Biomedical subjects

G Rollason

Publications and source records attributed to G Rollason.

5 recordsLinked to original sources

PubMed1993

Preliminary report on cell culture on a thermally reversible copolymer.

Anchorage-dependent cells (NRK 49F) were grown at 37 degrees C in serum containing medium on glass coated with a N-isopropyl acrylamide/N-t-butyl acrylamide copolymer (NIPAaM/NTBAM) with a lower critical solution temperature (LCST) of 8 degrees C. After incubating the dishes and adherent cells at 4 degrees C for 1 h, the cells had started to detach in 'sheets', without the use of trypsin; by 2 h the cells had fully detached and formed aggregates. We presume that at 4 degrees C, below the LCST of the coating, the polymer swelled and eventually dissolved, leaving the cells unattached to the solid substrate. We speculate that the 'sheet' morphology of the detaching cells implies that the extracellular matrix produced by the cells during culture was left intact, which may then alter the nature of their subsequent behaviour. The polymer was not optimum for cell attachment and optimization would be needed to realize the potential of this novel mode of cell culture and recovery.

Acrylamides↗

PubMed1992

Measurement of the rate of thrombin production in human plasma in contact with different materials.

Thrombin production in plasma in contact with various materials was consistent with a first-order autocatalytic model (d[T]/dt = kp[T]; [T] = thrombin concentration, t = time, kp = thrombin production rate constant) since the initial portion of a semilogarithmic plot of thrombin concentration against time was linear. Thrombin concentration was measured in clotting plasma (phospholipid enhanced or platelet-rich plasma) using a fluorogenic substrate (BMCA) by aliquot sampling at various intervals or more conveniently by monitoring cumulative fluorescence. The latter was generated by the action, on BMCA incubated in the clotting plasma, of the thrombin as it was generated. The thrombin concentration was determined from the first derivative of the S-shaped cumulative fluorescence curve. kp was greater for glass (7.92 x 10(-3) cm/s) than for the other materials (polypropylene, polystyrene, polyethylene and PVA; kp approximately 3.1 x 10(-3) cm/s) in plasma with cephalin without flow. A kp for heparin-PVA could not be determined since the thrombin concentration was too low to be quantified. A larger difference between polyethylene and PVA was noted with platelet-rich plasma without flow while lower values (1.0 x 10(-3) cm/s) were noted in a flow system but at a higher surface to volume ratio. The first-order rate constant can be used in simple models relating production of thrombin at a wall of a tube to its mass transfer away from the wall in flowing blood. One such model predicts that the concentration of thrombin at the wall should become infinite at the point in the tube when the mass transfer coefficient equals kp. According to this model, kp on the order of 10(-4) cm/s would be a useful target for a nonthrombogenic material.

Amino Acid Sequence↗

PubMed1989

Inactivation of thrombin in heparin-PVA coated tubes.

Heparin, immobilized to polyvinyl alcohol by reaction with glutaraldehyde (heparin-PVA), retained its ability to accelerate the antithrombin III inactivation of thrombin, in a recirculating flow loop using heparin-PVA coated polyethylene tubes. The extent of inactivation, for a constant flow time, was approximately constant over ten cycles of exposure to thrombin and antithrombin III, suggesting that the immobilized heparin was reusable, as expected from the catalytic nature of non-immobilized heparin. Assessment of the chromogenic substrate activity of adsorbed thrombin and the extent of displacement were less conclusive with the implication that thrombin is adsorbed to heparin-PVA or PVA without heparin in multiple states.

Adsorption↗

PubMed1986

Factor Xa inactivation by a heparinized hydrogel.

Incubating Factor Xa and plasma in the presence of "beads" of a heparinized hydrogel (heparin-PVA) resulted in a loss of Factor Xa activity as measured by clotting time or chromogenic substrate assays. This loss in activity was attributed to the activity of the heparin immobilized to the polyvinyl alcohol (PVA) hydrogel. The immobilized heparin retained an apparent activity approximately 1% that of a similar mass of heparin in solution. The loss in apparent activity was in part attributed to diffusion effects which limited the accessibility of the enzyme and inhibitor to the heparin immobilized in the interior of the gel "beads". The chromogenic substrate activity of adsorbed Factor Xa was also measured and was found to decrease after exposure to antithrombin III to similar extents regardless of the sequence of exposure. This suggests that Factor Xa and antithrombin III have similar affinities for this immobilized heparin, unlike the situation for thrombin (Thromb-Res., 20, 543-554, 1980).

Factor X↗

PubMed1983

Fate of thrombin and thrombin-antithrombin-III complex adsorbed to a heparinized biomaterial: analysis of the enzyme-inhibitor complexes displaced by plasma.

Heparin covalently-linked to polyvinyl alcohol (PVA) is a biomaterial which is of potential value as a non-thrombogenic coating. 125I-labelled thrombin adsorbed to heparin-PVA beads was not dislodged by phosphate-buffered saline, pH 7.4, although radioactivity was progressively displaced from the adsorbent by fibrinogen-free human plasma. Analysis by gel filtration and affinity chromatography showed that the released radioactivity was distributed between (thrombin-antithrombin-III) complex (approx. 70%) and, probably, (thrombin-alpha-2-macroglobulin) complex (approx. 30%). Less efficient thrombin displacement was obtained by either bovine serum albumin (5% w/v) or antithrombin-III-free human plasma: in the latter case, the dislodged enzyme was presumably associated with alpha-2-macroglobulin. Purified alpha-2-macroglobulin did not displace thrombin from heparin-PVA. The quantity of thrombin displaced by an alpha-2-macroglobulin-free plasma fraction compared well with fibrinogen-free plasma: The eluted enzyme was largely associated with antithrombin-III. Purified antithrombin-III did not displace thrombin from heparin-PVA despite causing greater than 70% inactivation of the bound enzyme. Subsequent treatment with fibrinogen-free plasma dislodged (thrombin-antithrombin-III) at a similar rate to that of bound thrombin. We conclude that plasma contains a component(s) which displaces (thrombin-antithrombin-III) complex from immobilised heparin: presumably this leaves the heparin sites free for further use in enzyme inactivation.

Antithrombin III↗