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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: 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.

We investigated antibodies to factor VII/VIIa (FVII/VIIa) and five other common target antigens in 33 patients with a history of anti-phospholipid syndrome (APS) and 50 healthy controls using an enzyme-linked immunosorbent assay (ELISA) technique. We found that antibody to FVII/VIIa, a previously unrecognized and common antigen in APS, was present in 67% of patients. Frequencies of antibodies to other target antigens were: anti-beta-2 glycoprotein 1 (anti-beta 2GP1), 88%; anti-cardiolipin (anti-CL), 76%; anti-phosphatidylethanolamine (anti-PE), 67%; anti-phosphatidylserine (anti-PS), 64%; and anti-phosphatidylcholine (anti-PC), 59%. Most patients had antibodies against multiple antigens, but a few were positive for only anti-beta 2GP1 (12%) or anti-CL (3%). Positivity for anti-FVII/VIIa was significantly associated with positivity for anti-PE, anti-PS and/or anti-PC (P < 0.05) but not anti-beta 2GP1. When frequencies of immunoglobulin G (IgG) versus immunoglobulin M (IgM) antibodies were compared, anti-beta 2GP1 IgG correlated with the lupus anticoagulant (P < 0.05) and was significantly more prevalent than IgM, but the reverse was seen for all other antigens. In arterial thrombosis, IgM was more prevalent for all antigens, and was significantly associated with FVII/VIIa, PE and PS, whereas in venous thrombosis, IgG was frequently prevalent, especially in association with FVII/VIIa, beta 2GP1 and CL. In summary, FVII/VIIa is a new and common antigen in APS. Anti-FVII/VIIa is often associated with anti-PE, anti-PS and anti-PC. The IgM class is more frequently associated with arterial thrombosis and the IgG class with venous thrombosis.

Although the presence of antiphospholipid antibodies (APLA) in immune thrombocytopenic purpura (ITP) has been reported, their clinical significance is not clear. The present study investigated APLA profiles in relation to the clinical stages of ITP. We studied APLA in 40 patients in three stages of ITP: exacerbation/relapse (n=7), stable (n=14) and remission (n=19). Both IgG and IgM APLA to six target antigens were measured by enzyme-linked immunosorbent assay: beta2-glycoprotein 1 (beta2GP1), cardiolipin, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine and factor VII/VIIa. The central finding was that APLA were common in ITP but differed significantly in disease stages, being highest in exacerbation (86% positive), intermediate in stable disease (57%) and lowest in remission (42%). In exacerbations, APLA were predominantly of IgG class, while in stable disease, IgM predominated. During remission, APLA often became undetectable. Both the frequency and titres of APLA were significantly higher during exacerbation than remission. An inverse correlation was found between platelet count and nearly all APLA (except beta2GP1). Sequential study of six patients revealed that APLA tended to emerge and rise with exacerbation, concurrently with new episodes of bleeding and became undetectable during remission. These findings raise the possibility that APLA may play a role in the exacerbation and remission of ITP or they may be a consequence of platelet destruction.

BACKGROUND: CD36 is a widely distributed transmembrane glycoprotein, called GpIV on platelets, involved in various physiologic processes. It has been implicated in thrombogenic pathologies such as diabetes, atherosclerosis and anti-phospholipid syndrome (APS). It is also an adhesion molecule capable of interacting with collagens and is considered a platelet collagen receptor. We investigated autoantibodies (Ab) against CD36 in TTP [Schultz et al, BrJH 103:849, 1998]. The present study was undertaken to explore relationships of anti-platelet Ab (aPlt-Ab), antiphospholipid Ab (APLA), and circulating cell-derived microparticles (MP) in patients with thrombosis. METHODS: Forty-five patients with documented thrombosis (TB) referred for hypercoagulable workups were recruited consecutively. Of them, 18 suffered from recurrent thrombosis (rTB) while 27 had a single event (sTB) within the past 5 years. We measured APLA, lupus anticoagulant (LA), antiplatelet antibody (aPlt-Ab) and MP. The APLA were IgG and IgM against cardiolipin (CL) and β2-glycoprotein-I (β2GP1) measured by ELISA. The aPlt-Abs were IgG and IgM reacting to GpIIb/IIIa (CD41b), GpIb/IX (CD42b), and GpIV (CD36), assayed by the PAICA method of Macchi et al [Thromb Haemost 76:1020, 1996]. The following types of MP were measured by flow cytometry: endothelial MP defined by CD31+/CD42− (EMP31) or by CD62E+ (EMP62); platelet MP defined by CD31+/CD42+ (PMP); leukocyte MP defined by CD45+ (LMP); and red blood cell MP by glycophorin+ (RMP). Lupus anticoagulant (LA) was measured in the hospital clinical laboratory and was considered positive if 2 of 3 different test methods were positive. Platelet activation was measured by CD62p expression in flow cytometry. RESULTS: Although both groups had increased prevalence of APLA and LA, there were no significant differences between the sTB and rTB groups. About 70% of patients in both groups were positive for at least one APLA. Of the aPlt-Ab, only aGpIV (aCD36) statistically discriminated between the groups, being more frequent in rTB for both IgG (p CONCLUSION: GpIV (CD36) antibodies are the most prominent risk factor for rTB to emerge from this study. This confirms a previous report [Pelegri et al, Clin Exp Rheum 21:221, 2003]. EMP, LMP, RMP and platelet activation (CD62p) are also increased in the recurrent TB group vs single TB. EMP62 were associated with aCD36. Since CD36 is found also on endothelial cells, this suggests that aCD36 could be responsible for the high EMP62 in the rTB group. The present study demonstrates an association between high EMP62 and aCD36. We suggest that persisting autoantibodies against GpIV activate EC to predispose to recurrent thrombosis.

Introduction: Antiphospholipid syndrome (APS) is characterized clinically by thrombotic events and the presence of antiphospholipid antibodies (aPLA) and/or lupus anticoagulant (LA). It is frequently associated with thrombocytopenia and anti-platelet antibodies have been implicated by some. However the roles of anti-platelet antibodies in APS have not been elucidated. We previously reported that platelet activation, but not endothelial activation, was associated with thrombosis in aPLA+ patients [Blood, 104:143a, 2004] but the cause of platelet activation was not addressed. In the present study, we investigated the prevalence of anti-platelet antibodies in APS patients, as well as platelet and endothelial activation. Material and Methods: We evaluated 47 patients with primary APS. Anti-platelet antibodies against GP IIb/IIIa (CD41b), GP Ib/IX (CD 42b) and GP IV (CD36) for IgG and IgM class were measured by PAICA assay [Thromb Haemost76:1820, 1996]. We also measured platelet and endothelial activation markers by flow cytometry: CD62P on platelets, CD31+/CD42+ platelet microparticles (PMP), and CD31+/CD42- endothelial microparticles (EMP). Results: Of the 47 patients, 34 (72%) were positive for at least one anti-platelet antibody. Looking first at IgG, 18/34 (53%) were positive for GP IV; 17/34 (50%) for GP IIb/IIIa; and 16/34 (47%) for GP Ib/IX. IgM antibodies were 47% (14/30) for GP Ib/IX, 38%(13/34) for GP IIb/IIIa, and 24% (8/33) for GP IV. Platelet and endothelial markers were significantly more common in the anti-platelet antibodies positive group: 40% vs. 21% for CD62P, 40% vs. 28.5% for EMP, and 23% vs. 5% for PMP, respectively. We found that CD62P associated significantly with IgM anti-GP IIb/IIIa (p< 0.05), and PMP with IgM anti-GP IIb/IIIa (p< 0.05), and IgM anti-GP IV (p< 0.05). Conclusions: Anti-platelet antibodies are common in APS, confirming previous reports. We found that anti-platelet antibodies IgM anti-GP IIb/IIIa, and IgM anti-GP IV were often associated with platelet activation, suggesting that these antibodies may activate platelets to play an important role in the thrombogenesis of APS. These antibodies were also associated with endothelial activation. It remains to be determined which antibodies, APLA and/or anti-platelet antibodies, play a dominant role in the activation of platelet or endothelial cells and contibute most to the pathogenesis of thrombosis in APS.

INTRODUCTION: Splenectomy has been widely employed in the treatment of immune thrombocytopenic purpura (ITP) and various other disorders. Increased life threatening infection is well recognized but the long-term safety of splenectomy has not yet been delineated. Splenectomy has been associated with increased incidence of cardiovascular death [Lancet, 1977; 2 (8029):127–9] and ischemic small vessel disease leading to recurrent TIA and cognitive impairment [Thromb Res, 2000; 107: 337–44]. Recent studies have demonstrated that procoagulant MP play an important role in the pathogenesis of thrombosis and cardiovascular complications (JTH, 2005; 3: 884–7). In the present study, we investigated profiles of cell-derived microparticles (MP) and thrombotic events in ITP patients with / without splenectomy. METHODS: Fifty-seven patients meeting diagnostic criteria of ITP were studied. Excluded were those with HIV, viral hepatitis, or lupus anticoagulant. Fifteen had splenectomy (ITPs) while 42 had not (ITPns). The ITPs group comprised 11F, 4M, while ITPns group had 28F, 14M. The 2 groups were comparable in sex, ages and platelet counts. In the latter, 9 failed to undergo remission, having active ITP (ITPs-A) while the other 6 were in remission (ITPs-R) following splenectomy. In the ITPns group, 23 responded poorly to therapies (ITPns-A) while the remaining 19 were in remission (ITPns-R). CBC and platelet counts, blood chemistry and PT, aPTT were measured. Flow-cytometry was employed to assay cell-derived MP from platelets (PMP) by anti-CD41, from leukocytes (LMP) by anti-CD45, from red cells (RMP) by anti-glycophorin, and from endothelial cells (EMP) by anti-CD31+/anti-CD41-. All patients were followed and incidence of thrombotic events was evaluated. RESULTS: Data are summarized in Table 1. Mean values of EMP, LMP, PMP and RMP were all significantly higher except PMP in ITPs compared to ITPns: RMP (p=0.002), EMP (p=0.008), LMP (p=0.03). In addition, aPTT was significantly shortened in ITPs vs ITPns (p=0.0059), as well as in ITPs-A vs. ITPns-A (p=0.0025). In the ITPs-R group, the aPTT was shorter than ITPns-R but did not reach statistical significance. Thrombotic events were recorded in 6 of the 15 ITPs group (40%) but in only 4 of the 42 ITPns (10%), p CONCLUSIONS: (i) Cell-derived MP, which are known to be procoagulant and implicated in the pathogenesis of thrombosis and inflammation, are significantly elevated, (ii) along with significantly shortened aPTT and (iii) thrombotic events were more prevalent in those without spleen compared to those with spleen among ITP patients. These observations support the hypothesis that spleen is the main site of clearance of procoagulant MP, and that accumulation of procoagulant MP may increase risk of thrombotic complications following splenectomy. A larger-scale study is warranted to clarify the long term safety of splenectomy in ITP and other disorders. Table 1 ITPs (no spleen) ITPns (with spleen) p value Patients; n= 15 42 EMP 730 323 0.008 PMP 16147 10995 0.11 LMP 1810 1238 0.03 RMP 2777 1759 0.002 aPTT 22.5 25.7 0.006 Thrombosis 40% 10% 0.002

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

BACKGROUND. Although the etiology of TTP remains controversial, some notable associated conditions have been identified. These include genetic predisposition, infections, drugs, pregnancy, systemic lupus erythematosus (SLE) and deficiency of ADAMTS13. These associations underscore the suspected immunological nature of this disorder. Some authors have reported the presence of antiphospholipid antibodies (APLA) with onset of fulminate severe TTP. Also, platelet and endothelial activation has been described in TTP but their possible relation with APLA is unknown. METHODS. We measured APLA in 19 patients with acute TTP and 17 in remission. Fifteen were female and four were male, mean age of 37 ± 15. All met the diagnostic criteria of TTP and were treated with standard therapy including exchange plasmapheresis. The severity of TTP was classified based on duration of hospitalization and numbers of plasmapheresis needed. Recurrence of TTP was assessed by history and review of medical records. CBC, platelet counts, blood chemistry and LDH were monitored. We investigated APLA antibodies IgG and IgM against 6 antigens (β2GP1, FVII/VIIa, CL, PC, PS, PE) by ELISA. To investigate platelet activation, we measured by flow cytometry: CD62P on platelets, and platelet microparticles (PMP) by CD41 (PMP41). We also measured endothelial microparticles (EMP) using CD62E. RESULTS. Lab study : In acute TTP, 42% (8/19) were APLA positive for at least one IgG antibody. The most frequent was aPS (33%) followed by aCL(28%), aPC(28%), aβ2GP1 (28%), FVII/VIIa (21%) and PE (21%). IgM antibodies were not detected. APLA was two-fold more prevalent in acute TTP (42%) than in remission (21%). In acute TTP multiple antibodies (two or more) were seen in 75% (6/8) while in remission only one (12.5%) had multiple antibodies. Platelet activation measured by CD62P was higher in APLA+ than in APLA− (102 ± 25 vs 33 ± 8 fluorescence units, p Clinical observations : When comparing the two groups (APLA+ vs APLA−), APLA+ group had higher incidence of recurrence of TTP than APLA− (75% vs 36%), p CONCLUSIONS: APLA are frequently seen in acute TTP (42%) and they tend to decline in remission. Their presence is associated with increased activation of platelets and endothelial cells, and also with clinical recurrence of TTP. APLA may play a role in excerbation and recurrence of TTP.