Fibrinogen Longmont. A heterozygous abnormal fibrinogen with B beta Arg-166 to Cys substitution associated with defective fibrin polymerization.

B beta Arg166 to Cys substitution was identified in an abnormal fibrinogen named fibrinogen Longmont. The proband, a young woman, and her mother were heterozygous; both experienced episodes of severe hemorrhage at childbirth. The neo-Cys residues were found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Thrombin and batroxobin induced fibrin polymerization were impaired, despite normal release of fibrinopeptides A and B. Moreover, the polymerization defect was not corrected by removing the dimeric species or adding calcium. Fibrinogen Longmont had normal polymerization site a, as evidenced by normal GPRP-peptide binding. Thus, the sites A and a can interact to form protofibrils, as evidenced by dynamic light scattering measurements. These protofibrils, however, do not associate laterally in a normal manner, leading to an abnormal clot formation.

Polymerization site a function dependence on structural integrity of its nearby calcium binding site.

To explore the functional relationship between the polymerization site a and the nearby high affinity calcium binding site, we analyzed four variant fibrinogens with substitutions at these sites: gamma D364A in the a site and gamma D318A, gamma D320A, and gamma D318 + gamma D320A in the Ca2+ site. In all cases fibrinopeptide A release was normal and thrombin catalyzed polymerization was markedly impaired (unpublished observations). We examined the functional connection between the Ca2+ site and the a site by testing for plasmin protection in the presence of Ca2+ or the a site peptide ligand GPRP. SDS-PAGE analysis of the products showed that gamma D364A fibrinogen was protected from plasmin cleavage by Ca2+ but not by the GPRP peptide. In contrast, neither Ca2+ nor the GPRP peptide protected gamma D318A, gamma D320A, or gamma D318 + gamma D320A fibrinogens from complete plasmin cleavage. These results suggest that the structural integrity of the calcium binding site is required for expression of the a site. In contrast, the structural integrity of the a site has no functional consequence on Ca2+ binding to this high affinity site.

Mutations on fibrinogen (gamma 316-322) are associated with reduction in platelet adhesion under flow conditions.

In this paper we report on studies of platelet adhesion to several fibrinogen gamma chain variants under physiological flow conditions. Reduced platelet adhesion was found to patient dysfibrinogen Vlissingen and its recombinant form (deletion of gamma 319-320). Furthermore, substitutions of the amino acids 318, 320, or both in the recombinant fibrinogen gamma chain showed a strong decrease in platelet adhesion under flow conditions in our perfusion system. Antibodies raised against peptides covering these sequences inhibited platelet adhesion completely, which suggested that the gamma 316-322 sequence could be involved in platelet adhesion in flowing blood.

The impaired polymerization of fibrinogen Longmont (Bbeta166Arg-->Cys) is not improved by removal of disulfide-linked dimers from a mixture of dimers and cysteine-linked monomers.

This study identified a new substitution in the Bbeta chain of an abnormal fibrinogen, denoted Longmont, where the residue Arg166 was changed to Cys. The variant was discovered in a young woman with an episode of severe hemorrhage at childbirth and a subsequent mild bleeding disorder. The neo-Cys residues were always found to be disulfide-bridged to either an isolated Cys amino acid or to the corresponding Cys residue of another abnormal fibrinogen molecule, forming dimers. Removing the dimeric molecules using gel filtration did not correct the fibrin polymerization defect. Fibrinogen Longmont had normal fibrinopeptide A and B release and a functional polymerization site "a." Thus, the sites "A" and "a" can interact to form protofibrils, as evidenced by dynamic light-scattering measurements. These protofibrils, however, were unable to associate in the normal manner of lateral aggregation, leading to abnormal clot formation, as shown by an impaired increase in turbidity. Therefore, it is concluded that the substitution of Arg166-->Cys-Cys alters fibrinogen Longmont polymerization by disrupting interactions that are critical for normal lateral association of protofibrils. (Blood. 2001;98:661-666)

Mineralocorticoid hormone signaling regulates the 'epithelial sodium channel' in fibroblasts from human cornea.

We investigated the regulation of sodium absorption by steroid hormones in embryologically diverse cells from the human eye. A cell extract from human corneal fibroblasts was positive for both the epithelial sodium channel (ENaC) and the mineralocorticoid receptor (MCR) as 82- to 85-kD and 102-kD bands, respectively, by the Western blot technique. In fluorescent, confocal and electron microscopy, the MCR was revealed as a nucleocytoplasmic protein, whereas the ENaC was almost exclusively membrane bound; both appeared aligned along actin filaments of corneal keratocytes, and both were widely colocalized in various cell types of human cornea in situ. Following reverse transcription and amplification of total RNA isolated from corneal fibroblasts, the ENaC and MCR genes in the PCR product were evident as predicted bands of 520 and 843 bp, respectively, whose sequence exhibited 100% identity with those from known human sources. The multiplication of corneal fibroblasts was influenced by both the MCR-specific antagonist RU 26752 and the natural hormone aldosterone, and these steroids also stimulated protein phosphorylation. In quantitative PCR, both the basal and aldosterone-induced levels of ENaC were diminished by the MCR-specific antagonist ZK 91587. Consequently, the ocular sodium channel appears to be regulated by steroid signalling in cells of diverse embryological origins, contrary to the existing notions where (a) this process would be limited exclusively to the epithelial cells and (b) ocular sodium transport would be regulated via the Na(+)-K(+)-ATPase in the basolateral membrane.

Fibrinogen Alès: a homozygous case of dysfibrinogenemia (gamma-Asp(330)-->Val) characterized by a defective fibrin polymerization site "a".

Congenital homozygous dysfibrinogenemia was diagnosed in a man with a history of 2 thrombotic strokes before age 30. His hemostatic profile was characterized by a dramatically prolonged plasma thrombin clotting time, and no clotting was observed with reptilase. Complete clotting of the abnormal fibrinogen occurred after a prolonged incubation of plasma with thrombin. The release of fibrinopeptides A and B by thrombin and of fibrinopeptide A by reptilase were both normal. Thrombin-induced fibrin polymerization was impaired, and no polymerization occurred with reptilase. The polymerization defect was characterized by a defective site "a," resulting in an absence of interaction between sites A and a, indicated by the lack of fragment D(1) (or fibrinogen) binding to normal fibrin monomers depleted in fibrinopeptide A only (Des-AA fm). By SDS-PAGE, the defect was detected on the gamma-chain and in its fragment D(1). The molecular defect determined by analysis of genomic DNA showed a single base change (A-->T) in exon VIII of the gamma-chain. The resulting change in the amino acid structure is gamma 330 aspartic acid (GAT) --> valine (GTT). It is concluded that the residue gamma-Asp(330) is essential for the normal functioning of the polymerization site a on the fibrinogen gamma-chain.

Recombinant fibrinogen Vlissingen/Frankfurt IV. The deletion of residues 319 and 320 from the gamma chain of firbinogen alters calcium binding, fibrin polymerization, cross-linking, and platelet aggregation.

We synthesized a variant, recombinant fibrinogen modeled after the heterozygous dysfibrinogen Vlissingen/Frankfurt IV, a deletion of two residues, gammaAsn-319 and gammaAsp-320, located within the high affinity calcium-binding pocket. Turbidity studies showed no evidence of fibrin polymerization, although size exclusion chromatography, transmission electron microscopy, and dynamic light scattering studies showed small aggregates. These aggregates did not resemble normal protofibrils nor did they clot. Fibrinopeptide A release was normal, whereas fibrinopeptide B release was delayed approximately 3-fold. Plasmin cleavage of this fibrinogen was not changed by the presence of calcium or Gly-Pro-Arg-Pro, indicating that both the calcium-binding site and the "a" polymerization site were non-functional. We conclude that the loss of normal polymerization was due to the lack of "A-a" interactions. Moreover, functions associated with the C-terminal end of the gamma chain, such as platelet aggregation and factor XIII cross-linking, were also disrupted, suggesting that this deletion of two residues affected the overall structure of the C-terminal domain of the gamma chain.

Fibrinogen Bastia (gamma 318 Asp-->Tyr) a novel abnormal fibrinogen characterized by defective fibrin polymerization.

A new congenital dysfibrinogen, Fibrinogen Bastia, was discovered in a 20-year-old woman with no clinical symptoms. The plasma thrombin-clotting time was severely prolonged. The functional plasma fibrinogen concentration was low (0.2 mg/ml), whereas the immunological concentration was normal (2.9 mg/ml). Purified fibrinogen Bastia displayed a markedly prolonged thrombin-clotting time related to a delayed thrombin-induced fibrin polymerization. Both the thrombin-clotting time and the fibrin polymerization were partially corrected by the addition of calcium ions. The anomaly of fibrinogen Bastia was found to be located in the gamma-chain since by SDS-PAGE performed according to the method of Laemmli two gamma-chains were detected, one normal and one with an apparently lower molecular weight. Furthermore, analysis of plasmin degradation products demonstrated that calcium ions only partially protect fibrinogen Bastia gamma-chain against plasmin digestion, suggesting that the anomaly is located in the C-terminal part of the gamma-chain. Sequence analysis of PCR-amplified genomic DNA fragments of the propositus demonstrated a single base substitution (G-->T) in the exon VIII of the gamma chain gene, resulting in the amino acid substitution 318 Asp (GAC)-->Tyr (TAC). The PCR clones were recloned and 50% of them contained the mutation, indicating that the patient was heterozygous. These data indicate that residue Asp 318 is important for normal fibrin polymerization and the protective effect of calcium ions against plasmin degradation of the C-terminal part of the gamma-chain.

Defective cell migration in an ovarian cancer cell line is associated with impaired urokinase-induced tyrosine phosphorylation.

The urokinase receptor (u-PAR), a protein anchored to cell membrane by a glycosyl phosphatidylinositol, plays a central role in cancer cell invasion and metastasis by binding urokinase plasminogen activator (u-PA), thereby facilitating plasminogen activation. Plasmin can promote cell migration either directly or by activating metalloproteinases that degrade some of the components of the extra cellular matrix. However, the IGR-OV1-Adria cell line contains the u-PAR but does not migrate even in the presence of exogenous u-PA, although the parental IGR-OV1 cell line migrates normally in the presence of u-PA. We therefore investigated the role of cell signalling for u-PA induced cell locomotion. We show that cell migration induced by u-PA-u-PAR complex is always associated with tyrosine kinase activation for the following reasons: (1) the blockade of the u-PAR by a chimeric molecule (albumin-ATF) inhibits not only the u-PA-induced cell migration, but also the signalling in IGR-OV1 line; (2) the binding of u-PA to u-PAR on non-migrating IGR-OV1-Adria cells was not associated with tyrosine kinase activation; (3) the inhibition of tyrosine kinase also blocked cell migration of IGR-OV1. Therefore tyrosine kinase activation seems to be essential for the u-PA-induced cell locomotion possibly by the formation of a complex u-PAR-u-PA with a protein whose transmembrane domain can ensure cell signalling. Thus, IGR-OV1 and IGR-OV1-Adria cell lines represent a good model for the analysis of the mechanism of u-PA-u-PAR-induced cell locomotion.