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Uso de Modelos In Vitro e In Vivo en el Diseňo de Nuevas Estrategias Uso de Modelos In Vitro e In Vivo en el Diseňo de Nuevas Estrategias Terapéuticas Dirigidas a Blancos Moleculares En Enfermedades Cardiovasculares. SILVIA S. PIERANGELI, Ph. D. Professor

What is the Antiphospholipid Antibody Syndrome ? u An acquired autoimmune thrombophilia, characterized by: What is the Antiphospholipid Antibody Syndrome ? u An acquired autoimmune thrombophilia, characterized by: a) vascular thrombosis. b) recurrent pregnancy losses. c) thrombocytopenia. d) laboratory evidence for: -antibodies against phospholipids or phospholipid-binding protein cofactors.

APS morbidity u u u u APS is the most common cause of acquired APS morbidity u u u u APS is the most common cause of acquired thrombophilia. Prevalence in general population: 2 -4% 15 -20% of all DVT with or without PE. 1/3 of new strokes in patients < 50 years age. 10 -15% women with recurrent pregnancy losses. APS: significant proportion of thromboembolic disease and pregnancy loss in SLE. APL Abs present in 30 -40% SLE. One third of those patients have clinical manifestations of APS. a. CL positivity may precede a more severe form of SLE.

Multiple Strokes in a Young Woman (Brain MRI) Occlusion of Right Middle Cerebral Artery Multiple Strokes in a Young Woman (Brain MRI) Occlusion of Right Middle Cerebral Artery In a 3 Years Old Child with Severe Headache and Hemiparesis With a. CL Antibodies +

Digital Necrosis and Gangrene Digital Necrosis and Gangrene

Diagnostic Tests u Anticardiolipin u Lupus Test Anticoagulant Test Diagnostic Tests u Anticardiolipin u Lupus Test Anticoagulant Test

ANTI-CARDIOLIPIN TEST u Advantages – Overwhelming majority of APS patients are anti cardiolipin positive ANTI-CARDIOLIPIN TEST u Advantages – Overwhelming majority of APS patients are anti cardiolipin positive – Test can be performed reproducibly. – Clinicians and laboratories generally familiar with units of measurement. u Disadvantages – Relatively nonspecific (particularly low positive, Ig. M positive). – Intra-laboratory and Inter-laboratory variability. – Problems with false positive results: a. CL positive in a wide variety of infectious diseases and in non-APS related autoimmune diseases.

Predictive value of Ig. G a. CL for thrombosis in patients with SLE (Escalante Predictive value of Ig. G a. CL for thrombosis in patients with SLE (Escalante et al) u Ig. G a. CL levels below 21. 4 = probability of thrombosis 0. 07 u Ig. G a. CL levels >21. 4 and < 65. 0 GPL = probability of thrombosis 0. 20 u Ig. G a. CL levels >65. 1 GPL units = probability of thrombosis 0. 75

Anti-ß 2 glycoprotein I u More specific than anticardiolipin test for diagnosis of Antiphospholipid Anti-ß 2 glycoprotein I u More specific than anticardiolipin test for diagnosis of Antiphospholipid Syndrome (but not 100% specific) u Not as sensitive as anticardiolipin test (70 -90% sensitivity) u Efforts of standardization continuing u Useful in diagnosis of doubtful cases of APS. Some APS patients negative for a. CL and positive for anti 2 GPI.

APh. L ELISA - Principle u Based on observation that antiphospholipid antibodies crossreact with APh. L ELISA - Principle u Based on observation that antiphospholipid antibodies crossreact with negatively charged phospholipids but syphilis and other infectious diseases sera largely limited to cardiolipin binding (no cross-reactivity) u Construction of a kit with negatively charged phospholipids might eliminate non-specific binding.

APh. L ELISA kit u Antigen composed fo mixture of phospholipids instead of cardiolipin APh. L ELISA kit u Antigen composed fo mixture of phospholipids instead of cardiolipin u Sensitivity of APS (greater than 90%) u More specific than anticardiolipin test and at least as specific (or more) compared to anti-ß 2 GPI u Incorporation of an in-house positive control u Can be utilized for first line testing, and certainly important in confirmation of APS

Treatment of thrombosis: Current Recommendations Vascular Thrombosis prevention Asymptomatic a. PL No treatment or Treatment of thrombosis: Current Recommendations Vascular Thrombosis prevention Asymptomatic a. PL No treatment or ASA? Venous thrombosis Warfarin INR 2. 0 -3. 0 Arterial thrombosis Warfarin INR 3. 0 Recurrent thrombosis Warfarin INR 3. 0 -4. 0+ASA CAPS Anticoagulation + corticosteroid + IVIG or plasmapheresis Erkan and Lockshin, Rheum Dis Clin North Am 2006; 32: 129 -48 Lim et al. JAMA 2006; 295: 1050 -1057

Erkan study u Aspirin for primary thrombosis prevention in the antiphospholipid syndrome: double-blind, placebo-controlled Erkan study u Aspirin for primary thrombosis prevention in the antiphospholipid syndrome: double-blind, placebo-controlled trial in asymptomatic antiphospholipid antibody-positive individuals. – APLASA: multicenter, randomized, double-blind, placebocontrolled clinical trial – Observational parallel study. u Conclusions: – a. PL-positive individuals do not benefit from low-dose aspirin for primary thrombosis prophylaxis – have low overall annual incidence rate of acute thrombosis and – develop vascular events when additional thrombosis risk factors are present. (Erkan D et al. Arthritis Rheum 2007; 56: 2382 -2391).

Treatment of thrombosis in APS u Oral anticoagulation: problems – Bleeding – Frequent monitoring Treatment of thrombosis in APS u Oral anticoagulation: problems – Bleeding – Frequent monitoring – Patient compliance with medication and diet

Prevention of thrombosis in APS: Current problems u In patients with previous thrombotic event(s): Prevention of thrombosis in APS: Current problems u In patients with previous thrombotic event(s): – Oral anticoagulation at high INR vs. moderate INR ? ? – Most recommendations based on retrospective studies. – (Khamashta et al, NEJM, 1995; Krnic et al, Arch Intern Med, 1997 – Prospetive studies: Crowther et al, NEJM, 2003; Finazzi et al, JTH, 2005) u Patients with a. PL and no thrombosis (low dose aspirin vs. no treatment? )

Unresolved questions u Do patients with stroke require same level of anticoagulation vs. those Unresolved questions u Do patients with stroke require same level of anticoagulation vs. those with DVT only? u Is aspirin or other anti-platelet agents alone, sufficient? u Do we discontinue oral anticoagulation in some patients when an additional risk factor is no longer a problem (i. e. contraceptives)?

There is a need for more safer and efficacious modalities of treatment for thrombosis There is a need for more safer and efficacious modalities of treatment for thrombosis in APS. Understanding the molecular and intracellular events triggered by antiphospholipid antibodies is important in designing new modalities of targeted therapies for treatment of APS.

Do a. PL antibodies cause/induce thrombosis? Do a. PL antibodies cause/induce thrombosis?

Exposed femoral vein and fiber-optic light positioned under the vein in a mouse Pierangeli, Exposed femoral vein and fiber-optic light positioned under the vein in a mouse Pierangeli, S. S. et al. Circulation 1996; 94: 1746 -1751

Photograph taken from the video monitor demonstrating the method for analysis of thrombus size Photograph taken from the video monitor demonstrating the method for analysis of thrombus size Pierangeli, S. S. et al. Circulation 1996; 94: 1746 -1751

Thrombus formation Thrombus formation

METHODS a. PL-Ig. G (500µg) or Control Ig. G 0 hr 48 hr 72 METHODS a. PL-Ig. G (500µg) or Control Ig. G 0 hr 48 hr 72 hr Thrombus Dynamics Analysis

Antiphospholipid Antibodies Promote Clot Formation in Mice Antiphospholipid Antibodies Promote Clot Formation in Mice

Un Visitante Ilustre… Un Visitante Ilustre…

Micropoint laser to induce thrombogenic injury. Micropoint laser to induce thrombogenic injury.

How has the animal model of induced thrombosis helped us in understanding a. PL How has the animal model of induced thrombosis helped us in understanding a. PL pathogenic effects? u Induction of thrombosis in a mouse model by Ig. G, Ig. M and Ig. A immunoglobulins from patients with the Antiphospholipid Syndrome. Pierangeli et al. Thrombosis Haemost. 1995; 74: 1361 -1367. u Generation and characterization of Monoclonal Ig. G Anticardiolipin Antibodies from a Patient with the Antiphospholipid Syndrome. Olee et al. Proc Nat Acad Sci. (USA). 1996; 93: 8606 -8611. u Identification of an Fc- receptor independent mechanism by which intravenous immunoglobulin (IVIG) ameliorates antiphospholipid antibody-induced thrombogenic phenotype. Pierangeli et al. Arthritis Rheum 2001; 44: 876 -883. u Arginine residues are important in determining the binding of human monoclonal antiphospholipid antibodies to clinically relevant antigens. Giles et al. J Immunol 2006; 177: 1729 -1736. u A human monoclonal anti-prothrombin antibody is thrombogenic. in vivo and upregulates expression of tissue factor and E-selectin on endothelial cells. Vega-Ostertag et al. Br. J. Haematology. 2006;

APL antibodies and platelets • APL antibodies bind to platelet membranes. (Khamashta et al. APL antibodies and platelets • APL antibodies bind to platelet membranes. (Khamashta et al. Ann Rheum Dis 1988; 47: 849 -854). • Prothrombotic properties of antiphospholipid (a. PL) antibodies may be explained in part by their ability to enhance the activation of platelets pre-treated with low doses of ADP, thrombin or collagen (Campbell et al. Thromb Haemost 73: 519 -524, 1995). • APL antibodies increase expression of GPIIb/IIIa and GPIIIa on platelets pre-treated with low doses of a thrombin receptor agonist peptide (TRAP) in a dose-dependent fashion. Hydroxychloroquine reverses those effects in vitro (Espinola et al. Thromb Haemost, 2002; 87: 518 -522).

Do APL antibodies affect platelet activation in vivo? • Infusions of anti-GPIIb/IIIa (1 B Do APL antibodies affect platelet activation in vivo? • Infusions of anti-GPIIb/IIIa (1 B 5) antibodies affect a. PLmediated enhanced thrombus formation in a mouse model of thrombosis. • a. PL-antibodies do not enhance thrombosis in ß 3 -null mice. • Hydroxychloroquine diminishes platelet activation and thrombus formation induced by a. PL antibodies. Edwards M et al. Circulation. 1997; 96: 4380 -4384. Thrombogenicity of 2 glycoprotein I-dependent antiphospholipid antibodies in a photochemically induced thrombosis model in the hamster. Jankowski et al. Blood 2003; 101: 157 -162.

Hydroxychloroquine in APS u Yoon KH. Sufficient evidence to consider hydroxychloroquine as an adjunct Hydroxychloroquine in APS u Yoon KH. Sufficient evidence to consider hydroxychloroquine as an adjunct therapy in antiphospholipid (Hughes’) syndrome. J Rheumatol 2002; 29: 1222 -1226. u Wallace DJ. Does hydroxychloroquine protect against clot formation in systemic lupus erythematosus? Arthritis Rheum 1987; 30: 11435 -1436. u Petri M. Hydroxychloroquine use in the Baltimore Lupus Cohort: effects on lipids, glucose and thrombosis. Lupus 1996; 5: S 16 -22. u Mc. Carty GA and Cason TE. Use of hydroxychloroquine in antiphospholipid antibody syndrome at three academic rheumatology units over two years: improvement in antibody titer and symptoms management (abstract). 7 th International Congress on SLE and Related condictions. Abstracts Book, NY 2004. . p. M 17 A.

Question u a. PL antibodies enhance platelet activation in vitro and in vivo. u Question u a. PL antibodies enhance platelet activation in vitro and in vivo. u What are the intracellular pathways involved in a. PL-mediated platelet activation?

Intracellular events mediated by a. PL on platelets. u APL induce platelet activation and Intracellular events mediated by a. PL on platelets. u APL induce platelet activation and thromboxane formation and platelet-derived thromboxane urinary metabolites. (Martinuzzo ME et al. Thromb Haemost 1993; 70: 667671 and Forastiero R et al. Thromb Haemost 1998; 79: 42 -45) u APL/anti- 2 GPI Abs induce production of thromboxane A 2 that is inhibited by cyclic-AMP agonists. Indomethacine and phosphodiesterase inhibitors such as theophylline inhibit TXA 2. (Robbins DL et al. J Rheumatol. 1998; 25: 51 -56 and Opara E et al. 2003; 30: 5559).

Phosphorylation of p 38 MAPK and ERK 1/ERK 2 by a. PL and thrombin Phosphorylation of p 38 MAPK and ERK 1/ERK 2 by a. PL and thrombin 43 PBS +Thrombin 1 U/ml F(ab)’ 2 a. PL 5 F(ab)’ 2 a. PL 4 F(ab)’ 2 a. PL 3 F(ab)’ 2 a. PL 2 Kda F(ab)’ 2 a. PL 1 F(ab)’ 2 Ig. GNHS +Thrombin 0. 005 U/ml p 38 - P A p 38 B ERK 1/ERK 2 -P C 43 43 Vega-Ostertag M et al. Arthritis Rheum 2004; 50: 2911 -2919

Vega-Ostertag M et al. Arthritis Rheum 2004; 50: 2911 -2919 Vega-Ostertag M et al. Arthritis Rheum 2004; 50: 2911 -2919

Vega-Ostertag M et al. Arthritis Rheum 2004; 50: 2911 -2919 Vega-Ostertag M et al. Arthritis Rheum 2004; 50: 2911 -2919

L Anti-GPIIb. IIIa antibody Receptor Antagonist a. P Fibrinogen GPIbβ f so se do L Anti-GPIIb. IIIa antibody Receptor Antagonist a. P Fibrinogen GPIbβ f so se do , ow mbin ADP L o , r Th lagen l co PS, PE EXPOSITION PLC gp IIIa GPIbα GPV gp. IIb GPIX Apo-E 2 R c DG a. PL (+) IP 3 (+) Dimerized PKC Ca 2+ PS PE P 38 (-) Full activation Ca 2+ c. PLA 2 AA TXB 2 TX Rc (-) Hydroxy. Aspirin chloroquine 2 -glycoprotein I SB 203580

Receptor recognized by a. PL on platelets u APO 33838. ER 2’ Lutters BC Receptor recognized by a. PL on platelets u APO 33838. ER 2’ Lutters BC et al. J Biol Chem 2003; 2778: 33831 - u Glycoprotein 2558 -2567. Ib/IX-V. Shi et al. Arthritis Rheum 2006; 54:

Dimers of 2 GPI. u u u In previous studies, a chimeric fusion protein Dimers of 2 GPI. u u u In previous studies, a chimeric fusion protein was constructed by the dimerization domain (apple 4) of ß 2 GPI. As a control the monomeric protein apple 2 - ß 2 GPI – which is not able to form dimer - was constructed. The authors demonstrated that dimeric ß 2 GPI mimics in vitro the effects of ß 2 GPI anti- ß 2 GPI antibodies complexes. [Lutters, et al. JBiol Chemistry 2001; 276: 5 , 3060 -3067]

Effects of B 1 APOER 2’ on thrombus formation and platelet aggregation in vivo Effects of B 1 APOER 2’ on thrombus formation and platelet aggregation in vivo Treatment Thrombus size Platelet aggregation Dimer of 2 GPI 3629 ± 562 μm 2 56. 3 % (n=3) Monomer control 690 ± 50 μm 2 10% (n=3) B 1 D APOER 2’ 23% (n=3) 532 ± 147 μm 2

Antiphospholipid Antibodies and Endothelial Cells Antiphospholipid Antibodies and Endothelial Cells

APL antibodies activate endothelial cells in vitro and in vivo. a. PL or anti- APL antibodies activate endothelial cells in vitro and in vivo. a. PL or anti- 2 GPI antibodies upregulate EC adhesion molecules and this effect is related to a. PL binding to EC (Del Papa N et al Arthritis Rheum 1997; 40: 551 -561. a. PL-induced upregulation of ICAM-1, VCAM-1 and Eselectin on HUVEC and increased adhesion of monocytes to EC in the presence of 2 GPI (Simantov R et al J Clin Invest 1995; 96: 2211 -2219). u Soluble levels of VCAM-1 significantly increased in plasma of patients with APS and recurrent thrombosis (Kaplanski G et al 2000; 43: 55 -64).

Activation of endothelial cells in vivo u Assessed by adhesion of leukocytes in the Activation of endothelial cells in vivo u Assessed by adhesion of leukocytes in the microcirculation of the cremaster muscle. u The number of leukocytes sticking within five different venules is determined. u Adhesion is defined as leukocytes remained stationary for at least 30 seconds. Peter FW, et al. Microsurgery. 1998; 18: 23 -28.

APL antibodies enhance thrombus formation and this correlates with activation of endothelial cells in APL antibodies enhance thrombus formation and this correlates with activation of endothelial cells in vivo Antiphospholipid antibodies from patients with Antiphospholipid Syndrome activate endothelial cells in vitro and in vivo. Pierangeli et al Circulation, 1999; 99: 1997 -2002. GDKV-induced antiphospholipid antibodies enhance thrombosis and activate endothelial cells in vivo. Gharavi et al J Immunol 1999; 163: 2922 -2927. Functional analysis of patient-derived Ig. G monoclonal anticardiolipin antibodies using in vivo thrombosis and in vivo microcirculation Models. Pierangeli et al. Thrombosis Haemost 2000; 84: 388 -395. • Thrombogenic effects of antiphospholipid (a. PL) antibodies are mediated by intercellular cell adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and P-selectin. Pierangeli et al. Circ Res 2001; 88: 245 -250. • E-selectin mediates pathogenic effects of antiphospholipid antibodies. Espinola et al Thromb Haemost. 2003; 1: 843 -848.

a. PL antibodies upregulate tissue factor expression Upregulation of tissue factor may account for a. PL antibodies upregulate tissue factor expression Upregulation of tissue factor may account for arterial and venous thrombosis. Increased expression of TF on monocytes by a. PL. (Dobado-Barrios M et al Thromb Haemost 1999; 82: 1578 -1582) Inhibition of TF upregulation in monocytes by dilazep (Zhou H et al, Blood 2004; 104: 2353 -2358). Increased s. TF and VEGF in plasma of patients with APS (Williams FM et al Thromb Haemost 2000; 84: 742 -746; , Forastiero RR et al J Thromb Haemost 2003; 10: 2250 -2251; Cuadrado MJ et al J Thromb Haemost 2006; 4: 2461 -2469)

Upregulation of TF by a. PL on EC Summary of our results u a. Upregulation of TF by a. PL on EC Summary of our results u a. PL increased TF expression on EC (1. 8 -4. 2 fold increase) u The increase in TF expression was dependent on the dose of antibody utilized. u TF function was increased by a. PL (1. 4 to 3. 8 fold increase) u TF function increase was dependent on the dose of antibody utilized. u TF expression was inhibited by MG 132 (10 -100%) and SB 203580 (50 -100%). u TF function was inhibited by SB 203580 (34 -54%). u a. PL induce upregulation of IL-6 and IL-8. u Vega-Ostertag ME et al Arthritis Rheum 2005; 52: 1545 -1554.

Effects of a. PL on phosphorylation of p 38 MAPK Effects of a. PL on phosphorylation of p 38 MAPK

Effects of a. PL on TF m. RNA expression (RT-PCR) in EC) Effects of a. PL on TF m. RNA expression (RT-PCR) in EC)

Conclusions – a. PL induce phosphorylation of p 38 MAPK – a. PL induce Conclusions – a. PL induce phosphorylation of p 38 MAPK – a. PL induce i. NOS – a. PL induce transcription of TF m. RNA and this effect is inhibited by SB 203580 in a dosedependent fashion. (Vega Ostertag M, et al. Arthritis Rheum, 2005; 52: 5: 1545 -1554)

Involvement of p 38 MAPK in a. PL-mediated effects in platelets, EC and monocytes. Involvement of p 38 MAPK in a. PL-mediated effects in platelets, EC and monocytes. Publication Cell type Vega-Ostertag et al. Arthritis Rheum 2004; 50: 2911 -2919 Lutters BC et al. J Biol Chem 2003; 278: 33831 -33838 Vega-Ostertag ME et al. Arthritis Rheum. 2005; 52: 1545 -1554 Simoncini S et al. Int Immunol 2005; 17: 489 -500. Bohgaki M et al. Int Immunol 2004; 16: 1632 -1641 López-Pedrera C et al. Arthritis Rheum. 2006; 54: 301 -311. Platelets Endothelial cells Monocytes

Experimental Design II Injection schedule Ig. G- APS (500µg) i. p. 0 hr In Experimental Design II Injection schedule Ig. G- APS (500µg) i. p. 0 hr In some experiments, mice were treated i. p. with 25 mg/Kg of SB 203580 or saline 30 min. before the Ig. G injections 48 hr 72 hr Ig. G- NHS(500µg) i. p. 1. Thrombus dynamics analysis 2. Adhesion of WBC to EC in cremaster 3. TF activity in carotid artery homogenates 4. TF activity in mouse peritoneal macrophages 5. ACL activity

THROMBOSIS MODEL IN MICE THROMBOSIS MODEL IN MICE

Effects of a p 38 MAPK inhibitor on a. PLinduced thrombosis in vivo. u Effects of a p 38 MAPK inhibitor on a. PLinduced thrombosis in vivo. u Vega-Ostertag ME et al J Thromb Haemost 2007; 5: 1828 -1834.

Effects of a p 38 MAPK inhibitor on a. PLmediated endothelial cell activation in Effects of a p 38 MAPK inhibitor on a. PLmediated endothelial cell activation in vivo. u Vega-Ostertag ME et al J Thromb Haemost 2007; 5: 1828 -1834.

Determination of TF activity in mouse peritoneal macrophages u Procedure done in the animals Determination of TF activity in mouse peritoneal macrophages u Procedure done in the animals immediately after the surgical procedures and after they were sacrificed. Peritoneal macrophages obtained after lavage of the peritoneal cavity with 5 ml sterile PBS. u Two x 106 peritoneal cells were washed twice with PBS and resuspended in 1 ml of Tris buffer saline-0. 1% Triton X-100 p. H 7. 4 and centrifuged at 14, 000 rpm during 30 minutes. The cells were then washed twice and then resuspended in 50 µl TBS-0. 1% Triton X-100 and sonicated. u The TF activity of peritoneal cells lysates determined using a commercial chromogenic assay (Actichrome TF, American Diagnostica, Stamford, CT) that measures factor Xa after activation by the TF-Factor VII complex. The amount of factor Xa generated is measured by its ability to cleave Spectrozyme Xa, a highly specific chromogenic substrate for factor Xa. u Results expressed in p. M/100 µg tissue.

Effects of a p 38 MAPK inhibitor on a. PL-induced TF activity in mouse Effects of a p 38 MAPK inhibitor on a. PL-induced TF activity in mouse peritoneal macrophages u Vega-Ostertag ME et al J Thromb Haemost 2007; 5: 1828 -1834.

Determination of TF activity in carotid artery homogenates. v Pieces of approximately 5 mm Determination of TF activity in carotid artery homogenates. v Pieces of approximately 5 mm of uninjured carotid arteries were dissected from both sides in each animal and were collected in a TBS 0. 1% Triton. X-100 buffer containing heparin as anticoagulant. v The samples were homogenized. Homogenates of pooled carotid artery from four animals in each group were washed once with the same buffer and twice with TBS-0. 1% Triton. X-100. Finally the preparations were resuspended in 50 L of this buffer and sonicated. v The TF activity of lysates was determined using a commercial chromogenic assay (Actichrome TF, American Diagnostica, Stamford, CT).

Effects of a p 38 MAPK inhibitor on a. PL-induced TF activity in carotid Effects of a p 38 MAPK inhibitor on a. PL-induced TF activity in carotid artery homogenates of mice u Vega-Ostertag ME et al J Thromb Haemost 2007; 5: 1828 -1834.

Platelet aggregation v Mouse blood was obtained in acid citrate dextrose anticoagulant (9/1 volume/volume) Platelet aggregation v Mouse blood was obtained in acid citrate dextrose anticoagulant (9/1 volume/volume) by cardiac puncture. Platelet rich plasma (PRP) was obtained by centrifugation for 20 min at 120 g. v Aggregation of platelet in PRP was measured turbidimetrically using a dual channel aggregometer (Minigator II) following calibration with platelet-poor plasma (PPP) at a stirring speed of 800 rpm. PRP was adjusted to 240, 00 platelets/ L with PPP. v Aliquots (250 L) of PRP were placed in cuvettes containing magnetic stir bars, warmed at 370 C and stirred for 1 min to obtain a stable baseline. v Aggregation was induced by addition of 0. 005 U/m. L of thrombin in light transmission was recorded for 5 min.

Effects of a p 38 MAPK inhibitor on a. PL-induced platelet aggregation u Vega-Ostertag Effects of a p 38 MAPK inhibitor on a. PL-induced platelet aggregation u Vega-Ostertag ME et al J Thromb Haemost 2007; 5: 1828 -1834.

Experimental Design Ex vivo experiments: Determination of VCAM-1 in flat aorta preparations of mice Experimental Design Ex vivo experiments: Determination of VCAM-1 in flat aorta preparations of mice using Qdot conjugates and dual photon confocal microscopy. v Mouse arteries were pressure-perfused with 10% formalin. After fixation, the arteries were washed three times with PBS. v The immunohistochemical procedure was done on a 24 well-plate. Blocking was done with 2% BSA/5% goat serum for one hour. Primary antibodies were incubated overnight at 4ºC. Washing steps were done to remove primary antibody excess. Qdot-conjugated secondary antibody (Qdot Corp) was incubated for one hour. Finally, nuclear visualization was done with Hoechst stain. v Image collection was done using a Zeiss LSM 510 Meta two-photon microscope equipped with a near-infrared (NIR) titanium-sapphire femtosecond laser (Mira 900 Ti: S Coherent) tuned and mode-locked at 750 nm. The separation of the emission signals was performed by acquisition of lambda stacks with posterior selection of reference spectra using the META detector. v The following primary antibodies were used: , monoclonal rat anti-mouse VCAM-1 Ig. G (1: 50 dilution, BD Pharmingen) and a nonimmune primary used to address the contribution of nonspecific Fc receptor-mediated binding (nonimmune purified rat Ig. G 2 a (BD Pharmingen™). The following Qdot-bioconjugate was used in the experiments: Qdot® 655 goat F(ab')2 anti-rat Ig. G Conjugate

Effects of a p 38 MAPK inhibitor on a. PL-induced VCAM-1 expression in aorta Effects of a p 38 MAPK inhibitor on a. PL-induced VCAM-1 expression in aorta of mice ex vivo: nano crystals Q dot conjugates and dual photon confocal microscopy. u Vega-Ostertag ME et al J Thromb Haemost 2007; 5: 1828 -1834.

Conclusions u. A p 38 MAPK inhibitor (SB 203580): u Effectively diminished in vivo Conclusions u. A p 38 MAPK inhibitor (SB 203580): u Effectively diminished in vivo Ig. G-APS- induced – thrombus formation – endothelial cell activation – platelet aggregation – tissue factor activity (carotid EC and peritoneal macrophages) – VCAM-1 expression (aorta EC)

NF- B u NF- B is a complex group of heterodimeric and homodimeric transcription NF- B u NF- B is a complex group of heterodimeric and homodimeric transcription factors that are trapped in the cytoplasm as an inactive complex by I- B. u NF- B involved in transcription of inflammatory genes such as: IL-6, IL-8, TNF- and IL-1 and in induction of adhesion molecules on EC (VCAM-1, E-sel and ICAM-1) and in recruitment of inflammatory cells to extravascular sites. u NF- B associated with rheumatoid arthritis and other autoimmune diseases.

APL antibodies and NF- B u Intracellular events in EC induced by a. PL APL antibodies and NF- B u Intracellular events in EC induced by a. PL antibodies: – a. PL induce activation of NF- B and correlates with EC activation in vitro and in vivo and with thrombosis in vivo. • Espinola RG et al: J Thromb Haemost, 2003; 1: 843 -848. • Dunoyer-Geindre S. et al. Thromb Haemost. 2002; 88: 851 -857. • Bohgaki M, et al. Int Immunol. 2004; 16: 1632 -1641.

MG 132 u NF- B inhibitors used in RA and other autoimmune and inflammatory MG 132 u NF- B inhibitors used in RA and other autoimmune and inflammatory diseases. u MG 132 = Carbobenzoxyl-leucinylleucinal (Z-Leu-Leu-aldehyde; ZLLL-CHO). u MG 132 is a potent 20 S proteasome inhibitor that has been shown effective in suppressing NF- B activation in different cellular systems. u Several studies have shown beneficial effects of MG 132 on models of rheumatoid arthritis, suggesting that this inhibitor may provide a new approach in the treatment of this autoimmune disease.

Objectives u Are NF- B inhibitors effective in reversing pro -inflammatory and pro-thrombotic effects Objectives u Are NF- B inhibitors effective in reversing pro -inflammatory and pro-thrombotic effects of a. PL in vivo?

Experimental Design Ig. G -APS (500µg) i. p + 10 µM MG 132 or Experimental Design Ig. G -APS (500µg) i. p + 10 µM MG 132 or DMSO: PBS (60: 40). 0 hr 48 hr Ig. M - NHS (500µg) i. p. + DMSO: PBS (60: 40) 72 hr 1. Thrombus dynamics analysis 2. Adhesion of WBC to EC in cremaster 3. TF activity in carotid artery homogenates 4. TF activity in mouse peritoneal macrophages 5. ACL activity

Inhibition of a. PL-induced thrombus formation by MG 132 Inhibition of a. PL-induced thrombus formation by MG 132

Inhibition of a. PL-induced EC activation in vivo by MG 132 Inhibition of a. PL-induced EC activation in vivo by MG 132

Effects of MG 132 on TF activity on mononuclear cells of mice treated with Effects of MG 132 on TF activity on mononuclear cells of mice treated with a. PL Antibodies

Effects of MG 132 on TF activity on homogenates of carotid artery of mice Effects of MG 132 on TF activity on homogenates of carotid artery of mice treated with a. PL Antibodies

Conclusions u Ig. G-APS enhanced thrombosis, endothelial cell activation and tissue factor in vivo Conclusions u Ig. G-APS enhanced thrombosis, endothelial cell activation and tissue factor in vivo in mice. u These effects were significantly diminished by pre-treatment of the mice with MG 132. u Montiel-Manzano et al. NY Acad Med 2007; 1108: 540 -553.

Inhibitors of p 38 MAPK and NF- B for APS? u P 38 MAPK Inhibitors of p 38 MAPK and NF- B for APS? u P 38 MAPK and NF- B inhibitors effective in diminishing in vitro and in vivo effects of a. PL Abs. u Can we use specific inhibitors of p 38 MAPK or NF- B to revert/ameliorate pathogenic effects of a. PL Abs? u Clinical trials are needed.

Antiphospholipid Antibodies and the statins Antiphospholipid Antibodies and the statins

Pleiotropic effects of statins u - TPA and PA inhibitior-1 expression u - Expression Pleiotropic effects of statins u - TPA and PA inhibitior-1 expression u - Expression of adhesion molecules u - Pro-inflammatory cytokines u - Expression of tissue factor u - Thromboxane A 2 synthesis and platelet reactivity u - Endothelin 1 synthesis u - NF- B activation u - MHC class II antigen expression

a. PL antibodies and fluvastatin u Fluvastatin reduces a. PL-induced adhesion of leukocytes and a. PL antibodies and fluvastatin u Fluvastatin reduces a. PL-induced adhesion of leukocytes and expression of adhesion molecules on EC in vitro. (Meroni PL, et al. Arthritis and Rheum 2001; 44: 2870 -2878). u Fluvastatin abrogated thrombogenic effects of a. PL antibodies in vivo (Ferrara DE et al, Arthritis Rheum, 2003; 48: 3272 -3279) u Fluvastatin inhibited the effects of the Ig. G-APS on tissue factor expression. The effect was dependent on the dose of fluvastatin. Mevalonate abrogated the inhibitory effects of fluvastatin on expression of TF by a. PL on endothelial cells. (Ferrara DE. et al, J Thromb Haemost, 2004; 2: 1558 -1563).

Implications u These findings may have important implications in designing new modalities of treatment Implications u These findings may have important implications in designing new modalities of treatment and prevention of recurrent thrombosis in patients with APS. u Well designed clinical trials are needed to investigate and confirm these findings in APS patients

Objectives of the study To determine the effects of statins on prothrombotic and pro-inflammatory Objectives of the study To determine the effects of statins on prothrombotic and pro-inflammatory markers in patients with a. PL Abs

Patients u Four u A) groups of 20 patients each PAPS u B) Asymptomatic Patients u Four u A) groups of 20 patients each PAPS u B) Asymptomatic APS with persistently positive a. CL, LA and/or anti- 2 GPI tests. u C) u D) SAPS Asymptomatic SAPS with persistently positive a. CL, LA and/or anti- 2 GPI tests.

Inclusion/Exclusion criteria u a. CL titers > 40 GPL or MPL units u Anti- Inclusion/Exclusion criteria u a. CL titers > 40 GPL or MPL units u Anti- 2 GPI > 99 th percentile of normal controls. u >18 years of age. u No pregnant women. u Exclude if they are on statins or with “pulse” therapy with steroids. u Not excluded if they are on HQ, aspirin, heparin, warfarin, low dose prednisone (5 -10 mg/day). u Exclude patients with liver problems.

Study Intervention u Fluvastatin 40 mg/day for 5 months. u Blood will be collected Study Intervention u Fluvastatin 40 mg/day for 5 months. u Blood will be collected at the screening visit, at one, three, six and seven months later. u At five months patients will be instructed to stop fluvastatin.

Outcome measures u Determination of a. CL, LA and anti- 2 GPI u Determination Outcome measures u Determination of a. CL, LA and anti- 2 GPI u Determination of PCA and TF m. RNA in titers. PBMC. u Determination of VEGF, s. TF, s. VCAM-1, IL-6, IL-8 and TNF-.

a. PL, complement, endothelial cell activation and thrombosis a. PL, complement, endothelial cell activation and thrombosis

Complement and a. PL Abs. u A murine C’ inhibitor (crry) reverses a. PL-mediated Complement and a. PL Abs. u A murine C’ inhibitor (crry) reverses a. PL-mediated pregnancy loss, thrombosis and endothelial cell activation in vivo. (Holers W et al. J Exp Med; 2002; 2: 211 -220) u Recent studies have shown that uncontrolled complement activation leads to fetal death in a. PL-antibody-treated mice. (Girardi G et al. J Clin Invest. 2003; 112: 1644 -1654) u u Heparin seems to prevent obstetrical complications by a. PL by blocking activation of complement and not by preventing placental thrombosis(Girardi G et al. Nature Medicine. 2004; 10: 1222 -1226) C 3 and C 5 Deficient mice are resistant to thrombosis and endothelial cell activation induced by a. PL antibodies (Pierangeli SS et al. Arthritis Rheum 2005; 52: 2120 -2124). u Hypocomplementemia has been reported in patients with APS in three studies. (Carbone J. et al. Lupus; 1999. 8: 274 -278; Munakata Y. Thromb Haemost. 2000; 83: 728 -731. Davis WD & Brey RL. 1992. Clin Exp Immunol 1992; 10: 455 -460).

Objective u Does an anti-C 5 Mo. Ab prevent a. PLmediated thrombosis in mice? Objective u Does an anti-C 5 Mo. Ab prevent a. PLmediated thrombosis in mice?

Does an anti-C 5 Mo. Ab prevent a. PL-mediated thrombosis in mice? u Pierangeli Does an anti-C 5 Mo. Ab prevent a. PL-mediated thrombosis in mice? u Pierangeli SS et al. Arthritis Rheum 2005; 52: 2120 -2124.

Effects of a. PL Abs on thrombosis in C 5 a. R deficient mice. Effects of a. PL Abs on thrombosis in C 5 a. R deficient mice. C 5 a. R-/- + Ig. G-APS 3400 ± 1681 108. 9 ± 33. 4 C 5 a. R-/- + Ig. G-NHS 777. 3 ± 270. 4 0. 8 ± 0. 4 C 5 a. R +/+ + Ig. G-APS 3507 ± 965 80. 3 ± 17. 6 C 5 a. R+/+ + Ig. G-NHS 1321 ± 798 0. 8 ± 0. 5 C 5 a. R -/- + Ig. M-APS *676 ± 690 96. 4 ± 30. 8 C 5 a. R-/- + Ig. M-NHS 958 ± 388 0. 0 ± 0. 0 C 5 a. R +/+ + Ig. M-APS 3198 ± 2361 99. 8 ± 31. 4 C 5 a. R+/+ + Ig. M-NHS 585 ± 460 00. 0 ± 0. 1 Romay-Penabad Z et al. NY Acad Sci 2007; 1108: 554 -566.

CONCLUSIONS u We demonstrated that complement activation is a central mechanism contributing to a. CONCLUSIONS u We demonstrated that complement activation is a central mechanism contributing to a. PL antibody-induced thrombophilia using three approaches: • a specific complement inhibitor (Crry-Ig) • genetically deficient mice (C 3 -/- and C 5 -/-) • Using specific anti-C 5 Monoclonal antibodies. • Using C 5 a. R deficient mice.

Further evidence of complement involvement u Thrombus formation induced by antibodies to 2 glycoprotein Further evidence of complement involvement u Thrombus formation induced by antibodies to 2 glycoprotein I is complement dependent and requires a priming factor (Fischetti et al Blood 2005; 106: 2340 -2346).

IMPLICATIONS u Pathogenic a. PL antibodies, in addition to their direct effects on platelet IMPLICATIONS u Pathogenic a. PL antibodies, in addition to their direct effects on platelet and endothelial cell targets, induce complement activation, generating complement split products which attract inflammatory cells that may induce then thrombosis and tissue injury u u Activation of complement may be a critical proximal effector mechanism in a. PL-associated thrombosis. u In APS patients, due to a. PL Ig. G deposition targeted to the endothelium, complement activation is increased locally and overwhelms normally adequate inhibitory mechanisms. u Therefore, inhibition of complement activation should ameliorate a. PL -mediated vascular thrombosis.

APL antibodies in EC. APL antibodies in EC.

Receptor for a. PL in EC? Human 2 GPI binds to endothelial cells through Receptor for a. PL in EC? Human 2 GPI binds to endothelial cells through a cluster of lysine residues that are critical for anionic phospholipids binding and offers epitopes for anti- 2 GPI antibodies (Del Papa et al, 1997)

Effects of recombinant Domain I of 2 glycoprotein I on a. PL-mediated thrombosis Treatment Effects of recombinant Domain I of 2 glycoprotein I on a. PL-mediated thrombosis Treatment a. CL titer in-vivo Thrombus size (GPLU) ( m 2) APS-Ig. G + r. DI 90. 7± 42. 4 568. 4± 325. 0 APS-Ig. G + control 70. 1± 18. 3 7959. 0± 2647 u Five mice per group u DI/control infused at 40 mg i. v 30 minutes before thrombosis dynamics analysis

TIFI and a. PL Abs u TIFI is a a 20 amino acid synthetic TIFI and a. PL Abs u TIFI is a a 20 amino acid synthetic peptide (derived from CMV) that shares similarity with the PL- (membrane binding) region of 2 GPI. u TIFI reduced a. PL-mediated thrombosis and EC activation in vivo. u TIFI reduced the binding of FITC- 2 GPI to target cells (EC, and monocytes). u (Vega-Ostertag et al. Lupus 2006; 597: 247 -256)

TLR-4: Receptor for a. PL on EC? u My. D 88 signaling cascade - TLR-4: Receptor for a. PL on EC? u My. D 88 signaling cascade - associated to TLR-4 - is triggered by a. PL reacting with 2 GPI on the endothelial cell surface membrane (Raschi et al. Blood 2003; 101: 3495 -3500). u APL Abs are not thrombogenic in LPS -/- mice and EC activation and TF are diminished. (Pierangeli SS et al Ann Rheum Dis. 2007; epub ahead of press)

Effects of various inhibitors on TF upregulation by 2 GPI dimer. Effects of various inhibitors on TF upregulation by 2 GPI dimer.

Effects of various inhibitors on ICAM-1 expression induced by dimers of 2 GPI Effects of various inhibitors on ICAM-1 expression induced by dimers of 2 GPI

Conclusions u Animal models of thrombosis and endothelial cell activation in APS have been Conclusions u Animal models of thrombosis and endothelial cell activation in APS have been instrumental in – Elucidating the pathogenic mechanisms induced by a. PL antibodies – Determining specific targets recognized by a. PL antibodies. – Examining possible ways by which a. PL antibodies are induced. – Test new targeted therapies to prevent a. PL-effects in vivo. – WE NEED HUMAN STUDIES!

Animal models in APS New Approaches to Prevention of Thrombosis in APS? u Statins: Animal models in APS New Approaches to Prevention of Thrombosis in APS? u Statins: Fluvastatin reversed EC activation and TF upregulation by a. PL antibodies in vitro and abrogated enhanced thrombus formation and EC in vivo. In mice u Hydroxychloroquine: Decreased platelet activation induced by a. PL antibodies in vitro and inhibited a. PL-mediated thrombosis in mice in vivo. u Antiplatelet agents: GPIIb. IIIa inhibitors decreased a. PL-mediated enhancement of platelet activation and abrogated a. PL-induced thrombus formation in mice. u p 38 MAPK inhibitors: In vitro effects on a. PL-induced TF upregulation in EC. Effects on a. PL-mediated thrombus formation, EC activation, TF upregulation and platelet activation in vivo. u NF- B inhibitors: In vitro effects on a. PL-mediated upregulation of TF. Significant decrease in some a. PL-enhanced thrombosis, TF upregulation, u Specific complement inhibitors: anti-C 5 Monoclonal antibody decreased a. PLmediated thrombus formation. u Specific inhibitors/blocking agents to the receptor(s) in target cells: TLR-4 inhibitors? Peptides that mimic regions of ß 2 GPI?

New Approaches to Prevention of Thrombosis in APS? u ACE inhibitors: Inhibit monocyte TF New Approaches to Prevention of Thrombosis in APS? u ACE inhibitors: Inhibit monocyte TF expression u Dilazep, dipyridamole: Adenosine uptake inhibitor; antiplatelet; inhibits monocyte TF expression. u LJP 1082: ß 2 GPI-specific B cell toleragen; decreases anti-ß 2 GPIspecific B cell toleragen; decreases anti- ß 2 GPI antibody levels. u Rituximab: Anti CD 20.

New Approaches to Prevention of Thrombosis in APS? u ACE inhibitors: Inhibit monocyte TF New Approaches to Prevention of Thrombosis in APS? u ACE inhibitors: Inhibit monocyte TF expression u Dilazep, dipyridamole: Adenosine uptake inhibitor; antiplatelet; inhibits monocyte TF expression. u LJP 1082: ß 2 GPI-specific B cell toleragen; decreases anti-ß 2 GPIspecific B cell toleragen; decreases anti- ß 2 GPI antibody levels. u Rituximab: Anti CD 20.

Collaborators u Mariano Vega-Ostertag, MS Morehouse School of Medicine u Zurina Romay-Penabad, Ph. D Collaborators u Mariano Vega-Ostertag, MS Morehouse School of Medicine u Zurina Romay-Penabad, Ph. D UTMB u Guadalupe Montiel, B. S. UTMB u Elizabeth Papalardo, B. S. UTMB u Dardo E. Ferrara, MD Morehouse School of Medicine u R. G. Espinola, MD Morehouse School of Medicine u X. Liu, MD Morehouse School of Medicine u Ian P. Giles University College London u Robert Swerlick, MD Emory University School of Medicine u Pier Luigi Meroni, MD University of Milan u Guillermina Girardi, Ph. D Hosp Spec Surgery u Jane Salmon, MD Hosp Spec Surgery u VM Holers, MD Univ Colorado, Denver u Philip de. Groot Utrecht University.

The 13 th International Congress on antiphospholipid antibodies: Galveston, TX. Spring 2010. The 13 th International Congress on antiphospholipid antibodies: Galveston, TX. Spring 2010.

Acknowledgements u These studies were partially funded by a Research Centers in Minority Institutions Acknowledgements u These studies were partially funded by a Research Centers in Minority Institutions National Institutes of Health grant # G 12 RR 03034 and a Minority Biomedical Research Support Grant from the National Institutes of Health (GM 58268 -02) and a multidisciplinary clinical research grant NIH grant #: 2 P 60 AR 047785 -06.