Fibrinogen and the early stages of polymerization to fibrin as studied by dynamic laser light scattering

Human fibrinogen and the polymerization of fibrin after activation by the enzymes, thrombin and Batroxobin, were studied by means of dynamic laser light scattering (DLS). The apparent diffusion constant, D, for fibrinogen was measured and has a value of (1.80 +/- 0.42) X 10(-7) cm2 X s-1. D was found to contain contributions from the translational diffusion constant (Dt) as well as from the rotational diffusion constant (Dr). A comparison between experimental and calculated values of Dr and Dt suggests that fibrinogen in the absence of added Ca2+ expresses a certain degree of flexibility, while it is straightened in the presence of added Ca2+. The time dependence of D showed periodic oscillations, while the average D values decreased with time. Thrombin and Batroxobin caused similar behaviour of D. The period length was related to the enzyme concentration, clotting time (Ct) and the rate of release of fibrinopeptide A (FPA). No periodic oscillations were observed in experiments where the enzyme was replaced by saline, or in experiments using a dysfunctional fibrinogen (fibrinogen Aarhus) which displayed slow rates of FPA-release and polymerization. We propose that the periodic oscillations in a system far from equilibrium may be explained by conformational changes occurring in the fibrinogen molecule during enzyme activation and polymerization.     

The polymerization and thrombin-binding properties of des-(B beta 1-42)-fibrin

Multiple factors affect the thrombin-catalyzed conversion of fibrinogen to fibrin, including: fibrinopeptide (FPA and FPB) release leading to exposure of two types of polymerization domains ("A" and "B," respectively) in the central portion of the molecule, and exposure of a noncatalytic "secondary" thrombin-binding site in fibrin. Fibrinogen containing the FPA sequence but lacking the B beta 1-42 sequence ("des-(B beta 1-42)-fibrinogen"), was compared to native fibrinogen (containing both FPA and FPB) to investigate the role played by B beta 1-42 in the polymerization of alpha-fibrin (i.e. fibrin lacking FPA), to compare reptilase and thrombin cleavage of FPA from fibrinogen, and to explore the location and function of the secondary thrombin-binding site. Electron microscopy of evolving polymer structures (mu, 0.14; pH 7.4) plus turbidity measurements, showed that early thin fibril formation as well as subsequent lateral fibril associations were impaired in des-(B beta 1-42)-alpha-fibrin, thus indicating that the B beta 1-42 sequence contributes to the A polymerization site. Reptilase-activated des-(B beta 1-42)-alpha-fibrin polymerized even more slowly than thrombin-activated des-(B beta 1-42)-alpha-fibrin, differences that disappeared when repolymerization of preformed fibrin monomers was carried out. Since existing data indicate that thrombin releases FPA in a concerted manner, resulting in relatively rapid evolution of fully functional divalent alpha-fibrin monomers, it can be inferred that delayed fibrin assembly of reptilase fibrin is due to slower formation of divalent alpha-fibrin monomers. Thrombin-activated des-(B beta 1-42)-alpha-fibrin polymerized more rapidly at low ionic strength (mu, 0.04) than did native alpha,beta-fibrin, a reversal of their behavior at physiological ionic strength (mu, 0.14). Concomitant measurement of FPA release revealed modest slowing of release at low ionic strength from des-(B beta 1-42)-fibrinogen (t1/2, 36.5 versus 21.5 min) and marked slowing from native fibrinogen (t1/2, 138 versus 22.2 min). This behavior correlated with increased thrombin binding to native alpha,beta-fibrin at low ionic strength, coupled with weak thrombin binding to des-(B beta 1-42)-alpha-fibrin, and indicates that secondary thrombin binding plays an important role in regulating thrombin diffusion and catalytic activity. Des-(B beta 1-42)-fibrinogen lacks or has a markedly defective secondary thrombin-binding site, from which we conclude that the B beta 15-42 sequence in fibrin plays a major role in forming or providing this site.     

Recombinant fibrinogen studies reveal that thrombin specificity dictates order of fibrinopeptide release

During cleavage of fibrinogen by thrombin, fibrinopeptide A (FpA) release precedes fibrinopeptide B (FpB) release. To examine the basis for this ordered release, we synthesized A'beta fibrinogen, replacing FpB with a fibrinopeptide A-like peptide, FpA' (G14V). Analyses of fibrinopeptide release from A'beta fibrinogen showed that FpA release and FpA' release were similar; the release of either peptide followed simple first-order kinetics. Specificity constants for FpA and FpA' were similar, demonstrating that these peptides are equally competitive substrates for thrombin. In the presence of Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB release from normal fibrinogen was reduced 3-fold, consistent with previous data; in contrast, the rate of FpA' release from A'beta fibrinogen was unaffected. Thus, with A'beta fibrinogen, fibrinopeptide release from the beta chain is similar to fibrinopeptide release from the alpha chain. We conclude that the ordered release of fibrinopeptides is dictated by the specificity of thrombin for its substrates. We analyzed polymerization, following changes in turbidity, and found that polymerization of A'beta fibrinogen was similar to that of normal fibrinogen. We analyzed clot structure by scanning electron microscopy and found that clots from A'beta fibrinogen were similar to clots from normal fibrinogen. We conclude that premature release of the fibrinopeptide from the N terminus of the beta chain does not affect polymerization of fibrinogen.     

Formation of soluble fibrin oligomers in purified systems and in plasma.

The kinetic parameters for release of fibrinopeptide A (FPA) from human fibrinogen by thrombin are: Km = 2.3 X 10(-6)M and Vmax. = 1.1 X 10(-10)mol of FPA/s per unit of thrombin; for fibrin formation, Km is similar to that for FPA release, but, the conditions of the present study, Vmax. was approximately half of that for FPA release. The formation of fibrin polymer before the sol-gel transition was studied by gel-permeation chromatography combined with effluent analysis for fibrinogen antigen and residual FPA. Polymer formation in purified fibrinogen incubated with thrombin proceeded as a bimolecular association of exposed sites in a manner predicted by probability calculations and assuming random FPA cleavage. Each oligomer consisted of n molecules of fibrin monomer and two fibrinogen molecules, each of the latter lacking one FPA molecule, i.e. each oligomer, regardless of molecular size, retains two FPA molecules. The addition of 5 mM-CaCl2 to the reaction mixture changed the rate of polymer formation, so that dimer was no longer the prevalent oligomer; in the presence of Ca2+, the trimer was the oligomer in highest concentration. The polymers formed in the presence of calcium were similar in composition to those without, i.e. 2 mol of FPA/mol of oligomer. EDTA-treated plasma samples incubated for short periods of time, 30s or less, with thrombin ranging in concentration up to 1 N.I.H. unit/ml did not form clots during the 10-15 min period of observation until they were applied to the column, though a large proportion of the available FPA was cleaved (maximum 45%). The soluble polymers in plasma were mostly of the high-Mr variety (tetramer and greater); these high-Mr polymers contained less than 2 mol of FPA/mol of polymer, whereas dimer and trimer in plasma were similar to those in the purified systems, i.e. 2 mol of FPA/mol.     

Catalytic competence of human alpha- and gamma-thrombin in the activation of fibrinogen and factor XIII

Steady-state kinetic parameters were compared for the action of alpha- and gamma-thrombin on the physiologically important thrombin substrates fibrinogen and factor XIII at 37 degrees C, pH 7.4, and 0.14 M NaCl. gamma-Thrombin, an alpha-thrombin derivative proteolytically cleaved at R-B73 and K-B154, was observed to catalyze the release of fibrinopeptide A (FPA) from fibrinogen with a specificity constant (kcat/Km) of 5 X 10(3) M-1 s-1. This value was approximately 2400-fold lower than the specificity constant for the corresponding alpha-thrombin-catalyzed reaction. The low specificity constant was attributed to an increase in Km and a decrease in kcat for gamma-thrombin-catalyzed release of FPA from fibrinogen. Conversion of alpha-thrombin to gamma-thrombin also resulted in an approximately 800-fold reduction in the specificity constant for thrombin-catalyzed release of fibrinopeptide B (FPB) from fibrin I, as well as a loss in discriminatory power. Whereas alpha-thrombin preferentially released FPA from intact fibrinogen, gamma-thrombin released FPA and FPB from intact fibrinogen at similar rates. In contrast to the large difference in specificity constants observed for alpha- and gamma-thrombin catalysis with fibrin(ogen) as substrate, the specificity constant (2.6 X 10(4) M-1 s-1) observed for gamma-thrombin-catalyzed release of activation peptide from factor XIII was only 5-fold lower than the corresponding value for the alpha-thrombin-catalyzed reaction. Additionally, the promotion of factor XIII activation by fibrin characteristic of the alpha-thrombin-catalyzed reaction did not occur in the gamma-thrombin-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombin-induced fibrinopeptide B release from normal and variant fibrinogens: influence of inhibitors of fibrin polymerization

Thrombin preferentially cleaves fibrinopeptides A (FPA) from fibrinogen resulting in the formation of desAA-fibrin from which most of the fibrinopeptides B (FPB) are then released with an enhanced rate. Kinetics of fibrinopeptide release from normal and dysfunctional fibrinogens were investigated in order to further characterize the mechanism of accelerated FPB release during desAA-fibrin polymerization. Dysfunctional fibrinogens London I and Ashford, exhibiting primary polymerization abnormalities (i.e., an abnormality present when all fibrinopeptides have been cleaved), which in the case of fibrinogen London I is believed to be caused by a defect in the D-domain, were shown to exhibit a decreased rate of FPB release compared with normal fibrinogen. While Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, was shown to decrease the rate of FPB release from normal fibrinogen by a factor of 5, normal fragment D1, although inhibiting clot formation of normal fibrinogen, did not influence the acceleration of FPB release. On the other hand, the presence of fragment D1 did not enhance FPB release from fibrinogen London I, suggesting that interaction of D-domains in functional isolation with desAA-fibrin E-domains is not sufficient to enhance FPB release. Although clot formation was inhibited by the concentrations of fragment D1 used, the formation of small desAA-fibrin oligomers was hardly affected. Thus, small fibrin polymers, but not desAA-fibrin monomers, act as optimal substrates for the release of FPB by thrombin.     

Characterization of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin

The time dependence of the release of fibrinopeptides from fibrinogen was studied as a function of the concentration of fibrinogen, thrombin, and Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The release of fibrinopeptides during fibrin assembly was shown to be a highly ordered process. Rate constants for individual steps in the formation of fibrin were evaluated at pH 7.4, 37 degrees C, gamma/2 = 0.15. The initial event, thrombin-catalyzed proteolysis at Arg-A alpha 16 to release fibrinopeptide A (kcat/Km = 1.09 X 10(7) M-1s-1) was followed by association of the resulting fibrin I monomers. Association of fibrin I was found to be a reversible process with rate constants of 1 X 10(6) M-1s-1 and 0.064 s-1 for association and dissociation, respectively. Assuming random polymerization of fibrin I monomer, the equilibrium constant for fibrin I association (1.56 X 10(7) M-1) indicates that greater than 80% of the fibrin I protofibrils should contain more than 10 monomeric units at 37 degrees C, pH 7.4, when the fibrin I concentration is 1.0 mg/ml. Association of fibrin I monomers was shown to result in a 6.5-fold increase in the susceptibility of Arg-B beta 14 to thrombin-mediated proteolysis. The 6.5-fold increase in the observed specificity constant from 6.5 X 10(5) M-1s-1 to 4.2 X 10(6) M-1s-1 upon association of fibrin I monomers and the rate constant for fibrin association indicates that most of the fibrinopeptide B is released after association of fibrin I monomers. The interaction between a pair of polymerization sites in fibrin I dimer was found to be weaker than the interaction of fibrin I with Gly-Pro-Arg-Pro and weaker than the interaction of fibrin I with fibrinogen.     

Decreased lateral aggregation of a variant recombinant fibrinogen provides insight into the polymerization mechanism

We analyzed the polymerization of BbetaA68T fibrinogen, the recombinant counterpart of fibrinogen Naples, a variant known to have decreased thrombin binding. When polymerized with equal thrombin concentrations, BbetaA68T fibrinogen had a longer lag time and lower rate of lateral aggregation, V(max), than normal recombinant fibrinogen, but a similar final turbidity. At thrombin concentrations that equalized the rates of fibrinopeptide A release, BbetaA68T fibrinogen polymerized with a lag time and V(max) similar to normal, but reached a significantly lower final turbidity. Similar results were produced when BbetaA68T was polymerized with Ancrod, which cleaves fibrinopeptide A at the same rate from either fibrinogen, and when BbetaA68T desA monomers were polymerized. The polymerization of desAB fibrin monomers, which circumvents fibrinopeptide release, was the same for both fibrinogens. We confirmed that turbidity was indicative of fiber thickness by scanning electron microscopy of fibrin clots. Here, we present the first experimental evidence of fibrin polymerization with a normal period of protofibril formation and rate of lateral aggregation, but with a significantly decreased extent of lateral aggregation. We conclude that the decreased lateral aggregation seen in BbetaA68T fibrinogen is due to an altered step in the enzymatic phase of its polymerization process. We propose that during normal polymerization a subtle conformational change in the E domain occurs, between the release of FpA and FpB, and that this change modulates the mechanism of lateral aggregation. Without this change, the lateral aggregation of BbetaA68T fibrinogen is impaired such that variant clots have thinner fibers than normal clots.     

Thrombin-induced fibrinopeptide release from a fibrinogen variant (fibrinogen Sydney I) with an Aalpha Arg-16----His substitution

Fibrinogen, purified from a recently identified case of dysfibrinogenaemia, fibrinogen Sydney I, was shown by thrombin digestion, high-performance liquid chromatography (HPLC) and amino acid analysis to be a heterozygous case of an A alpha Arg-16----His substitution. Kinetic studies have been carried out on the thrombin-induced release of fibrinopeptide A (FPA), fibrinopeptide B (FPB) and the variant peptide [His16]FPA. When thrombin was added to fibrinogen Sydney I at a concentration of 0.2 U/ml release of FPA was rapid and there was a 79-fold reduced rate of release of [His16]FPA, but the rate of release of FPB was not appreciably reduced. In contrast, at lower thrombin concentrations the rate of FPB release was reduced in proportion to the rate of total FPA release, supporting the view that release of fibrinopeptides is a sequential process. The second-order kinetic constant kcat/Km for hydrolysis of the abnormal A alpha chain by thrombin was calculated from Lineweaver-Burk plots to be 16-30-fold less than that for the normal A alpha chain. Molecular modelling studies, using a refined model of the trypsin-pancreatic-trypsin-inhibitor complex have been used to suggest how the histidine at the P1 site can be accommodated within the enzyme hydrophobic active-site pocket.     

人凝血酶和浙江蝮蛇毒类凝血酶凝聚纤维蛋白原及释放血纤肽的不同机制

人纤维蛋白原经人凝血酶和浙江蝮蛇毒类凝血酶作用后,释放出血纤纤肽A和血纤肽B,二者可用高效液相色谱法分离鉴定。人凝血酶首先释放出血纤肽A,浙江蝮蛇毒类凝血酶则先释放出血纤肽B,甚至在纤维蛋白凝聚之前,血纤肽B的释放量早已达到极值,即使凝块形成后,血纤肽A与血纤肽B之比仍为1:3。人凝血酶在钙离子存在下,作用于纤维蛋白原时,其凝聚时间缩短,血纤肽A释放量不变,血纤肽B释放量则增高。在同样条件下,浙江蝮蛇毒类凝血酶作用时,血纤肽A释放量无明显变化,血纤肽B释放量却降低近1/3。无论是人凝血酶还是浙江蝮蛇毒类凝血酶,当与纤维蛋白原在2M尿素存在下反应时,均无可见凝块形成,但在37℃下用350nm光吸收监测其聚合过程仍可见光吸收上升,溶液呈乳白色。本文首次报道用电子显微镜观察比较了人凝血酶和浙江蝮蛇毒类凝血酶作用人纤维蛋白原后所形成的凝块结构。前者形成的结构致密,纤维长而细;后者形成的结构松散,较透明,纤维短而粗,这种结构更易为体内纤溶系统所降解。     

Fibrin assembly after fibrinopeptide A release in model systems and human plasma studied with magnetic birefringence.

Magnetically induced birefringence was used to monitor fibrin polymerization after the release of the small negatively charged A fibrinopeptides from human fibrinogen by the action of the snake-venom-derived enzymes reptilase and ancrod. A range of conditions was investigated. Fibrin polymerization in solutions of purified fibrinogen shows a distinct break near the gelation point. On addition of Ca2+ or albumin the lag period is shortened, fibre thickness is increased and the break in assembly almost vanishes, probably because both of these additives promote lateral aggregation. There are minor differences in the kinetics, depending on the venom enzyme used. The kinetics of fibrin assembly in model systems containing either Ca2+ or albumin and in human plasma with a largely dormant coagulation cascade are very similar. Therefore in the latter condition there is no significant alteration in the assembly process due to interaction between fibrin or the venom enzymes and any of the plasma proteins. When the cascade is activated, the polymerization progress curves have a character that resembles a combination of the reactions observed when the venom enzymes and endogenously generated thrombin separately induce coagulation, except for a region near gelation where, paradoxically, polymerization appears to be slower on activation. The low-angle neutron-diffraction patterns from oriented gels made with thrombin or reptilase are identical. Therefore at low resolution the packing of the monomers within fibres is the same when fibrinopeptide A only or both fibrinopeptides A and B are removed.     

Clotting of bovine fibrinogen. Calcium binding to fibrin during clotting and its dependence on release of fibrinopeptide B

Polymerization of bovine fibrinogen acted upon by thrombin is accompanied by binding of Ca2+ and a concomitant decrease of the free Ca2+ concentration. The latter can be recorded by a Ca2+-selective electrode as a shift in the electrode potential. The shift shows marked dependence on the initial free Ca2+ concentration, being maximal at about 10(-4.1) M and decreasing sharply on either side of this. Thus, the effect is limited to the 10(-3)-10(-5) M free Ca2+ concentration range. From the initial and the final value of the electrode potential during a clotting experiment, the amount of Ca2+ bound to fibrinogen and fibrin, respectively, can be calculated. The difference between the two, plotted against free Ca2+ concentration, gives a bell-shaped curve. This indicates that the reason for the Ca2+ binding is a shift of the pK of some groups from a lower to higher value. The recordings can be used for evaluation of the kinetics of the Ca2+ uptake. However, they have to be corrected for the effect of the continuous shift in the free Ca2+ concentration during the experiment. The reaction does not follow simple kinetics, showing a lag period. Therefore, rates were estimated from inverse half-reaction times. Half-times of the corrected curves show that the reaction is first order with respect to thrombin. Moreover, the rate of Ca2+ uptake is identical with that of the conformational change seen in differential scanning calorimetry [Donovan, J.W., & Mihalyi, E. '1985) Biochemistry 24, 3434]. The inverse rate and the final corrected Ca2+ uptake increase linearly with the initial fibrinogen concentration. Concomitant estimates of fibrinopeptide A and B release showed that the Ca2+ uptake runs parallel to the release of fibrinopeptide B. Fibrinopeptide A was released largely during the lag period of the Ca2+ uptake. In agreement with this, clotting with Ancrod, an enzyme that liberates only fibrinopeptide A, was not accompanied by binding of Ca2+. Thus, polymerization is not sufficient for the Ca2+ uptake to occur; liberation of fibrinopeptide B seems to be obligatory. Further support for this was obtained with experiments with the polymerization inhibitor Gly-Pro-Arg-Pro. The tetrapeptide inhibits polymerization and also, proportional to this, release of fibrinopeptide B [Hurlet-Jensen, A., Cummins, H.Z., Nossel, H.L., & Liu, C.Y. (1982) Thromb. Res. 27, 419; Lewis, S.D., Shields, P.P., & Shafer, J.A. (1985) J. Biol. Chem. 260, 10192]. Calcium uptake was also depressed by the tetrapeptide in a way similar to its effect upon fibrinopeptide B release.     

The incipient stage in thrombin-induced fibrin polymerization detected by FCS at the single molecule level.

We used fluorescence correlation spectroscopy (FCS) to study the activation of fibrinogen by thrombin and the subsequent aggregation of fibrin monomers into fibrin polymers at a very low and at physiological fibrinogen concentrations. In the labeling procedure used the fibrinogen was randomly labeled and the label was bound to the fibrinopeptide A and/or to the part of fibrinogen which after activation takes part in fibrin formation. We measured a diffusion coefficient for fibrinogen of 2.48 x 10(-7) +/- 0.10 x 10(-7) cm2/s. After activation with thrombin both fibrinopeptide A and fibrin polymerization products could be demonstrated. From our findings we suggest a model for the formation of a three-dimensional network as two parallel processes, elongation and branching and that fibrin oligomers are not only intermediates in the polymerization process but also are substrates for branching.     

The influence of calcium ions on fibrin polymerization

The influence of calcium ions on the polymerization induced in fibrinogen solutions by thrombin and by Reptilase has been investigated by meansof static and dynamic light scattering in combination with measurements of the release of the fibrinopeptide A. The calcium concentration was varied in the range between 0.3 and 103 calcium ions per fibrinogen molecule. The enzyme concentration was chosen sufficiently low so that it was possible to make quantitative observations as a function of time, in particular, beforethe onset of gelation. Likewise, the influence of calcium ions on the enzymatically induced polymerization of fragment X was studied. The results indicate that there are at least three mechanisms by which calcium can influence the evolution of the polymer system on the path to gelation and clotting. Which mechanism dominates depends upon the calcium concentration.     

Review of some unusual effects of calcium binding to fibrinogen

Calcium binding curves of human and bovine fibrinogen were obtained by using a calcium sensitive electrode. The two were identical and showed 2 high, 2-3 medium and more than 15 low affinity sites. Differential scanning calorimetry at neutral pH demonstrated the presence of the D and E domains of fibrinogen; however, at pH 3.5 the D-domain was split into two. The presence of the subdomains was demonstrated also by digestion by pepsin at this pH. Combination of digestion of fibrinogen and of its fragments with different enzymes and temperatures identified up to 12 subdomains in the original molecule. Clotting of fibrinogen by thrombin at pH 7.0 was investigated also by differential scanning calorimetry. In the absence of Ca2+ clotting elicited a 40% increase in the enthalpy of thermal denaturation of the D domain of fibrinogen, but the position of the peak increased only by 0.4 degrees C. However, with clotting in the presence of 10(-3) M calcium the former increased by 70-75% and the latter by 11.0 degrees C, while these parameters of the E-domain remained unchanged. Changes of bound calcium during clotting were also measured with the calcium sensitive electrode. These had to be corrected, because the drop in free calcium was partly compensated by release of some calcium that was already bound to fibrinogen. Log of the half time of calcium uptake plotted against log thrombin concentration indicated a first order process with respect to thrombin concentration, moreover, the rate determined corresponded to that of the conformation change measured by calorimetry. The calcium uptake was correlated with release of the fibrinopeptides. Release of fibrinopeptide B follows parallel to binding of calcium and that of fibrinopeptide A is about fourfold faster. Polymerization and formation of thick bundles of fibrin is connected with release of fibrinopeptide A. Clotting with Ancrod, an enzyme that releases only fibrinopeptide A, showed only minimal binding of calcium. The polymerization inhibiting tetrapeptide Gly-Pro-Arg-Pro also depressed binding of calcium. These data suggest that a calcium-binding site must be in the proximity of the site of release of fibrinopeptide B and of a polymerization site.     

Inhibition of the enzymatic activity of thrombin by concanavalin A

Concanavalin A, a carbohydrate lectin derived from the jack bean, prolongs the thrombin clotting time of human plasma or purified fibrinogen. Prolongation is due to delay in peptide release from fibrinogen. The rate of fibrin monomer polymerization is not affected. Hydrolysis of protamine sulfate by thrombin is inhibited by concanavalin A. All inhibitory effects are prevented by α-methyl-D-mannoside. Concanavalin A does not delay clotting of fibrinogen by reptilase (releases fibrinopeptide A only) or by Ancistrodon contortrix contortrix (releases fibrinopeptide B initially followed by a small amount of A). It is concluded that concanavalin A binds to a carbohydrate on the thrombin molecule thus inhibiting its enzymatic activity.     

Localization of a fibrin polymerization site

The formation of a fibrin clot is initiated after the proteolytic cleavage of fibrinogen by thrombin. The enzyme removes fibrinopeptides A and B and generates fibrin monomer which spontaneously polymerizes. Polymerization appears to occur though the interaction of complementary binding sites on the NH2-terminal and COOH-terminal (Fragment D) regions of the molecule. A peptide has been isolated from the gamma chain remnant of fibrinogen Fragment D1 which has the ability to bind to the NH2-terminal region of fibrinogen as well as to inhibit fibrin monomer polymerization. The peptide reduces the maximum rate and extent of the polymerization of thrombin or batroxobin fibrin monomer and increases the lag time. The D1 peptide does not interact with disulfide knot, fibrinogen, or Fragment D1, but it binds to thrombin-treated disulfide knot with a Kd of 1.45 X 10(-6) M at approximately two binding sites per molecule of disulfide knot. Fibrin monomer formed either by thrombin or batroxobin binds approximately two molecules of D1 peptide per molecule of fibrin monomer, indicating that the complementary site is revealed by the loss of fibrinopeptide A. The NH2-terminal sequence (Thr-Arg-Trp) and COOH-terminal sequence (Ala-Gly-Asp-Val) of the D1 peptide were determined. Therefore the gamma 373-410 region of fibrinogen contains a polymerization site which is complementary to the thrombin-activated site on the NH2-terminal region of fibrinogen.     

Characterization of the kinetic pathway for fibrin promotion of alpha-thrombin-catalyzed activation of plasma factor XIII

Kinetic and thermodynamic studies are presented showing that the cofactor activity of fibrin I (polymerized des-A fibrinogen) in the alpha-thrombin-catalyzed proteolysis of activation peptide (AP) from plasma factor XIII can be attributed to formation of a fibrin I-plasma factor XIII complex (Kd = 65 nM), which is processed by alpha-thrombin more efficiently (kcat/Km = 1.2 x 10(7) M-1 s-1) than free, uncomplexed plasma factor XIII (kcat/Km = 1.4 x 10(5) M-1 s-1). The increase in the specificity constant (kcat/Km) is shown to be largely due to an increase in the apparent affinity of alpha-thrombin for the complex of plasma factor XIII and fibrin I, as reflected by the 30-fold decrease in the Michaelis constant observed for fibrin I bound plasma factor XIII relative to that for uncomplexed plasma factor XIII. Analysis of the initial rates of alpha-thrombin-catalyzed hydrolysis of fibrinopeptide B (FPB) from fibrin I polymer in the presence of plasma factor XIII indicated that alpha-thrombin bound to fibrin I in the ternary complex of alpha-thrombin, plasma factor XIII, and fibrin I polymer is competent to catalyze cleavage of both FPB from fibrin I and AP from plasma factor XIII. This observation is consistent with the view that alpha-thrombin within the ternary complex is anchored to fibrin I polymer through a binding site distinct from the active site (an exosite) and that the active site is alternatively complexed with the AP moiety of plasma factor XIII or the FPB moiety of fibrin I. This conclusion is supported by the observation that a 12-residue peptide, which binds to an exosite of alpha-thrombin and blocks the interaction of alpha-thrombin with fibrinogen and fibrin, competitively inhibits alpha-thrombin-catalyzed release of both FPB and AP from the fibrin I-plasma factor XIII complex.     

Interactions of thrombin with fibrinogen adsorbed on methyl-, hydroxyl-, amine-, and carboxyl-terminated self-assembled monolayers

We have examined the initial phase of fibrin formation, thrombin-catalyzed fibrinopeptide cleavage, from adsorbed fibrinogen using surface plasmon resonance and liquid chromatography-mass spectrometry. Fibrinogen adsorption impaired thrombin-fibrinogen interactions compared to the interactions of thrombin with fibrinogen in solution. The properties of the underlying substrate significantly affected the extent and kinetics of fibrinopeptide cleavage, and the conversion of adsorbed fibrinogen to fibrin. Fibrinogen adsorbed on negatively charged surfaces (carboxyl-terminated self-assembled monolayers) released a smaller amount of fibrinopeptides, at a reduced rate relative to those of hydrophobic, hydrophilic, and positively charged surfaces (methyl-, hydroxyl-, and amine-terminated self-assembled monolayers, respectively). Additionally, the conversion of adsorbed fibrinogen to fibrin was comparatively inefficient at the negatively charged surface. These data correlated well with trends previously reported for fibrin proliferation as a function of surface properties. We conclude that thrombin interactions with adsorbed fibrinogen determine the extent of subsequent fibrin proliferation on surfaces.     

Molecular mechanisms of the polymerization of fibrin and the formation of its three-dimensional network

The results of biochemical, immunochemical, and X-ray studies of the structures of fibrinogen and fibrin molecules were analyzed. The mechanisms of the successive formation of the fibrin three-dimensional network were described: the polymerization of monomeric molecules with the formation of bifilar protofibrils, the lateral association of protofibrils, and the embranchment of the forming fibrils. Data on the electron and confocal microscopy of the polymeric fibrin were considered. The role of the known polymerization centers of fibrin which participated in the formation of protofibrils and their lateral association was discussed. Data on the existence of the previously unknown polymerization centers were given. In particular, the experimental results demonstrated that one of such centers which participated in the formation of protofibrils was located in the Bβ12–46 fragment, and did not require the cleavage of fibrinopeptide B for its functioning. The results of the computer modeling of the spatial structure of the fibrin(ogen) molecule and the intermolecular interactions in the course of the fibrin polymerization were presented. The location of the αC domains in the fibrin(ogen) molecule and their role in the polymerization process were discussed. Information on the structure of the calciumbinding sites of fibrin(ogen) and the functional role of Ca²⁺ in fibrin polymerization was published. The structure of factor XIII(a) and the mechanisms of fibrin stabilization by this factor were briefly described.