Varju, I and Kolev, K and Keresztes, Z and Pap, A and Tenekedjiev, K and Machovich, RA, Fractal kinetic models of plasmin-catalyzed dissolution of fibrin, Journal of the International Society on Thrombosis and Haemostasis, 11 (S2), 29 June - 4 July 2013, Amsterdam, The Netherlands, pp. 791-792. ISSN 1538-7933 (2013) [Conference Extract]
Background: Intravascular fibrin clots are resolved through the proteolytic action of plasmin acting at the interface of gel-phase substrate and fluid-borne enzyme. The classic Michaelis-Menten (MM) kinetic scheme cannot describe satisfactorily this heterogeneous-phase proteolysis, because it assumes homogeneous well-mixed conditions. A more suitable model for these spatial constraints, known as fractal kinetics, includes a time-dependence of the Michaelis constant Km = Km0.(1 + t)exp(hF), where hF is a fractal exponent of time, t. Furthermore, a realistic kinetic model should take into account sequestration of plasmin due to kringle binding to C-terminal lysines (CTL), newly exposed during fibrin degradation. Aim: The aim of the present study is to build up and experimentally validate a mathematical model that adequately describes the kinetics of plasmin-catalyzed fibrin dissolution and thus contributes to a better understanding of the factors that influence plasmin efficiency at the fluid-gel interface.
Method: Two modifications of the basic MM scheme were introduced: a term reducing the enzyme concentration through rapid equilibrium binding of plasmin to continuously increasing unproductive sites including an association constant Ka and a fractal exponent, hF resulting in apparent Km, which accounts for the time-dependent clustering of the enzyme. A broad range of biochemical (fibrin turbidimetry, densitometry of electrophoretic samples, amidolytic assay on synthetic plasmin substrate) and imaging (atomic force microscopy, AFM; confocal laser microscopy, CLM) techniques were applied to test the predictions of the fibrinolytic model. The power of the predictions was assessed using known modifiers of fibrinolysis; e-amino caproic acid (EACA) which blocks the kringle-dependent binding and carboxypeptidase B (CPB) which removes CTLs. The clustering of plasmin was evaluated with AFM using nanogold-labeled anti-plasmin antibodies in an experimental setup where plasmin was applied to a mica surface decorated with fibrinogen using microcontact printing and with CLM using fluorescent protein-fusion derivative of plasminogen.
Results: Using a range of fibrin and plasmin concentrations, variants
of the kinetic model were fitted to the turbidimetric data for lysis of
fibrin by plasmin applied to the surface of the clots. A global fit to 32
time-course curves with 90 measured points each yielded four model
parameters with optimal values Km0 = 1.5 然, hF = 0.25,
Ka = 1.3 然-1 and kcat = 32.4 min-1. Addition of 1 mM EACA or
8 U/mL CPB increased the lysis rate, which could be satisfactorily
explained with unchanged Km0 and kcat model parameters accompanied
by a decrease in hF to 0.031 and in
Conclusion: These data, from multi-faceted, complementary approaches, support a mechanism for time-dependent loss of plasmin activity resulting from spatial redistribution in this heterogeneous system. Thus, plasmin-CTL binding retards lysis, opposing the stimulatory effect of CTLs in plasminogen activation.
|Item Type:||Conference Extract|
|Keywords:||mathematical modelling, simulation, fractal kinetic models, plasmin-catalyzed dissolution|
|Research Division:||Mathematical Sciences|
|Research Field:||Applied Statistics|
|Objective Division:||Expanding Knowledge|
|Objective Group:||Expanding Knowledge|
|Objective Field:||Expanding Knowledge in the Information and Computing Sciences|
|Author:||Tenekedjiev, K (Professor Kiril Tenekedjiev)|
|Deposited By:||NC Maritime Engineering and Hydrodynamics|
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