Intracellular events in platelet activation induced by antiphospholipid antibodies in the presence of low doses of thrombin

Abstract

Objective. Thrombosis and thrombocytopenia are features of the antiphospholipid syndrome (APS), suggesting that antiphospholipid antibodies (aPL) may bind platelets, causing activation and aggregation of platelets and thrombosis. The intracellular events involved in aPL-mediated platelet activation are not fully understood and are therefore the subject of this study.Methods. IgG fractions and their F(ab) 2 fragments were purified from the sera of 7 patients with APS and from the pooled sera of 10 healthy subjects (as controls). Phosphorylation of p38 MAPK, ERK-1/2, and [Ca 2؉ ]-dependent cytosolic phospholipase A 2 (cPLA 2 ) was determined in lysates of washed platelets pretreated with low doses of thrombin and aPL or control IgG or their F(ab) 2 fragments, by immunoblot. The effects of aPL on platelet aggregation in the presence or absence of a p38 MAPK inhibitor, SB203580, were examined. Thromboxane B 2 (TXB 2 ) production was detected by enzyme-linked immunosorbent assay on gel-filtered platelets treated with aPL and thrombin, with or without SB203580. Calcium mobilization studies were done utilizing a fluorometric assay.Results. Treatment of platelets with IgG aPL, or their F(ab) 2 fragments, in conjunction with subactivating doses of thrombin resulted in a significant increase in phosphorylation of p38 MAPK. Neither the IgG aPL nor their F(ab) 2 fragments increased significantly the phosphorylation of ERK-1/2 MAPKs. Furthermore, pretreatment of platelets with SB203580 completely abrogated the aPL-mediated enhanced aggregation of the platelets. Platelets treated with F(ab) 2 aPL and thrombin produced significantly larger amounts of TXB 2 when compared with controls, and this effect was completely abrogated by treatment with SB203580. In addition, cPLA 2 was also significantly phosphorylated in platelets treated with thrombin and F(ab) 2 aPL. There were no significant changes in intracellular [Ca 2؉ ] when platelets were treated with aPL and low doses of thrombin.Conclusion. The data strongly indicate that aPL in the presence of subactivating doses of thrombin induce the production of TXB 2 mainly through the activation of p38 MAPK and subsequent phosphorylation of cPLA 2 . The ERK-1/2 pathway does not seem to be involved in this process, at least in the early stages of aPL-mediated platelet activation.

Beta-2 GPI induced tissue factor and placental apoptosis for the pathophysiology of pregnancy loss in antiphospholipid syndrome

“…These processes are triggered by the interaction of pathogenic aPL with monocytes, endothelial cells (EC), platelets, trophoblast and endometrial cells, as well as plasma components of the coagulation cascade. 16,17,[26][27][28] Therefore, understanding the proposed hypothesis would stand for a growing evidence for the importance of placental apoptosis through TLRs in the pathogenesis of aPL-mediated pregnancy loss. Pathogenic aPL bind to phospholipid binding proteins of which β2GPI is the best characterized.…”

CMS links the tail

“…3 To this end, pre-TCR signaling may result in the arrest of pT␣ and the triggering of TCR␣ transcription. This, together with the higher pairing efficiency of TCR␤ with TCR␣ rather than with pT␣, may finally favor the switch from pre-TCR to the ␣␤TCR expression, thus sustaining differentiation progression.…”

“…For example, in experimental autoimmune encephalomyelitis (a model of multiple sclerosis), it has been shown that the order of epitope spreading can be a consistent and predictable process. 2,3 In this issue of Blood, Hall and colleagues have expanded this question to AIHA, using a murine model in which NZB mice spontaneously generate autoantibodies against Band 3. To test whether AIHA requires Band 3 to occur, Hall and colleagues monitored the development of AIHA in NZB mice with a deletion of the Band 3 gene.…”

In search for a receptor for antiphospholipid antibodies on target cells

“…Recently, some of the intracellular pathways activated by APL antibodies in platelets, monocytes and endothelial cells have been examined. Studies have conclusively shown that APL antibodies induce phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) in platelets, monocytes and endothelial cells, and activate the transcriptional factor nuclear factor-kappa B (NF-jB) in monocytes and endothelial cells [1][2][3][4][5].Although there is convincing evidence that APL antibodies can stimulate endothelial cells, monocytes and platelets, relatively little is known about the cell surface receptors that are engaged or crosslinked to induce intracellular signaling. In endothelial cells, there is indirect evidence that annexin A2, a receptor for tissue plasminogen activator (t-PA) and plasminogen, and toll-like receptor 4 (TLR-4), a receptor for bacterial lipopolysaccharide (LPS), may be binding b 2 -GPI and triggering intracellular signaling.…”

“…Recently, some of the intracellular pathways activated by APL antibodies in platelets, monocytes and endothelial cells have been examined. Studies have conclusively shown that APL antibodies induce phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) in platelets, monocytes and endothelial cells, and activate the transcriptional factor nuclear factor-kappa B (NF-jB) in monocytes and endothelial cells [1][2][3][4][5].…”