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Introduction. CD30, member of the TNF-receptor family, is expressed on activated T and B cells. Upon CD30 signaling and the downstream event of cytokine release of IL-4 and IL-13, CD30 is cleaved by a metalloprotease, resulting in soluble CD30 (sCD30). sCD30 has been shown to be a reliable proxy for Th2 activity and is elevated in Th2-polarized disorders such as atopic dermatitis, rheumatoid arthritis, and systemic lupus erythematosus (SLE) but is subnormal in patients with Th1-polarized disease such as X-linked agammaglobulinemia (Eur J Immunol2001; 31:1927–1934). Elevated sCD30 levels were also found in patients with Hodgkin’s lymphoma or anaplastic large cell lymphoma. We investigated sCD30 levels in patients with chronic idiopathic thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA). Method. We measured plasma sCD30 levels in 35 patients with ITP, 12 with AIHA, 8 with SLE, 9 with lymphoma and 18 healthy controls. sCD30 was measured by ELISA (Bender Medsystems) in platelet poor plasma from citrated blood. Results. Compared to controls (19.0 ± 6.2 units/mL), sCD30 levels were significantly higher in AIHA (mean 145.6, p=0.03) but not in ITP (22.7, p=0.1). sCD30 levels were also elevated in SLE (mean 73.6, p=0.001) and lymphoma (mean 107.4, p=0.04), confirming previously published studies. In AIHA, sCD30 levels were not significantly different in patients with active/chronic disease versus those in clinical remission, although levels trended higher during the acute episodes of hemolysis. In ITP, sCD30 levels were also similar between active disease versus remission and splenectomized versus unsplenectomized patients. Conclusion. Our results support the hypothesis that AIHA and SLE are Th2-polarized disorders, whereas ITP is not. Lack of significant differences in sCD30 between active disease and remission in AIHA and ITP may suggest that imbalances of Th1/Th2 immunity persist regardless of clinical course, consistent with Panitsas et al (BMC Blood Disord2004; 4:4). Discussion. Prior studies have examined serum cytokine concentrations such as IFN-γ and IL-4 in patients with ITP and reached inconclusive results. But recent studies using more sensitive techniques such as RT-PCR indicate that ITP is a Th1-polarized disorder (Blood2004; 103:2645–2647). Our findings of elevated sCD30 levels in AIHA but not ITP lead to a provocative question: what is it about the immunological destruction of platelets that makes it associated with a markedly different Th1/Th2 milieu in comparison to the destruction of red blood cells? We propose that the key differences are 1) platelets are more potent in modulating immunity via mechanisms including CD40L (CD154) release upon platelet activation (Immunity2003; 19:9–19) and 2) platelets express MHC I molecules whereas red blood cells do not and hence they are processed by macrophages in a different manner (Transfusion2002; 24:958–961).

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.

Introduction. Rituximab, humanized anti-CD20, was initially approved for the treatment of CD20-positive B cell lymphoma, but has recently been shown to be effective in the treatment of a wide range of autoimmune disorders. In a cohort of 25 patients, Stasi et al reported a 52% overall response rate in patients with chronic idiopathic thrombocytopenia (ITP) (Blood2001; 98:952–957). Overall response rates reported since then have been concordant, about 50%. Although rituximab has been increasingly employed for the treatment of ITP, the mechanism of immunological response is not clear. We investigated the effect of rituximab on titers of anti-platelet glycoprotein-specific antibodies in patients with refractory ITP. Methods. We studied 12 patients with chronic ITP (9 females, 3 males) of ages ranging from 22–87 years, mean age 43 years. All had been treated with glucocorticoids and failed to sustain remission. Three patients failed splenectomy. Their previous medications were continued without major alteration of the dosages during the period of followup. Rituximab was given 375 mg/m2 weekly for 4 courses. Once remission was achieved, previous medications were gradually tapered and stopped completely if remission was sustained. Complete remission (CR) was defined as normalization of platelet counts that lasted longer than 4 months. Partial remission (PR) was defined as a doubling of platelet count if initial counts were ≥20,000 or an increase to ≥30,000 if counts were 20,000 or below. Non-responders (NR) showed neither. Lab studies included CBC, platelets, and blood chemistries. IgM and IgG antibodies against platelet glycoproteins (GP IIb/IIIa, GP Ib/IX, GP IV) were assayed by PAICA prior to and following rituximab therapy. Results. Ten of 12 patients showed improvement of platelet counts following rituximab therapy. There were 6 CR, 4 PR and 2 NR. Among the 6 CR, remissions lasted for a range of 6–29 months with a mean of 17.0 months. Four of the 6 were able to stop previous therapy for ITP, and the other two were able to reduce their dosages by at least half. Among the 4 PR, remissions were maintained for 4–16 months (mean, 7.3 months). Anti-platelet antibodies became negative in 6 of 6 CR, in 3 of 4 PR and in 0 of 2 NR. Although on average, all anti-platelet antibodies tested were reduced after rituximab therapy, IgM anti-GP IIb/IIIa, IgM and IgG anti-GP Ib/IX and IgM anti-GP IV reached statistical significance (p Conclusions. Rituximab frequently induces long lasting clinical remissions that do not require further therapy for ITP and reduction or disappearance of autoantibodies in ITP, unusual features compared to other measures. This finding indicates that a putative mechanism of action of rituximab is inhibition or destruction of autoantibody-producing B cells in ITP. Rituximab is highly effective with minimal side effects and should be considered as primary therapy in patients with chronic ITP.

BACKGROUND. ITP is an autoimmune disorder in which autoantibodies (Ab) reacting to platelet glycoproteins (Gp) mediate immune destruction of platelets. In most cases the cause is unknown, but chronic infections such as hepatitis C, studied here, are often associated with ITP (ITP-C). In ITP-C, it is not clear whether platelets are destroyed by viral immune complex or by Gp Ab, as in classic active ITP (ITP-A). Antiviral therapy may not induce remission in ITP-C, so its management remains problematic. We investigated Gp Ab in ITP-C vs . ITP-A, and evaluated efficacy of IL-11 in a group of patients (pts) with ITP-C. A previous study found that IL-11 is ineffective in treatment of ITP-A [Am J Hematol, 2001; 66: 172–7]. METHODS. We studied 35 pts with ITP-A (19F, 16M, mean age 53yr, mean platelet count 61,000/μL) and 15 with ITP-C (9F, 6M, mean age 61yr, mean platelet count 66,0000/μL). Lab tests included CBC, platelet counts, liver function tests, HCV-RNA, and clotting factor activity (FVIII & vWF). Ab against Gp were assayed by PAICA. Six pts with ITP-C were treated with IL-11 (50 mg/kg) for 7 days to 9 mo’s. In four, clinical courses were followed after discontinuing IL-11. RESULTS. As shown in Table 1, the prevalence of all 3 Gp Ab (IIb/IIIa, Ib/IX, and IV) was significantly more frequent in ITP-C than ITP-A, for both IgG (p≤ 0.007) and IgM (p=0.005). Mean titers of Ab were also higher in ITP-C than ITP-A, but only IgM Ab were significant (p

BACKGROUND. Although APLA are a known risk factor for thrombosis, clear understanding of this association remains elusive. In recent years our laboratory has conducted surveys of APLA by ELISA of several immune, inflammatory and other disorders, here compared. METHODS. IgG and IgM to six target antigens (B2GP1, FVII, CL, PE, PS, PC) were measured. In most cases, platelet activation (by CD62P), endothelial activation (by endothelial microparticles, EMP) and lupus anticoagulant (LAC) were also measured. Patient groups compared were: 124 autoimmune disorders (40 ITP, 80 APLA, 24 multiple sclerosis, MS) and 54 hematologic disorders (35 thrombocytosis, 19 TTP). RESULTS. (1) ITP (n=40, of which 19 were remission, 14 stable, 7 acute). Although APLA are known to be common in ITP, no relation to symptoms was identified. We discovered that APLA rise with onset of acute ITP, and decline or disappear in remission, p=0.007 [Br J Haem 128:366, 2005]. APLA was associated with bleeding, not thrombosis. (2) Multiple sclerosis (MS) (n=24, of which 7 were in remission, 24 exacerbation). APLA were known to be common in MS but no clinical correlations had been shown. We found that APLA in MS arise mainly in exacerbations, p=0.002, suggesting a link to the pathology. [Neurology 2005, 64(6, supl 1):A194.]. APLA in MS were exclusively IgM. (3) TTP (n=19). A role of APLA in TTP had been conjectured but evidence was equivocal. We found that 42% of acute cases were APLA+, and these had higher frequency of recurrence, p CONCLUSIONS. These findings, together with work from others, point increasingly to a link between APLA and pathology in a wider range of inflammatory and hemostatic disorders than usually considered, not limited to thrombosis (TB). In autoimmune disorders such as ITP, MS and TTP, APLA tend to arise in exacerbation and remit in remission, suggesting role of APLA in exacerbation of autoimmunity and inflammation. Also of interest is our frequent finding of association between APLA and activated platelets and/or endothelium. Patterns appear disease-specific but specificity of the APLA involved may be subtle and beyond routine methods. Our findings suggest role of APLA in pathology is not limited to preponderately thrombotic disorders, but extends to other manifestations of autoimmune and inflammatory disorders. Clear understanding of meaning of APLA in these widespread associations could be of potentially great significance.

MPD is a clonal stem cell disorder encompassing essential thrombocythemia (ET), polycythemia vera (PV), myelofibrosis (MF), chronic myelogenous leukemia (CML). Chemotherapy has been a mainstay of therapy for the treatment of MPD but new agents including anegrelide, interferon, imatinib mesylate and thalidomide have been recently introduced. In most cases, continuous therapy is required to maintain remission and relapses are the rule when the treatment is discontinued. We report long-term remission of MPD with combined therapy of danazol and busulfan and an unusually long remission following discontinuation of therapy. Nine patients (4 women, 5 men) with MPD (7 MF, 1 PV, 1 ET) were studied. Danazol (200–800mg/day) was given with dosage adjustment for liver function abnormalities. Busulfan (2–4mg/day) was administered intermittently for 3 months to 2 yrs depending on blood counts and status of remission. Spleen size was evaluated with physical exam and CAT scans. The responses were classified as “excellent” [E] or complete remission (CR) when blood counts were normal with absence of splenomegaly, “good” [G] when spleen size was reduced > 50% with normal blood counts, “poor” [P] for no improvement in blood counts and splenomegaly and “fair” [F] for responses between P and G. Erythropoietin was administered when anemia developed. RESULT. Overall response of E and G was 77% (7/9). There were 2 E, 5 G, 1F and 1P responses. Table 1 summarizes data on 2 E responders. Table 1 Age/Sex Duration of MPD *Rx No (Rx yrs) ** Spleen Remission (Rm) Rm after D/C Rx * Rx=Danazol + Busulfan; No=Numbers of RX; (Yrs)=Duration of Rx. ** Size of spleen before and after each RX 72/F 2.5 yrs Rx1 (2.5 yrs) 6cm → 0 6.5 yrs 4yrs Rx2 (0.5 yrs) 2.5 yrs 2 yrs Rx3 (1 yr) 4cm → 0 6 yrs 5 yrs 72/F 3 yrs Rx1 (1.5 yrs) 25cm → 0 4 yrs 2.5 yrs Rx2 (0.5 yr) 20cm → 0 5/6 yrs 1/3 yrs The duration of CR in 2 E responders (1 PV, 1 MF) was 15 years in the first and approximately 5 years in the second. The remissions lasted over 2 –4 years even after therapy was discontinued. CAT scans confirmed reduced or normal sized spleen. All 5 “good” responders showed a remarkable clinical improvement initially but when the treatment was withheld, disease recurred. Re-treatment induced remissions but responses were seldom complete and required more uninterrupted therapy. In all responders, re-treatment was as effective as initial therapy, indicating that the benefit was due to therapy, not a coincidence. Compared to E responders, G and F responders had more advanced disease and longer duration of MPD, ranging from 2–15 yrs (average 9.2 years). Therapy was tolerated well but bone marrow suppression was seen in 3 cases, all of which recovered without serious complications. Danazol was withheld or reduced in 4 patients due to liver function abnormalities but was safely resumed in lower doses. SUMMARY. Danazol therapy with busulfan is very effective in MPD and offers a good alternative with potential clinical cure in some patients with MPD. Although clinical remissions were observed in most patients with MPD, those with shorter duration (

INTRODUCTION. Rituximab, a chimeric monoclonal antibody against CD20 expressed on B cells, has been approved for the treatment of B cell neoplasms. Recently, studies have supported the use of rituximab in various autoimmune disorders such as systemic lupus erythematosus (SLE), thrombotic thrombocytopenic purpura (TTP), and idiopathic thrombocytopenic purpura (ITP). We studied its efficacy in the treatment of antiphospholipid syndrome (APS) and APLA-associated TTP and ITP, and measured antiphospholipid antibodies (APLA) and lupus anticoagulant (LA) before and after rituximab therapy. METHOD. The study included 12 patients, 2 with antiphospholipid syndrome (APS) refractory to all known measures, 6 with TTP, and 4 with ITP, all of whom had positive tests for LA and/or APLA. Rituximab was given 375mg/m2 weekly 4 times. We studied clinical remission and duration of remission and the effect of rituximab on APLA and LA. Laboratory investigations included routine blood counts, IgM and IgG APLA to cardiolipin (aCL) and B2GPI, and LA. RESULT. I. Clinical Observation. (a) Two patients (a 29 and 44 year old female) with APS refractory to all measures including glucocorticoids, monthly chemotherapy with IV cyclophosphamide, and plasmapheresis suffered from recurrent thromboses despite anticoagulation with warfarin, and became disabled, spending most of their time in the hospital. Both went into clinical remission following rituximab, resumed full activities, and were able to be weaned off of glucocorticoids and treated with anticoagulation alone. The remissions lasted 15 and 7 months. LA became negative and APLA decreased or became negative in both. (b) Six patients with TTP and positive APLA and/or LA were treated with rituximab in conjunction with exchange plasmapheresis. Five achieved complete remission and one failed. Remissions of TTP lasted 0.5–3 years. (c) Four patients with ITP and positive tests for APLA or LA were treated and 2 went into clinical remission for 0.5 and 2 years, and 2 did not respond. Therapy was well tolerated but one (TTP) developed herpes zoster and another (ITP) had a TIA following remission with rituximab. Laboratory Observation. APLA and LA were studied in selected patients before and after rituximab treatment. LA became negative in 6 out of 8 patients and it remained negative for over 15 months in one patient. Among APLA, IgM B2GPI became negative in 71% (5/7), IgM ACLA became negative in 50% (4/8), IgG APLA, IgG B2GP1 became negative in 50% (2/4) and IgG aCLA became negative in 30% (2/6). In our series, IgM antibodies are more readily decreased by rituximab than IgG antibodies. Decrease in APLA was evident 3–4 months post therapy. SUMMARY. Although rituximab is widely used in autoimmune disorders, it has not been tested in APS. Our data indicate that it is an effective and life saving measure in refractory APS. Rituxan-induced, long lasting clinical remissions along with serological remission are unusual compared to other immunosuppressive therapies. Similar findings were also observed in APLA-associated TTP and ITP. Further prospective study is recommended to evaluate its full potential in APLA-associated disorders.

Introduction. CD30, member of the TNF-receptor family, is expressed on activated T and B cells and is cleaved by a metalloprotease following CD30 signaling, but the function of CD30 remains elusive. CD30 was initially described on the surface of Reed-Sternberg cells, and elevated levels of soluble CD30 (sCD30) have been described in patients with lymphoma, HIV and autoimmune diseases. Higher levels have been associated with unfavorable prognosis in lymphoma and HIV, and are thought to represent a Th2-polarized response. Relatively little is known about sCD30 profiles in many hematologic disorders. Method . We measured plasma sCD30 levels in patients with 1) non-malignant [27 ITP, 12 autoimmune hemolytic anemia (AIHA), 5 sickle cell, 5 PNH, 8 SLE] and 2) malignant [13 lymphoma, 8 multiple myeloma (MM), 11 MDS, 19 MPD] hematological disorders, as well as 3) thrombotic disorders [11 DVT, 17 hypercoagulable states] and 18 healthy controls. sCD30 was measured by ELISA using platelet poor plasma from citrated blood as well as lysates of patient red blood cells following osmotic lysis with water (1:10 dilution) versus supernatant of RBC’s diluted in PBS control. Results . In non-malignant disorders, sCD30 was significantly higher in AIHA (142.7±195.1 u/ml, p=0.006) and SLE (73.6±33.9, p=0.0000003), but not in other diseases associated with hemolysis such as sickle cell (18.7±12.3, p=0.4) and PNH (40.8±41.1, p=0.06) nor in another autoimmune disease, ITP (23.1±16.5, p=0.2) when compared to controls (19.0±6.2). In malignant disorders, all diseases tested had significantly higher sCD30 versus control: lymphoma (94.3±108.2, p=0.003), MM (47.3±28.5, p=0.0002), MDS (47.1±35.1, p=0.001), and MPD (35.3±31.6, p=0.02). In thrombotic disorders, none were significantly different from control. The surprising difference between AIHA and ITP sCD30 levels was statistically significant as well (p=0.001). In AIHA, sCD30 was similar in both active disease and remission, although sCD30 trended higher during acute hemolysis. The source of sCD30 in AIHA was first evaluated by FACS analysis of peripheral lymphocytes, which was negative. A small subset of patients had glycophorin A+, CD30+ red blood cells, but it did not correlate with sCD30 levels. However, RBC lysates from patients with AIHA contained significant levels of CD30 by ELISA in 5 of 5 patients (10.7–170.4 u/ml) with AIHA versus 0 of 4 patients with ITP (0–9.0). Discussion . High sCD30 levels in AIHA is a novel finding, and it is of interest to find that sCD30 is not elevated in ITP, a disorder once thought to be similar in pathophysiology. The possible explanations are: 1) AIHA may reflect a highly polarized Th2 disease whereas ITP is a more Th1-mediated disorder 2) hemolyzed RBC’s in AIHA release CD30 acquired by absorption or pinocytosis or 3) CD30 is produced and stored by RBC precursors. The negative findings of sCD30 in other hemolytic diseases has yet to be explained. Further delineation of the role of CD30 in hematologic disorders will provide new light on its elusive function. The new finding of high CD30 in AIHA and intracellular CD30 in RBC’s offers a new avenue of research on CD30 in immune-mediated hemolysis.

Background: Atrial fibrillation (AF) is associated with thromboemboli, which is due to blood stasis in the left atrium and hypercoagulability. Several studies have shown that both platelet and leukocyte activation are increased in AF. Formation of platelet-leukocyte conjugates (PLC) is an important mechanism by which leukocytes contribute to thrombosis. Increased levels of P selectin expression in platelets and platelet-leukocyte conjugates (PLC) have been correlated with the risk of cerebral infarction in AF (Circulation. 1998; 98:1721–7). The mechanisms driving the formation of PLC in AF is unclear. We investigated the levels of PLC, platelet expression of P selectin, and leukocyte activation in patients with non-valvular AF. Methods. Patient population: Fourteen patients with chronic AF (8 men and 6 women; mean age = 62±9 years) were studied. AF was documented by a 12-lead electrocardiogram and all patients underwent an echocardiogram to assess cardiac function and rule out valvular AF. The mean left ventricular ejection fraction was 49±16%. Comorbidities included hypertension (85%), diabetes mellitus (21%), coronary artery disease (35%) and congestive heart failure (28%). The mean heart rate was 80±13 bpm. The mean BP was 127±9 / 75±6 mmHg. All patients were anticoagulated with warfarin and 78% were taking aspirin. Since we have previously shown that digitalis use is associated with platelet activation in this population, patients taking digitalis were excluded. Laboratory methods: Venous blood from patients with AF and from healthy volunteers was examined by flow cytometry using fluorescent mAbs. We measured platelet expression of P selectin and leukocyte expression of CD11b. Coexpression of CD41 and CD45 was used to measure PLC. Results: Patients with AF had markedly increased levels of P selectin (22.1 vs 5.0 fluorescence units; p=0.008), PLC (61.2±13.6% vs 43.1±21.6%; p=0.001) and CD11b expression in leukocytes (16.4 vs. 7.9 fluorescence units; p=0.0003). PLC strongly correlated with P selectin expression (R= 0.80; R2=0.64; p=0.0006) but not with leukocyte activation as measured by CD11b expression (R=0.41; R2= 0.17; p=0.14). Conclusion: Our findings indicate that (1) Patients with AF demonstrate increased levels of platelet and leukocyte activation; (2) Increased PLC formation occurs in AF; (3) Levels of PLC closely correlate with platelet activation but not with leukocyte activation, suggesting than PLC formation is driven by platelet activation rather than by leukocyte activation in this population. Aspirin has been shown to be only marginally effective to prevent thromboembolism in AF. Clopidogrel, but not aspirin, has been shown to decrease the formation of PLC (J Am Coll Cardiol2004;43(11):1982–8). Our data suggests that aspirin use in AF patients would not be anticipated to normalize platelet activation and PLC formation in AF patients, but other therapies aimed at decreasing PLC formation might decrease the thrombotic risk in AF.

BACKGROUND . EMP have emerged as sensitive biomarkers of endothelial dysfunction. Little is known of their function. EMP interact with leukocytes and platelets and exert prothrombotic and proinflammatory effects. Recently we reported that vWf is expressed in EMP. The present study extends this observation to characterize EMP-bound vWf (EMP/vWf), specifically their multimer size and proaggregatory activity in ristocetin-induced platelet aggregation. METHODS . EMP, released from human renal microvascular endothelial cells 24hr following stimulation with TNF-α, were isolated by centrifugation (10,000g x 30min) and washed twice for vWf multimer analysis and proaggregatory activity assay. vWf multimer size was analyzed by 0.8% argarose gel electrophoresis and Western blotting. The proaggregatory activity of vWf was assessed flow ctyometrically by a two-step method: first, washed platelets were incubated with either EMP or soluble vWf such as plasma or Humate P (a commercial vWf multimers) in the presence of ristocetin for 10 min, then disappearance of free platelets as they formed small aggregates was measured. Second, the formed platelet aggregates were diluted 20-fold with buffer and degree of disaggregation was determined at intervals to assess the stability of PltAg. RESULTS . (i) Electrophoresis showed that vWf bands from normal plasma do not exceed 1,000 kDa and Humate P about 2,000 kDa while vWf bands on EMP extends much higher than 2,000 kDa. The protein banding pattern of EMP/vWf also differed from free soluble vWf (plasma) or humate P, the latter both showing clear ladder-like banding while EMP/vWf often appeared as a continuous smear of sizes in the high molecular weight regions, probably due to covalent bonds to transmembrane structures. (ii) Incubation of EMP (1 x 107 in 100 μL) with humate P (0.3 U) for 2hr (rotary shaker, 100 rpm) followed by washing the EMP revealed that significant additional vWf was adsorbed to the EMP: the vWf in the supernatant was decreased by 30% – 40%, and Western blotting showed additional high MW band on the treated EMP. This demonstrates that EMP express unoccupied sites capable of binding free vWf. (iii) Incubating EMP/vWf with washed platelets in the presence of ristocetin resulted in formation of platelet aggregates (PltAg). When PltAg were diluted 1:20, the PltAg began dissociating into free platelets in a time-dependent manner. The time for 50% dissociation of PltAg was 15–20min with soluble vWf (plasma or Humate P), but was prolonged to 45–60min with EMP/vWf. We attribute this mainly to the presence of ULvWf on EMP. (iv) Plasma from 4 acute TTP patients showed elevated EMP and enhanced formation of PltAg resistant to disaggregate. While 5 vWD plasma showed the opposite effect and their deficiency was corrected by added EMP. CONCLUSIONS . A new role of EMP is indicated in the present study. EMP-bound vWf is functionally active, inducing platelet aggregates of greater stability than free vWf in a ristocetin-based assay. EMP are capable of adsorbing additional vWf from plasma, indicating presence of extra binding sites. EMP enhance the stability of aggregates probably through ULvWf on the EMP, or to additional platelet-EMP-platelet adhesion molecules, or both. Through their high-affinity interaction with ULvWf, EMP likely play a role in vWf-dependent thrombotic disorders such as TTP and other platelet mediated thrombosis.

Background: The association between antiphospholipids antibodies (APLA) and thrombosis is controversial. Positive APLA are frequently noted in patients without thrombosis. Many mechanisms have been proposed to explain how APLA might induce thrombosis. A leading hypothesis is that platelet activation is central to thrombosis in APLA positive patients, but endothelial injury is also widely proposed. We aimed to evaluate these two theories by measuring endothelial microparticles (EMP) and platelet microparticles (PMP), which are sensitive markers of endothelial and platelet activation respectively, in patients with positive APLA, with and without thrombosis to determine whether these biomarkers correlate with thombotic risk in this population. Study Subjects and Methods: We studied 80 patients referred to our clinic having positive APLA. Positive IgM or IgG against cardiolipin or β2 glycoprotein I were confirmed at least twice by ELISA. Sixty of them had a history of obstetric complications or thrombosis, including TIA, stroke, myocardial infarction, deep venous thrombosis or pulmonary embolism. The remaining 20 patients had positive APLA without history of thrombosis. We excluded all patients who had a thrombotic event within 90 days prior to obtaining the sample to avoid the possible effect of acute thrombus formation on EMP and PMP. We measured EMP, PMP, and P-selectin expression by flow cytometry. PMP were defined as CD31+/CD42+ particles and EMP were defined as CD62E+ particles Results: Levels of EMP were significantly higher in patients with thrombosis (681 counts/μL) compared to normal controls (195 counts/μL; p Conclusion: EMP are elevated in patients with positive APLA regardless of whether they had thrombosis or not. However, only APLA positive patients with thrombosis had elevated PMP. These results suggest that endothelial injury is widely present in APLA positive patients, but that risk of thrombosis depends mainly on platelet activation