114 research outputs found
Evidence of platelet activation in multiple sclerosis
This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens
Antiphospholipid antibodies: Paradigm in transition
OBJECTIVES: This 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. ORGANIZATION: After 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. CONCLUSION: The 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
Clinical and neuroimaging correlates of antiphospholipid antibodies in multiple sclerosis: a preliminary study
<p>Abstract</p> <p>Background</p> <p>The presence of antiphospholipid antibodies (APLA) in multiple sclerosis (MS) patients has been reported frequently but no clear relationship between APLA and the clinical and neuroimaging features of MS have heretofore been shown. We assessed the clinical and neuroimaging features of MS patients with plasma APLA.</p> <p>Methods</p> <p>A consecutive cohort of 24 subjects with relapsing-remitting (RR) MS were studied of whom 7 were in remission (Rem) and 17 in exacerbation (Exc). All subjects were examined and underwent MRI of brain. Patients' plasma was tested by standard ELISA for the presence of both IgM and IgG antibodies using a panel of 6 targets: cardiolipin (CL), β2 glycoprotein I (β2GPI), Factor VII/VIIa (FVIIa), phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidylethanolamine (PE).</p> <p>Results</p> <p>In exacerbation up to 80% of MS subjects had elevated titers of IgM antibodies directed against the above antigens. However, in remission, less than half of MS patients had elevated titers of IgM antibodies against one or more of the above antigens. This difference was significant, p < 0.01, for all 6 target antigens. Interestingly, none of the MS patients had elevated plasma titers of IgG against any of the target antigens tested. Correlation analysis between MRI enhancing lesions and plasma levels of APLA revealed high correlation for aPC, aPS and aFVIIa (p ≤ 0.0065), a trend for aPE and aCL (p = 0.056), and no correlation for aβ2GP1. The strongest correlation was for aFVIIa, p = 0.0002.</p> <p>Conclusion</p> <p>The findings of this preliminary study show that increased APLA IgM is associated with exacerbations of MS. Currently, the significance of this association in pathogenesis of MS remains unknown. However, systematic longitudinal studies to measure APLA in larger cohorts of patients with relapsing-remitting MS, particularly before and after treatment with immunomodulatory agents, are needed to confirm these preliminary findings.</p
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Idiopathic thrombocytopenic purpura: pathophysiology and management
Our understanding of the pathophysiology of ITP owes to pioneering work of W J Harrington in 1951, delineating the immunologic nature of platelet destruction. In ITP, antibody-coated platelets are destroyed by macrophages of RES. However, other mechanisms are also implicated: C-mediated platelet lysis and newly described C-independent peroxide injury. Both induce platelet fragmentation and lysis, generating procoagulant platelet microparticles (PMP). A third mechanism of platelet consumption in the microvasculature is proposed, based on overlapping syndromes of ITP and TTP in some patients. In assessing hemostasis in ITP, platelet counts alone is not sufficient. Evaluation of platelet clumping, giant platelets, and platelet activation, marked by increased PMP is useful. Patients with platelet activation or giant platelets bleed less and detection of clumping prevents unwarranted therapy. Thrombotic complications may develop in ITP. A syndrome, characterized by recurrent TIA-like symptoms, progressive memory loss due to ischemic small vessel disease is described. The management of ITP should include the search for and elimination of underlying causes and careful evaluation of hemostasis. Therapy is divided into definitive vs symptomatic measures. The former including splenectomy, danazol, chemotherapy offers lasting remission after therapy was stopped, while the later including glucocorticoids, gammaglobuin, antiD antibodies and others increases platelet counts but seldom sustains remission upon withdrawal. Danazol therapy is up-dated since it is an effective and safe definite measure in ITP
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Microparticle Size and Its Relation to Composition, Functional Activity, and Clinical Significance
ABSTRACT
It is emerging that cell-derived microparticles (MP) have multiple functional activities in areas including hemostasis, thrombosis, inflammation, and as messengers in the transport of bioactive lipids, cytokines, complement, and immune signaling. Some of these activities may be performed by distinct phenotypic subsets of MP, even if derived from the same cell type. The focus of this article concerns the size classes of MP, covering methods of MP size measurement, differences in composition between size classes, and relation of size to functional (procoagulant) activity. Some of the issues considered remain to be resolved, such as whether the MP known as exosomes are truly a distinct class of MP, as well as the detailed mechanisms underlying the release of MP of different size ranges
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Increased Acetylcholinesterase Activity of Microparticles Derived from Red Cells (RMP) Compared to Platelets (PMP)
Abstract
INTRODUCTION. Cell-derived microparticles (MP) such as from platelets (PMP), endothelia (EMP) and leukocytes (LMP) are increasingly recognized as useful biomarker and important mediators of thrombosis and inflammation. However, little attention has been paid to the possible role of MP from RBC (RMP) in vascular disorders. RMP were identified by glycophorin (GPH) in flow cytometry in most studies. We reported heterogeneity of RMP in size and phenotypes and that GPH is expressed predominately in larger RMP, not in smaller RMP and that GPH+ RMP are more active than GPH- RMP in thrombin generation. Since acetylcholinesterase (AChE) activity has been measured on RMP, and was recently proposed as a marker of some inflammatory states, we investigated AChE activity of RMP compared to platelet-derived MP (PMP). AChE of PMP has not previously been reported.
METHODS. RMP were prepared from intact washed RBC at 18% Ht exposed to calcium ionophore (4uM) in presence of calcium (2mM) for 30 min. PMP were prepared from 20 mL citrated blood, and exposing the platelet-rich plasma to 1 uM calcium ionophore (without added Ca2+) and collagen, 4ug/mL, for 20 min. AChE assay was based on Ellman’s method and reagent (DTNB), run in 96-well plates, 300uL. Substrate was acetylthiocholine iodide (1 mM f.c.). DTNB was used at 0.67 mM f.c. Tests were run +/− quinidine (Q) (1.2 uM) and some tests were in presence of saponin 0.01%. Q is known to inhibit AChE of plasma but RBC activity is insensitive. Activity is expressed in umols substrate cleaved /min per 108 MP, with provisos below. Flow cytometry using FITC labeled lectin, Ulex europaeus (Ulex) was used to quantitate RMP and PMP.
RESULTS.
As expected, Q inhibited AChE in plasma by >90% but not AChE of RMP. On contrary, RMP were consistently stimulated by Q, up to 150% activity +Q; some preparations of PMP were also stimulated. Saponin, which has been used in assay of RBC AChE, had little effect on PMP or RMP activity. In 12 experiments, AChE of PMP exhibited marked concentration-dependence. The apparent activity per mL of suspension was greater with lesser volumes, by as much as 3-fold between 2.5uL and 20uL added. This could not be explained by substrate inhibition since the effect varied in different preparations, was absent in particle-free plasma, and did not diminish in low substrate. This suggests the presence of a natural inhibitor. Calculation of specific activity of the MP was complicated by the dependence of apparent activity on volume assayed. However, when equal dilutions were compared, a representative experiment showed RMP had about 6-fold greater activity than PMP per 108 MP: 36.0 vs. 5.88 for 2.5uL suspension; and 29.0 vs. 3.9 for 20 uL assayed, in units above.
CONCLUSIONS / DISCUSSION. The AChE activity of RMP is about 6-fold greater than PMP. Weaker activity on PMP is possibly attributed to a previously unreported natural inhibitor. Blood AChE activity has been shown to reflect inflammatory states. Since AChE is a GPI-anchored protein, it is preferentially depleted from cells on the MP shed off. Assay of this activity in patient cell-free plasma, +/− Q, may be a useful biomarker. It is well known that hemolytic anemia, where RMP are elevated, is often associated with thrombotic complications, whereas ITP, where PMP are frequently elevated, rarely is. Further study to characterize AChE in RMP and other MP, and to clarify the physiological role of MP- and cell-associated AChE in thrombosis, inflammation, and cardiovascular disease is in progress
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Preferential Stimulation of Contact Pathway Factors By Red Cell-Derived Microparticles: New Perspective on Hemostasis
Abstract
BACKGROUND / OBJECTIVES. Cell-derived microparticles (MP) are known to promote thrombin generation, and more recently have been shown to contribute substantially to hemostasis. This activity derives mainly from platelet MP (PMP) but recent evidence implicates also red cell MP (RMP). Traditionally, MP-mediated procoagulant activity has been ascribed to presentation of negatively charged surfaces for the assembly of tenase and prothrombinase. However, the mechanism of this activity is becoming more complicated as it is emerging that MP-mediated procoagulant activity includes augmentation of the contact pathway [van der Meijden et al,J Thromb. Haemost., 2012; 10:1355-62, Rubin et al, Transfusion, 2013; 53:1744-54] and of ADP-induced platelet aggregation [Jy et al, Thromb. & Haemost., 2013; 110:751-60]. Accordingly, we undertook examination of interactions of RMP with specific elements of the contact factor pathway, and related issues such as whether or not RMP can initiate contact pathway by activating FXII directly.
METHODS. Plasmas depleted in each of 8 factors (F’s II, V, VII, VIII, IX, X, XI, XII), obtained from commercial sources, were mixed with pooled normal plasma to produce depletion levels of 75% to 95%, i.e., from 25% to 5% of normal. These plasmas were then assayed by thromboelastography (TEG) with / without addition of RMP. The RMP was prepared by high-pressure extrusion of washed RBC [Jy et al, Thromb. & Haemost., 2013; 110:751-60]. Blood was drawn into citrated Vacutainers in presence / absence of 140 µg/mL corn trypsin inhibitor (CTI) and run in TEG. Controls were obtained from healthy staff volunteers (n=8) to establish baseline values, +/- addition of RMP. Tissue factor (TF) at 20 or 200 pg/mL was added to standardize extrinsic pathway activation. Anti-FVIIa (Seksui Diagnostics) was added in certain experiments to abolish the TF pathway. Finally, when contact factor activation was fully blocked (no fibrin generation within 2 hrs), kaolin was added in presence / absence of RMP.
RESULTS.
(1) In the factor depletion experiments, we found that RMP most strongly potentiated depletion of factors of the contact pathway, namely FVIII, FIX, FXI, and FXII, as judged by shortening of R (lag time), p<0.01 to p<0.05, at depletions as low as 5% normal. For example, FVIII at 15% of normal level responded to RMP by 50% reduction in R time. In contrast, RMP had little or no augmenting effect on plasmas depleted of F’s II, V, X. This suggests that RMP promote the contact pathway preferentially.
(2) RMP were capable of enhancing the rate of thrombin generation at low concentration of CTI (40 µg/mL), but at high concentration (140 µg/mL) this effect was completely abolished. When kaolin was added to the high concentration CTI-treated blood, it initiated thrombin generation after long delay (~30 min). Upon addition of RMP, the rate of thrombin generation was potentiated considerably (by ~50%). These data indicate that unlike kaolin, RMP are not capable of activating FXII directly but RMP can potentiate FXIIa-mediated thrombin generation.
(3) In whole blood treated with low concentration TF (20 pg/mL) and high concentration CTI, RMP increased rate of thrombin generation by 25% compared to controls (p< 0.05). When blood was treated with anti-FVIIa, RMP retained the ability to enhance thrombin generation by roughly 30% (p<0.05). This demonstrates that RMP can potentiate thrombin generation via the TF pathway, although not as robustly as by contact pathway.
CONCLUSIONS. First, these results demonstrate highly selective stimulation of elements of the contact pathway by RMP, as opposed to non-specific augmentation of coagulation. However, we do not have evidence of direct activation of FXII to FXIIa by RMP. Instead, reslults can be explained by marked potentiation of small amounts of activated enzyme. Second, it is shown that this activity is entirely independent of the TF pathway. Third, results support the potential value of RMP for use as an infusible hemostatic agent [Jy et al, Thrombosis & Haemostais, 110:751-60 (2013)], for treating a wide variety of clinical conditions involving excessive bleeding. Finally, in view of new evidence showing that MP-mediated procoagulant activity is diverse in action, new avenues of MP-mediated hemostasis are open to exploration.
Disclosures
No relevant conflicts of interest to declare
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Tissue Factor-Negative Cell-Derived Microparticles Play a Distinctive Role in Hemostasis: A Viewpoint Review
Circulating cell-derived microparticles (MPs) exhibit procoagulant activity and have been investigated for a possible role in some human pathologies. However, their potential role in hemostasis has been neglected and often denied. This review brings to attention a specific body of direct clinical evidence supporting an important but distinctive role of MPs in hemostasis. Evidence for a role of MPs in hemostasis includes: (1) two congenital bleeding disorders attributed to impaired release of MPs; (2) two recent studies of trauma patients relating naturally elevated endogenous MPs at admission to reduced transfusion requirements and better outcomes; (3) a study of coronary surgery patients showing that elevated MP before surgery reduces transfusion requirements during surgery; and (4) a clinical study of patients with immune thrombocytopenia demonstrating that those with high circulating MP have reduced bleeding compared to patients with similar platelet counts but lower MP levels. Mechanisms involving potentiating the contact factor pathway are thought to play a key role and are probably synergistic with polyphosphate released from activated platelets at sites of endothelial injury. Hemostatic defect of patients with deficient MP-mediated coagulation resembles deficiency of FXI (hemophilia C), distinct from hemophilia A or B, so can be termed type C hemostasis. A better understanding of this proposed hemostatic pathway may lead to improved methods for controlling excessive bleeding in surgery, trauma, and other clinical settings
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Endothelial microparticles as markers of endothelial dysfunction
Endothelial microparticles (EMP) are small vesicles released from disturbed endothelial cells (EC). Owing to the central importance of EC injury in thrombotic and inflammatory conditions, assay of EMP as a marker of EC disturbance has come under intensive development by several laboratories. The review begins with established markers of EC injury, commonly soluble markers such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, von Willebrand factor (vWF), etc., pointing out that many of these are in fact mixtures of true soluble molecules with membrane-bound forms, for example, EMP. Assays of EMP from different labs are reviewed and standardization of assay is recommended. EMP are heterogeneous: those released in activation vs. apoptosis are distinctive in phenotypic markers and procoagulant properties. Application of EMP phenotype analysis can distinguish EC state of activation from apoptosis. Some EMP carry functional vWF with properties different from soluble vWF. Certain EMP bind to and activate monocytes; EMP-monocyte conjugates were found to be a marker of inflammatory disease such as multiple sclerosis (MS), and to enhance transendothelial migration of leukocytes in vitro. Clinical studies have revealed elevated plasma levels of EMP in lupus anticoagulant (LA), multiple sclerosis (MS), thrombotic thrombocytopenic purpura (TTP), coronary artery disease (CAD), hypertension, preeclampsia, and diabetes. Further refinement of EMP assay could open new windows for evaluating and monitoring endothelial injury in thrombotic and inflammatory disorders
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