Paola Pradella

Platelet production and destruction in liver cirrhosis

BACKGROUND & AIMS Thrombocytopenia is common in liver cirrhosis (LC) but the mechanisms are not fully understood. The purpose of our work was to evaluate platelet kinetics in LC with different etiologies by examining platelet production and destruction. METHODS Ninety-one consecutive LC patients (36 HCV, 49 alcoholics, 15 HBV) were enrolled. As controls, 25 subjects with idiopathic thrombocytopenic purpura, 10 subjects with aplastic anemia, and 40 healthy blood donors were studied. Plasma thrombopoietin (TPO) was measured by ELISA. Reticulated platelets (RP) were determined using the Thiazole Orange method. Plasma glycocalicin (GC) was measured using monoclonal antibodies. Platelet associated and serum antiplatelet antibodies were detected by flow cytometry. B-cell monoclonality in PBMC was assessed by immunoglobulin fingerprinting. RESULTS Serum TPO was significantly lower in LC (29.9±18.1 pg/ml) compared to controls (82.3±47.6 pg/ml). The GC levels were higher in LC (any etiology) than in healthy cases. Conversely, the absolute levels of RP were lower in LC (any etiology) than in healthy controls. The platelet-associated and serum anti-platelet antibodies were higher in HCV+ LC compared to healthy subjects (p<0.0064), alcoholic LC (p<0.018), and HBV+ LC (p<0.0001). B-cell monoclonality was found in 27% of the HCV+LC, while it was not found in HBV+ or alcoholic LC. CONCLUSIONS Patients with LC present decreased plasma TPO, accelerated platelet turnover, and reduced platelet production. This indicates that LC thrombocytopenia is a multifactorial condition involving both increased platelet clearance and impaired thrombopoiesis.

Vascular PG-M/versican variants promote platelet adhesion at low shear rates and cooperate with collagens to induce aggregation

We have identified a novel von Willebrand factor/fibrinogen/selectin‐independent, platelet adhesion‐promoting function of vascular PG‐M/versicans that may be relevant in normal venous thrombosis and critical in atherosclerotic conditions. A purification scheme was devised to obtain vascular versicans, which by biochemical, immunochemical, and ultrastructural means were asserted to be 1) composed primarily of isoforms V1 and V2; 2) free of contaminants; 3) prevalently substituted with chondroitin‐4‐sulfate and dermatan sulfate (DS) chains; and 4) capable of binding hyaluronan to form link protein‐stabilized ternary complexes. Real‐time analysis of human platelet perfused under diverse shear forces showed that they largely failed to bind to several vascular and nonvascular proteoglycans (PGs). In contrast, they bound in a dose‐ and shear rate‐dependent manner to vascular versicans, exhibiting a unique attachment‐detachment kinetics and establishing a firm substrate tethering characterized with no significant aggregation. Digestion of these PGs with lyases and competition experiments with purified glycosaminoglycans revealed that platelet adhesion to vascular versicans was primarily mediated by their DS chains. Incorporation of the versicans into fibrillar collagen substrates augmented their adhesive activity and strongly promoted platelet aggregation at low and high shear rates. Affinity chromatography of platelet surfaces on DS columns identified a 120–140 kDa polypeptide complex that behaved as a specific vascular versican binding membrane ligand in solid‐phase binding assays. These findings indicate that selective versican variants of the subendothelium may serve as ancillary GPIba/integrin/selectin‐independent platelet ligands in healthy and diseased vascular beds and may be directly responsible for the platelet accruing after rupture of atherosclerotic plaques.—Mazzucato, M., Cozzi, M. R., Pradella, P., Perissinotto, D., Malmström, A., Mörgelin, M., Spessotto, P., Colombatti, A., de Marco, L., Perris, R. Vascular PG‐M/ versican variants promote platelet adhesion at low shear rates and cooperate with collagens to induce aggregation. FASEB J. 16, 1903–1916 (2002)

Clinical significance of positive direct antiglobulin test in patients with HIV infection

SummaryA direct antiglobulin test (DAT) was performed in 70 patients with anti-HIV antibodies (group A: seropositive patients without or with minimal disease and group B: AIDS patients with or without malignancies). A positive DAT was found in 24 of 70 patients (34%, significantly higher compared to 0.1% in healthy controls) and a higher prevalence of positive DAT was observed in group B than in group A patients (55% versus 21% p<0.01). When comparing DAT-positive and negative patients within the same clinical group, no significant difference is seen in haemoglobin levels. There is no difference in serum bilirubin, haptoglobin or reticulocyte count between DAT-positive and negative patients altogether or in the same clinical group. AZT therapy seems to exert no significant influence on the onset of a positive DAT. The results confirm a high prevalence of positive DAT in patients with HIV antibodies, mainly in worse clinical conditions, and suggest that a positive DAT might be a prognostic factor in the clinical course of the disease.ZusammenfassungBei 70 Patienten mit anti-HIV-Antikörpern wurde ein direkter Antiglobulin-Test durchgeführt. In Gruppe A waren seropositive Patienten ohne oder mit minimalen Krankheitszeichen, in Gruppe B AIDS-Patienten mit oder ohne maligne Erkrankung. Bei 24 von 70 Patienten (34%) war der DAT positiv. Im Vergleich dazu waren bei gesunden Kontrollen nur 0,1% positiv. Neben diesem signifikanten Unterschied fanden sich auch signifikante Unterschiede zwischen den beiden Gruppen mit 55% positivem DAT in Gruppe B und 21% in Gruppe A (p<0,01). DAT-positive und -negative Patienten in denselben klinischen Gruppen zeigten in ihren Hämoglobinspiegeln keine signifikanten Unterschiede. Auch fanden sich insgesamt und in den beiden klinischen Gruppen keine Unterschiede in den Werten von Serumbilirubin. Haptoglobin oder Retikulozytenzahlen bei DAT-positiven und -negativen Patienten. Die Behandlung mit AZT hat offensichtlich keinen wesentlichen Einfluß darauf, wann der DAT positiv wird. Unsere Ergebnisse bestätigen, daß bei Patienten mit HIV-Antikörpern eine hohe Rate an positiven DAT besteht und sich vor allem bei den ungünstigen klinischen Stadien positive Testergebnisse einstellen, so daß der positive DAT als prognostischer Faktor für den klinischen Verlauf der Erkrankung angesehen werden kann.A direct antiglobulin test (DAT) was performed in 70 patients with anti-HIV antibodies (group A: seropositive patients without or with minimal disease and group B: AIDS patients with or without malignancies). A positive DAT was found in 24 of 70 patients (34%, significantly higher compared to 0.1% in healthy controls) and a higher prevalence of positive DAT was observed in group B than in group A patients (55% versus 21% p<0.01). When comparing DAT-positive and negative patients within the same clinical group, no significant difference is seen in haemoglobin levels. There is no difference in serum bilirubin, haptoglobin or reticulocyte count between DAT-positive and negative patients altogether or in the same clinical group. AZT therapy seems to exert no significant influence on the onset of a positive DAT. The results confirm a high prevalence of positive DAT in patients with HIV antibodies, mainly in worse clinical conditions, and suggest that a positive DAT might be a prognostic factor in the clinical course of the disease. Bei 70 Patienten mit anti-HIV-Antikörpern wurde ein direkter Antiglobulin-Test durchgeführt. In Gruppe A waren seropositive Patienten ohne oder mit minimalen Krankheitszeichen, in Gruppe B AIDS-Patienten mit oder ohne maligne Erkrankung. Bei 24 von 70 Patienten (34%) war der DAT positiv. Im Vergleich dazu waren bei gesunden Kontrollen nur 0,1% positiv. Neben diesem signifikanten Unterschied fanden sich auch signifikante Unterschiede zwischen den beiden Gruppen mit 55% positivem DAT in Gruppe B und 21% in Gruppe A (p<0,01). DAT-positive und -negative Patienten in denselben klinischen Gruppen zeigten in ihren Hämoglobinspiegeln keine signifikanten Unterschiede. Auch fanden sich insgesamt und in den beiden klinischen Gruppen keine Unterschiede in den Werten von Serumbilirubin. Haptoglobin oder Retikulozytenzahlen bei DAT-positiven und -negativen Patienten. Die Behandlung mit AZT hat offensichtlich keinen wesentlichen Einfluß darauf, wann der DAT positiv wird. Unsere Ergebnisse bestätigen, daß bei Patienten mit HIV-Antikörpern eine hohe Rate an positiven DAT besteht und sich vor allem bei den ungünstigen klinischen Stadien positive Testergebnisse einstellen, so daß der positive DAT als prognostischer Faktor für den klinischen Verlauf der Erkrankung angesehen werden kann.

Labeling of platelet surface glycoproteins with biotin derivatives

We describe a non-radioactive method for the labeling of platelet surface proteins, consisting of platelet protein biotinylation by means of N-hydroxysuccinimido-biotin (NHS-B) and biotin-hydrazide (H-B); NHS-B labels proteins amino residues while H-B binds to periodate-modified sialoglycoproteins. Washed platelets were biotinylated and protein bands were detected after SDS-electrophoresis and western-blot using avidin-peroxidase and luminol as substrate to enhance the signal which was then detected by X-ray film. Biotin-labeled platelet proteins were also immunoprecipitated with monoclonal antibodies against glycoproteins Ib and the IIb-IIIa complex. The use of periodate induced biotinylation is the method of choice for labeling platelet surface glycoproteins while NHS-B also labels internal proteins. The sensitivity of this new procedure is similar to that obtained with radiolabeling techniques; biotinylation does not interfere with the antigenic properties of Ib and IIb-IIIa glycoproteins.

Relationship Between Human Leucocyte Antigen Class I and Class II and Chronic Idiopathic Urticaria Associated With Aspirin and/or NSAIDs Hypersensitivity

Background. HLA genes play a role in the predisposition of several diseases. The aim was to analyze the prevalence of HLA class I phenotypes and HLA-DRB1* genotype in patients with CIU associated with ASA and NSAIDs hypersensitivity (AICU). Methods. 69 patients with AICU, and 200 healthy subjects. Results. Subjects with HLA-B44 and HLA-Cw5 antigens were more represented in patients with AICU than in control group. Subjects with HLA-A11, HLA-B13, HLACw4, and HLA-Cw7 antigen were more represented in control group than in patients with AICU. Multiple logistic regression demonstrated an association of HLA-Cw4 and HLA-Cw7 with a lower risk of AICU, whereas carriers of HLA-B44 phenotype had a higher risk of AICU. No differences were found between patients and controls as regards to HLA-DRB1* genotype. Conclusions. We observed an association between some HLA class-I antigens and AICU. To the best of our knowledge this is the first description of such association.

Mixed‐Type Auto‐Immune Haemolytic Anaemia in a Patient with HIV Infection

A young HIV‐infected patient presented with a severe auto‐immune haemolytic anaemia with both warm and cold auto‐antibodies, an infrequent category of anti‐erythrocyte auto‐immunity. Serological findings were compatible with the presence of a low‐titre, high‐thermal‐amplitude anti‐I cold‐reacting antibody and a pan‐reactive warm‐reactive auto‐antibody. Immunochemical characterisation of the warm antibody failed to identify any membrane protein acting as auto‐antigen. This is, to our knowledge, the first reported case of mixed‐type autoimmune haemolytic anaemia in a patient with HIV infection. Overt haemolysis is a very rare complication in HIV‐infected patients, despite the high prevalence of a positive direct antiglobulin test reported in these patients. This suggests that HIV infection is a condition in which anti‐erythrocyte auto‐immunity is a serological finding without haemolytic effects in the large majority of cases.

On the Mechanism of the Spermine-Exerted Inhibition on α-Thrombin-Induced Platelet Activation*

Abstract Previous reports have shown that various amines inhibited platelet activation, but no definitive conclusions on their action mechanism were drawn. We have further investigated the action of spermine on platelet responses evoked by α-thrombin and other agonists. Spermine inhibited in a concentration-dependent manner (1–10 mM), and more efficiently than spermidine and putrescine, the α-thrombin-induced (1.5 nM) platelet activation. Spermine added at a concentration that inhibited completely aggregation only partially affected the thrombin-induced increase in cytosolic Ca 2+ concentration, protein phosphorylation, and ATP secretion. The polyamine had little effect on the morphology of resting platelets, as measured by electron microscopy, thrombin hydrolytic activity, and fibrinogen clotting capacity but decreased the thrombin binding to platelets and isolated glycocalicin. Spermine partially inhibited the aggregation elicited by ADP, vasopressin, platelet-activating factor, thrombin receptor-activating peptide, fluoroaluminate, ionomycin, and dioctanoylglycerol but did not affect the cytosolic Ca 2+ increase induced by these agonists. The polyamine bound to both glycocalicin and platelets, and it inhibited the fibrinogen binding to stimulated platelets. The amount of 14 C-spermine bound to resting cells decreased in the presence of the glycoprotein GPIb-antibody LJIB1, whereas the polyamine bound to activated platelets, which was higher than that tied to resting cells, was markedly reduced by LJCP8 or decorsin, a GPIIb/IIIa antibody and antagonist-peptide, respectively. These results indicate that spermine specifically inhibits the thrombin binding to GPIb of resting platelets and the fibrinogen binding to GPIIb/IIIa (integrin α IIb β 3 ) of activated platelets.

Glycocalicin in the diagnosis and management of immune thrombocytopenia

Abstract: We studied glycocalicin (GC), expressed as plasma GC concentration and as GC index (ratio to platelet count), in 129 thrombocytopenic patients (platelet count <100×109/1) and 60 sex‐ and age‐matched controls. Seventy‐two patients had idiopathic immune thrombocytopenia, 32 secondary immune thrombocytopenia, 8 microangiopathic thrombocytopenia and 17 thrombocytopenia secondary to bone marrow aplasia. Patients with immune thrombocytopenia (ITP) were also subclassified, according to their clinical behaviour, as having active disease or being in spontaneous or therapy‐induced partial remission. A significant correlation was found between glycocalicin levels and platelet count both in normals and in patients with bone marrow aplasia (r = 0.75). ITP patients showed a GC index significantly higher than controls (6.02±7.87 vs. 0.9±0.2, p<0.001). When ITP patients with similar platelet count (30–50×109/l) were studied, the mean level of GC and the GC index were significantly higher in those patients with active disease than in those in remission (0.97±0.38 vs. 0.58±0.17 μg/ml, p<0.05; 6.41±2.64 vs. 3.44±0.94, p<0.05, respectively). A longitudinal study performed in 10 patients with different subtypes of ITP suggested a positive correlation between GC index and the activity of the disease. The GC value and GC index were significantly higher in patients with microangiopathic thrombocytopenia than in controls (1.44±0.73 vs. 0.8±0.16 μg/ml, p<0.01; and 18.77±22.23 vs. 0.9±0.2, p<0.001, respectively). The GC value was significantly lower in bone marrow failure (0.15±0.04 μg/ml, p<0.01) compared to controls, while no difference was observed in the GC index. Our data confirm that the GC index is helpful in differentiating thrombocytopenia due to increased platelet destruction from the one due to impaired production. In addition, the assay has been proven useful in the differential diagnosis of different ITP subtypes and their follow‐up.

In vitro Proteolysis of the Red Cell Membrane in Patients with HIV Infection

It has been suggested that acquired abnormalities of the red cell membrane due to various injuries [azidothymidine (AZT) therapy, immunoglobulin coating of red cells, differentiation abnormalities of erythroid precursors] contribute to the onset of anaemia in HIV-infected patients. In vitro proteolysis of erythrocyte membrane proteins is regarded as a molecular marker of membrane damage induced in vivo by different agents. We therefore investigated in vitro proteolysis of ghosts derived from red blood cells of 30 HIV-infected patients. Considered collectively, there was no significant increase in in vitro proteolysis in ghosts from anaemic HIV patients. However, a significantly higher degree of in vitro self-digestion of RBC membrane proteins was evident in HIV-infected patients with spleen enlargement, but not in splenomegalic patients suffering from liver cirrhosis. Neither AZT therapy nor the presence of a positive direct antiglobulin test seemed to be directly associated with increased in vitro protein breakdown. The results seem to suggest damage of the red cell membrane in HIV infection, induced by injuries on red cells during their prolonged retention inside an enlarged spleen, while it seems unlikely that AZT therapy or immunoglobulin coating of red cells play major roles in red cell damage.