Red Cell-derived Microparticles Research Articles

Presurgical circulating platelet-derived microparticles level as a risk factor of blood transfusion in patients with valve heart disease undergoing cardiac surgery

BackgroundCell-derived microparticles (MPs) are membrane vesicles that have emerged as a potential biomarker for various diseases and their clinical complications. This study investigates the role of MPs as a risk factor for blood transfusion in patients with valve heart disease undergoing cardiac surgery. MethodsForty adult patients undergoing heart valve surgery with cardiopulmonary bypass (CPB) were enrolled, and venous blood samples were collected prior to surgical incision. Plasma rich in MPs was prepared by double centrifugation, and the concentration of MPs was determined using the Bradford method. Flow cytometry analysis was performed to determine MPs count and phenotype. Patients were divided into “with transfusion” (n = 18) and “without transfusion” (n = 22) groups based on red blood cell (RBC) transfusion. ResultsThere was no significant difference in MPs concentration between the “with transfusion” and “without transfusion” groups. Although the count of preoperative platelet-derived MPs (PMPs), monocyte-derived MPs (MMPs), and red cell-derived MPs (RMPs) was higher in “without transfusion” group, these differences were not statistically significant. The preoperative PMPs count was negatively correlated with RBC transfusion (P = 0.005, r = -0.65). Multivariate logistic regression analysis revealed that the count of CD41+ PMPs, Hemoglobin (Hb), and RBC count were risk factors for RBC transfusion. ConclusionThis study suggests that the presurgical levels of PMPs, Hb, and RBC count can serve as risk factors of RBC transfusion in patients with valve heart disease undergoing cardiac surgery. The findings provide insights into the potential use of MPs as biomarkers for blood transfusion prediction in cardiac surgery.

Annexin V− and tissue factor+ microparticles as biomarkers for predicting deep vein thrombosis in patients after joint arthroplasty

ObjectiveVenous thromboembolism (VTE) is a common and severe complication of joint arthroplasty. Microparticles (MPs) containing phosphatidylserine (PS) and tissue factor (TF) can trigger coagulation in VTE. This study aims to measure and compare MP levels in joint arthroplasty patients with and without VTE. MethodsThis prospective cohort study enrolled 181 patients who underwent joint arthroplasty. Ultrasound examination was used to diagnose VTE on preoperative day 0 and postoperative day 6. MPs were analysed using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. The levels of platelet-derived microparticles (PMPs), endothelial cell-derived microparticles (EMPs), granulocyte-derived microparticles (GMPs), red cell-derived microparticles (RMPs), monocyte-derived microparticles (MMPs), Annexin V+ MPs (AV+ MPs), and tissue factor+ MPs (TF+ MPs) derived from five kinds of MPs were measured on day 0 (before surgery), 1, 2, 3, 4, 5, and 6 after surgery. ResultsThe levels of AV-TF+ EMPs and AV-TF+ MMPs were significantly increased in patients with VTE on postoperative day 5 compared to those without VTE (P = 0.031 and P = 0.031, respectively). ConclusionAV-TF+ MPs may indicate the development of VTE and serve as predictive markers in joint arthroplasty patients.

Impact of storage, leukofiltration, and ascorbic acid fortification on red cell-derived microparticles in stored packed red blood cells: A flow cytometric and procoagulant study

INTRODUCTION: During blood storage, red blood cells (RBCs) undergo changes termed “storage lesion” including shape modification and microparticles (MPs) production, as well as oxidative injury to lipids and proteins. OBJECTIVE: We studied the impact of storage on RBCs by quantification of red cell MPs and their potential procoagulant activity (PCA). The antioxidant effect of ascorbic acid (AA) was assessed, as a proposed additive, to improve stored RBC quality. METHODS: Forty randomly selected packed red cell units (filtered and nonfiltered) were sampled weekly over 35 days of storage. Flow cytometric and coagulation tests were performed on MPs isolated from erythrocyte concentrates. RBCs were either in citrate phosphate dextrose adenine-1 (CPDA1) only (standard storage) or CPDA1 with AA (Vitamin C) in three concentrations. RESULTS: Red cell MPs progressively increased over 35 days of storage. Filtration efficiently reduced the production of MPs (P < 0.001). PCA increased significantly on storage assessed by the shortened coagulation time (P < 0.001). A strong inverse correlation between coagulation time and Annexin V positive MPs was found, (r = −0.723, P < 0.001), and the dual-positive population. This highlights the strong correlation between the functional assay (PCA) and the direct quantitative assay (flow cytometry). The red cells fortified with AA demonstrated a reduction in the accumulation of RBC MPs by almost 50% compared to standard storage in CPDA-1 alone in an evident dose-dependent manner. CONCLUSION: We concluded that MPs increase on storage with increased PCA. AA reduced MPs production which can be possible additive for further research in the future

Microparticles in red cell concentrates prime polymorphonuclear neutrophils and cause acute lung injury in a two-event mouse model

Red cell-derived microparticles (RMPs) are potential mediators of transfusion-related acute lung injury (TRALI). The aim of this study was to investigate the effects of microparticles present in red cell concentrates (RCC) on polymorphonuclear neutrophil (PMN) respiratory burst and acute lung injury (ALI) in mice. Microparticles (MPs) in RCC supernatant were quantified using flow cytometry. The priming activity of either isolated MPs or RCC supernatant toward human PMN was measured in vitro. Mice were injected with lipopolysaccharide (LPS), followed by an infusion of either isolated MPs or heat-treated RCC supernatant. The lungs were harvested to assess myeloperoxidase (MPO) activity, histology and pulmonary edema. Protein content in bronchoalveolar lavage fluid (BALF) was measured. The number of RMPs increased significantly during storage. Both isolated MPs and the supernatants from RCCs that had been stored for 28 and 35days effectively primed the PMN respiratory burst. The infusion of isolated MPs or supernatants that had been stored for >28days into LPS-treated mice caused ALI. The filtered supernatant resulted in significantly ameliorated mouse ALI. MPs that accumulate during RCC storage prime the PMN respiratory burst and cause ALI in a two-event mouse model.

Evidence for Anti-Fibrinolytic Activity of Red Cell-Derived Microparticles (RMP): A Novel Hemostatic Action of RMP

BACKGROUND. RMP have been developed as novel hemostatic agents for treatment of bleeding disorders [Thromb Haemost 110:751,2013]. RMP generate thrombin in vitro through activation of contact pathway independent of tissue factor. They correct coagulopathy associated with most factor deficient plasma, indicating promotion of secondary hemostasis. In addition, they promote primary hemostasis by enhancing platelet adhesion and aggregation. Recent literature suggests that excessive levels of tPA are responsible for the uncontrollable bleeding seen in trauma-related coagulopathies [J Trauma and Acute Care Surg 80:1,2016]. In this study, we used thromboelastography (TEG) to investigate the effects of RMP on tPA, a relation hitherto unexplored.METHODS. The RMP were made by high-pressure extrusion of washed red cells. TEG was used to assess coagulation and fibrinolysis in fresh whole blood (diluted to 80%) in presence vs. absence of RMP (2.78x107/mL final conc. based on flow cytometry of PE-labeled mAb to CD235a). To hasten fibrinolysis for ease of measurement, tPA was added in all runs at final conc. 0.4 ug/mL. Calcium chloride (0.2 M) was added to each trial to induce coagulation. Additions to cup were: 325 uL of 80% blood + 10 uL tPA + 10 uL saline or RMP + 20 uL CaCl2. The tPA was stored in 50% glycerol at -20°C.RESULTS. Representative TEG curves of tPA-induced fibrinolysis in the presence or absence of RMP are shown in Figure 1 below. In the presence of tPA without RMP, the MA is small and the amplitude will decline to the baseline in 30 - 60 min, reflecting ongoing fibrinolysis. The MA was increased by about 50% by addition of RMP, from 25mm to 37mm (p=0.016). Judging from fibrinolysis measured by the-built-in LY30 parameter (% lysed at 30 min after MA was achieved), the mean of n=6 paired t-tests was 61.3% lysed in absence of RMP vs. 36.3% with RMP, indicating 40% inhibition of fibrinolysis (p=0.046).To assess the overall anti-fibrinolytic effect of RMP, we used the integral of the 1st derivative of the TEG curve, called TG (thrombus generation), which amounts to the total or net hemostatic-like activity. By this measure, addition of RMP gave TG = 457 vs. 319mm/min in absence of RMP (p=0.0044 by paired t test, P=0.037 by unpaired t test).DISCUSSION. These findings suggest that RMP exert significant anti-fibrinolytic activity, enhancing its hemostatic activity. Uncontrollable bleeding due to fibrinolysis is a major component of many bleeding disorders, including acute coagulopathy of trauma which is seen in about 20% of cases and is associated with poor outcome. The mechanistic basis of this activity is not fully understood. It was suggested that RMP are incorporated into the fibrin network to consolidate its structure thus stabilizing the network and impeding fibrinolysis. Full exploration of this activity is warranted since treatment for excessive fibrinolysis would have great impact in clinical management of many bleeding disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Platelet-, leucocyte- and red cell-derived microparticles in stored whole blood, with and without leucofiltration, with and without ionising radiation.

Storage lesion, including microparticle formation, has been partially characterised in whole blood, but not in all combinations of pre-storage leucofiltration and/or irradiation. Single-donor whole blood products were processed into four subunits: with and without leucofiltration, with and without X-irradiation (25 Gy). Platelet-, leucocyte-, and erythrocyte-derived microparticles and free haemoglobin were measured periodically throughout 42 days of storage. Pre-storage leucofiltration substantially reduced platelet- and leucocyte-derived microparticle counts throughout storage. Irradiation, in contrast, had no significant effect on microparticle counts. A gate for all microparticles showed a substantial time-dependent increase in unfiltered whole blood. A time-dependent increase in free haemoglobin was greatest in unfiltered, irradiated whole blood. This study indicates that leucofiltration can prevent the formation of leucocyte- and platelet-derived microparticles, and might reduce haemolysis in irradiated whole blood, either by removing factors that provoke haemolysis, or by selective retention of senescent or effete red cells most prone to haemolysis.

Microparticles variability in fresh frozen plasma: preparation protocol and storage time effects.

Extracellular vesicles or microparticles exhibiting procoagulant and thrombogenic activity may contribute to the haemostatic potential of fresh frozen plasma. Fresh frozen plasma was prepared from platelet-rich plasma at 20 °C (Group-1 donors) or directly from whole blood at 4 °C (Group-2 donors). Each unit was aseptically divided into three parts, stored frozen for specific periods of time, and analysed by flow cytometry for procoagulant activity immediately after thaw or following post-thaw storage for 24 h at 4 °C. Donors' haematologic, biochemical and life-style profiles as well as circulating microparticles were analysed in parallel. Circulating microparticles exhibited a considerable interdonor but not intergroup variation. Fresh frozen plasma units were enriched in microparticles compared to plasma in vivo. Duration of storage significantly affected platelet- and red cell-derived microparticles. Fresh frozen plasma prepared directly from whole blood contained more residual platelets and more platelet-derived microparticles compared to fresh frozen plasma prepared from platelet-rich plasma. Consequently, there was a statistically significant difference in total, platelet- and red cell-derived microparticles between the two preparation protocols over storage time in the freezer. Preservation of the thawed units for 24 h at 4 °C did not significantly alter microparticle accumulation. Microparticle accumulation and anti-oxidant capacity of fresh frozen plasma was positively or negatively correlated, respectively, with the level of circulating microparticles in individual donors. The preparation protocol and the duration of storage in the freezer, independently and in combination, influenced the accumulation of microparticles in fresh frozen plasma units. In contrast, storage of thawed units for 24 h at 4 °C had no significant effect on the concentration of microparticles.

Pharmacokinetics of Red Cell-Derived Microparticles (RMP) in Rabbits and Rats: Effects of Administration Regimen and Labels for Assay

Background: Circulating cell-derived microparticles (MP) are currently of interest in the fields of hemostasis, thrombosis, and inflammation. Administration of MP has been proposed for use as a hemostatic agent. Efficacy of red cell microparticles (RMP) in reducing blood loss has been documented in rat and rabbit bleeding models [Thromb. & Haemost., 2013;110:751-60]. One of the challenges in developing MP as a hemostatic agent is uncertainty of half-life (T1/2) in circulation, reportedly ranging from several minutes to hours. The purpose of this study was to assess the pharmacokinetics of RMP in rabbits vs. rats, and comparing alternative dosing regimens and assay markers. Although this work was aimed at optimizing the dosage regimen of RMP as a hemostatic agent, it has wider interest because very few studies have addressed the question of MP half-life in circulation.Methods. (i) HumanRMP were produced by high-pressure extrusion [Thromb. & Haemost., 2013; 110:751-60]. The resulting product was washed twice with isotonic saline, lyophilized, and stored at -80°C. (ii) Pharmacokinetics of RMP were measured using either bolus infusion of RMP (3x109 counts/kg) during 1 min., or a combination of bolus (1/3 of total RMP) followed by continuous infusion (2/3 of total RMP) for 30 min to the anesthetized rabbits or rats, via cannulated jugular vein. Blood samples (0.4 mL each) were collected from the cannulated carotid artery at intervals: 5 min pre-injection, and at 1, 2, 3, 5, 7.5, 10, 15, 20, 25, 30, 45, and 60 min post-injection. A sample size of 8 animals was used for each infusion regimen. Levels of RMP were assayed by flow cytometry with 2 separated labels: anti-CD235a-PE (anti-glycophorin) and annexin V-FITC (AnV). The former is specific for human RMP and does not label rabbit or rat RMP. The latter is not species-sensitive, and labels both human and host MP. (iii) The procoagulant activity (PCA) of RMP was assayed by thromboelastogram (TEG) ex vivo.Results: (1) BolusInfusion of RMP (3 x109 MP/kg) resulted in a rapid rise of RMP levels peaking at 1.0 min post-infusion, followed by slower decline to baseline by 10 - 15 min. The half-life (T1/2) of clearance determined by anti-CD235a was 6.2 ±0.7 and was 4.2 ±0.5 min for rabbits and rats, respectively. In contrast, the T1/2 measured by AnV was 4.4 ±1.0 and 2.6 ±0.4 min, respectively. These results indicate that these two MP phenotypes were cleared by different mechanisms, i.e., one or more mechanisms predominates in clearing CD235a(+) MP vs. AnV(+) MP. The RMP enriched in phosphatidylserine (AnV+) are cleared faster than those expressing CD235a. (2) Two-step administration was bolus (1/3 total dosage) followed by continuous infusion (2/3 total dosage), and resulted in a smaller initial spike followed by rapid decline to ≈25-30%, then recovering to a nearly steady-state level of 0.3 -0.6 x107 MP/mL after 20 min. The RMP levels returned to baseline within 15 min after cessation of infusion. The area under the curve (AUC) of the PK curve for bolus infusion was about 40 -60% less than that of two-step administration of the same dose. (4) Procoagulant Activity (PCA): For bolus infusion, the ex vivo TEG data revealed some correlation between circulating RMP and PCA at the early phase (1 to 6 min). At later phase (>10 min), although RMP had dropped to almost baseline, the PCA remained active considerably longer than presence of RMP (T1/2= 9.3±1.2 min for PCA). However, bolus followed by continuous infusion resulted in maintaining stable PCA over the course of infusion.Conclusion/Discussion. (i) The clearance rate of RMP in rats is faster than that in rabbits. (ii) When T1/2 is determined by the two different markers, we found significant differences in half-life, indicating differential of mechanisms of clearance. (iii) The TEG data show that the PCA of RMP persists after detectable RMP have entirely cleared. We interpret this to mean that RMP continue to exert PCA by being bound to blood cells, even after free circulating RMP have cleared. (iv) Continuous infusion is expected to perform better than bolus infusion in controlling excessive bleeding in clinical patients. DisclosuresNo relevant conflicts of interest to declare.

Preferential Stimulation of Contact Pathway Factors By Red Cell-Derived Microparticles: New Perspective on Hemostasis

BACKGROUND / OBJECTIVES. Cell-derived microparticles (MP) are known to promote thrombin generation, and more recently have been shown to contribute substantially to hemostasis. This activity derives mainly from platelet MP (PMP) but recent evidence implicates also red cell MP (RMP). Traditionally, MP-mediated procoagulant activity has been ascribed to presentation of negatively charged surfaces for the assembly of tenase and prothrombinase. However, the mechanism of this activity is becoming more complicated as it is emerging that MP-mediated procoagulant activity includes augmentation of the contact pathway [van der Meijden et al,J Thromb. Haemost., 2012; 10:1355-62, Rubin et al, Transfusion, 2013; 53:1744-54] and of ADP-induced platelet aggregation [Jy et al, Thromb. & Haemost., 2013; 110:751-60]. Accordingly, we undertook examination of interactions of RMP with specific elements of the contact factor pathway, and related issues such as whether or not RMP can initiate contact pathway by activating FXII directly.METHODS. Plasmas depleted in each of 8 factors (F’s II, V, VII, VIII, IX, X, XI, XII), obtained from commercial sources, were mixed with pooled normal plasma to produce depletion levels of 75% to 95%, i.e., from 25% to 5% of normal. These plasmas were then assayed by thromboelastography (TEG) with / without addition of RMP. The RMP was prepared by high-pressure extrusion of washed RBC [Jy et al, Thromb. & Haemost., 2013; 110:751-60]. Blood was drawn into citrated Vacutainers in presence / absence of 140 µg/mL corn trypsin inhibitor (CTI) and run in TEG. Controls were obtained from healthy staff volunteers (n=8) to establish baseline values, +/- addition of RMP. Tissue factor (TF) at 20 or 200 pg/mL was added to standardize extrinsic pathway activation. Anti-FVIIa (Seksui Diagnostics) was added in certain experiments to abolish the TF pathway. Finally, when contact factor activation was fully blocked (no fibrin generation within 2 hrs), kaolin was added in presence / absence of RMP.RESULTS.(1) In the factor depletion experiments, we found that RMP most strongly potentiated depletion of factors of the contact pathway, namely FVIII, FIX, FXI, and FXII, as judged by shortening of R (lag time), p<0.01 to p<0.05, at depletions as low as 5% normal. For example, FVIII at 15% of normal level responded to RMP by 50% reduction in R time. In contrast, RMP had little or no augmenting effect on plasmas depleted of F’s II, V, X. This suggests that RMP promote the contact pathway preferentially.(2) RMP were capable of enhancing the rate of thrombin generation at low concentration of CTI (40 µg/mL), but at high concentration (140 µg/mL) this effect was completely abolished. When kaolin was added to the high concentration CTI-treated blood, it initiated thrombin generation after long delay (~30 min). Upon addition of RMP, the rate of thrombin generation was potentiated considerably (by ~50%). These data indicate that unlike kaolin, RMP are not capable of activating FXII directly but RMP can potentiate FXIIa-mediated thrombin generation.(3) In whole blood treated with low concentration TF (20 pg/mL) and high concentration CTI, RMP increased rate of thrombin generation by 25% compared to controls (p< 0.05). When blood was treated with anti-FVIIa, RMP retained the ability to enhance thrombin generation by roughly 30% (p<0.05). This demonstrates that RMP can potentiate thrombin generation via the TF pathway, although not as robustly as by contact pathway.CONCLUSIONS. First, these results demonstrate highly selective stimulation of elements of the contact pathway by RMP, as opposed to non-specific augmentation of coagulation. However, we do not have evidence of direct activation of FXII to FXIIa by RMP. Instead, reslults can be explained by marked potentiation of small amounts of activated enzyme. Second, it is shown that this activity is entirely independent of the TF pathway. Third, results support the potential value of RMP for use as an infusible hemostatic agent [Jy et al, Thrombosis & Haemostais, 110:751-60 (2013)], for treating a wide variety of clinical conditions involving excessive bleeding. Finally, in view of new evidence showing that MP-mediated procoagulant activity is diverse in action, new avenues of MP-mediated hemostasis are open to exploration. DisclosuresNo relevant conflicts of interest to declare.

Interaction Of Platelets With Red Cell-Derived Microparticles (RMP): RMP Increase Platelet Aggregate Size In a Shear-Dependent Manner

BackgroundRed cell microparticles (RMP) have come to recent attention as putative mediators of hemostasis. We reported that RMP improve hemostatic defect of blood samples of thrombocytopenia and thrombocytopathy and augment platelet function. To investigate possible mechanisms of this activity, we measured the effect of RMP on shear-dependent platelet adhesion and aggregation in whole blood. MethodsRMP were produced by high-pressure extrusion of washed, packed RBC. The RMP produced in this way are similar to natural circulating RMP in phenotype and most functional assays. Blood was collected in citrate Vacutainers from normal healthy staff volunteers, and first 3 mL discarded to minimize artifact of platelet activation due to tissue factor. It was tested within 2 hours of drawing. Variable shearing rates were applied by a cone-and-plate device, the DiaMed Impact-R, which yields photomicrographs of objects adhering to the plate, and data including percent surface coverage (SC), number objects adhering (OBJ) per mm2, and mean aggregate size (AS) in μm2. Initially, 4μL of either RMP (1.0 x 108/μL) or saline were mixed with 126μL of whole blood obtained as above, and incubated in a microcentrifuge tube for 60s. The mixtures were pipetted to a well of the device and run at a selected shear rate (range 900s-1 to 2700s-1) for 2 minutes. Blood was then carefully drawn off and the wells were washed gently with deionized water. Wells were stained with May-Grünwald stain for 60s, and excess removed. When dry, micrographs of the well surfaces were taken. The most promising shear rates (1125 s-1, 1575s-1, 1800s-1, 2025s-1) were retested with higher numbers of RMP in the 130μL volume. ResultsAddition of RMP vs. saline (control) induced increased AS over a range of shear rates. For example, 4μL RMP (1x108/μL) running at 2250s-1 increased AS by 34.8%, from 59.2 ±25.1μm2 to 79.8 ±24.7μm2 (n=15, p=0.01). To investigate effect of RMP concentration at fixed shear rate of 1575s-1, we found that 8μL RMP induced increase of 12.0 μm2, 12μL RMP by 24.8 μm2, 16μL by 26.7 μm2, and 20μL by 26.3 μm2; p<0.05 and n=4 replicates for all. As seen by the trend, this effect on AS reached a plateau at 20 μL RMP. With fixed volume of RMP added and varying shear rate, we found the largest RMP-induced increase in AS occurred at 1125s-1 shear rate: 16μL of RMP increased aggregate size to 172% of control (from 41.3 ±10.2μm2 to 71.0 ±2.6μm2; n=3, p=0.02). With 1/4 as much RMP (4 μL), the peak effect occurred at 1125s-1. We did not observe any significant differences in SC at any shear rate or RMP volume. OBJ was generally lower as AS increased, but this did not reach significance. Conclusion/DiscussionRMP increased the size of platelet aggregates under shear, indicating enhanced platelet adhesion by RMP. This effect varied with shear rate: larger amounts of RMP had maximum effect at lower shear rates; smaller amounts of RMP required higher shear to exert maximum effect. These effects are seen at physiologically relevant shear rates (200 - 2000s-1 or higher in pathology such as hypertension or valve defects). Due to the absence of endothelia from this model, high shear was required to induce maximum adhesion. Previous study has indicated the important role of GPIIb/IIIa in platelet adhesion and aggregate size under high shear [Varon, et al; Am Heart J, 1998]. We postulate that RMP may be acting in a GPIIb/IIIa dependent manner to enhance aggregate size. This was consistent with the findings that RMP-induced augmentation of platelet aggregation at relevant levels of ADP or AA concentrations in heparinized blood [Jy, et al; Thromb Haemost, in press]. We are continuing to investigate conditions affecting the interactions between RMP and platelets. Disclosures:No relevant conflicts of interest to declare.

Red cell-derived microparticles (RMP) as haemostatic agent

Among circulating cell-derived microparticles, those derived from red cells (RMP) have been least well investigated. To exploit potential haemostatic benefit of RMP, we developed a method of producing them in quantity, and here report on their haemostatic properties. High-pressure extrusion of washed RBC was employed to generate RMP. RMP were identified and enumerated by flow cytometry. Their size distribution was assessed by Doppler electrophoretic light scattering analysis (DELSA). Interaction with platelets was studied by platelet aggregometry, and shear-dependent adhesion by Diamed IMPACT-R. Thrombin generation and tissue factor (TF) expression was also measured. The effect of RMP on blood samples of patients with bleeding disorders was investigated ex vivo by thromboelastography (TEG). Haemostatic efficacy in vivo was assessed by measuring reduction of blood loss and bleeding time in rats and rabbits. RMP have mean diameter of 0.45 µm and 50% of them exhibit annexin V binding, a proxy for procoagulant phospholipids (PL). No TF could be detected by flow cytometry. At saturating concentrations of MPs, RMP generated thrombin robustly but after longer delay compared to PMP and EMP. RMP enhanced platelet adhesion and aggregation induced by low-dose ADP or AA. In TEG study, RMP corrected or improved haemostatic defects in blood of patients with platelet and coagulation disorders. RMP reduced bleeding time and blood loss in thrombocytopenic rabbits (busulfan-treated) and in Plavix-treated rats. In conclusion, RMP has broad haemostatic activity, enhancing both primary (platelet) and secondary (coagulation) haemostasis, suggesting potential use as haemostatic agent for treatment of bleeding.

Red Cell-Derived Microparticles (RMP) As Hemostatic Agent: Evaluation in a Rabbit Ear Bleeding Model.

Abstract 2238 Introduction:We are developing RMP for use as an infusible hemostatic agent to treat bleeding conditions. Preliminary evaluations in vitro, such as by thromboelesatography (TEG), were very promising, and were supported by in vivo studies in a small animal (rat) [Haemophilia 2011; 17: 44]. It was shown that RMP not only accelerate coagulation and correct hemostatic aberration of factor-deficient plasma but also enhance platelet aggregation and adhesion, suggesting its efficacy in both primary and secondary hemostasis [Blood 2011; 118:3263]. Here we report results of initial studies in a large animal, the rabbit ear bleeding model. Methods:RMP were produced by high-pressure extrusion of human RBC, converting >99% of RBC to RMP. Hemostatic efficacy was evaluated in a rabbit ear bleeding model. New Zealand male white rabbits of 2.5–3.5 kg were used. After sedation, the ear was prepared by chlorhexidime scrub, then a standard incision 6 mm wide was made to penetrate the ear (#11 scalpel blade), in a region of the ear devoid of macroscopic (visible) vessels. The ear was maintained at 37°C with a heating pad. Bleeding time (BT) was defined as the time required for the disappearance of blood stains on a filter paper blot. To reliably measure reduction of BT by the RMP product, bleeding was prolonged by administration of busulfan (20 mg/kg SC) twice each at days 1 and 3, to suppress bone marrow, resulting in thrombocytopenia after 7–12 days. BT was significantly prolonged in animals when platelet counts fell to <60,000/μL. This regimen allowed all animals to survive, but higher doses of busulfan was toxic and caused severe pancytopenia, unacceptable for experimentation. Results:In normal (untreated) rabbits, the BT was 90 ±20 seconds (n = 12). Injection of RMP (3×109/kg, IV) in untreated rabbits resulted in no significant changes in BT, and no major adverse events were seen (n = 6). In thrombocytopenic rabbits, the BT was prolonged to 620 ±130 sec., and prolongation of BT correlated with decrease in platelet counts, 30,000 – 60,000/μL (n = 12). In this range of platelet counts, injection of RMP (at either 1 × 109 or 3 × 109/kg, IV) induced significant shortening of BT, to 440 ±90 sec (n = 8) at the lower dose (p<0.01), and to 280 ±65 sec (n = 8), at the higher dose (p < 0.001). When platelet counts dropped below 10,000/μL and BT >15 min, RMP had no significant effect. The hemostatic effects disappeared if >30 min elapsed between RMP infusion and making the incision, indicating a short half-life of RMP in circulation. Conclusions:These data confirm efficacy of RMP in thrombocytopenic rabbits. Hemostatic efficacy of RMP was evident in moderate thrombocytopenia, and was dose-dependent. However, efficacy fell off if platelets <10,000/μL, indicating that RMP augment platelet activity but do not replace platelets. In both thrombocytopenic and non-thrombocytopenic rabbits, no major adverse reactions were observed with infusion of RMP. Disclosures:No relevant conflicts of interest to declare.

Red Cell-Derived Microparticles (RMP) Correct or Improve Abnormal Coagulation Profiles in TEG in a Variety of Disorders of Different Pathology

Abstract 2277 Introduction:Thromboelastography (TEG) reports several viscoelatstic changes during clot formation, giving useful information on multiple parameters, and can give information on effects of therapeutic measures. We previously demonstrated that RMP can improve clotting parameters in selected bleeding disorders, judging by TEG. In this report we characterize the abnormalities seen in TEG in several disease states, and how RMP can modify or correct the abnormal parameters. Methods: (i)The following patient groups were studied by TEG: Patients with ITP (n=10), other non immune TP (n=10), thrombocytopenia from aplastic anemia (AA, n=3), antiphospholipid syndrome (APS, n=10), systemic lupus erythmatosus (SLE, n=8), hemophilia (n=3), hemolytic anemia (HA, n=4) and healthy controls (n=84). (ii) RMP were produced from washed, packed RBC by high-pressure extrusion and were added at 1×10E8/mL f.c. to the test samples. (iii) TEG was performed on whole citrated blood, each in presence and absence (+/−) of added RMP. Parameters measured were R (lag time), k (clot formation time), A, angle (initial rate of clot growth), MA (maximum amplitude, or clot strength), G (shear elastic modulus, another measure of clot strength) and CI (coagulation index), a composite measure. Results:(1) Baseline findings (absence of RMP). All disease groups except HA had longer R (lag) time than controls (p≤0.03), but only AA, ITP, and SLE had longer k times (p≤0.03). HA had a significantly shorter k time and higher angle A (initial rate) compared to controls, both at p<0.01. Angle A was lower in Hemophilia, TP, ITP, AA and SLE (all p≤0.05 or better). APS did not differ from controls. Finally, MA (amplitude) and G (elastic modulus) were lower in TP, ITP, and AA than controls (p≤0.04), while the other diseases did not differ from controls in this measure. (2) Effect of RMP. The R and k parameters of all disease groups except HA were significantly shortened upon addition of RMP (p≤0.05 or better). RMP also shortened R and k of healthy controls (p<0.01, n=84). TP, ITP, AA, hemophilia APS, and controls also had shorter R+k time, and steeper angle A (p≤0.05 or better for all measures). HA had a shorter R+k time (p<0.05) but angle A remained unaffected. Additionally, modest increases in MA and G were seen in hemophilia and APS in the presence of RMP (p<0.04 or better). (3) Global measures. The CI (coagulation index) was significantly improved (p<0.05 or better) by addition of RMP in TP, ITP, APS, and SLE, indicating a better global coagulation profile in those diseases. (4) Combined groups. When all of the hematologic disorders were combined, significant differences in R, k, R+k, A, and G were observed in the presence of RMP (p≤0.02), but no difference in MA was seen, although it was very close to significance (p=0.051). CI was also markedly improved by RMP (p=0.02). (5) General observation. It is important to add that in nearly all groups, some patients responded strongly to RMP but others weakly or not at all, indicating different status of the disorder, possibly reflecting a state of platelet activation, inflammation, or disease activity. Conclusions/Discussion:RMP appear to have broad efficacy in correcting abnormal coagulation profiles in several disorders with differing pathologies. The parameter most strongly affected was time for initial fibrin formation, R. Effect on clot strength (G, MA) was statistically significant but varied among individual patients. RMP could correct abnormal clotting parameters in blood from differing pathologies, although there were individual variations. These data support the hypothesis that RMP can serve as a useful hemostatic agent to reduce bleeding in a variety of conditions of differing etiology. However, the factors responsible for such variations remain unknown. Elucidation of underlying factors influencing the effect of RMP will further improve and refine its therapeutic benefit and indications of its application. Disclosures:No relevant conflicts of interest to declare.

The absolute counting of red cell‐derived microparticles with red cell bead by flow rate based assay

Activation of red blood cell is associated with the formation of red cell-derived microparticles (RMPs). Analysis of circulating RMPs is becoming more refined and clinically useful. A quantitative Trucount tube method is the conventional method uses for quantitating RMPs. In this study, we validated a quantitative method called "flow rate based assay using red cell bead (FCB)" to measure circulating RMPs in the peripheral blood of healthy subjects. Citrated blood samples collected from 30 cases of healthy subjects were determined the RMPs count by using double labeling of annexin V-FITC and anti-glycophorin A-PE. The absolute RMPs numbers were measured by FCB, and the results were compared with the Trucount or with flow rate based calibration (FR). Statistical correlation and agreement were analyzed using linear regression and Bland-Altman analysis. There was no significant difference in the absolute number of RMPs quantitated by FCB when compared with those two reference methods including the Trucount tube and FR method. The absolute RMPs count obtained from FCB method was highly correlated with those obtained from Trucount tube (r(2) = 0.98, mean bias 4 cell/microl, limit of agreement [LOA] -20.3 to 28.3 cell/microl), and FR method (r(2) = 1, mean bias 10.3 cell/microl, and LOA -5.5 to 26.2 cell/microl). This study demonstrates that FCB is suitable and more affordable for RMPs quantitation in the clinical samples. This method is a low cost and interchangeable to latex bead-based method for generating the absolute counts in the resource-limited areas.