Freda Passam

Protein disulfide isomerase inhibitors constitute a new class of antithrombotic agents.

Thrombosis, or blood clot formation, and its sequelae remain a leading cause of morbidity and mortality, and recurrent thrombosis is common despite current optimal therapy. Protein disulfide isomerase (PDI) is an oxidoreductase that has recently been shown to participate in thrombus formation. While currently available antithrombotic agents inhibit either platelet aggregation or fibrin generation, inhibition of secreted PDI blocks the earliest stages of thrombus formation, suppressing both pathways. Here, we explored extracellular PDI as an alternative target of antithrombotic therapy. A high-throughput screen identified quercetin-3-rutinoside as an inhibitor of PDI reductase activity in vitro. Inhibition of PDI was selective, as quercetin-3-rutinoside failed to inhibit the reductase activity of several other thiol isomerases found in the vasculature. Cellular assays showed that quercetin-3-rutinoside inhibited aggregation of human and mouse platelets and endothelial cell-mediated fibrin generation in human endothelial cells. Using intravital microscopy in mice, we demonstrated that quercetin-3-rutinoside blocks thrombus formation in vivo by inhibiting PDI. Infusion of recombinant PDI reversed the antithrombotic effect of quercetin-3-rutinoside. Thus, PDI is a viable target for small molecule inhibition of thrombus formation, and its inhibition may prove to be a useful adjunct in refractory thrombotic diseases that are not controlled with conventional antithrombotic agents.

How we diagnose the antiphospholipid syndrome

The antiphospholipid syndrome (APS) is an acquired thrombophilia, characterized by the occurrence of venous and arterial events. This article examines the laboratory and key clinical aspects of APS. Particular focus is given to anti-beta 2-glycoprotein I (beta(2)GPI) antibodies in view of their recent inclusion in the APS classification criteria. The clinical utility of using the beta(2)GPI enzyme-linked immunosorbent assay, in conjunction with the established lupus anticoagulant assays and cardiolipin enzyme-linked immunosorbent assay, for diagnosing and risk stratifying patients suspected of having APS is discussed. The relative importance of the various assays in diagnosing obstetric APS (early and late gestation miscarriages) is explored. The implications of recent epidemiologic findings for possibly understanding the underlying pathophysiologic mechanisms of obstetric APS are highlighted. Insights into which patients with obstetric APS may be at most risk of thrombotic complications are presented.

Monocyte tissue factor-dependent activation of coagulation in hypercholesterolemic mice and monkeys is inhibited by simvastatin.

Hypercholesterolemia is a major risk factor for atherosclerosis. It also is associated with platelet hyperactivity, which increases morbidity and mortality from cardiovascular disease. However, the mechanisms by which hypercholesterolemia produces a procoagulant state remain undefined. Atherosclerosis is associated with accumulation of oxidized lipoproteins within atherosclerotic lesions. Small quantities of oxidized lipoproteins are also present in the circulation of patients with coronary artery disease. We therefore hypothesized that hypercholesterolemia leads to elevated levels of oxidized LDL (oxLDL) in plasma and that this induces expression of the procoagulant protein tissue factor (TF) in monocytes. In support of this hypothesis, we report here that oxLDL induced TF expression in human monocytic cells and monocytes. In addition, patients with familial hypercholesterolemia had elevated levels of plasma microparticle (MP) TF activity. Furthermore, a high-fat diet induced a time-dependent increase in plasma MP TF activity and activation of coagulation in both LDL receptor-deficient mice and African green monkeys. Genetic deficiency of TF in bone marrow cells reduced coagulation in hypercholesterolemic mice, consistent with a major role for monocyte-derived TF in the activation of coagulation. Similarly, a deficiency of either TLR4 or TLR6 reduced levels of MP TF activity. Simvastatin treatment of hypercholesterolemic mice and monkeys reduced oxLDL, monocyte TF expression, MP TF activity, activation of coagulation, and inflammation, without affecting total cholesterol levels. Our results suggest that the prothrombotic state associated with hypercholesterolemia is caused by oxLDL-mediated induction of TF expression in monocytes via engagement of a TLR4/TLR6 complex.

Levels of serum cytokines and acute phase proteins in patients with essential and cancer-related thrombocytosis.

&NA; Essential thrombocytosis (ET) is a myeloproliferative disorder resulting in an increased production of abnormal platelets. Reactive thrombocytosis (RT) is occasionally observed in clinical situations including chronic inflammation and malignancy. The aim of the present study was to evaluate the discriminatory efficiency of various laboratory tests in patients with ET and cancer‐related RT. Forty‐five patients with ET, 52 patients with RT, and 25 age‐matched normal individuals comprised the study population. Plasma interleukin‐1 alpha (IL‐1a), IL‐2, IL‐6, tumor necrosis factor alpha (TNF‐a), platelets, hematocrit, hemoglobin, erythrocyte sedimentation rate (ESR), C‐reactive protein (CRP), lactate dehydrogenase (LDH) and ferritin were determined. We found increased levels of ferritin, LDH, CRP, ESR, IL‐1a, and IL‐6 in RT compared with ET (p < 0.01 to p < 0.0005). Hemoglobin, hematocrit, and platelets were significantly lower in RT than in ET (p < 0.0005). Furthermore, ferritin and ESR were negatively correlated with Hct, hemoglobin, and TNF‐a, whereas ferritin was positively correlated with ESR, IL‐1a, IL‐6, and CRP, and IL‐1a was positively correlated with IL‐6. We consider that the aforementioned parameters should be included in the investigation of unexplained thrombocytosis for the differentiation of essential from cancer related thrombocytosis.

Naturally occurring free thiols within β2-glycoprotein I in vivo: nitrosylation, redox modification by endothelial cells, and regulation of oxidative stress-induced cell injury

β2-Glycoprotein I (β2GPI) is an evolutionary conserved, abundant circulating protein. Although its function remains uncertain, accumulated evidence points toward interactions with endothelial cells and components of the coagulation system, suggesting a regulatory role in vascular biology. Our group has shown that thioredoxin 1 (TRX-1) generates free thiols in β2GPI, a process that may have a regulatory role in platelet adhesion. This report extends these studies and shows for the first time evidence of β2GPI with free thiols in vivo in both multiple human and murine serum samples. To explore how the vascular surface may modulate the redox status of β2GPI, unstimulated human endothelial cells and EAhy926 cells are shown to be capable of amplifying the effect of free thiol generation within β2GPI. Multiple oxidoreductase enzymes, such as endoplasmic reticulum protein 46 (ERp 46) and TRX-1 reductase, in addition to protein disulfide isomerase are secreted on the surface of endothelial cells. Furthermore, one or more of these generated free thiols within β2GPI are also shown to be nitrosylated. Finally, the functional significance of these findings is explored, by showing that free thiol-containing β2GPI has a powerful effect in protecting endothelial cells and EAhy926 cells from oxidative stress-induced cell death.

Activated peripheral blood and endothelial cells in thalassemia patients

Abstract. Background and objectives: Thalassemia patients have alterations in the expression of some activation and adhesion molecules on peripheral blood lymphocytes. We studied cell surface antigens on peripheral blood cells associated with the activation of these cells and soluble molecules produced by activated endothelium. Design and methods: We investigated the expression of CD11b, CD18, CD35, CD43, CD44, and CD69 on the peripheral blood monocytes, Cd11b, CD18, CD35, CD43, CD44, CD67 on peripheral blood neutrophils and CD38 and CD69 on peripheral blood lymphocytes. We studied 68 transfusion-dependent thalassemics (group A),10 transfusion non-dependent thalassemics (group B), 18 β-thalassemia carriers (group C), and 28 normal individuals. Relative fluorescence intensity was used to determine the antigen density. Analysis was performed with an EPICS ELITE flow cytometer. Furthermore, soluble intercelullar adhesion molecule 1 (sICAM-1), soluble vascular adhesion molecule 1 (sVCAM-1), and E-selectin, tumor necrosis factor (TNF) α, and interleukin (IL) 1β were measured in the plasma of patients by enzyme-linked immunometric assay. Results: The expression of CD11b, CD18, and CD69 on the monocytes of group A was significantly greater than in groups B and C and in controls, while CD44 was significantly downregulated in group A. CD11b, CD18, CD35, CD44, and CD67 on the surface of neutrophils and CD38 and CD69 on the surface of lymphocytes were also overexpressed in group A. CD44 was downregulated on the monocytes and upregulated on the neutrophils of the patients compared to controls. The levels of sICAM-1, sVCAM-1, E-selectin, TNF-α, and IL-1β in the serum of patients in groups A and B were higher than those in group C and the controls. Conclusion: Endothelial activation markers are significantly increased in thalassemia patients, and activated blood cells circulate in the peripheral blood. These may be related to the vascular complications in these patients and might be useful markers for the follow-up of the vascular disease.

The relation between bone marrow angiogenesis and the proliferation index Ki-67 in multiple myeloma

Aim: Angiogenesis correlates with disease progression in various haematological malignancies. This study investigated the association between microvascular density (MVD) and the Ki-67 proliferation index (Ki-67 PI), bone marrow infiltration, and C reactive protein (CRP) in patients with multiple myeloma. Methods: Bone marrow MVD was examined in 44 biopsies at diagnosis and 15 in plateau phase by immunostaining the endothelial cells with a monoclonal antibody to CD34. The Ki-67 PI was evaluated by a double immunostaining technique using the monoclonal antibodies MIB-1 and CD38. Results: MVD, Ki-67 PI, bone marrow infiltration, and CRP were significantly higher in pretreatment patients than in controls and decreased in patients achieving plateau phase. MVD significantly correlated with Ki-67 PI and infiltration, and Ki-67 correlated with infiltration. Conclusion: In multiple myeloma, apart from being a marker of proliferative activity, Ki-67 is also associated with bone marrow angiogenesis and tumour burden.

Beta 2 glycoprotein I is a substrate of thiol oxidoreductases

To the editor: Beta 2 glycoprotein I (β2GPI) is an abundant plasma protein recognized as the major autoantigen in the antiphospholipid syndrome. Although the crystal structure of β2GPI has been resolved,[1][1],[2][2] its normal function remains unknown. We have been intrigued by the presence of a

Redox control of β2-glycoprotein I-von Willebrand factor interaction by thioredoxin-1.

Summary.  Background: β2‐Glycoprotein I (β2GPI) is an abundant plasma protein that is closely linked to blood clotting, as it interacts with various protein and cellular components of the coagulation system. However, the role of β2GPI in thrombus formation is unknown. We have recently shown that β2GPI is susceptible to reduction by the thiol oxidoreductases thioredoxin‐1 and protein disulfide isomerase, and that reduction of β2GPI can take place on the platelet surface. Methods: β2GPI, reduced by thioredoxin‐1, was labeled with the selective sulfhydryl probe Na‐(3‐maleimidylpropionyl)biocytin and subjected to mass spectrometry to identify the specific cysteines involved in the thiol exchange reaction. Binding assays were used to examine the affinity of reduced β2GPI for von Willebrand factor (VWF) and the effect of reduced β2GPI on glycoprotein (GP)Ibα binding to VWF. Platelet adhesion to ristocetin‐activated VWF was studied in the presence of reduced β2GPI. Results:  We demonstrate that the Cys288–Cys326 disulfide in domain V of β2GPI is the predominant disulfide reduced by thioredoxin‐1. Reduced β2GPI in vitro displays increased binding to VWF that is dependent on disulfide bond formation. β2GPI reduced by thioredoxin‐1, in comparison with non‐reduced β2GPI, leads to increased binding of GPIbα to VWF and increased platelet adhesion to activated VWF. Conclusions:  Given the importance of thiol oxidoreductases in thrombus formation, we provide preliminary evidence that the thiol‐dependent interaction of β2GPI with VWF may contribute to the redox regulation of platelet adhesion.

Molecular pathophysiology of the antiphospholipid syndrome: the role of oxidative post‐translational modification of beta 2 glycoprotein I

Summary.  It has been well established that antiphospholipid antibodies and specifically those directed against beta 2 glycoprotein I (β2GPI) are pathogenic for the development of thrombosis in the antiphospholipid syndrome (APS). Several groups have shown that anti‐β2GPI antibodies, in complex with β2GPI, elicit effects on blood cells and coagulation‐fibrinolysis proteins, which prime the arterial and venous vasculature for the development of thrombosis. However, much less is known about the mechanism initiating the production of autoantibodies against β2GPI, a physiological abundant protein of blood. In the current review, novel findings are presented regarding the structure and oxidative post‐translational modifications of β2GPI, which trigger the immune response. The majority of circulating β2GPI exists in a form containing unpaired cysteines (free thiols), which constitutes the reduced form of β2GPI. The free thiols exposed on β2GPI are involved in the interaction with platelets and endothelial cells. We propose that this abundant pool of free thiols may serve as an antioxidant reservoir protecting cells or critical molecules from oxidative stress. Oxidation of β2GPI confers an increase in its immunogenicity through a Th1 immunological mechanism. The clinical significance of these observations is that serum from patients with APS, assessed by a novel ELISA assay, have a significant increase in oxidised β2GPI. These findings hold promise, not only for the delineation of the role of β2GPI as an immunological target, but also for the development of improved diagnostic and prognostic assays for APS.