J. J. Jimenez

Elevated plasma endothelial microparticles in multiple sclerosis

Objective: To assess endothelial dysfunction in patients with MS and to investigate whether plasma from patients with MS induces endothelial cell dysfunction in vitro. Background: Endothelial cell dysfunction may contribute to the pathogenesis of MS. Elevations of soluble adhesion molecules intracellular adhesion molecule, vascular cell adhesion molecule, and platelet-endothelial cell adhesion molecule-1 (CD31) have been reported as markers of blood–brain barrier (BBB) damage in MS, but direct assay of endothelium has been difficult. Endothelial cells release microparticles <∼1.5 μm (EMP) during activation or apoptosis. The authors developed a flow cytometric assay of EMP and studied EMP as markers of endothelial damage in MS. Methods: Platelet-poor plasma (PPP) from 50 patients with MS (30 in exacerbation and 20 in remission) and 48 controls were labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD31 and anti-CD51 (vitronectin receptor) antibodies, and two classes of EMP (CD31+ and CD51+) were assayed by flow cytometry. For in vitro studies, patients’ plasma was added to the microvascular endothelial cell (MVEC) culture and release of CD31+ and CD51+ EMP were measured in the supernatant. Results: Plasma from patients in exacerbation had 2.85-fold elevation of CD31+ EMP as compared with healthy controls, returning to near control value during remission. The CD31+ EMP concentration showed a positive association with gadolinium enhancement in patients with MS. In contrast, CD51+ EMP remained elevated in both exacerbation and remission. This suggests that CD31+ EMP is a marker of acute injury, whereas CD51+ EMP reflects chronic injury of endothelium. MS plasma induced release of both CD31+ and CD51+ EMP from MVEC culture in vitro. Conclusion: Endothelial dysfunction is evident during exacerbation of MS, evidenced by shedding of EMP expressing PECAM-1 (CD31). The in vitro data indicate contribution of one or more plasma factors in endothelial dysfunction of MS.

Multiple sclerosis as a vascular disease

Abstract Multiple sclerosis (MS) has traditionally been viewed and researched as an immune-mediated disease with principal emphasis on the role of activated inflammatory cells, oligodendrocytes and astrocytes in its pathogenesis. Abnormalities of cerebral endothelial cells (CECs) is an under explored facet of MS pathogenesis and vascular abnormalities play a crucial role in formation of the MS lesions and disease progress, at least in the initial stages of disease. This review will focus on MS as a central nervous system (CNS) disease with a strong vascular constituent and examines abnormalities within CECs in MS and their role in the loss of blood–brain barrier and transendothelial migration of activated leukocytes into the CNS. One goal of this paper is to persuade and promote research on the endothelial abnormalities in pathogenesis of MS and to exploit existing knowledge on endothelial injury. A deeper understanding of endothelial pathophysiology in MS may help develop effective treatments through stabilization of endothelial function, translating into delay or arrest of MS disease onset and disability in MS patients.

Endothelial microparticles induce formation of platelet aggregates via a von Willebrand factor/ristocetin dependent pathway, rendering them resistant to dissociation.

Summary.  Endothelial microparticles (EMP) released from activated or apoptotic endothelial cells (EC) are emerging as useful markers for detection of EC dysfunction. Our recent observation that EMP carry von Willebrand factor (vWf) led us to investigate their interaction with platelets. EMP were incubated with normal washed platelets in the presence or absence of ristocetin, then platelet aggregates were measured by flow cytometry. In the absence of ristocetin, negligible EMP conjugated with platelets (< 5%) but in the presence of ristocetin (1 mg mL−1), EMP induced up to 95% of platelets to aggregate. EMP‐platelet interaction was 80% blocked by anti‐CD42b, or by 0.1 μm filtration to remove EMP. Platelet aggregates induced by normal plasma or high molecular weight vWf (Humate‐P) dissociated 50% within 15–25 min following 1 : 20 dilution. In contrast, aggregates formed with EMP persisted two‐ to threefold longer with the same treatment, indicating greater stability. A similar degree of prolongation of dissociation was observed using plasma from thrombotic thrombocytopenic purpura (TTP) patients compared with normal plasma. Addition of EMP to plasma from severe von Willebrand disease restored his ristocetin‐induced platelet aggregation. Multimer analysis of vWf on EMP showed unusually large vWf (ULvWf). In summary, EMP carries ULvWf multimers, promote platelet aggregates, and increase the stability of the aggregates thus formed.

Microparticle-mediated thrombin generation assay: Increased activity in patients with recurrent thrombosis

Summary.  Background: Circulating cell‐derived microparticles (MP) are important players in thrombogenesis, attributed in part to tissue factor (TF) carried on them. We developed MP‐mediated thrombin generation assay (TGA) and measured a series of patients with thrombosis (TBS) and normal controls (NC). Methods: MP were isolated from plasma of 66 patients with TBS and 34 NC. The MP were resuspended in normal pooled particle‐free plasma (PFP) containing corn trypsin inhibitor (to inhibit contact pathway). MP mediated TGA yields three parameters: lag time, peak and rate. This method is not influenced by anticoagulant therapy. Of the TBS patients, 41 had only a single thrombosis (S‐TBS) and 25 had recurrences (R‐TBS) within a 5‐year period. In parallel, MP were quantitated by flow cytometry, and cell origin was determined: endothelial cells (EMP), leukocytes (LMP), red cells (RMP) and platelets (PMP). Results: MP from all TBS patients exhibited higher thrombin generation than NC by all three TGA parameters. R‐TBS had significantly greater TGA values than S‐TBS, reflected in higher peak and rate, and shorter lag time. MP numbers were also higher in TBS vs. NC, for all MP subtypes, and were significantly higher in R‐TBS than S‐TBS (except LMP). All MP levels correlated with thrombin generation (P < 0.0001), most closely between PMP and peak (R = 0.47) and rate (R = 0.43). Conclusions: MP‐mediated TGA is a novel way to assess functional procoagulant activity of MP. Enhanced MP‐mediated TGA was demonstrated in TBS patients, and significantly higher activity in R‐TBS. These findings support a major role of MP in thrombogenesis.

More on: cellular microparticles: what are they bad or good for?: Letters to the Editors

We read with great interest the review article, Cellular microparticles: what are they bad or good for? , by Freyssinet in the Journal [1]. It opens with a succinct review of the hypothesis that a necessary prelude to vesiculation and shedding of microparticles (MP) is migration of phosphatidylserine (PS) from the inner to the outer leaflet of the membrane bilayer, implying by the illustration that surface exposure of PS is a defining feature or signature of MP. Freyssinet supported the concept that the role of MP is not limited to procoagulant activities with thrombotic potential but extends to inflammation, and in addition they can behave as true diffusible vectors in transcelluar signaling. He discusses at some length evidence that MP can bind to and influence leukocyte activation. It should be added that our laboratory was the first to demonstrate clearly that platelet microparticles (PMP) bind to neutrophils, activating them and causing formation of grape-like clusters of PMP– neutrophil complexes [2]. In that paper we advanced the concept that PMP can play an important role as a messenger linking thrombosis and inflammation through interaction with neutrophils [2]. That work was not cited in the review. He then concisely reviews some of the pertinent clinical publications, citing two of ours, namely, our original observation on PMP in patients with idiopathic thrombocytopenic purpura (ITP) in 1992 [3] and our comprehensive review of PMP in 1999 [4]. In our 1992 report, we demonstrated that PMP are hemostatically functional because ITP patients with high PMP do not bleed in spite of severe thrombocytopenia—certainly an example of good MP—but we also found that those with unusually high MP suffered from ischemic small vessel diseases of the central nervous system, manifesting as recurrent transient ischemic attack, confirmed by magnetic resonance imaging [3], some patients progressing to advanced vascular dementia [3,5]; hence in this setting, they are certainly bad MP. Our reports certainly fit into the dichotomy of MP into good and bad as Freyssinet proposed. However, good or bad may not be qualitative but quantitative in some clinical settings. Among the most exciting recent developments in MP studies is the burgeoning field of endothelial microparticle (EMP) analysis, not covered in his review. We have recently reviewed this field [6]. An important issue arising in our EMP studies is the distinction between MP-bound and true soluble circulating markers of endothelial activation: we have shown that many of the markers assumed to be soluble are in reality MP-bound, at least in part (as discussed in [6]). The pathophysiological mechanisms of release of true soluble and MP-bound species are quite different and the two forms may be functionally distinct as well (because true soluble forms generally lack transmembrane domains). It remains to be seen which form is the better marker of disease activity. Meanwhile, assays by ELISA methods measure the sum of both forms. Finally, it is important to add our observation that not all MP are positive for PS. For example, the majority of EMP from activated endothelial cells were not positive for PS as judged by annexin V (AnV) binding, since many more EMP were counted by CD62E than by AnV. Even EMP from apoptotic cells, which are much richer in AnV binding, still gave only about half as many positives for AnV as for CD31 [6,7]. Therefore, assay methods which define total MP in terms of positivity for PS, as employed by others [8] and illustrated in the review [1], can give grossly misleading results; for example, will tend to underestimate MP arising from cellular activation as distinct from apoptosis. Thus the critical question of how best to measure MP deserves further investigation. Our recent study of the mechanism of EMP generation indicated that capping precedes release of EMP from the membrane in a well-orchestrated manner, as opposed to random release from the membrane [9]. There may be many diverse routes governing release of MP, the floppase hypothesis being only one of them. The review of Freyssinet is timely and informative, bringing to attention a number of interesting papers and raising many questions. We hope it will initiate momentum towards a forum to refine and standardize MP assay methods, and to further elucidate the true role(s) of MP in health and disease. Methods to identify good and bad MP would certainly be welcome, as that could substantially improve the early diagnosis of thrombotic and inflammatory disorders, which are by far the most common and devastating illnesses in the modern world. Correspondence: Yeon S. Ahn, Director, Wallace H. Coulter Platelet Laboratory, Division of Hematology/Oncology [R-36 A], University Miami School of Medicine, 1600 NW 10th Ave, Miami, FL 33136, USA. Tel.: +1 305 243 6703; fax: +1 305 243 5975; e-mail: yahn@med. miami.edu

Endothelial microparticles correlate with high-risk angiographic lesions in acute coronary syndromes

BACKGROUND Endothelial Microparticles (EMP) are small fragments of endothelial cell membrane shed during apoptosis or activation. Our group has previously reported elevations of EMP in patients with coronary artery disease (CAD), thrombotic thrombocytopenic purpura (TTP), pre-eclampsia, multiple sclerosis (MS), and severe hypertension (HTN). In the present study, we evaluate the possible relationship between EMP levels and the angiographic severity and characteristics of coronary obstructive lesions. METHODS We studied a total of 43 patients undergoing coronary angiography. Fifteen had presented with acute myocardial infarction (MI), 20 with unstable anginas (UA), 5 with stable angina (SA) and 3 with congestive heart failure. Coronary angiography was reviewed and coronary lesions were classified using the Ambrose classification. Coronary stenoses were classified as high and low risk. High-risk included lesions with eccentric appearance (type II), presence of thrombi, or multiple irregularities. Low-risk lesions were defined as concentric or type I. Lesions were also analyzed by degree of stenosis and history of acute coronary syndrome (ACS). EMP in plasma was assayed by flow cytometry. RESULTS EMP in eccentric type II or multiple irregular lesions (high-risk) were 2.5-fold higher than in type I or concentric (low-risk) lesions, p<0.05. Lesions with thrombi had three-fold higher EMP than those without (p=0.05). Mild stenosis (>20%-<45%) had three-fold higher EMP than more severe (>45%), and five-fold higher than those without stenosis (p<0.01). Among patients with type II lesions, those with first ACS episode had four-fold higher EMP levels than those with recurrent ACS (p<0.01). CONCLUSION High EMP was associated with high-risk angiographic lesions including eccentric type II, multiple irregular, and lesions with thrombi. Mild to moderate stenosis was associated with higher EMP levels than severe stenosis. EMP may be a useful marker in detecting endothelial injury and risk of ACS as defined by angiography.