Abstract
Abstract Antiphospholipid antibodies include lupus anticoagulants and anticardiolipin antibodies (ACLA) which are the serological markers for antiphospholipid syndromes (APS). Patients of APS have at least one of the clinical features of venous thrombosis, arterial thrombosis, recurrent pregnancy loss, and thrombocytopenia. Platelet activation may have a role in APS, particularly in arterial thrombosis. This study aims to elucidate the mechanism of human platelet activation by ACLA and the synergistic effect of ACLA on low concentrations of physiological agonists including ADP and collagen. The function properties of anti-phosphatidylserine antibodies (APSA) and ACLA in the signaling pathways of platelet activation were investigated. APSA and ACLA showed the similar results to ACLA in several respects including: 1) the affinity to antigens CL and PS in ELISA test; 2) independent of □2GP1 for phospholipid binding in ELISA test. 3) activation of platelet aggregation; 4) biochemical events of platelet activation; 5) binding to a platelet membrane protein with an apparent molecular weight 67 kDa as revealed by Western blotting. Therefore, APSA and ACLA may have the similar functional capacity. Biochemical events studies showed that platelets activation by ACLA in: 1) PA formatioin; 2) Ca2+-mobilization; 3) phosphorylations of myosin-light chain and pleckstrin; 4) secretion of serotonin, □-thromboglobulin. Immunoprecipitation study showed that ACLA did not cause tyrosine phosphorylation of PLC□2. ACLA induced protein-tyrosine phosphorylation of platelets in a time-dependent manner similar to those of activation by thrombin and collagen. Ro 31-8220, a inhibitor of protein kinase C and BAPTA/AM, a Ca2+-chelator, inhibited ACLA-induced protein-tyrosine phosphorylation in the fibrinogen-dependent and aggregation-dependent steps. However, these two agents did not abolish these two steps of protein-tyrosine phosphorylation in high concentration of collagen. ACLA also inhibited prostaglandin E1-induced cyclic AMP formation by 30%. These results suggested that ACLA-signaling might be mediated by two signaling pathways including the receptor-inhibitory guanidine triphosphatase (receptor-Gi) pathway and phospholipase C□ (PLC□) pathway. Low concentrations of ADP and ACLA cooperatively potentiated the activation of human gel-filtrated platelets (GFP). The addition of fibrinogen was required to cause synergistic aggregation induced by ADP and ACLA. Biochemical events studied showed that the synergism occurred in phosphorylation of both myosin light chain and pleckstin, whereas there was only additive increase in [Ca2+]i and granule secretions. were observed. Furthermore, tyrosine kinases activities and the exposure of fibrinogen receptors □IIb□3 were synergistically increased when platelet stimulated by ADP and ACLA. A2P5P, an antagonist of ADP P2Y1 receptor, attenuated the exposure of □IIb□3 induced by ADP and ACLA. The additive inhibition of prostaglandin E1-induced cyclic AMP formation by ACLA and ADP were also observed. These results suggested platelet activation by ACLA and ADP was mediated by elevation the activity of tyrosine kinase and protein kinase C, therefore exposing the fibrinogen receptor. Low concentrations of collagen and ACLA also cooperatively potentiated human GFP. GFP were treated with ACLA for 5 min prior to exposure to collagen (ACLA + Col) exerting strong responses in cytosolic Ca2+-mobilization and cell aggregation; However, cells were treated with collagen for 1 min prior to ACLA (Col + ACLA) exhibiting responses to a lesser extent. The same pattern also occurred in biochemical events such as: 1) phosphorylation of pleckstrin and myosin light chain; and 2) secretion of □- and dense granule. Indomethacin inhibited Ca2+-mobilization, pleckstrin phosphorylation and cell aggregation in platelet stimulated by (ACLA + Col). The thromboxane B2 level in platelets induced by (ACLA + Col) was similar to that stimulated by low concentration of collagen alone. (ACLA + Col) increased the activities of phospholipases C (PLC) as determined by formation of PA, whereas indomethacin and A2P5P, abrogated the PA formation. These results suggest that ACLA, thromboxane A2 derived from the collagen pathway and secreted ADP cooperatively augmented the PLC’s activities leading to platelet aggregation. In conclusion, the receptor-inhibitory guanidine triphosphatase (receptor-Gi) pathway and the phospholipase C□ (PLC) pathway were complicated in the ACLA signaling increases in the activity of tyrosine kinases and protein kinase C triggered the exposure of fibrinogen receptor which may be the key points for platelet activation by ACLA and ADP. The studies of synergistic activation of ACLA and collagen also suggest ACLA, thromboxane A2 derived from the collagen pathway and secreted ADP cooperatively augment the PLC’s activities and lead to platelet aggregation.