Abnormal expression of C3ORF9 gene in patients with myelodysplastic syndromes.

Myelodysplastic Syndrome (MDS) cells present genetic instability and dysregulation of the gene C3ORF9, which was isolated from an MDS cDNA library and codes for a putative protein. We studied the expression of C3ORF9 in MDS syndromes to contribute to the understanding of the pathophysiology of MDS. One hundred and thirty-one patients and 35 healthy controls were involved in our study. Bone marrow aspirates and isolated CD34+ cells were used. Gene expression was estimated by quantitative PCR. C3ORF9 was found to be down-regulated in patients with CMML compared to the controls (p<0.01). There was no difference between RARS and the controls (p=0.1), while increased expression was found in RA, RAEB and RAEB-T (p<0.01 for all). No mutations or polymorphism were detected in our population. CD34+ cells expressed higher levels of C3ORF9 (p<0.01) in patients. The gene expression was correlated to the percentage of +cells in RAEB and RAEB-T (r=0.64). The altered C3ORF9 expression was possibly due to different gene regulation in these patients and/or to the increased CD34+ cells.

Plasma fibronectin in normal subjects and in various disease states.

Plasma fibronectin was determined in 121 normal adults and in 149 patients. Fibronectin levels in normals were strongly influenced by sex and age. The mean value of the protein in cancer patients did not differ from that in normal controls; however, patients with cryofibrinogenaemia or extensive liver metastases had lower values whereas those with obstructive jaundice due to pancreatic carcinoma had higher values than normal controls. Fibronectin levels were greatly increased in patients with primary biliary cirrhosis and moderately elevated in nephrotic syndrome. In patients with severe infection or sepsis, plasma fibronectin did not show a consistent pattern. Patients with overt disseminated intravascular coagulation, irrespective of its cause, had the lowest plasma fibronectin concentrations.

Characterization of cryofibrinogen isolated from patients plasma.

The cryofibrinogen of 27 patients was studied by SDS-polyacrylamide gel electrophoresis and immunochemical methods. Electrophoretic analysis of the isolated cryofibrinogens, as well as the proteins left after heat or thrombin defibrination, showed that cryofibrinogen is composed of two proteins, fibrin(ogen) and cold-insoluble globulin (CIg). A proportion of the fibrin(ogen) component formed stabilized oligomers interlinked through gamma-gamma dimerization. The degree of fibrin(ogen) proteolysis, as judged by measuring the alpha:gamma ratio of the reduced samples, was very similar to that of the fibrinogen of the original plasma. The CIg:fibrin(ogen) molar ratio in the cryofibrinogens was 0.04 +/- 0.018. The CIg and the fibrin(ogen) content of the cryofibrinogens were strongly correlated with the plasma CIg levels.

Soluble fibrin complexes and fibrinogen heterogeneity in diabetes mellitus.

The presence of soluble fibrin complexes (SFC) measured by gel filtration of plasma on 4% agarose columns, fibrinogen heterogeneity on 3.5% SDS-polyacrylamide gels and the concentrations of several plasma proteins were evaluated in 39 patients with diabetes mellitus (DM) and 19 matched control subjects. A small but significant increase of SFC was found in DM (p less than 0.01). On individual basis 51.2% of the patients had increased SFC (greater than M + 2 SD of the controls). Polyacrylamide gel electrophoresis of the SFC showed no evidence of cross-linking or proteolysis. Plasma clots formed in the presence of EDTA and trasylol were analysed in SDS-polyacrylamide gels in a normal and two lower molecular weight fibrin bands (band I, II, III). The percentage of band I fibrinogen was in diabetics (65.3 +/- 4.7%) lower than that of the controls (71.8 +/- 4.5%) (p less than 0.01). Fibrinogen levels, antithrombin III, alpha 1-antitrypsin, alpha 2-macroglobulin and plasminogen were significantly increased in DM. We suggest that in DM there is an enhancement of intravascular fibrin formation and accelerated fibrinogen degradation to lower molecular weight forms.

Interactions among heparin, cold-insoluble globulin, and fibrinogen in formation of the heparin-precipitable fraction of plasma.

Fibrinogen and the cold-insoluble globulin of plasma (CIg) are the main protein components of the heparin-precipitable fraction of normal plasma. The interactions among these proteins and heparin were examined. Heparin formed a cold-precipitable complex with purified CIg or with mixtures of CIg and fibrinogen but not with purified fibrinogen alone. Cryoprecipitation was augmented by addition of Ca(++) or by selection of optimal heparin levels; it was reduced or even abolished by raising the ionic strength or pH or both, or by raising the heparin concentration above that for maximum precipitation of CIg. Fibrinogen reduced the threshold for heparin-induced CIg cryoprecipitation and, by coprecipitating with heparin and CIg, increased the amount of precipitate that formed. In contrast to the heparin-precipitable fraction of normal plasma which contained both fibrinogen and CIg, that from a patient with congenital afibrinogenemia contained CIg but lacked fibrinogen. Normal plasma depleted of CIg by immunoabsorption failed to form a heparin-induced cryoprecipitate. Thus, CIg is essential for heparin-induced cryoprecipitation to occur. Fibrinogen, as assessed by chromatographic experiments with heparin-Sepharose columns, had a considerably lower binding affinity for heparin than did CIg, suggesting that it participates in precipitate formation mainly, if not entirely, by virtue of its affinity for CIg. The region of the fibrinogen molecule accounting for its precipitation with CIg appears to be localized in the carboxy-terminal segment of the Aalpha-chain; fibrinogen subfractions lacking this region failed to augment cryoprecipitation of heparin-CIg mixtures and, even though such species were present in normal plasma, they failed to coprecipitate in the heparin-induced complex.

Platelet function, blood coagulation and fibrinolysis in Behcet's syndrome.

In 10 patients with Behcet's syndrome, various parameters of platelet function, blood coagulation and fibrinolysis were studied. With varying frequency the following abnormalities were found: increased retention of platelets in glass bead column, reduced platelet aggregation to low concentrations of adenosine diphosphate, elevated plasma levels of fibrinogen concentration and factor VIII activity, increased plasma antiheparin activity and impairment of fibrinolytic activity. The above abnormalities were found long after the last thrombotic episode and were more frequent in patients with a history of thrombophlebitis. It is suggested that certain hemostatic abnormalities accompany or form part of Behcet's syndrome and that they are related to the thrombotic complications characteristic of this syndrome.

Familial thrombosis due to antithrombin III deficiency in a Greek family.

A Greek family with hereditary antithrombin III (AT III) deficiency associated with venous thrombosis is reported. 5 members of the family were affected. In these patients, AT III and heparin cofactor activities were decreased. Immunoreactive AT III showed a positive correlation to both AT III and heparin cofactor activities. alpha2-Macroglobulin and alpha1-antitrypsin were normal. The pattern of inheritance of the defect is autosomal dominant.

Refractory anemia with hyperplastic bone marrow: subclassification based on responsiveness to erythropoietin in vitro.

The responsivenes to erythropoietin of cultured bone marrow cells, obtained from 7 patients with refractory anemia with hyperplastic marrow, was studied. 5 of these patients' marrows also exhibited sideroblasti changes. Heme synthesis in cultured bone marow cells was either responsive to stimulation by erythropoietin, or completely refractory. The sensitivity of the bone marrow cells to the hormone was not related to either the clinical or laboratory findings.

Postoperative platelet function in patients on small subcutaneous doses of heparin.

Several platelet function tests were performed on 31 patients undergoing major operations, who recieved prophylactically small subcutaneous doses of heparin. A group of 15 similar patients without heparin served as controls. It was found that postoperatively in both groups (a) the platelet retention in glass bead column was significantly increased (p less than 0.001), (b) the platelet aggregation by ADP 1 micronM was slightly increased, (c) the collagen-induced aggregation, ADP release and the bleeding time remained unchanged and (d) the platelet counts decreased in the first 2 postoperative days and increased thereafter. There was no difference between patients on heparin and controls.

Hypercoagulability and hypofibrinolysis in sickle-cell disease.

Fifty-two patients with sickle-cell (SC) disease (48 with SC-beta-thalassaemia and 4 with homozygous SC-anaemia) were studied as regards blood coagulation and fibrinolysis. It was found that the thrombin and the reptilase times of the patients' plasma were significantly shorter than normal. The mean values of platelet count, fibrinogen level and factor VIII activity of patients with SC disease were higher than normal; however, in the group of patients transfused, with less than 50% haemoglobin S (HbS), the fibrinogen level and the factor VIII activity were significantly lower compared to the other patients. Antithrombin-III (At-III) activity was normal in all. The fibrinolytic activity was normal in patients with asymptomatic SC disease, but reduced in patients on painful crises. Plasminogen and fibrinogen/fibrin degradation product (FDP) levels were normal in all patients. Two patients on painful crises with complications had additional abnormal findings, namely prolonged prothrombin time, reduced At-III level and elevated FDP.