Biomedical subjects

Miriam Galbusera

Publications and source records attributed to Miriam Galbusera.

10 recordsLinked to original sources

PubMed2006

Thrombotic thrombocytopenic purpura--then and now.

Thrombotic thrombocytopenic purpura (TTP) is a potentially life-threatening disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and formation of microthrombi in several organs. The disease may manifest once in a lifetime or may relapse after complete recovery of the initial episode; in these recurrent cases, death or neurological sequelae are common final outcomes. Accumulation of unusually large (UL) von Willebrand factor (VWF) multimers was described in the plasma of patients with TTP. Such ULVWF multimers are synthesized in endothelial cells and megakaryocytes and are secreted into the blood upon stimulation. However, in healthy individuals ULVWF multimers do not normally circulate because they are rapidly reduced into smaller multimers soon after their secretion due to cleavage by a plasma metalloprotease, ADAMTS13. Deficiency of ADAMTS13 has been reported consistently in patients with TTP. Such defect may be constitutive, due to homozygous or double heterozygous mutations in the corresponding gene, or acquired, due to the presence of circulating inhibitory antibodies. It follows that in TTP patients, the absent or severely depressed plasma ADAMTS13 activity limits the cleavage of ULVWF multimers, which remain anchored to the endothelial cells in long strings. Particularly under conditions of high shear stress, the multimers may promote the adhesion of circulating platelets, initiating thrombus formation. The clinical implications of these findings to the diagnosis and treatment of TTP are discussed.

ADAM Proteins↗

PubMed2006

In-vitro and in-vivo consequences of mutations in the von Willebrand factor cleaving protease ADAMTS13 in thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a disease characterized by microvascular thrombosis, often associated with deficiency of the vonWillebrand factor (VWF) cleaving protease ADAMTS13. We investigated the spectrum of ADAMTS13 gene mutations in patients with TTP and congenital ADAMTS13 deficiency to establish the consequences on ADAMTS13 processing and activity. We describe five missense (V88M, G1239V, R1060W, R1123C and R1219W), 1 nonsense (W1016Stop) and 1 insertion (82_83insT) mutations. In two patients no mutation was identified despite undetectable protease activity. Expression in HEK293 mammalian cells (V88M, G1239V, R1123C and R1219W) documented that three missense mutants were not secreted, whereas theV88M was secreted at low levels and with reduced activity. We also provide evidence that impaired secretion of ADAMTS13 mutants observed in vitro translates into severely reduced ADAMTS13 antigen levels in patients in vivo. To evaluate whether the small amounts of mutant protease present in the circulation of patients had VWF cleaving activity, WT and mutant rADAMTS13 were stably expressed in Drosophila S2 cells under the influence of the Drosophila BiP protein signal sequence, which allows protein secretion. Drosophila expression system showed a 40-60% protease activity in the mutants. Several single nucleotide polymorphisms (SNPs) within exons and intron boundaries were found in patients, suggesting that the interplay of SNPs could at least in part account for ADAMTS13 functional abnormalities in patients without mutations. In conclusion, defective secretion and impaired activity of the mutants concur to determine an almost complete deficiency of ADAMTS13 activity in patients with a homozygous or two heterozygous ADAMTS13 mutations.

ADAM Proteins↗

PubMed2005

ADAMTS13 autoantibodies in patients with thrombotic microangiopathies and other immunomediated diseases.

Autoantibodies neutralizing human ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type 1 motif), the metalloprotease that physiologically cleaves von Willebrand factor, are a major cause of severe deficiency of the protease and of acquired thrombotic thrombocytopenic purpura (TTP). We evaluated prevalence of anti-ADAMTS13 antibodies in 59 patients with thrombotic microangiopathies (TMAs) and in 160 patients with immunologic or thrombocytopenic diseases different from TTP, using an enzyme-linked immunosorbent assay (ELISA). Immunoglobulin G (IgG) antibodies directed against ADAMTS13 were found in 97% of untreated patients with acute acquired TMA who had plasma levels of ADAMTS13 activity below 10%. The corresponding prevalence of IgM antibodies was 11%. In contrast, anti-ADAMTS13 antibodies of G or M isotypes were detected in 20% of patients with TMA with ADAMTS13 activity above 10%. The ELISA was more sensitive than the standard functional inhibitor assay for detecting antibodies against ADAMTS13. Patients with thrombocytopenia from various causes (n = 50), systemic lupus erythematosus (SLE; n = 40), and the antiphospholipid antibody syndrome (APS; n = 55) had prevalences of IgG antibodies of 8%, 13%, and 5% respectively, only slightly higher than the prevalence in 111 healthy donors (4%). A rather high prevalence of anti-ADAMTS13 IgM antibodies was found in patients with SLE and APS (18% each). The clinical significance of IgM antibodies in these groups is unclear. In conclusion, the ELISA method detected anti-ADAMTS13 IgG antibodies in a very large proportion of patients with acquired TMA associated with severe ADAMTS13 deficiency, and was more sensitive than the inhibitor assay.

ADAM Proteins↗

PubMed2005

Rituximab prevents recurrence of thrombotic thrombocytopenic purpura: a case report.

Thrombotic thrombocytopenic purpura (TTP) is a rare disorder of small vessels that is associated with deficiency of the von Willebrand factor-cleaving protease, ADAMTS13. The presence of anti-ADAMTS13 autoantibodies is considered a factor predisposing to relapses. Despite close monitoring and intensive plasma treatment, in these patients acute episodes are still associated with substantial morbidity and mortality rates, and the optimal therapeutic option should be prevention of relapses. This study was conducted in a patient with recurrent TTP due to high titers of ADAMTS13 inhibitors, who used to have 2 relapses of TTP a year. The study compared the standard treatment plasma exchange with rituximab. Results documented that plasma exchange had only a small transient effect on ADAMTS13 activity and inhibitors; on the contrary, prophylaxis with rituximab was associated with disappearance of anti-ADAMTS13 antibodies, a progressive recovery of protease activity, and it allowed the patient to maintain a disease-free state during a more than 2-year follow-up.

ADAM Proteins↗

PubMed2005

Complement factor H mutation in familial thrombotic thrombocytopenic purpura with ADAMTS13 deficiency and renal involvement.

Thrombotic thrombocytopenic purpura is a rare disorder of small vessels that is associated with deficiency of the von Willebrand factor-cleaving protease ADAMTS13, which favors platelet adhesion and aggregation in the microcirculation. The disease manifests mainly with central nervous system symptoms, but cases of renal insufficiency have been reported. Presented are findings of the genetic basis of phenotype heterogeneity in thrombotic thrombocytopenic purpura in two sisters within one family. The patients had ADAMTS13 deficiency as a result of two heterozygous mutations (causing V88M and G1239V changes). In addition, a heterozygous mutation (causing an S890I change) in factor H of complement was found in the patient who developed chronic renal failure but not in her sister, who presented with exclusive neurologic symptoms.

ADAM Proteins↗

PubMed2005

In response to protein load podocytes reorganize cytoskeleton and modulate endothelin-1 gene: implication for permselective dysfunction of chronic nephropathies.

Effacement of podocyte foot processes occurs in many proteinuric nephropathies and is accompanied by rearrangement of the actin cytoskeleton. Here, we studied whether protein overload affects intracellular pathways, leading to cytoskeletal architecture changes and ultimately to podocyte dysfunction. Mouse podocytes bound and endocytosed both albumin and IgG via receptor-specific mechanisms. Protein overload caused redistribution of F-actin fibers instrumental to up-regulation of the prepro-endothelin (ET)-1 gene and production of the corresponding peptide. Increased DNA-binding activity for nuclear factor (NF)-kappaB and Ap-1 nuclear proteins was measured in nuclear extracts of podocytes exposed to excess proteins. Both Y27632, which inhibits Rho kinase-dependent stress fiber formation, and jasplakinolide, an F-actin stabilizer, decreased NF-kappaB and Ap-1 activity and reduced ET-1 expression. This suggested a role for the cytoskeleton, through activated Rho, in the regulation of the ET-1 peptide. Focal adhesion kinase (FAK), an integrin-associated nonreceptor tyrosine kinase, was phosphorylated by albumin treatment via Rho kinase-triggered actin reorganization. FAK activation led to NF-kappaB- and Ap-1-dependent ET-1 expression. These data suggest that reorganization of the actin cytoskeletal network in response to protein load is implicated in modulation of the ET-1 gene via Rho kinase-dependent FAK activation of NF-kappaB and Ap-1 in differentiated podocytes. Increased ET-1 generation might alter glomerular permselectivity and amplify the noxious effect of protein overload on dysfunctional podocytes.

Actins↗

PubMed2005

Activation of porcine endothelium in response to xenogeneic serum causes thrombosis independently of platelet activation.

BACKGROUND: Endothelial cell activation and microvascular thrombosis are hallmarks of hyperacute xenograft rejection. However, the molecular determinants of platelet-endothelial interaction and thrombus formation are poorly understood. This study investigated whether: (i) xenogeneic human serum (HS), as a source of xenoreactive antibodies and complement, activates porcine aortic endothelial cells (PAEC) to promote thrombus formation under high shear stress; (ii) the endothelial adhesive proteins vitronectin receptor and P-selectin are involved in the von Willebrand factor (VWF)-platelet interaction during the thrombotic process under flow; (iii) reactive oxygen species (ROS) are activated by complement and served as intracellular signals for adhesive protein up-regulation. METHODS: The PAEC were pre-exposed for 90 min in static conditions to medium plus 10, 20, and 50% HS or 20% porcine serum (PS), as control, then cells were perfused at 50 dynes/cm2 in a parallel plate flow chamber with human blood and area occupied by thrombi was measured. The role of complement in HS-induced thrombus formation was assessed by incubating PAEC with 20% HS in the presence of soluble complement receptor type 1 (sCR1) before blood perfusion. The effect of platelet activation was assessed using human blood treated or not with ADP and then flowed over PAEC pre-exposed to 20% HS or 20% PS as control. To identify the endothelial adhesive proteins involved in thrombus formation PAEC treated with 20% HS were then incubated with anti-vitronectin receptor antibody, anti-P-selectin antibody or P-selectin glycoprotein ligand-1 (PSGL-1), the soluble ligand of P-selectin, before the adhesion assay. Confocal microscopy was used to detect changes in endothelial adhesive protein expression. VWF interaction with platelet receptors GPIb and alphaIIbbeta3 was assessed adding aurin tricarboxylic acid (ATA) and anti-alphaIIbbeta3 antibody to blood before perfusion. The ROS involvement in xenogeneic serum-induced thrombus formation was determined studying the intracellular production of hydrogen peroxide (H2O2). The effect of antioxidants and metal chelators on HS-induced thrombus formation was evaluated treating PAEC with pyrrolidine dithiocarbamate (PDTC) or 1,3-dimethyl-2-thiourea (DMTU) before and during incubation with 20% HS followed by blood perfusion. The effect of antioxidants and sCR1 on ROS generation was investigated treating PAEC with PDTC or DMTU before and during incubation with 20% HS. Intracellular ROS generation was measured by fluorescence spectroscopy using the probe dihydrorhodamine 123 (DHR-123). RESULTS: Human serum but not PS caused thrombus formation on PAEC under high shear stress. Blockade of complement activation by sCR1 prevented xenogeneic serum-induced thrombus formation. Activated platelets did not promote thrombus formation on resting endothelium, and did not further increase platelet deposition on xenogeneic serum-treated PAEC. Vitronectin receptor and P-selectin were up-regulated on the endothelial surface by HS. Their functional blockade by specific antibodies prevented platelet deposition and thrombus formation. H2O2 production significantly increased when PAEC were exposed to the xenogeneic condition. Antioxidants and sCR1 completely prevented thrombus formation by reducing excessive ROS production and the expression of vitronectin receptor and P-selectin. CONCLUSIONS: Xenogeneic complement induces endothelial cell activation and thrombosis which is independent of platelet activation. Complement deposition elicits a rapid generation of ROS that lead to overexpression of endothelial adhesive molecules instrumental for platelet deposition.

Animals↗

PubMed2005

Complement activation: the missing link between ADAMTS-13 deficiency and microvascular thrombosis of thrombotic microangiopathies.

Endothelial injury is the central factor in the events leading to thrombotic microangiopathy (TMA); however, the mechanisms involved are not fully understood. Here we investigate the role of neutrophils (PMNs) and of complement activation in inducing microvascular damage and loss of thromboresistance in TMA associated with ADAMTS-13 deficiency. PMNs isolated during the acute phase of the disease released excessive amounts of reactive-oxygen species (ROS), N-derived oxidants and proteinases and induced damage and thromboresistance loss in human microvascular endothelial cell line (HMEC-1) ex vivo. Endothelial cytotoxicity and thromboresistance loss was also induced by TMA serum. Complement-derived products were responsible for the above effects: in fact, TMA serum caused C3 and Membrane Attack Complex (MAC) deposition on HMEC-1 and its cytotoxic effect was abolished by complement inhibition. TMA serum caused surface expression of P-selectin on HMEC-1 which may promote PMN adhesion and resulted in increased PMN cytotoxicity, indicating that complement may have a role in PMN activation. In addition, TMA serum stimulated control PMNs to release ROS and proteinases, and to cause endothelial cell cytotoxicity. All of the above effects were abrogated by complement inactivation. These data document for the first time that complement-initiated PMN activation and endothelial injury may have a crucial role in microvascular thrombosis of TMA associated with ADAMTS-13 deficiency.

ADAM Proteins↗

PubMed2004

Platelet dysfunction in renal failure.

Patients with end-stage renal disease suffer from complex hemostatic disorders. Uremic patients show a bleeding diathesis that is mainly due to abnormalities of primary hemostasis; in particular, platelet dysfunction and impaired platelet-vessel wall interaction. However, despite decreased platelet function, these patients have a high prevalence of cardiovascular and thrombotic complications. Platelet dysfunction in uremic patients is partially due to uremic toxins present in circulating blood. Dialysis improves platelet abnormalities and reduces, but does not eliminate, the risk of hemorrhage. Hemodialysis can even contribute to the bleeding through the continuous platelet activation induced by the interaction between blood and artificial surfaces. Thrombocytopenia, glomerular thrombosis, and thrombi in small arteries and glomerular capillaries are common pathological features in many renal diseases. Platelets are also involved directly in the pathogenesis of glomerular diseases through a variety of mechanisms, including release of active molecules, by enhancing immune complex deposition, and by altering glomerular permeability.

Blood Platelet Disorders↗

PubMed2002

von Willebrand factor cleaving protease (ADAMTS13) is deficient in recurrent and familial thrombotic thrombocytopenic purpura and hemolytic uremic syndrome.

Whether measurement of ADAMTS13 activity may enable physicians to distinguish thrombotic thrombocytopenic purpura (TTP) from hemolytic uremic syndrome (HUS) is still a controversial issue. Our aim was to clarify whether patients with normal or deficient ADAMTS13 activity could be distinguished in terms of disease manifestations and multimeric patterns of plasma von Willebrand factor (VWF). ADAMTS13 activity, VWF antigen, and multimeric pattern were evaluated in patients with recurrent and familial TTP (n = 20) and HUS (n = 29). Results of the collagen-binding assay of ADAMTS13 activity were confirmed in selected samples by testing the capacity of plasma to cleave recombinant VWF A1-A2-A3. Most patients with TTP had complete or partial deficiency of ADAMTS13 activity during the acute phase, and in some the defect persisted at remission. However, complete ADAMTS13 deficiency was also found in 5 of 9 patients with HUS during the acute phase and in 5 patients during remission. HUS patients with ADAMTS13 deficiency could not be distinguished clinically from those with normal ADAMTS13. In a subgroup of patients with TTP or HUS, the ADAMTS13 defect was inherited, as documented by half-normal levels of ADAMTS13 in their asymptomatic parents, consistent with the heterozygous carrier state. In patients with TTP and HUS there was indirect evidence of increased VWF fragmentation, and this occurred also in patients with ADAMTS13 deficiency. In conclusion, deficient ADAMTS13 activity does not distinguish TTP from HUS, at least in the recurrent and familial forms, and it is not the only determinant of VWF abnormalities in these conditions.

ADAM Proteins↗