PubMed Health⌕ Search

Biomedical subjects

J W Weisel

Publications and source records attributed to J W Weisel.

At least 37 records · Page 2Linked to original sources
PubMed1995

Conformational transitions in the cell binding domain of fibronectin.

Plasma fibronectin readily changes shape in response to environmental conditions which may, in turn, lead to differential expression of its multiple functional sites. To test this possibility, the expression of two of the type III modules within cell binding domain of fibronectin was assessed with monoclonal antibodies (mAb). Utilizing proteolytic and recombinant fragments of plasma fibronectin, the epitopes recognized by mAbIII-9 and mAbIII-10 were localized to the ninth and tenth (RGD-containing) type III repeats of fibronectin, respectively. Both mAb inhibited the adhesion of platelets to immobilized fibronectin, suggesting that the recognized epitopes resided in close spatial proximity to the cell binding sites. Radioimmunoassay and Scatchard analyses showed that, in solution, each dimeric fibronectin molecule bound two mAbIII-9 but only one mAbIII-10 molecule (ionic strength 0.15, pH 7.4). The binding of a single mAbIII-10 per fibronectin molecule was verified by electron microscopy. Heparin, heparan sulfate, gangliosides (but not chondroitin sulfates A and B and hyaluronic acid), and self-association increased the apparent affinity of mAbIII-10 for soluble fibronectin. Adsorption of fibronectin onto a polystyrene surface resulted in the appearance of an additional binding site for mAbIII-10. MAbIII-9 binding also was altered by fibronectin immobilization. These results suggest that the deposition of fibronectin and its interaction with components of the extracellular matrix can modulate the expression of the cell binding domains including the RGDS-containing type III repeat. Exposure of the second tenth type III repeat within the fibronectin dimer, as a result of unfolding on a surface, could contribute to the enhanced adhesiveness of adsorbed fibronectin.

Antibodies, Monoclonal↗
PubMed1995

Three-dimensional reconstruction of fibrin clot networks from stereoscopic intermediate voltage electron microscope images and analysis of branching.

Fibrin polymerizes to produce branching fibers forming a three-dimensional network, which has been difficult to visualize by conventional microscopy. Three-dimensional images of whole clots at high resolution were obtained from stereo-pair intermediate-voltage electron micrographs. Computer software was developed to produce three-dimensional reconstructions of the networks in the form of a pattern of links that connect branching junctions. Network parameters were measured and analyzed to characterize the clots quantitatively. Models in which all links were moved to the origin, while preserving their orientation, allowed visualization of some network parameters and facilitated comparison of networks. Fibrin clots formed in three different conditions were analyzed and compared by these methods. Clots formed in 0.20 M saline buffer consist of fibers of uniform size, and most of the branching junctions consist of three links. Fibrin clots formed in 0.05 M saline buffer are made up of very large diameter fiber bundles with far fewer branching junctions and correspondingly longer links. Clots formed in 0.40 M saline buffer consist of very fine fibers with numerous branching junctions and very short links. In summary, the extent of lateral aggregation is directly related to the distance between branching junctions and inversely related to the total number of branching junctions. These observations must be considered in defining possible mechanisms of fibrin branching.

Biophysical Phenomena↗
PubMed1994

Role of the alpha C domains of fibrin in clot formation.

The role of the carboxyl-terminal portion of the alpha chains of fibrin (alpha C domains) in clot formation was investigated by transmission and scanning electron microscopy and turbidity studies of clots made from preparations of molecules missing one or both of these domains. Highly purified and entirely clottable preparations of bovine fragment X monomer, one containing primarily molecules missing a single alpha C domain (fragment X1) and the other consisting of molecules missing both alpha C domains (fragment X2), were used for these experiments. These preparations were characterized by various methods, including the complete determination of the amino- and carboxyl-termini of all peptides and fragments. These preparations formed clots on dilution to neutral pH. In all cases, clots observed by either scanning or transmission electron microscopy were made up of a branched network of fibers, similar to those formed by thrombin treatment of intact fibrinogen, suggesting that the alpha C domains are not necessary for protofibril and fiber formation or branching. However, both the fiber and clot structure varied with the different fractions, indicating that the alpha C domains do participate in polymerization. The rate of assembly, as indicated by the lag period and maximum rate of turbidity increase, as well as the final turbidity, was decreased with removal of the alpha C domains, suggesting that they accelerate polymerization. preparations of isolated alpha C fragment added to fibrin monomer have striking effects on the turbidity curves, showing a decrease in the rate of polymerization in a dose-dependent manner but not complete inhibition. Electron microscopy of fibrin monomer desA molecules at neutral pH showed that most of the alpha C domains, like those in fibrinogen, remain associated with the central region. Thus, it appears that normally with thrombin cleavage of fibrinogen the effects of the interactions of alpha C domains observed here will be most significant for lateral aggregation.

Amino Acid Sequence↗
PubMed1994

The shape of high molecular weight kininogen. Organization into structural domains, changes with activation, and interactions with prekallikrein, as determined by electron microscopy.

Knowledge of the organization of the kininogen gene and protein structure and function correlations has allowed the development of a model of high molecular weight kininogen. Domains 1-3 on the heavy chain are evolutionarily related to cystatin and the latter two are inhibitors of cysteine proteases. Proteolytic cleavage in domain 4 to release bradykinin causes a conformational change, exposing a surface-binding region (domain 5) on the disulfide-linked light chain. The carboxyl-terminal domain 6 contains a zymogen binding sequence for factor XI and prekallikrein which, with domain 5, accounts for its cofactor activity. To explore further the domain structure, we have determined the shapes of high molecular weight kininogen and prekallikrein by electron microscopy of rotary shadowed preparations and computer image processing. High molecular weight kininogen appears to be a linear array of three linked globular regions about 16 nm long, with the two ends also connected by another thin strand. Both prekallikrein and kallikrein have a compact globular shape, with a subdivision that is sometimes visible. Different functional domains of high molecular weight kininogen were identified by monoclonal antibodies against these regions, as well as ligand binding of prekallikrein. These studies indicate that one end globular region is the prekallikrein-binding domain, the other comprises the cysteine protease inhibitor domains and the smaller central nodule is the surface-binding domain. Cleavage of high molecular weight kininogen with plasma kallikrein to yield two-chain high molecular weight kininogen results in a striking change in conformation: the central surface-binding domain swings out so that it is still adjacent to the prekallikrein-binding domain but no longer in the middle. These structural studies provide insight into the interactions of these proteins and aspects of the mechanisms of their actions.

Amino Acid Sequence↗
PubMed1994

Interactions of plasminogen with polymerizing fibrin and its derivatives, monitored with a photoaffinity cross-linker and electron microscopy.

Localization of the plasminogen binding sites on fibrin has been difficult since these interactions occur on polymerizing fibrin, and studies with fragments can be misleading because of multiple carboxyl-terminal lysines that may bind to plasminogen. A hetero-functional photoaffinity cross-linker was used to study these interactions. Following attachment of the cross-linker to plasminogen in the dark, a clot was formed by addition of fibrinogen or fragment X and thrombin, and then the plasminogen was cross-linked to adjacent parts of fibrin by exposure to light. There was more Glu1-plasminogen bound to fibrin than to fibrinogen and more to fragment X polymer than to fibrin. Electron microscopy of rotary shadowed individual molecules reveals that Glu1-plasminogen appears to be more compact than Lys78-plasminogen or Glu1-plasminogen with 6-aminohexanoic acid. Cross-linked complexes from the dissolved clot observed by electron microscopy reveal plasminogen bound to the end of fibrin or bridging the ends of two fibrin molecules; larger complexes were also observed. Analysis of changes in the appearance of negatively contrasted fibers with plasminogen bound also indicates the probable locations of binding sites, yielding results consistent with the cross-linking studies. The photoaffinity probe was also used to study interactions between plasminogen and fibrin or its derivatives in the course of tissue plasminogen activator-mediated fibrinolysis. Samples cross-linked at various times indicate that complexes with fragment X are particularly dominant during the rapid phase of plasminogen activation. In conclusion, these studies indicate that plasminogen binds to the pocket at the end-to-end junction between two fibrin or fragment X molecules in the protofibril; from this position, it can reach all of the sites that are cleaved during fibrinolysis.

Affinity Labels↗
PubMed1993

Determination of the topology of factor XIIIa-induced fibrin gamma-chain cross-links by electron microscopy of ligated fragments.

After fibrin polymerizes to form a clot, the transglutaminase Factor XIIIa cross-links the gamma and alpha chains to stabilize the clot. There has been conflicting evidence on whether the gamma chain isopeptide bonds occur between molecules that are interacting in a longitudinal (end-to-end) manner or transverse (half-staggered) manner between the two strands of the protofibril. Since the topology of the cross-links has important consequences for fibrin structure, as well as for its stability and susceptibility to and pattern of fibrinolysis, cross-linked fibrin fragments were examined by electron microscopy to distinguish between these two possibilities for the arrangement of the ligated molecules. Cross-linked fibrin clots were produced by prolonged incubation of fibrinogen with thrombin and Factor XIII, and then digested with plasmin. The resulting soluble cross-linked fibrin complexes were rotary-shadowed with tungsten and examined by electron microscopy, revealing protofibril-like structures consisting of clusters of globular domains with a repeat of 22.5 nm. Longer plasmin digestion times yielded increasingly shorter structures. Rotary-shadowed cross-linked fibrin fragments, produced by dilution of the complexes into 0.125% acetic acid at pH 3.5 to dissociate all non-covalently linked fragments, showed uniformly single-stranded structures with a characteristic spacing of nodules, consistent with longitudinal cross-linking. Long, thin strands were seen at short digestion times, while shorter strands appeared with longer digestion. The smallest structures observed included two nodules together, and two such nodules with another nodule at a short distance from one or both ends, compatible with fragments DD, DY, and YY. Longer strands had the appearance of fibrin molecules that were linked end-to-end, usually with a fragment D or Y at each end. In conclusion, these results are consistent with previously proposed structures of these derivatives and clearly demonstrate that the interactions between cross-linked gamma chains are longitudinal (end-to-end) and not transverse.

Cross-Linking Reagents↗
PubMed1993

Long range propagation of conformational changes in integrin alpha IIb beta 3.

Integrin adhesion receptors participate in two-way transfer of information across the plasma membrane. For example, cytoplasmic events, such as activation of protein kinase C, cause an increase in the fibrinogen (Fg) binding affinity of the extracellular domain of integrin alpha IIb beta 3 ("inside-out signaling"). Conversely, ligand binding to alpha IIb beta 3 results in the generation of intracellular signals. We used anti-LIBS2, an anti-beta 3 monoclonal antibody, to understand potential mechanisms of this bidirectional signaling. Anti-LIBS2 bound to alpha IIb beta 3 with low affinity (Kd = 7.4 microM), and mimicked inside-out signaling by promoting Fg binding. The affinity of anti-LIBS2 binding was increased 20-fold (Kd = 326 nM) by addition of an Fg-mimetic synthetic peptide, RGDS. Thus, anti-LIBS2 and ligands (Fg and Fg-mimetic peptides) bind cooperatively to integrin alpha IIb beta 3, indicating a functional linkage between the ligand-binding site and the antibody-binding site. The anti-LIBS2-binding site was mapped by its binding to proteolytic and recombinant fragments of the beta 3 subunit. The epitope was located within an 89-residue region immediately adjacent to the transmembrane domain and 400 residues carboxyl-terminal to the known ligand-binding site(s). Electron microscope images of rotary shadowed ternary complexes of Fg, anti-LIBS2, and alpha IIb beta 3 revealed that the ligand-binding site and anti-LIBS2 epitope are separated by about 16 nm. This indicates that propagated long distance conformational changes can occur in alpha IIb beta 3. Such changes are likely to be involved in the bidirectional signaling function of this integral membrane protein.

Allosteric Regulation↗
PubMed1993

The sequence of cleavage of fibrinopeptides from fibrinogen is important for protofibril formation and enhancement of lateral aggregation in fibrin clots.

Thrombin cleaves A fibrinopeptides from fibrinogen faster than B; cleavage of the B fibrinopeptides has been associated with enhanced lateral aggregation in the fibrin clot. We show that protofibrils from soluble desA fibrin examined by electron microscopy appear to be more loosely organized than those from desAB fibrin. Protofibril lengths were measured from the micrographs; histograms of the distribution of protofibril sizes at different points in the lag period are different for the two species and indicate that the associations leading to oligomer formation are not random. A kinetic model for oligomer formation was developed to investigate the implications of these histograms for protofibril assembly; the model suggests that oligomer-oligomer reactions are important and that some reactions occur preferentially. The overall appearance of negatively contrasted clots made from fibrin monomer missing both fibrinopeptides was similar to that of clots from molecules missing only the A fibrinopeptide, in contrast to the large differences in clots produced by the action of the enzymes on fibrinogen. However, examination of the band patterns by electron microscopy reveals that fibers from desAB fibrin are less well ordered than those from desA fibrin and often aperiodic. In agreement with the electron microscope results, maximum turbidity values are similar for both types of clots, although the initial turbidity rise is more rapid for desAB soluble fibrin clots. If thrombin is added to desA soluble fibrin during the lag period, there is increased lateral aggregation of fibers observed by electron microscopy and the maximum turbidity is higher. These results indicate that there must be a delay in the cleavage of the B fibrinopeptides for the enhancement of lateral aggregation. Changes that occur upon fibrinopeptide B cleavage from molecules in a fiber, such as an increase in calcium ion binding or conformational changes, cause fibers to aggregate with each other or additional protofibrils to be added to a fiber. On the other hand, if these differences are present initially, as they are on soluble desAB fibrin, these results indicate that all steps occur more rapidly, producing relatively thin fibers that are not well ordered. In conclusion, the usual delay in fibrinopeptide B cleavage appears to be necessary both for normal protofibril and fiber assembly, so that the changes that accompany removal of these peptides preferentially affect lateral aggregation rather than earlier steps of fibrin clot assembly.

Animals↗
PubMed1993

Carboxyl-terminal portions of the alpha chains of fibrinogen and fibrin. Localization by electron microscopy and the effects of isolated alpha C fragments on polymerization.

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.

Amino Acid Sequence↗
PubMed1992

Examination of the platelet membrane glycoprotein IIb-IIIa complex and its interaction with fibrinogen and other ligands by electron microscopy.

The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), is a calcium-dependent heterodimer that binds fibrinogen, von Willebrand factor, and fibronectin after platelet activation. We examined GPIIb-IIIa alone and bound to these ligands by electron microscopy after rotary shadowing with platinum/tungsten. We found, as observed previously, that in the presence of detergent and 2 mM Ca2+, GPIIb-IIIa consists of an 8 x 12-nm globular head with two 18-nm flexible tails extending from one side. We also found that in the presence of EDTA, GPIIb-IIIa dissociates into two similar comma-shaped subunits, each containing a portion of the globular head and a single tail. Using monoclonal antibodies to GPIIb, GPIIIa, and the GPIIb-IIIa heterodimer, we found that the tails contained the carboxyl termini of each subunit, while the nodular head was composed of amino-terminal segments of both subunits. Electron microscopy of GPIIb-IIIa bound to fibrinogen revealed a highly specific interaction of the nodular head of GPIIb-IIIa with the distal end of the trinodular fibrinogen molecule and with the tails of GPIIb-IIIa extended laterally at an angle of approximately 98 degrees with respect to the long axis of fibrinogen. When a GPIIb-IIIa was bound to each end of a single fibrinogen, the tails were oriented to opposite sides of fibrinogen, enabling fibrinogen to bridge two adjacent platelets. Electron microscopy of GPIIb-IIIa bound to fibronectin revealed GPIIb/IIIa-binding sites approximately two-thirds of the distance from the amino terminus of each end of the fibronectin molecule, while GPIIb-IIIa was found to bind to von Willebrand factor protomers along a rod-like region near the central nodule of the molecule.

Detergents↗
PubMed1992

Anti-idiotypic antibodies against an antibody to the platelet glycoprotein (GP) IIb-IIIa complex mimic GP IIb-IIIa by recognizing fibrinogen.

Binding of the adhesive ligand fibrinogen and the monoclonal antibody PAC1 to platelet glycoprotein (GP) IIb-IIIa is dependent on cell activation and inhibited by Arg-Gly-Asp (RGD)-containing peptides. Previously, we identified a sequence in a hypervariable region of PAC1 (mu-CDR3) that mimics the activity of the antibody. Here we examine whether monoclonal antibodies to this idiotypic determinant in PAC1 can mimic GP IIb-IIIa by binding to fibrinogen. Mice were immunized with a peptide derived from the mu-CDR3 of PAC1. Four antibodies were obtained that recognized fibrinogen as well as a recombinant form of the variable region of PAC1. However, they did not bind to other RGD-containing proteins, including von Willebrand factor, fibronectin, and vitronectin. Several studies suggested that these anti-PAC1 peptide antibodies were specific for GP IIb-IIIa recognition sites in fibrinogen. Three such sites have been proposed: two RGD-containing regions in the A alpha chain, and the COOH terminus of the gamma chain (gamma 400-411). Two of the antibodies inhibited fibrinogen binding to activated platelets, and all four antibodies bound to the fibrinogen A alpha chain on immunoblots. Antibody binding to immobilized fibrinogen was partially inhibited by monoclonal antibodies specific for the two A alpha chain RGD regions. However, the anti-PAC1 peptide antibodies also bound to plasmin-derived fibrinogen fragments X and D100, which contain gamma 400-411 but lack one or both A alpha RGD regions. This binding was inhibited by an antibody specific for gamma 400-411. When fragment D100 was converted to D80, which lacks gamma 400-411, antibody binding was reduced significantly (p less than 0.01). Electron microscopy of fibrinogen-antibody complexes confirmed that each antibody could bind to sites on the A alpha and gamma chains. These studies demonstrate that certain anti-PAC1 peptide antibodies mimic GP IIb-IIIa by binding to platelet recognition sites in fibrinogen. Furthermore, they suggest that the gamma 400-411 region of fibrinogen may exist in a conformation similar to that of an A alpha RGD region of the molecule.

Amino Acid Sequence↗
PubMed1992

Computer modeling of fibrin polymerization kinetics correlated with electron microscope and turbidity observations: clot structure and assembly are kinetically controlled.

Although much is known about fibrin polymerization, because it is complex, the effects of various modifications are not intuitively obvious and many experimental observations remain unexplained. A kinetic model presented here that is based on information about mechanisms of assembly accounts for most experimental observations and allows hypotheses about the effects of various factors to be tested. Differential equations describing the kinetics of polymerization were written and then solved numerically. The results have been related to turbidity profiles and electron microscope observations. The concentrations of intermediates in fibrin polymerization, and fiber diameters, fiber and protofibril lengths have been calculated from these models. The simplest model considered has three steps; fibrinopeptide A cleavage, protofibril formation, and lateral aggregation of protofibrils to form fibers. The average number of protofibrils per fiber, which is directly related to turbidity, can be calculated and plotted as a function of time. The lag period observed in turbidity profiles cannot be accurately simulated by such a model, but can be simulated by modifying the model such that oligomers must reach a minimum length before they aggregate. Many observations, reported here and elsewhere, can be accounted for by this model; the basic model may be modified to account for other experimental observations. Modeling predicts effects of changes in the rate of fibrinopeptide cleavage consistent with electron microscope and turbidity observations. Changes only in the rate constants for initiation of fiber growth or for addition of protofibrils to fibers are sufficient to account for a wide variety of other observations, e.g., the effects of ionic strength or fibrinopeptide B removal or thrombospondin. The effects of lateral aggregation of fibers has also been modeled: such behavior has been observed in turbidity curves and electron micrographs of clots formed in the presence of platelet factor 4. Thus, many aspects of clot structure and factors that influence structure are directly related to the rates of these steps of polymerization, even though these effects are often not obvious. Thus, to a large extent, clot structure is kinetically determined.

Biophysical Phenomena↗
PubMed1990

Polymerization of fibrinogen-derived fragment X and subsequent rearrangement of fibers.

Polymerization of purified fragment X preparations, obtained from a late stage 2 plasmin digest of fibrinogen, was studied by turbidimetry and electron microscopy. The thrombin-induced polymerization of fragment X caused a rapid increase in turbidity followed by a slow decrease. The initial turbidity increase of fragment X polymers was considerably greater than that of normal fibrin. Electron microscopy carried out at different time points in the reaction revealed that initially there was a great deal of lateral aggregation of fragment X fibers. At later times, electron microscopy showed that there was more dispersal or disruption of the fibers, corresponding to the decrease in turbidity. The slow decrease in turbidity was apparently caused by the rearrangement and/or splitting apart of fragment X fibers, starting from a state similar to a very coarse clot, with much aggregation of fibers, to a delicate meshwork, similar to a fine clot. These changes in clot and fiber structure may result from cleavages partly in the carboxy-terminal region of the gamma chains and the amino-terminal region of the B beta chain of fragment X or the influence of other non-clottable fragments present in these preparations.

Biopolymers↗
PubMed1989

Fibrinogen sialic acid residues are low affinity calcium-binding sites that influence fibrin assembly.

Calcium ions occupy low (n congruent to 10; Kd congruent to 1 mM) and high (n = 3; Kd congruent to 1 microM) affinity sites on fibrinogen and facilitate fibrin monomer polymerization. We have previously localized two of the three high affinity Ca2+ sites to gamma 311-gamma 336. However, optimal enhancement of fibrin monomer polymerization occurs only at physiological millimolar Ca2+ concentrations which are two orders of magnitude higher than the concentration required for occupancy of the high affinity Ca2+-binding sites. In this study, we show that removal of fibrinogen sialic acid residues results in loss of low affinity Ca2+-binding sites. Clotting of asialofibrinogen appears to be Ca2+-independent and results in fiber bundles thicker in diameter than normal fibrin bundles as determined by turbidometry and scanning and transmission electron microscopy. By using a Ca2+-sensitive electrode, free sialic acid is shown to bind Ca2+ (Kd congruent to 1 mM). These observations suggest that the high affinity fibrinogen D-domain Ca2+-binding sites may play a role in the tertiary structure of the D-domain, whereas, sialic acid residues are low affinity sites whose occupancy by Ca2+ at physiological calcium concentration facilitates fibrin polymerization.

Asialoglycoproteins↗
PubMed1989

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.

Binding Sites↗
PubMed1988

Deglycosylation of fibrinogen accelerates polymerization and increases lateral aggregation of fibrin fibers.

Fibrinogen, the major structural precursor of blood clots, was deglycosylated by peptide-N-(N-acetyl-beta-glucosaminyl)asparagine amidase without denaturation of the polypeptide chains. Deglycosylated fibrinogen behaved normally in clinical coagulation assays, although it is less soluble than normal fibrinogen. However, the turbidity of clots formed from deglycosylated fibrinogen always rose faster and higher than that of clots from normal fibrinogen. Scanning and transmission electron microscopy demonstrated that fibrin made from clots of deglycosylated fibrinogen consisted of thicker, less-branched fiber bundles in a more porous network. Moreover, the degree of lateral aggregation was directly related to clot turbidity and inversely related to branching. Deglycosylation promoted turbidity development, lateral aggregation, and porosity of clots under all conditions tested. All other steps in the coagulation pathways appeared to be unaffected by the absence of carbohydrate. These results suggest that carbohydrate constitutively affects the behavior of deglycosylated fibrinogens by 1) contributing a repulsive force that promotes fibrinogen solubility and limits fibrin assembly and 2) sensitizing fibrin to conditions that influence assembly and clot structure.

Factor XII↗
PubMed1987

Involvement of the COOH-terminal portion of the alpha-chain of fibrin in the branching of fibers to form a clot.

A modified fibrinogen molecule which is missing the COOH-terminal portion of the A alpha chain has been used in structural investigations of the mechanism of assembly of the fibrin clot. Brief plasmin digestion of human fibrinogen, followed by ammonium sulfate fractionation and column chromatography, yielded a highly clottable fragment X-like preparation. Molecules in this preparation contain mostly intact B beta and gamma chains, but are missing the COOH-terminal two-thirds of the A alpha chain. Clots formed by addition of thrombin to this fragment were mechanically unstable and easily dispersed. Electron microscopy showed that the clots consist mainly of a suspension of individual fibers, in contrast to clots made from native fibrinogen, which are highly branched. It appears, therefore, that a part of the COOH-terminal two-thirds of the alpha chain is necessary for branching of fibers to form a stable three-dimensional gel. Intermolecular interactions of this portion of the alpha chain are consistent with certain of its unusual features, such as its apparent existence, in part, as a single polypeptide chain and its involvement in Factor XIIIa-mediated ligation between molecules.

Blood Coagulation↗