Lisocabtagene Maraleucel in Relapsed/Refractory Mantle Cell Lymphoma: Primary Analysis of the Mantle Cell Lymphoma Cohort From TRANSCEND NHL 001, a Phase I Multicenter Seamless Design Study.

PURPOSE: To report the primary analysis results from the mantle cell lymphoma (MCL) cohort of the phase I seamless design TRANSCEND NHL 001 (ClinicalTrials.gov identifier: NCT02631044) study. METHODS: Patients with relapsed/refractory (R/R) MCL after ≥two lines of previous therapy, including a Bruton tyrosine kinase inhibitor (BTKi), an alkylating agent, and a CD20-targeted agent, received lisocabtagene maraleucel (liso-cel) at a target dose level (DL) of 50 × 106 (DL1) or 100 × 106 (DL2) chimeric antigen receptor-positive T cells. Primary end points were adverse events (AEs), dose-limiting toxicities, and objective response rate (ORR) by independent review committee per Lugano criteria. RESULTS: Of 104 leukapheresed patients, liso-cel was infused into 88. Median (range) number of previous lines of therapy was three (1-11) with 30% receiving ≥five previous lines of therapy, 73% of patients were age 65 years and older, 69% had refractory disease, 53% had BTKi refractory disease, 23% had TP53 mutation, and 8% had secondary CNS lymphoma. Median (range) on-study follow-up was 16.1 months (0.4-60.5). In the efficacy set (n = 83; DL1 + DL2), ORR was 83.1% (95% CI, 73.3 to 90.5) and complete response (CR) rate was 72.3% (95% CI, 61.4 to 81.6). Median duration of response was 15.7 months (95% CI, 6.2 to 24.0) and progression-free survival was 15.3 months (95% CI, 6.6 to 24.9). Most common grade ≥3 treatment-emergent AEs were neutropenia (56%), anemia (37.5%), and thrombocytopenia (25%). Cytokine release syndrome (CRS) was reported in 61% of patients (grade 3/4, 1%; grade 5, 0), neurologic events (NEs) in 31% (grade 3/4, 9%; grade 5, 0), grade ≥3 infections in 15%, and prolonged cytopenia in 40%. CONCLUSION: Liso-cel demonstrated high CR rate and deep, durable responses with low incidence of grade ≥3 CRS, NE, and infections in patients with heavily pretreated R/R MCL, including those with high-risk, aggressive disease.

Circulating microparticles in patients with antiphospholipid antibodies: characterization and associations.

The antiphospholipid syndrome is characterized by venous or arterial thrombosis and/or recurrent fetal loss in the presence of circulating antiphospholipid antibodies. These antibodies cause activation of endothelial and other cell types leading to the release of microparticles with procoagulant and pro-inflammatory properties. The aims of this study were to characterize the levels of endothelial cell, monocyte or platelet derived, and tissue factor-bearing microparticles in patients with antiphospholipid antibodies, to determine the association of circulating microparticles with anticardiolipin and anti-ß2-glycoprotein antibodies, and to define the cellular origin of microparticles that express tissue factor. Microparticle content within citrated blood from 47 patients with antiphospholipid antibodies and 144 healthy controls was analyzed within 2hours of venipuncture. Levels of Annexin-V, CD105 and CD144 (endothelial derived), CD41 (platelet derived) and tissue factor positive microparticles were significantly higher in patients than controls. Though levels of CD14 (monocyte-derived) microparticles in patient plasma were not significantly increased, increased levels of CD14 and tissue factor positive microparticles were observed in patients. Levels of microparticles that stained for CD105 and CD144 showed a positive correlation with IgG (R=0.60, p=0.006) and IgM anti-beta2-glycoprotein I antibodies (R=0.58, p=0.006). The elevation of endothelial and platelet derived microparticles in patients with antiphospholipid antibodies and their correlation with anti-ß2-glycoprotein I antibodies suggests a chronic state of vascular cell activation in these individuals and an important role for ß2-glycoprotein I in development of the pro-thrombotic state associated with antiphospholipid antibodies.

Platelet CD36 mediates interactions with endothelial cell-derived microparticles and contributes to thrombosis in mice.

CD36 is a scavenger receptor that binds multiple ligands, including phosphatidyl serine (PS). Although CD36(-) mice do not have a bleeding diathesis, we show here that they do have significantly prolonged thrombotic occlusion times in response to FeCl(3)-induced vascular injury. Because cell-derived microparticles (MPs) are generated in response to vascular injury and circulate in patients with prothrombotic diseases, we hypothesized that PS exposed on their surfaces could be an endogenous CD36 ligand that transmits an activating signal to platelets. We found that MPs prepared from human ECs, monocytes, or platelets or isolated from blood of normal subjects bound to platelets. Binding was not observed with platelets from CD36(-) donors and was inhibited by an anti-CD36 antibody or by blockade of exposed PS by annexin V or anti-PS IgM. Preincubation of platelets with MPs led to CD36-dependent augmentation of platelet activation in response to low doses of ADP, as assessed by measuring alpha(2b)beta(3) activation, P-selectin expression, and aggregation. Immunofluorescence confocal microscopy of murine carotid thrombi from CD36(-) mice showed a significant decrement in endothelial antigen accumulation, which suggests that CD36 plays a role in MP recruitment into thrombi. These results provide what we believe to be a novel role for CD36 in thrombosis.

The inhibition of tube formation in a collagen-fibrinogen, three-dimensional gel by cleaved kininogen (HKa) and HK domain 5 (D5) is dependent on Src family kinases.

Cleaved high molecular weight kininogen (HKa), as well as its domain 5 (D5), inhibits migration and proliferation induced by angiogenic factors and induces apoptosis in vitro. To study its effect on tube formation we utilized a collagen-fibrinogen, three-dimensional gel, an in vitro model of angiogenesis. HKa, GST-D5 and D5 had a similar inhibitory effect of tube length by 90+/-4.5%, 86+/-5.5% and 77+/-12.9%, respectively. D5-derived synthetic peptides: G440-H455 H475-H485 and G486-K502 inhibited tube length by 51+/-3.7%, 54+/-3.8% and 77+/-1.7%, respectively. By a comparison of its inhibitory potency and its sequences, a functional sequence of HKa was defined to G486-G496. PP2, a Src family kinase inhibitor, prevented tube formation in a dose-dependent manner (100-400 nM), but PP3 at 5 microM, an inactive analogue of PP2, did not. HKa and D5 inhibited Src 416 phosphorylation by 62+/-12.3% and 83+/-6.1%, respectively. The C-terminal Src kinase (Csk) inhibits Src kinase activity. Using a siRNA to Csk, expression of Csk was down-regulated by 86+/-7.0%, which significantly increased tube length by 27+/-5.8%. The addition of HKa and D5 completely blocked this effect. We further showed that HKa inhibited Src family kinase activity by disrupting the complex of uPAR, alphavbeta3 integrin and Src. Our results indicate that the anti-angiogenic effect of HKa and D5 is mediated at least in part through Src family kinases and identify a potential novel target for therapeutic inhibition of neovascularization in cancer and inflammatory arthritis.

High-molecular-weight kininogen fragments stimulate the secretion of cytokines and chemokines through uPAR, Mac-1, and gC1qR in monocytes.

OBJECTIVE: Plasma high-molecular-weight kininogen (HK) is cleaved in inflammatory diseases by kallikrein to HKa with release of bradykinin (BK). We postulated a direct link between HKa and cytokine/chemokine release. METHODS AND RESULTS: HKa, but not BK, releases cytokines tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and chemokines IL-8 and MCP-1 from isolated human mononuclear cells. At a concentration of 600 nM, glutathione-S-transferase (GST) fusion proteins of kininogen domain 3 (D3), a fragment of domain 3, E7P (aaG255-Q292), HK domain 5 (D5), the D5 recombinant peptides HG (aa K420-D474) and HGK (aa H475-S626) stimulated secretion of IL-1beta from mononuclear cells. Monoclonal antibodies (MAbs) specific for D5 or specific for D3 blocked release of IL-1beta by HKa, supporting the importance of both domains. Antibodies to HK receptors on leukocytes including Mac-1, LFA-1, uPAR, and C1qR inhibited IL-1beta secretion induced by tKa 98%, 89%, 85%, and 62%, respectively. Fractionation of mononuclear cells identified the responsible cell, a blood monocyte. Inhibitors of signaling pathways NFkB, JNK, and p38 but not extracellular signal-regulated kinase (ERK) decreased cytokine release from mononuclear cells. HKa increased the synthesis of IL-1beta as deduced by an increase of IL-1beta mRNA at 1 to 2 hours. CONCLUSIONS: HKa domains 3 and 5 may contribute to the pathogenesis of inflammatory diseases by releasing IL-1beta from human monocytes using intracellular signaling pathways initiated by uPAR, beta2 integrins and gC1qR.

Frequency of antiphospholipid antibodies in patients with infectious diseases using three different ELISA methods

OBJETIVO: O ensaio de enzyme-linked immunosorbent assay (ELISA) para a pesquisa de anticorpos anticardiolipina (aCL) é o mais importante teste para o diagnóstico da síndrome antifosfolipídica (SAF). Entretanto esse teste também pode ser positivo em algumas doenças infecciosas. Tem sido sugerido que a detecção de anticorpos para uma mistura de fosfolípides ou para b2-glicoproteína I (b2-GP I) teria uma maior especificidade para a SAF que o teste de ELISA-padrão para aCL. O objetivo do presente estudo é comparar a especificidade de três testes para anticorpos antifosfolípides (aFL) em pacientes com doenças infecciosas. MÉTODOS: Anticorpos antifosfolípides foram pesquisados por três técnicas de ELISA, ou seja, o teste-padrão para aCL, o kit de ELISA APhL® e o teste para anti-b2-GP I em pacientes com doenças infecciosas, tais como sífilis (69), leptospirose (33) e Calazar (30). RESULTADOS: A freqüência de positividade de aFL da classe IgG em pacientes com sífilis, leptospirose e Calazar foi de 13/69 (19 por cento), 9/33 (27 por cento) e 2/30 (6 por cento), respectivamente, com o ELISA-padrão para aCL versus 1/69 (1,4 por cento), 0/33 (0 por cento) e 0/30 (0 por cento) com o kit de ELISA APhL®. A positividade do isotipo IgM foi de 10/69 (14 por cento), 4/33 (12 por cento) e 1/30 (3 por cento), respectivamente, com o ELISA-padrão para aCL, e 1/69 (1,4 por cento), 0/33 (0 por cento) e 0/30 (0 por cento) com o kit de ELISA APhL®. Anticorpos da classe IgG contra b2GPI foram detectados em 14/69 casos de sífilis (20 por cento), 6/33 casos de leptospirose (18 por cento) e 16/30 casos de Calazar (53 por cento). Assim, o kit de ELISA APhL® apresentou uma maior especificidade: 97 por cento (95 por cento CI: 92 por cento-99 por cento) comparado com 81 por cento (95 por cento CI: 74 por cento-87 por cento) para o teste de aCL-padrão e 72 por cento (95 por cento CI: 64 por cento-79 por cento) para o teste de anticorpos anti-b2 GPI. CONCLUSÕES: O kit de ELISA APhL® parece ser m...

Multiple myeloma in a murine syngeneic model:modulation of growth and angiogenesis by a monoclonal antibody to kininogen.

Multiple myeloma (MM), a B-cell malignancy characterized by proliferation of monoclonal plasma cells remains incurable. Murine plasma cell tumors share common features with human MM. We used two cell lines (B38 and C11C1) derived from P3X63Ag8 myeloma cells. The new cell lines were implanted subcutaneously in the strain of origin (Balb/c mice) and used as a model to monitor the effects of C11C1 monoclonal antibody (mAb) to kininogen (HK). We assessed their behavior by intraperitoneal and subcutaneous implantation, by implanting them together and by treating B38-MM with purified mAb C11C1. We evaluated growth, microvascular density (MVD), and cellular expression of urokinase-type plasminogen activator-receptor (uPAR), fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), bradykinin-1 receptor (B1R), bradykinin-2 receptor (B2R) and HK. We found that both MM-cell-lines are uPAR positive, that mAb C11C1 inhibits its own tumor growth in vivo, slows down B38-MM growth rate when both MM are implanted together and when mAb C11C1 is injected intraperitoneally. MAb C11C1-treated-MM showed decreased MVD and HK binding in vivo without FGF-2, B1R or B2R expression changes. We propose that the B38-extramedullary-myeloma-model is a useful tool to study the interactions of this hematopoietic tumor and its environment and that mAb C11C1 may improve the efficacy of conventional MM treatment with minimal side effects.

Requirement of activation of complement C3 and C5 for antiphospholipid antibody-mediated thrombophilia.

OBJECTIVE: Antiphospholipid antibodies (aPL) have been shown to induce thrombosis, activate endothelial cells, and induce fetal loss. The pathogenesis of aPL-induced thrombosis, although not completely understood, may involve platelet and endothelial cell activation as well as procoagulant effects of aPL directly on clotting pathway components. Recent studies have shown that uncontrolled complement activation leads to fetal death in aPL-treated mice. In this study, we tested the hypothesis that aPL are responsible for activation of complement, thus generating split products that induce thrombosis. METHODS: To study thrombus dynamics and adhesion of leukocytes we used in vivo murine models of thrombosis and microcirculation, in which injections of aPL were used. RESULTS: Mice deficient in complement components C3 and C5 were resistant to the enhanced thrombosis and endothelial cell activation that was induced by aPL. Furthermore, inhibition of C5 activation using anti-C5 monoclonal antibodies prevented thrombophilia induced by aPL. CONCLUSION: These data show that complement activation mediates 2 important effectors of aPL, induction of thrombosis and activation of endothelial cells.

A monoclonal antibody to high-molecular weight kininogen is therapeutic in a rodent model of reactive arthritis.

We reported that high-molecular weight kininogen is proangiogenic by releasing bradykinin and that a monoclonal antibody to high-molecular weight kininogen, C11C1, blocked its binding to endothelial cells. We now test if this antibody can prevent arthritis and systemic inflammation in a Lewis rat model. We studied 32 animals for 16 days. Group I (negative control) received saline intraperitoneally. Group II (disease-treated) received peptidoglycan-polysaccharide simultaneously with C11C1. Group III (disease-untreated) received peptidoglycan-polysaccharide simultaneously with isotype-matched mouse IgG. Group IV (disease-free-treated) and group V (disease-free isotype-treated) received saline and C11C1 or mouse IgG. Analysis of joint diameter changes showed a decrease in the C11C1 disease-treated group compared to the disease-untreated group. The hind paw inflammatory score showed a decrease in the intensity and extent of inflammation between the disease-untreated and the C11C1 disease-treated group. Prekallikrein, high-molecular weight kininogen, factor XI, and factor XII were decreased in the disease-untreated group compared to the C11C1 disease-treated group. T-kininogen was increased in the disease-untreated group when compared with the C11C1 disease-treated group. Disease-free groups IV and V did not show any sign of inflammation at any time. This study shows that monoclonal antibody C11C1 attenuates plasma kallikrein-kinin system activation, local and systemic inflammation, indicating therapeutic potential in reactive arthritis.

A peptide that shares similarity with bacterial antigens reverses thrombogenic properties of antiphospholipid antibodies in vivo.

OBJECTIVE: The factors causing production of antiphospholipid (aPL) antibodies remain unidentified. Recently, studies have shown that aPL and anti-beta2Glycoprotein I (anti-beta2GPI) antibodies with pathogenic properties can be generated with peptides from bacterial and viral origin, that mimic regions of beta2GPI. These data suggest a molecular mimicry between bacterial/viral antigens and self-proteins. In this study we examined the ability of a synthetic peptide (named peptide A, NTLKTPRVGGC) that shares similarity with common bacterial antigens, to reverse aPL-mediated thrombosis in mice in vivo. Peptide A is also found in region I/II of beta2GPI. A scrambled form of peptide A (named scA, GTKGCPNVRLT) was used as a control. METHODS AND RESULTS: Sera from 29 patients with APS bound to peptide A but not to peptide scA by ELISA in a dose-dependent fashion. Cardiolipin (CL) liposomes inhibited the binding of IgG-APS by ELISA to peptide A by 35% and to CL by 56%. The inhibition of binding to cardiolipin and to peptide A was enhanced by addition of beta2GPI to the liposomes. CL/peptide A liposomes but not peptide A alone inhibited the binding of IgG-APS to peptide A. beta2GPI alone did not inhibit binding of IgG-APS to peptide A, to beta2GPI or to CL. For the in vivo experiments, CD1 mice in groups of 20 were injected with affinity purified aPL antibodies or with control IgG-NHS twice intraperitoneally. Seventy hours after the first injection, and 30 min before the surgical procedure (induction of experimental thrombus) mice were infused i.v. in each group with either peptide A or with peptide scA. The femoral vein of the anesthetized mice were dissected to examine the dynamics of an induced thrombus in treated and control mice. The mean aCL titer of mice injected with aPL was 60 GPL units. Mice treated with aPL and infused with peptide scA produced significantly larger thrombi when compared to mice treated with IgG-NHS and peptide scA (2466+/-462 microm2 vs 772.5+/-626.4 microm2). Treatment with peptide A significantly decreased thrombus size in mice injected with aPL antibodies (1063+/-890 microm2 compared to 2466+/-462 microm2). CONCLUSION: The data indicates that a synthetic peptide that shares similarity with common bacterial antigens and with regions of beta2GPI is capable to inhibit thrombogenic properties of aPL in mice. This may have important implications in designing new modalities of prevention and/or treatment of thrombosis in APS.

Diagnostic performance of anti-beta2 glycoprotein I and anticardiolipin assays for clinical manifestations of the antiphospholipid syndrome.

The objective of the present study was to analyse the performance of the tests for detection of anti-beta2 glycoprotein I (beta2 GP I) and anticardiolipin (aCL) antibodies for identification of clinical manifestations of the antiphospholipid syndrome (APS). Patients with systemic lupus erythematosus (SLE) as well as carriers of infectious diseases such as Kala-azar, syphilis and leptospirosis were studied. Particular interest was given to the presence of clinical complications related to APS. Anticardiolipin and anti-beta2 GP I antibodies were searched using an enzyme-linked immunosorbent assay (ELISA) assay. Clinical manifestations of APS were observed in 34 of the 152 patients (22.3%) with SLE and no patient with infectious disease had such manifestations. Antibodies to cardiolipin in moderate or high levels and anti-beta2 GP I were detected in 55 of 152 (36.1%) and 36 of 152 (23.6%) patients with SLE, respectively, and in 2 of 30 (6.6%) and 16 of 30 (53.3%) patients with Kala-azar, in 9 of 39 (23%) and 6 of 34 (17.6%) patients with leptospirosis, and 14 of 74 (18.9%) and 8 of 70 (11.4%) cases of syphilis, respectively. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and likelihood ratio (LR) of the anti-beta2 GP I test for the identification of the clinical manifestation of APS were, respectively, 29% [95% confidence interval (CI) = 24%-34%], 78% (95% CI = 73-83%), 15% (95% CI = 11-19%), 89% (95% CI = 85-93%) and 1.38. Regarding the aCL assay, the figure was 29% (95% CI = 24-34%), 76% (95% CI = 71-81%), 14% (95% CI = 10-18%), 89% (95% CI = 86-92%) and 1.26. As the validity and performance of the anti-beta2 GP I assay were similar to the aCL in demonstrating the presence of clinical phenomena associated with APS and due to the difficulties in performing as well as the lack of standardisation of the anti-beta2 GP I test, we suggest that the test for aCL should continue to be the first one performed when the presence of APS is suspected.

Inhibition of the thrombogenic and inflammatory properties of antiphospholipid antibodies by fluvastatin in an in vivo animal model.

OBJECTIVE: Antiphospholipid antibodies (aPL) have thrombogenic properties in vivo, through their interactions with soluble coagulation factors and their ability to modulate the functions of cells involved in coagulation homeostasis. These antibodies have also been shown to enhance the adhesion of leukocytes to endothelial cells (ECs) in vivo. New lipophilic statins such as fluvastatin have antiinflammatory and antithrombogenic effects. This study uses an in vivo mouse model to investigate whether fluvastatin has an effect on decreasing both the adhesion of leukocytes to ECs and the thrombus formation induced by aPL. METHODS: Two groups of CD-1 male mice, each comprising approximately 18 mice, were fed either normal saline solution or 15 mg/kg fluvastatin for 15 days. Each of the 2 groups was further subdivided to receive either purified IgG from patients with the antiphospholipid syndrome (IgG-APS) or normal IgG from healthy subjects. Analysis of thrombus dynamics was performed in treated and control mice, using a standardized thrombogenic injury procedure, and the area (size) of the thrombus was measured. Adhesion of leukocytes to ECs was analyzed with a microcirculation model of exposed cremaster muscle. Baseline and posttreatment soluble intercellular adhesion molecule 1 (sICAM-1) levels were determined by enzyme-linked immunosorbent assay. RESULTS: IgG-APS mice treated with fluvastatin showed significantly smaller thrombi, a reduced number of adherent leukocytes, and decreased levels of sICAM-1 compared with IgG-APS animals treated with placebo. CONCLUSION: These findings indicate that fluvastatin significantly diminishes aPL-mediated thrombosis and EC activation in vivo. These results may have important implications for the design of new treatment strategies aimed at preventing recurrent thrombosis in patients with APS.

Prevalence of antiphospholipid and antiplatelet antibodies in human immunodeficiency virus (HIV)-infected Chilean patients.

Antiphospholipid (aPL) and antiplatelet (aPlt) antibodies, found in patients with autoimmune diseases, are also detected in infectious diseases. The purpose of this study was to examine the prevalence of these antibodies in HIV patients and to evaluate an association of these antibodies with thrombocytopenia and/or thrombosis. Sixty-three HIV-seropositive patients and 52 normal controls were studied. Anti-cardiolipin (aCL), anti-beta(2) glycoprotein I (anti-beta(2)GPI), and antiprothrombin (aPT) antibodies were determined and the lupus anticoagulant (LA) test was performed. Antiplatelet antibodies (aPlt) were also determined. Seven out of 63 (12.7%) HIV patients were positive for aCL, four of 63 (6.3%) for anti-beta(2)GPI, and five of 63 (7.9%) for aPT. No patients studied were LA positive. Six out of 63 (9.5%) patients were positive for aPlt. One of them showed weak reactivity for GPIb-IX. The platelet count of patients (202+/-63 x 10(3) platelets/microL) was significantly lower than in the controls (343+/-6 x 10(3) platelets/microL) (P<0.001). There was no correlation between the presence of aPL and/or aPlt and thrombocytopenia. Of the HIV-infected patients, 22.2% presented aPL and 9.4% aPlt antibodies. In this study, the presence of aPL and aPlt antibodies was not associated with the development of thrombosis and/or thrombocytopenia.

Antiphospholipid antibodies in Chilean patients with systemic lupus erythematosus.

Antiphospholipid antibodies (aPLs) are a heterogeneous family of antibodies found in autoimmune disorders, infectious diseases, and other situations. The presence of different aPLs has been associated with various clinical manifestations of the antiphospholipid syndrome (APS). The objective of this study was to investigate the prevalence of aPLs in a group of 90 Chilean patients with systemic lupus erytematosus (SLE) and 90 healthy controls. We measured anticardiolipin antibodies (aCLs), antiphosphatidylserine antibodies (aPSs), anti-beta(2) glycoprotein I antibodies (anti-beta(2)GPIs), and antiprothrombin antibodies (aPTs) with an enzyme-linked immunosorbent technique using "in-house" assays. Fifty-four of 90 SLE patients (60.0%) had some type of aPL. Forty of 90 (44.4%) were positive for aCLs, 9 of 61 (14.8%) had aPSs, 21 of 90 (23.3%) had anti-beta(2)GPIs, and 18 of 90 (20.0%) had aPTs. In the control group, prevalences were as follows: aCLs, 3.3%; aPSs, 1.1%; anti-beta(2)GPIs, 1.1%; aPTs, 2.2%. In most cases, values were in the low-positive range. Of all aPL detected, 29.5% was of the IgG isotype, 37.5% IgM, and 33.0% IgA. We observed a correlation between aCLs and aPSs and of these antibodies with anti-beta(2)GPIs and aPTs but not between anti-beta(2)GPIs and aPTs. Our results show a high prevalence of aPLs in SLE patients. An association between different specificities and isotypes of aPLs was also observed.

Hydroxychloroquine reverses platelet activation induced by human IgG antiphospholipid antibodies.

Prothrombotic properties of antiphospholipid (aPL) antibodies may be explained in part by their ability to enhance the activation of platelets pre-treated with low doses of ADP or thrombin. The antimalarial drug hydroxychloroquine (HQ) has been used successfully in prevention of postoperative thrombosis and in treatment of patients with SLE or APS. In one study, administration of HQ reversed the thrombogenic properties of aPL in mice. However, the mechanism of action of HQ in preventing thrombosis is not clearly understood. In order to explore this further, the effects of HQ on activation of platelets by aPL in the presence of a thrombin agonist was studied. The changes in the expression of GPIIb/IIIa (CD41a) and GPIIIa (CD61) on platelet membrane by flow cytometry were used as indicators of platelet activation. Citrated whole blood from a healthy donor was treated at room temperature with suboptimal doses of a thrombin agonist receptor peptide (TRAP) and affinity-purified aPL antibodies, in the presence and in the absence of hydroxychloroquine (1 mM). TRAP increased the expression of GPIIb/IIIa and GPIIIa on platelet surface. The treatment of the platelets with the six aPL antibodies in the presence of 12 nMol/ml TRAP further increased the expression of GPIIb/IIIa by 42.3+/-12.3% and the expression of GPIIIa was further incremented by 46.8+/-13.5%. The effects of aPL and TRAP on expression of platelet surface markers of activation was completely abrogated by HQ in a dose-dependent fashion and was effective at concentrations of HQ as low as 25 microg/ml (0.0125 mM). This suggests at least one possible mechanism by which HQ may prevent thrombosis. This may have important implications in treatment of thrombosis in APS patients.

Antiphospholipid antibodies induced in mice by immunization with a cytomegalovirus-derived peptide cause thrombosis and activation of endothelial cells in vivo.

OBJECTIVE: To characterize the binding and functional properties of antiphospholipid antibodies (aPL) induced by immunization with a viral peptide and to determine whether aPL are pathogenic in vivo. METHODS: Ten murine monoclonal aPL were generated from spleen cells of PL/J mice immunized with TIFI, a phospholipid-binding peptide spanning Thr(101)-Thr(120) of ULB0-HCMVA from human cytomegalovirus (CMV), which shares structural similarity with the phospholipid-binding site of beta(2)-glycoprotein I (beta(2)GPI). RESULTS: The antibodies generated had aPL activity that was inhibited by cardiolipin liposomes, and this inhibition was enhanced in the presence of beta(2)GPI. Some of the antibodies exhibited binding to cultured endothelial cells in vitro, and some had lupus anticoagulant activity. Injection with 2 of the monoclonal aPL in mice resulted in a significant increase in the number of leukocytes adhering to endothelial cells and enhanced thrombus formation in vivo. CONCLUSION: These results indicate that aPL induced by immunization with a phospholipid-binding CMV peptide are pathogenic in vivo. The results also suggest a mechanism (molecular mimicry) by which pathogenic aPL may be generated in patients with antiphospholipid syndrome.

Complement C3 activation is required for antiphospholipid antibody-induced fetal loss.

The antiphospholipid syndrome (APS) is characterized by recurrent fetal loss, vascular thrombosis, and thrombocytopenia occurring in the presence of antiphospholipid (aPL) antibodies. The pathogenesis of fetal loss and tissue injury in APS is incompletely understood, but is thought to involve platelet and endothelial cell activation as well as procoagulant effects of aPL antibodies acting directly on clotting pathway components. Recent studies have shown that uncontrolled complement activation in the placenta leads to fetal death in utero. We hypothesized that aPL antibodies activate complement in the placenta, generating split products that mediate placental injury and lead to fetal loss and growth retardation. To test this hypothesis, we used a murine model of APS in which pregnant mice are injected with human IgG containing aPL antibodies. We found that inhibition of the complement cascade in vivo, using the C3 convertase inhibitor complement receptor 1-related gene/protein y (Crry)-Ig, blocks fetal loss and growth retardation. Furthermore, mice deficient in complement C3 were resistant to fetal injury induced by aPL antibodies. While antigenic epitopes recognized by aPL antibodies are important in the pathogenesis of APS, our data show that in vivo complement activation is required for aPL antibody-induced fetal loss and growth retardation.