ALBERT

Description: Abstract 3435 Introduction Packed red blood cells in blood bank undergo a series of changes and this so-called “storage lesions” increases with time. It is believed that longer storage is associated with adverse transfusion-related complications, but the reason for this is not clear. Many bioactive substances are generated during blood storage and one or more of them may be responsible for adverse events. Among them, red cell microparticles (RMP) are a leading candidate for adverse effects, but conditions influencing their release are not well identified. Elucidation of these conditions is an important step toward minimizing storage lesions. Methods (I) MP generation: Non-leukoreduced and leukoreduced PC of known blood types (A+, B+, AB+, O+) were obtained from the blood bank within 2–4 days of collection, then stored at 4°C. Time of receipt was defined as day 0. At days 0, 10, 20, 30, and 40, forty mL samples were centrifuged at 1000×g for 20 min to remove cells. The supernatants were then assayed for subtypes of MP by flow cytometry comprising (a) RMP assessed by CD235a, (b) leukocyte MP (LMP) by CD45, (c) platelet MP (PMP) by CD41, and (d) generic MP by Ulex Europaeus (Ulex) or annexin V (AnV); MP-mediated procoagulant and proinflammatory activities were determined by TEG and CD11b expression, respectively. (II) Storage under anaerobic conditions: Anaerobic test units were processed by an OCDD (Oxygen Carbon dioxide Depletion Device) to deplete O2 and CO2, then stored anaerobically in an Anaerobic Storage Bag (ASB) [Transfusion 2011:51S, SP89]. After 42 days, MP were assayed as above. Results (a) Time of storage: In non-leukoreduced RBC, multiple MP types (PMP, LMP, RMP) were detected and seen to increase with time, but at distinctive rates. RMP increased little until day 10 when they rose exponentially; PMP counts rose steadily from day 0 and peaked at day 20; LMP showed little change until day 20 when they began to increase, then rose sharply after day 30. Levels of PMP (days 0 to 20) and RMP (days 20 to 40) correlated with increasing MP-mediated procoagulant and inflammatory markers. (b) Leukoreduction: Pre-storage leukoreduction decreased RMP generation by 20–40% and completely suppressed PMP and LMP generation. Leukoreduction decreased total MP-mediated procoagulant and inflammatory markers by 40–60%. This suggests that residual leukocytes and/or platelets potentiate RMP generation. (c) Residual platelet concentrations: We added increasing numbers of platelets to leukoreduced RBC, and then evaluated RMP generation during storage. The levels of RMP released were proportional to platelet counts in the storage bags. (d) Residual oxygen: We observed that storage of leukoreduced RBC under anaerobic conditions resulted in 40 – 60% reductions in RMP and annexin V+ MP generation measured at 42 days. Conclusions Multiple MP species (RMP, PMP, LMP) are released during storage of non-leukoreduced PC and increased with time. Procoagulant and proinflammatory activities increased in parallel. Leukoreduction eliminates LMP and PMP generation and reduces RMP generation by 40–50%. This was accompanied by reduction of procoagulant and proinflammatory activities by 60%. We have identified residual platelets, leukocytes, and oxygen levels as important factors governing MP release in stored blood. Reduction or elimination of factors influencing MP generation such as residual platelets, leukocytes and oxygen levels would improve future blood storage condition. Disclosures: No relevant conflicts of interest to declare.

Description: INTRODUCTION: ITP is an autoimmune disease in which antiplatelet antibodies induce immune-mediated platelet destruction. Cell-derived microparticles (C-MP) are microvesicles released from blood cells as well as endothelial cells (EMP) upon activation or apoptosis of parent cells. MP released from platelets (PMP), leukocytes (LMP), red cells (RMP) and endothelial cells (EMP) are believed to participate in hemostasis, thrombosis and inflammation. We previously reported C-MP profiles in ITP and that platelet counts correlated directly with PMP, and inversely with RMP [V. Fontana et al, ASH 2006, Abst #1087]. This suggests that RMP reflect severity of platelet destruction in ITP. IVIG is widely employed in the treatment of ITP and is believed to ameliorate platelet destruction by down-regulating Fc receptors of macrophages. In this study, we investigated how IVIG infusion affects C-MP levels in ITP patients. METHODS: Nineteen patients with ITP (7M/12 F, mean age 57.5yr) who received IVIG (20–30 gm/day × 2–5 days) for treatment of ITP were studied. Platelet counts and C-MP were measured before and 24hr post-infusion of IVIG, and platelet counts were monitored for 2–3wks thereafter. Patients were classified as responders (R) or non-responders (NR) to IVIG. Criteria for R was a rise in platelet counts to ≥70,000 and ≥50% increase over pre-treatment values; NR had ≤70,000 and ≤50% incremental increase within 3 weeks of IVIG. Flow cytometry was employed to assay C-MP using fluorescent markers of CD45 (LMP), CD41 (PMP), CD31+/CD41− (LMP), and glycophorin (RMP). RESULTS: Table 1 summarizes mean values of platelet counts and C-MP in the R and NR groups. The R group had n = 10 patients (7F/3M, mean age 56.4 yr; mean platelets 35,000μL pre, 118,000μL post, p = 0.001), and the NR group had n = 9 patients (4M/5F, mean age 58.8 yr, mean platelets 44,000μL pre, 51,000μL post, p〉0.05, n.s.). Mean levels of PMP increased significantly in the R group (p = 0.02) but no change was seen in the NR group. RMP decreased significantly in both groups but the decrease was more significantly in the R group (p=0.002) than the NR group (p=0.01). No significant change in EMP or LMP was seen pre- vs. post-IVIG. CONCLUSION/DISCUSSION. The present study demonstrates that IVIG modulates C-MP levels in ITP. IVIG decreased RMP significantly in both groups but more so in R than NR groups. It increased PMP in R but not in NR, consistent with our previous report that PMP levels correlated with platelet counts. However there is no effect of IVIG on LMP and EMP. These findings are consistent with the concept that IVIG down-regulates immune-mediated platelet destruction and has little effect on activation of leukocyte or endothelium. Since RMP appear to reflect severity of ITP, the observed decrease of RMP in both groups post-IVIG may reflect amelioration of immune-mediated platelet destruction by IVIG therapy. The mechanism by which RMP are produced in ITP, and how IVIG reduces RMP, remains to be elucidated. Table 1. Data on patients and C-MP RESPONDERS NON-RESPONDERS Pre IVIgG Post IVIgG p value Pre IVIgG Post IVIgG p value Plt (count/microL) 35,000 118,000 0.001 44,000 51,000 n. s. C-MP (count/microL): EMP 204 223 n. s. 227 325 n. s. PMP 1,780 2,693 0.02 1,963 2,071 n. s. LMP 1,109 1,116 n. s. 1,056 1,957 n. s. RMP 1,597 1,030 0.002 1,456 603 0.01

Description: BACKGROUND . EMP have emerged as sensitive biomarkers of endothelial dysfunction. Little is known of their function. EMP interact with leukocytes and platelets and exert prothrombotic and proinflammatory effects. Recently we reported that vWf is expressed in EMP. The present study extends this observation to characterize EMP-bound vWf (EMP/vWf), specifically their multimer size and proaggregatory activity in ristocetin-induced platelet aggregation. METHODS . EMP, released from human renal microvascular endothelial cells 24hr following stimulation with TNF-α, were isolated by centrifugation (10,000g x 30min) and washed twice for vWf multimer analysis and proaggregatory activity assay. vWf multimer size was analyzed by 0.8% argarose gel electrophoresis and Western blotting. The proaggregatory activity of vWf was assessed flow ctyometrically by a two-step method: first, washed platelets were incubated with either EMP or soluble vWf such as plasma or Humate P (a commercial vWf multimers) in the presence of ristocetin for 10 min, then disappearance of free platelets as they formed small aggregates was measured. Second, the formed platelet aggregates were diluted 20-fold with buffer and degree of disaggregation was determined at intervals to assess the stability of PltAg. RESULTS . (i) Electrophoresis showed that vWf bands from normal plasma do not exceed 1,000 kDa and Humate P about 2,000 kDa while vWf bands on EMP extends much higher than 2,000 kDa. The protein banding pattern of EMP/vWf also differed from free soluble vWf (plasma) or humate P, the latter both showing clear ladder-like banding while EMP/vWf often appeared as a continuous smear of sizes in the high molecular weight regions, probably due to covalent bonds to transmembrane structures. (ii) Incubation of EMP (1 x 107 in 100 μL) with humate P (0.3 U) for 2hr (rotary shaker, 100 rpm) followed by washing the EMP revealed that significant additional vWf was adsorbed to the EMP: the vWf in the supernatant was decreased by 30% – 40%, and Western blotting showed additional high MW band on the treated EMP. This demonstrates that EMP express unoccupied sites capable of binding free vWf. (iii) Incubating EMP/vWf with washed platelets in the presence of ristocetin resulted in formation of platelet aggregates (PltAg). When PltAg were diluted 1:20, the PltAg began dissociating into free platelets in a time-dependent manner. The time for 50% dissociation of PltAg was 15–20min with soluble vWf (plasma or Humate P), but was prolonged to 45–60min with EMP/vWf. We attribute this mainly to the presence of ULvWf on EMP. (iv) Plasma from 4 acute TTP patients showed elevated EMP and enhanced formation of PltAg resistant to disaggregate. While 5 vWD plasma showed the opposite effect and their deficiency was corrected by added EMP. CONCLUSIONS . A new role of EMP is indicated in the present study. EMP-bound vWf is functionally active, inducing platelet aggregates of greater stability than free vWf in a ristocetin-based assay. EMP are capable of adsorbing additional vWf from plasma, indicating presence of extra binding sites. EMP enhance the stability of aggregates probably through ULvWf on the EMP, or to additional platelet-EMP-platelet adhesion molecules, or both. Through their high-affinity interaction with ULvWf, EMP likely play a role in vWf-dependent thrombotic disorders such as TTP and other platelet mediated thrombosis.

Description: Injured endothelial cells (EC) are believed to play a critical role in the pathophysiology of TTP. Soluble markers of endothelial disturbance measured by enzyme-linked immunoassay (ELISA) have been found elevated in TTP. We have recently demonstrated an increase in the release of CD31/42b- EMP, and CD62E+ EMP. Moreover, we have observed that CD62E+ EMP also express vWF. The aim of this study was to quantitate soluble (s) vs. EMP-bound CD62E (bCD62E) in vitro and in vivo, in relation to the functional activity of vWF+ EMP. METHODS: Brain and renal microvascular endothelial cells (MVEC) were cultured and treated with 10ng/mL TNF-α to induce activation, or deprived of serum and growth factors (GFD) to induce apoptosis. Culture supernatants were collected and evaluated in a time-dependent manner. For in vivo studies, platelet-poor plasma was obtained from 4 TTP patients during the acute phase and upon remission. Filtration through 0.1μm, which retains most EMP, was employed to discriminate between (s) and bCD62E. sCD62E was measured by ELISA post-filtration and bCD62E by ELISA pre-filtration. Additionally, CD62E+ and CD62E+/vWF+ EMP were measured by flow cytometry. To assess pro-aggregatory function, EMP were added to washed platelets in the presence of 1 mg/mL ristocetin and aggregates were measured by flow cytometry. RESULTS: In vitro: Activation did not induce release of sCD62E at 3 hours, although bCD62E was present (1.5±0.5X106 EMP/mL). At 6 hours, some sCD62E was detected in the filtrate (0.09±0.02 ng/mL), but most was present in the unfiltered medium (3.5±0.85 ng/mL), signifying that the majority was bCD62E, confirmed by a doubling of CD62E+ EMP (3.0±0.6X106/mL). Subsequently, sCD62E levels were 1.0±0.2 ng/mL at 12 hr, 3.5±0.7 ng/mL at 18 hr, and 5±0.9 ng/mL at 24 hr. In contrast, EMP counts at 12, 18 and 24 hours were 4.6±1, 7±1.3 and 9±1.8 X106/mL (p=0.01, p=0.01, p=0.02, respectively). For all time periods, 40-60% of CD62E were positive for vWF. In control or GFD cultures, there was not a significant increase in sCD62E or CD62E+ EMP at any time period. MVEC from renal gave similar results. In acute TTP plasma samples, CD62E measured by ELISA was significantly increased (65±22 ng/mL) vs. remission (30±6 ng/mL). bCD62E accounted for 50% in acute and 15% in remission. CD62E+/vWF+ EMP were significantly elevated in plasma from acute TTP patients vs. remission (15±4.5 vs. 3±0.5, p=0.01). Sample filtration resulted in a decrease of 〉95% EMP in both acute and remission TTP plasma. MVEC-derived CD62E+/vWF+ EMP resulted in a dose-dependent increase in platelet aggregation. Additionally, plasma from 4 TTP patients with elevated CD62E+/vWF+ EMP obtained during the acute phase enhanced the formation of platelet aggregates by 48±12% (p=0.02) above remission plasma with low EMP counts. CONCLUSIONS: The results demonstrate that CD62E heretofore regarded as a soluble marker of endothelial dysfunction, in reality exists in both a soluble and EMP-bound form. Indeed, this distinction is highly relevant because CD62E+ EMP also express vWF and are pro-aggregatory to platelets. These EMP have been shown to be elevated during the acute phase of TTP and decrease upon remission. Thus, CD62E+/vWF+ EMP may be active participants in the formation of platelet-rich thrombi in TTP.

Description: BACKGROUND.. The endothelium plays a central role in sepsis, but monitoring endothelial function is difficult because it is inaccessible for direct sampling. EMP, small vesicles released from activated or apoptotic endothelim, can serve as a useful proxy, but it has been established that they are heterogenous in phenotype and function. Recently, we studied different species of EMP in severe sepsis, and demonstrated that certain subsets are good predictors of survival as early as day 1 of admission, and that survival correlated with elevation of inflammatory indicators. The present study refines that work by examining the differential levels of two phenotypic species of EMP, those positive for CD62E (EMP62E) vs. CD31 (EMP31), because of evidence that the latter is associated mainly with inflammation, the former with thrombosis. Evidence for this includes (i) in inflammatory disorders such as acute multiple sclerosis, EMP31 are predominant species [Neurology 56:1319, 2001]; (ii) in thrombotic disorders such as acute TTP, EMP62E are usually much higher than EMP31 [BJH 123:896, 2003]; (iii) in vitro experiments showed that EMP31 were 3-fold more active than EMP62E in binding to and activating leukocytes [Jy et al Frontiers Biosci, in press]. METHODS. Thirty-five patients who met the criteria of severe sepsis were recruited for the study. Laboratory and clinical evaluations were performed in day 1, 2, 3 and weekly for total 28 days. Patients were divided into survivors (S) and nonsurvivors (NS) according to the 28 days all-cause mortality. EMP were analyzed by flow cytometry using fluorescent mAb to CD62E and CD31 as described [BJH 123:896, 2003]. Results were log-normalized, analyzed using repeated measures ANOVA, and are expressed as geometric means. RESULTS . EMP31 levels expressed as geometric means were significantly higher in S than NS patients, 668 and 309 respectively for day 1 and the difference persisted through days 2 (634 vs. 247 counts/μL) and 3 (631 vs. 284 counts/μL). (p0.05). CONCLUSIONS . In survivors, EMP31 were significantly elevated compared to EMP62E as early as day 1 of enrollment, and this difference persisted. In nonsurvivors, both phenotypes were nearly equal and neither increased with time. This tends to confirm that EMP31, not EMP62E, is an inflammation-specific marker, and may have proinflammatory functional activity beneficial to immune response in sepsis. These findings also confirm heterogeneity of EMP in various clinical disorders. Our data suggest that clinical application of EMP assay may be improved by measuring different species of EMP in different diorders.

Description: INTRODUCTION. Rituximab, a chimeric monoclonal antibody against CD20 expressed on B cells, has been approved for the treatment of B cell neoplasms. Recently, studies have supported the use of rituximab in various autoimmune disorders such as systemic lupus erythematosus (SLE), thrombotic thrombocytopenic purpura (TTP), and idiopathic thrombocytopenic purpura (ITP). We studied its efficacy in the treatment of antiphospholipid syndrome (APS) and APLA-associated TTP and ITP, and measured antiphospholipid antibodies (APLA) and lupus anticoagulant (LA) before and after rituximab therapy. METHOD. The study included 12 patients, 2 with antiphospholipid syndrome (APS) refractory to all known measures, 6 with TTP, and 4 with ITP, all of whom had positive tests for LA and/or APLA. Rituximab was given 375mg/m2 weekly 4 times. We studied clinical remission and duration of remission and the effect of rituximab on APLA and LA. Laboratory investigations included routine blood counts, IgM and IgG APLA to cardiolipin (aCL) and B2GPI, and LA. RESULT. I. Clinical Observation. (a) Two patients (a 29 and 44 year old female) with APS refractory to all measures including glucocorticoids, monthly chemotherapy with IV cyclophosphamide, and plasmapheresis suffered from recurrent thromboses despite anticoagulation with warfarin, and became disabled, spending most of their time in the hospital. Both went into clinical remission following rituximab, resumed full activities, and were able to be weaned off of glucocorticoids and treated with anticoagulation alone. The remissions lasted 15 and 7 months. LA became negative and APLA decreased or became negative in both. (b) Six patients with TTP and positive APLA and/or LA were treated with rituximab in conjunction with exchange plasmapheresis. Five achieved complete remission and one failed. Remissions of TTP lasted 0.5–3 years. (c) Four patients with ITP and positive tests for APLA or LA were treated and 2 went into clinical remission for 0.5 and 2 years, and 2 did not respond. Therapy was well tolerated but one (TTP) developed herpes zoster and another (ITP) had a TIA following remission with rituximab. Laboratory Observation. APLA and LA were studied in selected patients before and after rituximab treatment. LA became negative in 6 out of 8 patients and it remained negative for over 15 months in one patient. Among APLA, IgM B2GPI became negative in 71% (5/7), IgM ACLA became negative in 50% (4/8), IgG APLA, IgG B2GP1 became negative in 50% (2/4) and IgG aCLA became negative in 30% (2/6). In our series, IgM antibodies are more readily decreased by rituximab than IgG antibodies. Decrease in APLA was evident 3–4 months post therapy. SUMMARY. Although rituximab is widely used in autoimmune disorders, it has not been tested in APS. Our data indicate that it is an effective and life saving measure in refractory APS. Rituxan-induced, long lasting clinical remissions along with serological remission are unusual compared to other immunosuppressive therapies. Similar findings were also observed in APLA-associated TTP and ITP. Further prospective study is recommended to evaluate its full potential in APLA-associated disorders.

Description: Abstract 3698 Background. Thromboelastography (TEG) is a method of analyzing coagulation commonly used to assess pre- and perioperative bleeding risk. We have been interested in the role of cell-derived microparticles (MP) in coagulation and in this study, examined correlations between TEG parameters and MP levels as described in Methods. Methods. Patients: Whole citrated blood from a total of 43 patients was examined by TEG (Haemoscope TEG 5000), within 5 hours of drawing. The study subjects consisted of 12 ITP, 5 TTP, 26 thrombocytopenia (TP) of various causes and 21 healthy controls. TEG: Parameters analyzed were maximum amplitude (MA), clot strength (G), lag time (R), time to formation of clot of amplitude 20 mm (k), rate of growth (a), coagulation index (CI). MP Assay: MP from platelets (PMP) were defined flow cytometrically by CD31+/CD41+; from red cells (RMP) by glycophorin A (CD235); from endothelia (EMP) by CD62E; and from leukocytes (LMP) by CD45. In addition, we employed generic markers, lectin FITC-Ulex europaeus (Ulex) as a measure of “total MP”; and annexin V+ (AnV+) MP, a marker of procoagulant phospholipids. Platelet counts were obtained from CBC at the clinical laboratory. Results. We assessed correlations between each TEG parameter, and each MP parameter, as well as platelet counts. Only those which were significant (p

Description: Introduction: Endothelial microparticle binding to monocytes has been shown to induce monocyte activation in vitro. In this study, we examined the correlations between EMP binding to monocytes and monocyte levels of nitric oxide (NO), as a marker of monocyte activation in different clinical conditions. Methods: We studied 186 subjects with acute venous thromboembolism (n=25), atrial fibrillation (n=48), metabolic syndrome (n=37), congestive heart failure (n=44), and normal controls (n=32). Using flow cytometry, we measured monocyte levels of NO by flow cytometry after loading monocytes with the membrane permeable NO-selective fluorescent indicator DAF-DA. Two different populations of EMP-monocyte conjugates were also measured. EMP62E+-monocyte and EMP54+-monocyte conjugates were measured based on the detection of E-selectin (CD62E) or CD54, respectively, coexpressed with CD45 in monocytes. Results: Pearson correlation coefficients between monocyte NO levels and EMP-monocyte conjugates are shown in the Table. A highly significant correlation was found between EMP62E+-monocyte conjugates and monocyte NO levels in patients with congestive heart failure (r=0.43; p=0.003). In contrast, EMP54+-monocyte conjugates strongly correlated with monocyte NO in patients with venous thromboembolism (r=0.58; p=0.009) and metabolic syndrome (r=0.49; p=0.002). No correlation was found between these conjugates and monocyte NO levels in atrial fibrillation or normal controls. Conclusions: The binding of different species of EMP to monocytes correlates with monocyte NO levels in clinical states such as congestive heart failure, metabolic syndrome, and venous thromboembolism. However, this correlation was not found in atrial fibrillation or normal controls. Our findings support the concept that the binding of different species of EMP exert different biological effects and/or reflect different biologic processes in specific disease states. Further research is needed to determine whether the binding of EMP species to monocytes regulates nitric oxide production by monocytes and whether monocytes themselves regulate the binding of different species of EMP in different disease states. Correlation between Monocyte NO levels and EMP-Monocyte Conjugates in different conditions. EMP54+-Monocyte Conjugates EMP62E+-Monocyte Conjugates Pearson r p value Pearson r p value Metabolic Syndrome 0.49 0.002 0.13 0.54 Venous Thromboembolism 0.51 0.009 0.12 0.57 Congestive Heart Failure 0.22 0.14 0.43 0.003 Atrial Fibrillation 0.22 0.14 0.17 0.24 Normal Controls 0.03 0.85 0.27 0.13

Description: Introduction: PMP, EMP and LMP are known to be sensitive markers of thrombotic and inflammatory disorders, but their respective functional activities are obscure. Recent data indicate that these microparticles (MP) possess procoagulant, proaggregatory, and proinflammatory activities. In this study, we have compared these activities in MP derived from different cells. Methods: PMP were prepared by stimulating platelet-rich plasma with 10 μM ADP plus 5 μg/mL collagen. EMP were prepared by incubating renal endothelial cells (EC) with 10 ng/mL of TNF-α for 24 hrs. LMP were prepared by incubating U937 monocytic cells or neutrophils with 10 ng/mL LPS for 1 hr. Cells were removed by centrifugation (1000xg for 10 min), MP were sedimented (15,000xg for 30 min), pellets were washed twice, then resuspended to equal MP concentration (1 x 108 counts/μL, final concentration) based on counts by flow cytometry. The MP were then tested for (ia) tissue factor (TF) antigen expression (TF:Ag) by flow cytometry, (ib) TF activity by recalcified clotting time in presence of corn trypsin inhibitor, (ii) platelet factor 3 (PF3) procoagulant activity by RVVT [Thromb Res 80:471, 1995], (iii) von willebrand factor (vWF)-dependent platelet aggregating activity by a ristocetin flow cytometric method [J Thromb Haemost 3:1301, 2005], and (iv) binding of MP to leukocytes, induced expression of CD11b and enhanced transendothelial migration (TEM) [Front Biosci 9:3137, 2004]. Results: In vivo, we found that the relative abundance of PMP, EMP, and LMP numbers in normal plasma is 50–70%, 5–10%, and 5–15% of total MP, respectively. In vitro results were as follows: (i) LMP exhibited the highest TF:Ag per MP followed by EMP, 〉PMP. However, PMP produced the shortest recalcified clotting time, indicating PMP had the highest apparent TF activity. (ii) PF3 activity was highest in PMP, followed by EMP, 〉 LMP. In view of the abundance of circulating PMP (50–70% of total MP), it appears that PMP are mainly responsible for hemostatic activity in cell-free coagulation. (iii) On the other hand, EMP had the highest specific activity in promoting vWF-dependent platelet aggregation, 〉 PMP, 〉 LMP. (iv) Both PMP and EMP showed high affinity in binding monocytes and neutrophils, and inducing expression of CD11b, as well as promoting TEM. LMP had little effect on leukocyte activation. Discussion: Our results show that MP of different cell origins have distinctive activities in promoting coagulation, platelet aggregation, leukocyte activation, and TEM. These differences may reflect their distinctive membrane compositions. Overall, PMP and EMP seem to play more active roles in hemostasis and inflammation, as judged by these measures. Since LMP expressed the highest TF:Ag, they may serve to initiate coagulation during leukocyte activation. But their net procoagulant and proinflammatory activities are relatively small. A better understanding of the functional activities of the growing number of recognized MP species is expected to provide new insights on their roles in hemostasis and inflammation.

Description: BACKGROUND: Cell derived MPs are small membrane vesicles released during cell activation or apoptosis. They express an inside-out membrane, which exposes the negatively charged phospholipid layer outside, allowing clotting factors to be anchored and generate thrombin. Among various species of MPs, RMP (red cell MPs), PMP (platelet MPs), LMP (leukocytes), EMP (endothelial cells) are of special interest. They play an important role in hemostasis, thromboses, and inflammation. MPs mirror early injury of parent cells and are sensitive early biomarkers of underlying disorders. TTP is a microangiopathy mediated by antibody-induced depletion of ADAMTS13, a von Willebrand factor-cleaving protease. Endothelial injury promotes platelet clumping and formation of platelet rich microthrombi in the microcirculation. Subsequent platelet sequestration leads to impaired microcirculation, thrombocytopenia and red cell fragmentation with a microangiopathic hemolytic anemia. Exchange plasmapheresis (EPP) is the standard therapy. It removes antibodies to ADAMTS13, replacing it with ADAMTS13 rich plasma. It is possible that EPP removes thrombogenic MPs to improve the clinical course of TTP. In this study, we investigated MP profiles in active and remission phase of TTP and the effect of EPP on MP profiles. We also aimed to determine if MP profiles may be a useful measure for monitoring clinical course and tracking progress of therapy. METHODS: A retrospective study was conducted evaluating MP assays in patients with TTP. MP profiles were reviewed in acute and remission phases of TTP. Acute phase was defined as thrombocytopenia, clinical evidence of microangiopathy and hemolytic anemia and low ADAMTS13 activity. Remission was defined as sustained normalization of laboratory parameters and no further microangiopathy for at least one month. Patients were studied longitudinally, with MP assays before and after EPP. EMP were measured by CD31+/CD42b− (EMP31), CD62E+ (EMP62); PMP by CD31+/CD42b+ (PMP42) and CD41+ (PMP41). All were measured in platelet-poor plasma by flow cytometry. All MP data are presented in units of x105/µL. The differences in MP patterns among TTP patients in active and remission phases of disease, as well as the effect of EPP on MP profiles were assessed. RESULTS: Among 20 patients with TTP, 8 (40%) were in acute phase and 12 (60%) in remission. An average of 10.7 EPP were performed. The average platelet count prior to EPP was 50.6x103/µL, which increased to 248 x103/µL following the last EPP. ADAMTS13 activity was generally