Raimondo De Cristofaro - Profile on Academia.edu (original) (raw)
Papers by Raimondo De Cristofaro
Biochemistry, Feb 1, 1992
The amidase activity of human a-thrombin has been studied at steady state in the pH range 6-10, a... more The amidase activity of human a-thrombin has been studied at steady state in the pH range 6-10, as a function of NaCl concentration from 1 mM to 1 M and temperature from 10 to 40 OC. The Michaelis-Menten constant, K,, shows a bell-shaped dependence over this pH range with a minimum around pH 7.5 in the presence of 0.1 M NaCl at 25 OC. The catalytic constant, kat, also has a bell-shaped pH dependence with multiple inflection points that are more evident at low NaCl concentrations and a maximum around pH 8.2 in the presence of 0.1 M NaCl at 25 OC. A detailed analysis of the results in terms of a general linkage scheme has allowed a thorough characterization of the linkage between proton and substrate binding and its dependence on NaCl concentration, as well as the relevant entropic and enthalpic contributions to binding and catalytic events. Formulation of detailed partition functions for each enzyme intermediate involved in the catalytic cycle suggests that (at least) three groups are responsible for the control of thrombin amidase activity as a function of pH. One group is to be identified with the active site His, due to its pK values in the free enzyme and the adduct and its enthalpy of ionization. The effect of NaCl concentration on amidase activity seems to be extremely specific. Comparative steady-state measurements carried out in the presence of NaCl, NaBr, NaI, KC1, and MgClz show that human a-thrombin is capable of discriminating among different cations and anions. This suggests that small ions participate as allosteric effectors in the regulation of thrombin activity. The linkage with NaCl is strongly pH dependent and increases with decreasing pH. The present results provide information on the basic aspects of human a-thrombin activity and regulation and enable a rigorous thermodynamic approach to other important regulatory interactions in human a-thrombin and its structurally perturbed derivatives.
Effect of protons and chloride on the amidase activity in human α-thrombin
Thrombosis Research, 1991
Temperature-Dependence and PH-Dependence of the Oxygen-Binding Reaction of Human-Fetal Hemoglobin
no abstrac
Turkish journal of hematology, Jul 23, 2019
Thrombotic microangiopathies (TMAs) are multiple disease entities with different etiopathogeneses... more Thrombotic microangiopathies (TMAs) are multiple disease entities with different etiopathogeneses, characterized by thrombocytopenia, microangiopathic hemolytic anemia (MAHA) with schistocytosis, variable symptoms including fever, and multi-organ failure such as mild renal impairment and neurological deficits. The two paradigms of TMAs are represented on one hand by acquired thrombotic thrombocytopenic purpura (TTP) and on the other by hemolytic uremic syndrome (HUS). The differential diagnosis between these two paradigmatic forms of TMA is based on the presence of either frank renal failure in HUS or a severe deficiency (<10%) of the zincprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) in TTP. ADAMTS13 is an enzyme involved in the proteolytic processing of von Willebrand factor (vWF), and its deficiency results in formation of highmolecular-weight vWF-rich microthrombi in the environment of the microvasculature. The presence of these ultra-large vWF multimers in the microcirculation can recruit platelets, promoting multi-organ ischemic lesions. The presence of ADAMTS13 activity at >10% could rule out the presence of a TTP form. However, it is often difficult to differentiate either a TTP or HUS clinical scenario presenting with typical symptoms of TMA. There are in fact several additional diagnoses that should be considered in patients with ADAMTS13 activity of >10%. Widespread inflammation with endothelial damage and adverse reactions to drugs play a central role in the pathogenesis of several forms of TMA, and in these cases, the differential diagnosis should be directed at the underlying disease. Hence, a correct etiologic diagnosis of TMA should involve a critical illness, cancer-associated TMA, drug-induced TMA, and hematopoietic transplant-associated TMA. A complete assessment of all the possible etiologies for TMA symptoms, including acquired or congenital TTP, will allow for a more accurate diagnosis and application of a more appropriate treatment.
Journal of Clinical Medicine
Background. Regular treatment to prevent bleeding and consequent joint deterioration (prophylaxis... more Background. Regular treatment to prevent bleeding and consequent joint deterioration (prophylaxis) is the standard of care for persons with severe hemophilia A, traditionally based on intravenous infusions of the deficient clotting FVIII concentrates (CFCs). In recent years, extended half-life (EHL) CFCs and the non-replacement agent emicizumab, subcutaneously administered, have reduced the treatment burden. Methods. To compare and integrate the opinions on the different therapies available, eight hemophilia specialists were involved in drafting items of interest and relative statements through the Estimate-Talk-Estimate (ETE) method (“mini-Delphi”), in this way reaching consensus. Results. Eighteen items were identified, then harmonized to 10, and a statement was generated for each. These statements highlight the importance of personalized prophylaxis regimens. CFCs, particularly EHL products, seem more suitable for this, despite the challenging intravenous (i.v.) administration. L...
Marked von Willebrand factor and factor VIII elevations in severe acute respiratory syndrome coronavirus-2-positive, but not severe acute respiratory syndrome coronavirus-2-negative, pneumonia: a case–control study
Blood Coagulation & Fibrinolysis, 2021
Patients with novel coronavirus pneumonia show increased thrombotic risk. Although hemostatic alt... more Patients with novel coronavirus pneumonia show increased thrombotic risk. Although hemostatic alterations have been described in novel coronavirus pneumonia patients, case-control studies of von Willebrand factor (VWF), factor VIII (FVIII), and a disintegrin-like and metalloprotease with thrombospondin type I motif, member 13 (ADAMTS13) are lacking. VWF, ADAMTS13, FVIII, D-dimer, C-reactive protein, and routine blood cells and chemistry were measured in 10 novel coronavirus pneumonia patients and 10 non-novel coronavirus pneumonia controls. Hemostatic factors were measured less than 48 h of hospital admission in patients without invasive ventilation. D-Dimer, C-reactive protein, and fibrinogen concentrations, high in both groups, did not differ significantly in novel coronavirus pneumonia vs. non-novel coronavirus pneumonia patients. Median VWF-antigen (324 vs. 153 IU/dl, P < 0.0001), VWF-Rco (342 vs. 133 IU/dl, P < 0.001), and FVIII-activity levels (203 vs. 123 IU/dl, P < 0.0001) were significantly higher in novel coronavirus pneumonia cases vs. controls ADAMTS13-activity was normal in both groups. Coronavirus pneumonia cases vs. non-novel coronavirus pneumonia controls showed marked VWF/FVIII elevation, suggesting specific virus-induced endothelial activation causing VWF/FVIII hypersecretion, which may represent a therapeutic target in novel coronavirus pneumonia.
Journal of Clinical Medicine, 2020
Heparin-induced thrombocytopenia (HIT) is a prothrombotic immune drug reaction caused by platelet... more Heparin-induced thrombocytopenia (HIT) is a prothrombotic immune drug reaction caused by platelet-activating antibodies that in most instances recognize platelet factor 4 (PF4)/polyanion complexes. Platelet activation assays (i.e., functional assays) are more specific than immunoassays, since they are able to discern clinically relevant heparin-induced antibodies. All functional assays used for HIT diagnosis share the same principle, as they assess the ability of serum/plasma from suspected HIT patients to activate fresh platelets from healthy donors in the presence of several concentrations of heparin. Depending on the assay, donors’ platelets are stimulated either in whole blood (WB), platelet-rich plasma (PRP), or in a buffer medium (washed platelets, WP). In addition, the activation endpoint studied varies from one assay to another: platelet aggregation, membrane expression of markers of platelet activation, release of platelet granules. Tests with WP are more sensitive and sero...
Localization and Function of Platelet ADAMTS-13
Blood, 2005
The haemostatic activity of von Willebrand factor (VWF) is strongly dependent on its multimeric s... more The haemostatic activity of von Willebrand factor (VWF) is strongly dependent on its multimeric size, with the highest activity in ‘unusually large’ multimers (ULVWF) secreted from endothelial cells. The multimeric size is regulated by a plasma metalloprotease, ADAMTS-13. Since 15–25% of circulating VWF is stored in platelets, the presence and function of ADAMTS-13 in platelets could be an important issue to be investigated. In our study, we showed the presence of ADAMTS-13 in human platelets consistently with observations reported by Suzuki M. et al (Biochem Biophys Res Commun, 2004). Total mRNA isolated from platelet lysates was reverse-transcribed and amplified with specific oligonucleotide primers spanning from exon 2 to 5 of ADAMTS-13 cDNA. The nucleotide sequence of amplified fragment corresponded to ADAMTS-13 cDNA reported in the NCBI database. The immunoblot analysis performed on platelet lysates, using a monoclonal antibody against ADAMTS-13 CUB domains (13E2/75), revealed ...
COX-2 Inhibition Suppresses the Interferon-γ-Induced Expression of Indoleamine 2,3-Dioxygenase (IDO) in Human Leukemia Cell Lines
Blood, 2008
Avoidance of immunosurveillance is presently considered as a hallmark of cancer. Indoleamine 2,3-... more Avoidance of immunosurveillance is presently considered as a hallmark of cancer. Indoleamine 2,3-dioxygenase (IDO) is a cytosolic enzyme which metabolizes L-tryptophan to kynurenines and induces T cell suppression either directly on T cells, or by altering antigen presenting cell function and generating regulatory T cells (Treg). Recently, IDO expression by cancer cells and/or cancer-associated stromal cells has been correlated with the induction of immune dysfunction by human solid tumors, such as ovarian and colon cancer. It has been previously shown that IDO may be expressed by human acute myeloid leukemia, where it promotes the differentiation of Treg cells (Curti A et al. Blood2007;109:2871–7). Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins and exists in two isoforms, COX-1 and COX-2. COX-2 is over-expressed by several tumor types and reportedly affects multiple pathways involved in tumorigenesis, including angiogenesis, invasion, and tumor-...
![Research paper thumbnail of Increased platelet-fibrinogen affinity in patients with myeloproliferative disorders [see comments]](https://attachments.academia-assets.com/105129181/thumbnails/1.jpg)
](Blood, 1988
Patients with myeloproliferative disorders (MPD) are known to have some abnormalities of platelet... more Patients with myeloproliferative disorders (MPD) are known to have some abnormalities of platelet glycoproteins (Gp). Quantitative changes of the Gp Ib, IIb-IIIa, and/or their glucidic content have been reported. Since the Gp IIb-IIIa complex plays a major role in fibrinogen binding by activated platelets, we measured the platelet fibrinogen affinity in nine patients with polycythemia vera (PV) and one subject with chronic myeloid leukemia (CML) by the aggregometric method of Marguerie. In all patients the Kd of the platelet fibrinogen reaction was significantly decreased as compared to controls, with evidence in two cases with PV of a heterogeneity of platelet-fibrinogen receptor sites. The measurement of 125I-labeled fibrinogen-platelet binding, performed in seven patients (five PV and two CML), showed receptor populations with increased (Kd1 = 0.58 + 0.3 X 10(7) mol/L) and normal affinity (Kd2 = 5.12 + 3.1 X 10(7) mol/L). These results demonstrate a heterogeneity of platelet-fibr...
Blood, 1996
The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder originally described as a cl... more The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder originally described as a clinical triad of thrombocytopenia with small platelets, eczema, and immunodeficiency. Impaired CD43 glycoprotein expression on lymphocytes is a typical hallmark of this disorder. The CD43 gene is located on chromosome 16, and the WAS gene, WASP, was recently isolated from the chromosome X p11.22-p11.23. This gene, mutated in WAS patients, encodes a protein that is likely to play a role in controlling the expression of CD43. However, the molecular mechanism(s) causing WAS are not yet known. Herein, we describe a three- generation family in which clinical and laboratory WAS features were expressed in six of nine subjects available for study. At variance with classic X-linked WAS, this disorder was characterized by the presence of thrombocytopenia with a broad spectrum of platelet size, including giant platelets, and was inherited as an autosomal dominant trait. This last finding led us to h...
Thrombosis and Haemostasis, 1988
Inherited Macrothrombocytopenia with Distinctive Platelet Ultrastructural and Functional Features
Thrombosis and Haemostasis, 2000
SummaryWe report a family with inherited macrothrombocytopenia and characteristic large membrane ... more SummaryWe report a family with inherited macrothrombocytopenia and characteristic large membrane complexes in the platelets. Two affected subjects had platelet counts of 40 and 65X109/L respectively as assessed by contrast phase microscopy. Ultrastructural studies revealed giant spheroid platelets with characteristic large membrane complexes and/or giant vacuoles containing platelet organelles. Immunohistochemical studies of actin and tubulin showed a disorganization of the microtubule and actin systems. These abnormalities were absent in leukocytes, indicating a platelet-specific cytoskeleton disorder.Platelet autoantibodies were repeatedly absent. Nevertheless, in the peripheral blood we observed several figures of platelet phagocytosis by macrophages and neutrophils. The in vitro aggregometric response of platelets to ADP, collagen, thrombin, ristocetin was present, but shape change was absent. The urinary excretion of thromboxane A2 metabolites of the affected subjects were appr...
Thrombin Interaction with Platelet GPIB: Role of the Heparin Binding Domain
Thrombosis and Haemostasis, 1997
SummaryThe platelet membrane glycoprotein lb (Gplb) has a high affinity binding site for α-thromb... more SummaryThe platelet membrane glycoprotein lb (Gplb) has a high affinity binding site for α-thrombin whose occupancy is thought to positively modulate the thrombin-induced platelet activation. In this study, aimed at further characterizing the thrombin-GpIb interaction, two thrombin anion exosites referred to as “heparin binding site” (HBS) and “fibrino#gen recognition site” (FRS) were investigated as the possible domains involved in Gplb binding. The role of thrombin HBS was explored by performing binding measurements of 125I-α-thrombin to purified glycocalicin (GC), the extracytoplasmic portion of Gplb, in the presence of heparin as well as after chemical modifications of the thrombin heparin binding site (thrombin-HBS phosphopyridoxylation). These studies showed that a) thrombin binding to GC could be competitively inhibited by heparin and b) the equilibrium association constant for thrombin-GC interaction was reduced up to ten-fold by chemical modifications at the HBS. On the oth...
Oxidation of Human α-Thrombin by the Myeloperoxidase-H2O2-chloride System: Structural and Functional Effects
Thrombosis and Haemostasis, 2000
SummaryThe myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to gener... more SummaryThe myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to generate reactive oxygen species in many processes involved in the pathogenesis of inflammation and atherothrombosis. This study investigated the biochemical and functional effects of α-thrombin oxidation by MPOS. This system, in the presence of 100 µM L-tyrosine, caused in the thrombin molecule loss of tryptophan and lysine residues and formation of dityrosine, chloramine and carbonyl groups. The same changes could be directly induced by thrombin incubation with reagent HOCl, but not with H2O2 alone. Exposure to either MPOS or HOCl caused major functional abnormalities in human α-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being the kcat/Km value for Protein C hydrolysis roughly reduced 13-fold and the affinity for the antithrombin III-heparin complex decreased approximately 15-fold. Ox-thrombin int...
Plasma Protein Oxidation Is Associated with an Increase of Procoagulant Markers Causing an Imbalance between Pro- and Anticoagulant Pathways in Healthy Subjects
Thrombosis and Haemostasis, 2002
SummaryThe aim of the present study was to investigate whether the overall oxidation state of pla... more SummaryThe aim of the present study was to investigate whether the overall oxidation state of plasma proteins is associated with changes of circulating pro- and anticoagulant markers in healthy subjects (n = 99, 49 males, 50 females, aged from 6 to 91 yrs.). The carbonyl content of plasma proteins was measured and validated as an ex vivo index of the overall protein oxidation state due to its correlation with the plasma level of o-tyrosine (r = 0.87, P <0.0001), which is a well known oxidized product of L-phenylalanine. Using a multivariate analysis the carbonyl content of plasma protein was positively associated with procoagulant markers such as prothrombin F1 + 2 (r = 0.28, P = 0.0019) and fibrinopeptide A, (FpA) (r = 0.278, P = 0.003), as well as with the soluble derivative of the endothelial protein thrombomodulin (TM) (r = 0.469, P <0.0001). The procoagulant marker of thrombin activity, FpA, was significantly and positively correlated with the anticoagulant product of thr...
Blood transfusion = Trasfusione del sangue, May 1, 2018
Thrombelastography (TEG) and rotational thromboelastometry (ROTEM) are viscoelastic haemostatic a... more Thrombelastography (TEG) and rotational thromboelastometry (ROTEM) are viscoelastic haemostatic assays (VHA) which exploit the elastic properties of clotting blood. The aim of this systematic review and meta-analysis was to evaluate the usefulness of these tests in bleeding patients outside the cardiac surgical setting. We searched the Cochrane Library, MEDLINE, EMBASE and SCOPUS. We also searched clinical trial registries for ongoing and unpublished studies, and checked reference lists to identify additional studies. We found 4 randomised controlled trials (RCTs) that met our inclusion criteria with a total of 229 participants. The sample size was small (from 28 to 111 patients) and the follow-up periods very heterogenous (from 4 weeks to 3 years). Pooled data from the 3 trials reporting on mortality (199 participants) do not show any effect of the use of TEG on mortality as compared to standard monitoring (based on the average treatment effect from a fixed-effects model): Risk Rat...
Annals of the Rheumatic Diseases, 2013
TNFalpha, IFNgamma) using flowcytometry. Results: In 21 SSc patients with the diffuse form lower ... more TNFalpha, IFNgamma) using flowcytometry. Results: In 21 SSc patients with the diffuse form lower proportions of naive T-cells were found (SSc: 57.8%, HD: 64.2%). Significantly higher proportions of CCR7+ cells in CD4+ TEMRA were found in SSc patients (66.5%) compared to HD (50.4%). Patients with diffuse SSc had higher proportions of CD4+CCR6+RORgt+ T-cells (SSc: 4.8%, HD: 3.2%) with higher IL-17 production (5.6%) compared to HD (4.7%). SSc patients showed a higher production of TNFalpha (64.4%) compared to HD (54.6%), concomitantly expressing CCR7 (SSc: 46.1%, HD: 37.1%). There are nearly equal proportions of IFNgamma producing CD4 T-cells in SSc patients (10.3%) and HD (11.0%). Conclusions: Our preliminary results showed that SSc patients had a different cytokine profile in T-cells that includes abundance of CCR6+ and CCR7+ memory T-cells and more TNFalpha and IL-17 production. These findings may account for the chronic T-cell stimulation and inflammation in the pathogenesis in SSc.
Liver international : official journal of the International Association for the Study of the Liver, Aug 7, 2017
Internal and Emergency Medicine, 2017
The direct oral anticoagulants (DOACs) are drugs used in clinical practice since 2009 for the pre... more The direct oral anticoagulants (DOACs) are drugs used in clinical practice since 2009 for the prevention of stroke or systemic embolism in non-valvular atrial fibrillation, and for the treatment and secondary prevention of venous thromboembolism. The four DOACs, including the three factor Xa inhibitors (rivaroxaban, apixaban and edoxaban) and one direct thrombin inhibitor (dabigatran) provide oral anticoagulation therapy alternatives to Vitamin K antagonists (VKAs). Despite their clear advantages, the DOACs require on the part of the internist a thorough knowledge of their pharmacokinetic and pharmacodynamic characteristics to ensure their correct use, laboratory monitoring and the appropriate management of adverse events. This document represents a consensus paper on the use of DOACs by representatives of three Italian scientific societies: the Italian Society of Internal Medicine (SIMI), the Federation of the Associations of Hospital Managers (FADOI), and the Society for the Study of Haemostasis and Thrombosis (SISET). This document formulates expert opinion guidance for pragmatic managing, monitoring and reversing the anticoagulant effect of DOACs in both chronic and emergency settings. This practical guidance
Biochemistry, Feb 1, 1992
The amidase activity of human a-thrombin has been studied at steady state in the pH range 6-10, a... more The amidase activity of human a-thrombin has been studied at steady state in the pH range 6-10, as a function of NaCl concentration from 1 mM to 1 M and temperature from 10 to 40 OC. The Michaelis-Menten constant, K,, shows a bell-shaped dependence over this pH range with a minimum around pH 7.5 in the presence of 0.1 M NaCl at 25 OC. The catalytic constant, kat, also has a bell-shaped pH dependence with multiple inflection points that are more evident at low NaCl concentrations and a maximum around pH 8.2 in the presence of 0.1 M NaCl at 25 OC. A detailed analysis of the results in terms of a general linkage scheme has allowed a thorough characterization of the linkage between proton and substrate binding and its dependence on NaCl concentration, as well as the relevant entropic and enthalpic contributions to binding and catalytic events. Formulation of detailed partition functions for each enzyme intermediate involved in the catalytic cycle suggests that (at least) three groups are responsible for the control of thrombin amidase activity as a function of pH. One group is to be identified with the active site His, due to its pK values in the free enzyme and the adduct and its enthalpy of ionization. The effect of NaCl concentration on amidase activity seems to be extremely specific. Comparative steady-state measurements carried out in the presence of NaCl, NaBr, NaI, KC1, and MgClz show that human a-thrombin is capable of discriminating among different cations and anions. This suggests that small ions participate as allosteric effectors in the regulation of thrombin activity. The linkage with NaCl is strongly pH dependent and increases with decreasing pH. The present results provide information on the basic aspects of human a-thrombin activity and regulation and enable a rigorous thermodynamic approach to other important regulatory interactions in human a-thrombin and its structurally perturbed derivatives.
Effect of protons and chloride on the amidase activity in human α-thrombin
Thrombosis Research, 1991
Temperature-Dependence and PH-Dependence of the Oxygen-Binding Reaction of Human-Fetal Hemoglobin
no abstrac
Turkish journal of hematology, Jul 23, 2019
Thrombotic microangiopathies (TMAs) are multiple disease entities with different etiopathogeneses... more Thrombotic microangiopathies (TMAs) are multiple disease entities with different etiopathogeneses, characterized by thrombocytopenia, microangiopathic hemolytic anemia (MAHA) with schistocytosis, variable symptoms including fever, and multi-organ failure such as mild renal impairment and neurological deficits. The two paradigms of TMAs are represented on one hand by acquired thrombotic thrombocytopenic purpura (TTP) and on the other by hemolytic uremic syndrome (HUS). The differential diagnosis between these two paradigmatic forms of TMA is based on the presence of either frank renal failure in HUS or a severe deficiency (<10%) of the zincprotease ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) in TTP. ADAMTS13 is an enzyme involved in the proteolytic processing of von Willebrand factor (vWF), and its deficiency results in formation of highmolecular-weight vWF-rich microthrombi in the environment of the microvasculature. The presence of these ultra-large vWF multimers in the microcirculation can recruit platelets, promoting multi-organ ischemic lesions. The presence of ADAMTS13 activity at >10% could rule out the presence of a TTP form. However, it is often difficult to differentiate either a TTP or HUS clinical scenario presenting with typical symptoms of TMA. There are in fact several additional diagnoses that should be considered in patients with ADAMTS13 activity of >10%. Widespread inflammation with endothelial damage and adverse reactions to drugs play a central role in the pathogenesis of several forms of TMA, and in these cases, the differential diagnosis should be directed at the underlying disease. Hence, a correct etiologic diagnosis of TMA should involve a critical illness, cancer-associated TMA, drug-induced TMA, and hematopoietic transplant-associated TMA. A complete assessment of all the possible etiologies for TMA symptoms, including acquired or congenital TTP, will allow for a more accurate diagnosis and application of a more appropriate treatment.
Journal of Clinical Medicine
Background. Regular treatment to prevent bleeding and consequent joint deterioration (prophylaxis... more Background. Regular treatment to prevent bleeding and consequent joint deterioration (prophylaxis) is the standard of care for persons with severe hemophilia A, traditionally based on intravenous infusions of the deficient clotting FVIII concentrates (CFCs). In recent years, extended half-life (EHL) CFCs and the non-replacement agent emicizumab, subcutaneously administered, have reduced the treatment burden. Methods. To compare and integrate the opinions on the different therapies available, eight hemophilia specialists were involved in drafting items of interest and relative statements through the Estimate-Talk-Estimate (ETE) method (“mini-Delphi”), in this way reaching consensus. Results. Eighteen items were identified, then harmonized to 10, and a statement was generated for each. These statements highlight the importance of personalized prophylaxis regimens. CFCs, particularly EHL products, seem more suitable for this, despite the challenging intravenous (i.v.) administration. L...
Marked von Willebrand factor and factor VIII elevations in severe acute respiratory syndrome coronavirus-2-positive, but not severe acute respiratory syndrome coronavirus-2-negative, pneumonia: a case–control study
Blood Coagulation & Fibrinolysis, 2021
Patients with novel coronavirus pneumonia show increased thrombotic risk. Although hemostatic alt... more Patients with novel coronavirus pneumonia show increased thrombotic risk. Although hemostatic alterations have been described in novel coronavirus pneumonia patients, case-control studies of von Willebrand factor (VWF), factor VIII (FVIII), and a disintegrin-like and metalloprotease with thrombospondin type I motif, member 13 (ADAMTS13) are lacking. VWF, ADAMTS13, FVIII, D-dimer, C-reactive protein, and routine blood cells and chemistry were measured in 10 novel coronavirus pneumonia patients and 10 non-novel coronavirus pneumonia controls. Hemostatic factors were measured less than 48 h of hospital admission in patients without invasive ventilation. D-Dimer, C-reactive protein, and fibrinogen concentrations, high in both groups, did not differ significantly in novel coronavirus pneumonia vs. non-novel coronavirus pneumonia patients. Median VWF-antigen (324 vs. 153 IU/dl, P < 0.0001), VWF-Rco (342 vs. 133 IU/dl, P < 0.001), and FVIII-activity levels (203 vs. 123 IU/dl, P < 0.0001) were significantly higher in novel coronavirus pneumonia cases vs. controls ADAMTS13-activity was normal in both groups. Coronavirus pneumonia cases vs. non-novel coronavirus pneumonia controls showed marked VWF/FVIII elevation, suggesting specific virus-induced endothelial activation causing VWF/FVIII hypersecretion, which may represent a therapeutic target in novel coronavirus pneumonia.
Journal of Clinical Medicine, 2020
Heparin-induced thrombocytopenia (HIT) is a prothrombotic immune drug reaction caused by platelet... more Heparin-induced thrombocytopenia (HIT) is a prothrombotic immune drug reaction caused by platelet-activating antibodies that in most instances recognize platelet factor 4 (PF4)/polyanion complexes. Platelet activation assays (i.e., functional assays) are more specific than immunoassays, since they are able to discern clinically relevant heparin-induced antibodies. All functional assays used for HIT diagnosis share the same principle, as they assess the ability of serum/plasma from suspected HIT patients to activate fresh platelets from healthy donors in the presence of several concentrations of heparin. Depending on the assay, donors’ platelets are stimulated either in whole blood (WB), platelet-rich plasma (PRP), or in a buffer medium (washed platelets, WP). In addition, the activation endpoint studied varies from one assay to another: platelet aggregation, membrane expression of markers of platelet activation, release of platelet granules. Tests with WP are more sensitive and sero...
Localization and Function of Platelet ADAMTS-13
Blood, 2005
The haemostatic activity of von Willebrand factor (VWF) is strongly dependent on its multimeric s... more The haemostatic activity of von Willebrand factor (VWF) is strongly dependent on its multimeric size, with the highest activity in ‘unusually large’ multimers (ULVWF) secreted from endothelial cells. The multimeric size is regulated by a plasma metalloprotease, ADAMTS-13. Since 15–25% of circulating VWF is stored in platelets, the presence and function of ADAMTS-13 in platelets could be an important issue to be investigated. In our study, we showed the presence of ADAMTS-13 in human platelets consistently with observations reported by Suzuki M. et al (Biochem Biophys Res Commun, 2004). Total mRNA isolated from platelet lysates was reverse-transcribed and amplified with specific oligonucleotide primers spanning from exon 2 to 5 of ADAMTS-13 cDNA. The nucleotide sequence of amplified fragment corresponded to ADAMTS-13 cDNA reported in the NCBI database. The immunoblot analysis performed on platelet lysates, using a monoclonal antibody against ADAMTS-13 CUB domains (13E2/75), revealed ...
COX-2 Inhibition Suppresses the Interferon-γ-Induced Expression of Indoleamine 2,3-Dioxygenase (IDO) in Human Leukemia Cell Lines
Blood, 2008
Avoidance of immunosurveillance is presently considered as a hallmark of cancer. Indoleamine 2,3-... more Avoidance of immunosurveillance is presently considered as a hallmark of cancer. Indoleamine 2,3-dioxygenase (IDO) is a cytosolic enzyme which metabolizes L-tryptophan to kynurenines and induces T cell suppression either directly on T cells, or by altering antigen presenting cell function and generating regulatory T cells (Treg). Recently, IDO expression by cancer cells and/or cancer-associated stromal cells has been correlated with the induction of immune dysfunction by human solid tumors, such as ovarian and colon cancer. It has been previously shown that IDO may be expressed by human acute myeloid leukemia, where it promotes the differentiation of Treg cells (Curti A et al. Blood2007;109:2871–7). Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins and exists in two isoforms, COX-1 and COX-2. COX-2 is over-expressed by several tumor types and reportedly affects multiple pathways involved in tumorigenesis, including angiogenesis, invasion, and tumor-...
Blood, 1988
Patients with myeloproliferative disorders (MPD) are known to have some abnormalities of platelet... more Patients with myeloproliferative disorders (MPD) are known to have some abnormalities of platelet glycoproteins (Gp). Quantitative changes of the Gp Ib, IIb-IIIa, and/or their glucidic content have been reported. Since the Gp IIb-IIIa complex plays a major role in fibrinogen binding by activated platelets, we measured the platelet fibrinogen affinity in nine patients with polycythemia vera (PV) and one subject with chronic myeloid leukemia (CML) by the aggregometric method of Marguerie. In all patients the Kd of the platelet fibrinogen reaction was significantly decreased as compared to controls, with evidence in two cases with PV of a heterogeneity of platelet-fibrinogen receptor sites. The measurement of 125I-labeled fibrinogen-platelet binding, performed in seven patients (five PV and two CML), showed receptor populations with increased (Kd1 = 0.58 + 0.3 X 10(7) mol/L) and normal affinity (Kd2 = 5.12 + 3.1 X 10(7) mol/L). These results demonstrate a heterogeneity of platelet-fibr...
Blood, 1996
The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder originally described as a cl... more The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder originally described as a clinical triad of thrombocytopenia with small platelets, eczema, and immunodeficiency. Impaired CD43 glycoprotein expression on lymphocytes is a typical hallmark of this disorder. The CD43 gene is located on chromosome 16, and the WAS gene, WASP, was recently isolated from the chromosome X p11.22-p11.23. This gene, mutated in WAS patients, encodes a protein that is likely to play a role in controlling the expression of CD43. However, the molecular mechanism(s) causing WAS are not yet known. Herein, we describe a three- generation family in which clinical and laboratory WAS features were expressed in six of nine subjects available for study. At variance with classic X-linked WAS, this disorder was characterized by the presence of thrombocytopenia with a broad spectrum of platelet size, including giant platelets, and was inherited as an autosomal dominant trait. This last finding led us to h...
Thrombosis and Haemostasis, 1988
Inherited Macrothrombocytopenia with Distinctive Platelet Ultrastructural and Functional Features
Thrombosis and Haemostasis, 2000
SummaryWe report a family with inherited macrothrombocytopenia and characteristic large membrane ... more SummaryWe report a family with inherited macrothrombocytopenia and characteristic large membrane complexes in the platelets. Two affected subjects had platelet counts of 40 and 65X109/L respectively as assessed by contrast phase microscopy. Ultrastructural studies revealed giant spheroid platelets with characteristic large membrane complexes and/or giant vacuoles containing platelet organelles. Immunohistochemical studies of actin and tubulin showed a disorganization of the microtubule and actin systems. These abnormalities were absent in leukocytes, indicating a platelet-specific cytoskeleton disorder.Platelet autoantibodies were repeatedly absent. Nevertheless, in the peripheral blood we observed several figures of platelet phagocytosis by macrophages and neutrophils. The in vitro aggregometric response of platelets to ADP, collagen, thrombin, ristocetin was present, but shape change was absent. The urinary excretion of thromboxane A2 metabolites of the affected subjects were appr...
Thrombin Interaction with Platelet GPIB: Role of the Heparin Binding Domain
Thrombosis and Haemostasis, 1997
SummaryThe platelet membrane glycoprotein lb (Gplb) has a high affinity binding site for α-thromb... more SummaryThe platelet membrane glycoprotein lb (Gplb) has a high affinity binding site for α-thrombin whose occupancy is thought to positively modulate the thrombin-induced platelet activation. In this study, aimed at further characterizing the thrombin-GpIb interaction, two thrombin anion exosites referred to as “heparin binding site” (HBS) and “fibrino#gen recognition site” (FRS) were investigated as the possible domains involved in Gplb binding. The role of thrombin HBS was explored by performing binding measurements of 125I-α-thrombin to purified glycocalicin (GC), the extracytoplasmic portion of Gplb, in the presence of heparin as well as after chemical modifications of the thrombin heparin binding site (thrombin-HBS phosphopyridoxylation). These studies showed that a) thrombin binding to GC could be competitively inhibited by heparin and b) the equilibrium association constant for thrombin-GC interaction was reduced up to ten-fold by chemical modifications at the HBS. On the oth...
Oxidation of Human α-Thrombin by the Myeloperoxidase-H2O2-chloride System: Structural and Functional Effects
Thrombosis and Haemostasis, 2000
SummaryThe myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to gener... more SummaryThe myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to generate reactive oxygen species in many processes involved in the pathogenesis of inflammation and atherothrombosis. This study investigated the biochemical and functional effects of α-thrombin oxidation by MPOS. This system, in the presence of 100 µM L-tyrosine, caused in the thrombin molecule loss of tryptophan and lysine residues and formation of dityrosine, chloramine and carbonyl groups. The same changes could be directly induced by thrombin incubation with reagent HOCl, but not with H2O2 alone. Exposure to either MPOS or HOCl caused major functional abnormalities in human α-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being the kcat/Km value for Protein C hydrolysis roughly reduced 13-fold and the affinity for the antithrombin III-heparin complex decreased approximately 15-fold. Ox-thrombin int...
Plasma Protein Oxidation Is Associated with an Increase of Procoagulant Markers Causing an Imbalance between Pro- and Anticoagulant Pathways in Healthy Subjects
Thrombosis and Haemostasis, 2002
SummaryThe aim of the present study was to investigate whether the overall oxidation state of pla... more SummaryThe aim of the present study was to investigate whether the overall oxidation state of plasma proteins is associated with changes of circulating pro- and anticoagulant markers in healthy subjects (n = 99, 49 males, 50 females, aged from 6 to 91 yrs.). The carbonyl content of plasma proteins was measured and validated as an ex vivo index of the overall protein oxidation state due to its correlation with the plasma level of o-tyrosine (r = 0.87, P <0.0001), which is a well known oxidized product of L-phenylalanine. Using a multivariate analysis the carbonyl content of plasma protein was positively associated with procoagulant markers such as prothrombin F1 + 2 (r = 0.28, P = 0.0019) and fibrinopeptide A, (FpA) (r = 0.278, P = 0.003), as well as with the soluble derivative of the endothelial protein thrombomodulin (TM) (r = 0.469, P <0.0001). The procoagulant marker of thrombin activity, FpA, was significantly and positively correlated with the anticoagulant product of thr...
Blood transfusion = Trasfusione del sangue, May 1, 2018
Thrombelastography (TEG) and rotational thromboelastometry (ROTEM) are viscoelastic haemostatic a... more Thrombelastography (TEG) and rotational thromboelastometry (ROTEM) are viscoelastic haemostatic assays (VHA) which exploit the elastic properties of clotting blood. The aim of this systematic review and meta-analysis was to evaluate the usefulness of these tests in bleeding patients outside the cardiac surgical setting. We searched the Cochrane Library, MEDLINE, EMBASE and SCOPUS. We also searched clinical trial registries for ongoing and unpublished studies, and checked reference lists to identify additional studies. We found 4 randomised controlled trials (RCTs) that met our inclusion criteria with a total of 229 participants. The sample size was small (from 28 to 111 patients) and the follow-up periods very heterogenous (from 4 weeks to 3 years). Pooled data from the 3 trials reporting on mortality (199 participants) do not show any effect of the use of TEG on mortality as compared to standard monitoring (based on the average treatment effect from a fixed-effects model): Risk Rat...
Annals of the Rheumatic Diseases, 2013
TNFalpha, IFNgamma) using flowcytometry. Results: In 21 SSc patients with the diffuse form lower ... more TNFalpha, IFNgamma) using flowcytometry. Results: In 21 SSc patients with the diffuse form lower proportions of naive T-cells were found (SSc: 57.8%, HD: 64.2%). Significantly higher proportions of CCR7+ cells in CD4+ TEMRA were found in SSc patients (66.5%) compared to HD (50.4%). Patients with diffuse SSc had higher proportions of CD4+CCR6+RORgt+ T-cells (SSc: 4.8%, HD: 3.2%) with higher IL-17 production (5.6%) compared to HD (4.7%). SSc patients showed a higher production of TNFalpha (64.4%) compared to HD (54.6%), concomitantly expressing CCR7 (SSc: 46.1%, HD: 37.1%). There are nearly equal proportions of IFNgamma producing CD4 T-cells in SSc patients (10.3%) and HD (11.0%). Conclusions: Our preliminary results showed that SSc patients had a different cytokine profile in T-cells that includes abundance of CCR6+ and CCR7+ memory T-cells and more TNFalpha and IL-17 production. These findings may account for the chronic T-cell stimulation and inflammation in the pathogenesis in SSc.
Liver international : official journal of the International Association for the Study of the Liver, Aug 7, 2017
Internal and Emergency Medicine, 2017
The direct oral anticoagulants (DOACs) are drugs used in clinical practice since 2009 for the pre... more The direct oral anticoagulants (DOACs) are drugs used in clinical practice since 2009 for the prevention of stroke or systemic embolism in non-valvular atrial fibrillation, and for the treatment and secondary prevention of venous thromboembolism. The four DOACs, including the three factor Xa inhibitors (rivaroxaban, apixaban and edoxaban) and one direct thrombin inhibitor (dabigatran) provide oral anticoagulation therapy alternatives to Vitamin K antagonists (VKAs). Despite their clear advantages, the DOACs require on the part of the internist a thorough knowledge of their pharmacokinetic and pharmacodynamic characteristics to ensure their correct use, laboratory monitoring and the appropriate management of adverse events. This document represents a consensus paper on the use of DOACs by representatives of three Italian scientific societies: the Italian Society of Internal Medicine (SIMI), the Federation of the Associations of Hospital Managers (FADOI), and the Society for the Study of Haemostasis and Thrombosis (SISET). This document formulates expert opinion guidance for pragmatic managing, monitoring and reversing the anticoagulant effect of DOACs in both chronic and emergency settings. This practical guidance