Dysfibrinogen Kagoshima with the amino acid substitution gammaThr-314 to Ile: analyses of molecular abnormalities and thrombophilic nature of this abnormal molecule.

INTRODUCTION: Emerging lines of evidence have suggested that certain dysfibrinogens present a significant risk of thrombosis. PATIENT/METHODS: The thrombophilic nature of a new-type of dysfibrinogen Kagoshima identified in a 36-year-old female with deep vein thrombosis during the postpartum period was studied. RESULTS/DISCUSSION: Based on the analyses of the patient fibrinogen and the fibrinogen genes, fibrinogen Kagoshima was shown to have the amino acid substitution of gammaThr-314 to Ile that resulted in impaired function and hypofibrinogenemia. Polymerization of fibrin monomers derived from patient fibrinogen was severely impaired with a partial correction in the presence of calcium ions, causing very low clottability and delayed cross-linking of patient fibrin catalyzed by activated factor XIII. Because of the low clottability, a large amount of soluble fibrin was formed upon thrombin treatment, resulting in an increase of thrombin in the soluble fraction. Additionally, tPA-mediated plasmin generation on fibrin was impaired and calcium-ion-dependent integrity of the gamma-chain D domain of Kagoshima fibrinogen was perturbed. The presence of many tapered-fiber ends inside the tangled fibrin networks, observed by scanning electron microscopy, suggested early termination of fibrin polymerization and the structural alteration. CONCLUSION: These data suggest that fibrinogen Kagoshima is dysfunctional, giving rise to formation of fibrinolysis-resistant soluble fibrin polymers and entrance of soluble fibrin associating with thrombin to the circulation, partly accounting for the thrombophilic nature of the affected fibrinogen and fibrin molecules.

Protection of plasminogen activator inhibitor-1-deficient mice from nasal allergy.

This study was performed to clarify the relationship between fibrinolytic components and the pathology of allergy, particularly that during the development of nasal allergy and nasal tissue changes. Intranasal OVA challenge after sensitization by i.p. administration of OVA induced a higher level of excess subepithelial collagen deposition in wild-type (WT) C57BL/6J mice than in plasminogen activator inhibitor (PAI)-1-deficient (PAI-1(-/-)) mice. The excess PAI-1 induction in the nasal mucosa and higher level of active PAI-1 in the nasal lavage fluid of WT-OVA mice compared with those in WT-control mice suggested that the decrease of proteolytic activity inhibits the removal of subepithelial collagen. The frequency of sneezing, nasal rubbing, nasal hyperresponsiveness, production of specific IgG1 and IgE in the serum, and production of IL-4 and IL-5 in splenocyte culture supernatant increased significantly in WT-OVA mice. In PAI-1(-/-) mice, these reactions were absent, and specific IgG2a in serum and IFN-gamma in splenocyte culture medium increased significantly. Histopathologically, there were marked goblet cell hyperplasia and eosinophil infiltration into the nasal mucosa in WT-OVA mice, but these were absent in PAI-1(-/-) mice. These results indicate that the immune response in WT-OVA mice can be classified as a dominant Th2 response, which would promote collagen deposition. In contrast, the Th2 response in PAI-1(-/-) mice was down-regulated, and the immune response shifted from Th2-dominant reaction to a Th1-dominant one. Taken together, these findings suggest that PAI-1 plays an important role not only in thrombolysis but also in immune response.

Expression profiles of fibrinolytic components in nasal mucosa.

Components of the fibrinolytic pathway contribute to diverse pathways in many tissues, in addition to their well-recognized role in degradation of fibrin clots. In this study of nasal mucosa, we investigated the presence of mRNA of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), plasminogen activator inhibitor-1 (PAI-1), and plasminogen activator inhibitor-2 (PAI-2) using reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization, and compared these results with their localization in immunostained tissues. According to real-time RT-PCR results, t-PA, u-PA, PAI-1, and PAI-2 mRNA were noted in human nasal mucosa. Particularly, expression of u-PA and PAI-1 mRNA was significantly high in allergic nasal mucosa in comparison with normal mucosa. t-PA mRNA was detected in endothelial cells and epithelium in normal nasal mucosa. t-PA mRNA was detected in mucous cells of allergic submucosal glands, but not in normal glands. In allergic rhinitis, u-PA and PAI-2 mRNA were detected in mucinous cells and epithelium, and PAI-1 mRNA was detected in serous cells and epithelium. Expression of u-PA and PAI-1 mRNA in normal nasal tissues was decreased in contrast to that in allergic nasal tissues. u-PA staining was observed in mucous cells of allergic submucosal glands and the staining pattern of PAI-2 was similar to that of u-PA. PAI-1 was present in serous cells of submucosal glands from allergy samples, while epithelial cells were almost devoid of stain. In contrast, with allergy, immunohistochemical staining of t-PA was negative in submucosal glands, though positive in endothelial cells and epithelium. However, the expression of t-PA mRNA in allergic nasal mucosa was noted in mucous cells. In fibrin autography of nasal discharge, u-PA was markedly activated in the allergic patient. These results suggest that t-PA synthesized in mucous cells is promptly secreted and modifies watery nasal discharge in allergic rhinitis, and that u-PA activity may help the passage of large amounts of rhinorrhea by also reducing its viscosity. A lot of cellular infiltration (eosinophils in particular) was recognized in allergic nasal mucosa. It is most likely that the modifications in expression of PAs and PAIs are due to the local release of cytokines or growth factors from these inflammatory and immune cells.

Thrombophilic dysfibrinogen Tokyo V with the amino acid substitution of gammaAla327Thr: formation of fragile but fibrinolysis-resistant fibrin clots and its relevance to arterial thromboembolism.

Thrombophilic dysfibrinogen Tokyo V was identified in a 43-year-old man with recurrent thromboembolism. Based on analyses of the patient fibrinogen genes, the amino acid sequence of the aberrant fibrinogen peptide, and deglycosylation experiments, fibrinogen Tokyo V was shown to have an amino acid substitution of gamma Ala327Thr and possibly extra glycosylation at gamma Asn325 because the mutation confers the N-linked glycosylation consensus sequence Asn-X-Thr. The mutation resulted in impaired function and hypofibrinogenemia (hypodysfibrinogen). Polymerization of fibrin monomers derived from patient fibrinogen was severely impaired with a partial correction in the presence of calcium, resulting in very low clottability. Additionally, a large amount of soluble cross-linked fibrin was formed upon thrombin treatment in the presence of factor XIII and calcium. However, Tokyo V-derived fibrin was resistant to degradation by tissue plasminogen activator (tPA)-catalyzed plasmin digestion. The structure of Tokyo V fibrin appeared severely perturbed, since there are large pores inside the tangled fibrin networks and fiber ends at the boundaries. Taken together, these data suggest that Tokyo V fibrin clots are fragile, so that fibrinolysis-resistant insoluble fibrin and soluble fibrin polymers may be released to the circulation, partly accounting for the recurrent embolic episodes in the patient.

Defective sorting to secretory vesicles in trans-Golgi network is partly responsible for protein C deficiency: molecular mechanisms of impaired secretion of abnormal protein C R169W, R352W, and G376D.

Three thrombophilic patients with protein C (PC) deficiency were found to have independent mutations in the PC gene. These mutations resulted in single amino acid substitutions of R169W, R352W, and G376D in the affected PC molecules. These abnormal PC molecules were expressed in CHO-K1 cells in the presence or absence of vitamin K, and their synthesis, posttranslational modification, and secretion were studied. PC G376D was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of PC R169W and PC R352W, but most of these molecules were not secreted but were degraded intracellularly. On the basis of pulse-chase, immunofluorescence, and endo-beta-N-acetylglucosaminidase H digestion experiments, the majority of wild-type PC molecules localize not in the Golgi apparatus but in the rough endoplasmic reticulum inside the cells. This suggests that wild-type PC molecules are secreted immediately after gamma-carboxylation and modification at the Golgi apparatus. In contrast, the mutant PC molecules were retained inside the cells even after modification of oligosaccharides at the trans-Golgi apparatus, which was probably due to impaired conformation of the abnormal molecules. Data suggest that these abnormal PC molecules were not sorted to secretory vesicles in the trans-Golgi network because of conformational defects in addition to the transport defect from the rough endoplasmic reticulum to the Golgi apparatus and were degraded inside the cells, thereby resulting in a PC deficiency in the affected patients.

Structure and function of human fibrinogen inferred from dysfibrinogens.

Fibrinogen is a 340-kDa plasma protein that is composed of two identical molecular halves, each consisting of three non-identical subunit polypeptides designated as A alpha, B beta- and gamma-chains held together by multiple disulfide bonds. Fibrinogen has a trinodular structure, i.e., one central E domain comprizing the amino-terminal regions of paired individual three polypeptides, and two identical outer D domains. These three nodules are linked by two coiled-coil regions [1,2]. After activation with thrombin, a tripeptide segment consisting of Gly-Pro-Arg is exposed at the amino-terminus of each alpha-chain residing at the center of the E domain and combines with its complementary binding site, called the 'a' site, residing in the carboxyl-terminal region of the gamma-chain in the outer D domain of another molecule. By crystallographic analysis [3], the alpha-amino group of alpha Gly-1 is shown to be juxtaposed between the carboxyl group of gamma Asp-364 and the carboxyamide of Gln-329 in the 'a' site. Half molecule-staggered, double-stranded fibrin protofibrils are thus formed [4,5]. Upon abutment of two adjacent D domains on the same strand, D-D self association takes place involving Arg-275, Tyr-280 and Ser-300 of the gamma-chain on the surface of the abutting two D domains [3]. Thereafter, carboxyl-terminal regions of the fibrin a-chains are thought to be untethered and interact with those of other protofibrils leading to the formation of thick fibrin bundles and interwoven networks after appropriate branching [6-9]. Although many enigmas still remain regarding the mechanisms of these molecular interactions, fibrin assembly proceeds in a highly ordered fashion. In my talk, I would like to discuss these molecular interactions of fibrinogen and fibrin based on the up-date data provided by analyses of normal as well as hereditary dysfibrinogens, particularly in the latter by introducing representative molecules at each step of fibrin clot formation.

Structural alterations in hereditary dysfibrinogens.

Dysfibrinogens can be grossly divided in two groups: (1) defective thrombin-catalyzed conversion of fibrinogen molecules to fibrin monomers, and (2) defective fibrin polymerization due to structural alterations in polymerization sites, that include "A" and "a" sites, end-to-end D:D abutment surfaces, and lateral association sites involving the carboxyl terminal region of the fibrin alpha-chain. Recently, a number of mutations in the fibrinogen genes have been identified, and many of these encode changes that occur in regions of fibrinogen that have been elucidated by high-resolution structural studies. Here we focus on the structure-function relationships of fibrinogen that can be inferred from studies involving these abnormal molecules.

Substitution of Gly-548 to Ala in the substrate binding pocket of prothrombin Perijá leads to the loss of thrombin proteolytic activity.

Prothrombin Perijá is a dysprothrombin derived from a homozygous patient that manifests low thrombin activity upon activation in a one-stage assay. Purified prothrombin Perijá showed normal appearance on SDS-PAGE. and was cleaved normally to form alpha-thrombin by the prothrombinase complex. The activated form, thrombin Perijá, however, did not show any proteolytic activity towards native substrates, fibrinogen, protein C or various synthetic substrates for alpha-thrombin, but it was able to bind to antithrombin III, although the binding capacity was markedly reduced even in the presence of heparin. Thrombin Perijá showed full reactivity toward a small inhibitor, DFP, indicating that the molecular defect is in the substrate binding site in the thrombin molecule but not in the active site itself. By DNA sequence analysis of the patient prothrombin gene, we identified a G to C mutation at nucleotide 20016 in exon 14, which predicts a Gly-548 to Ala substitution in the prothrombin Perijá molecule. The structural modeling of thrombin Perijá suggests that Ala-548 is located close to the limb of the cavity wall of the substrate binding pocket, and that the methyl group blocks protrusion of the guanidino group of Arg into the cavity. This steric hindrance may well inhibit the access of Arg-containing substrates to the catalytic Ser-525 leading to the loss of proteolytic activity.