PMP Levels Research Articles

Impact of Early Microparticle Release during Isolated Severe Traumatic Brain Injury: Correlation with Coagulopathy and Mortality.

Microparticles (MPs) have been implicated in thrombosis and endothelial dysfunction. Their involvement in early coagulopathy and in worsening of outcomes in isolated severe traumatic brain injury (sTBI) patients remains ill defined. We sought to quantify the circulatory MP subtypes derived from platelets (PMPs; CD42), endothelial cells (EMPs; CD62E), and those bearing tissue factor (TFMP; CD142) and analyze their correlation with early coagulopathy, thrombin generation, and in-hospital mortality. Prospective screening of sTBI patients was done. Blood samples were collected before blood and fluid transfusion. MP enumeration and characterization were performed using flow cytometry, and thrombin-antithrombin complex (TAT) levels were determined using enzyme-linked immunosorbent assay (ELISA). Circulating levels of procoagulant MPs were compared between isolated sTBI patients and age- and gender-matched healthy controls (HC). Patients were stratified according to their PMP, EMP, and TFMP levels, respectively (high ≥HC median and low < HC median). Isolated sTBI resulted in an increased generation of PMPs (456.6 [228-919] vs. 249.1 [198.9-404.5]; P = 0.01) and EMPs (301.5 [118.8-586.7] vs. 140.9 [124.9-286]; P = 0.09) compared to HCs. Also, 5.3% of MPs expressed TF (380 [301-710]) in HCs, compared to 6.6% MPs (484 [159-484]; P = 0.87) in isolated sTBI patients. Early TBI-associated coagulopathy (TBI-AC) was seen in 50 (41.6%) patients. PMP (380 [139-779] vs. 523.9 [334-927]; P = 0.19) and EMP (242 [86-483] vs. 344 [168-605]; P = 0.81) counts were low in patients with TBI-AC, compared to patients without TBI-AC. Our results suggest that enhanced cellular activation and procoagulant MP generation are predominant after isolated sTBI. TBI-AC was associated with low plasma PMPs count compared to the count in patients without TBI-AC. Low PMPs may be involved with the development of TBI-AC.

Megakaryocyte- and Platelet-Derived Microparticles as Novel Diagnostic and Prognostic Biomarkers for Immune Thrombocytopenia.

Altered cell-derived microparticles (MPs) have been reported in multiple autoimmune diseases. However, the roles of megakaryocyte- and platelet-derived MPs (MKMPs and PMPs) in immune thrombocytopenia (ITP) have not been investigated. In this study, we examined plasma MKMP and PMP levels in patients with ITP and evaluated their potential diagnostic values. Plasma MKMP and PMP levels were analyzed by flow cytometry in a discovery set of ITP patients (n = 78), non-immune thrombocytopenia (TP) patients (n = 69), and age- and gender-matched healthy controls (n = 88). Samples from a therapy set of ITP patients (n = 21) were used to assess the response to thrombopoietin receptor agonist (TPO-RA) treatment. Spearman correlation analysis was performed between MP levels and disease parameters. Receiver operator characteristic (ROC) curves were generated to evaluate the diagnostic values of the MPs. We found that plasma MKMP and PMP levels were significantly lower in ITP patients than those in healthy controls (p values &lt; 0.0001) but higher than in those in TP patients (p &lt; 0.002 and p &lt; 0.0002, respectively). After normalization to platelet counts, PMP/Platelet ratios in ITP patients were higher than those in TP patients and healthy controls (p values &lt; 0.001). PMP/Platelet ratios had a diagnostic value for ITP (area under the curve = 0.808, p &lt; 0.0001) with 73.1% sensitivity and 77.3% specificity. MKMP levels can be used to discriminate ITP from TP with a cut-off value of 112.5 MPs/μL and a sensitivity of 74.4%. Moreover, both MKMP and PMP levels were elevated in ITP patients who responded to TPO-RA treatment. Plasma PMP levels positively correlated with platelet counts in the responders (r = 0.558, p &lt; 0.01). Our results indicate that plasma MKMP and PMP levels are decreased in ITP patients and that plasma MKMP and PMP levels may serve as biomarkers for ITP diagnosis and prediction of TPO-RA treatment response.

Persistent circulating platelet and endothelial derived microparticle signature may explain on-going pro-thrombogenicity after acute coronary syndrome

AimsMicroparticles (MPs) are submicron vesicles, released from activated, and apoptotic cells. MPs are elevated in the circulation of patients with coronary artery disease (CAD) and have pro-thrombotic potential. However, limited data exists on MP signature over time following an acute coronary event. Methods & resultsCirculating total annexin v + (Anv+) MPs of endothelial (EMP), platelet (PMP), monocyte (MMP), neutrophil (NMP) and smooth muscle cell (SMMP) origin were quantified by flow cytometry. 13 patients with acute coronary syndrome (ACS) were prospectively enrolled and 12 patients with stable angina (SA) were included as a comparator group. A panel of MP was measured at baseline, after percutaneous coronary intervention (PCI) and at days 1, 7, 30 and 6 months. Intra & inter group comparison was made between various time points. MP mediated thrombin generation was measured by recording lag phase, velocity index, peak thrombin and endogenous thrombin potential at these time points and compared with healthy controls. The total AnV+ MP levels were similar in ACS and SA groups at baseline, peaked immediately after PCI and were at their lowest on day 1. PMP & EMP levels remained significantly elevated in ACS patients at 6 months when compared to SA. No such difference was noted with NMP, MMP and SMMP. Patients with coronary artery disease showed abnormal thrombograms when compared to controls. Peak thrombin (nano moles) was significantly higher in CAD when compared to controls (254 IQR [226, 239] in ACS, 255 IQR [219, 328] in SA and 132 IQR [57, 252] in controls; p = 0.006). Differences in thrombin generation between ACS and SA were not significant (p = 1). Furthermore, thrombin parameters remained abnormal in ACS & SA patients at 6 months. ConclusionsTotal MP and individual MP phenotypes were significantly elevated after PCI reflecting endothelial injury. Elevated PMP and EMP levels at 6 months in ACS patients is suggestive of on-going inflammation, endothelial injury and may explain on-going pro-thrombogenicity seen up to 6 months after ACS despite dual antiplatelet therapy.

Increased Levels of Platelets and Endothelial-Derived Microparticles in Patients With Non-Valvular Atrial Fibrillation During Rivaroxaban Therapy.

It is known that atrial fibrillation (AF) is associated with the procoagulant state. Several studies have reported an increase of circulating microparticles in AF, which may be linked to a hypercoagulable state, atrial thrombosis and thromboembolism. We evaluated in our study alterations in both platelet (PMP, CD42b) and endothelial-derived (EMP, CD144) microparticle levels on anticoagulant therapy with rivaroxaban in nonvalvular AF. After administration of rivaroxaban, PMP levels were increased (median, [IQR] 35.7 [28.8-47.3] vs. 48.4 [30.9-82.8] cells/µL; P = 0.012), along with an increase in EMP levels (14.6 [10.0-18.6] vs. 18.3 [12.9-37.1] cells/µL, P < 0.001). In the multivariable regression analysis, the independent predictor of post-dose change in PMPs was statin therapy (HR −0.43; 95% CI −0.75,−0.10, P = 0.011). The post-dose change in EMPs was also predicted by statin therapy (HR −0.34; 95% CI −0.69, −0.01, P = 0.046). This study showed an increase in both EMPs and PMPs at the peak plasma concentration of rivaroxaban. Statins have promising potential in the prevention of rivaroxaban-related PMP and EMP release. The pro-thrombotic role of PMPs and EMPs during rivaroxaban therapy requires further study.

Thrombin Generation and other hemostatic parameters in patients with atrial fibrillation in use of warfarin or rivaroxaban.

Patients with atrial fibrillation (AF) present hyperactivation of both platelets and coagulation leading to a hypercoagulable state which contributes to an increased risk of thromboembolism. Therefore, one of the main strategies for treatment of AF is prevention of these events through the use of oral anticoagulants (OAC). The aim of this study was to evaluate hemostasis as a whole in patients with non-valvular AF undergoing warfarin or rivaroxaban by thrombin generation test (TGT), in addition to monocyte-platelet aggregates (MPA), glycoprotein IIb/IIIa (GPIIb/IIIa), and platelet (PMP) and endothelium (EMP) microparticles, compared to age and sex matched controls. PT/INR for OAC use was also determined. In patients taking OAC, compared to control group, a decrease in TGT (p = 0.000 for all parameters) were observed. Patients taking warfarin showed to be more hypocoagulable, presenting lower levels of ETP (p = 0.000) and peak (p = 0.002) than patients using rivaroxaban. Patients on warfarin use with INR > 3 had also lower levels of ETP (p = 0.01) and peak (p = 0.006). A decrease in ETP (p = 0.03) and peak (p = 0.02) values was also observed in patients using rivaroxaban with PT > 21.4s. Patients using warfarin (p = 0.000) and rivaroxaban (p = 0.000) presented lower levels of MPA in relation to control group. It was also observed in patients using warfarin, lower GPIIb/IIIa levels in relation to control group (p = 0.011). Patients taking rivaroxaban (p = 0.003) and warfarin (p = 0.001) had higher PMP levels compared to control group. There was no difference in levels of EMP between the groups (p = 0.0536). The present study reinforces the usefulness of OAC in AF, which decisively contribute to a better management of the disease preventing possible complications.

P6602Plasma signature of apoptotic microparticles in acute coronary syndromes is associated with endothelial dysfunction and plaque rupture

Abstract Background Circulating microparticles (MP) are surrogate biomarkers of atherosclerosis and are elevated in acute coronary syndromes (ACS) but its exact biological role remains unknown. Purpose To explore the diagnostic and biological significance of circulating apoptotic MP signature in patients with ACS. Methods We enrolled 122 ACS patients: n=38 with unstable angina (UA), n=49 with STEMI, 35 with NSTEMI. Flow cytometry analysis was used to quantify circulating apoptotic (annexin+) endothelial cell (EMP), red blood cell (RMP) and platelet (PMP) derived microparticles. Endothelial function was estimated with flow mediated dilation (FMD), and inflammatory status with C-reactive protein (hsCRP). Results The association between EMP, RMP and PMP is shown on a cloud plot (A). Using an unbiased approach, we performed hierarchical clustering (A) of the total population of patients with ACS by using the circulating levels of EMP, RMP and PMP (B). Hierachical clustering identified two discreet clusters of patients (Cluster A and B) without any differences in the presence of traditional risk factors (not shown), but significant differences in the distribution of ACS type (C). STEMI subtype (a surrogate for definite plaque rupture) was significantly increased in Cluster B, which also had significantly decreased FMD (D), but not hsCRP (p=NS). Conclusions Apoptotic MP are involved in the pathogenesis of acute coronary syndromes via promotion of endothelial dysfunction and plaque rupture. The diagnostic and/or predictive value of microparticles' profiling for plaque vulnerability should be explored in future studies.

Platelet Glycoprotein VI Haplotypes and the Presentation of Paediatric Sepsis.

Sepsis triggers a complex series of pathophysiologic events involving inflammatory responses and coagulation abnormalities. While circulating blood platelets are well-characterized for their contributions to coagulation, increasingly platelet-dependent effects on inflammation are being recognized. Here, we focus on the platelet membrane receptor, glycoprotein VI (GPVI), and its role in platelet microparticle (pMP) release. The GPVI receptor is a platelet-specific collagen membrane receptor that, upon ligand binding, facilitates the release of pMPs. As membrane-bound platelet fragments of less than 1 μm, pMPs are known to have both pro-inflammatory and pro-coagulant properties. Thus, pMPs are potentially impacting sepsis at multiple stages of the inflammatory response. Studies are presented documenting the impact of the most common GPVI haplotypes, GPVIa and GPVIb, on pMP levels and release in healthy individuals (n = 49). The GPVIa haplotype corresponds to an approximately twofold increase in circulating pMPs as a percentage of total microparticles in healthy individuals along with a heightened in vitro release of pMPs. Additionally, patients admitted to a paediatric intensive care unit (ICU) (n = 73) with an initial diagnosis of sepsis were recruited and their GPVI haplotypes determined. Septic patients of the GPVIa haplotype (n = 59) were statistically more likely to present with a diagnosis of severe sepsis or septic shock, as compared with GPVIb individuals (n = 14). Independent disease classification via PELOD-2 and Pediatric Risk of Mortality III scores confirmed individuals with the GPVIa haplotype were more likely to have significant organ failure. Thus, GPVI haplotypes influence pMP levels in the circulation and are predictive of sepsis severity when presenting to the ICU.

Diagnostic value of platelet-derived microparticles in pulmonary thromboembolism: A population-based study.

An early and accurate diagnosis of pulmonary thromboembolism (PTE) remains challenging. The present study aimed to evaluate the diagnostic value of platelet-derived microparticles in PTE based on a population study. A total of 102 patients with PTE, 102 healthy controls and 40 patients suspected with PTE were enrolled in this study. The platelet count, mean platelet volume and platelet distribution width were assessed using an automated hematology analyzer, P-selectin was assessed using an ELISA kit and PMPs were explored using flow cytometry using Megamix beads. Receiver operating characteristic curves were established to evaluate the diagnostic values of PMPs, D-dimer, PMPs combined with D-dimer, and multiple parameters (including PMPs, platelet distribution width, P-selectin and D-dimer in PTE). The PMP levels were significantly higher in the patients with PTE (609.10/µl) compared with those in the healthy controls (230.60/µl) and patients with suspicious PTE (166.70/µl; P<0.01). The accuracy (72.06%) of PMPs in the diagnosis of PTE was similar to those of D-dimer (P>0.05). The combination of D-dimer and PMPs significantly increased the sensitivity (86.27%) of D-dimer and the specificity of PMP for the diagnosis of PTE (P<0.01). The combination of PMPs, platelet distribution width, P-selectin and D-dimer exhibited high sensitivity (88.24%), specificity (91.18%) and accuracy (89.71%) in the diagnosis of PTE. These findings suggest that elevated PMP levels are an effective predictor of PTE. The combination of PMPs, platelet distribution width, P-selectin and D-dimer may be used in the diagnosis of PTE with high sensitivity and specificity.

Microparticle subpopulations are potential markers of disease progression and vascular dysfunction across a spectrum of connective tissue disease

ObjectiveMicroparticles (MPs) are membrane-bound vesicles derived from vascular and intravascular cells such as endothelial cells (EMPs) and platelets (PMPs). We investigated EMP and PMP numbers across a spectrum of autoimmune rheumatic diseases (AIRDs) with the aim of comparing the levels of, and relationship between, EMPs and PMPs. MethodsPatients with Systemic Lupus Erythematosus (SLE) (n=24), Systemic Sclerosis (SSc) (n=24), Primary Raynauds Phenomenon (RP) (n=17) and “other CTD” (n=15) (Primary Sjogrens Syndrome, UCTD or MCTD) as well as 15 healthy controls were recruited. EMPs and PMPs were quantified using flow cytometry. Associations between MP levels and objective functional vascular assessments were evaluated. ResultsSLE patients had significantly higher EMPs compared with healthy controls and SSc patients. Higher PMP levels were noted in SSc and primary RP when compared to healthy controls and ‘other CTD’ patients. A modest correlation was noted between EMP and PMP levels in healthy controls (Spearman r=0.6, p=0.017). This relationship appeared stronger in SLE (r=0.72, p<0.0001) and other CTD patients (r=0.75, p<0.0001). The association between EMPs and PMPs was notably less strong in SSc (r=0.45, p=0.014) and RP (r=0.37, p=0.15). A significantly lower EMP/PMP ratio was detected in SSc/RP patients in comparison to both healthy controls and SLE/other CTD patients. Higher EMP and PMP levels were associated with higher digital perfusion following cold challenge in SSc. In contrast, higher PMP (but not EMP) levels were associated with lower digital perfusion at both baseline and following cold challenge in primary RP. Higher PMP levels were associated with greater endothelial-independent dilation in patients with SLE. ConclusionMP populations differ across the spectrum of AIRDS, possibly reflecting differences in vascular cell injury and activation. MP levels are associated with functional assessments of vascular function and might have a role as novel vascular biomarkers in AIRDs. Significance and innovationsLevels of circulating endothelial and platelet microparticles differ between SSc/primary RP compared with SLE and other CTDs (UCTD, MCTD and Primary Sjogrens). MP release may occur within different vascular sites across these disease groups (macrovascular and microvascular).The association between circulating MP levels and objective assessment of macro- and microvascular dysfunction within these disease areas suggests that MPs might have a useful role as novel circulating biomarkers of vascular disease within the CTDs.

Relationship between renal function and circulating microparticles, soluble P-selectin and E-selectin levels in atrial fibrillation

Atrial fibrillation (AF) and chronic kidney disease are closely related, and any associated risk of stroke and thromboembolism due to AF is increased by concurrent renal dysfunction. The mechanism(s) for this include abnormalities in platelets and endothelial cells. We hypothesized relationships between levels of circulating platelet microparticles (PMPs, defined by CD42b), soluble P selectin (both reflecting platelet activation), soluble E-selectin (reflecting endothelial activation) and endothelial/platelet microparticles (EPMPs, defined by CD31) with progressive renal dysfunction. Blood samples were obtained from 160 anticoagulated AF patients. Microparticles were measured by flow cytometry, soluble E and P selectin levels by ELISA. Renal function was determined by estimated glomerular filtration rate (eGFR). EPMP levels demonstrated a linear increased trend across quartiles of eGFR (p = 0.034) and CKD stage (p < 0.001), and correlated with eGFR and serum creatinine (p < 0.01). PMPs, P-selectin and E-selectin levels were not significantly different across groupings of renal dysfunction, and no significant correlations with eGFR were evident (p = 0.186, p = 0.561, p = 0.746 respectively). Stepwise multivariable regression analysis demonstrated that worsening renal function was an independent predictor of EPMP levels (p < 0.001). In well-anticoagulated AF patients, there is potential relationship between endothelial function (as judged by elevated EPMP levels, with no change in PMPs) and renal function. Other markers of prothombotic state or cellular activation (PMP, P-selectin and E-selectin levels) were not significantly different across the various degree of renal dysfunction. Renal function must be addressed when measuring EPMP levels.

Presurgical levels of circulating cell-derived microparticles discriminate between patients with and without transfusion in coronary artery bypass graft surgery

Improved understanding of presurgical risk factors for transfusions will lead to reduction in their number and related complications. The goal of this study is to identify these factors in coronary artery bypass graft (CABG) surgery. Presented herein are results of analyses of data from an ongoing study of transfusion in CABG surgery. Of 122 patients, 81 received transfusion (Tx) and 41 did not (NoTx). In addition to routine tests, presurgical levels of microparticles from platelets (PMPs), red cells (RMPs), and other lineages were assayed. The Tx and NoTx groups were similar with respect to most presurgical variables but differed in distribution of gender, blood type, diabetes prevalence, activated partial thromboplastin time (aPTT), hemoglobin (HGB), and microparticle levels. Stepwise multiple logistic regression was used to evaluate presurgical variables and to develop a model to assess risk factors for transfusion. CD41(+) PMP and CD235(+) RMP levels were found to be the main risk factors for transfusion. The Model's discriminating ability was assessed using receiver operating characteristic curve analysis, which showed that the area under the model curve (± standard error) was 0.86 ± 0.04 (95% confidence interval, 0.77-0.94). According to the model, patients with higher presurgical levels of circulating CD41(+) PMP, CD235a(+) RMP, and HGB, as well as a shorter aPTT, are less likely to receive transfusion(s). Presurgical levels of CD41(+) PMPs and CD235a(+) RMPs are the main risk factors for transfusion in CABG, followed by HGB and aPTT.

Flow cytometric assessment of endothelial and platelet microparticles in preeclampsia and their relation to disease severity and Doppler parameters

ObjectivePlatelet (P) and endothelial (E) microparticle (MP) levels increase in preeclampsia. However, their relation to the severity of the disease needs to be clarified. The objectives of this study were to compare the levels of EMP and PMP in severe and mild preeclampsia to healthy gravidas to find possible correlations to severity of the disease, Doppler changes, and complications.MethodsA comparative prospective clinical trial (Canadian Task Force II-1) was conducted on 135 pregnant women divided into three groups: 35 women with severe preeclampsia (group 1), 40 with mild preeclampsia (group 2), and 60 healthy gravids (group 3). Assessment of EMP and PMP was done by flow cytometry using anti-CD31 and anti-CD42b antibodies.ResultsExpression of CD31 and CD42b (EMPs) was higher in group 1 compared to groups 2 and 3 with P < 0.001, while expression of CD42b alone (PMPs) did not show a statistically significant difference (P = 0.957). EMPs were correlated positively to umbilical and middle cerebral artery resistance index. There was a significant negative correlation between platelet count and EMPs. Also, EMPs were correlated positively to aspartate transferase and bilirubin levels and were significantly higher with neonatal death.DiscussionThe present study revealed a significant association between plasma levels of EMPs and severity of preeclampsia together with poor neonatal outcome as regards birth weight and percent of neonatal death. So, EMPs assay could be a good predictor of maternal and fetal outcomes and in cases with preeclampsia.

Pre-Surgical Levels Of Circulating Cell-Derived Microparticles Are Most Significant Predictors Of Transfusion Requirement In Coronary Artery Bypass Graft Surgery

IntroductionBlood transfusion is associated with serious adverse events (SAE) such as poor surgical outcome, higher surgical mortality. The cost of transfusion is escalating due to increasing demand and limited blood supply. There is a worldwide growing interest in improving blood utilization methods and storage. Identification of reliable predictors of surgical bleeding is of upmost importance in order to reduce the number of unnecessary transfusions, save limited blood resources, and lower the related costs. Cell derived microparticles (MP) are small membrane vesicles of 0.1 µm in size, released during cell activation and apoptosis. They play an important role in hemostasis and thrombosis. MethodsData presented herein are from an NIH funded prospective randomized study to investigate role of cell derived MP in surgical complications in CABG. Of a total of 122 patients, 81 received transfusion during and/or after surgery (Tx group) whereas the remaining 41 did not (NoTx group). In addition to routine laboratory tests, special assays were performed pre-surgery including concentrations of MP from platelets (PMP), red cells (RMP), endothelia (EMP), and leukocytes (LMP). The two groups were compared with respect to pre-surgical variables in order to assess potential predictors of the need for surgery. ResultsThere were no significant differences between the two groups with respect to most of the pre-surgical demographic, clinical, and lab variables, except that Tx group was associated with higher incidence of female gender, type O blood group, and heparin use. On the other hand, the NoTx group was associated with higher pre-surgical levels of RMP and PMP. Using stepwise logistic regression analyses, four factors were identified as the most significant predictors of the need for transfusion. As depicted by an the areas under the curve (AUC) of the ROC curve, the 95% Confidence Interval of the AUC, and their corresponding p-value for each factor, were as follows.For the model combining all four variables, the AUC and its 95% CI were 0.86 [0.78 - 0.94]. In addition, analysis of post-surgery data revealed better outcomes for the NoTx group in terms of complications such as hours on mechanical ventilation (mean ± SD: 9.2 ± 7.0 vs 15.5 ± 17.1, p = 0.005), number of SAE other than death (0 vs 9 (11.1%), p =0.028), and number of in-hospital deaths (0 vs 5 (6.1%), p= 0.167).FactorAUC[95% C.I]p-valueHGB0.66[0.55 - 0.78]0.005aPTT0.68[0.57 - 0.79]0.015RMP0.69[0.58 - 0.80]0.005CD41+PMP0.77[0.67 - 0.86]<0.001 DiscussionThis is the first report to show that pre-surgical levels of circulating MP are highly significant predictors of need for transfusion in CABG. This finding is consistent with other evidence that MP play a major role in hemostasis. Implementation of pre-surgery MP assay will greatly imporove assessment of risk of bleeding in surgery, and requirements for transfusion. This could reduce the number of transfusions, with corresponding improvement in patient outcomes, as well as reduced costs and strains on limited blood supplies. Furthermore, it may be feasible to manipulate pre-surgical MP levels in patients to minimize bleeding and Tx requirements. This could be done by increasing rate of MP generation or retarding rate of clearance, or both. Alternatively, MP could be produced in vitro (autologous or not) for infusion during surgery to minimize blood loss. Such approach is in progress [Jy, et al Thromb Haemost, in press]. Disclosures:No relevant conflicts of interest to declare.

Exchange Plasmapheresis Is Effective In Removing Procoagulant Microparticles and Has Additional Therapeutic Benefit In The Treatment Of TTP

IntroductionIn thrombotic thrombocytopenic purpura (TTP), autoantibodies bind and deplete ADAMTS13, a vWF cleaving protease, leading to accumulation of large molecular weight vWF multimers, excessive platelet adhesion and aggregation in microvasculature, formation of platelet rich thrombi and disruption of microcirculation. This results in microangiopathic hemolytic anemia, severe thrombocytopenia and multi-organ dysfunction. Under higher shear stress, red cells undergo shape changes, fragmentation and release of red cell microparticles (RMP). Similarly, MP are released from activated platelets (PMP) and endothelium (EMP). It is now well established that cell derived MP play an important role in hemostasis and thrombosis. Roles of circulating MP in TTP are not well elucidated. Exchange plasmapheresis and infusion of FFP is now a standard therapy for TTP. It is assumed that plasmapheresis removes autoantibodies against ADAMTS 13 and plasma infusion supplies the deficient protease to resume normal hemostasis. It is possible that plasmapheresis also removes procoagulant MP, contributing to remission. We studied MP profiles in patients with acute TTP and investigated efficacy of exchange plasmapheresis to remove procoagulant MP. MethodsThree patients presenting acute TTP were admitted for exchange plasmapheresis. In addition to routine CBC, platelet counts, reticulocyte counts, blood chemistry, LDH, special assays of MP derived from platelets (PMP), red cells (RMP), leukocytes (LMP) and endothelium (EMP) were performed daily and before and after each exchange plasmapheresis. Samples were drawn immediately before and immediately after plasmapheresis into citrated vacutainer tubes. Microparticles were assayed by flow cytometry using fluorophore conjugated mAb's specific to endothelial, platelet, leukocyte, or erythrocyte lineage. Thromboelastography (TEG) was employed to study thrombus formation in vitroand correlate microparticles and clinical parameters. Results1) Effects of plasmapheresis on MP levels: Circulating RMP and PMP levels fell rapidly following ExPh. Each round of plasmapheresis reduced RMP by 45% (p=0.001) from starting baselines. Repeated plasmapheresis over one week removed 74% of RMP from the peak level (p<0.01). By day 6, RMP were, on average, reduced from 4111/μL to a normal level of 1100/μL. Each round of plasmapheresis also reduced PMP by 53% from baseline (p=0.001). Over one week period, PMP were reduced by 69% from peak levels.2) Effects of plasmapheresis on LDH, HGB, and platelets: LDH was reduced by 30% on average over a week period, from 845 ±211U/L to 545 ±33U/L, but this represented only 45% normalization. Platelet count normalized completely, increasing almost fivefold from an average of 46 ±22 to 219 ±68 after seven days (p<0.05). HGB fell an average of 10% over the course of treatment but this result was not significant.3) Effect of plasmapheresis on TEG parameters: Exchange plasmaphresis tended to prolong the R time (8.4 ±2.2 minutes pre vs. 10.9 ±6.1 minutes post). It also increased K time by 38% and reduced CI by 42%. Although these changes did not reach statistical significance due to high degree of variation in the post-plasmapheresis group, the trend indicated plasmapheresis reduced procoagulance and hypercoagulability. ConclusionExchange plasmapheresis is very effective in reducing procoagulant and proinflammatory RMP and PMP. Clearance of microparticles resulted in improved TEG parameters and reduced hypercoagulability in TTP. Benefits were pronounced even in the first round of plasmapheresis. Exchange plasmaphresis has additional therapeutic benefit in the treatment of TTP by removing procoagulant microparticles and reducing hypercoagulability in TTP. Disclosures:No relevant conflicts of interest to declare.

Climacteric Lowers Plasma Levels of Platelet-Derived Microparticles: A Pilot Study in Pre- versus Postmenopausal Women

Background: Climacteric increases the risk of thrombotic events by alteration of plasmatic coagulation. Up to now, less is known about changes in platelet- (PMP) and endothelial cell-derived microparticles (EMP). Methods: In this prospective study, plasma levels of microparticles (MP) were compared in 21 premenopausal and 19 postmenopausal women. Results: No altered numbers of total MP or EMP were measured within the study groups. However, the plasma values of CD61-exposing MP from platelets/megakaryocytes were higher in premenopausal women (5,364 × 10⁶/l, range 4,384–17,167) as compared to postmenopausal women (3,808 × 10⁶/l, range 2,009–8,850; p = 0.020). This differentiation was also significant for the subgroup of premenopausal women without hormonal contraceptives (5,364 × 10⁶/l, range 4,223–15,916; p = 0.047; n = 15). Furthermore, in premenopausal women, higher plasma levels of PMP exposing CD62P were also present as compared to postmenopausal women (288 × 10⁶/l, range 139–462, vs. 121 × 10⁶/l, range 74–284; p = 0.024). This difference was also true for CD63+ PMP levels (281 × 10⁶/l, range 182–551, vs. 137 × 10⁶/l, range 64–432; p = 0.015). Conclusion: Climacteric lowers the level of PMP but has no impact on the number of EMP in women. These data suggest that PMP and EMP do not play a significant role in enhancing the risk of thrombotic events in healthy, postmenopausal women.

Intracoronary microparticles and microvascular obstruction in patients with ST elevation myocardial infarction undergoing primary percutaneous intervention

Microparticles (MP) are cell-derived fragments known to be increased in the blood of patients with acute coronary syndromes. We aimed to assess, in ST elevation myocardial infarction (STEMI), the systemic and local (in the culprit coronary artery) levels of platelet-derived MP (PMP, CD42+CD31+) and endothelial-derived MP (EMP, CD42-CD31+) and their relation to indexes of microvascular obstruction (MVO). In 78 STEMI patients undergoing successful primary percutaneous coronary intervention, blood samples were sequentially drawn from the aorta and the culprit coronary artery for cytofluorimetric MP detection. Thrombolysis in myocardial infarction (TIMI) flow, thrombus score (TS), corrected TIMI frame count (cTFC), myocardial blush grade (MBG), quantitative blush evaluator (QuBE) score, and 90 min ST resolution (ΣSTR) were calculated. Both PMP and EMP levels were significantly higher in the intracoronary than in the aortic blood samples. Intracoronary PMP and EMP levels were positively related to TS and cTFC and inversely related to MBG and QuBE. Aortic PMP (but not EMP) levels were related to TS and cTFC and, inversely, to QuBE. Intracoronary PMP were independently related to angiographic and electrocardiographic MVO in a multivariate model. The correlations of intracoronary EMP and of both systemic and intracoronary PMP levels with TS support the role of MP as markers of ongoing thrombosis. Moreover, the correlation of intracoronary MP with indexes of microvascular dysfunction suggests, for the first time, a possible direct role of MP in the pathogenesis of MVO.

Circulating endothelial and platelet derived microparticles reflect the size of myocardium at risk in patients with ST-elevation myocardial infarction

ObjectivesMicroparticles (MP) are small membrane vesicles, released from activated, damaged and apoptotic endothelial cells (EMP) or platelets (PMP) that may actively modulate inflammation, coagulation and vascular function. We tested the hypothesis that the number of circulating EMP or PMP in acute myocardial infarction correlates with the myocardium at risk (MaR) and infarct size (IS). MethodsEMP were quantified in plasma samples of 36 patients (age: 63±10 years) with first time ST-elevation myocardial infarction (STEMI) using flow cytometry. EMP were defined as CD31+/CD42− MP and CD144+ MP and PMP as CD31+/CD42+ MP. MaR and IS was determined by cardiovascular magnetic resonance imaging one week after the index event. ResultsPlasma levels of CD31+/CD42− EMP were 251.0±178.8/μl and CD144+ 106.3±33.7/μl. PMP levels were 579.2±631.8/μl. MaR was 31.0±11.2% of the left ventricle and IS was 11.4±7.1% of the left ventricle. Patients with STEMI in the left anterior descending artery had higher levels of CD31+/CD42− EMP and PMP than those with other infarct-related arteries (p<0.05). The numbers of CD31+/CD42− EMP and PMP correlated to MaR, but not to IS. ConclusionsCirculating EMP and PMP correlate to the size of MaR in patients with STEMI suggesting that they reflect the severity of the endothelial injury and platelet activation during myocardial ischemia.

Influence of Cell-Derived Microparticles on Thromboelastography.

AbstractAbstract 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<0.05) are reported below. (1) RMP levels correlated positively and strongly with lag time, R (r=0.737, p<0.0001). LMP levels correlated positively with k (r=0.553, p=0.0001) moderately. Interestingly, we found moderate but consistent positive correlation between PMP levels and MA, G, and CI. These values were r=0.314, r=0.317, and r=0.346, respectively, and p<0.05 in each case. (2) Normal control values had an average G of 7.86 ±1.70 Kd/cm2 (mean ±SD), in contrast to ITP, TTP, and TP patients which had average G's of 8.62 ±3.96, 10.34 ±1.97, and 5.96 ±3.98 Kd/cm2, respectively. However, no statistically significant differences of means between patient groups and controls were found for any parameter in these small populations. (3) Platelet count was found to have a moderate correlation with G in TP patients (r=0.427, p<0.05) and with k (r= -0.459, p=0.018), but neither variable (G, k) correlated with platelet counts in ITP or TTP, possibly owing to the smaller populations in those groups. (4) Examining all patients combined for correlation between platelet count and G, we found r=0.633 (p<0.0001), confirming previous studies suggesting a relation between platelet count and G in both healthy and diseased patients. High platelet count was reasonably well correlated to high PMP (r=0.506, p<0.001). This may indicate that platelet activation as well as platelet count influences TEG, as it has been shown that PMP levels are a proxy of platelet activation. Conclusions:We conclude that MP contributes significantly to multiple variables in TEG profiles of patients with ITP, TTP, and TP, some positively, some negatively; and that there is wide variability in MP levels among these diseases. Platelets clearly also modulate TEG parameters, although the interplay between platelets and MP in forming a stable clot bears further research. PMP have the largest array of effects, positively correlating with MA, G, and CI, and negatively correlating with R+k, while having no significant effect on the other parameters. The data suggest that nearly all the MP affect the TEG parameters significantly. The most commonly affected parameter was MA, which is correlated to EMP, LMP, PMP, and Ulex. G and CI were also frequently affected by various MPs. This study is limited by the small patient population, various medications, and varying degree of severity of diseases. Disclosures:No relevant conflicts of interest to declare.

Red-Cell Microparticles Released From Stored Packed Cells: Possible Contributing Factor to Adverse Responses to Transfusion

AbstractAbstract 342 Background.Packed red cells (PC) stored in blood bank undergo a series of changes, so-called “storage lesion”, which increase with time. In view of some but not all recent studies, it is widely believed that transfusion with younger blood carries less risk of adverse reactions than older blood. However, there is no agreement on the “safe” age of blood, nor is it clearly understood why older blood may carry increased risks. The purpose of this study was to identify microparticle-related factors in stored PC at different time intervals that might pose risk of adverse effects. We investigated profiles of cell-derived microparticles (MP), particularly RBC-derived MP (RMP), in stored PC and assessed their procoagulant and inflammatory property. Methods.Twelve bags of fresh packed red cells (PC) of known blood types (A+, B+, AB+, O+) were obtained from blood bank (2-4 days since drawing). All were non-leuko depleted and were stored at 4°C. Time of receipt was considered day 0. At intervals of 0, 10, 20, and 30 days, 40 mL was withdrawn and centrifuged at 1000xg for 20 min to remove cells. The supernatants were then assayed for (1) quantity of different species of cell-derived MP by flow cyteometry comprising (a) RMP defined by CD235b; (b) LMP by CD45; (c) PMP by CD41; (d) EMP by CD144; (e) generic MP by Ulex Europaeus (Ulex) or Annexin V (AnV), (2) procoagulant activity by MP-mediated thrombin generation assay (TGA); (3) MP-mediated proinflammatory activity by CD 11b expression in neutrophils following incubation with RMP. Results.(1) MP Profiles. The time-course of generation of the MP subtypes varied considerably. For RMP, there was little increase before day 10, but then rose rapidly with time, to 180% at 20 days, and to 450% at 30 days. Small amounts of MP derived from leukocytes (LMP), platelets (PMP), and endothelia (EMP) were present in all bags at day 0, generally <10% by number compared to RMP. For LMP, there was no significant change in the first 20 days but was increased significantly at day 30, to 160% of day 0. For PMP, counts rose steadily from day 0 and peaked to 220% of baseline at day 20. For EMP, counts were very low (<1% of RMP) and no change was observed over 30 days. For total MP, defined by Ulex counts and total protein concentrations, the time course was similar to RMP. Results with AnV+ MP showed significant increase from day 10 to day 20. We found no influence of blood type on MP generation. (2) Procoagulant Activity. There was little change in MP-mediated thrombin generation in the first 10 days, but it rose significantly from day 10 to day 20, correlating well with counts of AnV+ MP or RMP. (3) Proinflammatory Activity. Leukocyte CD11b expression induced by MP from the PC bags showed a nearly linear rate of increase from day 0 to day 30, correlating closely with PMP. (4) Exceptions. In 2 of the 12 PC bags, we observed exceptionally high levels of both RMP and PMP (4-8 fold higher than average) from day 0 to day 30. CBC assay showed that all bags contained similar counts of RBC and WBC, with exception of high concentrations of platelets (>200,000/μL) in the two exceptional bags vs. the others (20,000 – 45,000/μL). In the two exceptional bags, coagulant activities and inflammatory potential were also highly elevated compared to the values in the other bags from day 0 to day 30. Conclusions.(i) RMP are the predominant MP species in stored RBC, increasing slowly from day 0 to 10 and thereafter rising exponentially to day 30. (ii) PMP were also present in significant amounts. The time course of RMP and PMP release correlated well with procoagulant and proinflammatory indicators in stored RBC. (iii) Two of the 12 bags (17%) exhibited exceptionally high platelet content and RMP, PMP. The significance of this new finding remains to be clarified. These preliminary results indicate that procoagulant, proinflammatory MP levels increased significantly after 10 days of storage, and that contaminating platelets exacerbate RMP generation. The increase in MP in stored PC constitutes one aspect of the storage lesion and may pose prothrombotic and/or proinflammatory risks in blood transfusions. (Supported by NIH Grant 5R01HL098031-02) Disclosures:No relevant conflicts of interest to declare.

Increased platelet, leukocyte and endothelial microparticles predict enhanced coagulation and vascular inflammation in pulmonary hypertension

Pulmonary hypertension (PH) is associated with platelet activation, vascular inflammation and endothelial dysfunction leading to often life threatening thrombo-embolic complications. Microparticles (MPs) are cell vesicles with strong coagulatory and inflammatory effects being released during cell activation and apoptosis. As there are currently no established surrogate markers predicting platelet activation and pro-coagulation in PH patients, the aim of the study was to analyze different pro-coagulatory MP populations that might be related to thrombo-embolic complications in PH patients. Circulating MPs from platelet- (PMP, CD31(+)/61(+)), leukocyte- (LMP, CD11b(+)) and endothelial- (EMP, CD62E(+)) origin were measured by flow cytometry in 19 PH patients and were compared to 16 controls. PH patients had increased levels of PMP (PH vs. control 1,016 ± 201 vs. 527 ± 59 counts per min [cpm], P = 0.032), LMP (PH vs. control 31 ± 3 cpm vs. 18 ± 2 cpm, P = 0.001) and EMP (PH vs. control 99 ± 14 cpm vs. 46 ± 6 cpm, P = 0.001). Furthermore, PMP correlated to LMP (PMP vs. LMP: r = 0.75, P < 0.001) and LMP correlated to EMP levels (LMP vs. EMP, r = 0.74, P < 0.001) indicating a functional interaction between the different types of MP. In comparison to non-embolic PH patients, patients with a thrombo-embolic PH suffered from enhanced endothelial cell dysfunction as represented by significantly increased EMP levels (thrombo-embolic PH vs. non-embolic PH 137 ± 27 vs. 72 ± 10, P = 0.02). PH patients have increased levels of platelet-, leukocyte- and endothelial MP indicating an increased vascular pro-coagulation and inflammation which might be related to thrombo-embolic complications as well as PH progression.