Yeon-Soong Ahn

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.

Increased procoagulant cell-derived microparticles (C-MP) in splenectomized patients with ITP ☆

BACKGROUND Splenectomy is frequently employed for therapeutic and diagnostic purposes in various clinical disorders. However its long-term safety is not well elucidated. Although risk of infection by encapsulated organisms is widely recognized, less well-known are risks of thrombosis and cardiovascular disease. METHODS We investigated levels of cell-derived microparticles (C-MP) in 23 splenectomized ITP (ITP-S) and 53 unsplenectomized ITP patients (ITP-nS). Assay of C-MP derived from platelets (PMP), leukocytes (LMP), red cells (RMP) and endothelial cells (EMP) were performed by flow cytometry. Coagulation parameters included PT, aPTT and activities of FVIII, IX and XI. Results of all measures were compared between the two groups, ITP-S vs ITP-nS. RESULTS Levels of all C-MP were higher in ITP-S than ITP-nS but only RMP and LMP reached statistical significance (p = 0.0035 and p < 0.0001, respectively). The aPTT was significantly shorter in ITP-S (p = 0.029). Interestingly, correlation analysis revealed that RMP, but not other C-MP, were associated with shortening of aPTT (p = 0.024) as well as with increased activities of factors VIII (p = 0.023), IX (p = 0.021) and XI (p = 0.0089). CONCLUSIONS RMP and LMP were significantly elevated in splenectomized compared to non-splenectomized ITP patients. This suggests that the spleen functions to clear procoagulant C-MP, and that elevation of C-MP might contribute to increased risk of thrombosis, progression of atherosclerosis and cardiovascular disease following splenectomy.

Original Article: Electrophoretic Properties of Platelets from Normal, Thrombotic and ITP Patients by Doppler Electrophoretic Light Scattering Analysis

At fixed pH and ionic environment platelet net charge depends on the composition of surface glycoproteins and phospholipids. It is believed that this composition changes, becoming more negative in the course of activation, and that certain thrombotic disorders such as transient ischemic attacks (TIA) may be associated with platelet activation. To investigate this hypothesis we measured the electrophoretic mobility (U) of normal platelets, activated normal platelets and platelets from patients with TIA and immune thrombocytopenic purpura (ITP). Normal platelets gave U = -0.91 ± 0.05 pm cmh, while normal platelets activated by ADP gave -1.25 and by collagen -1.70. TIA patients (n = 22) had mean U = -1.14 ± 0.10 and the ITP patients (n = 37) had U= -1.07 ± 0.13. The U of both patient groups differed significantly from normal controls, P < 0.001. Indeed, only 2 of the 22 TIA cases had U within 2 SD of the control value. The ITP group included 10 patients with TIA-Like symptoms; the mean U of this subgroup was identical to that of the TIA group without ITP. We conclude that U is a useful measure for research on platelet abnormalities and may even be useful as a routine clinical tool. This work was greatly facilitated by the use of an automated instrument, the DELSA 440, allowing determination of U of a given sample in as little as 5 min.

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