Synthesis and study of biological activity of stereoisomeric dipeptides containing tyrosine and arginine residues

Series of methyl esters of stereoisomeric dipeptides of the sequences Tyr-Arg and Arg-Tyr has been synthesized by classic methods of the peptide chemistry. The study of their reactivity towards thrombin and trypsin has shown that the kinetic parameters of enzyme-catalyzed hydrolyses of stereoisomeric compounds differ in values essentially. Testing the synthetic peptides on analgic effect, on inhibition of the reaction fibrinogen with thrombin or on influence upon the process of fibrin-monomer polymerization has shown that these biological effects depend on peptide structure and on configuration of amino acid residues forming the peptides.     

Peptide-derivatized albumins that inhibit fibrin polymerization

Synthetic peptides patterned on sequences that appear during thrombin proteolysis of fibrinogen are known to influence fibrin formation in very different ways. A-Knob sequences (GPR-) inhibit polymerization, but B-knob sequences (GHR-) can actually enhance the process. We now report that when such peptides are attached to albumin carriers, both knob conjugates inhibit fibrin formation. In contrast, the 2-aminoethylthiol-albumin conjugate control enhances the polymerization to the same degree as albumin. The peptide AHRPam, which is known to bind exclusively to the βC holes of fibrinogen/fibrin, nullifies the inhibitory effects of the GHRPYGGGCam-albumin conjugate on fibrin polymerization, indicating that the inhibition was exclusively due to interactions with βC holes. AHRPam was much less effective in countering inhibition by the GPRPGGGGCam-albumin conjugate, suggesting that the observed effects with this conjugate involve mainly the γC holes of fibrin/fibrinogen. This study demonstrates that peptides modeled on fibrin polymerization knobs tethered to albumin retain their capacity to interact with fibrinogen/fibrin and may prove useful as inhibitors of clotting in vivo.     

Gel formation by fibrin oligomers without addition of monomers

Soluble fibrin oligomers were formed by reacting fibrinogen with thrombin under fine clotting conditions where the action of thrombin is the rate-determining step for polymerization, and by inhibiting the reaction shortly before gelation. Oligomeric fibrin was separated from unreacted fibrinogen and small oligomers by gel permeation chromatography. Electron microscopy revealed that the largest soluble fibrin oligomers resemble the protofibrils present in fine clots, but are somewhat shorter and entirely lack the twisted, trifunctional junctions that contribute to the elastic properties of fine clots. When thrombin was added to the soluble fibrin oligomers, polymerization resumed and clots were formed at a more rapid rate than from fibrinogen at the same concentration and resulted in a less-opaque clot under coarse clotting conditions. The results confirm a prediction of a theory for the polymerization of fibrin and provide additional evidence that the final state of a coarse fibrin clot depends on the mobility of protofibrils during its formation.     

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.     

Effect of heparin on fibrinogen formation during experimental toxic syndrome of disseminated intravascular coagulation

The formation of circulation components of fibrinogen pool in toxigenic syndrome of disseminated intravascular blood coagulation (DBC-syndrome) caused by the Vipera lebetina turanica venom has been examined by the gel-filtration method. Simultaneously it has been studied what components of fibrinogen pool are removed in paracoagulation tests and with addition of the coagulating enzymes (thrombin, reptilase and the Echis multisguamatus venom) to the plasma. The preliminary heparinization of animals poisoned by Vipera lebetina turanica venom was found to prevent the fibrinogen formation mainly at the fibrin-monomer formation stage. Besides, the effect of polymerization inhibition was revealed in the plasma of such animals.     

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.     

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 impact of delayed fibrinopeptide-A release on fibrin structure. Studies of an abnormal fibrinogen

The impact of delayed fibrinopeptide-A release on polymerization and structure of fibrin gels was studied utilizing a heterozygously transmitted variant fibrinogen. An arginine to histidine substitution at position 16 of the alpha chain of the abnormal fibrinogen delayed release of an abnormal fibrinopeptide-A (A) by thrombin and completely blocked release of A by reptilase. When clotted with thrombin, patient fibrin formed more slowly than normal fibrin, but clottability was normal and gel fiber mass/length ratios were decreased less than 10%. Gels formed with reptilase clotted slowly, demonstrated reduced clottability, but had normal fiber mass/length ratios. Reptilase clotted the normal but not the variant component of the patient fibrinogen. Thrombin-induced cleavage of fibrinopeptide-B prior to A occurred in these experiments, but polymerization of this species beyond trimers has been reported to be minimal under the conditions used. With time, A is removed by thrombin resulting in the slow production of normal fibrin monomer from the abnormal component. These monomers subsequently polymerize. The minimal change in gel fiber size caused by slow A release implies that fibrin fiber size is primarily a function of ionic environment and not of the sequence of peptide release.     

The role of gamma A/gamma ' fibrinogen in plasma factor XIII activation

Factor XIII zymogen activation is a complex series of events that involve fibrinogen acting in several different roles. This report focuses on the role of fibrinogen as a cofactor in factor XIII activation by thrombin. We demonstrate that fibrinogen has two distinct activities that lead to an increased rate of factor XIII activation. First, the thrombin proteolytic activity is increased by fibrin. The cleavage rates of both a small chromogenic substrate and the factor XIII activation peptide are increased in the presence of either the major fibrin isoform, gammaA/gammaA fibrin, or a minor variant form, gammaA/gamma' fibrin. This enhancement of thrombin activity by fibrin is independent of fibrin polymerization and requires only cleavage of the fibrinopeptides. Subsequently, gammaA/gamma' fibrinogen accelerates plasma factor XIII activation by a non-proteolytic mechanism. This increased rate of activation results in a slightly more rapid cross-linking of fibrin gammaA and gamma' chains and a significantly more rapid cross-linking of fibrin alpha chain multimers. Together, these results show that although both forms of fibrin increase the rate of activation peptide cleavage by thrombin, gammaA/gamma' fibrinogen also increases the rate of factor XIII activation in a non-proteolytic manner. A revised model of factor XIII activation is presented below.     

Effect of fibronectin on fibrinogen conversion into fibrin

The effects of fibronectin on fibrinogen clotting induced by thrombin or reptilase and on fibrin monomer polymerization in a pure system in the absence of factor XIIIa were studied. It was shown that within a broad range of concentrations and molar ratios of the mixed proteins, fibronectin does not alter significantly the fibrinogen clotting time either under thrombin or under reptilase action. The effect of fibronectin on the fibrin self-assembly consists in a slight acceleration of this process, whose degree is directly dependent on the fibronectin/fibrin monomer molar ratio as well as on the absolute fibrin monomer content at a constant molar ratio. The stimulating effect of fibronectin is amplified by Ca2+. The experimental results suggest that fibronectin can noncovalently bind the fibrin monomer and/or intermediate polymers in the non-enzymatic phase of fibrinogen conversion to fibrin.     

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.     

The Non-catalytic B Subunit of Coagulation Factor XIII Accelerates Fibrin Cross-linking

Covalent cross-linking of fibrin chains is required for stable blood clot formation, which is catalyzed by coagulation factor XIII (FXIII), a proenzyme of plasma transglutaminase consisting of catalytic A (FXIII-A) and non-catalytic B subunits (FXIII-B). Herein, we demonstrate that FXIII-B accelerates fibrin cross-linking. Depletion of FXIII-B from normal plasma supplemented with a physiological level of recombinant FXIII-A resulted in delayed fibrin cross-linking, reduced incorporation of FXIII-A into fibrin clots, and impaired activation peptide cleavage by thrombin; the addition of recombinant FXIII-B restored normal fibrin cross-linking, FXIII-A incorporation into fibrin clots, and activation peptide cleavage by thrombin. Immunoprecipitation with an anti-fibrinogen antibody revealed an interaction between the FXIII heterotetramer and fibrinogen mediated by FXIII-B and not FXIII-A. FXIII-B probably binds the γ-chain of fibrinogen with its D-domain, which is near the fibrin polymerization pockets, and dissociates from fibrin during or after cross-linking between γ-chains. Thus, FXIII-B plays important roles in the formation of a ternary complex between proenzyme FXIII, prosubstrate fibrinogen, and activator thrombin. Accordingly, congenital or acquired FXIII-B deficiency may result in increased bleeding tendency through impaired fibrin stabilization due to decreased FXIII-A activation by thrombin and secondary FXIII-A deficiency arising from enhanced circulatory clearance.     

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.     

Fibrin digestion by thrombin. Comparison with plasmin-digested fibrinogen.

Solutions of plasminogen-free human fibrinogen alone or (1) treated with sodium p-chloromercuribenzoate in order to inactivate factor XIII, or (2) enriched with factor XIII, cysteine and CaC12, were clotted with plasmin-free human thrombin and incubated under sterile conditions. The clots dissolved gradually within 2 days (fibrin from sodium p-chloromercuribenzoate-treated fibrinogen) to 15 days (fibrin from factor XIII-enriched fibrinogen). This proteolytic process was not affected by soybean trypsin inhibitor but was completely inhibited by hirudin. Gel electrophoresis of the thrombin digests indicated the formation of bands equivalent to bands X, Y, D and E of plasmin digests of fibrinogen. The two latter bands, whose identity was confirmed by immunoelectrophoresis, appeared at a more advanced stage of proteolysis than the corresponding bands of plasmin digests. The number of isopeptide bonds present did not appear to affect the rate of release of acid-soluble peptides. Gel electrophoresis and the rate of release of acid-soluble peptides indicated that fewer bonds are hydrolysed by thrombin at the time of the complete solubilization of the clot than are split by plasmin when fibrinogen becomes unclottable by thrombin.     

Roles of low specificity and cofactor interaction sites on thrombin during factor XIII activation. Competition for cofactor sites on thrombin determines its fate

Factor XIII is activated by thrombin, and this reaction is enhanced by the presence of fibrin(ogen). Using a substrate-based screening assay for factor XIII activity complemented by kinetic analysis of activation peptide cleavage, we show by using thrombin mutants of surface-exposed residues that Arg-178, Arg-180, Asp-183, Glu-229, Arg-233, and Trp-50 of thrombin are necessary for direct activation of factor XIII. These residues define a low specificity site known to be important also for both protein C activation and for inhibition of thrombin by antithrombin. The enhancing effect of fibrinogen occurs as a consequence of its conversion to fibrin and subsequent polymerization. Surface residues of thrombin further involved in high specificity fibrin-enhanced factor XIII activation were identified as His-66, Tyr-71, and Asn-74. These residues represent a distinct interaction site on thrombin (within exosite I) also employed by thrombomodulin in its cofactor-enhanced activation of protein C. In competition experiments, thrombomodulin inhibited fibrin-enhanced factor XIII activation. Based upon these and prior published results, we propose that the polymerization process forms a fibrin cofactor that acts to approximate thrombin and factor XIII bound to separate and complementary domains of fibrinogen. This enables enhanced factor XIII activation to be localized around the fibrin clot. We also conclude that proximity to and competition for cofactor interaction sites primarily directs the fate of thrombin.     

Coarse-grained molecular dynamics simulations of fibrin polymerization: effects of thrombin concentration on fibrin clot structure

Studies suggest that patients with deep vein thrombosis and diabetes often have hypercoagulable blood plasma, leading to a higher risk of thromboembolism formation through the rupture of blood clots, which may lead to stroke and death. Despite many advances in the field of blood clot formation and thrombosis, the influence of mechanical properties of fibrin in the formation of thromboembolisms in platelet-poor plasma is poorly understood. In this paper, we combine the concepts of reactive molecular dynamics and coarse-grained molecular modeling to predict the complex network formation of fibrin clots and the branching of fibrin monomers. The 340-kDa fibrinogen molecule was converted into a coarse-grained molecule with nine beads, and using our customized reactive potentials, we simulated the formation and polymerization process of a fibrin clot. The results show that higher concentrations of thrombin result in higher branch-point formation in the fibrin clot structure. Our results also highlight many interesting properties, such as the formation of thicker or thinner fibers depending on the thrombin concentration. To the best of our knowledge, this is the first successful molecular polymerization study of fibrin clots to focus on thrombin concentration.     

Three-dimensional structural studies on fragments of fibrinogen and fibrin

Fibrinogen is a 340 kDa glycoprotein found in the blood plasma of all vertebrates. It is transformed into a fibrin clot by the action of thrombin. Recent X-ray structures of core fragments of both fibrinogen and fibrin have revealed many details about this polymerization event. These include structures of a 30 kDa recombinant γC domain, an 86 kDa fragment D from human fibrinogen and a cross-linked double-D fragment from fibrin.     

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     

Differences in the clotting of lamprey fibrinogen by lamprey and bovine thrombins

1. Improved methods for the purification of lamprey thrombin and fibrinogen are presented. 2. Lamprey thrombin releases two fibrinopeptides from lamprey fibrinogen during the transformation into fibrin. Bovine thrombin releases only one of these, a peptide referred to as fibrinopeptide B. The differences in the by-products of fibrin formation are reflected in the different N-terminal amino acid compositions of the two types of fibrin. 3. The fibrinopeptide that is not removed from the lamprey fibrinogen by bovine thrombin can subsequently be released by treatment of that fibrin with lamprey thrombin. 4. Under the conditions used, lamprey thrombin releases both fibrinopeptides at about the same rate. 5. The differences in interaction among these pairs of related proteins are extreme manifestations of the phenomenon loosely referred to as `species specificity'.     

Hirudin-based peptides block the inflammatory effects of thrombin on endothelial cells

Thrombin is a serine protease that plays an essential role in blood coagulation and also induces various responses in endothelial cells. The actions of thrombin on the conversion of fibrinogen to fibrin are inhibited by peptides based on the amino acid sequence of hirudin, a natural anticoagulant from leeches. We show in these studies that the peptides Hir45-64 and sulfated Hir53-64 block the effects of thrombin on endothelial cells. These peptides inhibited, in a concentration-dependent manner, the synthesis of prostaglandin I2 and platelet-activating factor, and the acquisition of an adhesive surface for leukocytes that occur in response to thrombin. These actions of the peptides occurred even though the catalytic site of thrombin was not blocked.