A gamma methionine-310 to threonine substitution and consequent N-glycosylation at gamma asparagine-308 identified in a congenital dysfibrinogenemia associated with posttraumatic bleeding, fibrinogen Asahi.

In an abnormal fibrinogen with severely impaired polymerization of fibrin monomers, we identified a methionine-to-threonine substitution at position 310 of the gamma chain. Furthermore, asparagine at position 308 was found to be N-glycosylated due to a newly formed consensus sequence, asparagine(308)-glycine(309)-threonine(310). The two structural defects in the mutant gamma chain may well perturb the conformation required for fibrin monomer polymerization that is specifically assigned to the D domain of fibrinogen. This alteration also seems to affect the intermolecular gamma chain cross-linking of fibrin and fibrinogen, although the amine acceptor gamma glutamine-398 was found to function normally. These functional abnormalities may well be related to posttraumatic hemorrhage as observed in a 33-yr-old man with moderate hemorrhagic diathesis related to injuries since his early adolescence. The structure of the extra carbohydrate moiety attached to asparagine-308 was found to be identical with those derived from the normal B beta and gamma chains as evidenced by HPLC.

Fibrinogen Lima: a homozygous dysfibrinogen with an A alpha-arginine-141 to serine substitution associated with extra N-glycosylation at A alpha-asparagine-139. Impaired fibrin gel formation but normal fibrin-facilitated plasminogen activation catalyzed by tissue-type plasminogen activator.

An A alpha-arginine-141 to serine substitution has been identified in a homozygous dysfibrinogen, fibrinogen Lima, associated with impaired fibrin polymerization. The point mutation created an asparagine-X-serine-type glycosylation sequence, and indeed, extra, mainly disialylated biantennary oligosaccharides have been isolated from A alpha asparagine-139 of the patient's fibrinogen. This type of glycosylation sequence is unique for human fibrinogen, because the sequences shown for normal and abnormal fibrinogens are all asparagine-X-threonine types. The terminal sialic acids of the extra oligosaccharides seem to have largely contributed to the impaired fibrin gel formation, as evidenced by its correction to a near normal level by desialylation. Nevertheless, the polymerizing fibrin facilitated tissue-type plasminogen activator-catalyzed plasmin formation in a normal fashion, indicating that the initial two-stranded fibrin protofibrils had been constructed normally. Thus the impaired fibrin gel formation could be attributed to the delay in their subsequent lateral association, most probably because of the repulsive forces generated by the negative electric charge of the extra sialic acids. The substitution of a basic residue arginine to a noncharged residue serine may also have contributed to the impaired function in a similar manner or by steric hindrance in association with bulky extra oligosaccharide chains.

Expression of p-STAT3 in human colorectal adenocarcinoma and adenoma; correlation with clinicopathological factors.

BACKGROUND: The signal transducer and activator of transcription 3 (STAT3) is a key signalling molecule implicated in the regulation of growth and malignant transformation. Constitutive activation of STAT3 is seen in several tumour derived cell lines, and in a wide variety of human malignancies. AIMS: To examine the relation between p-STAT3 (activated form of STAT3) expression and clinicopathological factors in human colorectal adenocarcinoma and adenoma. METHODS: Immunohistochemical analyses were carried out on tissues from 44 colorectal adenomas and 95 colorectal adenocarcinomas, comprising 18 intramucosal carcinomas and 77 invasive carcinomas. RESULTS: Seventy seven of these 139 samples (55.4%) showed immunoreactivity for p-STAT3. Positive staining for p-STAT3 was seen in 69 of the 95 carcinomas. Only eight of the 44 adenomas showed immunopositivity for p-STAT3, resulting in a significant difference between total adenocarcinomas and adenomas (p < 0.001). Among the 95 cases of colorectal adenocarcinoma, p-STAT3 immunoreactivity was significantly correlated with the depth of tumour invasion (p < 0.05), venous invasion (p < 0.05), lymph node metastasis (p < 0.05), and increasing stages of the Dukes' classification (p < 0.01). Expression of p-STAT3 was detected by Western blot analysis in two different cultured human colorectal carcinoma cell lines and six colon carcinoma tissue samples obtained at surgery. CONCLUSION: This is the first study to report a significant correlation of p-STAT3 expression with the depth of tumour invasion. These findings suggest that p-STAT3 expression is an important factor related to carcinogenesis and/or tumour invasion of colorectal adenocarcinoma.

Normal plasmic cleavage of the gamma-chain variant of "fibrinogen Saga" with an Arg-275 to His substitution.

We have identified a gamma-Arg-275 to His substitution in an abnormal fibrinogen designated as "fibrinogen Saga" characterized by impaired fibrin monomer polymerization. By chromatofocusing chromatography, we isolated normal and abnormal fragment D1 populations separately from the plasmic-calcium digests of fibrinogen derived from the propositus, a heterozygote for the abnormality. We found that both normal and abnormal fragment D1's were similarly protected from digestion by plasmin in the presence of calcium ions and further degraded to fragments D2 and D3 due to cleavage of the gamma-chain remnant when calcium ions were replaced by chelating agents. Abnormal fragment D1 failed to inhibit both thrombin-clotting of normal fibrinogen and polymerization of normal fibrin monomer, while normal D1 exhibited marked inhibitory activities. In an aberrant peptide comprising residues gamma-274-302 isolated by HPLC from the lysyl endopeptidase-digests of abnormal fragment D1, we identified a His substituting for an Arg at position 2, which corresponds to position 275 of the mutant gamma-chain.

Structure and function of fibrinogen: insights from dysfibrinogens.

The structure-function relationships of dysfibrinogens and their clinical implications are discussed on the basis of the data provided from representative molecules.

An activated state of blood coagulation and fibrinolysis in patients with abdominal aortic aneurysm.

BACKGROUND: Relationships between blood coagulation and the fibrinolysis system and morphology of aneurysms in patients with abdominal aortic aneurysm (AAA) are unknown. METHODS: Preoperative and postoperative evaluations of hemostatic factors such as thrombin-antithrombin III complex (TAT), D-dimer, fibrinogen/fibrin degradation products (FDP), and platelet count were performed in 36 patients with atherosclerotic AAA. As control subjects, 25 age- and sex-matched healthy volunteers were analyzed for these hemostatic factors. In all patients, morphological evaluation of AAA included the largest diameter, tortuosity, and the thickness of intraluminal thrombus to be compared with preoperative levels of hemostatic factors such as alpha-2 plasmin inhibitor-plasmin complex (PIC), thrombomodulin (TM), von Willebrand factor (vWF), tissue factor (TF), and free form of tissue factor pathway inhibitor (F-TFPI). RESULTS: The preoperative values of TAT, D-dimer, and FDP were significantly higher in AAA patients than in controls. Of all patients, 23 (64%) or 22 (58%) had TAT or D-dimer values greater than 8.2 ng/mL or 3.4 microg/mL (mean + 2SD of controls), respectively. The postoperative values of these hemostatic factors significantly improved, but were not normalized. The largest diameter of AAA correlated with the preoperative levels of TAT (r = 0.566, P = 0.001), D-dimer (r = 0.644, P = 0.0001), FDP (r = 0.561, P = 0.0009), PIC (r = 0.413, P = 0.0146), and F-TFPI (r = 0.408, P = 0.0158). We have also found that tortuosity of AAA has relation not only to the preoperative levels of fibrinolytic factors but also to the plasma F-TFPI antigen levels. On the other hand, the preoperative levels of a marker of endothelial damage, such as TM or vWF, and TF did not correlate with those of F-TFPI in all patients. The maximum thickness of thrombus in AAA significantly correlated not only with the preoperative levels of TAT, D-dimer, FDP, and PIC, but also with AAA size. CONCLUSIONS: We have found evidence that an activated state of both blood coagulation and fibrinolysis in AAA patients is associated with the morphological characteristics of aneurysms.

Delayed intermolecular gamma-chain cross-linking by factor XIIIa in fibrinogen Asahi characterized by a gamma-Met-310 to Thr substitution with an N-glycosylated gamma-Asn-308.

In an abnormal fibrinogen with gamma-Met-310 to Thr substitution accompanied by an extra oligosaccharide attached to gamma-Asn-308, factor-XIIIa-mediated intermolecular gamma-dimer formation of fibrin was found to be markedly delayed. The delayed gamma-dimer formation was not due to impaired fibrin polymerization because the fibrinogen gamma-chain also failed to be efficiently cross-linked by factor XIIIa. Since fluorescent amine was normally incorporated into the abnormal gamma-chain by factor XIIIa, we conclude that the abnormal molecules were unable to align their gamma-chains in an anti-parallel fashion because of inaccessibility of the molecules with a profoundly perturbed conformation near the carboxyl terminal region of the gamma-chain included in the D domain.

[A case of hepatic arterial infusion chemotherapy for multiple liver metastasis from breast cancer].

A 52-year-old woman underwent Auchinclass' operation for breast cancer. The histological type was papillotubular carcinoma. One year and 5 months after operation, multiple liver tumors were found on the CT scan and multiple bone metastasis on MRI. The former were treated by hepatic artery infusion chemotherapy with epirubicin and 5-FU using a subcutaneous implanted pump and the latter by 50 Gy irradiation. The patient began to complain of abdominal pain and discomfort after hepatic artery infusion, so all treatment was discontinued. Six months later the patient died of respiratory failure due to pleural dissemination. No liver mass was detected, and bone metastasis was not changed in section tissues. This suggested that the therapy for a breast cancer patient with distant metastasis must be considered according to the region of recurrence.

[Changes in the liver weight in the elderly].

To elucidate the effect of aging on liver weight in the elderly, 582 elderly cases (male 291, female 291), were selected from 2000 elderly autopsied cases on the basis of being free from pathological findings affecting liver weight. Both liver weight and its ratio to body weight decreased with age, and influenced by obesity; the former increased and the latter decreased in obese cases. Analysis according to the degree of obesity also showed decrease of liver weight and is ratio to body weight with age. Comparison between males and females of the same decade revealed the tendency that in females the liver weight was low, while liver weight.body weight ratio was high. The same results were obtained by analysis based on the degree of obesity.

Photoaffinity labeling of the primary fibrin polymerization site: localization of the label to gamma-chain Tyr-363.

Fragment D prepared from human fibrinogen was labeled specifically by photoactivation of the peptide [14C]Gly-Pro-Arg-N-(4-azido-2-nitrophenyl)Lys amide. The preparation was freed of excess labeling reagents and then reduced and alkylated. The component alpha, beta, and gamma chains were purified by chromatography on carboxymethylcellulose and the radioactivity was found to be restricted to the gamma chain. Isolated gamma chains were digested with various endopeptidases, both alone and in tandem, and the products were fractionated by gradient HPLC. The amino acid compositions of all labeled peptides led to the conclusion that the modification occurs exclusively on gamma-chain Tyr-363.

Fibrinogen Osaka IV: a congenital dysfibrinogenemia found in a patient originally reported in relation to surgery, now defined to have an A alpha arginine-16 to histidine substitution.

We discovered a congenital heterozygous dysfibrinogen in a patient and reported this case in relation to surgery some time ago (Jpn J Surg (1988) 18:43-46). Further studies on the isolated abnormal population of fibrinogen derived from this patient have revealed that fibrinopeptide A was not cleaved by ancrod, a snake venom-derived thrombin-like enzyme, but by thrombin, slowly but completely. The released fibrinopeptide A components, being the A, AY, and AP peptides, were all found to be abnormal, as evidenced by slightly earlier elution positions on high-performance liquid chromatography, compared with the normal counterparts. By analyzing their amino acid sequence, we have identified an arginine to histidine substitution at position 16 of the A alpha chain, the thrombin cleavage site. Utilizing insolubilized abnormal fibrinogen, we confirmed that the polymerization site assigned to the central E domain, the "A" site, was exposed by thrombin, but not by ancrod. This dysfibrinogen, designated as fibrinogen Osaka IV, is the second abnormal molecule with an A alpha arginine-16 to histidine substitution identified among Japanese families.

Fibrinogen Kyoto II, a new congenitally abnormal molecule, characterized by the replacement of A alpha proline-18 by leucine.

A new case of heterozygous dysfibrinogenemia characterized by an amino acid replacement in the NH2-terminal region of the fibrin alpha-chain was found in a 27-year-old woman with a bleeding problem. Her one-stage prothrombin time and activated partial thromboplastin time were slightly prolonged, and the purified fibrinogen from this patient had a markedly prolonged thrombin or reptilase time. Release of fibrinopeptides A and B was normal, but the polymerization of fibrin monomers was impaired. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified fibrinogen under the reduced condition showed no abnormalities in the apparent molecular weights of its three chains. Reverse-phase high performance liquid chromatography (HPLC) of the lysylendopeptidase-cleaved purified A alpha-chains showed a decrease in one peptide compared with the normal amount and the appearance of an abnormal peptide peak. These peptides were treated with thrombin and further separated on HPLC. Amino acid sequence analysis of the abnormal peptide indicated that A alpha proline-18, the second residue from the NH2-terminus of the fibrin alpha-chain, was replaced by leucine. The synthetic peptide Gly-Pro-Arg-Pro inhibited both thrombin- and reptilase-induced fibrin aggregation, but Gly-Leu-Arg-Pro showed little or no inhibition under the same conditions. The discovery of this abnormal fibrinogen supports the findings that A alpha proline-18 is important as part of the polymerization site in the NH2-terminus of the fibrin alpha-chain. The propositus' mother had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Kyoto II.

Retroperitoneal foregut cyst.

A foregut cyst is formed as a result of abnormal budding and pinching of the tracheobronchial tree when bronchial buds develop to form the primitive respiratory tree. Foregut cysts are clinically classified as bronchogenic, esophageal, enterogastric, or ciliated hepatic. We present a foregut cyst that occurred in the retroperitoneum and was difficult to distinguish from other retroperitoneal cystic mass lesions. Magnetic resonance imaging was useful in revealing the cyst's continuity to adjacent organs.

Fibrinogen Kyoto III: a congenital dysfibrinogen with a gamma aspartic acid-330 to tyrosine substitution manifesting impaired fibrin monomer polymerization.

A congenital dysfibrinogen characterized by impaired fibrin monomer polymerization was found in an asymptomatic 50-year-old woman and her two sons. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) run according to the method of Laemmli, we noticed two gamma chain species in fibrinogen and its plasmic fragments D1 and D2, consisting of a normal species and an apparently lower molecular weight (mol wt) variant in respective fractions. However, in fragment D3 only a single gamma chain remnant was observed. By chromatofocusing of the plasmic-CaCl2 digests of the abnormal fibrinogen, we separately isolated the normal and abnormal D1 species, the latter having been eluted in a slightly higher pH range. As expected, the abnormal D1 species failed to interfere with thrombin clotting of normal fibrinogen and normal fibrin monomer polymerization, whereas the normal D1 species inhibited them markedly. By analyzing the lysyl endopeptidase digests of the isolated gamma chain, we identified a replacement of aspartic acid by tyrosine at position 330 of the mutant gamma chain. The point mutation from an aspartic acid (pK for the beta-carboxyl = 3.86) to a tyrosine (pK for the aromatic hydroxyl = 10.07) may have perturbed the folding gamma chain structure in the D domain of fibrinogen specifically required for polymerization.