Interaction of an analog of the E1 site of tetrapeptide Gly-Pro-Arg-Pro with the D1 site of two forms of monomeric fibrin

Two monomeric fibrin forms differing in a set of polymerization sites (fibrin desAA and fibrin-desAABB) are inhibited to a different extent by tetrapeptide Gly-Pro-Arg-Pro which simulates a moiety of polymerization site E1. The lesser sensitivity of fibrin-desAABB polymerization to the inhibiting tetrapeptide is due to the presence of active site E2 in it. A shape of the concentration dependence curve of the inhibitory effect of tetrapeptide Gly-Pro-Arg-Pro on the polymerization of both fibrin types is similar to the previously found curve for fibrinogen and its fragments--specific inhibitors of polymerization. Ca2+ intensifies inhibition of fibrin-desAABB polymerization by tetrapeptide Gly-Pro-Arg-Pro twice as much as that of fibrin-desAA evidently due to the peptide blockage of sites D2. An increase of the ionic strength from 0.15 to 0.3 enhances the inhibitory effect of the tetrapeptide on polymerization of two monomeric fibrin forms.     

Interaction of fibrinogen and its derivatives with fibrin

The binding between complementary polymerization sites of fibrin monomers plays an essential role in the formation of the fibrin clot. One set of polymerization sites involved in the interaction of fibrin monomers is believed to pre-exist in fibrinogen, while the complementary set of binding sites is exposed after the cleavage of fibrinopeptides from fibrinogen. The polymerization sites present in fibrinogen and its derivatives mediate their binding to fibrin. Although the binding of fibrinogen and its derivatives to fibrin have been qualitatively studied, there has been no systematic, quantitative investigation of their interaction with forming or preformed clots. In the present study, the binding of fibrinogen and fragments DD, D1, and E1 was measured using a sonicated suspension of plasminogen- and thrombin-free human cross-linked fibrin as a model of a preformed clot. Dissociation constants of 0.056, 0.19, and 2.44 microM, and the number of binding sites corresponding to 0.10, 0.21, and 0.13/fibrin monomer unit of fibrin polymer were found for fibrinogen, fragment DD, and fragment D1, respectively. Fragment E1 did not bind to sonicated noncross-linked or cross-linked fibrin suspensions. However, it was bound to forming fibrin clots as well as to fibrin-Celite, suggesting that the binding sites on fibrin involved in the interaction with fragment E1 may have been altered upon sonication. Affinity chromatography of various fibrinogen derivatives on a fibrin-Celite column showed that only part of the bound fragment DD was displaced by arginine, whereas fragments D1 and E1 were completely eluted under the same conditions. The results indicate that interaction of fibrinogen with the preformed fibrin clots is characterized by affinity in the nanomolar range and that binding between fibrin monomers, in the process of clot formation, could be characterized by even a higher affinity.     

Calcium-binding site beta 2, adjacent to the "b" polymerization site, modulates lateral aggregation of protofibrils during fibrin polymerization

Structural analysis of recombinant fibrinogen fragment D revealed that the calcium-binding site (beta2-site) composed of residues BbetaAsp261, BbetaAsp398, BbetaGly263, and gammaGlu132 is modulated by the "B:b" interaction. To determine the beta2-site's role in polymerization, we engineered variant fibrinogen gammaE132A in which calcium binding to the beta2-site was disrupted by replacing glutamic acid at gamma132 with alanine. We compared polymerization of gammaE132A to normal fibrinogen as a function of calcium concentration. Polymerization of gammaE132A at concentrations of calcium 相似文献   

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.     

Fragment E derived from both fibrin and fibrinogen stimulates interleukin-6 production in rat peritoneal macrophages

Fibrin derived from fibrinogen after thrombin cleavage plays an essential role in forming blood clots. Fibrin as well as fibrinogen is also involved in the induction of platelet aggregation, leukocyte cell adhesion and phagocytosis. An additional biological role of fibrin and fibrinogen is presented in this study. One of the proteolytic peptides of fibrin/fibrinogen, fragment E, and not fragment D, was able to stimulate rat peritoneal macrophages to express interleukin-6 (IL-6). The stimulation of fibrin/fibrinogen fragment E on macrophages appeared to work in a dose- and time-dependent manner. Adherent fibrin fragment E was able to stimulate IL-6 expression as well as IL-6 protein production. The effect of fibrin fragment E was inhibited by the addition of an excess amount of GPRP tetrapeptide, but not by GHRP, which are the amino acids derived from the amino terminus of fibrin alpha and beta chains, respectively. These results suggest that fibrin as well as fibrinogen function as a stimulator to macrophages, and leukocyte integrin p150,95 (CD11c/ CD18), not Mac-I (CD11b/CD18), is involved in mediating fibrin stimulatory activity in macrophages.     

Mechanism of functioning of specific sites of interdomain D-E binding of fibrin molecules: interaction of fibrinogen with the N-terminal disulfide node of fibrin

The intermolecular noncovalent binding of complementary fibrin polymerization sites localized in fibrin domains D and E was investigated in the model system. In this system fibrinogen molecules represent the active D domains and the N-terminal disulphide knot of fibrin (N-DSK) represents the active E domain. Quantitative definition of insoluble fibrinogen and N-DSK copolymer and light scattering data of their mixtures before the appearance of visible precipitate show that complexing of these structures decreases with an increase of the temperature and ionic strength. The character of this dependence permits certain conclusions to be made on the functioning mechanism for two types of the D-E binding sites. These conclusions are based on an idea of their different affinity. The interdomain binding is primarily realized by D1-E1 sites which are characterized by a high affinity and work mainly on the basis of electrostatic forces. This binding directs the D2-E2 binding which is characterized by lower affinity and which determines the final degree of fibrinogen and N-DSK complexing. These sites function mainly by the H-binding.     

Gly-Pro-Arg-Pro modifies the glutamine residues in the alpha- and gamma-chains of fibrinogen: inhibition of transglutaminase cross-linking

During blood clotting Factor XIIIa, a transglutaminase, catalyzes the formation of covalent bonds between the epsilon-amino group of lysine and the gamma-carboxamide group of peptide-bound glutamine residues between fibrin molecules. We report that glycyl-L-prolyl-L-arginyl-L-proline (GPRP), a tetrapeptide that binds to the fibrin polymerization sites (D-domain) in fibrin(ogen), inhibits transglutaminase cross-linking by modifying the glutamine residues in the alpha- and gamma-chains of fibrinogen. Purified platelet Factor XIIIa, and tissue transglutaminase from adult bovine aortic endothelial cells were used for the cross-linking studies. Gly-Pro (GP) and Gly-Pro-Gly-Gly (GPGG), peptides which do not bind to fibrinogen, had no effect on transglutaminase cross-linking. GPRP inhibited platelet Factor XIIIa-catalyzed cross-linking between the gamma-chains of the following fibrin(ogen) derivatives: fibrin monomers, fibrinogen and polymerized fibrin fibers. GPRP functioned as a reversible, noncompetitive inhibitor of Factor XIIIa-catalyzed incorporation of [3H]putrescine and [14C]methylamine into fibrinogen and Fragment D1. GPRP did not inhibit 125I-Factor XIIIa binding to polymerized fibrin, demonstrating that the Factor XIIIa binding sites on fibrin were not modified. GPRP also had no effect on Factor XIIIa cross-linking of [3H]putrescine to casein. This demonstrates that GPRP specifically modified the glutamine cross-linking sites in fibrinogen, and had no effect on either Factor XIIIa or the lysine residues in fibrinogen. GPRP also inhibited [14C]putrescine incorporation into the alpha- and gamma-chains of fibrinogen without inhibiting beta-chain incorporation, suggesting that the intermolecular cross-linking sites were selectively affected. Furthermore, GPRP inhibited tissue transglutaminase-catalyzed incorporation of [3H]putrescine into both fibrinogen and Fragment D1, without modifying [3H]putrescine incorporation into casein. GPRP also inhibited intermolecular alpha-alpha-chain cross-linking catalyzed by tissue transglutaminase. This demonstrates that the glutamine residues in the alpha-chains involved in intermolecular cross-linking are modified by GPRP. This is the first demonstration that a molecule binding to the fibrin polymerization sites on the D-domain of fibrinogen modifies the glutamine cross-linking sites on the alpha- and gamma-chains of fibrinogen.     

Interaction between complementary polymerization sites in the structural D and E domains of human fibrin.

Plasmic degradation products of human fibrin, fragments DD, D, and E, bind to fibrin. It has been inferred from this observation that the binding occurs by attraction of complementary sites located in the NH2- and COOH-terminal domains of the fibrin molecule. The interaction between fragments D1 and E1 has been investigated in this work since it represents the first step in the process of fibrin clot formation. Fragment D1, that was initially as active as fragment DD, lost most of its anticoagulant activity after purification by cation-exchange chromatography. The lability of fragment D1 function explained the previous unsuccessful attempts to form a complex between fragments D1 and E1. The loss of fragment D1 anticoagulant activity was not associated with the cleavage of the gamma 63-85 chain segment, since fragments D1A and D1 identically inhibited the fibrin monomer polymerization rate. In order to demonstrate the formation of a complex between fragments D1 and E1, three lines of experiments were advanced. First, the anticoagulant activity of fragment D1 was neutralized by fragment E1 in a dose-dependent manner, demonstrating that the association between these fragments involved polymerization sites. Second, two products, D1.E1 and D1.E1.D1, were stabilized in a reaction with bifunctional cross-linking reagents, proving the formation of D.E complexes in aqueous solution. Third, immobilized fragment D1 bound fragments E1 and E2, but not fragment E3, showing that fragments E1 and E2 attached via a polymerization site to the complementary one in fragment D1, since this association was disrupted by fibrin polymerization inhibitory peptide GPRP. These results provided direct evidence for specific binding between the structural D and E domains of fibrin mediated through complementary polymerization sites. Thus, the initial formation of fibrin clot fibers appears to be driven by specific association of these sites.     

纤维蛋白单体D区与E区聚合后E区结构的变化

应用纤维蛋白单克隆抗体IF 5 3,观察当纤维蛋白的“A”位点与另一纤维蛋白D区域的“a”位点结合后纤维蛋白E区的变化 .纤维蛋白原Aα链经赖氨酰肽链内切酶消化后 ,应用反相HPLC分离纯化 ;通过ELISA法检测单克隆抗体IF 5 3与纤维蛋白原及其衍生物的反应情况 ;应用放射免疫法检测RGD合成肽抑制纤维蛋白单体与IF 5 3反应的情况 .发现IF 5 3能与纤维蛋白原Aα链的一个片段反应 ,该片段经氨基酸序列分析显示为纤维蛋白原Aα链氨基末端 (1～ 2 9) .该抗体能与酸溶解的纤维蛋白单体和可溶性纤维蛋白及XDP反应 ,但不能与酸化纤维蛋白原或GPRP反应 ,因此IF 5 3的抗原决定簇在Aα 2 0～ 2 9,与凝血酶作用于纤维蛋白肽A ,暴露出的聚合位点“A”(Aα17～19)紧邻 .当GPRP存在于纤维蛋白原溶液时 ,经凝血酶作用产生这种纤维蛋白单体不能与IF 5 3反应 .Aα(93～ 99) (ILRGDFS)合成肽部分抑制纤维蛋白单体与IF 5 3的反应 .实验结果提示 ,当纤维蛋白单体相互聚合 ,或纤维蛋白单体与纤维蛋白原聚合时 ,纤维蛋白单体结构会发生变化 ,其中Aα2 0～ 2 9片段成为新抗原暴露于E区表面 ,并且Aα2 0～ 2 9与纤维蛋白原细胞粘附区域RGD1片段邻近     

Interaction of fibrinogen with two forms of fibrin differing in the degree of activation by thrombin

The inhibitory effect of fibrinogen in the clotting of two fibrin monomer species--f-desAA and f-desAABB--was studied. The concentration dependence of this effect for two fibrin forms was found to be of the same character. This fact indicates that the modifying influence of fibrinogen proposed earlier in relation to f-desAABB also takes place in the case of f-desAA. However, an equal inhibitory effect is achieved for f-desAA at much higher fibrinogen concentrations than that for f-desAABB. The inhibitory effect of fibrinogen is greater at higher ionic strengths for both fibrin forms, but in the case of f-desAA this effect is more pronounced. The role of fibrin polymerization sites formed after fibrinopeptides B removal in initial fibrin polymerization and in F-f-desAABB interaction is discussed.     

Localization of a fibrinogen calcium binding site between gamma-subunit positions 311 and 336 by terbium fluorescence

Calcium is required for effective fibrin polymerization. The high affinity Ca2+ binding capacity of fibrinogen was directly localized to the gamma-chain by autoradiography of nitrocellulose membrane blots of fibrinogen subunits incubated with 45Ca2+. Terbium (Tb3+) competitively inhibited 45Ca2+ binding to fibrinogen during equilibrium dialysis, accelerated fibrin polymerization, and limited fibrinogen fragment D digestion by plasmin. The intrinsic fluorescence of Ca2+-depleted fibrinogen was maximally enhanced by Ca2+ and Tb3+, but not by Mg2+, at about 3 mol of cation/mol of fibrinogen. Protein-bound Tb3+ fluorescence at 545 nm was maximally enhanced by resonance energy transfer from tryptophan (excitation at 290 nm) at about 2 mol of Tb3+mol of fibrinogen and about 1 mol of Tb3+/mol of plasmic fragment D94 (Mr 94,000). Fibrinogen fragments D78 (Mr 78,000) and E did not show effective enhancement of Tb3+ fluorescence, suggesting that the Ca2+ site is located within gamma 303 to gamma 411, the peptide which is absent in fragment D78 but present in D94. When CNBr fragments of the carboxyamidated gamma-subunit were assayed for enhancement of Tb3+ fluorescence, peptide CBi (gamma 311-336) bound 1 mol of Tb3+/mol of CBi. Thus, the Ca2+ site is located within this peptide. The sequence between gamma 315 and gamma 329 is homologous to the calmodulin and parvalbumin Ca2+ binding sites.     

Conformational and structural modulation of the NH2-terminal regions of fibrinogen and fibrin associated with plasmin cleavage.

Conformational and structural modulations of the NH2-terminal region of fibrinogen and fibrin associated with plasmin cleavage have been examined utilizing specific antibody probes. The E region derived from the NH2-terminal aspects of fibrinogen undergoes complex structural and conformational changes throughout the cleavage process as indicated by differences in the quantitative and qualitative expression of antigenic determinants by the E region of each isolated cleavage fragment. When the range of antigenic determinants recognized by the antibody probe is limited to a specific molecular marker on the gamma chain within the E region, fg-E-neo, evidence for a systematic and progressive modulation of this site during plasmin cleavage is observed. Fg-E-neo undergoes progressive exposure as the cleavage of fibrinogen proceeds from X to Y to D:E complex. Separation of the D:E complex into its constituent, D and E fragments, is associated with further exposure of fg-E-neo determinants. The sequential cleavage of fibrin by plasmin also leads to progressive exposure of the fg-E-neo site; however, comparison of corresponding fragments derived from fibrinogen and fibrin reveals significant differences in the character of fg-E-neo expression. Immunochemical differences between fibrin and fibrinogen E fragments are not abolished by further exposure of the fragments to plasmin, are apparently not due to the presence or absence of fibrinopeptides, and are maintained following denaturation and renaturation of the fragments. These results suggest that the differential expression of fg-E-neo by the E fragments may be primarily dependent upon differences in amino acid compositions of the fragments.     

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.     

The role of D2-E2 centers of interdomain binding of fibrin monomer during fibrinogen co-polymerization with an NH2-terminal disulfide knot of fibrin

Copolymerization of fibrinogen with desAB- and desA-fibrin NH2-terminal disulfide knots (tN-DSK and rN-DSK, respectively) caused by interdomain D-E-binding was compared. It was shown that only tN-DSK effectively produces with fibrinogen soluble and insoluble forms of the copolymer characterized by a constant stoichiometry which, in turn, reflects its regular structure. Fibrinogen and rN-DSK complexing is weakly expressed. No soluble complexes were identified. A small quantity of insoluble complexes formed had no constant stoichiometry which points to the variability of their structure. It is concluded that the formation of the regular polymer structure during fibrinogen and fibrin N-DSK complex formation requires the participation of two types of complementary centers, namely: D1-E1 and D2-E2. This conclusion was confirmed by disturbances in fibrinogen and tN-DSK copolymerization at pH 6.5, when the function of the E2-center was inhibited. The significance of these findings for the understanding of the mechanisms of two types of D-E center function during fibrin clotting is discussed.     

Localization of a fibrin polymerization site complementary to Gly-His-Arg sequence

Dansyl-labeled tetrapeptide Gly-His-Arg-Pro which mimics the central fibrin polymerization site was used to investigate its binding to a number of fibrinogen fragments containing different numbers of domains. The tetrapeptide was found to bind to fragments D_H(95 kDa), D_L(82 kDa) and D_Y(63 kDa) but not to the TSD(28 kDa) fragment. The D_Y fragment differs from the TSD by the presence of β and βC domains. Therefore these domains, which are formed by the C-terminal part of the β chain, possess a polymerization site complementary to the Gly-His-Arg containing counterpart.     

Substitution of gamma Arg-275 by Cys in an abnormal fibrinogen, "fibrinogen Osaka II". Evidence for a unique solitary cystine structure at the mutation site

In an abnormal fibrinogen with impaired fibrin monomer polymerization designed as fibrinogen Osaka II, we have identified substitution of Arg by Cys at position 275 of the gamma chain. This Cys is linked to a free cysteine molecule by a disulfide link as evidenced by fast atom bombardment mass spectrometry. This finding was supported by identification of a single cysteine released from isolated abnormal fragment D1 upon reduction. This unique cystine structure at the mutation site has not been reported heretofore in any abnormal protein including fibrinogen. The substitution may well perturb the structure required for fibrin monomer polymerization, specifically that assigned to the carboxyl-terminal D domain of fibrinogen. Indeed, isolated fragment D1 with the Cys substitution failed to inhibit thrombin-mediated clotting of normal fibrinogen and normal fibrin monomer polymerization, while normal fragment D1 inhibited them markedly. Our data seem to provide supporting evidence that the putative polymerization site(s) assigned to the D domain of fibrinogen may be structure-dependent, including the carboxyl-terminal segment of the gamma chain as well as a contiguous region that contains the gamma 275 residue.     

Evidence that catalytically-inactivated thrombin forms non-covalently linked dimers that bridge between fibrin/fibrinogen fibers and enhance fibrin polymerization

Phe-pro-arg-chloromethyl ketone-inhibited alpha-thrombin [FPR alpha-thr] retains its fibrinogen recognition site (exosite 1), augments fibrin/fibrinogen [fibrin(ogen)] polymerization, and increases the incorporation of fibrin into clots. There are two 'low-affinity' thrombin-binding sites in each central E domain of fibrin, plus a non-substrate 'high affinity' gamma' chain thrombin-binding site on heterodimeric 'fibrin(ogen) 2' molecules (gamma(A), gamma'). 'Fibrin(ogen) 1' (gamma(A), gamma(A)) containing only low-affinity thrombin-binding sites, showed concentration-dependent FPR alpha-thr enhancement of polymerization, thus indicating that low-affinity sites are sufficient for enhancing polymerization. FPR gamma-thr, whose exosite 1 is non-functional, did not enhance polymerization of either fibrin(ogen)s 1 or 2 and DNA aptamer HD-1, which binds specifically to exosite 1, blocked FPR alpha-thr enhanced polymerization of both types of fibrin(ogen) (1>2). These results showed that exosite 1 is the critical element in thrombin that mediates enhanced fibrin polymerization. Des B beta 1-42 fibrin(ogen) 1, containing defective 'low-affinity' binding sites, was subdued in its FPR alpha-thr-mediated reactivity, whereas des B beta 1-42 fibrin(ogen) 2 (gamma(A), gamma') was more reactive. Thus, the gamma' chain thrombin-binding site contributes to enhanced FPR alpha-thr mediated polymerization and acts through a site on thrombin that is different from exosite 1, possibly exosite 2. Overall, the results suggest that during fibrin clot formation, catalytically-inactivated FPR alpha-thr molecules form non-covalently linked thrombin dimers, which serve to enhance fibrin polymerization by bridging between fibrin(ogen) molecules, mainly through their low affinity sites.     

Polymerization and gelation of fibrinogen in D2O

The solution properties of fibrinogen and the thrombin-induced activation and gelation of fibrinogen in 95% D2O at pH 7.4 were compared to those in H2O under similar conditions. The initial release rates of fibrinopeptides A and B in D2O were slightly slower than those in H2O. However, the values of the Michaelis-Menten parameters Km and V for the release of the two peptides in D2O and H2O in the presence of 0.5 M NaCl were about the same. From turbidity measurements at 450 nm it is obvious that fibrinogen is soluble in a slightly more narrow range of NaCl concentration and that the fibrin gels have a higher degree of lateral aggregation in D2O than in H2O. The variation of fibrinogen concentration, thrombin concentration, pH and ionic a strength have a similar dependence on the final gel structure and clotting time in D2O and H2O. SDS-gel electrophoresis on fibrin samples, which were cross-linked by factor XIII, yielded results where the cross-linking of the gamma-chain appeared to be the same in D2O and H2O. The alpha-chain cross-linking was somewhat faster in D2O than in H2O. When fibrinogen solutions in 95% D2O were incubated at 20 mM CaCl2, a slow gelation of fibrinogen was observed, which was found to be induced by trace amounts of factor XIII. The final gel turbidity appeared to be about the same for this gelation as for that induced by thrombin. The differences in solubility for fibrinogen, kinetics for the enzyme reaction and optical properties for the fibrin gels in D2O and H2O may be explained by differences in electrostatic interactions, hydrogen bonding and hydration of fibrinogen in these two media.     

Expression of primary polymerization sites in the D domain of human fibrinogen depends on intact conformation

Fragments D1 and DD, plasmic degradation products of human fibrinogen and cross-linked fibrin, respectively, originate from the COOH-terminal domain of the parent molecule. Since a specific binding site for fibrin resides in the COOH-terminal region of the gamma chain, the primary structure of the two fragments was compared and their affinity for fibrin monomer measured. Fragments D1 and DD contained the same segments of the three fibrinogen chains, corresponding to the sequences alpha 105-206, beta 134-461, and gamma 63-411. Fragment DD had a double set of the same chain remnants. Fragments D1 and DD inhibited polymerization of fibrin monomer in a dose-dependent manner; 50% inhibition occurred at a molar ratio of fragment to monomer of 1:1 and 0.5:1, respectively. To prevent fibrin monomer polymerization and render it suitable for binding studies in the liquid phase, fibrinogen was decorated with Fab fragments isolated from rabbit antibodies to human fragment D1. Fibrinogen molecules decorated with 6 molecules of this Fab fragment did not clot after incubation with thrombin, and the decorated fibrin monomer could be used to measure binding of fragments D1 and DD in a homogeneous liquid phase. The data analyzed according to the Scatchard equation and a double-reciprocal plot gave a dissociation constant of 12 nM for fragment D1 and 38 nM for fragment DD. There were two binding sites/fibrin monomer molecule for each fragment. After denaturation in 5 M guanidine HCl, the inhibitory function on fibrin polymerization was irreversibly destroyed. Denatured fragments also lost binding affinity for immobilized fibrin monomer. The preservation of the native tertiary structure in both fragments was essential for the expression of polymerization sites in the structural D domain.