Dok-2 adaptor protein regulates the shear-dependent adhesive function of platelet integrin αIIbβ3 in mice.

The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin αIIbβ3, raising the possibility that it participates in integrin αIIbβ3 outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase.

GPVI potentiation of platelet activation by thrombin and adhesion molecules independent of Src kinases and Syk.

Objective: The present study investigates the role of Src and Syk tyrosine kinases in signaling by G-protein coupled and platelet adhesion receptors.

Methods And Results: Using Syk-/- platelets or the Src kinase inhibitor PP2, we demonstrate a critical role for Src and Syk kinases in mediating lamellipodia formation on VWF, collagen, CRP, fibrinogen, and fibronectin. In all cases, the spreading defect was overcome by addition of thrombin.

Cellular pathology of atherosclerosis: smooth muscle cells promote adhesion of platelets to cocultured endothelial cells.

Although platelets do not ordinarily bind to endothelial cells (EC), pathological interactions between platelets and arterial EC may contribute to the propagation of atheroma. Previously, in an in vitro model of atherogenesis, where leukocyte adhesion to EC cocultured with smooth muscle cells was greatly enhanced, we also observed attachment of platelets to the EC layer. Developing this system to specifically model platelet adhesion, we show that EC cocultured with smooth muscle cells can bind platelets in a process that is dependent on EC activation by tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1.

A novel Syk-dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2.

The snake venom rhodocytin has been reported to bind to integrin alpha2beta1 and glycoprotein (GP) Ibalpha on platelets, but it is also able to induce activation independent of the 2 receptors and of GPVI. Using rhodocytin affinity chromatography, we have identified a novel C-type lectin receptor, CLEC-2, in platelets that confers signaling responses to rhodocytin when expressed in a cell line. CLEC-2 has a single tyrosine residue in a YXXL motif in its cytosolic tail, which undergoes tyrosine phosphorylation upon platelet activation by rhodocytin or an antibody to CLEC-2, but not to collagen, thrombin receptor agonist peptide (TRAP), or convulxin.

PI 3-kinase p110beta: a new target for antithrombotic therapy.

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin alpha(IIb)beta(3) (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110beta isoform in regulating the formation and stability of integrin alpha(IIb)beta(3) adhesion bonds, necessary for shear activation of platelets.

Selective impairment of platelet activation to collagen in the absence of GATA1.

Defects in the X-linked DNA-binding megakaryocyte transcription factor GATA1 cause thrombocytopenia and abnormal platelet function. However, detailed studies of GATA1 function in platelet activation are lacking. Here, we studied platelets from GATA1-deficient mice and from a male patient (S14) with a bleeding diathesis attributed to a single amino acid substitution (R216Q) in the N-terminal GATA1 zinc finger that alters binding to DNA.

Differential proteome analysis of TRAP-activated platelets: involvement of DOK-2 and phosphorylation of RGS proteins.

We have applied a proteomics approach to analyze signaling cascades in human platelets stimulated by thrombin receptor activating peptide (TRAP). By analyzing basal and TRAP-activated platelets using 2-dimensional gel electrophoresis (2-DE), we detected 62 differentially regulated protein features. From these, 41 could be identified by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) and were found to derive from 31 different genes, 8 of which had not previously been reported in platelets, including the adapter downstream of tyrosine kinase 2 (Dok-2).

Integrin alpha IIb beta 3-dependent calcium signals regulate platelet-fibrinogen interactions under flow. Involvement of phospholipase C gamma 2.

Platelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin alpha IIb beta 3 engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading.

RhoA sustains integrin alpha IIbbeta 3 adhesion contacts under high shear.

The small GTPase RhoA modulates the adhesive nature of many cell types; however, despite high levels of expression in platelets, there is currently limited evidence for an important role for this small GTPase in regulating platelet adhesion processes. In this study, we have examined the role of RhoA in regulating the adhesive function of the major platelet integrin, alpha(IIb)beta(3). Our studies demonstrate that activation of RhoA occurs as a general feature of platelet activation in response to soluble agonists (thrombin, ADP, collagen), immobilized matrices (von Willebrand factor (vWf), fibrinogen) and high shear stress.

Essential role for phosphoinositide 3-kinase in shear-dependent signaling between platelet glycoprotein Ib/V/IX and integrin alpha(IIb)beta(3).

Platelet adhesion and aggregation at sites of vascular injury are critically dependent on the interaction between von Willebrand factor (VWF) and 2 major platelet adhesion receptors, glycoprotein (GP) Ib/V/IX and integrin alpha(IIb)beta(3). GP Ib/V/IX binding to VWF mediates platelet tethering and translocation, whereas activation of integrin alpha(IIb)beta(3) promotes cell arrest. To date, the signaling pathways used by the VWF-GP Ib/V/IX interaction to promote activation of integrin alpha(IIb)beta(3), particularly under shear, have remained poorly defined.