Spin-polarised electrons in a one-magnet-only Mott spin junction.

The current flowing through a Mott spin junction depends on the relative spin orientation of the two ferromagnetic layers comprising the "source" and "drain" sides of the junction. The resulting current asymmetry is detected as giant or tunnelling magnetoresistance depending on whether the two ferromagnets are separated by a metal or an insulator. Based on the fundamental principles of reciprocity for spin-dependent electron scattering, one can envisage a one-magnet-only spin junction in which the source is non-magnetic, and the spin information is encoded by the spin polarisation of the electrons that have crossed or are backscattered from the drain magnetic layer. The practical significance of using an unpolarised source is that the state of the magnetic layer can be modified without affecting the process of probing it. Whether this reciprocity is realised in the actual junctions is not yet known. Here, we demonstrate a nano-sized, one-magnet-only Mott spin junction by measuring the finite spin polarisation of the backscattered electrons. Based on this finding, we conclude that since the junction acts as a spin filter, the magnetic layer must experience a spin transfer that could become detectable in view of the high current densities achievable in this technology.

Critical exponents and scaling invariance in the absence of a critical point.

The paramagnetic-to-ferromagnetic phase transition is classified as a critical phenomenon due to the power-law behaviour shown by thermodynamic observables when the Curie point is approached. Here we report the observation of such a behaviour over extraordinarily many decades of suitable scaling variables in ultrathin Fe films, for certain ranges of temperature T and applied field B. This despite the fact that the underlying critical point is practically unreachable because protected by a phase with a modulated domain structure, induced by the dipole-dipole interaction. The modulated structure has a well-defined spatial period and is realized in a portion of the (T, B) plane that extends above the putative critical temperature, where thermodynamic quantities do not display any singularity. Our results imply that scaling behaviour of macroscopic observables is compatible with an avoided critical point.

Thirty per cent contrast in secondary-electron imaging by scanning field-emission microscopy.

We perform scanning tunnelling microscopy (STM) in a regime where primary electrons are field-emitted from the tip and excite secondary electrons out of the target-the scanning field-emission microscopy regime (SFM). In the SFM mode, a secondary-electron contrast as high as 30% is observed when imaging a monoatomic step between a clean W(110)- and an Fe-covered W(110)-terrace. This is a figure of contrast comparable to STM. The apparent width of the monoatomic step attains the 1 nm mark, i.e. it is only marginally worse than the corresponding width observed in STM. The origin of the unexpected strong contrast in SFM is the material dependence of the secondary-electron yield and not the dependence of the transported current on the tip-target distance, typical of STM: accordingly, we expect that a technology combining STM and SFM will highlight complementary aspects of a surface while simultaneously making electrons, selected with nanometre spatial precision, available to a macroscopic environment for further processing.

Scaling theory of electric-field-assisted tunnelling.

Recent experiments report the current (I) versus voltage (V) characteristics of a tunnel junction consisting of a metallic tip placed at a distance d from a planar electrode, d varying over six orders of magnitude, from few nanometres to few millimetres. In the 'electric-field-assisted' (or 'field emission') regime, as opposed to the direct tunnelling regime used in conventional scanning tunnelling microscopy, all I-V curves are found to collapse onto one single graph when d is suitably rescaled, suggesting that the current I=I(V,d) is in reality a generalized homogeneous function of one single variable, i.e. [Formula: see text], where λ being some characteristic exponent and [Formula: see text] being a scaling function. In this paper, we provide a comprehensive explanation-based on analytical arguments, numerical simulations and further experimental results-for the scaling behaviour that we show to emerge for a variety of tip-plane geometries and thus seems to be a general feature of electric-field-assisted tunnelling.

Experimental phase diagram of perpendicularly magnetized ultrathin ferromagnetic films.

We image the domain patterns in perpendicularly magnetized ultrathin Fe films on Cu(100) as a function of the temperature T and the applied magnetic field H. Between the low-field stripe phase and the high-field uniform phase we find a bubble phase, consisting of reversed circular domains in a homogeneous background. The curvature of the transition lines in the H-T parameter space is in contrast to the general expectations. The pattern transformations show yet undetected scaling properties.

Finite-size effects in single chain magnets: an experimental and theoretical study.

The problem of finite-size effects in s=1/2 Ising systems showing slow dynamics of the magnetization is investigated introducing diamagnetic impurities in a Co2+-radical chain. The static magnetic properties have been measured and analyzed considering the peculiarities induced by the ferrimagnetic character of the compound. The dynamic susceptibility shows that an Arrhenius law is observed with the same energy barrier for the pure and the doped compounds while the prefactor decreases, as theoretically predicted. Multiple spin reversal has also been investigated.

Ising-type magnetic anisotropy in a cobalt(II) nitronyl nitroxide compound: a key to understanding the formation of molecular magnetic nanowires.

The compound [Co(hfac)2-(NITPhOMe)2] (2) (hfac = hexafluoroacetylacetonate, NITPhOMe = 4'-methoxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) crystallizes in the triclinic P1 space group, a= 10.870(5), b = 11.520(5), c = 19.749(5) A, alpha = 78.05(5), beta = 84.20(5), gamma = 64.51(5) degrees, Z = 2. It can be considered a model system for studying the nature of the magnetic anisotropy of [Co(hfac)2(NITPhOMe)] (1), which was recently reported to behave as a molecular magnetic wire. The magnetic anisotropy of 2 was investigated by EPR spectroscopy and SQUID magnetometry both in the polycrystalline powder and in a single crystal. The experimental magnetic anisotropy was related to the anisotropy of the central ion and to the exchange interaction between the cobalt(II) ion and the radicals.

N(omega)-arginine dimethylation modulates the interaction between a Gly/Arg-rich peptide from human nucleolin and nucleic acids.

We studied the interaction between a synthetic peptide (sequence Ac-GXGGFGGXGGFXGGXGG-NH(2), where X = arginine, N(omega),N(omega)-dimethylarginine, DMA, or lysine) corresponding to residues 676-692 of human nucleolin and several DNA and RNA substrates using double filter binding, melting curve analysis and circular dichroism spectroscopy. We found that despite the reduced capability of DMA in forming hydrogen bonds, N(omega),N(omega)-dimethylation does not affect the strength of the binding to nucleic acids nor does it have any effect on stabilization of a double-stranded DNA substrate. However, circular dichroism studies show that unmethylated peptide can perturb the helical structure, especially in RNA, to a much larger extent than the DMA peptide.

Identification of human DNA helicase V with the far upstream element-binding protein.

The properties of human DNA helicase V (HDH V) were studied in greater detail following an improved purification procedure. From 450 g of cultured cells, <0.1 mg of pure protein was isolated. HDH V unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand in an ATP- and Mg(2+)-dependent fashion. The enzyme is not processive and can also unwind partial RNA-RNA duplexes such as HDH IV and HDH VIII. The M:(r) determined by SDS-PAGE (66 kDa) corresponds to that measured under native conditions, suggesting that HDH V exists as a monomer in the nucleus. Microsequencing of the purified HDH V shows that this enzyme is identical to the far upstream element-binding protein (FBP), a protein that stimulates the activity of the c-myc gene by binding specifically to the 'FUSE' DNA region localized upstream of its promoter. The sequence of HDH V/FBP contains RGG motifs like HDH IV/nucleolin, HDH VIII/G3BP as well as other human RNA and DNA helicases identified by other laboratories.

Platelet glycoprotein Ib alpha binds to thrombin anion-binding exosite II inducing allosteric changes in the activity of thrombin.

The glycoprotein (GP) Ib-IX complex is a platelet surface receptor that binds thrombin as one of its ligands, although the biological significance of thrombin interaction remains unclear. In this study we have used several approaches to investigate the GPIb alpha-thrombin interaction in more detail and to study its effect on the thrombin-induced elaboration of fibrin. We found that both glycocalicin and the amino-terminal fragment of GPIb alpha reduced the release of fibrinopeptide A from fibrinogen by about 50% by a noncompetitive allosteric mechanism. Similarly, GPIb alpha caused in thrombin an allosteric reduction in the rate of turnover of the small peptide substrate d-Phe-Pro-Arg-pNA. The K(d) for the glycocalicin-thrombin interaction was 1 microm at physiological ionic strength but was highly salt-dependent, decreasing to 0.19 microm at 100 mm NaCl (Gamma(salt) = -4.2). The salt dependence was characteristic of other thrombin ligands that bind to exosite II of this enzyme, and we confirmed this as the GPIb alpha-binding site on thrombin by using thrombin mutants and by competition binding studies. R68E or R70E mutations in exosite I of thrombin had little effect on its interaction with GPIb alpha. Both the allosteric inhibition of fibrinogen turnover caused by GPIb alpha binding to these mutants, and the K(d) values for their interactions with GPIb alpha were similar to those of wild-type thrombin. In contrast, R89E and K248E mutations in exosite II of thrombin markedly increased the K(d) values for the interactions of these thrombin mutants with GPIb alpha by 10- and 25-fold, respectively. Finally, we demonstrated that low molecular weight heparin (which binds to thrombin exosite II) but not hirugen (residues 54-65 of hirudin, which binds to exosite I of thrombin) inhibited thrombin binding to GPIb alpha. These data demonstrate that GPIb alpha binds to thrombin exosite II and in so doing causes a conformational change in the active site of thrombin by an allosteric mechanism that alters the accessibility of both its natural substrate, fibrinogen, and the small peptidyl substrate d-Phe-Pro-Arg-pNA.

Role of residue Y99 in tissue plasminogen activator.

The crystal structure of the fibrinolytic enzyme tissue plasminogen activator (tPA) shows that the bulky side chain of Y99 hinders access to the active site by partially occluding the S2 site and may be responsible for the low catalytic activity of tPA toward plasminogen. We have tested the role of Y99 by replacing it with Leu, the residue found in more proficient proteases like trypsin and thrombin. The Y99L replacement results in an increase in the k(cat)/Km for chromogenic substrates due to enhanced diffusion into the active site. The increase is modest (threefold) for substrates specific for tPA that carry Pro or Gly at P2, but reaches 80-fold for less specific substrates carrying Arg at P2. On the other hand, the Y99L mutation has no effect on the activity of tPA toward the natural substrate plasminogen, that carries Gly at P2, and reduces more than 10-fold the inhibition of tPA by plasminogen activator inhibitor-1 (PAI-1), that carries Ala at P2. We conclude that the steric hindrance provided by Y99 in the crystal structure affects mostly nonphysiological substrates with bulky residues at P2. In addition, residue Y99 plays an active role in the recognition of PAI-1, but not plasminogen. Mutations of Y99 could therefore afford a resistance to inhibition by PAI-1 without compromising the fibrinolytic potency of tPA, a result of potential therapeutic relevance.

Incorporation of noncoded amino acids into the N-terminal domain 1-47 of hirudin yields a highly potent and selective thrombin inhibitor.

Hirudin is an anticoagulant polypeptide isolated from a medicinal leech that inhibits thrombin with extraordinary potency (Kd = 0.2-1.0 pM) and selectivity. Hirudin is composed of a compact N-terminal region (residues 1-47, cross-linked by three disulfide bridges) that binds to the active site of thrombin, and a flexible C-terminal tail (residues 48-64) that interacts with the exosite I of the enzyme. To minimize the sequence of hirudin able to bind thrombin and also to improve its therapeutic profile, several N-terminal fragments have been prepared as potential anticoagulants. However, the practical use of these fragments has been impaired by their relatively poor affinity for the enzyme, as given by the increased value of the dissociation constant (Kd) of the corresponding thrombin complexes (Kd = 30-400 nM). The aim of the present study is to obtain a derivative of the N-terminal domain 1-47 of hirudin displaying enhanced inhibitory potency for thrombin compared to the natural product. In this view, we have synthesized an analogue of fragment 1-47 of hirudin HM2 in which Val1 has been replaced by tert-butylglycine, Ser2 by Arg, and Tyr3 by beta-naphthylalanine, to give the BugArgNal analogue. The results of chemical and conformational characterization indicate that the synthetic peptide is able to fold efficiently with the correct disulfide topology (Cys6-Cys14, Cys16-Cys28, Cys22-Cys37), while retaining the conformational properties of the natural fragment. Thrombin inhibition data indicate that the effects of amino acid replacements are perfectly additive if compared to the singly substituted analogues (De Filippis V, Quarzago D, Vindigni A, Di Cera E, Fontana A, 1998, Biochemistry 37:13507-13515), yielding a molecule that inhibits the fast or slow form of thrombin by 2,670- and 6,818-fold more effectively than the natural fragment, and that binds exclusively at the active site of the enzyme with an affinity (Kd,fast = 15.4 pM, Kd,slow = 220 pM) comparable to that of full-length hirudin (Kd,fast = 0.2 pM, Kd,slow = 5.5 pM). Moreover, BugArgNal displays absolute selectivity for thrombin over the other physiologically important serine proteases trypsin, plasmin, factor Xa, and tissue plasminogen activator, up to the highest concentration of inhibitor tested (10 microM).

Energetic dissection of specificity in serine proteases.

Serine proteases of the chymotrypsin family share a similar fold and architecture, but they differ widely in specificity. The molecular origin of this difference remains for the most part elusive. A detailed understanding of the molecular origin of their specificity is of fundamental importance for structure-function and evolutionary studies. Current approaches put much emphasis on single site substitutions of ligand sequences or protein residues and neglect second- and higher-order coupling among residues leading to an incomplete and often misleading assessment of the underlying energetics. Information on how recognition sites interact is key to unveil structure-function links and to enable the development of more effective drugs for therapeutic purposes. A novel strategy has been recently developed for dissecting enzyme specificity using the principles of site-specific thermodynamics and is applied in the present work to thrombin, trypsin, tissue plasminogen activator. The results provide a much needed data base of information for computational studies of protease specificity and protein-ligand interaction. They suggest precise guidelines for the design of novel active-site inhibitors. Basic differences are also identified between thrombin, tPA, plasmin and trypsin in the energetic contribution of the specificity sites and the coupling between them.

Synthesis and characterization of more potent analogues of hirudin fragment 1-47 containing non-natural amino acids.

Hirudin is the most potent and specific inhibitor of thrombin, a key enzyme in the coagulation process existing in equilibrium between its procoagulant (fast) and anticoagulant (slow) form. In a previous study, we described the solid-phase synthesis of a Trp3 analogue of fragment 1-47 of hirudin HM2, which displayed approximately 5-fold higher thrombin inhibitory potency relative to that of the natural product [De Filippis, V., et al. (1995) Biochemistry 34, 9552-9564]. By combining automated and manual peptide synthesis, here we have produced in high yields seven analogues of fragment 1-47 containing natural and non-natural amino acids. In particular, we have replaced Val1 with tert-butylglycine (tBug), Ser2 with Arg, and Tyr3 with Phe, cyclohexylalanine (Cha), Trp, alpha-naphthylalanine (alphaNal), and beta-naphthylalanine (betaNal). The crude reduced peptides are able to fold almost quantitatively into the disulfide-cross-linked species, whose unique alignment (Cys6-Cys14, Cys16-Cys28, and Cys22-Cys37) has been shown to be identical to that of the natural fragment. The results of conformational characterization provide evidence that synthetic peptides retain the structural features of the natural species, whereas thrombin inhibition data indicate that the synthetic analogues are all more potent inhibitors of thrombin. In particular, Val --> tBug exchange leads to a 3-fold increase in binding, interpreted as arising from a favorable reduction of the entropy of binding, due to the presence of the more symmetric side chain of tBug relative to that of Val. The S2R analogue binds 24- and 125-fold more tightly than the natural fragment to the fast or slow form of thrombin. These results are explained by considering that Arg2 may favorably couple to Glu192, a key residue involved in the slow to fast transition, thus stabilizing the slow form. Replacement of Tyr3 with more hydrophobic residues having different side chain orientations and electronic structures improves binding by 2-40-fold, suggesting that nonpolar interactions and shape-dependent packing effects strongly influence binding at this position. Overall, these results provide new insights for elucidating the mechanism of hirudin-thrombin recognition at the molecular level and highlight new strategies for designing more potent and selective inhibitors of thrombin.

Role of P225 and the C136-C201 disulfide bond in tissue plasminogen activator.

The protease domain of tissue plasminogen activator (tPA), a key fibrinolytic enzyme, was expressed in Escherichia coli with a yield of 1 mg per liter of media. The recombinant protein was titrated with the Erythrina caraffa trypsin inhibitor (ETI) and characterized in its interaction with plasminogen and the natural inhibitor plasminogen activator inhibitor-1 (PAI-1). Analysis of the catalytic properties of tPA using a library of chromogenic substrates carrying substitutions at P1, P2, and P3 reveals a strong preference for Arg over Lys at P1, unmatched by other serine proteases like thrombin or trypsin. In contrast to these proteases and plasmin, tPA shows little or no preference for Pro over Gly at P2. A specific inhibition of tPA by Cu2+ was discovered. The divalent cation presumably binds to H188 near D189 in the primary specificity pocket and inhibits substrate binding in a competitive manner with a Kd = 19 microM. In an attempt to engineer Na+ binding and enhanced catalytic activity in tPA, P225 was replaced with Tyr, the residue present in Na+-dependent allosteric serine proteases. The P225Y mutation did not result in cation binding, but caused a significant loss of specificity (up to 100-fold) toward chromogenic substrates and plasminogen and considerably reduced the inhibition by PAI-1 and ETI. Interestingly, the P225Y substitution enhanced the ability of Cu2+ to inhibit the enzyme. Elimination of the C136-C201 disulfide bond, that is absent in all Na+-dependent allosteric serine proteases, significantly enhanced the yield (5 mg per liter of media) of expression in E. coli, but caused no changes in the properties of the enzyme whether residue 225 was Pro or Tyr. These findings point out an unanticipated crucial role for residue 225 in controlling the catalytic activity of tPA, and suggest that engineering of a Na+-dependent allosteric enhancement of catalytic activity in this enzyme, must involve substantial changes in the region homologous to the Na+ binding site of allosteric serine proteases.

Site-specific dissection of substrate recognition by thrombin.

Current approaches to enzyme specificity focus on the identification of consensus sequences from combinatorial chemistry libraries or phage display. These synthetic substrates can also be used as sensitive probes for the molecular environment of the enzyme specificity sites to determine how they contribute to recognition in the transition state. Libraries constructed to include all relevant species for a site-specific analysis contain a relatively small number of substrates and provide quantitative information on the energetics of recognition that can be exploited in studies of structure-function relations and rational drug design. We have constructed a library of substrates carrying substitutions at P1, P2, and P3 to probe the response of the specificity sites S1, S2, and S3 of thrombin. The library has been used to identify differences between the anticoagulant slow and procoagulant fast forms of thrombin and the structural origin of the effects. The results also offer new guidelines for the design of active-site inhibitors of thrombin.

Prevention of vascular and neural dysfunction in diabetic rats by C-peptide.

C-peptide, a cleavage product from the processing of proinsulin to insulin, has been considered to possess little if any biological activity other than its participation in insulin synthesis. Injection of human C-peptide prevented or attenuated vascular and neural (electrophysiological) dysfunction and impaired Na+- and K+-dependent adenosine triphosphate activity in tissues of diabetic rats. Nonpolar amino acids in the midportion of the peptide were required for these biological effects. Synthetic reverse sequence (retro) and all-D-amino acid (enantio) C-peptides were equipotent to native C-peptide, which indicates that the effects of C-peptide on diabetic vascular and neural dysfunction were mediated by nonchiral interactions instead of stereospecific receptors or binding sites.

Energetics of thrombin-thrombomodulin interaction.

Temperature and salt dependence studies of thrombin interaction with thrombomodulin, with and without chondroitin sulfate, and two fragments containing the EGF-like domains 4-5 and 4-5-6 reveal the energetic signatures and the mechanism of recognition of this physiologically important cofactor. Binding of thrombomodulin is affected drastically by the particular salt present in solution and is positively linked to Na+ binding to thrombin and the conversion of the enzyme from the slow to the fast form, but is opposed by Cl- binding to the fibrinogen recognition site and especially to the heparin binding site. Binding of thrombomodulin has an unusually large salt dependence (gamma(salt) = -4.8) contributed mostly by the polyelectrolyte-like nature of the chondroitin sulfate moiety that binds to the heparin binding site and increases the affinity of the cofactor by almost 10-fold. On the other hand, the chondroitin sulfate has no effect on the deltaCp of binding, which is determined predominantly by contacts made by the EGF-like domains 5 and 6 with the fibrinogen recognition site. The modest heat capacity change (-0.2 kcal mol(-1) K(-1)) observed when thrombomodulin binds to the fast form suggests a rigid-body association of the cofactor with the enzyme. In the slow form, however, the heat capacity change is significantly more pronounced (-0.5 kcal mol(-1) K(-1)) and signals the presence of a conformational transition of the enzyme linked to binding of the cofactor that mimics the slow-->fast conversion. These results demonstrate that recognition of thrombomodulin by thrombin is steered electrostatically by the highly charged regions of the fibrinogen recognition site and the heparin binding site, to which the chondroitin sulfate moiety binds and enhances the affinity of the interaction. The recognition event also involves conformational changes of the enzyme in the slow form mediated by binding of the EGF-like domains 5-6 to the fibrinogen recognition site. Consistent with this model, binding of thrombomodulin to the fast form has only a small effect on the hydrolysis of nine chromogenic substrates carrying substitutions at P1, P2, and P3 aimed at probing the environment of the specificity sites S1, S2, and S3 of the enzyme. Binding to the slow form, on the other hand, enhances the specificity toward all substrates up to 15-fold. For substrates carrying a Gly at P2, binding of thrombomodulin changes the relative specificity of the slow and fast forms and makes the slow form more specific. Interestingly, these effects are not specific of thrombomodulin and depend solely on binding to the fibrinogen recognition site of the enzyme. In fact, they are also observed with the hirudin C-terminal fragment 55-65. The characterization of the mechanism of thrombin-thrombomodulin interaction and the effects of the cofactor on the hydrolysis of chromogenic substrates probing the interior of the catalytic pocket bear on the thrombomodulin-induced enhancement of protein C cleavage by thrombin. We propose that this enhancement is due predominantly to an effect of thrombomodulin on the bound protein C in the ternary complex. Therefore, thrombomodulin would carry out its physiological function by making protein C a better substrate for thrombin, rather than making thrombin a better enzyme for protein C.

Release of fibrinopeptides by the slow and fast forms of thrombin.

The release of fibrinopeptides A and B by the slow and fast forms of thrombin was studied over the temperature range from 5 to 45 degrees C and the salt concentration range from 100 to 800 mM. The sequential mechanism for the release of fibrinopeptides originally proposed by Shafer was found to be obeyed under all conditions examined. The origin of preferential binding of fibrinogen and fibrin I to the fast form of thrombin in the transition state is in the second-order rate constant for association, k(l). In the case of fibrinogen, the values of k(l) for interaction with the fast and slow forms at 25 degrees C are 19 +/- 4 and 2.5 +/- 0.3 microM(-1) s(-1), with an activation energy of about 10 kcal/mol in both forms. In the case of fibrin I, the analogous values of k(l) are 9.1 +/- 0.7 and 2.5 +/- 0.2 microM(-1) s(-1), and the activation energy is about 4.5 kcal/mol in both forms. The mechanism of recognition of fibrinogen and fibrin I by thrombin entails a diffusion-controlled step with a small energy barrier. Analysis of the temperature dependence of the coupling free energy for allosteric switching indicates that the preferential interaction of fibrinogen and fibrin I with the fast form of thrombin in the transition state is entropy-driven, signaling a contribution of the hydrophobic effect to the slow-->fast transition. The salt dependence of the release of fibrinopeptides shows a constant coefficient Gamma(salt) = d ln(k(cat)/K(m))/d ln [salt] in the concentration range examined. Interestingly, the value of Gamma(salt) is independent of the salt used (NaCl, ChCl, or NaF) and is -1.5 +/- 0.1 for fibrinopeptide A and -2.5 +/- 0.1 for fibrinopeptide B. Hence, Gamma(salt) reflects predominantly the electrostatic contribution to the formation of the transition state, with a larger contribution seen in the interaction of thrombin with fibrin I. It is concluded that the interaction of thrombin with fibrinogen and fibrin I, leading to the release of fibrinopeptides A and B, is driven by electrostatic forces that presumably favor the correct preorientation of the enzyme and the substrate to form a productive complex in the transition state. This electrostatic-steering effect, also reported for thrombin-hirudin interaction, leads to a diffusion-controlled encounter with a very small energy barrier. Once the complex is formed, the enzyme switches to the fast form as a result of entropic factors presumably linked to water release from a more extended surface of recognition. While the release of fibrinopeptides as a function of salt concentration was being studied, an important observation was made on the role of Cl- in the formation of the fibrin clot. This anion drastically and specifically reduces the thickness of fibrin fibers, as judged by the 10-fold decrease in the equilibrium turbidity of clots developed in NaCl as compared to the turbidity of clots developed in NaF. Hence, the transition from a "coarse" to a "fine" clot induced by an increase in ionic strength as first described by Ferry is, instead, due to the specific binding of Cl- to intermediates in the ensuing polymerization. In fact, no change in the clotting curve is observed when the ionic strength is changed with NaF.