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Biomedical subjects

M W Mosesson

Publications and source records attributed to M W Mosesson.

At least 37 records · Page 2Linked to original sources
PubMed2000

Adhesion of microvascular endothelial cells to metallic implant surfaces.

The objective of this study was to explore the molecular mechanisms of adhesion of endothelial cells (ECs) to implant grades of titanium alloy (Ti) and stainless steel (SS), compared to tissue culture polystyrene (PS). The idea is that promotion of EC adhesion to implant surfaces during the initial stages of healing may be critical in the formation of a capillary bed intimately associated with the implant surface. Ultimately this could be expected in turn to promote bone formation close to the surface and a more stable implant/bone interface. Surfaces were coated with either peak 1 fibrinogen gammaAgammaA, fibrinogen Fr I-9, fibrinogen fragment D1, fibronectin, vitronectin, or fetal calf serum and then post-coated with bovine serum albumin (BSA) to block non-specific cell adhesion. Surfaces with BSA alone and no other protein coating were also evaluated. Fibronectin coating maximized cell adhesion on all three surfaces, and adhesion was highest on PS. BSA blocked cell adhesion to PS (and most adhesion to SS) much better than to Ti. These results provide evidence that BSA adsorption on the metal surface is unable to effectively block the adhesion of the cells to the Ti. These data may provide a basis for understanding in vivo observations that soft tissue becomes attached to a Ti surface more rapidly and with more bone formation than to SS. Evidence is also presented that alphavbeta3 plays an important role in adhesion of ECs to the Ti surface. These experiments also provide preliminary data which may reflect some of the features of initial EC adhesion to metal implants.

Journal Article↗
PubMed2000

Coexisting dysfibrinogenemia (gammaR275C) and factor V Leiden deficiency associated with thromboembolic disease (fibrinogen Cedar Rapids).

Fibrinogen Cedar Rapids is a heterozygous dysfibrinogenemia (gammaR275C) that was associated with thromboembolism during and following pregnancy in three second-generation family members who also were heterozygotic for factor V Leiden (V R506Q). Like other dysfibrinogenemias with substitutions at position 275 of the gamma-chain, fibrinogen Cedar Rapids is characterized by defective end-to-end intermolecular fibrinogen and fibrin 'D : D' associations, a fibrin network structure that is composed of thicker and more highly branched fibers, normal fibrin 'D: E' associations, and normal factor XIII-mediated crosslinking of fibrinogen and fibrin. In addition, Cedar Rapids fibrinogen and fibrin displayed delayed plasmin lysis rates. Compared with normal fibrinogen, platelet aggregation or platelet fibrinogen receptor clustering was defective in the presence of fibrinogen Cedar Rapids. Most subjects with gammaR275 mutations do not experience clinical thrombotic disorders, suggesting that the combination of a factor V Leiden defect and a gammaR275C dysfibrinogenemia predisposes to thromboembolic disease.

Adult↗
PubMed2000

Evidence for new endothelial cell binding sites on fibrinogen.

In vitro assays were used to characterize adhesion of human aortic, microvascular and umbilical vein endothelial cells to various forms of immobilized fibrinogen. All three types of endothelial cells adhered to fibrinogen in a manner that was independent of the Aalpha-chain 572-574 RGD cell binding site. In fact, all three adhered to a fragment of the molecule which is composed of only one D domain (D1) of fibrinogen. A time course study revealed that extensive adhesion of endothelial cells on the ligand coated surface occurred between one and two hours incubation. The anti-fibrinogen gammaA-chain monoclonal antibody 4A5 as well as 4A5 Fabs, blocked adhesion of endothelial cells to fibrinogen, not vitronectin. The inhibitory effects of 4A5 seemed to be indirect because the endothelial cells adhered to the recombinant fibrinogen gamma407 (which lacks the gamma-chain AGDV sequence of the carboxyl terminal 4A5 binding site) as well as they did to normal recombinant fibrinogen. A recombinant fibrinogen lacking the gamma-chain AGDV sequence, containing RGE in place of RGD at the gamma-chain 572-574 and 95-97 positions, also supported endothelial cell adhesion. The anti-alphavbeta3 antibody, LM609, blocked adhesion of endothelial cells to fibrinogen. The peptide GRGDSP inhibited endothelial cell adhesion on fibrinogen and vitronectin. These results demonstrate that alphavbeta3 mediated adhesion (attachment and spreading) of HUVECs to fibrinogen may use a site in the D domain of fibrinogen and is not dependent on the Aalpha-chain RGD (95-97 and 572-574) sequences, as has been shown in shorter term (where cells were rounded) experiments, or the alphaA-chain 408-411 cell binding sites. Thus, the data reveal the existence of another unidentified site(s) on fibrinogen which can support the irreversible adhesion (attachment and spreading) of endothelial cells.

Binding Sites↗
PubMed1999

Fibrinogen Niigata with impaired fibrin assembly: an inherited dysfibrinogen with a Bbeta Asn-160 to Ser substitution associated with extra glycosylation at Bbeta Asn-158.

A novel BbetaAsn-160 (TAA) to Ser (TGA) substitution has been identified in fibrinogen Niigata derived from a 64-year-old asymptomatic woman, who is heterozygotic for this abnormality. The mutation creates an Asn-X-Ser-type glycosylation sequence, and a partially sialylated biantennary oligosaccharide was linked to the BbetaAsn-158 residue. The functional abnormality was attributed to delayed lateral association of normally formed double-stranded protofibrils based on normal cross-linking of fibrin gamma-chains and tissue-type plasminogen activator-catalyzed plasmin generation by polymerizing fibrin monomers. Enzymatic removal of all the N-linked oligosaccharides from fibrinogen Niigata accelerated fibrin monomer polymerization that reached the level of untreated normal fibrin monomers, but the thrombin time was prolonged from 18.2 seconds to 113 seconds (normal: 11.2 seconds to 8.9 seconds). By scanning electron micrographic analysis, Niigata fibrin fibers were found to be more curvilinear than normal fibrin fibers. After deglycosylation, Niigata fibers became straight being similar to untreated normal fibrin fibers, whereas normal deglycosylated fibrin appeared to be less-branched than untreated normal or deglycosylated Niigata fibrin. Although normal and Niigata fibrins were similar to each other in permeation and compaction studies, deglycosylated normal and Niigata fibrins had much higher permeability and compaction values, indicating that deglycosylation had brought about the formation of more porous networks. The enzymatic deglycosylation necessitates an Asn to Asp change at position Bbeta-158 that is responsible for reducing the fiber thickness because of either local repulsive forces or steric hindrance in the coiled-coil region.

Amino Acid Substitution↗
PubMed1999

Dysfibrinogenemia and thrombosis.

Congenital abnormal fibrinogen molecules (dysfibrinogenemias) are due to structural defects in the molecule. The molecular structure of the fibrinogen molecule is to a great extent known and this has allowed identification of the abnormalities at a molecular level. While most patients with dysfibrinogenemia are clinically asymptomatic, some present with a bleeding diathesis, others with thrombophilia, and occasionally with both, bleeding and thromboembolism. In principle, the dysfibrinogenemias are due to either impaired release of the fibrinopeptides, defective fibrin polymerization, or abnormal cross-linking by factor XIIIa. Dysfibrinogenemias associated with thrombophilia have been reported in those related to abnormal fibrinopeptide release or defective polymerization. In addition, abnormal interactions with platelets, defective fibrinolysis, defective assembly of the fibrinolytic system, and abnormal calcium binding have been described. The presently identified dysfibrinogenemias associated with thrombosis and their molecular defects are described in this review. It must also be recognized that some patients with abnormal fibrinogen molecules have additional hemostasis defects, such as abnormalities of antithrombin, protein C, protein S, factor V Leiden, and others. On rare occasions, dysfibrinogenemias can be associated with hypofibrinogenemia.

Fibrinogens, Abnormal↗
PubMed1998

The location of the carboxy-terminal region of gamma chains in fibrinogen and fibrin D domains.

Elongated fibrinogen molecules are comprised of two outer "D" domains, each connected through a "coiled-coil" region to the central "E" domain. Fibrin forms following thrombin cleavage in the E domain and then undergoes intermolecular end-to-middle D:E domain associations that result in double-stranded fibrils. Factor XIIIa mediates crosslinking of the C-terminal regions of gamma chains in each D domain (the gammaXL site) by incorporating intermolecular epsilon-(gamma-glutamyl)lysine bonds between amine donor gamma406 lysine of one gamma chain and a glutamine acceptor at gamma398 or gamma399 of another. Several lines of evidence show that crosslinked gamma chains extend "transversely" between the strands of each fibril, but other data suggest instead that crosslinked gamma chains can only traverse end-to-end-aligned D domains within each strand. To examine this issue and determine the location of the gammaXL site in fibrinogen and assembled fibrin fibrils, we incorporated an amine donor, thioacetyl cadaverine, into glutamine acceptor sites in fibrinogen in the presence of XIIIa, and then labeled the thiol with a relatively small (0.8 nm diameter) electron dense gold cluster compound, undecagold monoaminopropyl maleimide (Au11). Fibrinogen was examined by scanning transmission electron microscopy to locate Au11-cadaverine-labeled gamma398/399 D domain sites. Seventy-nine percent of D domain Au11 clusters were situated in middle to proximal positions relative to the end of the molecule, with the remaining Au11 clusters in a distal position. In fibrin fibrils, D domain Au11 clusters were located in middle to proximal positions. These findings show that most C-terminal gamma chains in fibrinogen or fibrin are oriented toward the central domain and indicate that gammaXL sites in fibrils are situated predominantly between strands, suitably aligned for transverse crosslinking.

Fibrin↗
PubMed1998

Fibrinogen structure and fibrin clot assembly.

Fibrinogen is a complex multifunctional protein, which contains constitutive association sites (gammaXL, D:D, Da, Db) as well as cryptic sites that become exposed as a result of fibrinogen proteolysis by thrombin (EA, EB). Utilization of these sites by self-association (gammaXL or D:D) or by association with exposed complementary fibrin sites (Da:EA, Db:EB) produces an orderly process of molecular assembly to form linear and branched fibrils, concomitant with lateral fibril associations and factor XIIIa-mediated fibrin crosslinking that together result in the mature fibrin network.

Binding Sites↗
PubMed1998

Evaluation of the factors contributing to fibrin-dependent plasminogen activation.

Polymerized fibrin strongly enhances tissue plasminogen activator (tPA)-mediated plasminogen activation, concomitant with exposure of 'fibrin-specific' epitopes at 'Aalpha148-160' and 'gamma312-324'. To investigate which aspects of polymerization are involved in these activities, we explored the fibrin polymerization process by evaluating the ability of factor XIIIa-crosslinked fibrinogen polymers to expose 'fibrin-specific' epitopes and enhance plasminogen activation. Crosslinked normal fibrinogen, fibrinogen with deficient [des Bbeta1-42] or defective [Birmingham (AalphaR16H)] fibrin 'D:E' assembly sites ('E(A)'), or with defective end-to-end self-association sites ('D:D') [Cedar Rapids (gammaR275C)], exposed both 'fibrin-specific' epitopes and enhanced tPA-dependent plasminogen activation, whereas non-crosslinked fibrinogens showed minimal or no such activities. Epitope expression in crosslinked fibrinogen was retained in the presence of the fibrin E(A) site peptide homolog, gly-pro-arg-pro (GPRP), which inhibits fibrin D:E association, except for the Aalpha148-160 epitope in des Bbeta1-42 fibrinogen, which was not expressed. Fibrin prepared from crosslinked normal or abnormal fibrinogen, except for the des Bbeta1-42 fibrin epitopes, which were reduced or absent, expressed 'fibrin-specific' epitopes even in the presence of GPRP, which otherwise impairs such expression in non-crosslinked fibrin. Epitope exposure in fibrin prepared from non-crosslinked fibrinogen was nearly normal in Cedar Rapids fibrin (heterozygous D:D defect), but reduced in Birmingham fibrin (heterozygous E(A) defect), nil in des Bbeta1-42 fibrin (E(A) deficient), and absent in all cases in the presence of GPRP. In contrast, plasminogen activation stimulatory activity that had been exposed in crosslinked normal fibrinogen or in crosslinked des Bbeta1-42 or Cedar Rapids fibrin, was preserved to a large extent in the presence of GPRP, suggesting that once enhanced stimulatory activity and epitopes are exposed, they are not completely reversible. The findings indicate that end-to-end intermolecular associations (D:D) are not critical for 'fibrin-specific' epitope exposure, but that polymerization brought about in fibrinogen through factor XIIIa crosslinking, or in fibrin through 'D:E' interactions, is necessary for 'fibrin-specific' (more correctly, 'polymerization-specific') epitope exposure and enhancement of plasminogen activation.

Afibrinogenemia↗
PubMed1997

The role of putative fibrinogen Aalpha-, Bbeta-, and GammaA-chain integrin binding sites in endothelial cell-mediated clot retraction.

In this study, endothelial cell-mediated clot retraction was supported by fibrin generated from several purified fractions of plasma fibrinogen, purified proteolytic fragments of plasma fibrinogen, recombinant normal fibrinogen, and recombinant variant fibrinogen. These results were surprising because some of these fibrinogens lack domains that are known binding sites for the integrin receptors that support clot retraction. Specifically, fibrinogens lacking Aalpha-chain RGD residues at 572-574 or lacking the gamma-chain residues AGDV 408-411 supported endothelial cell-mediated clot retraction as well as intact fibrinogen. Thus, clot retraction mediated by endothelial cells is not dependent on either of these sites. A variety of monoclonal antibodies against the integrin alphavbeta3 partially inhibited the endothelial cell-mediated retraction of clots formed from plasma fibrinogen. As expected, an antibody to the platelet integrin alphaIIbbeta3 did not inhibit endothelial cell-mediated clot retraction. These results indicate that this retraction is mediated at least in part by alphavbeta3. These results support the conclusion that (a) neither of the two fibrinogen cell binding sites described above is required to support clot retraction or that (b) either site alone or in conjunction with other fibrin(ogen) region(s) can support clot retraction. Thus, endothelial cell-mediated clot retraction appears to be dependent on fibrinogen cell binding sites other than those required to support adhesion of resting platelets to immobilized fibrinogen and platelet aggregation.

Binding Sites↗
PubMed1997

Human fibroblasts bind directly to fibrinogen at RGD sites through integrin alpha(v)beta3.

Fibroblast migration into the blood clot initially filling a wound requires close interaction between fibroblasts and the matrix of the fibrin clot. However, very little is known about the specific receptor-ligand interactions that mediate fibroblast attachment to fibrin. Using an attachment assay developed to measure even relatively weak interactions, we demonstrate here that normal human dermal fibroblasts can attach to substrates coated with fibrinogen, fibrin, or the fibrinogen breakdown product I-9D. Fibroblast attachment to these ligands did not require the presence of fibronectin on the cell surface or as a component of the substrate. Cells treated with cycloheximide and monensin, to limit the synthesis and secretion of endogenous fibronectin, attached as well as untreated cells. The synthetic peptide GRGDS inhibited adhesion to fibrinogen, fibrin, and fibrinogen I-9D by about 60%, while the control peptide GRGES had no substantial effect. We conclude that attachment to these ligands is mediated at least partially by direct interactions between the substrates and one specific receptor, the integrin alpha(v)beta3. Affinity chromatography demonstrated that alpha(v)beta3 from detergent lysates of fibroblasts bound to a fibrinogen matrix and was eluted with EDTA. Furthermore, antibodies against the alpha(v)beta3 complex or against the alpha(v) subunit inhibited fibroblast attachment to fibrinogen and fibrin by 50-70%. An inhibitory antibody against the integrin beta1 subunit had no effect. The observation that integrin antagonists could not produce complete inhibition suggests that there may be other fibroblast cell surface proteins that can bind directly to fibrinogen.

Cell Adhesion↗
PubMed1997

Fibrinogen and fibrin polymerization: appraisal of the binding events that accompany fibrin generation and fibrin clot assembly.

Fibrinogen is a complex multifunctional protein comprised of three major domains (two outer D and one central E) which contains constitutive binding sites (e.g. Da, Db, gammaXL, D:D, gamma', thrombin substrate, platelet receptor) as well as binding sites that become exposed or expressed as a result of fibrinogen proteolysis by thrombin and/or that are exposed as a consequence of the polymerization process itself (tPA binding sites). Fibrin-dependent tPA-mediated activation of plasminogen is associated with exposure of polymerization-dependent epitopes (Aalpha148-160, gamma312-324) that are expressed in assembled fibrin and in crosslinked (polymerized) fibrinogen but not in unpolymerized fibrinogen or fibrin. Fibrin polymerization is initiated by thrombin cleavage of fibrinopeptide A from fibrinogen Aalpha chains, exposing two E domain E(A) sites. Cleavage of fibrinopeptide B from fibrinogen Bbeta chains exposes other E domain polymerization sites, termed E(B), that also interact with platelets, fibroblasts and endothelial cells. Fibrin generation is followed by an assembly process of intermolecular end-to-middle D to E associations to form linear and branched double-stranded fibrin fibrils, lateral fibril-fibril associations to form fibers and a branched fiber network. Binding sites in fibrinogen play their roles in fibrin assembly by self-association (gammaXL to gammaXL and D:D to D:D) or by complementary association with exposed sites in fibrin (Da to E(A) and Db to E[B]). Other binding sites in fibrinogen include thrombin substrate recognition sites in each E domain and a non-substrate high affinity thrombin binding site in the carboxy-terminal region of each gamma' chain, which also binds plasma factor XIII. Fibrin possesses low affinity thrombin binding sites in each E domain and retains the gamma' chain nonsubstrate thrombin-binding site.

Animals↗
PubMed1996

Identification and characterization of the thrombin binding sites on fibrin.

Thrombin binds to fibrin at two classes of non-substrate sites, one of high affinity and the other of low affinity. We investigated the location of these thrombin binding sites by assessing the binding of thrombin to fibrin lacking or containing gamma' chains, which are fibrinogen gamma chain variants that contain a highly anionic carboxyl-terminal sequence. We found the high affinity thrombin binding site to be located exclusively in D domains on gamma' chains (Ka, 4.9 x 10(6) M-1; n, 1.05 per gamma' chain), whereas the low affinity thrombin binding site was in the fibrin E domain (Ka, 0.29 x 10(6) M-1; n, 1.69 per molecule). The amino-terminal beta15-42 fibrin sequence is an important constituent of low affinity binding, since thrombin binding at this site is greatly diminished in fibrin molecules lacking this sequence. The tyrosine-sulfated, thrombin exosite-binding hirudin peptide, S-Hir53-64 (hirugen), inhibited both low and high affinity thrombin binding to fibrin (IC50 1.4 and 3.0 microM respectively). The presence of the high affinity gamma' chain site on fibrinogen molecules did not inhibit fibrinogen conversion to fibrin as assessed by thrombin time measurements, and thrombin exosite binding to fibrin at either site did not inhibit its catalytic activity toward a small thrombin substrate, S-2238. We infer from these findings that there are two low affinity non-substrate thrombin binding sites, one in each half of the dimeric fibrin E domain, and that they may represent a residual aspect of thrombin binding and cleavage of its substrate fibrinogen. The high affinity thrombin binding site on gamma' chains is a constitutive feature of fibrin as well as fibrinogen.

Amino Acid Chloromethyl Ketones↗
PubMed1996

Plasma factor XIII binds specifically to fibrinogen molecules containing gamma chains.

The difference between peak 1 and peak 2 fibrinogen lies in their gamma chains. Peak 1 molecules contain 2 gamma A chains; peak 2 molecules contain 1 gamma A and 1 gamma chain, the latter of which contains a 20 amino acid extension (gamma 408-427) replacing the carboxyl-terminal 4 amino acids of the gamma A chain (gamma A 408-411). While the existence of gamma chains in plasma fibrinogen molecules has been known for many years, their function remains unknown. When fibrinogen is purified from plasma, the factor XIII zymogen (A2B2) copurifies with it and is found only in the peak 2 fibrinogen when this fraction is separated from peak 1 fibrinogen by ion-exchange chromatography on DEAE-cellulose. Factor XIII alone applied to the same DEAE column elutes at a position between peak 1 and peak 2. When mixtures of peak 1 fibrinogen plus factor XIII or peak 2 fibrinogen plus factor XIII are applied to DEAE columns, the peak 1/factor XIII mixture elutes in two peaks, whereas the peak 2/factor XIII mixture elutes in the peak 2 fibrinogen position. Gel sieving on Superose 6 of peak 1/factor XIII mixtures results in two protein peaks, the first of which contains the fibrinogen. Most factor XIII activity elutes in the second peak with a small amount of activity emerging with the trailing end of the fibrinogen peak. Gel sieving of mixtures of peak 2 and factor XIII results in a single protein peak with all factor XIII activity emerging with the leading edge of the fibrinogen peak. The interaction between peak 2 fibrinogen and plasma factor XIII appears to be through binding to the B subunit of factor XIII since placental or platelet factor XIII (A2), which does not contain B subunits, elutes independently from peak 2 fibrinogen on DEAE-cellulose chromatography. The results indicate that peak 2 fibrinogen gamma chains have a physiologically significant affinity for the B subunits of plasma factor XIII and that through this interaction fibrinogen serves as a carrier for the plasma zymogen in circulating blood.

Binding Sites↗
PubMed1996

The relationship between the fibrinogen D domain self-association/cross-linking site (gammaXL) and the fibrinogen Dusart abnormality (Aalpha R554C-albumin): clues to thrombophilia in the "Dusart syndrome".

Cross-linking of fibrinogen at its COOH-terminal gamma chain cross-linking site occurs in the presence of factor XIIIa due to self-association at a constitutive D domain site ("gammaXL"). We investigated the contribution of COOH-terminal regions of fibrinogen Aalpha chains to the gammaXL site by comparing the gamma chain cross-linking rate of intact fibrinogen (fraction I-2) with that of plasma fraction I-9, plasmic fraction I-9D, and plasmic fragment D1, which lack COOH-terminal Aalpha chain regions comprising approximately 100, approximately 390, and 413 residues, respectively. The cross-linking rates were I-2 > I-9 > 1-9D = D1, and indicated that the terminal 100 or more Aalpha chain residues enhance gammaXL site association. Fibrinogen Dusart, whose structural abnormality is in the COOH-terminal "alphaC" region of its Aalpha chain (Aalpha R554C-albumin), is associated with thrombophilia ("Dusart Syndrome"), and is characterized functionally by defective fibrin polymerization and clot structure, and reduced plasminogen binding and tPA-induced fibrinolysis. In the presence of XIIIa, the Dusart fibrinogen gamma chain cross-linking rate was about twice that of normal, but was normalized in proteolytic fibrinogen derivatives lacking the Aalpha chain abnormality, as was reduced plasminogen binding. Electron microscopy showed that albumin-bound Dusart fibrinogen "alphaC" regions were located in the vicinity of D domains, rather than at their expected tethered location near the fibrinogen E domain. In addition, there was considerable fibrinogen aggregation that was attributable to increased intermolecular COOH-terminal Aalpha chain associations promoted by untethered Dusart fibrinogen aC domains. We conclude that enhanced Dusart fibrinogen self-assembly is mediated through its abnormal alphaC domains, leads to increased gammaXL self-association and gamma chain cross-linking potential, and contributes to the thrombophilia that characterizes the "Dusart Syndrome."

Dextrans↗
PubMed1996

Evidence of intramolecular cross-linked A alpha.gamma chain heterodimers in plasma fibrinogen.

A peptide band of approximately 105 kDa migrating near the gamma dimer position of disulfide bond reduced human plasma fibrinogen prepared from fresh single donor or outdated plasma was identified by SDS-PAGE. The band, amounting to approximately 2% of the total A alpha/gamma chain population, was thrombin and plasmin sensitive and reacted with antibodies to A alpha or gamma chains but not with antibodies to B beta chains, plasminogen, or factor XIII. Amino acid sequencing revealed a double sequence corresponding to that of A alpha and gamma chains, indicating that the band consists of covalently cross-linked A alpha.gamma chain heterodimers. A alpha.gamma heterodimers were identified as a component of monomeric fibrinogen by two-dimensional SDS-PAGE and by SDS-PAGE analysis of the monomer fraction isolated by gel sieving chromatography, thus indicating that A alpha.gamma heterodimers arise by intramolecular A alpha/gamma chain cross-linking.

Amino Acid Sequence↗
PubMed1995

The dimeric Aalpha chain composition of dysfibrinogenemic molecules with mutations at Aalpha 16.

In the last stage of fibrinogen synthesis, two Aalpha-Bbeta-gamma half-molecules are disulfide linked in their N-terminal regions to form a dimeric fibrinogen molecule. It is not known whether intracellular hepatocyte assembly of fibrinogen half-molecules occurs randomly or is a directed process. One analysis based on partitioning of coagulable components of fibrinogen from a heterozygous dysfibrinogenemic subject having a mutation at the thrombin cleavage site (Fibrinogen Louisville, Aalpha16 R-->H), suggested that only homodimeric molecules containing two normal fibrinopeptides A (FPA, FPA) or two abnormal fibrinopeptides A (FPA*, FPA*) were present in plasma, implying that fibrinogen dimer assembly is directed. The same type of analyses on Fibrinogen Birmingham (Aalpha16 R-->H) indicated that there were heterodimers as well as homodimers, suggesting that fibrinogen dimer assembly is random. To examine this question more directly, the composition of fibrinogen molecules from seven dysfibrinogenemic families with either R-->C (four) or R-->H (three) Aalpha16 mutations was determined. Following treatment with Atroxin to release normal FPA from fibrinogen, N-terminal disulfide knot ('N-DSK') cleavage fragments were prepared and subsequently separated by SDS-PAGE to resolve 'N-DSK' components with two FPA*'s (N-DSK homodimer), one FPA* (des A N-DSK heterodimer), or no FPA's (des AA N-DSK homodimer). Fibrinogen from subjects whose molecules contained both normal and abnormal Aalpha chains, yielded a heterodimeric des A N-DSK derivative, as well as smaller amounts of homodimeric N-DSK and des AA N-DSK. These results indicate that when both types of Aalpha chain are produced, both Aalpha chain alleles are expressed and the resulting fibrinogen dimers are assembled randomly.

Cyanogen Bromide↗
PubMed1995

The role of fibrinogen D domain intermolecular association sites in the polymerization of fibrin and fibrinogen Tokyo II (gamma 275 Arg-->Cys).

Intermolecular end-to-middle domain pairing between a thrombin-exposed 'A' polymerization site in the central 'E' domain of fibrin, and a constitutive complementary 'a' site in each outer 'D' domain ('D:E'), is necessary but not alone sufficient for normal fibrin assembly, as judged from previous studies of a congenital dysfibrinogen, Tokyo II (gamma 275 arg-->cys), which showed defective fibrin clot assembly and a normal D:E interaction (Matsuda, M., M. Baba, K. Morimoto, and C. Nakamikawa, 1983. J. Clin. Invest. 72:1034-1041). In addition to the 'a' polymerization site, two other constitutive intermolecular association sites on fibrinogen D domains have been defined: between gamma chain regions containing the carboxy-terminal factor XIIIa crosslinking site ('gamma XL:gamma XL'); and between sites located at the outer ends of each molecule ('D:D') (Mosesson, M. W., K. R. Siebenlist, J. F. Hainfeld, and J. S. Wall, manuscript submitted for publication). We evaluated the function of these sites in Tokyo II fibrinogen, and confirmed that there was a normal fibrin D:E interaction, as determined from a normal fibrin crosslinking rate in the presence of factor XIIIa. We also found a normal gamma XL: gamma XL interaction, as assessed by a normal fibrinogen crosslinking rate. Judging from electron microscopic images, factor XIIIa-crosslinked Tokyo II fibrinogen failed to form elongated double-stranded fibrils like normal fibrinogen. Instead, it formed aggregated disordered collections of molecules, with occasional short fibrillar segments. In addition, Tokyo II fibrin formed an abnormal, extensively branched clot network containing many tapered terminating fibers. These findings indicate that the Tokyo II fibrinogen defect results in a functionally abnormal D:D self-association site, and that a normal D:D site interaction is required, in addition to D:E, for normal fibrin or fibrinogen assembly.

Binding Sites↗