Thrombosis has been considered an uncommon complication in patients with AIDS. In a 42-month period, 28 adult male homosexuals with AIDS experienced 34 thrombotic events. All but three received HAART regimen, two a successful round of double nucleoside analog therapy, and one patient received no treatment. Median age of group was 38.5 years (range, 24 to 56 years). Median time from HIV infection to thrombosis was 40.5 months (range, 3 to 108 months). No patient had previous thrombosis, family history of thrombosis, or prothrombotic conditions. There were 31 deep vein thromboses, two pulmonary thromboembolisms, and one renal vein thrombosis. Six patients had two thrombotic events. The rate of thrombosis during the 42-month study period was 1.52% (cumulative incidence = 0.30%/year), while the rate of thrombosis in 600 patients before the era of protease inhibitor therapy was 0.33% (cumulative incidence approximately 0.055%/year) (p < 0.001). Due to high incidence of thrombotic recurrences and hemorrhagic complications while using oral anticoagulants, acetylsalicylic acid was initiated; no thrombotic episodes were recorded while using this drug. Protein C and protein S deficiency were found in nine and two patients, respectively. Two patients had lupus anticoagulant and two activated protein C resistance (APCR) without FV Leiden mutation (APCR test was negative after initial screening). Fifteen patients had no thrombophilic abnormalities. These data suggest that protease inhibitors could be a risk factor for venous thrombosis not due to thrombophilic abnormalities but likely related to abnormalities in platelets or endothelium.

It has been shown that endothelial cell (EC) activation and tissue factor (TF) upregulation in EC and monocytes by antiphospholipid antibodies (aPL Abs) leads to a prothrombotic state and involves translocation of nuclear factor-kappa B (NF-kappaB). Here we examined the effects of an NF-kappaB inhibitor on aPL-induced thrombosis, TF activity, and EC in vivo. We treated CD1 mice with IgG from a patient with antiphospholipid syndrome (IgG-APS) or with control IgG (IgG-NHS). The adhesion of leukocytes (number of white blood cells) to EC in cremaster muscle (as an indication of EC activation) as well as the size of an induced thrombus in the femoral vein of the mice were examined. Some mice in each group were infused with 10 microM MG132 (an inhibitor of NF-kappaB). TF activity was determined using a chromogenic assay in homogenates of carotid arteries and in peritoneal cells of mice. In vivo, IgG-APS increased significantly the number of white blood cells adhering to ECs (4.7 +/- 2.2) when compared to control mice (1.5 +/- 0.8), and these effects were significantly reduced when mice were pretreated with MG132 (0.8 +/- 0.2). IgG-APS increased significantly the thrombus size and MG132 inhibited that effect (93%). Treatment of the mice with IgG-APS also induced significantly increased TF function in peritoneal cells and in homogenates of carotid arteries. Pretreatment of the mice with MG132 abrogated those effects significantly. Mice injected with IgG-APS or with IgM-APS with or without the inhibitor had medium-high titers of anticardiolipin antibodies in serum at the time of the surgical procedures. The data show that prothrombotic and proinflammatory properties of IgG-APS and IgM-APS are downregulated in vivo by an NF-kappaB inhibitor. These findings may be important in designing new modalities of targeted therapies to treat thrombosis in patients with APS.

Recent findings indicate that complement activation--involving specifically C3 and C5--contributes to antiphospholipid (aPL)-mediated thrombosis. Two complement effector pathways are initiated by the cleavage of C5, C5a and C5b, which leads to the formation of the C5b-9 membrane attack complex. To delineate and distinguish the role of C5a from the C5b-9 membrane attack complex seeded by C5b, we examined the in vivo effects (thrombosis) of aPL on C5a receptor-deficient (C5aR-/-) mice. C5aR-/- and C5aR+/+ mice were injected with IgM or with IgG from two different patients with APS (IgM-APS or IgG-APS) or with control IgM or IgG (IgM-NHS or IgG-NHS) twice. Complement fixing activity of the Ig fractions and anticardiolipin activity in the sera of the mice were determined by enzyme-linked immunosorbent assay. Surgical procedures to study thrombus dynamics were performed. IgM-APS but not IgG-APS fixed C1q to cardiolipin-coated plates. IgM-APS significantly enhanced thrombus size in C5aR+/+ mice compared to C5aR+/+ mice treated with IgM-NHS (3198 +/- 2361 microm2 versus 585 +/- 460 microm2). C5aR-/- mice treated with IgM-APS showed a significant reduction in thrombi size as compared with C5aR+/+ mice (676 +/- 690 microm2 versus 3198 +/- 2361 microm2; P = 0.001). IgG-APS enhanced thrombus formation significantly in C5aR+/+ when compared to IgG-NHS-treated mice (3507 +/- 965 microm2 versus 1321 +/- 798 microm2), and these effects were not altered in C5aR-/- mice (3400 +/- 1681 microm2). The data indicate that C5aR-/- mice are protected from the thrombogenic effects of some aPL antibodies.

Platelet aggregation is inhibited by the polyamines putrescine, spermidine, and spermine. To date, the mechanism of action has not been clearly identified. Evidence suggests that polyamines may interact with the fibrinogen receptor (GP IIb/IIIa), interfering with platelet-platelet attachment. The effect of polyamines on human platelet aggregation and GP IIb/IIIa activation was evaluated. For the aggregation experiments, platelets were obtained from heparin- or citrate-collected blood. Our results indicate that the polyamines putrescine, spermidine, and spermine cause a dose-dependent inhibition of ADP- or collagen-mediated platelet aggregation with an order of potency spermine>spermidine>putrescine. In addition, spermine arrests or inhibits thrombin-, epinephrine-, arachidonate-, or ristocetin-induced platelet aggregation. Expression of platelet membrane glycoproteins IIb, IIIa, and IX is not reduced by polyamines. However, spermine inhibits the ADP- or thrombin-induced activation of GP IIb/IIIa. It is concluded that the final step in aggregation, common to all agonists, ie, fibrinogen binding to GP IIb/IIIa, is inhibited by spermine through inhibition of the agonist-induced activation of GP IIb/IIIa that precedes fibrinogen-ligand binding.

Background: Thrombosis is an important cause of morbidity and mortality in Antiphospholipid Syndrome (APS) and in SLE patients with antiphospholipid antibodies (aPL). APL recognize β2 glycoprotein I (β2GPI)-bound to receptor (s) in endothelial cells (EC) and other target cells (i.e. platelets, monocytes) and trigger an intracellular signalling and a pro-coagulant and pro-inflammatory phenotype [i e.expression of tissue factor (TF), vascular cell adhesion molecule-1 (VCAM-1)] that lead to thrombosis. There is in vitro evidence that annexin A2 (A2), a receptor for tissue plasminogen activator (tPA) and plasminogen – and possibly other proteins such as toll-like receptors or the receptor for apolipoprotein E2′ - may be binding β2GPI on the membrane of target cells. Here, we examined the involvement of A2 in aPL-mediated pathogenic effects in vivo. We studied the effects of aPL Abs on thrombus formation, VCAM-1 expression in aortas of mice, and TF function in carotid artery homogenates in annexin A2 deficient (−/−) mice. Methods: A2 (−/−) mice and the corresponding wild-type (WT) mice, in groups of 10, were injected i.p. twice (0 and 48 hours later) with IgG from a patient with APS (IgG-APS) or with control IgG (IgG-NHS). Seventy-two hours after the first injection, several procedures were done in each mice: dynamics of thrombus formation (thrombus size), TF function in homogenates of carotid arteries, and c) VCAM-1 expression in the aortas using quantum dot nano crystals and two-photon excitation laser scanning microscopy. In addition, we examined the effect of an anti-A2 antibody on aPL-induced expression of intercellular cell-adhesion molecule (ICAM-1), E-selectin and TF acvitity on cultured endothelial cells (EC). Results: The titers of aCL and anti-β2GPI Abs in the sera of the mice at the time of surgery were medium-high positive in A2 (−/−) mice and in wild type mice injected with IgG-APS. Thrombus sizes were significantly larger in WT mice injected with IgG-APS when compared to similar type of mice treated with IgG-NHS (p=0.003). The size of thrombus in A2 (−/−) mice injected with IgG-APS was significantly smaller than mean thrombus size in WT mice injected with IgG-APS (p:0.0005). However, thrombus size in A2 (−/−) mice was larger in mice injected with IgG-APS when compared to same type of mice treated with control IgG-NHS (p=0.003), indicating a partial but significant abrogation of the thrombogenic effect. TF activity was significantly larger in WT mice treated with IgG-APS when compared to mice injected with IgG-NHS. Importantly, TF activity in carotid arteries homogenates of annexin A2 (−/−) mice injected with IgG-APS was significantly decreased (by 52%) when compared to wild type mice treated with IgG-APS. The expression of VCAM-1 in aorta of annexin A2 (−/−) ex vivo was also significantly reduced compared to LPS-treated mice (positive control) (p= 0.01). Interestingly, anti-A2 antibody significantly decreased aPL-induced expression of ICAM-1, E-sel and TF on cultured EC. Conclusions: Altogether these data indicate for the first time that A2 is involved in vivo pathogenic effects of aPL Abs. These findings may have important implications to devise new targeted and more specific therapeutic approaches to block the pathogenic effects of aPL Abs in patients with APS and SLE.