Interferon-β1a reduces plasma CD31+ endothelial microparticles (CD31+EMP) in multiple sclerosis

BACKGROUND: A correlation between plasma CD31+ endothelial microparticles (CD31+EMP) levels and clinical, as well as brain MRI activity, in multiple sclerosis (MS) patients has been previously reported. However, the effect(s) of treatment with interferon-β1a (IFN-β1a) on plasma levels of CD31+EMP has not been assessed. In a prospective study, we measured plasma CD31+EMP levels in 30 patients with relapsing-remitting MS. METHODS: Using flow cytometry, in a blinded study, we measured plasma CD31+EMP in 30 consecutive patients with relapsing-remitting MS (RRMS) prior to and 4, 12, 24 and 52 weeks after initiation of intramuscular therapy with interferon-β1a (IFN-β1a), 30 micrograms weekly. At each visit, clinical examination was performed and expanded disability status scale (EDSS) scores were assessed. RESULTS: Plasma levels of CD31+EMP were significantly reduced from 24 through 52 weeks following initiation of treatment with IFN-β1a. CONCLUSION: Our data suggest that serial measurement of plasma CD31+EMP levels may be used as a surrogate marker of response to therapy with INF-β1a. In addition, the decline in plasma levels of CD31+EMP further supports the concept that IFN-β1a exerts stabilizing effect on the cerebral endothelial cells in pathogenesis of MS

Evidence of platelet activation in multiple sclerosis

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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

Calcium Handling Systems And Platelet Activation (quin 2, Chlorotetracycline, Hyperaggregation, Thrombosis, Channel)

Ca(\u272+) handling systems in platelets from normal donors and patients with vascular occlusive diseases were studied with fluorescent Ca(\u272+) probes chlorotetracycline (CTC) and Quin2. The CTC fluorescence signal is shown to be a linear measure of the level of free calcium in the dense tubules and in the mitochondria, with probe sensitivity in the millimolar range. The cytoplasmic free Ca(\u272+) concentration is measured with intracellularly trapped Quin 2. Experiments perturbing the system with Ca(\u272+) ionophore A23187 shows that the mitochondrial Ca(\u272+) uptake is slow, produces a large increase in CTC fluorescence and is inhibited by sodium azide plus oligomycin. Uptake by the dense tubules is more rapid, produces a smaller increase in CTC fluorescence and is inhibited by trifluoperazine.A quantitative comparison of the free cytoplasmic calcium concentration and the free Ca(\u272+) concentration in the lumen of the dense tubules of human platelets between normal donors and patients with vascular occlusive diseases have been made. Studies show that the resting cytoplasmic and dense tubular Ca(\u272+) concentrations were elevated in patients suffering from venous and arterial thrombosis and other diseases involving platelet hyperactivation. The Ca(\u272+) handling defect of platelets form thrombotic patients was identified as leakage through activated channels in the plasma membrane. The defect and the elevated resting Ca(\u272+) (,cyt) and Ca(\u272+) (,dt) are adequate to explain the observation of increased rates of collagen-activated platelet aggregation in the thrombotic patients.Upon thrombin stimulation, the calcium concentration in the cytoplasm rose rapidly from 0.1 (mu)M to approx. 1 (mu)M then slowly decayed to a lower level. The change of Ca(\u272+) (,cyt) induced by thrombin is due to the initially massive Ca(\u272+) influx and dense tubular Ca(\u272+) release followed by Ca(\u272+) sequestration, and Ca(\u272+) extrusion. In the presence of 2 mM of external Ca(\u272+), most of the elevated Ca(\u272+) in the cytoplasm is due to the thrombin induced Ca(\u272+) influx. Studies indicate that the thrombin induced Ca(\u272+) influx is mediated through the receptor operated Ca(\u272+) channels and can be enhanced by the thromboxane A(,2) which is formed by the trigger of elevated Ca(\u272+) (,cyt). The extent of platelet aggregation is shown to be closely dependent on the increase of Ca(\u272+) (,cyt)

Thrombin-induced calcium movements in platelet activation

The thrombin-induced Ca 2+ fluxes and their coupling to platelet aggregation of the human platelet were studied using quin2 as a measure of the cytoplasmic Ca 2+ concentration ([Ca 2+] cyt) and chlorotetracycline (CTC) as a measure of internally sequestered Ca 2+. Evidence is given that the CTC fluorescence change is proportional to the free internal Ca 2+ concentration in the dense tubular lumen. The intracellular quin2 concentration was 1 mM and analysis showed that it did not perturb the processes reported herein. The value of [Ca 2+] cyt at rest and during thrombin activation was analyzed in terms of Ca 2+ influx, Ca 2+ release, Ca 2+ sequestration, and Ca 2+ extrusion. Influx was distinguished from internal release by removing extracellular Ca 2+ 1 min before thrombin activation. In the presence of 2 mM external Ca 2+, the thrombin-induced Ca 2+ influx accounts for most of the increase in [Ca 2+] cyt (over 80%). Thrombin-induced Ca 2+ influx and release have somewhat different EC 50 values (0.17 U/ml vs. 0.35 U/ml). The contribution of influx can be inhibited by verapamil, bepridil and Cd 2+ (IC 50 values of 19 μM, 2 μM and 50 μM). The influx results were analyzed in terms of a thrombin-activated channel. Indomethacin pretreatment experiments suggest that activation of the arachidonic pathway accounts for approx. 50% of the influx-related [Ca 2+] cyt elevation. Elevation of [Ca 2+] cyt by intracellular release is not inhibited by verapamil or Cd 2+ but is inhibited by bepridil with a high IC 50 (25 μM). It is only 15–20% inhibited by indomethacin and is thus not dependent on thromboxane A 2 formation. The release reaction does not require Ca 2+ influx. The rate of thrombin-activated platelet aggregation is shown to have an approximately fourth-power dependence on [Ca 2+] cyt with an apparent K m of 0.4 μM. Comparisons of aggregation rates of the partially thrombin-activated vs. fully thrombin-activated, partially verapamil-inhibited conditions suggest that this dependence on [Ca 2+] cyt is the major determinant of the aggregation behavior. Analysis shows that calcium influx is the major pathway for elevating [Ca 2+] cyt by thrombin when physiological concentrations of external Ca 2+ are present

Calcium uptake and release characteristics of the dense tubules of digitonin-permeabilized human platelets

The kinetics of ATP-driven Ca 2+ uptake by the dense tubules were studied in digitonin-permeablized human blood platelets. Digitonin at 3 μg/ml was shown capable of permeablizing the plasma membrane to lactate dehydrogenase and the cytoplasmic Ca 2+ indicator Quin2 without increasing the passive permeability of the dense tubular membrane for Ca 2+. Experimentation was carried out with platelets treated with 3 μg/ml digitonin reisolated and resuspended in detergent-free medium (‘digitonin-permeablized’ platelets). Active Ca 2+ accumulation, which occurs over a period of minutes, was monitored by the increase in the fluorescence of chlorotetracycline after the addition of Mg-ATP (37°C). The active uptake is inhibited by 15 μM trifluoperazine. The process is saturable with respect to external [Ca 2+], with a K m of 180 ± 5 nM and a Hill coefficient (n) of 1.40 ± 0.05. Analysis of the maximal uptake in steady state gave similar results ( K m = 160 ± 5 nM, n = 1.50 ± 0.05). The rate of uptake at [Ca 2+] ≈ K m is increased when the digitonin-permeablized platelets are preincubated with 100 nM phorbol 12-myristate 13-acetate. Actively accumulated Ca 2+ is rapidly released (less than 1 min) by addition of d -myo- inositol triphosphate (IP 3). The maximal extent of release is 50%; the EC 50 for IP 3 is approx. 12 μM. The data are compared with findings for fractionated dense tubular membrane vesicles and for the intact platelet

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