É. Biró

Measuring circulating cell-derived microparticles

Cell-derived microparticles (MPs) are receiving increasing attention in recent years, both as a diagnostic aid and investigative tool [1–4]. Because they carry markers of the parent cell, including those induced by activation or apoptosis, endothelial MPs (EMPs) can provide valuable information on the status of the parent cell, obtainable in no other way. In addition, there is a growing belief that MPs can function as important diffusible vectors of specific adhesins and cytokines promoting cellular interactions and signal transmission [2]. ThusMP analysis constitutes a new avenue for investigation of pathologies in various diseases. Although still considered investigational [1–4], recent results from several laboratories suggest that MP analysis may be poised to enter the mainstream of clinical testing. However, a major impediment to that end is the wide variety ofmethodologies used by different laboratories in this field, few of which can be directly compared to the others, and results from which are sometimes inconsistent or conflicting. As a first step in addressing that problem, the Editor has organized this Forum article, consisting of a brief description of the preferred methods and rationality from each of six active laboratories in the field, including our own [5–10]. Table 1 lists some key features of the six methodological approaches. It is seen that major differences exist in the preparation of the MP samples (such as centrifugation), whether or not they are first sedimented and resuspended, means of generic MP detection (4 of 6 use annexin V), and cell lineage-specific antigenic markers. These differences probably account for some of the different findings among the groups.

Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor-dependent manner

Summary.  Background: Circulating microparticles of various cell types are present in healthy individuals and, in varying numbers and antigenic composition, in various disease states. To what extent these microparticles contribute to coagulation in vivo is unknown. Objectives: To examine the in vivo thrombogenicity of human microparticles. Methods: Microparticles were isolated from pericardial blood of cardiac surgery patients and venous blood of healthy individuals. Their numbers, cellular source, and tissue factor (TF) exposure were determined using flow cytometry. Their in vitro procoagulant properties were studied in a fibrin generation test, and their in vivo thrombogenicity in a rat model. Results: The total number of microparticles did not differ between pericardial samples and samples from healthy individuals (P = 0.786). In both groups, microparticles from platelets, erythrocytes, and granulocytes exposed TF. Microparticle‐exposed TF antigen levels were higher in pericardial compared with healthy individual samples (P = 0.036). Pericardial microparticles were strongly procoagulant in vitro and highly thrombogenic in a venous stasis thrombosis model in rats, whereas microparticles from healthy individuals were not [thrombus weights 24.8 (12.2–41.3) mg vs. 0 (0–24.3) mg median and range; P < 0.001]. Preincubation of pericardial microparticles with an inhibitory antibody against human TF abolished their thrombogenicity [0 (0–4.4) mg; P < 0.01], while a control antibody had no effect [19.6 (12.6–53.7) mg; P > 0.05]. The thrombogenicity of the microparticles correlated strongly with their TF exposure (r = 0.9524, P = 0.001). Conclusions: Human cell‐derived microparticles promote thrombus formation in vivo in a TF‐dependent manner. They might be the direct cause of an increased thromboembolic tendency in various patient groups.

The phospholipid composition and cholesterol content of platelet‐derived microparticles: a comparison with platelet membrane fractions

Summary.  Background: The processes that govern the distribution of molecules between platelets and the microparticles (MP) they release are unknown. Certain proteins are sorted selectively into MP, but lipid sorting has not been studied. Objectives: To compare the phospholipid composition and cholesterol content of platelet‐derived MP obtained with various stimuli with that of isolated platelet membrane fractions. Methods: Washed platelets from venous blood of healthy individuals (n = 6) were stimulated with collagen, thrombin, collagen plus thrombin, or A23187. Platelet activation, MP release and antigen exposure were assessed by flow cytometry. MPs were isolated by differential centrifugation. Platelet plasma‐, granule‐ and intracellular membranes were isolated from platelet concentrates (n = 3; 10 donors each) by pressure homogenization and Percoll density gradient fractionation. The phospholipid composition and cholesterol content of MPs and membrane fractions were analyzed by high performance thin layer chromatography. Results: The phospholipid composition of MPs was intermediate compared with that of platelet plasma‐ and granule membranes, and differed significantly from that of intracellular membranes. There were small but significant differences in phospholipid composition between the MPs produced by the various agonists, which paralleled differences in P‐selectin exposure in case of the physiological agonists collagen, thrombin, or collagen plus thrombin. The cholesterol content of MPs tended to be higher than that of the three‐platelet membrane fractions. Conclusions: Regarding its phospholipid content, the MP membrane is a composite of the platelet plasma‐ and granule membranes, showing subtle differences depending on the platelet agonist. The higher cholesterol content of MPs suggests their enrichment in lipid rafts.

Activated complement components and complement activator molecules on the surface of cell-derived microparticles in patients with rheumatoid arthritis and healthy individuals

Objectives: In vitro, microparticles can activate complement via the classical pathway. If demonstrable ex vivo, this mechanism may contribute to the pathogenesis of rheumatoid arthritis (RA). We therefore investigated the presence of activated complement components and complement activator molecules on the surface of cell-derived microparticles of RA patients and healthy individuals. Methods: Microparticles from synovial fluid (n = 8) and plasma (n = 9) of 10 RA patients and plasma of sex- and age-matched healthy individuals (n = 10) were analysed by flow cytometry for bound complement components (C1q, C4, C3) and complement activator molecules (C-reactive protein (CRP), serum amyloid P component (SAP), immunoglobulin (Ig) M, IgG). Results: Microparticles with bound C1q, C4, and/or C3 were abundant in RA synovial fluid, while in RA and control plasma much lower levels were present. Microparticles with bound C1q correlated with those with bound C3 in synovial fluid (r = 0.961, p = 0.0001), and with those with bound C4 in plasma (RA: r = 0.908, p = 0.0007; control: r = 0.632, p = 0.0498), indicating classical pathway activation. In synovial fluid, microparticles with IgM and IgG correlated with those with C1q (r = 0.728, p = 0.0408; r = 0.952, p = 0.0003, respectively), and in plasma, microparticles with CRP correlated with those with C1q (RA: r = 0.903, p = 0.0021; control: r = 0.683, p = 0.0296), implicating IgG and IgM in the classical pathway activation in RA synovial fluid, and CRP in the low level classical pathway activation in plasma. Conclusions: This study demonstrates the presence of bound complement components and activator molecules on microparticles ex vivo, and supports their role in low grade complement activation in plasma and increased complement activation in RA synovial fluid.

Plasma markers of coagulation and endothelial activation in Fabry disease: impact of renal impairment

BACKGROUND In Fabry disease, storage of globotriaosylceramide (Gb3) in arterial walls is one of the main pathogenetic factors that are thought to underlie the clinical manifestations of the disease. Abnormalities of the vessel wall, haemodynamics and pro- and anticoagulant factors may play a role, though the exact pathophysiology is incompletely understood. In this study, we try to clarify inconsistencies regarding coagulation activation, fibrinolysis, platelet activation and endothelial activation in 36 patients with Fabry disease. METHODS Cell-derived microparticles, markers for coagulation activation (F(1+2), TAT, sTF, sEPCR), fibrinolysis (D-dimer, tPA, alpha(2)-AP), platelet activation (beta-TG, PF4), endothelial activation (vWF) and acute phase response (IL-6, CRP) were studied in relation to renal function and severity of the disease and compared to data from 36 age- and sex-matched healthy controls (17 males). RESULTS Markers for endothelial activation and fibrinolysis were normal. Male patients had elevated levels of sTF and beta-TG, with an association between sTF and renal function and severity of the disease. In female patients, levels of TAT, beta-TG, PF4, CD63-positive platelet-derived microparticles and IL-6 were somewhat increased, with no correlation with renal function or disease severity. CONCLUSIONS Only minimal abnormalities in markers for platelet, endothelial activation and coagulation activation and fibrinolysis could be established in a large cohort of Fabry disease patients. The existing laboratory abnormalities are more likely related to renal insufficiency rather than to Fabry disease itself.

Complement activation on the surface of cell-derived microparticles during cardiac surgery with cardiopulmonary bypass - is retransfusion of pericardial blood harmful?

Objectives To investigate whether cell-derived microparticles play a role in complement activation in pericardial blood of patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and whether microparticles in pericardial blood contribute to systemic complement activation upon retransfusion. Methods Pericardial blood of 13 patients was retransfused in 9 and discarded in 4 cases. Microparticles were isolated from systemic blood collected before anesthesia (T1) and at the end of CPB (T2), and from pericardial blood. The microparticles were analyzed by flow cytometry for bound complement components C1q, C4 and C3, and bound complement activator molecules C-reactive protein (CRP), serum amyloid P-component (SAP), immunoglobulin (Ig)M and IgG. Fluid-phase complement activation products (C4b/c, C3b/c) and activator molecules were determined by ELISA. Results Compared with systemic T1 blood, pericardial blood contained increased C4b/c and C3b/c, and increased levels of microparticles with bound complement components. In systemic T1 samples, microparticle-bound CRP, whereas in pericardial blood, microparticle-bound SAP and IgM were associated with complement activation. At the end of CPB, increased C3b/c (but not C4b/c) was present in systemic T2 blood compared with T1, while concentrations of microparticles binding complement components and of those binding complement activator molecules were similar. Concentrations of fluid-phase complement activation products and microparticles were similar in patients whether or not retransfused with pericardial blood. Conclusions In pericardial blood of patients undergoing cardiac surgery with CPB, microparticles contribute to activation of the complement system via bound SAP and IgM. Retransfusion of pericardial blood, however, does not contribute to systemic complement activation.

Targeting Complement in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by polyarticular synovitis leading to cartilage, tendon and bone destruction, and pain and dysfunction of the joints. It is considered to be an immune-mediated inflammatory disorder, in which the complement system also plays a fundamental role. In the circulation of RA patients, increased levels of complement activation products have been found, often correlating with disease activity. In synovial fluid of the patients, decreased levels of native complement components and increased levels of activation products have been detected. Furthermore, in synovial tissue and cartilage, deposition of activated complement components has been demonstrated. As activators of the complement system in RA, immune complexes, C-reactive protein, and certain immunoglobulin G glycoforms have been identified. A role for complement activation in the pathogenesis of this disease is supported by studies showing an association between complement activation and inflammatory responses in the diseased joints or in individual cell types found in RA joints, and by extensive studies on animal models of the disease, utilizing for example animals deficient for certain complement components. Finally, several agents are under development to therapeutically influence the complement system, and some have already been tested in clinical trials of RA.