Int. J. Biochem. Vol. 16, No. 5, pp. 553-559, 1984 Printed in Great Britain. All rights reserved 0020-711X/84 $3.00 + 0.00 Copyright 0 1984 Pergamon Press Ltd zyxwvut A MECHANISM FOR FIBRIN MONOMER AGGREGATION BASED ON A KINETIC STUDY J. L. USERO, F. J. BDRGUILLO*, C. IZQULBRDO, A. DEL ARCO and M. A. HERR&Z Department0 de Quimica F&a, Facultad de Quimica, Universidad de Salamanca, Espaiia (Received 2.5 JuEy 1983) Abstract-l. A kinetic study was carried out of the aggregation of fibrin monomers under different reaction conditions. 2. At either acid or base pH values, second-order kinetic processes were observed throughout the concentration range studied. 3. At neutral pH (6.8 g pH < 7.3) the kinetics were of second order at fibrin monomer concentrations of less than 0.3-0.4 mg/ml, while at higher concentrations they changed to first order. 4. Both the second order rate constants and the opacity of the fibrin gels are highly dependent on pH, ionic strength, the concentration of calcium ions and on tem~rature. 5. A three-step reaction mechanism is proposed for the aggregation of fibrin monomers to explain the kinetic behaviour observed in the different reaction media. INTRODUCTION The aggregation of fibrin monomers, the finai stage in blood clotting, has been widely studied at molecu- lar level by methods such as electron microscopy, light scattering or X-ray diffraction (Hantgan et al., 1979, 1980; Fowler et al., 1981; Cavazza et al., 1981 and many others). This kind of research seems to agree with the proposition of a two-step mechanism. In the first step, the fibrin monomers polymerize linearly, giving rise to a first set of fib& called protofibers which after achieving a given length (~antgan, 1979) aggre- gate lateraliy in a second step to form the true clot-forming fibre. However, few works deal with the system from a kinetic point of view and as yet there is no agreement regarding the kinetic equations governing the pre- viously proposed mechanism. Thus, Nakanishi et aE. (1980), using a turbidimetric method proposed sec- ond order kinetics with respect to the fibrin mono- mers for their overah aggregation reaction, while Kaibara and Fukada (1980) using viscoelasticity measurements reported first order kinetics for each step of the reaction. In a previous paper (User0 et al., 1981) we reported kinetics whose overall order varied between 1.6 and 1.9, measuring opacity at 350 nm, though no reaction mechanism was proposed and neither was it possible to explain such variations in the overall order. The present work, based on a systematic kinetic study, proposes a reaction mechanism for the aggregation of fibrin monomers which does explain the discrepancies observed up to the present in the determination of a kinetic order for the overall reaction and for each step that consti~tes it. At the same time, studies were carried out on the effects of pH, ionic strength, *Present address: Departamento Flsicoquimica, Fact&ad Farmacia, Uni~rsid~ de Salamanca, Espaiia. concentration of calcium ions and temperature both on the aggregation kinetics of fibrin monomers and on the structure of the gels formed. MATERIALS AND METHODS zyxwvutsrqponmlk Preparation of fibrin monomers Fibrin monomers were obtained from Kabi grade L human fibrinogen (AB Kabi, Stockholm) previously treated for 6 hr at 0°C with a suspension of barium sulphate and EACA to remove possible contamination by plasminogen (Regaiion eb al., 1977a,b). Following this, the fibrinogen was subjected to successive dialyses against O.lM sodium chio- ride and then treated with Fibrindex human thrombin (Ortho Diagnostic Inc.) at a final concentration of 5 units (NIH)/ml for 24 hr at 0°C. The fibrin coagulate obtained kas centrifuged and washed repeatedly in dis&d water and was finally redissolved in 5 M urea. The resulting solution was dialyzed again for 72 hr at 0°C against IO-‘M AcOH/AcO- buffer, pH = 4.7 and divided into aliquots which were frozen at -20°C until they were used. The solution of fibrin monomers obtained in this way was examined s~c~ophotomet~cally at 28Onm, using an ex- tinction coefficient of 1.29 mg-’ ml cm-’ calculated pre- viously by a gravimetric method (User0 et al., 1981). The fibrin monomers thus prepared are stable for 24 hr at 4°C while at -20°C they remain stable for a minimum of 6 days. Kinetic procedure The aggregation of fibrin monomers was studied by continuous measurement at 350 nm of the opacity of the clot formed with a Schimadzu Q-V 50 spectrophotometer equipped with an automatic Servoscribe I-S register and a the~ostat~ble cuvette holder. After a short induction pe- riod, the kinetic curves obtained showed a sharp rise in the slope. Initial aggregation rates were always calculated from the maximum slope of the kinetic curves, these latter showing a standard deviation ranging between 4 and 7% in the rate range studied, as confirmed by experiments with 5 replicates at 7 different rates. The calculation of the slope was carried out by the least squares method. The rates were always referred to the concentration of fibrin monomers clotted during the time period in question by the use of the ~rres~n~ng extinction coefficient in each 553