Proc. Nati Acad. Sci. USA Vol. 80, pp. 1616-1620, March 1983 Biophysics Fibrinogen and fibrin structure and fibrin formation measured by using magnetic orientation (Cotton-Mouton effect/magnetic birefringence/secondary structure/fiber structure/ldnetics of polymerization) J.-M. FREYSSINET*, J. TORBETt, G. HUDRY-CLERGEON*, AND G. MARETt *Laboratoire d'H6matologie, Unite 217 Institut National de la Sante et de la Recherche Mdicale, D6partement de Recherche Fondamentale, Centre d'Etudes Nuclkaires, 85 X, F-38041 Grenoble Cedex, France; and tHochifeld-Magnetlabor des Max-Planck-Institutes fir Festkorperforschung, 166 X, F-38042 Grenoble Cedex, France Communicated by Manfred Eigen, December 21, 1982 ABSTRACT Accurate birefringence measurements show that packwith three-dimensionalorder, probably in atetragonal unit fibrinogen orients to a small degree in high magnetic fields. This cell with a = b = 185 A and c = 446 A and containing eight mol- effect can be explained as due to the molecule having about 30% ecules (14). The mechanism of assembly of fibrin has been ex- (by weight) a-helix oriented relatively parallel to the long axis. Bi- tensively studied (2, 15-18) but due to its complexity and the refringence measurements on fully oriented fibrin suggest that limitations of the experimental investigations many important aligned a-helical content is less than that estimated for fibrinogen. points remain to be elucidated. It is important to have as precise But because of limitations in the analysis this difference must be knowledge as possible about the fibrin polymerization process; viewed with caution. Highly oriented fibrin results when poly- not only is it of interest in itself but also it may be an excellent merization takes place slowlyin a strong magneticfield. Low-angle model for other aggregation processes of biological macromol- neutron diffraction patterns from oriented fibrin made in the ecules (19). presence of EDTA, made in the presence of calcium, or stabilized the orien- with factor XIIIa are very similar, showing that the packing of the The present study shows that measuring gradual molecules within the fibers is the same or very similar in these dif- tation of fibrin in a high magnetic field reveals kinetic features ferent preparations. The induced magnetic birefringence was used of the polymerization reaction, and it also demonstrates that to follow fibrin formation under conditions in which thrombin was structural details of the secondary structure of the fibrinogen rate limiting. The fiber network formed by approximately the ge- molecule or the fibrin monomer can be assessed by studying lation point constitutes a kind of matrix or frame that is largely their behavior in the field. built upon during the remaining ==85% of the reaction. After ge- lation the reaction is pseudo-first order. MATERIALS AND METHODS The arrest of blood loss from an injured vessel, hemostasis, re- Protein Preparation. Purified bovine fibrinogen (>98% quires the participation of several plasma proteins and also clottable protein) was obtained as described in ref. 20. Unless platelets, cells that form occlusive aggregates at the site of the specified otherwise the experiments were performed in 0.05 M rupture. The last stage of the blood clotting process is the en- Tris'HC1 buffer containing 0.1 M NaCl, 0.5 mM EDTA, and zyme-catalyzed activation of a soluble plasma protein, fibrin- 0.01% (wt/vol) NaN3 (pH7.5). Thrombin and reptilase (Both- ogen, which then undergoes polymerization to form an insol- rops atrox serine proteinase) were purchased from the Institut uble fibrin gel, thus mechanically reinforcing the platelet plug. de Serotherapie Hematopoietique (Paris) and Laboratoire Stago The limited cleavage of fibrinogen by thrombin, a serine pro- (Asnieres, France), respectively. All measurements were made teinase, is the result of a series of steps involving many other at 200C. clotting factors; much is known about this sequence of highly Samples for neutron diffraction experiments were made by regulated events (for a recent and exhaustive review see ref. 1). forming fibrin (polymerization time 1 hr) in a magnetic field Thrombin also converts factor XIII into factor XIIIa, the plasma that had an average value of about 15 teslas (1 tesla, T, = 104 transglutaminase which, in the presence of calcium, crosslinks gauss). The orientation was performed in lH20 buffer, which adjacent fibrin monomers of a fiber by forming E-(y-gluta- was subsequently replaced as required with 2H20 buffer by dif- myl)lysyl pseudo peptide bonds (2). fusion. The sample thickness was 0.1 or 0.2 cm. The trinodular elongated (450-A-long) structure for the fi- Measurements of the Magnetically Induced Birefringence. brinogen molecule proposed by Hall and Slayter (3) is the most The samples were contained in quartz cells that had an optical widely accepted model, and it has obtained additional support path length of3, 1, or 0.1 cm. These were placed in a temper- from recentwork on native fibrinogen (4-8) or slightly modified ature-stabilized (±0.1C) sample holder within a Bitter type fibrinogen (9-11). Fibrin monomers are produced by thrombin, magnet (maximal field 13.5 T) that had a small radial optical which releases the small negatively charged fibrinopeptides A bore. The magnetic birefringence An was sensitively measured and B. The monomers associate in a longitudinal half-staggered (resolution An 10-10, A = 6,328 A) by using a combined pho- arrangement to generate the two-stranded fibrin protofibril (12, toelastic modulation and compensation technique (21). Polar- 13), then these protofibrils associate laterally to form the thicker izer and analyzer were crossed and at 450 with respect to the fibrin fibers (12). In a recent study, we have shown that when field direction. A 50-kHz modulation of the birefringence was polymerization of fibrin takes place slowly in a high magnetic produced by a photoelastic modulator, resulting in a 100-kHz field one ends up with a highly oriented gel on which neutron intensity modulation of the photodiode output. Any superim- low-angle diffraction studies demonstrate that the protofibrils posed steady-state (magnetic) birefringence produced an ad- ditional 50-kHz photodiode output, which was phase-sensi- The publication costs of this article were defrayed in partby page charge tively detected, converted to dc, and used (as error signal in a payment. This article must therefore be hereby marked "advertise- feedback loop) to compensate the steady-state birefringence by ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. means of a Pockels cell. Hence in the compensated case the 1616 Downloaded by guest on September 25, 2021 Biophysics: Freyssinet et al. Proc. NatL Acad. Sci. USA 80 (1983) 1617 voltage across the Pockels cell was a direct measure of the mag- Lorentz-Lorenz formula netic birefringence An. In order to measure the large birefrin- gence given by fibrin polymerization the Pockels cell was re- 2ir (n2 + 2)2 CNA An, = -a. [3] placed by an electrically driven Babinet-Soleil compensator. 9 no Mr Neutron Diffraction Measurements. The neutron diffrac- tion patterns were obtained on the small-angle scattering cam- The Lorentz-Lorenz formula does not have afirm theoretical era D 11 (22) at the Institut Laue-Langevin (Grenoble). The basis for solutions or gels of anisotropic particles, but because scattered neutrons were detected on a two-dimensional (64 X the analysis is based on comparison with measurements of known 64) BF3 multidetector. The wavelength used was 10 A and AA/ structures this is not a handicap. A was 8% (full width, half maximum). The specimen-to-detector Now consider that polymerization and orientation occur si- distance was 2.55 m. Awater spectrum, which is isotropic under multaneously and that the An from the oriented polymer con- these conditions, was used to correct for detector response. centration, c2(t), is large compared to that of the unpolymerized material concentration cl(t). Both concentrations are dependent Theory on time, t, and add up to the total concentration c = cl(t) + c2(t). At the beginning (t = 0), c1(O) = c, c2(0) = 0, and An(O) 0, We are interested in a dilute solution of elongated molecules and at the end of polymerization (t -m oo), cl(oo) = 0, c2(oo) =c, that after limited proteolysis polymerize to form large fibers. and An(oo) = An,. Consider the molecules to be rotationally symmetric about their At intermediate times long axis. The following quantities are defined: c = concentra- tion; Mr = molecular weight; NA = Avogadro's number (cNA/ A'~t' - 21r(n- + 2)2 C2(t)NA Mr = number of molecules per unit volume); k = Boltzmann 9n. MrA, [4] constant; T = absolute temperature; A = wavelength of light; no = refractive index, which at lowconcentration is equal to that which, when combined with Eq. 3, gives of water (1.33); H = magnetic field strength; and Aa = all - a1, the optical anisotropy, and Ax = All - Xl, the diamagnetic An(t) = - c2(t) and Ans - An(t) = -s cl(t). [5] anistropy-i.e., the difference in their values parallel and per- C C pendicular to the axis of symmetry (XI and X1 are always neg- ative). The induced birefringence, An, is used to monitor the Thus An(t) is directly proportional to the polymer concen- orientation, An = nil - no, the difference in the refractive in- tration. This is valid even if the alignment is not complete, pro- dices of light linearly polarized parallel and perpendicular to vided the degree of orientation is the same throughout and, as the applied magnetic field direction. An, = birefringence at full above, the signal from the unpolymerized material is relatively orientation; in this study the symmetry axis orients parallel to weak, in which case Ans is not given by Eq. 3 but is equal to the the field direction. birefringence at the end of the reaction.