Polymerisation of fibrin αC-domains promotes endothelial cell migration and proliferation.

Upon conversion of fibrinogen into fibrin, fibrinogen αC-domains containing the RGD recognition motif form ordered αC polymers. Our previous study revealed that polymerisation of these domains promotes integrin-dependent adhesion and spreading of endothelial cells, as well as integrin-mediated activation of the FAK and ERK1/2 signalling pathways. The major goal of this study was to test the impact of αC-domain polymerisation on endothelial cell migration and proliferation during wound healing, and to clarify the mechanism underlying superior activity of αC polymers toward endothelial cells. In an in vitro wound healing assay, confluent endothelial cell monolayers on tissue culture plates coated with the αC monomer or αC polymers were wounded by scratching and wound closure was monitored by time-lapse videomicroscopy. Although the plates were coated with equal amounts of αC species, as confirmed by ELISA, wound closure by the cells occurred much faster on αC polymers, indicating that αC-domain polymerisation promotes cell migration and proliferation. In agreement, endothelial cell proliferation was also more efficient on αC polymers, as revealed by cell proliferation assay. Wound closure on both types of substrates was equally inhibited by the integrin-blocking GRGDSP peptide and a specific antagonist of the ERK1/2 signalling pathway. In contrast, blocking the FAK signaling pathway by a specific antagonist decreased wound closure only on αC polymers. These results indicate that polymerisation of the αC-domains enhances integrin-dependent endothelial cell migration and proliferation mainly through the FAK signalling pathway. Furthermore, clustering of integrin-binding RGD motifs in αC polymers is the major mechanism triggering these events.

[Family-school interaction in solving the problem of pupils' health preservation].

The paper covers the most methodological priority forms of a family-school interaction, which are necessary for effective collaboration in molding children's health. It gives the results of a study conducted at experimental schools, which showed the level of performance of heath-preserving, tutorial, and educational functions of a family.

Interaction of fibrin with VE-cadherin and anti-inflammatory effect of fibrin-derived fragments.

BACKGROUND: The interaction of the fibrin ßN-domain with VE-cadherin on endothelial cells is implicated in transendothelial migration of leukocytes, and the ß15-42 fragment representing part of this domain has been shown to inhibit this process. However, our previous study revealed that only a dimeric (ß15-66)(2) fragment, corresponding to the full-length ßN-domain and mimicking its dimeric arrangement in fibrin, bound to VE-cadherin. OBJECTIVE: To test our hypothesis that dimerization of ß15-42-containing fragments increases their affinity for VE-cadherin and ability to inhibit transendothelial migration of leukocytes. METHODS: Interaction of ß15-42-containing fragments with VE-cadherin was characterized by ELISA and surface plasmon resonance. The inhibitory effect of such fragments was tested in vitro with a leukocyte transendothelial migration assay and in vivo with mouse models of peritonitis and myocardial ischemia-reperfusion injury. RESULTS: First, we prepared the monomeric ß15-42 and ß15-64 fragments and their dimeric forms, (ß15-44)(2) and (ß15-66)(2) , and studied their interaction with the fibrin-binding domain of VE-cadherin, VE-cad(3). The experiments revealed that both dimeric fragments bound to VE-cad(3) with high affinity, whereas the affinities of ß15-42 and ß15-64 were significantly lower. Next, we tested the ability of these fragments to inhibit leukocyte transmigration in vitro and infiltration into the inflamed peritoneum in vivo, and found that the inhibitory effects of the dimers on these processes were also superior. Furthermore, (ß15-44)(2) significantly reduced myocardial injury induced by ischemia-reperfusion. CONCLUSION: The results confirm our hypotheses and indicate that (ß15-66)(2) and (ß15-44)(2) , which exhibited much higher affinity for VE-cadherin, are highly effective in suppressing inflammation by inhibiting leukocyte transmigration.

Evaluation of fibrinogen self-assembly: role of its αC region.

BACKGROUND: Exposure of cryptic, functional sites on fibrinogen upon its adsorption to hydrophobic surfaces of biomaterials has been linked to an inflammatory response and fibrosis. Such adsorption also induces ordered fibrinogen aggregation which is poorly understood. OBJECTIVE: To investigate hydrophobic surface-induced fibrinogen aggregation. METHODS: Contact and lateral force scanning probe microscopy, yielding topography, image dimensions and fiber elastic modulus measurements were used along with transmission and scanning electron microscopy. Fibrinogen aggregation was induced under non-enzymatic conditions by adsorption on a trioctyl-surface monolayer (trioctylmethylamine) grafted onto silica clay plates. RESULTS: A more than one molecule thick coating was generated by adsorption on the plate from 100 to 200 µg mL⁻¹ fibrinogen solutions, and three-dimensional networks formed from 4 mg mL⁻¹ fibrinogen incubated with uncoated or fibrinogen-coated plates. Fibrils appeared laterally assembled into branching and overlapping fibers whose heights from the surface ranged from approximately 3 to 740 nm. The elastic modulus of fibrinogen fibers was 1.55 MPa. No fibrils formed when fibrinogen lacking αC-domains was used as a coating or was incubated with intact fibrinogen-coated plates, or when the latter plates were sequentially incubated with anti-Aα529-539 mAb and intact fibrinogen. When an anti-Aα241-476 mAb was used instead, fine, long fibers formed. Similarly, sequential incubations of fibrinogen-coated plates with recombinant αC-domain (Aα392-610 fragment) or αC-connector (Aα221-372 fragment) and fibrinogen resulted in distinctly fine fiber networks. CONCLUSIONS: Adsorption-induced fibrinogen self-assembly is initiated by a more than one molecule-thick surface layer and eventuates in three-dimensional networks whose formation requires fibrinogen with intact αC-domains.

Plasma fibrinogen gamma' chain content in the thrombotic microangiopathy syndrome.

BACKGROUND: Human fibrinogen gamma chain variants, termed gamma' chains, contain a unique 20-residue sequence after gamma chain residue 407 that ends at gamma'427, and is designated gamma'(427L). Full-length (FL) gamma'(427L) chains are constituents of a fibrin-dependent thrombin inhibitory system known as antithrombin I, whereas a gamma' chain processed in vivo, termed gamma'(423P), lacks the C-terminal tetrapeptide EDDL, and does not bind thrombin. Together, the gamma'(423P) and gamma'(427L) chains comprise the total plasma fibrinogen gamma' chain content. OBJECTIVES: Lowered plasma gamma' chain content (i.e. gamma' chain-containing fibrinogen/total fibrinogen ratio) has been shown to correlate with susceptibility to venous thrombosis, thus prompting this study on the total and FL gamma' chain content in 45 subjects with thrombotic microangiopathy (TMA), a disorder characterized by microvascular thrombosis. METHODS: We measured by enzyme-linked immunosorbent assay the total gamma' chain-containing fibrinogen/total fibrinogen (Total gamma'-fgn/Total fgn) ratio and the FL gamma' chain-containing fibrinogen/total fibrinogen (FL gamma'-fgn/Total fgn) ratio in these plasmas and in healthy subjects (n = 87). RESULTS: In healthy subjects, the mean Total gamma'-fgn/Total fgn ratio was 0.127, whereas the FL gamma'-fgn/Total fgn ratio was somewhat lower at 0.099 (P < 0.0001), a difference reflecting the presence of gamma'(423P) chains. In TMA plasmas, both the Total gamma'-fgn and FL gamma'-fgn/Total fgn ratios (0.099 and 0.084, respectively) were lower than those of their healthy subject counterparts (P < 0.0001). CONCLUSIONS: These findings in TMA suggest that reductions in the gamma' chain content indicate reduced antithrombin I activity that may contribute to microvascular thrombosis in TMA.

[Etalon-free laser mass-spectrometry as a new method of the elementary analysis of bone tissue at identification]. / Bezétalonnaia lazernaia mass-spektometriia - novyi metod élementnogo analiza kostnoi tkani pri reshsenii identsifikatsionnykh zadach.

The results of an experimental study dealing with the elementary composition of bone tissue by using the method of laser mass spectrometry are described in the paper. The method ensures the quantification of concentration values of all elements from the Mendeleev periodic table that can be made within an extensive dynamic range, which makes it promising in the forensic medical identification.

Crystal structure of the central region of bovine fibrinogen (E5 fragment) at 1.4-A resolution.

The high-resolution crystal structure of the N-terminal central region of bovine fibrinogen (a 35-kDa E(5) fragment) reveals a remarkable dimeric design. The two halves of the molecule bond together at the center in an extensive molecular "handshake" by using both disulfide linkages and noncovalent contacts. On one face of the fragment, the Aalpha and Bbeta chains from the two monomers form a funnel-shaped domain with an unusual hydrophobic cavity; here, on each of the two outer sides there appears to be a binding site for thrombin. On the opposite face, the N-terminal gamma chains fold into a separate domain. Despite the chemical identity of the two halves of fibrinogen, an unusual pair of adjacent disulfide bonds locally constrain the two gamma chains to adopt different conformations. The striking asymmetry of this domain may promote the known supercoiling of the protofibrils in fibrin. This information on the detailed topology of the E(5) fragment permits the construction of a more detailed model than previously possible for the critical trimolecular junction of the protofibril in fibrin.

Structural and functional role of the beta-strand insert (gamma 381-390) in the fibrinogen gamma-module. A "pull out" hypothesis.

Study of the folding status of the fibrinogen gamma-module (residues gamma 148-411) revealed that its COOH-terminal beta-strand (residues gamma 381-390), that is normally inserted into its central domain, can be removed without destroying its compact structure. Based on this and other observations we propose a "pull out" hypothesis that suggests a mechanism for the formation of transverse gamma-gamma crosslinks in fibrin.

Conformational changes upon conversion of fibrinogen into fibrin. The mechanisms of exposure of cryptic sites.

Conformational changes upon conversion of fibrinogen to fibrin result in the exposure of multiple binding sites that provide its interaction with various proteins and cells and, thus, its participation in a number of physiological and pathological processes. Here we focus on conformational changes in the fibrinogen D regions (domains) and alpha C-domains that are directly involved in intermolecular interactions upon fibrin assembly. According to the current view, two alpha C-domains that interact intramolecularly in fibrinogen undergo an intra- to intermolecular switch to form alpha C-polymers in fibrin. The availability of recombinant fragments that correspond to the alpha C-domain made it possible to further clarify this mechanism and to reveal novel cryptic sites in this domain for plasminogen and its activator tPA, whose exposure may play an important role in the regulation of fibrinolysis. To elucidate the mechanism of exposure of cryptic sites in the D regions, we tested the accessibility of their fibrin specific epitopes (A alpha 148-160 and gamma 312-324) that are also involved in binding of plasminogen and tPA, in several fragments derived from fibrinogen (fragment D), and crosslinked fibrin (fragment D-D and its non-covalent complex with the E1 fragment, D-D:E1). Neither D nor D-D bound tPA, plasminogen, or anti-A alpha 148-160 and anti-gamma 312-324 monoclonal antibodies. At the same time both epitopes became accessible in the D-D:E1 complex. Melting of D and D-D revealed that their domains have the same stability while in the D-D:E1 complex they became more stable. These results indicate that upon fibrin assembly, driven primarily by the interaction between complementary binding sites of the E and two D regions, the latter undergo conformational changes that cause the exposure of their cryptic sites. They also suggest that the fibrin specific conformation of the D regions is preserved in the D-D:E1 complex.

The structure and function of the alpha C domains of fibrinogen.

The alpha C domains have been localized on fibrinogen and fibrin. Several model systems have been developed to study their functions. Analysis of the amino acid sequence of the alpha C domains suggested that each is made up of a globular and an extended portion. Microcalorimetry confirmed this result and showed that the two alpha C domains interact intramolecularly. Electron microscopy of fibrinogen with a monoclonal antibody to the alpha C domains demonstrated that these regions normally interact with the central portion of the molecule. In the conversion from fibrinogen to fibrin there is a large scale conformational change, such that the alpha C domains dissociate from the central region and are available for intermolecular interaction. Experiments with highly purified and well characterized fragment X monomer, missing either one or both of the alpha C domains, indicate that intermolecular interactions between alpha C domains are important for the enhancement of lateral aggregation during fibrin polymerization. Isolated alpha C fragments polymerized at neutral pH and interacted with the alpha C domains of fibrin monomer to influence clot formation. Several dysfibrinogenemias in which there are amino acid substitutions in, or truncations of, the alpha C domains revealed that these changes can have dramatic effects on polymerization and clot structure. The polymerization of A alpha 251 recombinant fibrinogen, that contains A alpha chains truncated at residue 251, was altered, as were the mechanical properties and the rate of fibrinolysis of the clots. Altogether, these results help to define the role of the alpha C domains in determining the structure and properties of clots.

Fibrinogen alpha C domains contain cryptic plasminogen and tPA binding sites.

Surface plasmon resonance and ELISA experiments revealed that recombinant fibrinogen alpha C fragment (residues A alpha 221-610) corresponding to the alpha C domain binds tPA and plasminogen with high affinity. This binding was found to be Lys-dependent and occurred via independent binding sites. Study with truncated variants of the alpha C fragment located these sites in its COOH-terminal half. Binding of tPA and plasminogen to these sites stimulated activation of the latter whereas proteolytic degradation of the alpha C fragment reduced this effect substantially, suggesting the importance of the alpha C domains in regulation of fibrinolysis.

Identification and characterization of novel tPA- and plasminogen-binding sites within fibrin(ogen) alpha C-domains.

Molecular defects in the alphaC-domains of some abnormal fibrinogens have been associated with impaired fibrin-mediated activation of plasminogen (Pg) by its activator tPA, suggesting the involvement of these domains in fibrinolysis. To test this suggestion, we expressed in E. coli the alphaC-fragment (residues Aalpha221-610) corresponding to the entire alphaC-domain as well as its NH(2)- and COOH-terminal halves (residues Aalpha221-391 and Aalpha392-610) and tested their effects on activation of Pg and their interaction with Pg and tPA. When the activation was monitored by cleavage of a chromogenic substrate with newly formed plasmin, the reaction was much more efficient in the presence of the alphaC-fragment. This stimulation was abolished upon digestion of the alphaC-fragment with plasmin. In surface plasmon resonance experiments, both tPA and Pg bound to the immobilized alphaC-fragment with K(d)s of 33 and 32 nM, respectively. Similar results were obtained by ELISA. This binding occurred via independent sites since saturating amounts of Pg did not prevent binding of tPA and vice versa. Both sites were localized in the COOH-terminal half of the alphaC-domain since the Aalpha392-610 fragment bound both tPA and Pg and was an effective stimulator whereas Aalpha221-391 was inactive. These results indicate that the fibrinogen alphaC-domains contain novel high-affinity tPA- and Pg-binding sites that play an important role in the regulation of fibrinolysis.

Role of the beta-strand insert in the central domain of the fibrinogen gamma-module.

The crystal structure of the fibrinogen gamma-module (residues gamma143-411) [Yee, V. C., et al. (1997) Structure 5, 125-138] revealed an unusual feature. Namely, residues gamma381-390 in the functionally important COOH-terminal region form a beta-strand that is inserted into an antiparallel beta-sheet of the central domain (gamma192-286), while the rest (gamma393-411) seems to be flexible. To clarify the structural and functional importance of this beta-strand insert, we analyzed the folding status of the plasmin-derived fibrinogen fragment D(3) and several truncated variants of the gamma-module expressed in Escherichia coli. It was found that D(3), in which most of the COOH-terminal domain of the gamma-module (gamma287-379) is removed proteolytically, retains a gamma374-405 peptide that seems to be associated noncovalently with the bulk of the molecule via its beta-strand insert region. A study of the denaturation-renaturation process of D(3) suggested that without this peptide its truncated gamma-module remains folded but is destabilized. This was confirmed directly with the truncated recombinant variants of the gamma-module, including residues gamma148-392, gamma148-373, and gamma148-286. They all were folded, but those devoid of the beta-strand insert were destabilized. The results indicate that although the beta-strand insert contributes to the stabilization of the gamma-module, it can be removed without destroying the compact structure of the latter. On the basis of this finding and some other observations, we propose a mechanism for the function-related conformational changes in the fibrin(ogen) gamma-modules.

Conversion of fibrinogen to fibrin: mechanism of exposure of tPA- and plasminogen-binding sites.

Conversion of fibrinogen into fibrin results in the exposure of cryptic interaction sites and modulation of various activities. To elucidate the mechanism of this exposure, we tested the accessibility of the Aalpha148-160 and gamma312-324 fibrin-specific epitopes that are involved in binding of plasminogen and its activator tPA, in several fragments derived from fibrinogen (fragment D and its subfragments) and fibrin (cross-linked D-D fragment and its noncovalent complex with the E(1) fragment, D-D. E(1)). Neither D nor D-D bound tPA, plasminogen, or anti-Aalpha148-160 and anti-gamma312-324 monoclonal antibodies, indicating that their fibrin-specific epitopes were inaccessible. The Aalpha148-160 epitope became exposed only upon proteolytic removal of the beta- and gamma-modules from D. At the same time, both epitopes were accessible in the D-D.E(1) complex, indicating that the DD.E interaction resulted in their exposure. This exposure was reversible since the dissociation of the D-D.E(1) complex made the sites unavailable, while reconstitution of the complex made them exposed. The results indicate that upon fibrin assembly, driven primarily by the interaction between complementary sites of the D and E regions, the D regions undergo conformational changes that cause the exposure of their plasminogen- and tPA-binding sites. These changes may be involved in the regulation of fibrin assembly and fibrinolysis.

Specific binding of integrin alpha v beta 3 to the fibrinogen gamma and alpha E chain C-terminal domains.

Integrin alpha v beta 3, a widely distributed fibrinogen receptor, recognizes the RGD572-574 motif in the alpha chain of human fibrinogen. However, this motif is not conserved in other species, nor is it required for alpha v beta 3-mediated fibrin clot retraction, suggesting that fibrinogen may have other alpha v beta 3 binding sites. Fibrinogen has conserved C-terminal domains in its alpha (E variant), beta, and gamma chains (designated alpha EC, beta C, and gamma C, respectively), but their function in cell adhesion is not known, except that alpha IIb beta 3, a platelet fibrinogen receptor, binds to the gamma C HHLGGAKQAGDV400-411 sequence. Here we used mammalian cells expressing recombinant alpha v beta 3 to show that recombinant alpha EC and gamma C domains expressed in bacteria specifically bind to alpha v beta 3. Interaction between alpha v beta 3 and gamma C or alpha EC is blocked by LM609, a function-blocking anti-alpha v beta 3 mAb, and by RGD peptides. alpha v beta 3 does not require the HHLGGAKQAGDV400-411 sequence of gamma C for binding, and alpha EC does not have such a sequence, indicating that the alpha v beta 3 binding sites are distinct from those of alpha IIb beta 3. A small fragment of gamma C (residues 148-226) supports alpha v beta 3 adhesion, suggesting that an alpha v beta 3 binding site is located within the gamma chain 148-226 region. We have reported that the CYDMKTTC sequence of beta 3 is responsible for the ligand specificity of alpha v beta 3. gamma C and alpha EC do not bind to wild-type alpha v beta 1, but do bind to the alpha v beta 1 mutant (alpha v beta 1-3-1), in which the CYDMKTTC sequence of beta 3 is substituted for the corresponding beta 1 sequence CTSEQNC. This suggests that gamma C and alpha EC contain determinants for fibrinogen's specificity to alpha v beta 3. These results suggest that fibrinogen has potentially significant novel alpha v beta 3 binding sites in gamma C and alpha EC.

Domain organization of the 39-kDa receptor-associated protein.

The 39-kDa receptor-associated protein (RAP) is an endoplasmic reticulum resident protein that binds to the low density lipoprotein receptor-related protein (LRP) as well as certain members of the low density lipoprotein receptor superfamily and antagonizes ligand binding. In order to identify important functional regions of RAP, studies were performed to define the domain organization and domain boundaries of this molecule. Differential scanning calorimetry (DSC) experiments revealed that the process of thermal denaturation of RAP is highly reversible and occurs in a broad temperature range with two well resolved heat absorption peaks. A good fit of the endotherm was obtained with four two-state transitions suggesting these many cooperative domains in the molecule. A number of recombinant fragments of RAP were expressed in bacteria, and their domain composition and stability were characterized by DSC, circular dichroism, and fluorescence spectroscopy. The results confirmed that RAP is composed of four independently folded domains, D1, D2, D3, and D4, that encompass residues 1-92, 93-163, 164-216, and 217-323, respectively. The first and the fourth domains preserved their structure and stability when isolated, whereas the compact structure of the fragment corresponding to D2 seems to be altered when isolated from the parent molecule. Isolated D3 was partially degraded during isolation from bacterial lysates. The isolated D4 was capable of binding with high affinity to LRP whereas neither D1 nor D2 bound. At the same time a fragment containing both D1 and D2 exhibited high affinity binding to LRP. These facts combined with the thermodynamic analysis of the melting process of the fragments containing D1 and D2 indicate that these two domains interact with each other and that the proper folding of the second domain into a native-like active conformation requires presence of the first domain.

Identification of a novel recognition sequence for integrin alphaM beta2 within the gamma-chain of fibrinogen.

The interaction of leukocyte integrin alphaMbeta2 (CD11b/CD18, Mac-1) with fibrinogen has been implicated in the inflammatory response by contributing to leukocyte adhesion to the endothelium and subsequent transmigration. Previously, it has been demonstrated that a peptide, P1, corresponding to residues 190-202 in the gamma-chain of fibrinogen, binds to alphaM beta2 and blocks the interaction of fibrinogen with the receptor and that Asp199 within P1 is important to activity. We have demonstrated, however, that a double mutation of Asp199-Gly200 to Gly-Ala in the recombinant gamma-module of fibrinogen, spanning region 148-411, did not abrogate alphaM beta2 recognition and considered that other binding sites in the gamma-module may participate in the receptor recognition. We have found that synthetic peptide P2, duplicating gamma377-395, inhibited adhesion of alphaM beta2-transfected cells to immobilized D100 fragment of fibrinogen in a dose-dependent manner. In addition, immobilized P2 directly supported efficient adhesion of the alphaM beta2-expressing cells, including activated and non-activated monocytoid cells. The I domain of alphaM beta2 was implicated in recognition of P2, as the biotinylated recombinant alphaMI domain specifically bound to both P2 and P1 peptides. Analysis of overlapping peptides spanning P2 demonstrated that it may contain two functional sequences: gamma377-386 (P2-N) and gamma383-395 (P2-C), with the latter sequence being more active. In the three-dimensional structure of the gamma-module, gamma190-202 and gamma377-395 reside in close proximity, forming two antiparallel beta strands. The juxtapositioning of these two sequences may form an unique and complex binding site for alphaM beta2.

Domain structure and functional activity of the recombinant human fibrinogen gamma-module (gamma148-411).

Human fibrinogen gamma-module comprising residues gamma148-411 was expressed in Escherichia coli and refolded in vitro. Differential scanning calorimetry revealed that in addition to the two previously identified independently folded thermolabile domains, one in each half of the module, the gamma-module also contains one or two thermostable domains that melt above 65 degrees C. To localize the latter, an NH2-terminal 6-kDa fragment was prepared by limited proteolysis of the recombinant gamma-module. It melted at high temperature, indicating that this portion is folded into a compact structure that represents a thermostable domain, also identified in the proteolytic fibrinogen fragment D1 which contains the natural gamma-module. Thus the NH2-terminal half of the gamma-module forms two domains, a thermostable one and a thermolabile one, leaving the rest of the module to be responsible for the formation of the other one or two domains. The thermal stability of some domains was lower in the recombinant gamma-module than in its natural counterpart in D1, reflecting most probably the loss of interactions with neighboring domains; however, the major functional sites were essentially preserved. The module bound Ca2+ and was stabilized by it against denaturation and proteolysis. It inhibited fibrin polymerization and was efficiently cross-linked by factor XIIIa. The gamma-module supported adhesion of platelets via their GP IIbIIIa (alpha(IIb)beta3) receptor in the same manner as D1 fragment. It also supported the adhesion of alpha(M)beta2- (Mac-1-) transfected cells and in the fluid phase was more effective than D1 as an inhibitor of that adhesion, suggesting that the Mac-1 binding site is better exposed.