Preparation of monomeric fibrin lacking intact alpha C-domains (monomeric X1-fragment), but fully clottable, is described. The assembly process of both monomeric fibrin and monomeric X1-fragment has been studied by electron microscopy and... more
Preparation of monomeric fibrin lacking intact alpha C-domains (monomeric X1-fragment), but fully clottable, is described. The assembly process of both monomeric fibrin and monomeric X1-fragment has been studied by electron microscopy and light scattering methods. It was shown that both proteins form similar fibrils with characteristic cross-banding. Upon dilution a sharp elevation of the differences between the assembly rates of monomeric X1-fragment and monomeric fibrin was revealed. The results obtained show that alpha C-domains take part in fibrin clot formation not as structural components but as the factor accelerating the ordered assembly of complex fibrin structure. The possible mechanism of alpha C-domains participation in fibrin clot formation are regarded.
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: Surface plasmon resonance and ELISA experiments revealed that recombinant fibrinogen αC fragment (residues Aα221–610) corresponding to the αC domain binds tPA and plasminogen with high affinity. This binding was found to be... more
: Surface plasmon resonance and ELISA experiments revealed that recombinant fibrinogen αC fragment (residues Aα221–610) corresponding to the αC 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 αC 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 αC fragment reduced this effect substantially, suggesting the importance of the αC domains in regulation of fibrinolysis.
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Research Interests: Algorithms, Chemistry, Cell Adhesion, Biological Chemistry, Medicine, and 15 moreBiological Sciences, Fibrinogen, Cell line, Humans, Biological, Peptides, CHEMICAL SCIENCES, Amino Acid Sequence, Ligands, Binding Site, Integrin, Molecular Sequence Data, DNA mutational analysis, binding sites, and Medical and Health Sciences
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Numerous studies have revealed the involvement of fibrinogen in the inflammatory response. To explain the molecular mechanisms underlying fibrinogen-dependent inflammation, two bridging mechanisms have been proposed in which fibrin(ogen)... more
Numerous studies have revealed the involvement of fibrinogen in the inflammatory response. To explain the molecular mechanisms underlying fibrinogen-dependent inflammation, two bridging mechanisms have been proposed in which fibrin(ogen) bridges leukocytes to endothelial cells. The first mechanism suggests that bridging occurs via the interaction of fibrinogen with the leukocyte receptor Mac-1 and the endothelial receptor ICAM-1 (intercellular adhesion molecule-1), which promotes leukocyte transmigration and enhances inflammation. The second mechanism includes bridging of leukocytes to the endothelium by fibrin degradation product E1 fragment through its interaction with leukocyte receptor CD11c and endothelial VE-cadherin to promote leukocyte transmigration. The role of E1 in promoting inflammation is inhibited by the fibrin-derived β15–42 fragment, and this has been suggested to result from its ability to compete for the E1–VE-cadherin interaction and to trigger signaling pathways through the src kinase Fyn. Our recent study revealed that the β15–42 fragment is ineffective in inhibiting the E1– or fibrin–VE-cadherin interaction, leaving the proposed signaling mechanism as the only viable explanation for the inhibitory function of β15–42. We have discovered that fibrin interacts with the very-low-density lipoprotein (VLDL) receptor, and this interaction triggers a signaling pathway that promotes leukocyte transmigration through inhibition of the src kinase Fyn. This pathway is inhibited by another pathway induced by the interaction of β15–42 with a putative endothelial receptor. In this review, we briefly describe the previously proposed molecular mechanisms underlying fibrin-dependent inflammation and their advantages/disadvantages and summarize our recent studies of the novel VLDL receptor-dependent pathway of leukocyte transmigration which plays an important role in fibrin-dependent inflammation.
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Research Interests: Chemistry and Fibrinogen
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Factor (F) VIII and FIX are two key plasma proteins that are deficient in the bleeding disorders hemophilia A and hemophilia B, respectively. FVIII is an inactive cofactor that normally circulates in complex with its carrier protein von... more
Factor (F) VIII and FIX are two key plasma proteins that are deficient in the bleeding disorders hemophilia A and hemophilia B, respectively. FVIII is an inactive cofactor that normally circulates in complex with its carrier protein von Willebrand factor (VWF) (for reviews see [1,2]). Upon injury within the vasculature, this cofactor is activated to FVIIIa by limited proteolysis resulting in its dissociation from VWF and subsequent assembly onto the membrane surface with an enzymatically active form of FIX (FIXa) to form a macromolecular Xase complex. This complex effectively activates FX, the next proenzyme in the coagulation cascade. The fact that deficiencies in both FVIIIa and FIXa lead to bleeding disorders attests to the significant role that the macromolecular Xase complex plays in the blood coagulation cascade. While a substantial amount of biochemical and structural information has been uncovered for each of these proteins, it is only recently that attention has begun to focus on mechanisms by which these two proteins are metabolized. Implicated in the metabolism of both of these critical proteins is the large endocytic receptor, the low-density lipoprotein receptor-related protein (LRP). LRP was first identified as a hepatic receptor that is responsible for removing complexes of alpha-2-macroglobulin with various proteases [3–6]. Subsequent work revealed that this receptor, which is widely expressed, also binds apoEenriched lipoproteins [7,8], plasminogen activators [9], matrix components such as thrombospondin [10] and fibronectin [11], serpin-enzyme complexes [12], bacterial toxins [13], and growth factors [14]. Deletion of the LRP gene reveals an essential, but still undefined role in development [15]. Recent work has implicated LRP in several signal transduction pathways and has expanded its realm of involvement to include protection of the vasculature [14,16,17], regulation of cell migration [18–20], and modulation of the integrity of the blood–brain barrier [21]. Recently, it appears that LRP may also play an important role in regulating the blood coagulation process by mediating the clearance of two key players. The first of these is blood coagulation FIX, a vitamin K-dependent zymogen that is proteolytically activated by FXIa or by FVIIa complexed to tissue factor to generate the active enzyme. Interestingly, the zymogen or inactive form of FIX does not bind to LRP. In contrast, when activated by FXIa, a site is exposed on the FIXamolecule that is recognized by LRP [22]. Mutagenesis studies revealed that the LRP binding site is located within its serine protease domain, namely, in a loop consisting of residues Phe342 to Asn346 [23]. Interestingly, soluble forms of LRP containing ligand-binding cluster IV inhibited the ability of FIXa to catalyze the activation of FX, both in the presence and absence of FVIIIa. Together, these studies reveal that LRP selectively binds and mediates the cellular catabolism of FIXa, and led to the suggestion that LRP may function as a regulator of blood coagulation by regulating FIXa levels and activity [23]. The second blood coagulation protein reported to bind to LRP is FVIII. The first reports that LRP might be involved in the clearanceofFVIII results from theworkof Saenko et al. [24] andLenting et al. [25]. Both investigators found that LRP could bind to FVIII with KD values estimated to be between 60 and 116 nM [24,25], which is well above the plasma levels of FVIII. Further, both studies found that cells were able to mediate the uptake of FVIII in an LRP-dependent manner. The in vivo significance of the observations was demonstrated by showing that RAP, a potent antagonist of LRP, blocked the in vivo clearance of I-labeled FVIII [24]. Importantly, VWF was shown to inhibit the LRP-mediated clearance of FVIII [25]. Correspondence: Dudley K. Strickland, Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, 800 West Baltimore Street, R219, Baltimore, MD 21201, USA. Tel.: +1 410 706 8010; fax: +1 410 706 8121; e-mail: [email protected] Journal of Thrombosis and Haemostasis, 4: 1484–1486
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Research Interests: Thermodynamics, Chemistry, Crystallography, Kinetics, Medicine, and 12 moreDifferential scanning calorimetry, Domains, Denaturation, Glycine, Streptokinase, Molecule, Hydrogen-Ion Concentration, Protein Denaturation, Biochemistry and cell biology, Domain Structure, Chymotrypsin, and Medical biochemistry and metabolomics
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Research Interests: Chemistry, Colorimetry, Fluorescence, Calcium, Biological Chemistry, and 12 moreMedicine, Biological Sciences, Humans, Osteocalcin, Recombinant DNA, CHEMICAL SCIENCES, Epidermal Growth Factor, Thermal Stability, Protein Conformation, Recombinant Proteins, Protein Denaturation, and Medical and Health Sciences
Research Interests: Chemistry, Medicine, Fibrinogen, Fibrin, Thrombin, and 3 moreClinical Sciences, Thrombosis, and plasmin
Research Interests: Biochemistry, Biophysics, Chemistry, Kinetics, Medicine, and 15 moreFibrinogen, Fibrin, Humans, Endothelial Cells, Endothelial cell, Amino Acid Sequence, Base Sequence, Cadherin, Binding Site, Extracellular, Biochemistry and cell biology, Molecular Sequence Data, Cadherins, binding sites, and Medical biochemistry and metabolomics
... Galina Tsurupa, Latchezar Tsonev, § and Leonid Medved* . Department of Biochemistry, The American Red Cross Holland Laboratory, Rockville, Maryland 20855, and Resuscitative Medicine Department, Transfusion and ...
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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... more
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.
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Research Interests: Chemistry, Biological Chemistry, Medicine, Biological Sciences, Humans, and 10 moreCHEMICAL SCIENCES, Fluorescence polarization, Protein Conformation, Amino Acid Sequence, Recombinant Proteins, Molecular Sequence Data, binding sites, fluorescent dyes, Medical and Health Sciences, and In Vitro Techniques
Research Interests: Biophysics, Chemistry, Crystallography, Electron Microscopy, Molecular Biology, and 15 moreBiology, Transmission Electron Microscopy, Medicine, Polymerization, Molecular, Fibrinogen, Fibrin, Animals, Blood Coagulation, Gels, Monomer, Biochemistry and cell biology, Electron Microscope, negative staining, and electron micrographs
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Research Interests: Biochemistry, Chemistry, Kinetics, Medicine, Structural Stability, and 13 moreProtein Stability, Fibrinogen, Humans, Circular Dichroism, Animals, Recombinant DNA, Cattle, Monomer, Thermal Stability, Protein Binding, Size Exclusion Chromatography, Biochemistry and cell biology, and Medical biochemistry and metabolomics
Research Interests: Biochemistry, Chemistry, Medicine, Fibrinogen, Fibrin, and 15 moreThrombin, Humans, Vascular endothelium, Surface plasmon resonance, Recombinant DNA, Endothelial cell, Amino Acid Sequence, Base Sequence, Recombinant Proteins, Binding Site, Biochemistry and cell biology, Molecular Sequence Data, Cadherins, binding sites, and Medical biochemistry and metabolomics
: The αC 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 αC domains suggested that each is made up of a globular and an... more
: The αC 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 αC domains suggested that each is made up of a globular and an extended portion. Microcalorimetry confirmed this result and showed that the two αC domains interact intramolecularly. Electron microscopy of fibrinogen with a monoclonal antibody to the αC 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 αC 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 αC domains, indicate that intermolecular interactions between αC domains are important for the enhancement of lateral aggregation during fibrin polymerization. Isolated αC fragments polymerized at neutral pH and interacted with the αC domains of fibrin monomer to influence clot formation. Several dysfibrinogenemias in which there are amino acid substitutions in, or truncations of, the αC domains revealed that these changes can have dramatic effects on polymerization and clot structure. The polymerization of Aα251 recombinant fibrinogen, that contains Aα 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 αC domains in determining the structure and properties of clots.
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Research Interests: Biochemistry, Computer Science, Chemistry, Crystallography, Medicine, and 14 moreFibrinogen, Sequence alignment, Notice, Animals, Citation, Cattle, Protein Secondary Structure Prediction, Altmetrics, Amino Acid Sequence, Recombinant Proteins, Biochemistry and cell biology, Molecular Sequence Data, random coil, and Medical biochemistry and metabolomics
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Research Interests: Chemistry, Biology, Cell Migration, Wound Healing, Cell Biology, and 15 moreMedicine, Signal Transduction, Fibrinogen, Fibrin, Video microscopy, Humans, Haemostasis and Thrombosis, Clinical Sciences, Time Factors, Cell Proliferation, Protein Kinase Inhibitors, Integrin, Focal Adhesion Kinase, Cardiovascular medicine and haematology, and Human Umbilical Vein Endothelial Cells
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The process of heat denaturation of recombinant factor XIII (rFXIII), as well as its C-terminal 24 kDA and 12 kDa elastase-produced fragments starting at Ser514 and Thr628, respectively, was investigated in a wide range of conditions by... more
The process of heat denaturation of recombinant factor XIII (rFXIII), as well as its C-terminal 24 kDA and 12 kDa elastase-produced fragments starting at Ser514 and Thr628, respectively, was investigated in a wide range of conditions by fluorescence, CD and differential scanning calorimetry (DSC). It was found that the intact protein melts in two distinct temperature regions reflecting unfolding of different parts of the molecule with different stability. The less stable structures unfold in a low temperature transition with a tm of 69 degrees C or lower depending on conditions. Unfolding of the more stable structures was observed at extremely high temperatures, tm > 110 degrees C at acidic pH < 3.5 and tm = 90 degrees C at pH 8.6 with 2 M GdmCL. Thermodynamic analysis of the low and high temperature DSC-obtained heat absorption peaks indicated unambiguously that the first represents melting of three thermolabile independently folded domains while two thermostable domains melt in the second one giving a total of five domains in each a subunit of rFXIII. Both 24 kDa and 12 kDa fragments exhibited a sigmoidal spectral transition at comparatively high temperature where the thermolabile structures are already denatured, indicating that two thermostable domains are formed by the C-terminal portion of rFXIII and correspond to the two beta-barrels revealed by crystallography. The remaining 56 kDa portion forms three thermolabile domains, one of which corresponds to the N-terminal beta-sandwich and the other two to the catalytic core. Fast accessible surface calculations of the X-ray model of rFXIII confirmed the presence of two structural subdomains in the core region with the boundary at residue 332. The thermolabile domains appear to interact with each other intra- and/or intermolecularly resulting in dimerization the a subunits. At acidic pH, where all domains became destabilized but still remained folded, interdomainial interactions seemed to be abolished, resulting in the reversible dissociation of the dimer as revealed by ultracentrifugation analysis.
Research Interests: Chemistry, Crystallography, Molecular Biology, Protein Folding, Fluorescence, and 15 moreMedicine, Molecular, Humans, Circular Dichroism, Differential scanning calorimetry, DSC, Domains, Denaturation, Protein Conformation, Calorimetry, Guanidines, Polyacrylamide Gel Electrophoresis, Protein Denaturation, Biochemistry and cell biology, and Factor XIII
Research Interests: Biochemistry, Computer Science, Chemistry, Polymers, Medicine, and 15 moreFibrinogen, Fibrin, Thrombin, Humans, Icon, Differential scanning calorimetry, Blood Coagulation, Citation, ICON, Alkylation, Amino Acid Sequence, Structure activity Relationship, Biochemistry and cell biology, Molecular Sequence Data, and Medical biochemistry and metabolomics
Research Interests: Chemistry, Biology, Inflammation, Medicine, In Vitro, and 15 moreFibrin, Humans, Endothelial Cells, Mice, Animals, Haemostasis and Thrombosis, Clinical Sciences, In Vivo, Peritonitis, Leukocytes, Dimerization, Cadherins, Cardiovascular medicine and haematology, Human Umbilical Vein Endothelial Cells, and In Vitro Techniques
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The gelatin-binding region of fibronectin is isolated easily as a stable and functional 42 kDa fragment containing four type I "finger" modules and two type II... more
The gelatin-binding region of fibronectin is isolated easily as a stable and functional 42 kDa fragment containing four type I "finger" modules and two type II "kringle-like" modules arranged in the order I6-II1-II2-I7-I8-I9. This fragment exhibits a single reversible melting transition near 64 degrees C in TBS buffer (0.02 M-Tris buffer containing 0.15 M-NaCl, pH 7.4). The transition is characterized by a calorimetric to van't Hoff enthalpy ratio of 1.6, suggesting a complex domain structure. A 30 kDa fragment with the same NH2 terminus (I6-II1-II2-I7) melts reversibly near 65 degrees C with delta Hcal/delta HvH = 1.3, also consistent with the presence of more than one domain. To elucidate further the domain structure, three non-overlapping subfragments were prepared and characterized with respect to their unfolding induced by heat and guanidinium chloride. The three subfragments, each containing two modules, are designated from amino or carboxyl-terminal location as 13 kDa (I6-II1) 16 kDa (II2-I7) and 21 kDa (I8-I9) according to their apparent Mr in SDS/polyacrylamide gel electrophoresis. All three subfragments exhibited reversible transitions in TBS buffer, behaving in the calorimeter as single co-operative units with delta Hcal/delta HvH close to unity. However, the specific enthalpies and changes in heat capacity associated with the melting of all fragments and subfragments in TBS buffer were low compared to those of most compact globular proteins, suggesting that not all modules are represented. When titrated with guanidinium chloride at 25 degrees C, all fragments exhibited monophasic reversible unfolding transitions detected by changes in fluorescence. Heating in the presence of 6 M-guanidinium chloride revealed three additional transitions not seen in the absence of denaturants. These transitions have been assigned to three of the four type I finger modules (I6, I7 and I9), one of which (I6) was isolated and shown to retain a compact structure as stable as that observed for this module within the parent fragments. Two other modules (II2 and I7) are destabilized when separated from their neighbors. Thus, despite their small size (50 to 60 amino acid residues), all six of the modules in the gelatin-binding region of fibronectin form independently folded domains, three of which (I6, I7 and I9) are unusually stable. Evidence is provided that four of the six modules interact with each other in the parent fragment. This interaction may explain previously noted disruptions in the otherwise uniform strand-like images seen in electron micrographs of fibronectin.
Research Interests: Chemistry, Crystallography, Molecular Biology, Biology, Medicine, and 15 moreMolecular, Humans, Differential scanning calorimetry, Enthalpy, Gelatin, Fibronectin, Gel electrophoresis, Guanidines, Amino Acid Sequence, Binding Site, Biochemistry and cell biology, Electrophoretic Mobility, Molecular Sequence Data, binding sites, and Guanidinium Chloride
ABSTRACT
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Research Interests: Chemistry, Cell Migration, Cell Adhesion, Cell Biology, Angiogenesis, and 15 moreFibrinogen, Fibrin, Humans, Animals, Blood, Clinical Sciences, Cattle, Endothelial cell, Binding Site, Integrin, Biochemistry and cell biology, Dimerization, binding sites, Guinea pigs, and Cardiovascular medicine and haematology
Research Interests: Thermodynamics, Chemistry, Crystallography, Molecular Biology, Medicine, and 10 moreFibrinogen, Differential scanning calorimetry, Temperature, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Macromolecule, Protein Conformation, Molecular weight, Molecule, Protein Denaturation, and Biochemistry and cell biology
The locations of the carboxyl-terminal two thirds of the A alpha chains, or the alpha C domains, were determined for fibrinogen and some of its derivatives by electron microscopy of rotary-shadowed preparations. A monoclonal antibody, G8,... more
The locations of the carboxyl-terminal two thirds of the A alpha chains, or the alpha C domains, were determined for fibrinogen and some of its derivatives by electron microscopy of rotary-shadowed preparations. A monoclonal antibody, G8, to the carboxyl-terminal 150 amino acids of the A alpha chain, binds near the central region of fibrinogen, indicating that the alpha C domains of most molecules are not normally visible because they are on or near the amino-terminal disulfide knot. At pH 3.5, fibrinogen and fibrin monomers appear to be similar, with a projection terminating in a small globular domain from each end of most molecules. In contrast, fragment X monomers, produced by cleavage of the alpha C domains from fibrinogen with plasmin, show no such projections. When fibrin monomer is brought to neutral pH under conditions where polymerization is delayed, individual molecules are still visible showing the alpha C domains as a single additional nodule near the central region. Moreover, analysis of clusters of molecules reveals some intermolecular associations via the alpha C domains. A 40-kDa fragment comprising the alpha C domain has been isolated from a plasmin digest of fibrinogen and characterized by SDS-polyacrylamide gel electrophoresis and determination of amino-terminal amino acid sequences. Electron microscopy of alpha C fragments reveals individual globular structures, as well as oligomeric aggregates. The addition of alpha C fragments to fibrin monomer followed by dilution to neutral pH to initiate polymerization results in lower turbidity, longer lag period, and slower maximum rate of turbidity increase. Also, electron microscopy reveals complexes of alpha C fragments with fibrin monomer at neutral pH. It appears that the free alpha C fragments can bind to the alpha C domains of fibrin, competing with the normal alpha C domain interactions involved in polymerization.