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Yeonsoong Ahn - One of the best experts on this subject based on the ideXlab platform.
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correlations of red cell derived micropareticles RMP with other mp species in hematological and thrombotic disorders
Blood, 2015Co-Authors: Lawrence L. Horstman, Carlos J. Bidot, Pamela Dudkiewicz, Mohamed El Dinali, Gabriel Tinoco, Yeonsoong AhnAbstract:Background : We have recently observed that increased levels of circulating RMP are associated with several hematological disorders and thrombophilic states, and that levels were significantly higher when thrombosis was present. However, other species of MP, such as from platelets (PMP), endothelia (EMP), leukocytes (LMP) and annixin V-binding (AnV) have also been shown to be associated with thrombophilic states. The purpose of this study was to determine if correlations exist between RMP and other MP. Methods : This is a retrospective analysis of 702 laboratory samples over an 8 year period, limited to elevated values: >2SD above normal controls (>2,000/uL).. About 87% were individuals, the remaining 13% were tested 2-3 times and all tests >3 per patient were excluded. The disorders were hemolytic anemia (HA, n=38), hypercoagulable state (HCS, n=64), immune thrombocytopenia (ITP, n=86), thrombocytopenia of all causes (TP, n=69), myeloprolifereative disorder (MPD, n=29), thrombotic thrombocytopenic purpura (TTP, n=29), antiphospholipid syndrome (APS, n=34), pulmonary embolism (PE, n=21), and all-cause thrombosis (TBS, n=251). Some were classified in more than one way. MP species assayed were RMP by glycophorinA, PMP by CD41 (PMP41), PMP by CD42 (PMP42), EMP by CD31+/42- (EMP31), EMP by CD62E (EMP62E), LMP by CD45, annexin V binding (AnV), and counts by lectin, FITC-Ulex, which efficiently detects total MP including very small. Results : In HCS, the RMP >2,000/uL correlated well with PMP41 (R=0.407, p 2,000/uL and any of the MP markers in APS, TP, or TTP. The MP species markers, EMP31, EMP62E, and AnV, failed to show correlation with RMP in any of the disorders analyzed. In addition, we tested for correlations between elevated RMP and other MP for the entire data set (all disorders combined) and found that only LMP was significant (p 0.05). Discussion : These correlation analyses shows that RMP correlated most frequently with PMP, as seen in HA, ITP, HCS, and TBS, followed by LMP, as seen in HA and TBS. These observations suggest that platelet or/and leukocyte activation may be involved in RMP generation. Of added interest is the finding that the overall data correlated well with LMP only at the higher LMP levels, not at all in the lower quintile of LMP. This suggests that RMP elevations are associated with immunolgic, inflammatory processes. In summary, correlation analysis reveals likely interaction between red cells and platelets or leukocytes during immunologic, inflammatory or in thrombophilic states, resulting in elevated RMP. Disclosures No relevant conflicts of interest to declare.
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Elevated Red Cell Microparticles (RMP) in Hematologic and Thrombotic Disorders
Blood, 2015Co-Authors: Gabriel Tinoco, Lawrence L. Horstman, Carlos J. Bidot, Pamela Dudkiewicz, Mohamed El Dinali, Yeonsoong AhnAbstract:Introduction. Circulating cell-derived microparticlesa (MP) from platelets (PMP), leukocytes (LMP), and endothelia (EMP) have been well-documented for their roles in hemostasis, thrombosis and inflammation but the clinical significance of RMP is less well understood. The purpose of this study is to study the relation of elevated RMP to selected hematologic and thrombotic disorders. Methods. This is a retrospective study on RMP profiles for patients referred to University of Miami Hospital and Clinics for hematologic consultation over the last 8 years. The patient population includes 51 hemolytic anemia (HA), 459 thrombocytopenia (TP), 26 myeloproliferative disorder (MPD), 413 hypercoagulable state (HCS), and 446 thrombotic disorders (TBS). Some patients were analyzed in more than one of the above disorders. Levels of RMP were measured by flow cytometry with PE-conjugated anti-CD235a labeling as previously described [Thromb. & Haemost., 2013;110:751-60]. Levels of RMP above mean +2SD of controls (> 2,000 counts/mL) are designated as "elevated RMP". Results. (I) Prevalence of elevated RMP in patient populartion: Elevated RMP were found in 31 of 51 HA (60.8%), 138 of 459 TP (30.1%), 20 of 26 MPD (78.1%), 175 of 413 HCS (42.4%), and 251 of 446 TBS (56.3%). (II) Association of elevated RMP with thrombosis: Of 31 HA patients with elevated RMP, 11 were positive for TBS, and the remaining 20 were negative. Levels of RMP (mean ±SD) for TBS(+) and TBS(-) were 5,824 ±3713 and 3,265 ±1,048, counts /mL, respectively (p Discussion. (i) The cause of RMP elevation in HA is reasonably attributed to red cell destruction. The cause in TP/ITP is likely due to products of platelet destruction or leukocyte activation. The cause in MPD could result from clearance of the high burden of red cells and platelets. (ii) Patients who were TBS(+) had higher RMP than TBS(-), seen in HCS, HA, TP, raising the question of whether high RMP is a cause or consequence of TBS. To answer this will require further study. (iii) The finding of exceptionally high RMP levels in recurrent TBS vs. non-recurrent TBS (HCS-3 vs. HC-2) indicates that RMP levels reflect severity of TBS. (iv) Finally, these data indicate that RMP may be a useful biomarker of thrombotic risk, particularly because some TBS patients had elevated RMP, yet tested negative by all conventional markers of HCS workups. Disclosures No relevant conflicts of interest to declare.
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red cell microparticles RMP and other cell derived microparticles c mp in hemolytic anemias
Blood, 2012Co-Authors: Max E Johansen, Pamela Dudkiewicz, Yeonsoong AhnAbstract:Abstract 5158 Introduction: The lifespan of red cells (RBC) are shortened by several mechanisms in hemolytic anemias (HA): Ab-mediated hemolysis in autoimmune hemolytic anemia (AIHA), complement-mediated lysis in paroxysmal nocturnal hemoglobinuria (PNH), microangiopathy in thrombotic thrombocytopenic purpura (TTP) and hemoglobinopathy in sickle cell anemia (SS) and thalassemia (Thal). During hemolysis, RMP are released along with MP from platelets (PMP), endothelia (EMP) and leukocytes (LMP). The role of MP in HA is unknown but may be involved in complications of HA. Methods: We investigated RMP and other MP profiles in the following hemolytic anemias in active phase: 14 patients with AIHA, 10 with TTP, 4 with PNH, 8 with SS or Thalassemia (SS - Thal), along with 60 healthy controls. Whole, citrated blood was centrifuged at 1600xgfor 10 minutes to yield platelet-poor plasma (PPP). Microparticles in the PPP were measured by FITC- or PE-conjugated mAb specific to the aforementioned cell types. Control plasma was prepared in an identical fashion from healthy volunteers. We compared RMP profiles and other MP in HA during active phase and in remission. Associations with clinical features were calculated using the R statistical software. Results: Means and standard deviations of C-MP in the different types of HA are shown in the TABLE. Mean RMP and PMP were highest in SS-Thal, followed by AIHA. Mean Annexin V binding procoagluant MP were higher in all subgroups of HA and highest in SS-Thal, followed by TTP. EMP in all subgroups was similar or lower than controls. Regarding clinical associations, high RMP levels were associated with low hemoglobin (p=0. 01), high reticulocytes (p=0. 003), and high LDH (p=0. 01) across all groups. High reticuloctye counts (>3. 0 %) were also associated with significantly elevated PMP (p=0. 04) and total MP counts by Ulex (p=0. 003). HAs in remission had marginally lower RMP levels than controls (p=0. 05). None of the subtypes had significantly higher EMP, LMP, or PMP compared to each other or controls. Conclusion: The small population yielded large standard deviations in the MP counts and did not allow statistical study among subgroups of patients with HA. However prothrombotic MPs such as Annexin V, PMP tend to be higher in HA. A larger scale future study will clarify this issue. The constellation of elevated LDH, reticulocytes, and decreased hemoglobin were associated with increased RMP. Therefore we suggest that elevated RMP may be viewed as an additional, new diagnostic marker of active hemolysis. Disclosures: No relevant conflicts of interest to declare.
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Increased Acetylcholinesterase Activity of Microparticles Derived from Red Cells (RMP) Compared to Platelets (PMP).
Blood, 2008Co-Authors: Lawrence L. Horstman, Jacob Esquenazi, Yeonsoong AhnAbstract: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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microparticle mediated thrombin generation assay increased activity in patients with recurrent thrombosis
Journal of Thrombosis and Haemostasis, 2008Co-Authors: L Bidot, Lawrence L. Horstman, Carlos Bidot, J J Jimenez, V Fontana, Yeonsoong AhnAbstract: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.
Lawrence L. Horstman - One of the best experts on this subject based on the ideXlab platform.
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correlations of red cell derived micropareticles RMP with other mp species in hematological and thrombotic disorders
Blood, 2015Co-Authors: Lawrence L. Horstman, Carlos J. Bidot, Pamela Dudkiewicz, Mohamed El Dinali, Gabriel Tinoco, Yeonsoong AhnAbstract:Background : We have recently observed that increased levels of circulating RMP are associated with several hematological disorders and thrombophilic states, and that levels were significantly higher when thrombosis was present. However, other species of MP, such as from platelets (PMP), endothelia (EMP), leukocytes (LMP) and annixin V-binding (AnV) have also been shown to be associated with thrombophilic states. The purpose of this study was to determine if correlations exist between RMP and other MP. Methods : This is a retrospective analysis of 702 laboratory samples over an 8 year period, limited to elevated values: >2SD above normal controls (>2,000/uL).. About 87% were individuals, the remaining 13% were tested 2-3 times and all tests >3 per patient were excluded. The disorders were hemolytic anemia (HA, n=38), hypercoagulable state (HCS, n=64), immune thrombocytopenia (ITP, n=86), thrombocytopenia of all causes (TP, n=69), myeloprolifereative disorder (MPD, n=29), thrombotic thrombocytopenic purpura (TTP, n=29), antiphospholipid syndrome (APS, n=34), pulmonary embolism (PE, n=21), and all-cause thrombosis (TBS, n=251). Some were classified in more than one way. MP species assayed were RMP by glycophorinA, PMP by CD41 (PMP41), PMP by CD42 (PMP42), EMP by CD31+/42- (EMP31), EMP by CD62E (EMP62E), LMP by CD45, annexin V binding (AnV), and counts by lectin, FITC-Ulex, which efficiently detects total MP including very small. Results : In HCS, the RMP >2,000/uL correlated well with PMP41 (R=0.407, p 2,000/uL and any of the MP markers in APS, TP, or TTP. The MP species markers, EMP31, EMP62E, and AnV, failed to show correlation with RMP in any of the disorders analyzed. In addition, we tested for correlations between elevated RMP and other MP for the entire data set (all disorders combined) and found that only LMP was significant (p 0.05). Discussion : These correlation analyses shows that RMP correlated most frequently with PMP, as seen in HA, ITP, HCS, and TBS, followed by LMP, as seen in HA and TBS. These observations suggest that platelet or/and leukocyte activation may be involved in RMP generation. Of added interest is the finding that the overall data correlated well with LMP only at the higher LMP levels, not at all in the lower quintile of LMP. This suggests that RMP elevations are associated with immunolgic, inflammatory processes. In summary, correlation analysis reveals likely interaction between red cells and platelets or leukocytes during immunologic, inflammatory or in thrombophilic states, resulting in elevated RMP. Disclosures No relevant conflicts of interest to declare.
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Elevated Red Cell Microparticles (RMP) in Hematologic and Thrombotic Disorders
Blood, 2015Co-Authors: Gabriel Tinoco, Lawrence L. Horstman, Carlos J. Bidot, Pamela Dudkiewicz, Mohamed El Dinali, Yeonsoong AhnAbstract:Introduction. Circulating cell-derived microparticlesa (MP) from platelets (PMP), leukocytes (LMP), and endothelia (EMP) have been well-documented for their roles in hemostasis, thrombosis and inflammation but the clinical significance of RMP is less well understood. The purpose of this study is to study the relation of elevated RMP to selected hematologic and thrombotic disorders. Methods. This is a retrospective study on RMP profiles for patients referred to University of Miami Hospital and Clinics for hematologic consultation over the last 8 years. The patient population includes 51 hemolytic anemia (HA), 459 thrombocytopenia (TP), 26 myeloproliferative disorder (MPD), 413 hypercoagulable state (HCS), and 446 thrombotic disorders (TBS). Some patients were analyzed in more than one of the above disorders. Levels of RMP were measured by flow cytometry with PE-conjugated anti-CD235a labeling as previously described [Thromb. & Haemost., 2013;110:751-60]. Levels of RMP above mean +2SD of controls (> 2,000 counts/mL) are designated as "elevated RMP". Results. (I) Prevalence of elevated RMP in patient populartion: Elevated RMP were found in 31 of 51 HA (60.8%), 138 of 459 TP (30.1%), 20 of 26 MPD (78.1%), 175 of 413 HCS (42.4%), and 251 of 446 TBS (56.3%). (II) Association of elevated RMP with thrombosis: Of 31 HA patients with elevated RMP, 11 were positive for TBS, and the remaining 20 were negative. Levels of RMP (mean ±SD) for TBS(+) and TBS(-) were 5,824 ±3713 and 3,265 ±1,048, counts /mL, respectively (p Discussion. (i) The cause of RMP elevation in HA is reasonably attributed to red cell destruction. The cause in TP/ITP is likely due to products of platelet destruction or leukocyte activation. The cause in MPD could result from clearance of the high burden of red cells and platelets. (ii) Patients who were TBS(+) had higher RMP than TBS(-), seen in HCS, HA, TP, raising the question of whether high RMP is a cause or consequence of TBS. To answer this will require further study. (iii) The finding of exceptionally high RMP levels in recurrent TBS vs. non-recurrent TBS (HCS-3 vs. HC-2) indicates that RMP levels reflect severity of TBS. (iv) Finally, these data indicate that RMP may be a useful biomarker of thrombotic risk, particularly because some TBS patients had elevated RMP, yet tested negative by all conventional markers of HCS workups. Disclosures No relevant conflicts of interest to declare.
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Release of Microparticles During Blood Storage Is Influenced by Residual Platelets, Leukocytes and Oxygen Levels
Blood, 2012Co-Authors: Wenche Jy, Ralph R. Vassallo, Max E Johansen, Orlando Gomez-marin, Tatsuro Yoshida, John Morgan, Lawrence L. Horstman, Carlos J. Bidot, Sherry ShariatmadarAbstract: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.
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Interaction of Red Cell Microparticles (RMP) with Platelets: Potential Role of RMP in Hemostasis and Thrombosis,
Blood, 2011Co-Authors: Max E Johansen, Lawrence L. Horstman, Carlos J. Bidot, Powei Chen, Yeon S. AhnAbstract:Abstract 3263 Introduction: We previously reported data indicating that RMP are well suited for use as hemostatic agent for treating bleeding disorders ([Jy et al, Hemophilia 17:4, 2011][1]). Previous studies have shown that RMP can contribute to RBC-related thrombotic complications such as sickle cell disease and PNH. Microparticles (MP) derived from platelets (PMP), endothelia (EMP), and leukocytes (LMP) are believed to play a role in hemostasis and thrombosis. They can adhere to blood cells and endothelia, facilitating prothrombotic and proinflammatory reactions. However, less is known about interaction of RMP with cells and their potential role in hemostasis and thrombosis. Here we report evidence of interaction of RMP with platelets resulting in enhanced platelet aggregation and increased size of adherent platelet aggregates induced by shear stress. Methods: (i) RMP were prepared by high-pressure extrusion of washed RBC. (ii) Platelet aggregation was performed in a Chrono-log aggregometer. PRP (490 μL) was mixed with 10 μL of RMP (1 × 108 /mL final conc.) for 5 min, then low-dose activating agent (ADP 0.2 μM, or arachidonic acid (AA) 0.3 mM) was added. (iii) Shear-induced platelet adhesion was measured in a cone-and-well device (Diamed Impact-R). Whole blood was pre-incubated with RMP as above for 10 min, then subjected to various shear rates (900, 1800, 2700 sec−1) for 1 min. The adherent platelets were then washed, stained, and quantitated by image analyzer. (iv) RMP-platelet interaction employed 2-color flow cytometry. RMP-platelet conjugates were identified by co-expression of α-CD41-FITC and α-glycophrin A-PE, in both the free platelet and micro-aggregated platelet populations. Results: (1) Platelet aggregation: Addition of RMP to PRP did not induce platelet aggregation. However, RMP enhanced platelet aggregation induced by low-dose ADP or AA. Low-dose ADP alone induced a transient increase of aggregation peaking at 25–35% followed by slow disaggregation to 0–5% at 10 min; but in presence of RMP, a similar rate (slope) of aggregation was seen but peaking at 50–60% and disaggregation was abolished. Using AA, the RMP also potentiated aggregation from 20–30% to 50–60%. These results were obtained with heparinized PRP. Interestingly, when citrated PRP was used, the RMP effect was negligible. (2) Shear-induced platelet adhesion: At 1800 sec−1 shear rate, which approximates venous blood flow, addition of RMP increases the adhered mean aggregate size from 47 to 53 μm2 (p
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Red Cell Microparticles (RMP) As Hemostatic Agent: Summary of Recent Advances
Blood, 2011Co-Authors: Yeon S. Ahn, Max E Johansen, Lawrence L. Horstman, Carlos J. BidotAbstract:Abstract 2260 Introduction: Microparticles (MP) derived from platelets (PMP), endothelia (EMP) and leukocytes (LMP) have received much attention as biomarkers in cardiovascular and inflammatory disorders. However MP derived from red cells (RMP) have been less well investigated. We previously presented evidence for the efficacy of RMP as a hemostatic agent in vivo and other effects such as on thrombin generation. Here we report data on in vitro proxies of hemostatic potential of RMP which further support its expected value as a hemostatic agent, and provide clues about mechanism of action. Methods: (i) RMP were produced by high-pressure extrusion of washed RBC. (ii) The effect of RMP on coagulation/clotting was assessed by thromboelastography (TEG) using whole blood or factor-deficient plasmas tested in presence vs. absence of added RMP (1×108/mL). Patient samples included those with coagulopathy, thrombocytopenia, thrombocytopathy. (iii) The effect of RMP on platelet aggregation was assessed by aggregometry (Chrono-Log). (iv) The effect of RMP on shear-induced platelet adhesion was by cone-and-well device (Diamed IMPACT-R) yielding measures of mean aggregate size (AS), percent surface coverage (SC) and number of objects (OBJ). Results: (1) RMP produced as above express procagulant phospholipids as judged by 20–30% of them binding annexin V. (2) RMP increased procoagulance in all factor-deficient plasmas tested (FII, V, VII, VIII, IX, X, XI, XII, XIII). The most pronounced effects were observed with FII, FVIII, FIX, FX deficiency, most markedly, FVIII and IX. RMP give significant correction for deficiencies as low as 5–10% normal levels. (3) RMP augmented platelet aggregation induced by low-dose ADP (0.2 μM) or arachidonic acid (0.3 mM). RMP largely eliminated dissociation of platelet aggregates after low-dose ADP. (4) At shear rate of 1800 sec−1, which approximates venous flow, aggregate size (AS) was significantly increased (p=0.02). (5) Using flow cytometry, RMP were observed to interact with platelet microaggregates expressing markers of activation, but not with free resting platelets. (6) RMP partly or completely corrected abnormal parameters seen on TEG in blood from patients with thrombocytopenia (aplastic anemia, ITP) as well as platelet dysfunctions induced by ASA or Plavix. It also improved or corrected abnormal parameters in coagulopathies such as hemophilia A with low inhibitor; and anticoagulant therapy with wafarin, LMWH, or Dabigatran. Summary/Discussion: (1) Increased procoagulance in all factor deficient plasma by RMP indicates that RMP provide anionic surface for clotting factors. Since this was most pronounced with FII, FVIII, FIX, and FX, and was strongest in FVIII, FIX, it appears that RMP have some specificity for assembly of tenase. (2) Correction of clotting abnormality in thrombocytopenia and platelet dysfunction by RMP suggests that RMP may provide additional catalytic surface for coagulation, as well as contributing modestly to clot strength. The latter effect may depend on presence of weakly activated platelets. (3) The increased maximum amplitude (MA) seen in TEG by RMP suggests that RMP may enhance platelet-fibrin interaction, increasing clot stability. Taken together, these data support the potential use of RMP as a universal hemostatic agent. Disclosures: No relevant conflicts of interest to declare.
Carlos J. Bidot - One of the best experts on this subject based on the ideXlab platform.
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correlations of red cell derived micropareticles RMP with other mp species in hematological and thrombotic disorders
Blood, 2015Co-Authors: Lawrence L. Horstman, Carlos J. Bidot, Pamela Dudkiewicz, Mohamed El Dinali, Gabriel Tinoco, Yeonsoong AhnAbstract:Background : We have recently observed that increased levels of circulating RMP are associated with several hematological disorders and thrombophilic states, and that levels were significantly higher when thrombosis was present. However, other species of MP, such as from platelets (PMP), endothelia (EMP), leukocytes (LMP) and annixin V-binding (AnV) have also been shown to be associated with thrombophilic states. The purpose of this study was to determine if correlations exist between RMP and other MP. Methods : This is a retrospective analysis of 702 laboratory samples over an 8 year period, limited to elevated values: >2SD above normal controls (>2,000/uL).. About 87% were individuals, the remaining 13% were tested 2-3 times and all tests >3 per patient were excluded. The disorders were hemolytic anemia (HA, n=38), hypercoagulable state (HCS, n=64), immune thrombocytopenia (ITP, n=86), thrombocytopenia of all causes (TP, n=69), myeloprolifereative disorder (MPD, n=29), thrombotic thrombocytopenic purpura (TTP, n=29), antiphospholipid syndrome (APS, n=34), pulmonary embolism (PE, n=21), and all-cause thrombosis (TBS, n=251). Some were classified in more than one way. MP species assayed were RMP by glycophorinA, PMP by CD41 (PMP41), PMP by CD42 (PMP42), EMP by CD31+/42- (EMP31), EMP by CD62E (EMP62E), LMP by CD45, annexin V binding (AnV), and counts by lectin, FITC-Ulex, which efficiently detects total MP including very small. Results : In HCS, the RMP >2,000/uL correlated well with PMP41 (R=0.407, p 2,000/uL and any of the MP markers in APS, TP, or TTP. The MP species markers, EMP31, EMP62E, and AnV, failed to show correlation with RMP in any of the disorders analyzed. In addition, we tested for correlations between elevated RMP and other MP for the entire data set (all disorders combined) and found that only LMP was significant (p 0.05). Discussion : These correlation analyses shows that RMP correlated most frequently with PMP, as seen in HA, ITP, HCS, and TBS, followed by LMP, as seen in HA and TBS. These observations suggest that platelet or/and leukocyte activation may be involved in RMP generation. Of added interest is the finding that the overall data correlated well with LMP only at the higher LMP levels, not at all in the lower quintile of LMP. This suggests that RMP elevations are associated with immunolgic, inflammatory processes. In summary, correlation analysis reveals likely interaction between red cells and platelets or leukocytes during immunologic, inflammatory or in thrombophilic states, resulting in elevated RMP. Disclosures No relevant conflicts of interest to declare.
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Elevated Red Cell Microparticles (RMP) in Hematologic and Thrombotic Disorders
Blood, 2015Co-Authors: Gabriel Tinoco, Lawrence L. Horstman, Carlos J. Bidot, Pamela Dudkiewicz, Mohamed El Dinali, Yeonsoong AhnAbstract:Introduction. Circulating cell-derived microparticlesa (MP) from platelets (PMP), leukocytes (LMP), and endothelia (EMP) have been well-documented for their roles in hemostasis, thrombosis and inflammation but the clinical significance of RMP is less well understood. The purpose of this study is to study the relation of elevated RMP to selected hematologic and thrombotic disorders. Methods. This is a retrospective study on RMP profiles for patients referred to University of Miami Hospital and Clinics for hematologic consultation over the last 8 years. The patient population includes 51 hemolytic anemia (HA), 459 thrombocytopenia (TP), 26 myeloproliferative disorder (MPD), 413 hypercoagulable state (HCS), and 446 thrombotic disorders (TBS). Some patients were analyzed in more than one of the above disorders. Levels of RMP were measured by flow cytometry with PE-conjugated anti-CD235a labeling as previously described [Thromb. & Haemost., 2013;110:751-60]. Levels of RMP above mean +2SD of controls (> 2,000 counts/mL) are designated as "elevated RMP". Results. (I) Prevalence of elevated RMP in patient populartion: Elevated RMP were found in 31 of 51 HA (60.8%), 138 of 459 TP (30.1%), 20 of 26 MPD (78.1%), 175 of 413 HCS (42.4%), and 251 of 446 TBS (56.3%). (II) Association of elevated RMP with thrombosis: Of 31 HA patients with elevated RMP, 11 were positive for TBS, and the remaining 20 were negative. Levels of RMP (mean ±SD) for TBS(+) and TBS(-) were 5,824 ±3713 and 3,265 ±1,048, counts /mL, respectively (p Discussion. (i) The cause of RMP elevation in HA is reasonably attributed to red cell destruction. The cause in TP/ITP is likely due to products of platelet destruction or leukocyte activation. The cause in MPD could result from clearance of the high burden of red cells and platelets. (ii) Patients who were TBS(+) had higher RMP than TBS(-), seen in HCS, HA, TP, raising the question of whether high RMP is a cause or consequence of TBS. To answer this will require further study. (iii) The finding of exceptionally high RMP levels in recurrent TBS vs. non-recurrent TBS (HCS-3 vs. HC-2) indicates that RMP levels reflect severity of TBS. (iv) Finally, these data indicate that RMP may be a useful biomarker of thrombotic risk, particularly because some TBS patients had elevated RMP, yet tested negative by all conventional markers of HCS workups. Disclosures No relevant conflicts of interest to declare.
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Release of Microparticles During Blood Storage Is Influenced by Residual Platelets, Leukocytes and Oxygen Levels
Blood, 2012Co-Authors: Wenche Jy, Ralph R. Vassallo, Max E Johansen, Orlando Gomez-marin, Tatsuro Yoshida, John Morgan, Lawrence L. Horstman, Carlos J. Bidot, Sherry ShariatmadarAbstract: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.
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Interaction of Red Cell Microparticles (RMP) with Platelets: Potential Role of RMP in Hemostasis and Thrombosis,
Blood, 2011Co-Authors: Max E Johansen, Lawrence L. Horstman, Carlos J. Bidot, Powei Chen, Yeon S. AhnAbstract:Abstract 3263 Introduction: We previously reported data indicating that RMP are well suited for use as hemostatic agent for treating bleeding disorders ([Jy et al, Hemophilia 17:4, 2011][1]). Previous studies have shown that RMP can contribute to RBC-related thrombotic complications such as sickle cell disease and PNH. Microparticles (MP) derived from platelets (PMP), endothelia (EMP), and leukocytes (LMP) are believed to play a role in hemostasis and thrombosis. They can adhere to blood cells and endothelia, facilitating prothrombotic and proinflammatory reactions. However, less is known about interaction of RMP with cells and their potential role in hemostasis and thrombosis. Here we report evidence of interaction of RMP with platelets resulting in enhanced platelet aggregation and increased size of adherent platelet aggregates induced by shear stress. Methods: (i) RMP were prepared by high-pressure extrusion of washed RBC. (ii) Platelet aggregation was performed in a Chrono-log aggregometer. PRP (490 μL) was mixed with 10 μL of RMP (1 × 108 /mL final conc.) for 5 min, then low-dose activating agent (ADP 0.2 μM, or arachidonic acid (AA) 0.3 mM) was added. (iii) Shear-induced platelet adhesion was measured in a cone-and-well device (Diamed Impact-R). Whole blood was pre-incubated with RMP as above for 10 min, then subjected to various shear rates (900, 1800, 2700 sec−1) for 1 min. The adherent platelets were then washed, stained, and quantitated by image analyzer. (iv) RMP-platelet interaction employed 2-color flow cytometry. RMP-platelet conjugates were identified by co-expression of α-CD41-FITC and α-glycophrin A-PE, in both the free platelet and micro-aggregated platelet populations. Results: (1) Platelet aggregation: Addition of RMP to PRP did not induce platelet aggregation. However, RMP enhanced platelet aggregation induced by low-dose ADP or AA. Low-dose ADP alone induced a transient increase of aggregation peaking at 25–35% followed by slow disaggregation to 0–5% at 10 min; but in presence of RMP, a similar rate (slope) of aggregation was seen but peaking at 50–60% and disaggregation was abolished. Using AA, the RMP also potentiated aggregation from 20–30% to 50–60%. These results were obtained with heparinized PRP. Interestingly, when citrated PRP was used, the RMP effect was negligible. (2) Shear-induced platelet adhesion: At 1800 sec−1 shear rate, which approximates venous blood flow, addition of RMP increases the adhered mean aggregate size from 47 to 53 μm2 (p
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Red Cell Microparticles (RMP) As Hemostatic Agent: Summary of Recent Advances
Blood, 2011Co-Authors: Yeon S. Ahn, Max E Johansen, Lawrence L. Horstman, Carlos J. BidotAbstract:Abstract 2260 Introduction: Microparticles (MP) derived from platelets (PMP), endothelia (EMP) and leukocytes (LMP) have received much attention as biomarkers in cardiovascular and inflammatory disorders. However MP derived from red cells (RMP) have been less well investigated. We previously presented evidence for the efficacy of RMP as a hemostatic agent in vivo and other effects such as on thrombin generation. Here we report data on in vitro proxies of hemostatic potential of RMP which further support its expected value as a hemostatic agent, and provide clues about mechanism of action. Methods: (i) RMP were produced by high-pressure extrusion of washed RBC. (ii) The effect of RMP on coagulation/clotting was assessed by thromboelastography (TEG) using whole blood or factor-deficient plasmas tested in presence vs. absence of added RMP (1×108/mL). Patient samples included those with coagulopathy, thrombocytopenia, thrombocytopathy. (iii) The effect of RMP on platelet aggregation was assessed by aggregometry (Chrono-Log). (iv) The effect of RMP on shear-induced platelet adhesion was by cone-and-well device (Diamed IMPACT-R) yielding measures of mean aggregate size (AS), percent surface coverage (SC) and number of objects (OBJ). Results: (1) RMP produced as above express procagulant phospholipids as judged by 20–30% of them binding annexin V. (2) RMP increased procoagulance in all factor-deficient plasmas tested (FII, V, VII, VIII, IX, X, XI, XII, XIII). The most pronounced effects were observed with FII, FVIII, FIX, FX deficiency, most markedly, FVIII and IX. RMP give significant correction for deficiencies as low as 5–10% normal levels. (3) RMP augmented platelet aggregation induced by low-dose ADP (0.2 μM) or arachidonic acid (0.3 mM). RMP largely eliminated dissociation of platelet aggregates after low-dose ADP. (4) At shear rate of 1800 sec−1, which approximates venous flow, aggregate size (AS) was significantly increased (p=0.02). (5) Using flow cytometry, RMP were observed to interact with platelet microaggregates expressing markers of activation, but not with free resting platelets. (6) RMP partly or completely corrected abnormal parameters seen on TEG in blood from patients with thrombocytopenia (aplastic anemia, ITP) as well as platelet dysfunctions induced by ASA or Plavix. It also improved or corrected abnormal parameters in coagulopathies such as hemophilia A with low inhibitor; and anticoagulant therapy with wafarin, LMWH, or Dabigatran. Summary/Discussion: (1) Increased procoagulance in all factor deficient plasma by RMP indicates that RMP provide anionic surface for clotting factors. Since this was most pronounced with FII, FVIII, FIX, and FX, and was strongest in FVIII, FIX, it appears that RMP have some specificity for assembly of tenase. (2) Correction of clotting abnormality in thrombocytopenia and platelet dysfunction by RMP suggests that RMP may provide additional catalytic surface for coagulation, as well as contributing modestly to clot strength. The latter effect may depend on presence of weakly activated platelets. (3) The increased maximum amplitude (MA) seen in TEG by RMP suggests that RMP may enhance platelet-fibrin interaction, increasing clot stability. Taken together, these data support the potential use of RMP as a universal hemostatic agent. Disclosures: No relevant conflicts of interest to declare.
Madleen Adel A Abdou - One of the best experts on this subject based on the ideXlab platform.
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increased circulating red cell microparticles RMP and platelet microparticles pmp in immune thrombocytopenic purpura
Thrombosis Research, 2013Co-Authors: Eman M Sewify, Douaa Sayed, Refat Abdel F Aal, Heba M Ahmad, Madleen Adel A AbdouAbstract:Abstract It has been suggested that patients with ITP have an increased thrombotic risk compared to the general population and compared to those with other causes of acquired thrombocytopenia. The pro-coagulant role of microparticles in some clinical situations has been reported, yet, very little data is available about microparticles in ITP and their effect. Aim of the work To assess the levels of red cell microparticles (RMP), platelet microparticles (PMP) and their possible relation to some haemostatic parameters in ITP patients Patients and methods The levels of RMP and PMP in addition to FVIII, FIX, FXI, PC and aPTT were assessed in 29 patients with chronic ITP (8 of them had splenectomy). Ten apparently healthy volunteers served as controls. We compared the levels of the studied parameters in ITP patients with that in controls. Correlations of these parameters with each other and with the platelet count were studied. Results RMP (p = 0.0001), PMP (p = 0.0001), D- dimer (p = 0.048), FVIII (p = 0.049), FIX (p = 0.0001) and FXI (p = 0.0001) were significantly higher in ITP patients compared to controls. aPTT was significantly longer in ITP patients (p = 0.0001) but PC showed no significant difference. However, RMP was associated with shorter aPTT. Generally, the coagulation factors were negatively correlated with platelet count in ITP patients. Compared to controls, ITP patients preserved higher levels of RMP and PMP even in those with near-normal platelet count. Splenectomy was associated with lower FIX (p = 0.0001) and FXI (p = 0.028) and higher RMP (p = 0.0001). In conclusion Chronic ITP was associated with increased levels of RMP and PMP. FVIII, FIX and FXI were increased in ITP patients but showed a negative correlation with platelet count. Splenectomy was associated with increased levels of RMP and lower levels of FIX and F XI. The high level of microparticles in ITP might point towards a prothrombotic tendency.
Vincenzo Fontana - One of the best experts on this subject based on the ideXlab platform.
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Elevated Red Cell Microparticles (RMP) as Risk Factor for Thrombosis in Patients with Renal Failure.
Blood, 2007Co-Authors: Vincenzo Fontana, Lawrence L. Horstman, Carlos J. Bidot, Dudkiewicz Pamela, Fernandez Carina, Ahn S. AhnAbstract:BACKGROUND: Cell-derived microparticles (MP) are microvesicles released during activation or apoptosis from endothelial cells (EMP), platelets (PMP), leukocytes (LMP) and red cells (RMP). Their roles in hemostasis and inflammation are increasingly coming to light. Chronic and acute renal failure (RF) is associated with increased risk of bleeding as well as thrombosis but the pathogenesis is unclear. Platelet hyperaggregation, elevated clotting factors, deficiency in natural anticoagulants, impaired fibrinolysis and hyperhomocysteinemia have all been implicated in thrombosis in RF. Recently high levels of EMP, PMP and LMP have been reported in RF but their clinical significance is obscure. We investigated these MP as well as RMP in patients with RF. METHODS: Twenty-seven patients (14M/13F, mean age 53yr) with RF were investigated. Incidence of thrombosis was assessed. Healthy controls were n=109. Flow cytometry was used to assay LMP by anti CD45, PMP by anti CD41, RMP by anti-glycophorin, and EMP by CD31+/CD41−. RESULTS: The RF patients were divided into groups with thrombosis (TB) and without (NTB). Nine patients (33%) had thrombosis, most often venous. When MP were compared between controls and all RF patients, EMP (p=0.0004) and RMP (P=0.01) were elevated in RF. However, PMP and LMP in RF did not differ from controls (FIG. 1). On the other hand, when we compared MP between TB and NTB, only RMP was significantly greater in TB (mean 2064/uL) than NTB (mean 972/uL), p=0.001. Mean levels of EMP, PMP and LMP did not differ significantly between TB and NTB in RF (FIG. 2). CONCLUSION / DISCUSSION: First, we confirm elevated EMP in RF compared to normal controls, and also report for the first time elevated RMP in RF. This indicates activation of endothelium and and red cells. Elevation of procoagulant MP likely contribute to risk of thrombosis in RF. Interestingly, PMP and LMP were not significantly increased in RF. Secondly, in comparing MP between the two groups of RF patients, only RMP, not other MP, were significantly higher in TB than NTB. The potential role of RMP in hemostasis and thrombosis is unknown, but the present study demonstrates a unique association of elevated RMP with thrombosis. Specifically, only RMP discriminate the TB from the NTB group in RF. This may suggest a role of RMP in thrombosis. RMP carry procoagulant phosphatidylserine (PS). Expression of PS was increased in RBC of patients with RF. The mechanism of increased RMP in RF is not known. Increased membrane fragility and decreased deformability of RBC seen in uremic patients, together with enhanced membrane lipid peroxidation from oxidative stress would contribute to increased hemolysis and RMP in RF. Thus, the present findings should further stimulate investigation into the causes and significance of RMP in RF and other TB disorders.
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cell derived microparticles in itp patients with vs without bleeding tendency
Blood, 2007Co-Authors: Vincenzo Fontana, Wenche Jy, Pamela Dudkiewicz, Lawrence L. HorstmanAbstract:INTRODUCTION: In immune thrombocytopenic purpura (ITP), autoantobodies mediate platelet destruction, leading to thrombocytopenia and a hemorrhagic diathesis. Although platelet counts are used to assess risk of bleeding in ITP, this is not dependable since some seldom bleed while others bleed excessively at the same level of thrombocytopenia. Cell-derived microparticles (C-MP) are microvesicles released upon activation or apoptosis from blood cells such as platelets (PMP), leucocytes (LMP) and red cells (RMP) as well as endothelial cells (EMP). Their roles in hemostasis, thrombosis and inflammation are increasingly appreciated. We investigated C-MP in ITP in patients with bleeding tendency vs. those without. METHODS: Thirty-seven patients (24 F/13M, mean age 53.8 yr) with chronic ITP (platelet counts history of spontaneous frequent mucosal bleeding (nose, gum etc), episodes of organ bleeding such as GI, GU, CNS bleeding, menorrhagia with recurring anemia, cutaneous bleeding characterized by gross petechiae on >2 sites (leg, arm, trunk etc) or multiple (>5) ecchymosis >3cm sizes or wet purpuras. Patients with similar platelet counts but not meeting any of these criteria were defined as non-bleeders (NBL). The BL group consisted of 17 pt (7M/10F, mean age 53.6 yr) while the NBL group comprised 20 pt (6M/14F, mean age 54.0 yr). The BL group contained 12 with skin bleeding and 5 with mucosal /organ bleeding. Pertinent data are summarized in Table. Coulter XL flowcytometer was employed to identify LMP by anti-CD45, PMP by anti-CD41, RMP by anti-glycophorin, and EMP by the combination CD31+/CD41-. Platelet counts and aPTT were also compared between the groups. RESULTS: Mean platelet count was similar in both groups (27,000/μL). When C-MP were compared between BL vs NBL, there was no significant difference in mean levels of EMP (203 vs 169, p > 0.05) or LMP (1342 vs 1422, p > 0.05). However, RMP were significantly higher in the NBL group (2878 vs 1310, p = 0.01). See Table. PMP were also higher in NBL (3498 vs 1771) but did not reach significance. The aPTT was shorter in the NBL (24.4s vs 26.5s) but not significantly. CONCLUSION / DISCUSSION: These data support the unexpected conclusion that RMP are significantly associated with hemostasis in ITP. PMP were also elevated in NBL compared to BL but did not reach significance in this study. Our previous study documenting elevated PMP in NBL vs BL [Jy et al, JLCM119:334, 1992] employed a different assay system [Coulter EPICS V (2 watt laser) and detection by light scatter with CD42 not CD41]. Other factors implicated in hemostasis in ITP include increased number of larger young platelets and activated platelets. Our data suggest that elevated RMP may be a significant hemostatic factor in thrombocytopenic states. However, we also note a trend of increased PMP, as well as shortened aPTT in NBL. Since RMP are known to express phosphatidylserine (binding sites for coagulation factors), they can promote coagulation to facilitate blood clotting, and thus may aid in prevention of bleeding in thrombocytopenic patients.
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cell derived microparticles c mp are elevated in splenectomized patients and depressed in those with hypersplenism suggesting spleen as scavenger of c mp
Blood, 2006Co-Authors: Vincenzo Fontana, Wenche Jy, Lawrence L. Horstman, Pamela Dudkiewicz, Carlos J. BidotAbstract:BACKGROUND: Cell-derived microparticles (C-MP) are microvesicles released during activation and apoptosis from platelets (PMP), leukocytes (LMP), endothelial cells (EMP) and red cells (RMP). They commonly express phosphatidylserine (PS) judged by binding annexin V (AnV) and carry markers of parent cells. C-MP are involved in thrombosis, inflammation and angiogenesis. Little is known how C-MP are cleared from circulation. To test that the spleen might be involved, we investigated C-MP profiles in patients who underwent splenectomy for various causes (group A), those with hypersplenism (group B), and healthy controls with spleen (group C). MATERIAL AND METHODS : Group A (Gr A) consisted of 44 patients (18 M and 26 F, mean age 57.2 yr) with splenectomy; group B (Gr B) consisted of 25 patients (12 M and 13 F, mean age 61.6 yr) with hypersplenism. Gr A had 25 ITP, 7 myeloproliferative disorders, 4 lymphoma, 3 hemolytic anemias, 4 trauma related splenectomy, 1 unknown reason. Group B had 19 chronic hepatitis C, 2 alcoholic, 2 idiopathic and 1 autoimmune hepatitis, 1 portal vein thrombosis. Group C (Gr C) were healthy controls with spleen (n=109). Flow cytometry was used to assay C-MP. PMP were identified by CD41+, LMP by CD45+, EMP by CD31+/CD41−, and RMP by glycophorin+. Profile of C-MP and blood counts were compared among the 3 groups. RESULTS: Table 1 summarizes C-MP data among the 3 groups. Gr A and B were well matched for ages and sex. Mean values of WBC and Hgb were normal in all groups while the platelet count was low in Gr B. Mean values of LMP, EMP and RMP were significantly higher in Gr A compared to Gr B and C, but PMP were not. All species of C-MP (PMP, LMP, EMP, RMP) in Gr B were significantly lower than Gr C (healthy control) and were markedly depressed compared to Gr A (see Table 1). Correlation analysis revealed that PMP correlated with platelet count (p DISCUSSION/COMCLUSIONS: Our data suggest that spleen sequesters and/or clears C-MP of all types (PMP, LMP, EMP, RMP). C-MP express PS (bind AnV), which is known to promote clearance from circulation. Splenectomy is known to be associated with thrombosis but its mechanism in not well elucidated. Elevated C-MP in splenectomized patients may contribute to their increased risk of thrombosis.
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Profile of Cell-Derived Microparticles (C-MP) in ITP: Red Cell Microparticles (RMP) Correlate with Severity of ITP.
Blood, 2006Co-Authors: Vincenzo Fontana, Lawrence L. Horstman, Pamela Dudkiewicz, Eugene Ahn, Myriam Yaniz, Yeonsoong AhnAbstract:BACKGROUND: The roles of cell-derived microparticles (C-MP) released from platelets (PMP), endothelial cells (EMP), leukocytes (LMP) and red cells (RMP) in hemostasis, thrombosis and inflammation have been appreciated in recent years. Although PMP were shown to be hemostatically active, the roles of other C-MP, especially RMP, have not been studied in ITP. We investigated C-MP in patients with ITP. MATERIAL AND METHODS: One-hundred-six patients with ITP were studied. They consisted of 73 pts with active ITP (ITP-A, 30M/43F, mean age 53.8 yr) and 33 pts in remission (ITP-R, 4M/29F, mean age 53.4 yr). ITP-A was defined by plt count 3 months; ITP-R was defined by plt count >140,000 for >3 months. Mean plt counts was 78,000 for ITP-A and 224,000 for ITP-R. The two groups were similar in ages. CBC with plt count, aPTT and activities of FVIII, FIX, FXI, were measured. Using flow cytometry, PMP were identified by CD41+, EMP by CD31+/CD41−, LMP by CD45+, and RMP by glycophorin+. Effects of intravenous immunoglobulins (IVIG) on C-MP were also evaluated. RESULTS: There was a strong inverse correlation between plt counts and RMP (p=0.002). RMP were higher when plt counts were lower. A significant correlation was found between platelet counts and PMP (p 0.05). The mean value of RMP was higher in ITP-A than ITP-R (p= 0.009). Conversely, PMP and EMP were higher in ITP-R (P DISCUSSION/CONCLUSIONS: (i) RMP inversely correlated with plt counts and appear to be a sensitive marker of severity of ITP. However mechanisms of RMP generation, their reduction following IVIG and their roles remain to be elucidated. (ii) Among C-MP, only RMP, not other C-MP, were associated with shortening aPTT and elevated FVIII, IX, XI. (iii) RMP are positive for annexin V, providing anionic phospholipids for clotting factors and generating thrombin (data not shown). These findings suggest that RMP associated with these factors are involved in hemostasis to prevent bleeding in severe ITP.
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Evidence of Different Subspecies of Microparticles (MP) Derived from Red Blood Cells (RMP) and Comparison of Their Phenotypes and Procoagulant Activity.
Blood, 2006Co-Authors: Jaehoon Bang, Vincenzo Fontana, Eugene Ahn, Loreta Bidot, Andrew Lin, Joaquin J. Jimenez, Yeonsoong AhnAbstract:BACKGROUND: The potential roles of cell derived microparticles (MP) such as those derived from platelets (PMP), endothelium (EMP), leukocytes (LMP), and red cells (RMP) have been receiving increasing attention in disorders of hemostasis/thrombosis and inflammation and they are emerging as valuable biomarkers. However among these MP, little is known about RMP. Our recent clinical studies indicate that RMP play a role in hemostasis and thrombosis in patients with thrombocytopenia and in thrombocytosis. However, the phenotypes and procoagulant activity of their subspecies remain unknown. We report evidence for heterogeneity of RMP following differential centrifugation. METHODS: RMP were prepared by exposure of washed RBC to the calcium ionophore, A23187, and the RBC were removed by low-speed centrifugation. The RMP were washed twice at 20,000xg for 15 min. Procoagulant activity of RMP was measured by the calibrated automated thrombogram (CAT) system (Hemker et al Pathophysiol Haemost Thromb.2002;32:249) using thrombin substrate Z-Gly-Gly-Arg-AMC on a fluorescence plate reader. The lag time and peak height (nM) of thrombin generation were recorded. Markers used for labeling RMP were PE-labeled anti-glycophorin (GlyP), FITC-anti-tissue factor (TF), FITC-annexin V (AnV), and/or FITC-lectin Ulex europeaus I (Ulex). RESULTS: In thrombin generation assay, RMP induced a long lag time (24±3 min) but high thrombin peak (330±37 nM). These data were consistent with the flow cytometric finding that RMP carried very little TF ( SUMMARY: The present study demonstrates that RMP are rich in anionic phospholipids and effective in generating thrombin in vitro. We have identified 2 distinct subpopulations of RMP by differential centrifugation: One larger RMP express binding of anti-GlyP, AnV and Ulex, and carry the majority of procoagulant activity. The smaller RMP expressing only Ulex binding exhibit much weaker procoagulant activity. The roles of these two species of RMP remain to be elucidated. We speculate that smaller RMP may represent the nanovesicles described by Allen et al [Biochem J 188:881, 1980] and that Ulex may be a novel and convenient means for the study of these small vesicles.
