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BACKGROUND: It has been shown that platelet microparticles (PMP) exert pro-inflammatory activity. It has also been suggested that red cell MP (RMP) are similarly pro-inflammatory, but the evidence is less clear. This issue has clinical significance for several reasons. For example, there is evidence that both PMP and RMP have hemostatic activity [Horstman et al, Semin Thromb Hemost, 2019] and that infusion of RMP is a viable strategy for control of excessive bleeding [Jy et al,Thromb Haemost, 2013]. For these reasons we undertook assay of inflammatory potential of PMP and RMP by the method of CD11b expression on neutrophils, using N-Formylmethionyl-leucyl-phenylalanine (fMLP) as a positive control. METHODS: RMP were isolated from leukoreduced, type O+ packed RBCs obtained from three different donors at storage time points of 3 and 6 weeks (RMPStor). In addition, RMP were prepared by high-pressure extrusion from type O+ packed RBCs stored for less than one week (RMPxt). Platelets were isolated from healthy volunteers and sonicated to produce PMP. MP concentrations were determined using flow cytometry. RMP were identified by PE-labeled anti-glycophorin and PMP were identified by FITC-labeled anti-CD41. Each MP species was added to separate 500 µL aliquots of fresh whole blood collected from healthy volunteers for equal final concentrations of 20,000/µL, 40,000/µL, 80,000/µL, and 200,000/µL. To one of each pair, fMLP was added for a final concentration of 1 µM. Fresh, whole blood treated with sterile saline was used as a negative control. A positive control was obtained by adding fMLP to whole blood without MP treatment. The blood samples were then incubated for one hour under gentle shaking. Two-color flow cytometry with PE-Cy5-anti-CD11b and FITC-anti-CD45 MAbs were employed to measure the ratio of CD11b to CD45 expression on neutrophils (identified through FS, SS, and CD45+ FL gating). The differences in CD11b expression were compared using the paired t test. The mean values presented below were determined using the results from at least 5 replicate experiments in which the two groups were directly assayed against one another using neutrophils from a single donor, resulting in slightly different mean values for each group depending on the comparison being analyzed. RESULTS: For all RMP species, there was no significant difference between concentration and their effect on neutrophil CD11b expression. In contrast, the concentration of PMP positively correlated with an increase in CD11b expression (R2=0.78). At a MP concentration of 200,000/µL, there was no statistical difference when comparing neutrophil CD11b expression in the negative control vs. incubation with RMPxt (27.5 vs. 27, p=0.7), or the negative control vs. incubation with RMPStor Wk 6 (29.0 vs. 28.4, p=0.6). However, neutrophil CD11b expression was lower when incubated with RMPStor Wk 3 compared to the negative control (25.1 vs. 28.8, p CONCLUSION: In sum, PMP exhibited robust pro-inflammatory activity, but RMP did not. On the contrary, RMP exhibit modest but significant anti-inflammatory activity in this assay method. It remains necessary to elucidate the mechanism and to assess if these findings apply in vivo. Disclosures Jy: RxMP Therapeutics: Consultancy, Equity Ownership, Patents & Royalties: US patents No. 8,150,632 and 9,155,764, Research Funding. Horstman:RxMP Therapeutics: Patents & Royalties: US patents No. 8,150,632 and 9,155,764. Ahn:RxMP Therapeutics: Consultancy, Equity Ownership, Patents & Royalties: US patents No. 8,150,632 and 9,155,764, Research Funding.

BACKGROUND: Immune Thrombocytopenic Purpura (ITP) is an autoimmune disorder in which the production of antiplatelet antibodies stimulates increased clearance of platelets by macrophages of the mononuclear phagocytic system, resulting in thrombocytopenia and bleeding diathesis. In contrast, Thrombotic Thrombocytopenic Purpura (TTP) is a disorder in which platelets are sequestered in microcirculation to form platelet-rich microthrombi in response to endothelial injury. This leads to thrombocytopenia, red cell fragmentation with hemolytic anemia, systemic microangiopathy, and multiorgan failure. Cell-derived microparticles (C-MP) are microvesicles released in cell activation or apoptosis. C-MP from endothelial cells (EMP), platelets (PMP), leukocytes (LMP), and erythrocytes (RMP) are of particular interest because they have emerged as useful biomarkers, reporting early stages of cellular injury prior to clinical manifestations of underlying cardiovascular, thrombotic, and inflammatory disorders. We investigated C-MP profiles in both ITP and TTP patients with the aim of gaining greater insight into the pathophysiology of these two distinct thrombocytopenic disorders. METHODS: A retrospective study was conducted comparing C-MP data of 29 ITP patients with those of 20 TTP patients. Both groups were stratified into active vs. remission phases. The ITP cohort consisted of 18 patients in active phase (7M/11F, mean age 49.3 yr, mean platelet count 48 x 103/µL) and 11 in remission (2M/9F, mean age 51.9 yr, mean platelet count 221 x 103/µL). The TTP cohort consisted of 8 patients in active phase (1M/7F, mean age 43.1 yr, mean platelet count 54 x 103/µL) and 12 in remission (2M/10F, mean age 43.7 yr, mean platelet count 223 x 103/µL). For ITP, the active phase was defined by platelet counts 3 months and the remission phase by counts >150 x 103/µL >3 months. None were splenectomized. For TTP, the active phase was defined as thrombocytopenia, clinical evidence of microangiopathy, elevated lactate dehydrogenase (LDH) and low ADAMTS13 activity prior to initiation of plasmapheresis. The remission phase was defined as sustained normalization of the laboratory parameters in the absence of further microangiopathy for at least one month. Flow Cytometry was employed to identify LMP by anti-CD45, RMP by anti-glycophorin, PMP by CD42+/CD31-, and EMP by anti-CD62E. The differences in C-MP patterns between ITP and TTP were evaluated. All C-MP data are presented in units of x103/µL. Statistical analysis was performed using the unpaired t test. RESULTS: When C-MP were compared between active ITP and TTP, TTP had far higher EMP (37 vs. 540, p CONCLUSION/DISCUSSION: PMP were much higher in active ITP than TTP, presumably reflecting the antibody-mediated platelet fragmentation inherent to ITP. This is further corroborated by the higher LMP. Interestingly, PMP in remission ITP was higher than in active ITP, although the PMP/platelet ratio remained constant. In contrast, the PMP/platelet ratio in TTP decreased from the active to remission phase, despite the increase in PMP. This suggests that the immunologic milieu in ITP favors a consistent generation of PMP throughout the different disease phases. EMP and RMP were much higher in active TTP than ITP, reflecting the endothelial injury and red cell fragmentation with hemolysis present in TTP. In addition, the elevated RMP in active TTP may contribute to the formation of microthrombi due to their inherent procoagulant activity. This study demonstrates that C-MP profiles can provide valuable insight on immune-mediated disorders which may lead to improvements in their diagnosis and management. Disclosures Ahn: RxMP Therapeutics, Inc.: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

BACKGROUND: Cell derived MPs are small membrane vesicles released during cell activation or apoptosis. They express an inside-out membrane, which exposes the negatively charged phospholipid layer outside, allowing clotting factors to be anchored and generate thrombin. Among various species of MPs, RMP (red cell MPs), PMP (platelet MPs), LMP (leukocytes), EMP (endothelial cells) are of special interest. They play an important role in hemostasis, thromboses, and inflammation. MPs mirror early injury of parent cells and are sensitive early biomarkers of underlying disorders. TTP is a microangiopathy mediated by antibody-induced depletion of ADAMTS13, a von Willebrand factor-cleaving protease. Endothelial injury promotes platelet clumping and formation of platelet rich microthrombi in the microcirculation. Subsequent platelet sequestration leads to impaired microcirculation, thrombocytopenia and red cell fragmentation with a microangiopathic hemolytic anemia. Exchange plasmapheresis (EPP) is the standard therapy. It removes antibodies to ADAMTS13, replacing it with ADAMTS13 rich plasma. It is possible that EPP removes thrombogenic MPs to improve the clinical course of TTP. In this study, we investigated MP profiles in active and remission phase of TTP and the effect of EPP on MP profiles. We also aimed to determine if MP profiles may be a useful measure for monitoring clinical course and tracking progress of therapy. METHODS: A retrospective study was conducted evaluating MP assays in patients with TTP. MP profiles were reviewed in acute and remission phases of TTP. Acute phase was defined as thrombocytopenia, clinical evidence of microangiopathy and hemolytic anemia and low ADAMTS13 activity. Remission was defined as sustained normalization of laboratory parameters and no further microangiopathy for at least one month. Patients were studied longitudinally, with MP assays before and after EPP. EMP were measured by CD31+/CD42b− (EMP31), CD62E+ (EMP62); PMP by CD31+/CD42b+ (PMP42) and CD41+ (PMP41). All were measured in platelet-poor plasma by flow cytometry. All MP data are presented in units of x105/µL. The differences in MP patterns among TTP patients in active and remission phases of disease, as well as the effect of EPP on MP profiles were assessed. RESULTS: Among 20 patients with TTP, 8 (40%) were in acute phase and 12 (60%) in remission. An average of 10.7 EPP were performed. The average platelet count prior to EPP was 50.6x103/µL, which increased to 248 x103/µL following the last EPP. ADAMTS13 activity was generally For patients in the acute phase, PMPs were low: PMP41 0.32 (±0.11), PMP42 3.11 (±2.56), but consistently increased following EPP to the point of statistical significance at the last treatment: PMP41 1.26 (±0.71), p=0.005 and PMP42 7.65 (±5.03), p=0.039. Moreover, levels were higher in the remission phase for both PMPs, but only statistically significant for PMP41: 1.39, p=0.034. Conversely, EMP62E was initially elevated on presentation, but declined with successive EPP: 6.07 (±3.02) prior to initiation of EPP to 4.5 (±3.1) upon the last day of EPP. Furthermore, this pattern continued into remission, with EMP62E of 2.72 (±2.11), p=0.009. Similar to the trend in EMP62E, RMP was elevated in the acute phase (25.1 [±18.5]) before steadily declining with EPP (19.65 [±12.53]). This progressive drop in RMP persisted for those in remission phase of TTP 10 (±5.42), p=0.015. DISCUSSION/CONCLUSION: Taken collectively, cell derived MP's were found to reflect disease activity and response to therapy. There was a linear correlation between PMP levels and platelet count. Low PMPs in the acute phase reflects the initial thrombocytopenia characteristic of TTP. When EPP was initiated, the disease improved and there was a rise in PMP which mirrored the rise in platelets. This rise was sustained in the remission phase. Conversely, EMP62E and RMP are increased in acute phase TTP due to endothelial activation, microthrombi deposition and red cell fragmentation. After EPP the disease is quiescent, hence the progressive decline of EMP62E and RMP. These results show promising utility of cell derived MP profiles as a clinical tool to surveil chronic TTP patients and to predict disease relapse at an early stage. After EPP is initiated, change in MP Profiles may be used to monitor response to therapy and determine the appropriate time to wean EPP. Disclosures No relevant conflicts of interest to declare.

BACKGROUND. Cell derived microparticles (MP), are small vesicle ( METHODS. For RMP-Stor, samples were taken at intervals up to 42 days of PC stored at 4oC, then centrifuged to recover the RMP released. For RMP-CaI, supernatants of fresh RBC exposed to Ca2+/ionophore (A23187) for 30 min were centrifuged. For RMP-HPE, washed RBC were subjected to high-pressure extrusion, and the resulting RMP were collected by centrifugation and washed. Flow cytometric counts were by CD235a (glycophorin A) and annexinV (AnV) binding. Thromboelastography (TEG) compared equal counts of each sample type, based on CD235a, diluted as needed with particle-free pooled plasma (PFP). RESULTS: (1) Yield per mL of initial packed RBC: The highest yield of RMP was seen in RMP-HPE, as expected since ≈100% of RBC convert to RMP-HPE. This was followed by RMP-CaI, which was about 3x more than RMP-Stor at 42 days. The RMP-Stor increased linearly from day 21 to 42. (2) AnV binding was expressed as ratio AnV+/CD235+, and is a marker of procoagulant phospholipid. This ratio was least in RMP-HPE (0.38) and more than doubled in both RMP-Stor (0.92) and RMP-CaI (1.0). (3) Size of RMP by forward scatter (FS) increased steadily with time in RMP-Stor, from day 21 (0.56 um) to day 42 (0.79 um). These values are somewhat larger than RMP-CaI (0.47 um) or RMP-HPE (0.52 um). The side-scatter signal correlated with the FS values. (4) Acetylcholinesterase (AChE) activity was by far highest in RMP-Stor (56 nM/min, per 106 particles) vs. 4.5 for RMP-CaI, and 1.5 for RMP-HPE. (5) Procoagulant activity: In TEG, the increase in MA (maximum amplitude) by addition of RMP-Stor was 64 mm, but was not increased by RMP-HPE or RMP-CaI. The R-time (lag) showed greatest shortening with RMP-Stor (10.2 min), followed by RMP-CaI (6.7 min), and RMP-HPE (3.6 min) (6)Anti-fibrinolytic activity: In presence of 0.4 ug/mL of tissue plasminogen activator (tPA), for the purpose of making fibrinolytic activity more detectable in a short time, all 3 species of RMP effectively inhibit t-PA-mediated fibrinolysis measured by TEG parameters, including MA, Ly30, and TTG (total thrombus formation). The RMP-HPE was most effective followed by RMP-Stor, and RMP-CaI. CONCLUSION/DISCUSSION. Results demonstrate clear and distinctive differences in functional and phenotypic properties of the 3 species compared. RMP-Stor and RMP-CaI are lager in size, higher in PS and AChE expression, and higher in procoagulant activity, compared to RMP-HPE. On the other hand, RMP-HPE are higher in number generated per mL packed RBC and exhibit stronger inhibition of tPA-mediated fibrinolysis. It is well known that RMP generated by storage and calcium ionophore are enriched in lipid rafts. In contrast, the RMP-HPE are not enriched in lipid rafts, as reflected in low AChE. We conjecture that the contents of lipid raft on RMP may play an important role in determining the functional properties of RMP. Disclosures No relevant conflicts of interest to declare.