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The amidolytic activity of plasmin (Pln) that spontaneously adsorbs from solutions to modified-graphite (GR) and glassy carbon (GL) surfaces was studied in the 10-45 degrees C temperature range in the presence of a chromogenic substrate. Surfaces were modified with a coating of fibrinogen, either electrochemically oxidized or not. The effect of an additional modification via deposition of Langmuir-Blodgett films of behenic acid (BA-LB) onto the former surfaces, thus leading to either hydrophobic or hydrophilic surfaces according to the number of deposed layers, was examined. In all cases results showed the occurrence of a first order transition which strongly and transiently increases the surface activity of Pln. At BA-LB surfaces, the most prominent change compared with other coatings was a significant enhancement of the critical temperature Tc that characterizes the beginning of the transition. When fibrinogen was present in the solution, the transition was no longer observable up to 37 degrees C as shown by the linear kinetics exhibited by surfaces bearing oxidized fibrinogen.

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Whether the in vitro activation of factor XI in plasma is mediated by thrombin or by auto-activation remains a controversial question. In this context, we have simulated theoretical activated partial thromboplastin time (aPTT) by means of a program based on a body of 22 essential elementary reactions implemented with rate constants quoted in current literature. To meet self-consistency in input data issued from varying sources, the results were optimized using the simplex treatment. The performance of the model was systematically evaluated considering the extent of the deviations observed between predicted aPTT and laboratory measurements conducted on normal and factor VIII, IX, XI and XII single-factor deficient plasma. The influence of the auto-activation or thrombin-mediated activation of factor XI on these aPTTs was tested separately after insertion of these reactions in the model. According to the best fits, a mechanism accounting for an auto-activation reaction of activated factor XI rather than a positive feedback reaction mediated by thrombin seemed more likely. Based on this conclusion, a chart of self-consistent rate constant values accounting for the intrinsic pathway of coagulation under static conditions is proposed.

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The effect of zinc ions on kinetic and equilibrium steps that may contribute to the activation and subsequent autoactivation of human blood coagulation factor XII in the presence of 500-kDa dextran sulfate was studied. To analyze the results, the expression of the overall autoactivation constant that had been established from the model presented in a previous study was used. Comparison of the kinetics obtained at different levels of zinc, which included amounts lower than the residual concentration introduced by NaCl in the incubation mixture, suggested that the addition of Zn2+ up to 5 microM lowered the mean number of sites available for the binding of factor XII to the surface from 220 to 172 and increased the rate of the first-order activation of factor XII by one order of magnitude, from (1.6 +/- 0.4) x 10(-4) s(-1) to (8.0 +/- 0.4) x 10(-4) s(-1) in the presence of 550 nM dextran sulfate. Neither the factor XII/surface dissociation constant (1 microM), the apparent catalytic constant, nor the apparent Michaelis-Menten constant associated with the postulated multi-stage kinetic sequence were affected by the presence of zinc. Most experimental trends induced by the presence of zinc could be successfully interpreted by using the model, thus reinforcing its reliability under different conditions.

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The autoactivation kinetics of purified factor XII (FXII) in the presence of dextran sulfate of 500000 Da was reexamined assuming the existence of two preceding activation steps. Kinetics were numerically simulated by using rate and equilibrium constants related to surface-bound species. Relevant feature parameters related to the polymer (number of binding sites and concentration, dissociation constant of FXII from the surface) and the zymogen (concentration. Michaelis-Menten constant of the autoactivation reaction, catalytic rate constant) were accordingly introduced in the mechanisms. Depending on the rate-limiting step i.e. whether the polymer or FXII predominates, numerical simulation analysis led to obtain for the observed autoactivation rate constant (kobs) two explicit expressions which included the contributing variables. One of the two proposed models was in good accordance with the experimental data obtained in this study and with others published previously. We were able to estimate the mean number of the FXII-activating sites supported by the polymer chains (220) and the equilibrium dissociation constant of FXII from the surface (1 microM). Further treatment led us to determine surface-concentration-independent constants (K(m) = 2510 nM and kcat = 0.01 s-1), as well as the rate constant (k1 = 1.6 x 10(-4) s-1) of the postulated first-order activation rate aimed at explaining the formation of the first trace amounts of FXIIa via an intramolecular mechanism. Overall, the treatment applied to the dextran sulfate case offers a quantitative tool by which data determined in the presence of other activating materials can be rationalized.

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Direct potential-mediated and time-controlled activation of purified human factor XII (FXII) immobilized on carbon has been demonstrated. Initial experiments were required to find a procedure for characterizing the immobilization of FXII and its activated form, factor XIIa (FXIIa). After achieving saturation of the carbon surface with FXII, surface catalytic activities could be generated under negative potential conditions. Activities depended on the duration and amplitude of the polarization applied to the immobilized FXII. The activities thus electrochemically induced in the surface-bound FXII molecules were tested biologically in the presence of normal human plasma and FXII-deficient plasma. The shortening of the activated partial thromboplastin time test suggested identity of the catalytic activities with that of activated FXII molecules. The electrochemical activation mechanism was consistently analysed according to first-order kinetics. The apparent rate constants increased in the presence of zinc ions.

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Until recently the investigation of length-dependent effects in cardiac muscle was restricted to multicellular preparations. We describe our experimental set-up which for the first time, in single cardiac myocytes, permits the effects of changes in cell length on auxotonic contractions (measured by carbon fibre transducers) to be simultaneously recorded with the effects on membrane potential and/or changes in intracellular calcium concentration (using indo-1 AM, acetoxylmethyl form). Consistent with previous findings (in experiments at 20-25 degrees C and 0.25 Hz) we report that following a stretch there was an increase in passive tension and contraction. A stretch which increased sarcomere length by approximately 3% had no significant effect on resting membrane potential or action potential amplitude. There was, however, a significant decrease in the action potential duration (P < 0.01, n = 8). No significant change in the amplitude of the intracellular calcium transient was seen following a stretch but a reduction in its duration was observed (P < 0.025, n = 11). Our observations on intracellular calcium transients are consistent with the hypothesis that, in mechanically loaded preparations, their time course is more dependent on changes in tension than changes in length.

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We prepared a derivative of dextran T40 (average M(r) 43,000) from which fractions of different M(r) but with equal charge density were obtained and tested for their ability to promote autoactivation of human blood coagulation factor XII. The mechanism of autoactivation appeared dependent upon the M(r) of the polymer used. Thus, with polymers of 38,000 M(r) or higher only alpha-factor XIIa was formed and the reaction could be completely described in terms of a simple second-order mechanism of autoactivation. With smaller polymer molecules beta-factor XIIa became a major reaction product and as a result of this the autoactivation kinetics did not adhere to the second-order mechanisms thus far described.

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The surface dependent contact activation of human plasma was studied as a function of the molecular weight of several synthetic macromolecular and watersoluble derivatives: polystyrene sulphonates, dextran methyl-benzylamide sulphonate/carboxylates as well as dextran sulphates as reference materials. Extent of the contact activation was determined by the amidolytic activities generated from cold activation of plasma in the presence of dilutions of the polymers for varying incubation times. The activating properties of the materials have been rationalized by means of a parameter calculated from the shapes of the amidolytic rates vs. incubation time variations. It was found that all the studied polymers behave similarly: contact stimulating properties begin to increase sharply passed a molecular weight of 25,000 and thereafter gradually reach a limiting plateau for Mw = 100,000. Activating capacities estimated for these limiting values have been expressed in term of specific capacities unrelated to Mw effects. These parameters were also compared to that of standard heparin estimated in the same conditions.

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Negatively charged surfaces are known to promote contact activation. The mechanism responsible for increasing affinity for surfaces is not yet quite understood, although the presence of negative charge densities is thought to be a prerequisite. With the availability of soluble dextran derivatives, varyingly substituted with charged methylcarboxylate, methylbenzylamide sulphonate and uncharged methylbenzylamide residues, we were able to discriminate between the contributions of these chemical moieties to contact activation, thus suggesting that the stimulating properties of synthetic negatively charged surfaces should also be described in terms of specific interactions instead of global negative charge density. This could be effected by quantifying the activating capacities as a function of the chemical group composition. A direct correlation linking activating capacities to anticoagulant properties has been observed.

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In-vitro determinations of purified kallikrein on the one hand, of human plasma and whole blood prekallikrein on the other hand have been conducted with the S-2648 electrogenic substrate using the electroanalytical technique. Plasmatic evaluations have been checked photometrically for assessing the reliability of the procedure. Whole blood levels of prekallikrein compare with plasma if the haematocrit of the sample is taken into account.

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