Arterial disease and thrombosis in the antiphospholipid syndrome (APS): A pathogenic role for endothelin-1 (ET-1)

Objective To explore a possible correlation between endothelin 1 (ET-1), the most potent endothelium-derived contracting factor that modulates vascular smooth muscle tone, and arterial disease in patients with the antiphospholipid syndrome (APS). Methods Plasma levels of ET-1 were measured in APS patients with (n = 16) and without (n = 11) arterial thrombosis and in non-APS patients with arterial thrombosis (n = 9). In addition, steady-state prepro-ET-1 messenger RNA (mRNA) levels were determined in endothelial cells treated with a range of human monoclonal anticardiolipin antibodies (aCL) (as anti-β2-glycoprotein I antibodies) by semiquantitative 32P-dCTP-labeled reverse transcription-polymerase chain reaction. Results Compared with healthy controls, markedly increased plasma levels of ET-1 were found in APS patients with arterial thrombosis (2.00 ± 0.87 versus 0.96 ± 0.37 pg/ml; P = 0.0001) but not in other groups. Three human monoclonal aCL induced prepro-ET-1 mRNA levels significantly more than did control monoclonal antibody lacking aCL activity. Conclusion Plasma ET-1 levels correlated significantly with a history of arterial thrombosis in patients with APS. Prepro-ET-1 mRNA was induced by human monoclonal aCL in the in vitro experimental system. The induction of ET-1 by antiphospholipid antibodies might contribute to increased arterial tone, leading to vasospasm and, ultimately, to arterial occlusion.

Gap junctions and connexin hemichannels underpin hemostasis and thrombosis

BACKGROUND: Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have now examined the role of connexins in platelets, blood cells that circulate in isolation, but upon tissue injury adhere to each other and the vessel wall to prevent blood loss and facilitate wound repair. METHODS AND RESULTS: We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses prior to platelet-platelet contact, and reduced laser induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion and clot retraction indicating an important role for Cx37 hemichannels and gap junctions in platelet thrombus function. CONCLUSIONS: Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of haemostasis and thrombosis and represent potential therapeutic targets.

Gap junctions and connexin hemichannels in the regulation of haemostasis and thrombosis

Platelets are involved in the maintenance of haemostasis but their inappropriate activation leads to thrombosis, a principal trigger for heart attack and ischemic stroke. Although platelets circulate in isolation, upon activation they accumulate or aggregate together to form a thrombus, where they function in a coordinated manner to prevent loss of blood and control wound repair. Recent reports indicate that the stability and functions of a thrombus are maintained through sustained, contact dependent signalling between platelets. Given the role of gap junctions in the coordination of tissue responses, it was hypothesized that gap junctions may be present within a thrombus and mediate intercellular communication between platelets. Therefore studies were performed to explore the presence and functions of connexins in platelets. In this brief review, the roles of hemichannels and gap junctions in the control of thrombosis and haemostasis and the future directions for this research will be discussed.

SLAP/SLAP2 prevent excessive platelet (hem)ITAM signaling in thrombosis and ischemic stroke in mice

Glycoprotein VI and C-type lectin-like receptor 2 are essential platelet activating receptors in hemostasis and thrombo-inflammatory disease, which signal through a (hem)immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway. The adapter molecules Src-like adapter proteins (SLAP and SLAP2) are involved in the regulation of immune cell surface expression and signaling, but their function in platelets is unknown. In this study, we show that platelets expressed both SLAP isoforms and that overexpression of either protein in a heterologous cell line almost completely inhibited glycoprotein VI and C-type lectin-like receptor 2 signaling. In mice, single deficiency of SLAP or SLAP2 had only moderate effects on platelet function, whereas double deficiency of both adapters resulted in markedly increased signal transduction, integrin activation, granule release, aggregation, procoagulant activity, and thrombin generation in response to (hem)ITAM-coupled, but not G protein-coupled, receptor activation. In vivo, constitutive SLAP/SLAP2 knockout mice displayed accelerated occlusive arterial thrombus formation and a dramatically worsened outcome after focal cerebral ischemia. This was attributed to the absence of both adapter proteins in platelets, as demonstrated by adoptive transfer of Slap(-/-)/Slap2(-/-) platelets into wild-type mice. Our results establish SLAP and SLAP2 as critical inhibitors of platelet (hem)ITAM signaling in the setting of arterial thrombosis and ischemic stroke.