Leukocyte-derived Microparticles Research Articles

Circulating Endothelial Microparticle Levels Predict Hemodynamic Severity of Pulmonary Hypertension

Circulating microparticles (MPs) are submicron membrane fragments shed from damaged or activated vascular cells. Endothelial MPs are a biological marker of dysfunctional endothelium. Vascular remodeling and endothelial dysfunction are involved in pulmonary hypertension (PH). We tested the hypothesis that circulating MPs are increased in patients with PH and that identifiable subgroups of MPs predict the hemodynamic severity of this condition progression. Patients (n = 24; age, 54 +/- 4 yr) undergoing right heart catheterization for precapillary PH without any endothelium-active vasodilator therapy participated in the study. Age- and sex-matched healthy control subjects (n = 20) were included. Endothelial (PECAM(+) [CD31(+)]/ CD41(-), VE-cadherin(+) [CD144(+)], and E-selectin(+) [CD62e(+)]), platelet (CD41(+)), leukocyte-derived (CD45(+)), and annexin V(+) MPs were measured by flow cytometry in platelet-free plasma from venous blood. Levels of circulating endothelial PECAM(+), VE-cadherin(+), E-selectin(+), and leukocyte-derived MPs, but not platelet and annexin V(+) MPs, were increased in subjects with PH compared with control subjects (P < 0.01 each). PECAM(+) and VE-cadherin(+) MP levels significantly correlated with mean pulmonary artery pressure (r = 0.92 and r = 0.87, respectively), pulmonary vascular resistance (r = 0.78 and r = 0.73), and mean right atrial pressure (r = 0.43, and r = 0.46) and correlated inversely with cardiac index (r = -0.59 and r = -0.52). These relationships were not observed for other MP subgroups, and persisted in multivariate analysis after adjustment for confounding factors. In subjects with precapillary PH, levels of circulating endothelial and leukocyte MPs were increased compared with control subjects. In addition, levels of PECAM(+) and VE-cadherin(+), but not E-selectin(+), endothelial MPs predicted hemodynamic severity of the disease.

Abstract 523: Expression, Activation and Function of Integrin α M β 2 on Neutrophil-derived Microparticles

The levels of circulating leukocyte-derived microparticles (MPs) are elevated in thrombosis and most cardiovascular diseases, and it has been suggested that MPs are not only markers of disease but also active contributors to pathogenesis via their interaction with various cell types. Such interactions will depend on the presence and activity of molecules expressed on the MP surface. In our study we have considered the presence and activation state of a major adhesion receptor on the surface of leukocytes, integrin α M β 2 (CD11b/18, Mac-1) on MPs derived from human neutrophils (PMN). MPs were obtained from resting or agonist-stimulated PMNs, and the presence of α M β 2 was assessed by FACS. α M β 2 expression was significantly enhanced on MPs derived from stimulated PMN (e.g., resting MPs: MFI = 3.5 ± 0.7 vs PMA-MPs: MFI = 20 ± 4, and fLMP-MPs: MFI = 24 ± 5). PSGL-1 was expressed by MPs but levels were not affected by stimulation. Of note, α M β 2 expressed on MPs obtained from stimulated PMNs was in active conformation as assessed using mAb (CBRM1/5) to activation-dependent epitope of this integrin. Recognition of a protein ligand, fibrinogen, by activated α M β 2 on the MPs was also demonstrable. With evidence of a functionally active integrin, we sought to evaluate its role in mediating MP interaction with platelets. MP binding to isolated, resting platelets was dose and time dependent. However, MPs expressing activated α M β 2 bound to more platelets, and only MPs obtained from stimulated PMNs induced platelet activation as assessed P-selectin expression (resting-MPs: MFI = 25.1 ± 6.5 vs PMA-MPS: MFI = 293 ± 60, n = 4). MP binding and platelet activation were reduced by ~40–50% by function-blocking mAbs to α M β 2 and GPIbα, implicated as a major counter-receptor for α M β 2 . Complete abrogation of these events was achieved by the combination of blocking reagents to both α M β 2 /GPIbα and to PSGL-1/P-selectin. In conclusion, MPs released by stimulated PMNs express activated α M β 2 . This integrin cooperates with PSGL-1 to mediate interaction with and activation of platelets.

Adenovirus-induced thrombocytopenia: the role of von Willebrand factor and P-selectin in mediating accelerated platelet clearance

AbstractThrombocytopenia has been consistently reported following the administration of adenoviral gene transfer vectors. The mechanism underlying this phenomenon is currently unknown. In this study, we have assessed the influence of von Willebrand Factor (VWF) and P-selectin on the clearance of platelets following adenovirus administration. In mice, thrombocytopenia occurs between 5 and 24 hours after adenovirus delivery. The virus activates platelets and induces platelet-leukocyte aggregate formation. There is an associated increase in platelet and leukocyte-derived microparticles. Adenovirus-induced endothelial cell activation was shown by VCAM-1 expression on virus-treated, cultured endothelial cells and by the release of ultra-large molecular weight multimers of VWF within 1 to 2 hours of virus administration with an accompanying elevation of endothelial microparticles. In contrast, VWF knockout (KO) mice did not show significant thrombocytopenia after adenovirus administration. We have also shown that adenovirus interferes with adhesion of platelets to a fibronectin-coated surface and flow cytometry revealed the presence of the Coxsackie adenovirus receptor on the platelet surface. We conclude that VWF and P-selectin are critically involved in a complex platelet-leukocyte-endothelial interplay, resulting in platelet activation and accelerated platelet clearance following adenovirus administration.

Circulating Leukocyte-Derived Microparticles Predict Subclinical Atherosclerosis Burden in Asymptomatic Subjects

To clarify circulating microparticles (MP) relationships with preclinical atherosclerosis. In 216 subjects without cardiovascular disease, we assessed: (1) annexin V-positive, platelet-derived, endothelium-derived and leukocyte-derived circulating MP by capture on annexin V, anti-GPIb, anti-CD105, and anti-CD11a antibody-coated wells, respectively; (2) Framingham risk, metabolic syndrome, and low-grade inflammation by risk factors measurement including hsCRP; and (3) subclinical atherosclerosis by ultrasound examination of carotid, abdominal aorta, and femoral arteries. Number of sites with plaque ranged from 0 to 3 and plaque burden was classified into 0 to 1 or 2 to 3 sites disease. Leukocyte-derived MP level was higher in the presence than in the absence of moderate to high Framingham risk (P<0.05), metabolic syndrome (P<0.01), high C-reactive protein (CRP) (P<0.05), or 2- to 3-sites disease (P<0.01), and correlated positively with number of metabolic syndrome components (P<0.001), tertiles of fibrinogen (P<0.001), and number of diseased sites (P<0.01). In multivariate analysis, 2- to 3-sites disease was independently associated with leukocyte-derived MP level (P<0.05), Framingham risk (P<0.001), and metabolic syndrome (P<0.01). None of the other MP types correlated with risk markers or atherosclerosis. Leukocyte-derived MP, identified by affinity for CD11a, are increased in subjects with ultrasound evidence of subclinical atherosclerosis, unveiling new directions for atherosclerosis research.

Microparticles as mediators of cellular cross-talk in inflammatory disease

Microparticles are a heterogeneous population of membrane-coated vesicles which can be released from virtually all cell types during activation or apoptosis. Release occurs from the cell surface in an exogenous budding process involving local rearrangement of the cytoskeleton. Given their origin, these particles can be identified by staining for cell surface markers and annexin V. As shown in in vitro studies, microparticles may represent a novel subcellular element for intercellular communication in inflammation. Thus, microparticles can transfer chemokine receptors and arachidonic acid between cells, activate complement, promote leukocyte rolling and stimulate the release of pro-inflammatory mediators. Under certain conditions, however, microparticles may also exert anti-inflammatory properties by inducing immune cell apoptosis and the production of anti-inflammatory mediators. Microparticles may play an important role in the pathogenesis of rheumatologic diseases as evidenced by their elevation in diseases such as systemic sclerosis (SSc), systemic vasculitis and antiphospholipid antibody syndrome and correlation with clinical events. A role in inflammatory arthritis is suggested by the finding that leukocyte-derived microparticles induce the production of matrix metalloproteinases and cytokines by synovial fibroblasts. Together, these findings point to novel signaling pathways of cellular cross-talk that may operate along the spectrum of soluble cytokines and mediators of direct cell–cell contact.

Plasma Tissue Factor (TF) Antigen, Cellular Microparticles and Markers of Coagulation and Platelet Activation in Localized Prostate Cancer.

Coagulation and platelet activation are involved in tumor growth and dissemination, and recent trials have demonstrated promising efficacy of low-molecular-weight heparin (LMWH) in cancer treatment. It is unclear, however, which subgroup of patients benefits most from anticoagulant therapy, although LMWH may be most effective in limited-stage malignancy. About 15–20% of patients with localized prostate cancer (PC) experience recurrent local and/or metastatic disease after radical prostatectomy. We conducted a prospective study to identify laboratory markers of hypercoagulability in early-stage PC, providing a potential rational for adjuvant anticoagulant treatment strategies in this tumor entity. In 98 consecutive patients with clinically localized PC (62±6 years), we found significantly higher preoperative plasma levels of TF (median, 95 vs. 0 pg/ml), prothrombin fragment F1+2 (1.6 vs. 1.1 nmol/l), plasmin-antiplasmin complex (339 vs. 238 ng/ml), and D-dimer (0.27 vs. 0.17 mg/l) than in 42 sex- and age-matched controls (P<0.001). Patients with organ-confined (pT2) and histologically more differentiated tumors (Gleason sum, <7) had lower D-dimer levels than patients with pT3 (P=0.06) and less differentiated tumors (P=0.02). No association was found between hemostatic parameters and preoperative PSA values, lymph node involvement, or positivity of resection margins. Since TF has been implicated in tumor angiogenesis and metastasis, additional studies were performed to elucidate the cellular origin of measured TF antigen levels. To this end, platelet- (PMP) and leukocyte-derived microparticles (LMP) were enumerated by flow cytometry in 18 controls and 36 patients using FITC-conjugated antibodies against CD41 for PMP detection and CD11b or CD14 for LMP detection. Background fluorescence was determined by IgG-FITC control antibody. Calibration microspheres were used to gate all FITC+ events according to their size (forward scatter) and to correct for variations in sample flow. Only FITC+ events <1 μm were included in the analysis. The intra- and inter-assay CVs for this methodology were <10%. Controls had TF levels <50 pg/ml, and patients had TF levels of either <50 pg/ml (low-TF, n=18) or >200 pg/ml (high-TF, n=18). Compared to controls, median PMP numbers were increased 2-fold in low-TF (P<0.05) and 5-fold in high-TF patients (P<0.001). PMP numbers but not whole blood platelet counts were significantly different between patient groups (P<0.05). Compared to low-TF patients, high-TF patients also had elevated plasma levels of sP-selectin (37±15 ng/ml vs. 23±7, P<0.01) and sCD40L (361±817 vs. 47±95 pg/ml, P=0.26), two markers of in vivo platelet activation. LMP were barely detectable in controls, and their numbers were only slightly increased in patients, representing not more than 5–10% of PMP counts. Using immunohistochemistry on paraffin-embedded specimens, TF was localized predominantly to tissue macrophages and adventitial fibroblasts but not to tumor cells, showing a similar staining pattern in both patient groups. In summary, laboratory evidence of coagulation and platelet activation is already present in early-stage PC. Although TF has been associated with a poor clinical outcome in various types of malignancy, its plasma antigen levels may not reflect tumor cell TF expression in localized PC.

Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall

AbstractLeukocytes and leukocyte-derived microparticles contain low levels of tissue factor (TF) and incorporate into forming thrombi. Although this circulating pool of TF has been proposed to play a key role in thrombosis, its functional significance relative to that of vascular wall TF is poorly defined. We tested the hypothesis that leukocyte-derived TF contributes to thrombus formation in vivo. Compared to wild-type mice, mice with severe TF deficiency (ie, TF–/–, hTF-Tg+, or “low-TF”) demonstrated markedly impaired thrombus formation after carotid artery injury or inferior vena cava ligation. A bone marrow transplantation strategy was used to modulate levels of leukocyte-derived TF. Transplantation of low-TF marrow into wild-type mice did not suppress arterial or venous thrombus formation. Similarly, transplantation of wild-type marrow into low-TF mice did not accelerate thrombosis. In vitro analyses revealed that TF activity in the blood was very low and was markedly exceeded by that present in the vessel wall. Therefore, our results suggest that thrombus formation in the arterial and venous macrovasculature is driven primarily by TF derived from the blood vessel wall as opposed to leukocytes.

Circulating procoagulant microparticles and soluble GPV in myocardial infarction treated by primary percutaneous transluminal coronary angioplasty. A possible role for GPIIb‐IIIa antagonists

Circulating procoagulant microparticles (MP) were measured as markers of vascular damage and prothrombotic risk in patients undergoing ST-segment myocardial infarction (STEMI) treated by primary percutaneous transluminal coronary angioplasty (PTCA) and additional GPIIb-IIIa antagonists. Cells possibly more responsive to GPIIb-IIIa (alpha(IIb)beta(3)) antagonists were evidenced through MP phenotypes by comparison with healthy volunteers (HV) and STEMI patients treated by PTCA without GPIIb-IIIa antagonist (CP). In 50 STEMI patients, blood samples were collected at day 1 and day 6. Circulating procoagulant MP were captured on annexin V and quantified by prothrombinase assay as nanomolar phosphatidylserine equivalents (nm PhtdSer). Platelet activation by thrombin was confirmed through independent measurement of soluble GPV (sGPV). With respect to HV, procoagulant MP levels were high in patients with STEMI or unstable angina, platelet-derived MP and elevated sGPV testifying to significant platelet activation. A substantial release of endothelial-derived MP was evidenced simultaneously. In abciximab-treated patients, procoagulant MP, mainly of platelet origin, decreased precociously at day 1 (4.2 +/- 0.6 vs. CP 15.5 +/- 2.1 nm PhtdSer; P = 0.001) together with sGPV (36 +/- 3 vs. CP 58 +/- 8 ng mL(-1); P = 0.02). Leukocyte-derived MP decreased at day 6 (0.12 +/- 0.04 vs. CP 0.56 +/- 0.12 nm PhtdSer; P = 0.01) suggesting a possible effect on underlying inflammatory status. In patients presenting cardiovascular events at 6-month follow-up, procoagulant MP levels at day 1 could be indicative of a worsened outcome. MP could constitute a relevant parameter for the follow-up of STEMI patients treated by GPIIb-IIIa antagonists.

Tissue factor in neutrophils: no

Tissue factor in neutrophils: no

P-selectin and leukocyte microparticles are associated with venous thrombogenesis.

P-selectin inhibition has been found to limit venous thrombosis. We hypothesize that elevated levels of P-selectin will amplify thrombosis, mediated by procoagulant microparticles (MPs). Male mice (Mus musculus, n659), 20 to 25 grams, underwent IVC ligation to induce thrombosis. Groups consisted of wild type (WT) C57BL/6 controls, mice with high circulating levels of soluble P-selectin (CT), P-selectin gene-interrupted knockout mice (PKO), and E- and P-selectin gene-interrupted mice (EPKO). Additional groups were used to evaluate the ability of a P-sel antagonist (rPSGL-Ig) and an antibody directed against PSGL-1 to downregulate the effects of P-sel in CT mice and WT mice administered soluble P-sel at time of thrombosis. Animals were sacrificed on days 2 and 6 after IVC ligation. Thrombus mass (TM), vein wall morphometrics, and serum leukocyte/platelet microparticles (MPs) were evaluated by means of double-stained fluorescence-activated cell scanning analysis, and soluble P- and E-sel protein determination by ELISA. At days 2 and 6 in phase I of the experiment, significant differences (P <.01) in TM were noted between groups, with CT animals having the largest thrombi (50% and 57% increase in TM compared to WT at days 2 and 6) while EPKO mice had the smallest thrombi. Statistically, greater levels of neutrophils and total inflammatory cells were noted in the vein walls of CT animals at day 2 compared with WT and PKO animals. A significant difference was noted between CT and EPKO for neutrophils, monocytes, and total inflammatory cells, also at day 2. At day 6, the only statistically significant difference was found for monocytes, with a higher number in the CT animals than in WT animals. The evaluation of MPs revealed that the CT mice had a mixed leukocyte (MAC-1) and platelet (CD41) MP population that was also present in WT and PKO mice on day 2 and day 6. EPKO mice revealed a primarily platelet-derived MP population. Of interest, the CT mice with the highest TM showed a high amount of mean channel fluorescence for MAC-1 (phycoerythrin) antibody, indicative of leukocyte MPs. CT mice revealed statistically higher levels of soluble P-selectin at days 2 and 6. In phase 2, an antibody directed against PSGL-1 was more effective than rPSGL-Ig in decreasing TM and limiting leukocyte-derived MP fluorescence. This study demonstrates that high circulating levels of P-selectin are associated with increased thrombosis, whereas a lack of P-selectin and E-selectin is associated with a lessening of thrombosis. Additionally, leukocyte MPs are associated with venous thrombus formation. These data suggest the importance of selectins to venous thrombogenesis and show that P-selectin and leukocyte-derived MPs should be good targets to limit venous thrombus formation.

Transfer of tissue factor from leukocytes to platelets is mediated by CD15 and tissue factor

AbstractWe describe thrombogenic tissue factor (TF) on leukocyte-derived microparticles and their incorporation into spontaneous human thrombi. Polymorphonuclear leukocytes and monocytes transfer TF+particles to platelets, thereby making them capable of triggering and propagating thrombosis. This phenomenon calls into question the original dogma that vessel wall injury and exposure of TF within the vasculature to blood is sufficient for the occurrence of arterial thrombosis. The transfer of TF+ leukocyte-derived particles is dependent on the interaction of CD15 and TF with platelets. Both the inhibition of TF transfer to platelets by antagonizing the interaction CD15 with P-selectin and the direct interaction of TF itself suggest a novel therapeutic approach to prevent thrombosis.

Transfer of tissue factor from leukocytes to platelets is mediated by CD15 and tissue factor

We describe thrombogenic tissue factor (TF) on leukocyte-derived microparticles and their incorporation into spontaneous human thrombi. Polymorphonuclear leukocytes and monocytes transfer TF(+) particles to platelets, thereby making them capable of triggering and propagating thrombosis. This phenomenon calls into question the original dogma that vessel wall injury and exposure of TF within the vasculature to blood is sufficient for the occurrence of arterial thrombosis. The transfer of TF(+) leukocyte-derived particles is dependent on the interaction of CD15 and TF with platelets. Both the inhibition of TF transfer to platelets by antagonizing the interaction CD15 with P-selectin and the direct interaction of TF itself suggest a novel therapeutic approach to prevent thrombosis.