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Biomedical subjects

Thomas W Stief

Publications and source records attributed to Thomas W Stief.

11 recordsLinked to original sources
PubMed2007

Thrombin generation by hemolysis.

Hemolysis is the fragmentation of erythrocytes into microparticles (Hb-MP). Clinical hemolysis can result in a severe procoagulant state. The influence of Hb-MP on thrombin generation was quantified. Unfrozen citrated normal plasma (five donors) was supplemented with 0 or 1 g/l Hb-MP obtained through erythrocyte destruction by hypotonic lysis, freezing/thawing, or blood oxidation with 1 or 2 mmol/l chloramine-T. Pooled normal plasma was supplemented with 0-10 g/l Hb-MP and with 0-1 IU/ml low-molecular-weight heparin (dalteparin). Samples (50 microl) were tested in the recalcified coagulation activity assay. At 10 min coagulation reaction time the hypotonic lysis of erythrocytes appears to be the most procoagulant condition, followed by twice freezing/thawing, three times freezing/thawing, and once freezing/thawing. Oxidation of whole blood with 1 or 2 mmol/l chloramine-T decreases thrombin generation by about 20 or 50%, respectively. The thrombin generation in 1 mmol/l chloramine-T or 2 mmol/l oxidized plasma decreases by about 70 or 85%, respectively. The 50% inhibitory concentrations of low-molecular-weight heparin against recalcified thrombin generation are 0.01, 0.025, or 0.035 IU/ml for plasma supplemented with 0, 0.1, or 1 g/l Hb-MP, respectively. The recalcified coagulation activity assay allows one to quantify thrombin generation in critical hemolytic samples. It is suggested to find the appropriate pharmacologic dose of low-molecular-weight heparin.

Blood Coagulation↗
PubMed2007

Influence of coagulation factors on intrinsic thrombin generation.

The intrinsic coagulation activity assay (INCA) is a new thrombin-generation test that imitates the intrinsic pathway of blood coagulation. The aim of the present study was to investigate the influence of the main coagulation factors on the INCA. The INCA was performed with citrated plasma samples supplemented with different amounts of fibrinogen. The INCA and activated partial thromboplastin time determination were performed with factor-depleted plasmas and with mixtures of depleted plasmas with normal plasma. Supplemented purified fibrinogen resulted in a decrease of intrinsic thrombin generation (50% inhibitory concentration = 0.8 g/l). The INCA depends on the intrinsic factors (factors VIII, IX, XI and XII) and on the factors of the common pathway (factors II, V and X): for normal thrombin generation, at least about 50% of normal factor II is necessary. For the majority of factors, the sensitivity of the INCA appears to be approximately one order of magnitude better than that of the activated partial thromboplastin time. The INCA allows one to diagnose defects in the intrinsic coagulation system and might be a useful test to support development and characterization of new drugs targeted at the intrinsic generation of thrombin.

Blood Coagulation↗
PubMed2007

The fibrinogen antigenic turbidimetric assay (FIATA): the X2x test--the corrected chi-square comparison against the control-mean.

Vancomycin precipitates fibrinogen. The turbidity induced by this vancomycin-fibrinogen interaction is used to establish a simple standardized antigenic assay for plasmatic fibrinogen, the FIATA. 1 mM vancomycin or 2 mM chloramine-T inactivates 50% of fibrinogen in human plasma. In contrast to chloramine-T, vancomycin does not react in NaJ-based photometric assay for chloramines,vancomycin does not inactivate the singlet oxygen-sensible antithrombin III, and the vancomycin action against fibrinogen is not changed in spite of the presence of the 1O2 quenchers methionine or ascorbic acid. The FIATA is performed as follows: to 25 microL plasma 50 microL PBS are added and the absorbance (A) at 405 nm is read. Then 50 microL FIATA-reagent, consisting of 4.4 mM vancomycin in PBS, are added. After 2 minutes (RT) DeltaA is determined and standardized against a plasma pool of 100% of norm (2.8 g/L) fibrinogen. The FIATA is nearly linear up to a fibrinogen concentration of about 150% of norm (4.2 g/L), resulting in a DeltaA of about 600 mA. The lower detection limit is 4% of norm (0.1 g/L). The intra-assay and interessay CV values are < 4%. The normal range of FIATA is 100% +/-20% (x- +/- 1 SD). In = 321 or 344 unselected patient plasmas the FIATA (x- = 130%; SD = 52% or 43%) correlated with the functional fibrinogen assays a) modified Clauss-Method (x- = 4.1 g/L; SD =1.7 g/L) with r = 0.755 and b) FIFTA (x- = 124%; SD = 40%) with r = 0.813. The vancomycin/fibrinogen interaction (binding of about 16 molecules of vancomycin/molecule of fibrinogen) can be used to purify fibrinogen out of plasma. Vancomycin also clouds dysfunctional fibrinogen (fibrinogen in presence of EDTA or chloramine-T)or soluble fibrin. Vancomycin-reacted fibrinogen stimulates tissue type plasminogen activator (t-PA) up to about 20-fold. The experimental data are analyzed by a new significance test: the two foldYates-corrected chi-square comparison against the mean value ofthe control-collective, called the Chi2x - Test. The P < .05 significance barrier calculated with the Chi2x - Test is equivalent to that calculated with the Fisher's Exact Test. The FIATA might be considered an interesting screening test for inactive fibrinogen forms or soluble fibrin, as eg in disseminated intravascular coagulation. Fibrinogen precipitation by vancomycin within the blood vessel might explain why vancomycin has to be infused slowly (< 10 mg/min) to prevent nephrotoxicity. The FIATA is of such a simplicity that the determination of fibrinogen antigen in plasma can be performed anywhere--even outside a hospital--within seconds. Thus, the presented FIATA might contribute to extra hospital testing of patients for assessing their risk for myocardial or cerebral ischemia/infarction.

Antigens↗
PubMed2006

The intrinsic coagulation activity assay.

A new assay for the contact-phase-mediated generation of thrombin activity has been developed - the intrinsic coagulation activity assay (INCA). Citrated plasma (50 microl) is incubated with 5 microl SiO2, 250 mmol/l CaCl2 in polystyrole flat-bottom wells. After exactly 4 and 5 min (37 degrees C) coagulation reaction times (INCA-4 and INCA-5), 100 microl of 2.5 mol/l arginine, pH 8.6, is added to inhibit hemostasis activation in the important ascending part of the thrombin generation curve and to depolymerize fibrin. After 20 min, 50 microl of 1 mmol/l (final concentration 0.24 mmol/l) chromogenic thrombin substrate CHG-Ala-Arg-pNA in 1.25 mol/l arginine, pH 8.7, is added. The increase in absorbance is determined at 405 nm using a microtiterplate photometer. The assay is calibrated against 1 IU/ml thrombin. The normal thrombin activity range of INCA-4 (main value) or INCA-5 (control value) is 100 +/- 30% of normal (mean value +/- 1 SD; 100% = 0.5 IU/ml for INCA-4 and 1.9 IU/ml for INCA-5). With the INCA the normal range of intrinsic hemostasis is reflected, low-molecular-weight heparins can be monitored, the plasma matrix is not changed significantly, and the assay results are a percentage of normal generated thrombin activity and not coagulation seconds.

Blood Coagulation Tests↗
PubMed2006

Inhibition of extrinsic hemostasis activation by low-molecular-weight heparin.

Low-molecular-weight heparins (LMWHs) are very important drugs; unfortunately, the routine global hemostasis assays activated partial thromboplastin time and prothrombin time are not sensitive to LMWHs. Here the 50% inhibitory concentration (IC(50)) values of heparin and LMWHs on extrinsic thrombin generation are determined. Pooled normal plasma was supplemented with 0-2 IU/ml unfractionated heparin, 0-2 IU/ml LMWH dalteparin, or 0-20 microg/ml pentosanpolysulfate in 5-ml polystyrole tubes (23 degrees C) and tested in the tissue-factor-triggered extrinsic coagulation activity assay (EXCA): 50 microl plasma + 5 microl tissue factor/CaCl(2), 1 and 2 min incubation time at 37 degrees C (coagulation reaction time for EXCA-1 and EXCA-2); + 100 microl of 2.5 mol/l arginine (pH 8.6), 20 min at room temperature; + 50 microl of 1 mmol/l CHG-Ala-Arg-pNA, 1.25 mol/l arginine; increase in absorbance/time at 23 degrees C; calibrator = 1 IU/ml bovine thrombin in 6.7% human albumin replacing the plasma sample; in EXCA-1, about 1 IU/ml thrombin is generated in pooled unfrozen normal citrated plasma. The IC(50) values in EXCA-1 are 0.1 IU/ml heparin, 0.02 IU/ml LMWH, and 4.7 microg/ml pentosanpolysulfate. In ECXA-2 the IC(50) values are 0.07 IU/ml, 0.01 IU/ml, and 4.6 microg/ml, respectively. The EXCA reflects the efficiency of anticoagulants on plasmatic coagulation. It is suggested to adjust the dosage of LMWH according to the EXCA value; about 30% of normal extrinsic thrombin generation might be the correct dose for prophylactic anticoagulation.

Blood Coagulation↗
PubMed2006

In vitro simulation of therapeutic thrombolysis with microtiter plate clot-lysis assay.

Only limited comparable data are available on the clot lysis power of the clinically used plasminogen activators (PA). Here the PA were used at different clinically relevant concentrations, and the lysis of the microclots was determined. A microclot lysis assay was used to study thrombolysis by urokinase, tissue-PA (t-PA), streptokinase, plasminogen-streptokinase activator complex (PSAC), reteplase, or tenecteplase. The clot turbidity served as a tool to determine clot mass: 100 microL fresh microclots were incubated with 25 microL PA in 6% bovine serum albumin (BSA)-phosphate-buffered saline (PBS) and 100 microL BSA-PBS or pooled normal human plasma; that is, the PA were in the liquid supernatant of a plasma clot and were not entrapped in the clot, an assay system comparable to normal physiology. The turbidity was determined after 0 to 5 hours (37 degrees C) by a microtiter plate reader. The lysable clot turbidity (clot mass) was expressed in percent of 100% lysable clot control. The clot lysis activity is 100% minus the clot mass in percent. The effective doses at 50% (ED(50)) of lysis of fresh clots after 4 hours (37 degrees C) with 6% BSA or pooled normal human plasma in the clot-supernatant were urokinase 128 or 180 IU/mL; t-PA 0.3 or 0.2 microg/mL; streptokinase 215 or 1371 IU/mL; PSAC 60 or 91 U/mL; reteplase 664 or 996 U/mL; tenecteplase 0.2 or 0.2 microg/mL. The presence of a plasma thrombus with plasma supernatant increases the activity of t-PA approximately 20-fold and that of tenecteplase approximately 400-fold after 4 hours (37 degrees C), when compared to urokinase; in contrast, the lytic activity induced by reteplase decreases; i.e., the plasmin generated by reteplase is hampered on its lytic action against a thrombus. When comparing the clot lysability of microclots of 29 different donors, the only correlation (r > 0.6) was that between u-PA and t-PA. The lysability of individual clots by PA can be measured with the present routine-suited technique. It is suggested that different thrombolytic agents or concentrations thereof would have a different clinical outcome in different individuals.

Dose-Response Relationship, Drug↗
PubMed2004

Regulation of hemostasis by singlet-oxygen (1DeltaO2*).

Hemostasis is the system of generation and destruction of thrombi. It consists of coagulation and thrombolysis and has a plasmatic part and a cellular one, the latter being the thrombocytes and endothelial cells for coagulation and the polymorphonuclear granulocytes (PMN) for thrombolysis. Main products of PMN are oxidants of the hypochlorite/chloramine-type that can generate the nonradical excited oxidant singlet molecular oxygen ((1)DeltaO(2)(*)). Physiologically, (1)DeltaO(2)(*) reacts with methionine and cysteine residues and with carbenic structures in lipids, generating dioxetanes, which upon disruption emit photons in the blue spectrum of light (380-450 nm). It modifies some important hemostasis components in blood: (1)DeltaO(2)(*) inactivates the factors I (fibrinogen), V, VIII, vWF, X, plasminogen activator inhibitor-1 (PAI-1), and alpha2-antiplasmin. (1)DeltaO(2)(*) oxidation of plasminogen and fibrin facilitates their specific cleavage by plasminogen activators and plasmin. Furthermore,(1)DeltaO(2)(*)downregulates thrombocyte-function and upregulates PMN-function. Chloramines seem to be the main physiologic generators of (1)DeltaO(2)(*): in concentrations of 0.1-2 mM in blood they strongly inhibit coagulation and enhance thrombolysis. The biogenesis and reaction pattern of (1)DeltaO(2)(*) is of importance to understand the PMN-physiology in hemostasis, giving rise to new therapy forms of thromboatherothrombosis in man.

Animals↗
PubMed2003

The physiology and pharmacology of singlet oxygen.

Reactive oxygen species (ROS) are generated by many different cells. Singlet oxygen (1O(2)) and a reaction product of it, excited carbonyls (C=O*), are important ROS. 1O(2) and C=O* are nonradicalic and emit light (one photon/molecule) when returning to ground state oxygen. Especially activated polymorphonuclear neutrophil granulocytes (PMN) produce large amounts of 1O(2). Via activation of the respiratory burst (NADPH oxidase and myeloperoxidase) they synthesize hypochlorite (NaOCl) and chloramines (in particular N-chlorotaurine). Chloramines are selective and stable chemical generators of 1O(2). In the human organism, 1O(2) is both a signal and a weapon with therapeutic potency against very different pathogens, such as microbes, virus, cancer cells and thrombi. Chloramines at blood concentrations between 1 and 2 mmol/L inactivate lipid enveloped virus and chloramines at blood concentrations below 0.5 mmol/L, i.e. at oxidant concentrations that do not affect thrombocytes or hemostasis factors, act antithrombotically by activation of the physiologic PMN mediated fibrinolysis; this thrombolysis is of selective nature, i.e. it does not impair the hemostasis system of the patient allowing the antithrombotic treatment in patients where the current risky thrombolytic treatment is contraindicated. The action of 1O(2) might be compared to the signaling and destroying gunfire of soldiers directed against bandits at night, resulting in an autorecruitment of the physiological inflammatory response. Chloramines (such as the mild and untoxic oxidant chloramine T (N-chloro-p-toluene-sulfonamide)) and their signaling and destroying reaction product 1O(2) might be promising new therapeutic agents against a multitude of up to now refractory diseases.

Chloramines↗
PubMed2003

Singlet oxygen (1O2)-oxidazable lipids in the HIV membrane, new targets for AIDS therapy?

Human immunodeficiency virus (HIV) is a lipid enveloped virus. The lipid envelope differs significantly from the lipid membrane of normal human cells: it contains high amounts of cholesterol, that is of importance for the virus-cell interaction (for entry and exit of the virus) at so-called lipid rafts. Cholesterol, as a R-C=C-R compound possesses an oxidazable carbenic bond. The present work suggests the inactivation of HIV by oxidation of viral cholesterol and/or unsaturated fatty acids. For oxidation, the relatively mild oxidant singlet oxygen (1O(2)) might be used. 1O(2) is generated by redoxcyclers (e.g., of the quinone type, such as vitamin K) or by chloramines (e.g., taurine-chloramine). At the 1O(2) concentrations necessary to inactivate lipid enveloped virus in human blood the oxidation-sensible critical hemostasis parameters such as thrombocytes and fibrinogen are only partly inactivated. Therefore, it is proposed to consider generators of 1O(2) as a new form of AIDS therapy.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Point of care: diagnostics in hemostasis--the wrong direction?

Point-of-care-testing (POCT) is performance of a laboratory assay outside the laboratory by nontrained personnel. The advantages of POCT are: more rapid medical decisions, avoidance of long sample transports, and small samples. The disadvantages of POCT are: no laboratory personnel, insufficient calibration, quality control and maintenance, poor documentation, high costs, difficult comparability POCT/central laboratory. Therefore, disposing of a 24-hour central laboratory, the POCT spectrum should be limited to the vital parameters: K+, Ca++, Na+, glucose, creatinine, blood gases, hemoglobin or hematocrit, NH3, lactate. POCT offers no advantages, if the hospital has a rapid transport system such as a pneumatic delivery to the central laboratory. The rapid diagnosis of the acute hemostasis state of a patient should be performed in the 24-hour central laboratory that is connected to all hospital wards via a good pneumatic delivery.

Biomarkers↗