Biomedical subjects

D Billy

Publications and source records attributed to D Billy.

6 recordsLinked to original sources

PubMed1997

Prothrombin conversion under flow conditions by prothrombinase assembled on adherent platelets.

Prothrombin activation by prothrombinase was investigated on platelets adhered onto a fibrinogen-coated coverslip mounted in a flow chamber. Once bound to the fibrinogen, platelets gradually changed their morphology: they developed pseudopods, spread over the surface and finally transformed into balloon-shaped cells. This last morphologic change required the presence of calcium and was accompanied by the exposure of procoagulant phospholipid at the outer membrane as detected by the capability of the platelets to bind fluorescein-labelled annexin V. Prothrombinase complexes were allowed to assemble on these adhered platelets by perfusion with factor Xa and varying concentrations of factor Va and prothrombin. The steady-state rate of thrombin formation during continuous flow increased with the prothrombin concentration but not with the factor Va between 0.05 and 0.5 nM. Once prothrombinase was assembled, factor Xa could be omitted from the perfusion mixture without affecting the steady state rate of thrombin production. Our study demonstrates the efficient ability of the procoagulant surface of adherent platelet to support the assembly of stable prothrombinase complexes. Thrombin production was limited by the rate of supply of prothrombin towards the catalytic surface.

Animals↗

PubMed1995

Prothrombin contributes to the assembly of the factor Va-factor Xa complex at phosphatidylserine-containing phospholipid membranes.

The activation of prothrombin is catalyzed by prothrombinase, a complex of factor Xa and factor Va assembled on a negatively charged phospholipid membrane. We used a tubular flow reactor to identify the relative contributions of factor Va, prothrombin, and the negatively charged phosphatidylserine to the assembly of prothrombinase. Perfusion of phospholipid-coated capillaries with a mixture of factor Xa, factor Va, and prothrombin resulted in a steady-state rate of thrombin production that increased with (i) the phosphatidylserine content of the phospholipid bilayer, (ii) the factor Va concentration, and, most interestingly, (iii) the prothrombin concentration of the perfusion solution. Incorporation of 20 mol % phosphoatidylethanolamine, a phospholipid with poor ability to promote prothrombinase activity, into a 5 mol % phosphatidylserine membrane also increased the steady-state rate of thrombin production. Direct measurements of the amount of prothrombinase in the flow reactor demonstrated that increased catalytic activities were the result of an increased steady-state amount of membrane-associated prothrombinase. Thus, similar turnover numbers of prothrombin activation (3100 min-1) were calculated, irrespective of the phosphatidylserine content of the membrane. We established for membranes with low phosphatidylserine content (< 10 mol%) a linear relationship between the prothrombinase activity and the arithematical product of the factor Va concentration in the perfusion solution and the prothrombin concentration near the catalytic surface. Our results indicate that, in addition to factor Va, prothrombin also is essential to the assembly of prothrombinase at macroscopic surfaces with low phosphatidylserine content. The data further suggest that the prothrombin concentration near the surface, controlled by the prothrombinase activity and mass transfer, is an important regulator of the prothrombinase surface density.

Factor Va↗

PubMed1995

Inhibition of prothrombinase at macroscopic lipid membranes: competition between antithrombin and prothrombin.

The kinetics of inhibition of prothrombinase during prothrombin conversion by antithrombin and antithrombin-heparin complexes was studied in a tubular flow reactor. Prothrombinase was assembled at a macroscopic phospholipid membrane, composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine, deposited on the inner wall of a glass capillary, by perfusion with a factor Xa-factor Va mixture. Measurement of thrombin production allowed estimation of the amount of prothrombinase present at the capillary wall. Perfusion with a mixture of prothrombin and antithrombin or antithrombin-heparin complexes caused a progressive decline of the prothrombinase activity. The rate of inactivation steeply decreased with increasing prothrombin concentrations, indicating competitive inhibition. Analysis of competitive inhibition data requires estimation of the time-dependent substrate concentration, Co, near the prothrombin converting surface using earlier developed transport theory [Billy, D., et al. (1995) J. Biol. Chem. 270, 1029-1034]. It appears that the inhibition rate is proportional to the fraction of enzyme, Km/(Km+Co), not occupied by substrate. The value of Km of prothrombinase estimated from the dependence of the inhibition rate on the prothrombin concentration (Km = 2-3 nM) is in excellent agreement with the value estimated from the substrate conversion rate (Km = 3 nM). Therefore inhibition of prothrombinase by antithrombin and antithrombin-heparin complexes is fully competitive with the substrate: prothrombin. Our results show that prothrombinase assembled on macroscopic lipid surfaces by virtue of its low Km value is protected for inhibition due to highly effective competition of prothrombin with antithrombin for the active site of factor Xa.

Animals↗

PubMed1995

Prothrombin activation by prothrombinase in a tubular flow reactor.

Thrombin production by the phospholipid-bound complex of blood clotting factors Xa and Va (prothrombinase) was studied in a tubular flow reactor. The inner wall of a glass capillary was coated with a phospholipid bilayer of 25% phosphatidylserine and 75% phosphatidylcholine. Prothrombinase was assembled on this bilayer by perfusion with a mixture containing an excess of factor Va (2 nM) and a limiting amount of factor Xa (1-100 pM), either in the absence or presence of prothrombin. The rate of assembly of prothrombinase in the presence of prothrombin appeared to be limited by the transfer rate of factor Xa to the phospholipid surface. A good agreement was found between the predicted mass transfer coefficient for factor Xa and the observed pre-steady state rate of thrombin production. The eventually obtained steady state rates of thrombin production were proportional to the prothrombin concentration and independent of the surface density of prothrombinase. The observed rate of thrombin production was in excellent agreement with the predicted mass transfer rate for prothrombin. Transport-limited prothrombin conversion was observed for prothrombinase densities exceeding 1 fmol/cm2, which corresponds to 0.05% occupation of available binding sites. The kinetic parameters of the reaction were determined at low prothrombinase densities (0.02-0.04 fmol/cm2). Even in this situation the Michaelis-Menten equation had to be corrected for substrate depletion near the catalytic surface. We hereto employed an accurate approximation of the mass transfer coefficient. The kinetic parameter kcat was 60 s-1 and the intrinsic Km had a surprisingly low value of 3 nM. Both parameters were not influenced by the wall shear rate.

Biotechnology↗

PubMed1995

Inhibition of prothrombinase by antithrombin-heparin at a macroscopic surface.

The antithrombin-dependent inhibition of prothrombinase, assembled at a macroscopic surface, was studied under flow conditions utilizing a tubular flow reactor that consists of a phospholipid-coated glass capillary. Prothrombinase activity was determined from steady-state rates of thrombin production upon perfusion with prothrombin and from factor Va-associated factor Xa activity present in the flow reactor. The prothrombinase density was maintained at a low level (0.03 fmol/cm2) to assure that the rate of thrombin production reflected the amount of prothrombinase present in the capillary. Perfusion of the flow reactor with antithrombin resulted in an exponential decrease of prothrombinase activity in time. The second order rate constant (8.5 x 10(4) M-1min-1) is comparable with the rate of inactivation of free factor Xa. Inhibition was much faster when antithrombin was complexed with heparin. The second order rate constants of inhibition decreased with decreasing heparin chain length: 9.6 x 10(7), 4.5 x 10(7) and 0.39 x 10(7) M-1min-1 for unfractionated heparin, low molecular weight heparin and synthetic pentasaccharide heparin, respectively. In the presence of prothrombin (0.2 microM), however, the heparin-dependent rate of inhibition of prothrombinase was about 50-fold lower. The heparin-independent inhibition of prothrombinase by antithrombin (4 microM) in the presence of prothrombin (0.2 microM) was virtually negligible. At a 70-fold higher surface density of prothrombinase (2 fmol/cm2) prothrombinase activity was much faster inactivated. The rate of thrombin production, however, was not affected. In conclusion, at low prothrombinase densities, prothrombin efficiently protects prothrombinase from inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombin III↗

PubMed1993

How does sodium lauryl sulfate alter the skin barrier function in man? A multiparametric approach.

The effects of topically applied sodium lauryl sulfate were studied in vivo, in man, through noninvasive methods and compared with those obtained in vitro, using human stratum corneum as a model. The results show that the very marked increase of transepidermal water loss obtained in vivo cannot be related to any removal of epidermal lipids. The most likely explanation of our findings lies in the hyperhydration of stratum corneum consecutive to the inflammation process and a possible disorganization of the lipid bilayers.

Adult↗