[Show abstract][Hide abstract] ABSTRACT: Platelets are anuclear cell fragments derived from bone marrow megakaryocytes that safeguard vascular integrity by forming thrombi at sites of vascular injury. Although the early events of thrombus formation-platelet adhesion and aggregation-have been intensively studied, less is known about the mechanisms and receptors that stabilize platelet-platelet interactions once a thrombus has formed. One receptor that has been implicated in this process is the signaling lymphocyte activation molecule (SLAM) family member CD84, which can undergo homophilic interactions and becomes phosphorylated upon platelet aggregation.
The role of CD84 in platelet physiology and thrombus formation was investigated in CD84-deficient mice.
We generated CD84-deficient mice and analyzed their platelets in vitro and in vivo. Cd84-/- platelets exhibited normal activation and aggregation responses to classical platelet agonists. Furthermore, CD84 deficiency did not affect integrin-mediated clot retraction and spreading of activated platelets on fibrinogen. Notably, also the formation of stable three-dimensional thrombi on collagen-coated surfaces under flow ex vivo was unaltered in the blood of Cd84-/- mice. In vivo, Cd84-/- mice exhibited unaltered hemostatic function and arterial thrombus formation.
These results show that CD84 is dispensable for thrombus formation and stabilization, indicating that its deficiency may be functionally compensated by other receptors or that it may be important for platelet functions different from platelet-platelet interactions.
PLoS ONE 12/2014; 9(12):e115306. DOI:10.1371/journal.pone.0115306 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Agonist-induced platelet activation involves different signaling pathways leading to the activation of phospholipase C (PLC) beta or PLCgamma2. Activated PLC produces inositol 1,4,5-trisphosphate and diacylglycerol, which trigger Ca(2+) mobilization and the activation of protein kinase C, respectively. PLCbeta is activated downstream of Gq-coupled receptors for soluble agonists with only short interaction times in flowing blood. In contrast, PLCgamma2 becomes activated downstream of receptors that interact with immobilized ligands such as the collagen receptor glycoprotein (GP) VI or activated integrins.
We speculated that PLCgamma2 activity might be optimized for sustained but submaximal signaling to control relatively slow platelet responses. To test this hypothesis, we analyzed platelets from mice heterozygous for a gain-of-function mutation in the Plcg2 gene (Plcg2(Ali5/+)).
Plcg2(Ali5/+) platelets showed enhanced Ca(2+) mobilization, integrin activation, granule secretion and phosphatidylserine exposure upon GPVI or C-type lectin-like receptor-2 stimulation. Furthermore, integrin alpha(IIb)beta(3) outside-in signaling was markedly enhanced in the mutant platelets, as shown by accelerated spreading on different matrices and faster clot retraction. These defects translated into virtually unlimited thrombus formation on collagen under flow in vitro and a prothrombotic phenotype in vivo.
These results demonstrate that the enzymatic activity of PLCgamma2 is tightly regulated to ensure efficient but limited platelet activation at sites of vascular injury.
Journal of Thrombosis and Haemostasis 03/2010; 8(6):1353-63. DOI:10.1111/j.1538-7836.2010.03838.x · 5.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Coronary artery thrombosis is often initiated by platelet activation on collagen-rich subendothelial layers in the disrupted atherosclerotic plaque. The activating platelet collagen receptor glycoprotein VI (GPVI) noncovalently associates with the Fc receptor gamma-chain (FcRgamma), which signals through its immunoreceptor-tyrosine-based activation motif (ITAM) via the adaptor LAT leading to the activation of phospholipase Cgamma2 (PLCgamma2). GPVI is a promising antithrombotic target as anti-GPVI antibodies induce the irreversible loss of the receptor from circulating platelets by yet undefined mechanisms in humans and mice and long-term antithrombotic protection in the latter. However, the treatment is associated with transient but severe thrombocytopenia and reduced platelet reactivity to thrombin questioning its clinical usefulness. Here we show that GPVI down-regulation occurs through 2 distinct pathways, namely ectodomain shedding or internalization/intracellular clearing, and that both processes are abrogated in mice carrying a point mutation in the FcRgamma-associated ITAM. In mice lacking LAT or PLCgamma2, GPVI shedding is abolished, but the receptor is irreversibly down-regulated through internalization/intracellular clearing. This route of GPVI loss is not associated with thrombocytopenia or altered thrombin responses. These results reveal the existence of 2 distinct signaling pathways downstream of the FcRgamma-ITAM and show that it is possible to uncouple GPVI down-regulation from undesired side effects with obvious therapeutic implications.