialysis, with little effect on the KCCT. It is concluded that the anti-factor Xa activity of Org 10172 may reflect its ability to inhibit fibrin during dialysis and that single bolus injection of Org 10172 may be a useful alternative method of achieving anticoagulation.

Anticoagulants↗
PubMed1986

Neutralization of heparin-related saccharides by histidine-rich glycoprotein and platelet factor 4.

Heparin and heparin oligosaccharides prepared by nitrous acid depolymerization were fractionated by affinity chromatography on immobilized antithrombin and by gel chromatography. The anticoagulant activities of high affinity heparin of Mr greater than or equal to 7,800 could be readily neutralized by the plasma protein histidine-rich glycoprotein (see also Lijnen, H.R., Hoylaerts, M., and Collen, D. (1983) J. Biol. Chem. 258, 3803-3808), whereas oligosaccharides falling below 18 saccharide units (Mr 5,400) became increasingly resistant to neutralization. An octasaccharide with characteristic marked ability to accelerate the inactivation of Factor Xa by antithrombin retained greater than 50% of its activity even at a histidine-rich glycoprotein/oligosaccharide molar ratio of 500:1. Histidine-rich glycoprotein, like the platelet-derived heparin neutralizing protein platelet factor 4 (Lane, D.A., Denton, J., Flynn, A.M., Thunberg, L. and Lindahl, U. (1984) Biochem J. 218, 725-732), therefore requires interaction with saccharide sequences in addition to the antithrombin-binding pentasaccharide of heparin in order to efficiently express its antiheparin activity. Heparan sulfate isolated from pig intestinal mucosa (HS I, Mr approximately 20,000) and from human aorta (HS II, Mr approximately 40,000) exhibited anti-Factor Xa activities of 180 and 20 units/micromol [corrected], respectively. A fraction corresponding to about 5% of HS I bound with high affinity to immobilized antithrombin and contained all of the anticoagulant activity of the starting material. While these heparan sulfates were readily neutralized by platelet factor 4, they were relatively resistant to neutralization by histidine-rich glycoprotein, although complete neutralization could be attained in the presence of molar excess of this protein. These findings may be of importance in relation (a) to the functional role of endogenous anticoagulant polysaccharides at the vascular wall and (b) to clinical situations in which heparin or heparin-related compounds are administered as exogenous anticoagulants.

Heparin↗
PubMed1986

On the evaluation of heparin and low molecular weight heparin in haemodialysis for chronic renal failure.

Anticoagulation during haemodialysis for chronic renal failure can be assessed by measurement of plasma fibrinopeptide A (FPA) levels as an objective method of monitoring the initial step in fibrin formation, in conjunction with visual inspection of the dialyser circuit for fibrin clot deposition. Employing this approach, unfractionated commercial heparin administered as an intravenous bolus followed by an intravenous maintenance dose (5,000 IU + 1,500 IU/h) was found to suppress almost completely fibrin formation and deposition during prolonged dialysis. Comparison of a low molecular weight heparin, Kabi 2165, revealed that it can inhibit fibrin formation in the extracorporeal circulation, that this property is largely reflected in its anti-factor Xa activity in plasma, and that a useful and effective dose of Kabi 2165 for haemodialysis may be 4,000 anti-factor Xa U intravenous bolus + 750 anti-factor Xa U/h intravenous maintenance infusion. This dose only minimally alters the KCCT and corresponds to approximately 60% of that of unfractionated heparin, which may be important in the long-term use of heparin in these patients.

Biological Availability↗
PubMed1986

Haemodialysis with low MW heparin: dosage requirements for the elimination of extracorporeal fibrin formation.

We have performed a dose ranging study of a low MW heparin, Kabi 2165, during haemodialysis in humans (n = 16) and compared it to a dose of unfractionated commercial heparin that has already been shown to inhibit fibrin formation. Low MW heparin administered as 5000 or 10,000 anti-factor Xa units, s.c., half an hour prior to the initiation of dialysis was unable to prevent fibrin formation in the dialyser circuit. A single bolus injection of 5000 anti-factor Xa units of low MW heparin given i.v. inhibited fibrin formation, as determined by FPA generation, for up to 4 h and permitted dialysis for 6 h. Such a bolus injection may be useful for short frequent dialyses. Infusion of low MW heparin in the same dosage regimen as unfractionated heparin, 5000 anti-factor Xa units bolus plus 1500 anti-factor Xa units/h, resulted in a progressive rise in heparin, caused by its longer half-life of elimination from the circulation, and almost completely suppressed both FPA generation and fibrin clot formation for 6 h dialysis. From these studies we calculate that infusion of this low MW heparin at a dose of approximately 4000 anti-factor Xa units bolus plus 750 anti-factor Xa units/h should be a useful regimen that will be effective in suppressing fibrin formation during prolonged dialysis, and the plasma anti-factor Xa level of low MW heparin may reflect its ability to inhibit fibrin formation, although exactly comparable anti-factor Xa levels of unfractionated commercial heparin and low MW heparins may not have identical inhibitory effects.

Clinical Trials as Topic↗
PubMed1985

Characterization of peptides cleaved by plasmin from the C-terminal polymerization domain of human fibrinogen.

The C-terminal region of the fibrinogen gamma chain is known to participate in several functional interactions including fibrin polymerization. This part of the molecule is retained on the gamma chain of fragment D (FgD) when fibrinogen is digested by plasmin in the presence of calcium to produce the fragment D-fragment E (FgD X FgE) complex but is lost if FgD is prepared in the absence of calcium. In an attempt to characterize the C-terminal polymerization domain we have used three techniques to examine this further degradation of FgD following the addition of EDTA and plasmin. Analysis of the digestion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a progressive cleavage of the gamma chain to two small remnants. The polymerization-inhibitory activity of the whole digest was studied using acid-solubilized fibrin. A progressive loss of inhibitory activity was associated with gamma chain shortening, reaching greater than a 120-fold reduction at the end of digestion. The cleavage of peptides was followed by reverse-phase high performance liquid chromatography and the release of a characteristic peptide triplet was associated with gamma chain cleavage. Manual sequencing, amino acid analysis, and fast atom bombardment mass spectrometry established the three peptides as gamma 303-356, 357-373, and 374-405. These peptides have sequences in common with those peptides recently reported by other investigators to be potent polymerization inhibitors. However, when a mixture of the three peptides was added in a 200-fold molar excess to polymerizing fibrin, no inhibitory activity could be demonstrated. It is concluded that the C-terminal polymerization domain of fibrinogen may be an extended region which includes the sequence gamma 303-405, when this is contiguous with the remainder of the gamma chain.

Amino Acid Sequence↗
PubMed1985

Invasive carcinoma of the cervix in young women.

The cases of 194 women aged 40 years of age or less who had been diagnosed as having invasive cervical cancer in Queensland during 1972-1981 were reviewed retrospectively. Medical records contained a previous cervical cytological report for 89 women, with 18 of these having had a negative result of a smear test within two years of the diagnosis of invasive cancer. Cytological review of five of the 18 cases revealed neoplastic cells in three cases, and possible sampling inadequacies in the other two. Gynaecological symptoms had been present in seven of the 18 patients at the time of the negative result of cytological examination, and these persisted until the diagnosis was eventually made. A high index of suspicion is required when symptoms persist in spite of negative results of cytological examination and we recommend a review of the smear, a repeat smear examination with adequate sampling and referral for colposcopic examination in these circumstances.

Adult↗
PubMed1985

Fibrinogen Manchester. Detection of a heterozygous phenotype in the intraplatelet pool.

Family members heterozygous for the congenitally abnormal fibrinogen designated fibrinogen Manchester, A alpha 16Arg----His, have previously been shown by h.p.l.c. and amino acid analysis to release a variant fibrinopeptide, [His16]fibrinopeptide A, from plasma fibrinogen after the addition of thrombin. The present study was designed to determine if the same abnormal phenotype was also present in the intraplatelet fibrinogen pool. Fresh platelets were washed in buffers containing EDTA until it could be shown that all washable plasma fibrinogen was removed. Normal platelets were then lysed by freezing and thawing to release their intracellular proteins, which were then treated with thrombin. The fibrinopeptides, cleaved from the intraplatelet fibrinogen, could be detected by an optimized h.p.l.c. technique. Quantification of the intraplatelet fibrinogen gave a result (means +/- S.D., n = 5) of 110 +/- 30 and 90 +/- 30 micrograms/10(9) platelets, when determined by h.p.l.c. quantification of fibrinopeptide B content and fibrinogen fragment E radioimmunoassay respectively. Examination of fibrinopeptides released from the platelet fibrinogen from the family with fibrinogen Manchester with the same techniques showed elution peaks in the same positions as both [His16]fibrinopeptide A and normal fibrinopeptide A. The identity of these peaks was further substantiated by analysis of the h.p.l.c. peaks by using specific radioimmunoassay to fibrinopeptide A. Our results therefore demonstrate that platelet fibrinogen expresses the heterozygous A alpha 16His phenotype. This supports the view that the A alpha chains of platelet and plasma fibrinogen are produced from a single genetic locus.

Blood Platelets↗
PubMed1985

Thrombin-induced fibrinopeptide release from a fibrinogen variant (fibrinogen Sydney I) with an Aalpha Arg-16----His substitution.

Fibrinogen, purified from a recently identified case of dysfibrinogenaemia, fibrinogen Sydney I, was shown by thrombin digestion, high-performance liquid chromatography (HPLC) and amino acid analysis to be a heterozygous case of an A alpha Arg-16----His substitution. Kinetic studies have been carried out on the thrombin-induced release of fibrinopeptide A (FPA), fibrinopeptide B (FPB) and the variant peptide [His16]FPA. When thrombin was added to fibrinogen Sydney I at a concentration of 0.2 U/ml release of FPA was rapid and there was a 79-fold reduced rate of release of [His16]FPA, but the rate of release of FPB was not appreciably reduced. In contrast, at lower thrombin concentrations the rate of FPB release was reduced in proportion to the rate of total FPA release, supporting the view that release of fibrinopeptides is a sequential process. The second-order kinetic constant kcat/Km for hydrolysis of the abnormal A alpha chain by thrombin was calculated from Lineweaver-Burk plots to be 16-30-fold less than that for the normal A alpha chain. Molecular modelling studies, using a refined model of the trypsin-pancreatic-trypsin-inhibitor complex have been used to suggest how the histidine at the P1 site can be accommodated within the enzyme hydrophobic active-site pocket.

Amino Acids↗
PubMed1985

Fibrinogen derivatives and platelet activation products in acute and chronic liver disease.

1. The concentration in plasma of fibrinogen derivatives fibrinopeptide A (FPA) and B beta 1-42 and the platelet release products beta-thromboglobulin (beta TG) and platelet factor 4 (PF4) have been determined in patients with acute and chronic liver disease. 2. In 21 patients with fulmiant hepatic failure on admission in grade III or IV coma the plasma FPA, B beta 1-42, beta TG and PF4 levels were significantly increased compared with those in normal control subjects. On heparinization before haemoperfusion the FPA levels returned to the normal range and during resin and charcoal haemoperfusion there were no significant changes in the coagulation or platelet factors, except for a small increase in FPA with charcoal haemoperfusion. 3. In ten patients with compensated chronic liver disease there was a significant increase in B beta 1-42 and beta TG levels but not FPA and PF4 as compared with normal controls. 4. Interpretation of the results is complicated by the possible reduced clearance of these proteins as a result of renal failure in some of the patients with fulminant hepatic failure and also by the damaged liver itself. However, these results have confirmed that disseminated intravascular coagulation can occur in both acute and chronic liver disease.

Acute Disease↗
PubMed1985

Haemodialysis with prostacyclin (Epoprostenol) alone.

Dialysis with prostacyclin (Epoprostenol, PGI2) alone prevents platelet activation and endothelial cell stimulation but not the elevation of fibrinopeptide A (FPA), a sensitive marker of fibrin generation. The generation of FPA may explain why some patients develop clot in the dialysis circuit during PGI2-only dialysis. In combination with heparin, PGI2 augments the anticoagulant effect of the heparin as well as providing platelet protection.

Blood Platelets↗
PubMed1985

Specific identification of fibrin polymers, fibrinogen degradation products, and crosslinked fibrin degradation products in plasma and serum with a new sensitive technique.

A new method is described for identifying low concentrations of circulating derivatives of fibrinogen and fibrin, even when present in heterogeneous mixtures. This technique is applicable to plasma and serum and uses electrophoresis in 2% agarose in the presence of sodium dodecyl sulfate (SDS) followed by immunological identification of separated derivatives, using radiolabeled antifibrinogen antiserum and autoradiography. Unique electrophoretic patterns distinguish plasmic derivatives of crosslinked fibrin from those of fibrinogen and also identify crosslinked fibrin polymers produced by the combined action of thrombin and factor XIII on fibrinogen. The assay is sensitive to a concentration of 0.1 micrograms/mL of fibrinogen in serum or plasma. Fibrin polymers, plasmic degradation products of fibrinogen, and plasmic degradation products of crosslinked fibrin were detected in the plasma or serum of a patient with disseminated intravascular coagulation. Plasmic derivatives of both fibrinogen and crosslinked fibrin appeared in serum in the course of fibrinolytic therapy for pulmonary embolism, whereas during acute myocardial infarction a marked increase in the proportion of fibrin polymers in plasma was found in comparison with normal controls. Thus, the procedure can distinguish between the simultaneous processes of fibrin polymer formation, fibrinogenolysis, and fibrinolysis, and is sufficiently sensitive to detect relevant quantities of derivatives in pathologic conditions.

Autoradiography↗
PubMed1984

Detection of enhanced in vivo platelet alpha-granule release in different patient groups--comparison of beta-thromboglobulin, platelet factor 4 and thrombospondin assays.

During the platelet release reaction beta-thromboglobulin (beta TG), platelet factor 4 (PF4) and thrombospondin (TSP) are released from the platelet into plasma and assays of these proteins can be used to monitor in vivo platelet activation. We have assessed their relative merits as markers of the in vivo platelet alpha-granule release reaction in a number of patient groups which have previously been shown to have elevated plasma beta TG and/or PF4 levels. It is concluded that in diseases or conditions not complicated by its reduced clearance, beta TG is the most sensitive marker of in vivo platelet alpha-granule release. However, the TSP assays may be the least ambiguous when monitoring the platelet alpha-granule release reaction in patients with renal failure who are undergoing haemodialysis with heparin anticoagulation. Under these circumstances plasma beta TG, but not PF4 or TSP, levels are elevated because of impaired renal catabolism, and the presence of a heparin-releasable reservoir of PF4 on the endothelium complicates the use of the PF4 assay. In liver failure none of these assays may accurately reflect platelet alpha-granule release because of impaired hepatic or renal elimination of the proteins.

Adolescent↗
PubMed1984

Experimental evidence for renal catabolism of fibrin fragment beta 15-42.

The elimination of labelled human fibrin fragment beta 15-42 from the circulation has been studied in an experimental rat model. When 125I beta 15-42 was injected as a bolus into a control group of rats, its elimination from the circulation could be fitted to a two compartment exponential model. A fast initial elimination had a t1/2 of less than 2 min and this was followed by a slower component with a calculated t1/2 of 135 min. The fast component was caused by equilibration of peptide throughout the intra and extra vascular spaces and the slow component reflected the action of catabolic processes once equilibration had been attained. Rats that had undergone bilateral nephrectomy eliminated significantly less of the labelled peptide than the control animals at a given time and the t1/2 of the slow component was prolonged, but not significantly, to 159 min. Rats with ligated ureters had statistically indistinguishable elimination curves from control rats. Examination of the heterogeneity of labelled peptide in plasma samples taken during the experiments by immunoprecipitation and by gel filtration revealed progressive extensive degradation in the control and ureteral ligated rats, but less degradation in nephrectomized rats. These results suggest that beta 15-42 is eliminated, in part, from the circulation by uptake and catabolism by the kidney. It is concluded that impaired renal function may results in elevated plasma levels of beta 15-42 antigen in human renal failure without the need for an increased rate of production of the peptide.

Animals↗