Carlos J. Bidot

ENDOTHELIAL MICROPARTICLES (EMP) BIND AND ACTIVATE MONOCYTES: ELEVATED EMP- MONOCYTE CONJUGATES IN MULTIPLE SCLEROSIS

Elevated plasma endothelial microparticles (EMP) have been documented in MS during exacerbation. However, the role of EMP in pathogenesis of MS remains unclear. We investigated the formation of EMP-monocyte conjugates (EMP-MoC) and their potential role in transendothelial migration of inflammatory cells in MS. EMP-MoC were assayed in 30 MS patients in exacerbation, 20 in remission and in 35 controls. EMP-leukocyte conjugation was investigated flowcytometrically by employing alpha-CD54 or alpha-CD62E for EMP, and alpha-CD45 for leukocytes. EMP-MoC were characterized by identifying adhesion molecules involved and their effect on monocyte function. In vivo (clinical): EMP-MoC were markedly elevated in exacerbation vs. remission and controls, correlating with presence of GD+ MRI lesions. Free CD54+ EMP were not elevated but free CD62E+ EMP were. In vitro: EMP bound preferentially to monocytes, less to neutrophils, but little to lymphocytes. Bound EMP activated monocytes: CD11b expression increased 50% and migration through cerebral endothelial cell layer increased 2.6-fold. Blockade of CD54 reduced binding by 80%. Most CD54+ EMP bound to monocytes, leaving little free EMP, while CD62+ EMP were found both free and bound. These results demonstrated that phenotypic subsets of EMP interacted differently with monocytes. Based on our observations, EMP may enhance inflammation and increase transendothelial migration of monocytes in MS by binding to and activating monocytes through CD54. EMP-MoC were markedly increased in MS patients in exacerbation compared to remission and may serve as a sensitive marker of MS disease activity.

Cell‐Derived Microparticles and Exosomes in Neuroinflammatory Disorders

All blood cells and the vascular endothelium shed microparticles (MP) from their plasma membranes when suitably stimulated, and assay of MP in patient blood has found increasing application to the monitoring of disease states. In addition, mounting evidence suggests that MP are not mere epiphenomena but play significant roles in the pathophysiology of thromboses, inflammation, and cancers. This chapter endeavors to summarize the limited number of studies thus far done on MP in neurological disorders such as multiple sclerosis (MS), transient ischemic attacks, and the neurological manifestations of antiphospholipid syndrome (APS). In addition, the chapter offers some plausible hypotheses on possible roles of MP in the pathophsyiology of these disorders, chiefly, the hypothesis that MP are indeed important participants in some neuropathologies, especially those which are ischemic in nature, but probably also inflammatory ones. The chapter also goes over the history and general principles of MP studies (e.g., assay methods and pitfalls), comparison with alternative methods (e.g., soluble markers of disease states), subclasses of MP (such as exosomes), and other topics aimed at helping readers to consider MP studies in their own clinical fields. Tables include a listing of bioactive agents known to be carried on MP, many of which were heretofore considered strictly soluble, and some of which can be transferred from cell to cell via MP vectors, for example certain cytokine receptors.

Endothelial microparticles (EMP) as vascular disease markers.

Publisher Summary Endothelial cells (ECs) have long been implicated in the pathogenesis and pathophysiology of a myriad of vascular, thrombotic, and inflammatory disorders. The prevalence of endothelial perturbation in vascular disease has underscored the need for noninvasive sensitive and specific markers to monitor endothelial status. The popularity of endothelial microparticles (EMPs) as markers of perturbed has increased because of its promising clinical applications in the area of noninvasive EC monitoring. An overview of the main aspects of endothelial dysfunction as they relate to thrombogenesis and inflammation is important to provide a background of the factors affecting EMP release and their phenotype. General properties of microparticles such as mechanisms of microparticles generation and antigenic characterization of EMP are also discussed in this chapter. EMPs have emerged as a preferred direct method for assessing EC injury in different disorders. EMP analysis could provide insight into the actual status of the endothelium in vivo by a simple blood analysis. The main challenge remains in the selection of specific and sensitive monoclonal antibodies that may yield consistent results among different laboratories. Clinical endothelial research has been hampered by the difficulty of accessing and sampling ECs in a noninvasive manner. EMPs in clinical disease such as cardiovascular disease, diabetes mellitus, hypertension and preeclampsia, inflammatory and infectious disease, autoimmune disorders, hematological disorders, and neurological disease are also discussed in this chapter.

Factor VII/VIIa: a new antigen in the anti‐phospholipid antibody syndrome

Summary. We investigated antibodies to factor VII/VIIa (FVII/VIIa) and five other common target antigens in 33 patients with a history of anti‐phospholipid syndrome (APS) and 50 healthy controls using an enzyme‐linked immunosorbent assay (ELISA) technique. We found that antibody to FVII/VIIa, a previously unrecognized and common antigen in APS, was present in 67% of patients. Frequencies of antibodies to other target antigens were: anti‐beta‐2 glycoprotein 1 (anti‐β2GP1), 88%; anti‐cardiolipin (anti‐CL), 76%; anti‐phosphatidylethanolamine (anti‐PE), 67%; anti‐phosphatidylserine (anti‐PS), 64%; and anti‐phosphatidylcholine (anti‐PC), 59%. Most patients had antibodies against multiple antigens, but a few were positive for only anti‐β2GP1 (12%) or anti‐CL (3%). Positivity for anti‐FVII/VIIa was significantly associated with positivity for anti‐PE, anti‐PS and/or anti‐PC (P < 0·05) but not anti‐β2GP1. When frequencies of immunoglobulin G (IgG) versus immunoglobulin M (IgM) antibodies were compared, anti‐β2GP1 IgG correlated with the lupus anticoagulant (P < 0·05) and was significantly more prevalent than IgM, but the reverse was seen for all other antigens. In arterial thrombosis, IgM was more prevalent for all antigens, and was significantly associated with FVII/VIIa, PE and PS, whereas in venous thrombosis, IgG was frequently prevalent, especially in association with FVII/VIIa, β2GP1 and CL. In summary, FVII/VIIa is a new and common antigen in APS. Anti‐FVII/VIIa is often associated with anti‐PE, anti‐PS and anti‐PC. The IgM class is more frequently associated with arterial thrombosis and the IgG class with venous thrombosis.

Red cell-derived microparticles (RMP) as haemostatic agent

Among circulating cell-derived microparticles, those derived from red cells (RMP) have been least well investigated. To exploit potential haemostatic benefit of RMP, we developed a method of producing them in quantity, and here report on their haemostatic properties. High-pressure extrusion of washed RBC was employed to generate RMP. RMP were identified and enumerated by flow cytometry. Their size distribution was assessed by Doppler electrophoretic light scattering analysis (DELSA). Interaction with platelets was studied by platelet aggregometry, and shear-dependent adhesion by Diamed IMPACT-R. Thrombin generation and tissue factor (TF) expression was also measured. The effect of RMP on blood samples of patients with bleeding disorders was investigated ex vivo by thromboelastography (TEG). Haemostatic efficacy in vivo was assessed by measuring reduction of blood loss and bleeding time in rats and rabbits. RMP have mean diameter of 0.45 µm and 50% of them exhibit annexin V binding, a proxy for procoagulant phospholipids (PL). No TF could be detected by flow cytometry. At saturating concentrations of MPs, RMP generated thrombin robustly but after longer delay compared to PMP and EMP. RMP enhanced platelet adhesion and aggregation induced by low-dose ADP or AA. In TEG study, RMP corrected or improved haemostatic defects in blood of patients with platelet and coagulation disorders. RMP reduced bleeding time and blood loss in thrombocytopenic rabbits (busulfan-treated) and in Plavix-treated rats. In conclusion, RMP has broad haemostatic activity, enhancing both primary (platelet) and secondary (coagulation) haemostasis, suggesting potential use as haemostatic agent for treatment of bleeding.

Platelet Activation in Helicobacter pylori-Associated Idiopathic Thrombocytopenic Purpura: Eradication Reduces Platelet Activation but Seldom Improves Platelet Counts

Introduction: It has been suggested that Helicobacter pylori eradication often increases platelet counts in patients with chronic idiopathic thrombocytopenic purpura (ITP). In addition, H. pylori has been shown to induce platelet activation (CD62p or P-selectin expression) in previous studies. We assessed the response of platelet count and CD62p expression after eradication therapy in patients with ITP and H. pylori infection. Methods and Results: We prospectively studied 15 ITP patients diagnosed with H. pylori infection by serology and breath test. A follow-up breath test was used to document eradication. Two out of 15 patients showed improvement in platelet counts after 6 months, 1 of which may have had drug-induced thrombocytopenia. Overall, certain platelet response rate in our series was 6.7% (1/15). We found that platelet CD62p expression by flow cytometry was elevated in 10/15 (66.7%) H. pylori-infected patients, which is a statistically significant difference when compared with 3/33 (9.1%) control ITP patients seronegative for H. pylori (p = 0.002). In addition, eradication therapy decreased CD62p expression (p = 0.04). However, reduction in platelet activation was not associated with an increase in platelet counts (mean 72.4 × 109/l before and 68.7 after therapy; p = 0.4). Conclusion: In our series, platelet activation was common in ITP patients with H. pylori, and eradication therapy decreased platelet activation but seldom increased platelet counts. Increased platelet CD62p expression is a putative link between chronic infections and atherosclerosis, but further study is needed to clarify the implications of our observation.

Antiphospholipid antibodies in immune thrombocytopenic purpura tend to emerge in exacerbation and decline in remission

Although the presence of antiphospholipid antibodies (APLA) in immune thrombocytopenic purpura (ITP) has been reported, their clinical significance is not clear. The present study investigated APLA profiles in relation to the clinical stages of ITP. We studied APLA in 40 patients in three stages of ITP: exacerbation/relapse (n = 7), stable (n = 14) and remission (n = 19). Both IgG and IgM APLA to six target antigens were measured by enzyme‐linked immunosorbent assay: β2‐glycoprotein 1 (β2GP1), cardiolipin, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and factor VII/VIIa. The central finding was that APLA were common in ITP but differed significantly in disease stages, being highest in exacerbation (86% positive), intermediate in stable disease (57%) and lowest in remission (42%). In exacerbations, APLA were predominantly of IgG class, while in stable disease, IgM predominated. During remission, APLA often became undetectable. Both the frequency and titres of APLA were significantly higher during exacerbation than remission. An inverse correlation was found between platelet count and nearly all APLA (except β2GP1). Sequential study of six patients revealed that APLA tended to emerge and rise with exacerbation, concurrently with new episodes of bleeding and became undetectable during remission. These findings raise the possibility that APLA may play a role in the exacerbation and remission of ITP or they may be a consequence of platelet destruction.

Antiphospholipid antibodies: Paradigm in transition

ObjectivesThis is a critical review of anti-phospholipid antibodies (aPL). Most prior reviews focus on the aPL syndrome (APS), a thrombotic condition often marked by neurological disturbance. We bring to attention recent evidence that aPL may be equally relevant to non-thrombotic autoimmune conditions, notably, multiple sclerosis and ITP.OrganizationAfter a brief history, the recent proliferation of aPL target antigens is reviewed. The implication is that many more exist. Theories of aPL in thrombosis are then reviewed, concluding that all have merit but that aPL may have more diverse pathological consequences than now recognized. Next, conflicting results are explained by methodological differences. The lupus anticoagulant (LA) is then discussed. LA is the best predictor of thrombosis, but why this is true is not settled. Finally, aPL in non-thrombotic disorders is reviewed.ConclusionThe current paradigm of aPL holds that they are important in thrombosis, but they may have much wider clinical significance, possibly of special interest in neurology.

Potential roles of cell-derived microparticles in ischemic brain disease

Abstract Purpose: The objective of this study is to review the role of cell-derived microparticles in ischemic cerebrovascular diseases. Materials and methods: An extensive PubMed search of literature pertaining to this study was performed in April 2009 using specific keyword search terms related to cell-derived microparticles and ischemic stroke. Some references are not cited here as it is not possible to be all inclusive or due to space limitation. Discussion: Cell-derived microparticles are small membranous vesicles released from the plasma membranes of platelets, leukocytes, red cells and endothelial cells in response to diverse biochemical agents or mechanical stresses. They are the main carriers of circulating tissue factor, the principal initiator of intravascular thrombosis, and are implicated in a variety of thrombotic and inflammatory disorders. This review outlines evidence suggesting that cell-derived microparticles are involved predominantly with microvascular, as opposed to macrovascular, thrombosis. More specifically, cell-derived microparticles may substantially contribute to ischemic brain disease in several settings, as well as to neuroinflammatory conditions. Conclusion: If further work confirms this hypothesis, novel therapeutic strategies for minimizing cell-derived microparticles-mediated ischemia are available or can be developed, as discussed.

Life-threatening hypercoagulable state following splenectomy in ITP: Successful management with aggressive antithrombotic therapy and danazol

A life-threatening hypercoagulable state (HCS) is reported that developed after splenectomy in idiopathic thrombocytopenic purpura (ITP). A 50-year-old active male was rejected for blood donation because of an incidental finding of low platelet counts, 40,000/uL. The diagnosis was ITP. Although asymptomatic, he underwent splenectomy because of poor response to steroids and intravenous (IV) gamma globulin. One month after splenectomy, he suffered pulmonary emboli without deep venous embolism (DVT), followed by bilateral DVT, threatening amputation of the legs. Emergency thrombolysis, insertion of stent, and IV heparin saved his legs. Extensive workup for HCS was negative. IV heparin was witheld for colonoscopy for possible gastrointestinal neoplasm, at which time DVT recurred, necessitating another thrombolysis and heparin infusion. He was discharged on enoxaparin, antiplatelet therapy, and danazol. Platelet hyperactivation, characterized by high platelet microparticles (PMP) and CD62P, was present throughout his course of active ITP, resolving when ITP went into remission with danazol therapy. ITP has remained in remission for 4 years after stopping enoxaparin and danazol. In vitro, his plasma in active ITP induced activation of normal platelets, generating PMP and inducing CD62p-positive platelets and platelet aggregates; his plasma from remission had no effect. This indicates the presence of a platelet activating factor, possibly anti-platelet antibodies. Splenectomy may have allowed procoagulant PMP to accumulate to high levels resulting in HCS. We advise awareness of thrombotic complications post-splenectomy in the subset of ITP patients who are largely asymptomatic and exhibit persisting platelet activation.