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Biomedical subjects

A Sturk

Publications and source records attributed to A Sturk.

At least 127 records · Page 7Linked to original sources
PubMed1987

The function of the human factor V carbohydrate moiety in blood coagulation.

Human factor V was subjected to desialation and deglycosylation to investigate the function of the molecular carbohydrate moiety. Removal of 90% of the sialic acid residues resulted in a 1.5-2-fold increase in clotting activity, and up to 70% deglycosylation in a concurrent decrease in clotting activity. Desialation had no effect on thrombin-induced activation, whereas deglycosylated factor V activation was impaired. Lectin-blot experiments with sialic-acid-specific Limax flavus agglutinin (LFA), galactose-specific Ricinus communis agglutinin (RCA-II) and mannose-specific concanavalin A on thrombin-induced factor V fragments revealed the presence of carbohydrate residues in fragments B, C1, D and F1F2. Interestingly, sialic acid was present in C1 whilst galactose was not detectable. Fragment F1F2 contained terminal galactose residues. LFA and RCA-II inhibited the procoagulant activity of native factor V and of desialated factor V respectively. These investigations distinctly indicate the important role of the human factor V carbohydrate moiety in the process of blood coagulation.

Blood Coagulation↗
PubMed1987

Analytical and clinical evaluation of commercial protein C assays.

Protein C deficiency is an important risk factor for thrombosis. Recently several commercial assays for this protein have become available. We evaluated two antigen and three activity assays in 50 healthy volunteers, 52 patients with deep venous thrombosis (DVT) proven by positive impedance plethysmography (IPG) and flebography, and 48 control patients referred to us for objective diagnosis of DVT but with repeatedly negative IPG. All subjects were less than 50 yr of age. Based upon assay performance, correlation of the assays, influence of heparin, and practical ease, an ELISA antigen assay and a chromogenic activity assay are recommended. The prevalence of protein C deficiency cannot be accurately determined from this study, because 3 DVT negative and 13 DVT positive patients were receiving coumarin therapy. However, based upon protein C:factor II antigen ratios, at least 7 of the DVT positive patients are likely to be protein C-deficient. Protein C determination should, therefore, be considered in all DVT patients less than 50 yr of age.

Chromogenic Compounds↗
PubMed1987

Tissue plasminogen activator activity and inhibition in acute myocardial infarction and angiographically normal coronary arteries.

Parameters of blood coagulation, blood platelet reactivity and fibrinolysis were analyzed in 18 patients with acute myocardial infarction (AMI) and angiographically normal coronary arteries. This study group was compared with a patient control group of 18 AMI patients with 1-vessel obstructive coronary artery disease. Patients were matched for sex, age and AMI date. A healthy control group consisted of 18 sex- and age-matched volunteers. Blood coagulation measurements and platelet reactivity were similar in the 3 groups, except for fibrinogen, which was significantly higher in the patient control group. Plasma activity of tissue plasminogen activator (tPA) was detectable in only 2 patients in the study group, 9 in the patient control group (p less than 0.02) and 12 in the healthy control group (p less than 0.0001). Median plasma tPA inhibitory activity was higher in the study group (10.3 IU/ml) and the patient control group (8.1 IU/ml) than in the healthy control group (2.7 IU/ml, p less than 0.0001 and p less than 0.03). Thus, reduced activity and enhanced inhibition of plasma tPA may be important factors in the origin of coronary thrombosis, especially in the absence of coronary artery disease.

Adult↗
PubMed1987

Association of vinculin to the platelet cytoskeleton during thrombin-induced aggregation.

Vinculin is a protein generally believed to be involved in membrane-cytoskeleton interaction, and its presence in platelets has been verified earlier. Here we show that in resting bovine platelets, vinculin is not associated with the Triton-insoluble cytoskeletal fraction but becomes incorporated into it during the thrombin-induced activation process. The incorporation starts around the same time as the release reaction and only after the shape change and the first phase of aggregation have taken place. Its time course parallels the cytoskeletal association of actin and certain other contractile proteins. Vinculin is a minor component of platelet cytoskeleton and only about 10% of the total platelet vinculin becomes incorporated into the Triton X-100 residue.

Animals↗
PubMed1987

Platelet-activating factor: mediator of the third pathway of platelet aggregation? A study in three patients with deficient platelet-activating factor synthesis.

Thrombin, collagen, and Ca2+-ionophore A23187 aggregate platelets in the presence of inhibitors of the first (ADP-mediated) and second (cyclooxygenase-dependent) pathway of platelet activation. This aggregation, via a third pathway, was hypothesized to be mediated by the alkoxyether lipid platelet-activating factor (PAF). We recently demonstrated virtual absence of plasmalogen-type alkoxyether lipids and deficiency in key enzymes of their biosynthesis in Zellweger patients. We hypothesized that PAF synthesis might also be impaired. We report two Zellweger patients with an undetectable A23187-induced PAF synthesis of leukocytes (patients, less than 3 pmol PAF/10(8) granulocytes (PMN); four age-matched controls, 249-2,757 pmol PAF/10(8) PMN; five adult controls, 291-5,433 pmol PAF/10(8) PMN). In a third patient, residual PAF synthesis was detected. However in all patients the thrombin-induced third mechanism of platelet aggregation was present. We therefore conclude that PAF may not be the mediator of the third pathway.

Abnormalities, Multiple↗
PubMed1987

Endotoxin testing in blood.

A chromogenic assay is presented for the determination of endotoxin (LPS) in blood. The assay is based upon the LPS-dependent activation of Limulus amebocyte lysate (LAL), and the subsequent measurement of the activated enzyme with a chromogenic substrate. Handling and stability of the reagents, details of the assay method in tubes or microtiter-plates, recovery of LPS from spiked blood in platelet-rich plasma (PRP), platelet-poor plasma (PPP) and serum, as well as the possibility to store plasma samples will be discussed. A clinical evaluation of the assay is provided by S. van Deventer et al. in this volume.

Chromogenic Compounds↗
PubMed1987

Age-related deficiency of the synthesis of platelet activating factor by leukocytes from Zellweger patients.

Ca2+-ionophore A23187-induced synthesis of the alkoxyether lipid platelet activating factor (PAF) by leukocytes from Zellweger patients was undetectable in two patients studied at 3 and 4 weeks of age, reduced in a third patient studied at 2 months of age, and in the low normal range in four patients studied between 4 months and 5 years of age. We have previously reported that plasmalogen-type phosphatidylethanolamine (PE) levels of erythrocytes are reduced in Zellweger patients up to 20 weeks of age, but normal in older patients. These levels were reduced in the three patients with abnormal PAF synthesis, and normal in the other four patients. The results suggest a close relationship between the age of the patients at sampling, and both the A23187-induced capacity of leukocytes to synthesize PAF and the plasmalogen PE levels in their erythrocytes.

Abnormalities, Multiple↗
PubMed1986

Heparin versus low molecular weight heparin K 2165 in chronic hemodialysis patients: a randomized cross-over study.

Ten patients on chronic intermittent hemodialysis treatment received either unfractionated heparin or low molecular weight (LMW) heparin K 2165 in a single-blinded randomized cross-over study to assess: effects on hemostasis and ex vivo platelet functions, and effectiveness, i.e. prevention of fibrin formation in the extracorporeal circuit. The 20 dialysis treatments were without untoward side effects, for both drugs used. The variation in the plasma anti-Xa activities was significantly less during K 2165 treatment than during heparinization. No differences between the drugs were observed regarding the Ivy bleeding time, platelet count and platelet aggregation (spontaneous, and induced by ADP and collagen). Plasma platelet factor 4 levels did not increase under K 2165 to such an extent as under heparin. Both drugs did not influence the plasma levels of beta-thromboglobulin, thromboxane B2 and platelet serotonin content. K 2165 did not affect platelet adhesion to collagen, in contrast to heparin which substantially inhibited platelet adhesion. Under both treatments, 4 minor clots were observed in 4 artificial kidneys, despite plasma anti-Xa levels in between 0.19 and 0.46 U/ml. K 2165 may therefore be considered as effective an anticoagulant as heparin, with less effects on ex vivo platelet functions.

Adult↗
PubMed1985

Synergistic effects of platelet-activating factor and other platelet agonists in human platelet aggregation and release: the role of ADP and products of the cyclooxygenase pathway.

The synergistic effects of platelet-activating factor (PAF) with ADP, collagen, thrombin, A23187, adrenaline, sodium arachidonate and ristocetin in human platelet aggregation and beta-thromboglobulin (beta-TG) release were investigated in citrated platelet-rich plasma (PRP). Synergism in both aggregation and release was present with all agonists except ristocetin. Upon oral intake of aspirin (ASA) the PAF-induced irreversible aggregation as well as the synergistic irreversible aggregation became reversible. Both prior to and after ASA ingestion ADP removal by creatine phosphate/creatine phosphokinase (CP/CPK) resulted in a reduced, reversible platelet aggregation induced by PAF alone or in combination with the other agonists. The ADP-removal and ASA-ingestion also strongly inhibited the beta-TG release. The synergistic aggregation and release were also inhibited by ASA and indomethacin in vitro as well as by the competitive ADP-inhibitor ATP. It is concluded that not only the activation of human platelets by low doses of PAF itself, but also the synergism of PAF and other platelet agonists is highly dependent upon ADP and products of the cyclooxygenase pathway.

Adenosine Diphosphate↗
PubMed1985

Optimalization of a chromogenic assay for endotoxin in blood.

Early detection of Gram-negative septicemia or endotoxemia may become feasible with sensitive and reliable endotoxin (LPS) measurements. We recently published an assay for LPS in blood, based upon the LPS dependent activation of Limulus amebocyte lysate (LAL) and the subsequent chromogenic measurement of the activated enzyme(s). Inhibitors and activated clotting factors potentially interfering in the assay were removed by dilution and heating. In the present study we describe the further improvement of the assay. Optimal conditions include: blood anticoagulated with 30 I.U./ml heparin; dilution of the platelet-rich plasma (PRP) in water; 5 min. heating at 75 degrees C; 15 mM Mg2+, 1.5 mM Ca2+, 125 mM Na+, 50 mM TRIS, pH 8.5 in the LAL activation step; substrate step without extra addition of Ca2+, Mg2+, or Na+, but in the presence of 50 mM TRIS at pH = 9.5. Under those optimal conditions less than 10 pg LPS per ml blood (PRP) can easily be detected. Prospective clinical trials are presently envisaged to investigate the clinical usefulness of this extremely sensitive LPS assay.

Chromogenic Compounds↗
PubMed1985

Validity of the endotoxin assay in post surgical patients.

In a prospective study 56 patients were studied after major surgery for 10 days to correlate endotoxemia with bacteremia and clinical septicemia. Endotoxemia was detected with a quantitative plasma endotoxin assay using limulus lysate and a chromogenic substrate. Clinical septicemia was defined using a set of predetermined criteria. In 29 patients both bacterial cultures and endotoxin assays were negative. One patient showed clinical signs of septicemia. In 14 patients, low grade endotoxemia (mean 47 +/- 15 pg/ml) was observed in the first three days after surgery. Only three of these patients also showed positive endotoxin tests after this period. Corresponding blood cultures were frequently negative (81%) most likely due to prior and concurrent antibiotic prophylaxis. Two endotoxin positive patients had clinical signs of septicemia. In a total of 16 patients, including two patients from the former group endotoxemia (mean 56 +/- 21 pg/ml) was noted beyond the third post-operative day. Samples for blood cultures collected simultaneously were positive for gram negative bacilli in six patients. Clinical septicemia was present in 12 of the 16 patients. The positive and negative predictive value of the endotoxin test for bacteremia and/or septicemia after the third day of surgery for all patients was found to be 75% and 98% respectively. The results of this small prospective study in surgical patients suggest that this rapid endotoxin test is a useful clinical adjunct for both the detection and exclusion of gram negative bacteremia/septicemia.

Adult↗
PubMed1984

Comparative investigation of a quantitative chromogenic endotoxin assay and blood cultures.

In a prospective study, the clinical relevance of a quantitative chromogenic endotoxin assay in plasma (detection limit 10 ng/L, assay time 2.5 hours) versus blood cultures was evaluated in 51 critically ill patients with increased susceptibility for infectious complications. Of the 400 samples tested, the endotoxin assay and bacterial culture both were negative in 342 samples. In 21 samples from 15 patients, gram-negative aerobic microorganisms were cultured. Corresponding endotoxin assays were positive in 14 samples (mean 100 ng/L). Twenty-three samples grew gram-positive bacteria. The associated endotoxin assays all were negative. Twelve samples were found to be endotoxin-positive without a corresponding gram-negative bacterial culture. In 7 of these 12 positive endotoxin assays, a laboratory or clinical explanation for these positive tests could be provided. In view of the high sensitivity, specificity, and predictive values obtained, the authors conclude that the endotoxin assay used is a useful clinical adjunct for both the detection and exclusion of gram-negative septicemia.

Bacterial Infections↗
PubMed1983

Identification of a contaminant protein in the purification of human factor V. Comparison of two purification methods.

Coagulation factor V has been isolated from human plasma by the methods of Bolhuis and of Kane to investigate reported differences in purity and specific activity. The specific activities of the preparations are 10 and 40 units/mg, respectively. The Kane preparation shows a single band (Mr 330 000) in polyacrylamide gel electrophoresis in the presence and absence of dithiothreitol, but the Bolhuis preparation shows an additional band at Mr 190 000 upon reduction with dithiothreitol. This suggests the presence of a contaminant which could account for the lower specific activity. Modification of the Bolhuis technique to include ion-exchange chromatography on DEAE-Sepharose resulted in separation of the two isolated proteins. The proteins were identified as factor V, with characteristics indistinguishable from Kane factor V, and a dimeric subunit of alpha 2-macroglobulin (Mr 350 000). Contamination by alpha 2-macroglobulin may explain the I2-component described in some preparations of bovine factor V, which is the basis of speculation that bovine factor V has a second polypeptide chain modulating stability and activity.

Blood Proteins↗
PubMed1983

Detection of carriers in Glanzmann's thrombasthenia.

Quantitative glycoprotein (GP) analysis of whole platelets or platelet membranes was performed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and periodic acid Schiff staining in the families of two unrelated Glanzmann's thrombasthenia (GT) patients. Each family consisted of two symptom free parents, a symptom free daughter and a GT daughter. All symptom free members had a normal bleeding time, clot retraction and platelet aggregation response to adenosine 5'-diphosphate (ADP), collagen and adrenalin. Platelet Zwa antigen was normally expressed in these subjects. GT patients, classified as a type I and II subject, showed reduced amounts of GP IIb and of GP IIIa. Analysis of isolated membranes in the non-reduced state, however, showed that the amount of GP IIIa was also reduced in three of the four parents, whereas one parent (of the GT type I patient) and the two unaffected daughters had normal amounts of GP IIIa. Quantitative SDS-PAGE may therefore provide a method for the detection of asymptomatic carriers in GT type I and II.

Blood Platelet Disorders↗