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Biomedical subjects

A Girolami

Publications and source records attributed to A Girolami.

At least 199 records · Page 11Linked to original sources
PubMed1996

Characterization of recombinant human coagulation factor XFriuli.

Naturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational gamma-carboxy-glutamic acid (Gla) and beta-hydroxy aspartic acid (beta-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel beta-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

1-Carboxyglutamic Acid↗
PubMed1996

Deep venous thrombosis and lupus anticoagulant. A case-control study.

BACKGROUND: A definite evidence in favour of an association of deep-vein thrombosis (DVT) with lupus anticoagulant (LA) in patients free from systemic lupus erythematosus is still lacking. METHODS: In a case-control study, LA was determined in 176 consecutive outpatients who underwent phlebography because of the first episode of clinically suspected DVT of lower limbs. The association between DVT and LA was described using odds ratios (OR). RESULTS: Contrast venography confirmed the clinical suspicion in 59 patients (33.5%). LA was detected in 5 of the 59 patients with DVT (8.5%), and in none of the 117 subjects with normal venogram (P = 0.007). The OR for having an acute DVT in patients with LA was 10.7 (95% CI: 1.2-94.2). CONCLUSIONS: LA is significantly associated with DVT in symptomatic patients. Further studies are needed to establish the clinical implications of this association.

Adult↗
PubMed1996

DDAVP infusion in haemophilia A carriers: different behaviour of plasma factor VIII and von Willebrand factor.

1-desamino-8-D-arginine vasopressin (DDAVP) increases factor VIII (FVIII) and von Willebrand factor (vWF) levels in patients with haemophilia A and in some patients with von Willebrand disease. It is generally held that the increase of FVIII is a consequence of the increase of vWF. Carriers of haemophilia A generally, but not always, show plasma FVIII levels lower than vWF due to an abnormality in one of the two alleles of the FVIII gene. We investigated the time-course of plasma FVIII:C and vWF:Ag levels in 25 obligate carriers of haemophilia A after DDAVP infusion. In carriers with a normal FVIII to vWF ratio (> 0.8), DDAVP induced a progressive ratio decrease that reached levels significantly lower than that taken as cut-off to discriminate between low and normal values (0.68 +/- 0.1 vs before 0.912 +/- 0.18). In carriers with a borderline (0.7-0.8) or reduced (< 0.7) ratio DDAVP induced a further decrease in the FVIII/vWF ratio, albeit with a different kinetic; after an initial increase, values were lower than pre-DDAVP figures. In all subjects, following the post-DDAVP peak, plasma FVIII progressively decreased while vWF contemporaneously continued to increase. In contrast, DDAVP did not induce significant changes in the FVIII/vWF ratio in normal females, and the two molecules appeared to increase similarly throughout the observation period. These findings indicate that after DDAVP, FVIII increases less or for a shorter time than vWF, also in haemophilia A carriers who have a normal FVIII/vWF ratio. Hence, DDAVP may help identify haemophilia A carriers, especially subjects with normal or borderline ratios. Even though molecular biology procedures at present are the best and more reliable tools to identify the carrier state, DDAVP seems to improve the accuracy of haemostatic parameters.

Adult↗
PubMed1996

Inhibitory effect of prostacyclin and nitroprusside on type IIB von Willebrand factor-promoted platelet activation.

Von Willebrand disease (vWD) of type IIB is a hereditary haemorrhagic disorder characterised by an excessive interaction of von Willebrand factor (vWF) with the platelet receptor GPIb which promotes platelet activation and aggregation through a phospholipase A2-mediated release of arachidonic acid. The present report shows that prostacyclin and nitroprusside, vasodilator-compounds that enhance the cAMP and cGMP concentration respectively, cause a drastic inhibition of the type IIB vWF-induced platelet responses including increase of cytosolic Ca2+ concentration, phosphorylation of pleckstrin (47 kDa) and myosin light chain (20 kDa), secretion of ATP and serotonin, and aggregation parallel to a decrease of arachidonic acid release. Type IIB vWF also elicits tyrosine phosphorylation of proteins with apparent molecular mass of 60, 74, 82 and 130 kDa. Prostacyclin, which induces per se tyrosine-phosphorylation of proteins of about 38 and 45 kDa, inhibits drastically the type IIB vWF-promoted tyrosine-phosphorylation of the 74 kDa protein while inhibits slightly that of 60 kDa band. The protein tyrosine-kinase inhibitor genistein causes a little decrease in the type IIB vWF-induced release of arachidonic acid. It is concluded that the inhibition exerted by prostacyclin and nitroprusside on type IIB vWF-elicited platelet activation seems to be largely ascribable to prevention of the phospholipase A2 activation with the ensuing decrease of the subsequent protein tyrosine phosphorylation.

Epoprostenol↗
PubMed1996

Hyperhomocysteinemia and deep-vein thrombosis. A case-control study.

In a case-control study, fasting total homocysteinemia was determined in 208 consecutive outpatients who underwent phlebography because of the first episode of clinically suspected deep-vein thrombosis (DVT) of lower limbs. Contrast venography confirmed the clinical suspicion in 60 patients (28.8%). Hyperhomocysteinemia was detected in 15 of the 60 patients with DVT (25.0%), and in 17 of the 148 subjects without thrombosis (11.5%; p = 0.025). The OR for having an acute DVT in patients with hyperhomocysteinemia was 2.6 (95% CI: 1.1-5.9). It is concluded that high plasma homocysteine levels are significantly associated with DVT in symptomatic patients. Further studies are needed to clarify the clinical implications of this association.

Adult↗
PubMed1996

Essential thrombocythemia following polycythemia vera: an unusual sequence.

Myeloproliferative disorders (MPD) are prone to modification and evolution during the progression of the disease. While post-polycythemia myeloid metaplasia and chronic myelogenous leukemia following polycythemia vera have been frequently described, no report is available about the evolution of polycythemia vera into essential thrombocythemia. Our case is probably the first report on this occurrence. In the course of a fortuitous observation of electrocardiographic alterations, a diagnosis of polycythemia vera was ruled out in accordance with polycythemia vera study group criteria. At the time of diagnosis, RBC was 6 x 10(12)/L, WBC 15 x 10(9)/L, Ht 59% and platelets 1000 x 10(9)/L. The patient was treated with phlebotomies and radioactive phosphorus achieving a good remission or the disease. Five years later, platelets rose to over 3300 x 10(9)/L without significant modification or RBC, WBC and Ht. The restaging or the disease was consistent for an essential thrombocythemia. In particular, RBC mass was within normal levels. During the last ten years, the patient has been followed recurrently and the blood picture remained stationary, without an increase in the hematocrit but with a platelet count between 658 and 800 x 10(9)/L. We conclude that this report may complete data concerning the evolution of MPD in others.

Aged↗
PubMed1995

Detection and characterization of seven novel protein S (PROS) gene lesions: evaluation of reverse transcript-polymerase chain reaction as a mutation screening strategy.

The molecular genetic analysis of protein S deficiency has been hampered by the complexity of the protein S (PROS) gene and by the existence of a homologous pseudogene. In an attempt to overcome these problems, a reverse transcript-polymerase chain reaction (RT-PCR) mutation screening procedure was developed. However, the application of this mRNA-based strategy to the detection of gene lesions causing heterozygous type I protein S deficiency appears limited owing to the high proportion of patients exhibiting absence of mRNA derived from the mutation-bearing allele ("allelic exclusion"). Nevertheless, this strategy remains extremely effective for rapid mutation detection in type II/III protein S deficiency. Using the RT-PCR technique, a G-to-A transition was detected at position +1 of the exon IV donor splice site, which was associated with type I deficiency and resulted in both exon skipping and cryptic splice site utilization. No abnormal protein S was detected in plasma from this patient. A missense mutation (Asn 217 to Ser), which may interfere with calcium binding, was also detected in exon VIII in a patient with type III protein S deficiency. A further three PROS gene lesions were detected in three patients with type I deficiency by direct sequencing of exon-containing genomic PCR fragments: a single base-pair (bp) deletion in exon XIV, a 2-bp deletion in exon VIII, and a G0to-A transition at position -1 of the exon X donor splice site all resulted in the absence of mRNA expressed from the disease allele. Thus, the RT-PCR methodology proved effective for further analysis of the resulting protein S-deficient phenotypes. A missense mutation (Met570 to Thr) in exon XIV of a further type III-deficient proband was subsequently detected in this patient's cDNA. No PROS gene abnormalities were found in the remaining four subjects, three of whom exhibited allelic exclusion. However, the father of one such patient exhibiting allelic exclusion was subsequently shown to carry a nonsense mutation (Gly448 to Term) within exon XII.

Base Sequence↗
PubMed1995

Type IIB von Willebrand factor induces phospholipase A2 activation and cytosolic Ca2+ increase in platelets.

Von Willebrand factor (vWF) is a large glycoprotein which plays a central role in thrombus formation and blood clotting. The type IIB variant of vWF is characterized by an abnormally high affinity for the platelet receptor GPIb. Type IIB vWF purified from plasma and added to a platelet suspension induced a rapid, dose-dependent (1.2-9 micrograms/ml) increase in the cytosolic Ca2+ concentration. ATP secretion and platelet aggregation also occurred with type IIB vWF concentrations higher than about 5 micrograms/ml, which corresponds to the original plasmatic level. The IIB vWF-evoked (3 micrograms/ml) cytosolic Ca2+ increase was negligibly affected by ADP scavengers or protein kinase C inhibitors; it was drastically reduced by EGTA, La3+, Ni2+ or acetylsalicylate and abolished by the phospholipase A2 inhibitors ONO-RS-082 or oleolyloxyethyl-phosphocholine. Platelet exposure to IIB vWF caused arachidonic acid release, thromboxane B2 and inositoltrisphosphate formation. LJIB1, a monoclonal antibody against GPIb, completely suppressed all platelet responses, whereas LJCP8, an antibody against the receptor GPIIb-IIIa (alpha IIb beta 3 integrin), or the tetrapeptide RGDS, caused a complete inhibition of the aggregation but a partial inhibition of the activation-linked parameters. It is concluded that type IIB vWF-binding to GPIb induces phospholipase A2 activation, arachidonic acid release and GPIIb-IIIa dependent cellular Ca2+ influx. These events may lead to platelet secretion and aggregation.

Arachidonic Acid↗
PubMed1995

Reduced fibrinolytic potential one year after kidney transplantation. Relationship to long-term steroid treatment.

Thromboembolic complications constitute an important risk in renal transplant patients, in whom a hypercoagulable state is associated with immunosuppressive treatment, and the presence of hypercoagulability and hypofibrinolysis specifically with cyclosporine. Hypercorticism secondary to steroid treatment has been associated with a thrombophilic state and the presence of a reduced fibrinolytic potential in particular. The aims of this study were to first evaluate the fibrinolytic potential by the venous occlusion (VO) test in 19 renal transplant (RT) patients, and then compare these findings with those obtained in similar groups of normal subjects and patients with Cushing's disease. The following tests were carried out before and after the VO test: euglobulin lysis time and t-PA and PAI-1 activities and antigen. Compared with normal controls, RT and Cushing's patients both showed a similar significant increase in PAI-1 activity and concentration. The VO test revealed a similar impairment in fibrinolytic potential in both the RT and Cushing groups. High and pathological PAI-1 levels before and after the VO test were consistent with a defective fibrinolytic potential due to the inhibitory effect of PAI-1 on plasminogen activation. A hypofibrinolytic state was found in 68.4% of RT patients. Our results suggest that an imbalance in the fibrinolytic system is a typical feature of RT patients one year after transplantation. Steroids appear to be the immunosuppressive drug mainly involved in determining thromboembolic risk after renal transplantation.

Adult↗
PubMed1995

Biotin-avidin immobilization of platelet glycoproteins (BAIPG): a new capture assay for the detection of anti-platelet antibodies.

Several 'capture' assays are currently employed to identify specific platelet antibodies, but all require the use of murine monoclonal antibodies (MoAbs) against the antigen of interest. We have developed a new antigen capture assay for the detection of platelet reactive antibodies, based on platelet surface sialoglycoprotein labelling with biotin hydrazide, and a following immobilization of the biotinylated platelet proteins to microtiter wells that had been coated with streptavidin. The resulting solid phase can then be used in a simple ELISA to detect serum and platelet associated antibodies. We describe here two versions of this biotin-avidin immobilization of platelet glycoproteins (BAIPG) assay. In BAIPG assay type I, the test sera are directly incubated in microtiter wells previously coated with streptavidin plus biotinylated platelet proteins. The BAIPG type II procedure involves the incubation of sera with biotinylated platelets before platelet solubilization, and, after platelet lysis, the immobilization of the immune complexes to streptavidin-coated wells. In both cases, the bound antibodies are determined by alkaline phosphatase conjugated anti-human IgG. Using BAIPG type I, positive results were obtained in 7/33 patients with idiopathic thrombocytopenic purpura (ITP), 1/10 patients with secondary immune thrombocytopenia (SIT) and 4/17 with non-immune thrombocytopenia (NIT). The BAIPG type II test was positive in 13 out of 33 patients with ITP, in six out of ten patients with SIT, and in three out of the 17 patients with NIT. A comparison between BAIPG and monoclonal antibody immobilization of platelet antigens (MAIPA) assays showed a high degree of correlation between the two methods. These results suggest that the BAIPG assay is a valuable new tool for the detection of anti-platelet antibodies.

Autoantibodies↗
PubMed1995

CD25-negative hairy cell leukaemia: intracytoplasmic detection of Tac antigen and interferon-induced surface expression.

Hairy cell leukaemia (HCL) is a chronic lymphoproliferative disease of B-cell lineage. One of the peculiar immunophenotypic markers is the strong expression of the p55 chain of the interleukin-2 receptor (IL2R), recognized by anti-CD25 (or anti-Tac) monoclonal antibody. However, it is known that in rare cases CD25 may not be detectable, even when variant forms of HCL are excluded. The possibility has not been investigated that in these situations CD25 is present in the cytoplasm of the neoplastic cells. This paper describes a case in which the clinical, histological, and electron microscopic features were consistent with a typical HCL. Immunophenotype analysis showed the whole spectrum of markers of HCL, except for the expression of IL2R. The soluble form of the molecule was, however, increased in the patient's serum. Cytospin staining of the neoplastic B cells with anti-CD25 clearly demonstrated the presence of IL2R in the cytoplasm of hairy cells. When the cells were cultivated in vitro in the presence of interferon-alpha 2b, CD25 was detectable at the membrane level. These findings suggest that at least some cases of CD25-negative HCL may express cytoplasmic IL2R.

Aged↗
PubMed1995

Failure of B cells of chronic lymphocytic leukemia in presenting soluble and alloantigens.

B-cell-type chronic lymphocytic leukemia (B-CLL) patients have immunological abnormalities of both B and T lymphocytes. Since T cell defects might depend upon a defective accessory function of neoplastic B lymphocytes, we analyzed the ability of peripheral blood B cells of seven B-CLL patients to stimulate allogenic normal T cells in mixed lymphocyte reaction (MLR) and to present tetanus toxoid (TT) to autologous T cells. In both systems, neoplastic B lymphocytes show a defective antigen-presenting function, which is more evident with disease progression. Such a defect cannot be ascribed to a decreased MHC class II molecule expression nor to an abnormal IL-1 beta production, but it can be partially accounted for by a low B7 expression. Pretreatment of neoplastic B cells with interleukin-4 (IL-4) restores primary MLR, but has little effect on the response to TT. The effect of IL-4 is not mediated by quantitative modifications of class II and B7 molecule expression or of IL-1 beta production.

Antigen Presentation↗
PubMed1995

Different organization of von Willebrand factor oligomers in type-2A and -2B von Willebrand disease variants: effects of DDAVP infusion and protease inhibitors.

Plasma von Willebrand factor (vWf) displays a complex pattern of repeating multimers, whose heterogeneous size distribution seems to depend on the proteolytic cleavage of the constituent vWf subunit. Smaller vWf multimers are thought to derive by proteolytic cleavage of the larger forms. To clarify the relationship between large multimer representation and the structure of small vWf oligomers, DDAVP was infused in patients with type-2A and -2B von Willebrand disease (vWd) variants which lack circulating high vWf forms. Before infusion, high-resolution multimer analysis demonstrated a more pronounced representation of the satellite bands of each oligomer, mainly concerning fast-moving components, especially in type 2B vWd. After DDAVP, in type-2A vWd each oligomer displayed a different organization depending on whether restoration of large vWf multimers occurred. The lack of large vWf multimer restoration, as shown in citrated samples, was associated with the fast band being significantly more represented than the slower, and almost similar to the central component. In contrast, when the high-molecular-weight vWf forms were restored, as occurred in the samples collected in the presence of protease inhibitors, the relative representation of the fast- and slow-moving bands was similar to that of normal samples. In type-2B vWd, regardless of the anticoagulant used, DDAVP infusion did not restore large vWf multimers, and each oligomer displayed a significant increase in both the central band and fast-moving satellite, the fast being even more highly represented. These findings suggest that, regardless of the origin, the disappearance of large circulating multimers in type-2A and -2B vWd induces an increased representation of the fast-moving satellite of the low-molecular-weight multimers. Moreover, the time course of large and low/intermediate multimer decrease and increase provides a further demonstration that low vWf multimers derive from the larger ones, and that mainly the fast-moving band of the oligomer is involved.

Deamino Arginine Vasopressin↗