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Biomedical subjects

W T Hermens

Publications and source records attributed to W T Hermens.

At least 73 records · Page 4Linked to original sources
PubMed1993

Adsorption and conversion of prothrombin on a rotating disc.

In most flow systems, the rate of protein transfer from bulk solution to a macroscopic surface is site-dependent. In studies on surface-mediated protein conversion, this hampers the comparison of a proposed expression for the conversion process, such as the Michaelis-Menten equation, which actually measured overall conversion rates. However, the rotating disc is a classical example of a uniformly accessible surface and therefore was used for a quantitative analysis of prothrombin conversion by the phospholipid-bound factor Xa/factor Va complex (prothrombinase complex). A simple design of a rotating disc, adapted for ellipsometric measurement of protein adsorption, is presented. Agreement between experiment and theory was obtained for the influence of rotation velocity on the initial, transport-limited, adsorption rates of lysozyme, prothrombin, and fibrinogen. After coverage of the disc with a 20% phosphatidylserine/80% phosphatidylcholine bilayer and preadsorption of factor Va, addition of excess factor Xa and prothrombin resulted in effective conversion of prothrombin. For high (10 fmol.cm-2) surface coverage of prothrombinase, the rate of conversion equals the transport limited adsorption rate of prothrombin. For low (0.1 to 0.5 fmol.cm-2) surface concentrations of prothrombinase, the conversion rate dropped below the transport limit and the intrinsic kinetic parameters could be estimated at Km = 7.1 +/- 1.2 nM and kcat = 25 +/- 1.0 s-1 (20 degrees C). At these low surface activities of prothrombinase, the effect of the rotation rate (6 to 225 rad.s-1) on prothrombin conversion could be explained by the rotation-rate dependent prothrombin transport. This indicates that the fluid shear rate has no drastic influence on the intrinsic kinetics of prothrombin conversion.

Adsorption↗
PubMed1993

Aggregation of phospholipid vesicles by a chimeric protein with the N-terminus of annexin I and the core of annexin V.

A chimeric protein was produced with the N-terminal domain (amino acids 1-45) of annexin I and the core of annexin V (amino acids 19-320). This protein, annexin IN-VC, has a similar Ca2+ requirement for binding to phospholipid bilayers of 20% phosphatidylserine (PS)/80% phosphatidylcholine (PC) as annexin V. In contrast to annexin V, this protein has a strong potency to aggregate phospholipid vesicles as is shown by turbidimetric measurements and cryo-electron microscopy. Ellipsometry was employed to study quantitatively the phenomenon of phospholipid vesicle adhesion to annexin IN-VC bound to a planar phospholipid bilayer. The amount of phospholipid vesicles bound by annexin IN-VC on the planar bilayer is proportional to its surface coverage and can be inhibited by coadsorption of annexin V on the planar bilayer or by shielding the phospholipid surface of the vesicles with blood coagulation factor Va. Annexin IN-VC, like annexin V, does not bind to pure PC bilayers, but its adsorption on anionic phospholipid bilayers brings about the capacity to bind pure PC vesicles. This suggests that annexin IN-VC generates or exposes after binding to anionic phospholipids another phospholipid binding site, that differs from the annexin V phospholipid binding site. Collectively, the data suggest that two-dimensional cluster formation of annexin IN-VC on a bilayer with anionic phospholipids is involved in vesicle adherence.

Adsorption↗
PubMed1993

Monitoring of unbound protein in vesicle suspensions with off-null ellipsometry.

In studies on the binding of proteins to small unilamellar phospholipid vesicles (SUV), the concentration of unbound protein usually remains unknown, because the vesicles cannot be separated from the bulk solution. In the present study, this limitation was overcome by addition of a supported planar phospholipid bilayer to the cuvette containing a vesicle suspension. Ellipsometric measurement of the protein adsorption velocities on this bilayer allowed determination of the concentrations of unbound protein. At high protein concentrations the adsorption is rapidly completed and the usual null-ellipsometry is too slow to obtain well-defined initial adsorption rates. Therefore, an off-null technique was developed, allowing measurement of the adsorbed protein mass at time intervals of 20 ms. Binding of prothrombin and coagulation factor Xa was measured in SUV suspensions prepared from a 20% dioleoylphosphatidylserine (DOPS) and 80% dioleoylphosphatidylcholine (DOPC) phospholipid mixture. For prothrombin, a dissociation constant Kd = 140 +/- 27 nM (mean +/- S.E.) and maximal surface concentration gamma max = (8.9 +/- 0.8) x 10(-3) mole of protein per mole of lipid, were obtained. For factor Xa, these values were Kd = 49.6 +/- 6.3 nM and gamma max = (23.0 +/- 1.4) x 10(-3) mole of protein per mole of lipid. These binding parameters are similar to those obtained earlier for planar bilayers. Apparently, the binding of factor Xa and prothrombin is not dependent on surface curvature.

Adsorption↗
PubMed1993

alpha MSH but not Org 2766 induces expression of c-fos in cultured rat spinal cord cells.

Alpha-melanocyte stimulating hormone (alpha MSH) and Org 2766 (an adrenocorticotrophic hormone (ACTH) MSH4-9 analogue) stimulate sprouting after nerve damage in vivo. In cultures of rat spinal cord cells only alpha MSH enhances neurite outgrowth. In order to analyse the mode of action of alpha MSH and to get a clue why its action differs from that of Org 2766, we studied whether expression of c-fos was induced in spinal cord cells by treatment with alpha MSH or Org 2766. We found a rapid and transient induction of both c-fos mRNA and protein after treatment with 100 microM alpha MSH. Treatment with Org 2766 (100 pM-100 microM) had no effect on c-fos expression. These results suggest that c-fos is involved in the signal transduction system of alpha MSH stimulated neuronal outgrowth in vitro.

Adrenocorticotropic Hormone↗
PubMed1993

Circulatory models in assessment of cardiac enzyme release in dogs.

Cardiac ischemia causes interstitial leakage of cellular enzymes followed by release of these enzymes into plasma. Quantitative interpretation of these data requires a specific circulatory model, and the performance of such models was investigated. Plasma activities of cardiac enzymes were measured for increasingly abrupt forms of ischemic heart injury in the dog: 1) permanent ligation of the left anterior descending coronary artery (LAD); 2) reperfusion after 2 h of ligation of the LAD; and 3) calcium-free perfusion of the LAD during 10 min (calcium-paradox injury). Release into plasma of a rapidly (41%/h) and a slowly (2.2%/h) catabolized enzyme was calculated from the plasma activities, using a detailed circulatory model with compartments for heart, plasma, muscle, skin, and viscera. The time course of cellular enzyme leakage into interstitial space in the heart was calculated from release into plasma and a range of reported values for transendothelial permeability. Simplification to one- and two-compartment models introduced, respectively, 10 and 2% error in calculated cumulative release. Considering the other sources of error, this implies adequate performance of the two-compartment model. Protein washout from the heart is strongly influenced by expansion of interstitial protein space with dead myocyte volume and depends on the microheterogeneity of necrotic tissue areas. Accelerated release of enzymes into plasma after reperfusion reflects accelerated cellular leakage rather than enhanced washout.

Alanine Transaminase↗
PubMed1992

Clustering of lipid-bound annexin V may explain its anticoagulant effect.

In 1985 we isolated a new vascular anticoagulant protein VAC alpha, now called annexin V, with a high binding affinity (Kd less than 10(-10) M) for phospholipids. Its anticoagulant effect was attributed to displacement of coagulation factors from the phospholipid membrane. The present study demonstrates that the inhibition of prothrombinase activity by annexin V strongly depends on the curvature of the membrane surface and on the calcium concentration. Half-maximal inhibition of prothrombinase on and binding of annexin V to small vesicles, composed of 20% phosphatidylserine and 80% phosphatidylcholine, requires 2-3 mM calcium. With large vesicles and planar bilayers considerably less calcium is required for inhibition of prothrombinase and for lipid binding. Half-maximal binding of annexin V to large vesicles and to planar bilayers occurs at 0.7 and 0.2 mM calcium, respectively. This seemingly confirms the displacement model. The displacement of coagulation factors, however, proved to be incomplete, with residual surface concentrations of factors Xa, Va, and prothrombin sufficient for effective production of thrombin. Cryoelectron microscopy revealed that annexin V binding to large vesicles caused planar facets, indicating the formation of large sheets of clustered annexin V. Apparently, the formation of these two-dimensional arrays is promoted by calcium and hampered by high surface curvature. It is speculated that the complete inhibition (greater than 99%) of prothrombinase activity by annexin V is caused by the reduced lateral mobility of prothrombin and factor Xa in rigid sheets of annexin V covering the membrane.

Annexin A5↗
PubMed1991

Membrane-mediated assembly of the prothrombinase complex.

Prothrombinase assembly was studied on macroscopic planar bilayers consisting of 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC). The dissociation constant for the binding of factor Xa to the bilayer, measured by ellipsometry, was Kd = 47 +/- 8 nM (mean +/- S.D.) and this value was lowered to Kd = 2.2 +/- 0.3 pM by preadsorption of factor Va. This latter value was determined from direct measurement of steady-state thrombin production. A comparable value of Kd = 1.0 +/- 0.1 pM was found by repeating these experiments in suspensions of phospholipid vesicles, and it was verified that prothrombinase assembly was not influenced by the addition of prothrombin. Using a minute amount (0.094 fmol cm-2) of preadsorbed factor Va, it was found that the rate of prothrombinase assembly exceeds the rate of collisions between Xa molecules from the buffer and the sparse Va molecules on the bilayer. Apparently, factor Xa adsorbs first to the membrane and then associates rapidly with factor Va by lateral diffusion. The data indicate almost instantaneous equilibrium of this complex formation on the surface with a lower limit for the bimolecular rate constant of kon = 2.8 x 10(13) (mol/cm2)-1 s-1. In suspensions of small phospholipid vesicles, prothrombinase assembly is collisionally limited and the value of kon should be proportional to vesicle diameter. This was verified with a method for estimation of kon values from thrombin generation curves. Values of 0.36 x 10(9) and 1.6 x 10(9) M-1 s-1 were found for vesicles of 20-30- and 60-80-nm diameter, respectively.

Animals↗
PubMed1991

Production of thrombin by the prothrombinase complex is regulated by membrane-mediated transport of prothrombin.

Production of thrombin by phospholipid-bound prothrombinase complexes has been described as being regulated by the prothrombin concentration in the buffer (free-substrate model) as well as by the concentration of prothrombin adsorbed to the phospholipid surface (bound-substrate model). We studied simultaneous adsorption and conversion of prothrombin on planar bilayers consisting of 20% dioleoylphosphatidylserine and 80% dioleoylphosphatidylcholine. A transport limitation in the conversion of prothrombin was prevented by using a very low (0.3 fmol cm-2) amount of prothrombinase on the bilayer. The Michaelis and catalytic constants thus found were Km = 5.8 +/- 0.7 nM and kcat = 33 +/- 1 s-1 (mean +/- S.D.). The apparent bimolecular rate constant Kcat/Km = 5.7 x 10(9) M-1 s-1 exceeds the theoretically maximal value for the free-substrate model. In contrast, kcat/Km is within the range expected for a diffusion-controlled bound-substrate model. A similar mechanism for prothrombin conversion in suspensions of phospholipid vesicles would imply increasing kcat/Km values for increasing vesicle diameter. This prediction was tested and a 3-fold increase in kcat/Km values was indeed found for vesicles 60-80 nm in diameter compared to vesicles of 20-30 nm diameter. It is concluded that thrombin production is dependent on protein fluxes rather than on protein concentrations.

Adsorption↗
PubMed1991

Surface exclusion and molecular mobility may explain Vroman effects in protein adsorption.

Data on protein adsorption usually show that for increasing surface coverage the adsorption velocity decreases much faster than linearly. This contrasts to the classical Langmuir model with an adsorption velocity proportional to the number of unoccupied binding sites. It has been shown that this non-linearity may explain phenomena like transient adsorption of different proteins from a protein mixture or dilution-dependent changes in binding properties, collectively called Vroman effects. However, the molecular mechanisms explaining this non-linear behavior remain to be established. A Monte Carlo simulation model is presented that incorporates steric hindrance, lateral mobility and mutual interactions of adsorbed molecules. Experimental data on the adsorption kinetics of prothrombin and annexin V, a recently discovered anticoagulant protein, at phospholipid bilayers are analyzed with this model. A major conclusion is that the steep decline in adsorption rates for increasing surface coverage can be explained, without assuming repulsive forces between adsorbed molecules, as a surface exclusion effect combined with lateral mobility of adsorbed molecules. The fact that annexin V shows this effect to a much lesser degree than prothrombin is tentatively explained by clustering of adsorbed annexin V molecules. A qualitative effect of lateral mobility on the adsorption characteristics, predicted by the model, is confirmed in experiments in which the fluidity of the bilayers was manipulated.

Adsorption↗
PubMed1991

Simulation model for thrombin generation in plasma.

A simulation model for the production of thrombin in plasma is presented. Values of the reaction rate constants as determined in purified systems are used and the model is tested by comparison of simulations of factor Xa, factor Va and thrombin generation curves with experimental data obtained in thromboplastin-activated plasma. Simulations of the effect of hirudin indicate that factor V is predominantly activated by thrombin and not by factor Xa. The model predicts a threshold value for the factor Xa production which, if exceeded, results in explosive and complete activation of prothrombinase. The dependence of this threshold value on different negative feedback reactions, e.g. the inactivation of thrombin and factor Xa by antithrombin III (+ heparin), is investigated. The threshold value, for control plasma in the range of 1-10 pM total factor Xa production, can be raised two orders of magnitude by accelerated inactivation of factor Xa and prothrombinase but is hardly affected by a tenfold increase in the rate of thrombin inactivation or by increased production of activated protein C. This latter effect, however, results in a more gradual input-response relation between factor Xa input and the extent of prothrombinase activation.

Computer Simulation↗
PubMed1990

Binding of vascular anticoagulant alpha (VAC alpha) to planar phospholipid bilayers.

Vascular anticoagulant alpha (VAC alpha, annexin V) is a member of the family of calcium and phospholipid binding proteins, the annexins. The binding properties of VAC alpha to phospholipid bilayers were studied by ellipsometry. Adsorption was calcium-dependent and completely reversible upon calcium depletion. Half-maximal adsorptions to phospholipid bilayers consisting of 100, 20, 5, and 1% dioleoyl-phosphatidylserine (DOPS) supplemented with dioleoyl-phosphatidylcholine (DOPC) were reached at Ca2+ concentrations of 0.04, 0.22, 1.5, and 8.6 mM. These surfaces all showed the same maximal adsorption of 0.22 +/- 0.01 micrograms of VAC alpha/cm2 (mean +/- S.D.). The adsorption to bilayers containing more than 10% DOPS was independent of VAC alpha concentrations in the range of 0.5-100 nM. Dissociation constants for VAC alpha binding to these surfaces were estimated to be below 2 x 10(-10) M. No adsorption was observed on pure DOPC bilayers at a Ca2+ concentration of 3 mM. The ability to mediate VAC alpha binding to 20% DOPS/80% DOPC bilayers was highly specific for Ca2+. The use of other divalent cations resulted in decreased binding in the order Cd2+ greater than Zn2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+. Zinc ions had a synergistic effect on Ca2(+)-dependent VAC alpha binding. The Ca2+ concentration needed for half-maximal binding to cardiolipin, dioleoyl-phosphatidylglycerol, DOPS, phosphatidylinositol, phosphatidic acid, dioleoyl-phosphatidylethanolamine, and sphingomyelin increased in that order. Adsorption was independent of the overall surface charge of the phospholipid membrane.

Adsorption↗
PubMed1990

Complete recovery in plasma of enzymes lost from the heart after permanent coronary artery occlusion in the dog.

Plasma activities of creatine kinase (CK) and alpha-hydroxybutyrate dehydrogenase (HBD) were measured after permanent coronary artery occlusion in the dog. Cumulative release of enzymes in plasma was calculated from these data by using a previously validated two-compartment model for circulating enzymes. Regional myocardial ischemia was measured by injection of radiolabeled microspheres. After 48 hours, the dogs were killed, and a detailed map of left ventricular enzyme activity was obtained from 108 tissue samples. Cumulative release into plasma of CK and HBD was 96 +/- 20% and 112 +/- 26%, respectively, of the total activities depleted from the heart (mean +/- SD, n = 11). The scatter in these values is inherent to the calculations, and it is concluded that both enzymes are recovered completely in plasma and, thus, can be used as quantitative markers of injury. Discrepancies between this result and earlier reports on the recovery of CK are only partly apparent and can be explained partly by underestimation of the elimination rate of CK from plasma, irregardless of tissue edema and incomplete extraction of enzyme activity from tissue.

Animals↗
PubMed1990

Time course of cellular enzyme release in dog heart injury.

The transport time of enzyme from heart to plasma was studied in two experimental models. First, the enzyme alanine aminotransferase was slowly infused into the left ventricular wall in open-chest dogs. The half-life for the washout of alanine aminotransferase activity into plasma was 20 +/- 4 minutes (mean +/- SEM, n = 8) and was not different in ischemic and normally perfused tissue. From measurements of arteriovenous differences in alanine aminotransferase activity and left ventricular blood flow, it was concluded that 77 +/- 14% of total enzyme washout from ischemic tissue occurred by direct entry into the bloodstream. The corresponding value for the vascular permeability-surface area product was 264 +/- 55 ml.kg-1.hr-1. For a second model, we studied myocardial enzyme release into plasma after abrupt heart injury induced by 10 minutes of calcium-free coronary perfusion followed by reintroduction of calcium (calcium-paradox mechanism). The half-life for the release into plasma was 1.9 +/- 0.2 hours (mean +/- SEM, n = 6) and was again not influenced by sustained ischemia. Slower washout, as observed for this second model, is consistent with increased interstitial protein space and corresponds to a permeability--surface area product between 135 and 285 ml.kg-1.hr-1. These results were used to calculate the time course of cellular enzyme leakage from the rate of enzyme release into plasma in various forms of heart injury. Significant shifts between the time curves of evolving cellular injury and enzyme release into plasma are observed after 2 hours of ischemia followed by coronary reperfusion, but not after permanent ischemia.

Alanine Transaminase↗