Effect of Shear Stress on Platelet Activation via the Glycoprotein VI Receptor

Cardiovascular diseases are the nation\u27s leading cause of death. Such diseases are caused by platelet response to collagen especially in the event of vascular injury leading to thrombosis. One of the platelet receptors known to bind to the collagen ligand is glycoprotein VI (GPVI) with co-receptor Fc receptor γ chain (FcRγ). By stably expressing the GPVI receptor in rat basophilic leukemia cells (RBL-2H3), which abundantly express FcRγ, but endogenously lack GPVI, studies have shown that GPVI-FcRγ is sufficient to confer adhesion as well as signaling responses to collagen as long as the receptor density is equivalent to that found on human platelets. While those investigations confirm that the GPVI receptor mediate binding to collagen under static conditions, they do not provide information on how the GPVI receptor interacts with collagen under dynamic conditions. In the present study we have used the GPVI-expressing RBL-2H3 cells to observe the kinetics of adhesion to collagen under hydrodynamic flow conditions in vitro using a parallel plate flow chamber coupled with video microscopy. We demonstrate that these cells do adhere to the surface at a low shear rate and do so at a greater adherent cell density than wild-type RBL-2H3 (WT-RBL) cells

Role of GPVI and alpha2beta1 in platelet collagen responses

In this thesis, we examine the functionality of two platelet collagen receptors, GPVI and α2β1, in their role in platelet collagen interactions as a result of vascular injury. Using rat basophilic leukemia cells transfected with the GPVI receptor, we show that GPVI is capable of mediating adhesion to collagen under shear stress in a density-dependent fashion that is independent of signaling. To extend these findings to a more physiological context, we used pharmacologic and genetic approaches to study human and mouse platelet adhesion to collagen under arterial shear rates. Our studies demonstrate that both GPVI and integrin α2β1 play significant roles for platelet adhesion to collagen under flow, and that the loss of both receptors completely ablates this response. Intracellular signaling mediated by the cytoplasmic adaptor SLP-76, but not by the transmembrane adaptor LAT, is critical for platelet adhesion to collagen under flow. In addition, reduced GPVI receptor density results in severe defects in platelet adhesion to collagen under flow. Defective adhesion to collagen under flow is associated with prolonged tail bleeding times in mice lacking one or both collagen receptors. These studies establish platelet collagen responses under physiologic flow as the consequence of a close partnership between two structurally distinct receptors and suggest that GPVI and α2β1 play significant hemostatic roles in vivo. With the establishment that integrin α2β1 is required for platelet collagen responses, we next address how it mediates bidirectional signaling in those responses. Specifically, we study whether or not the tyrosine residues of its β1 cytoplamic domain contribute to the response in outside-in signaling by inducing mutations, YF and YA, in the NPXY motifs. We find that YF mutant platelets maintain a near normal response in platelet adhesion and spreading on collagen but the YA results show a defective response. These results suggest that the tyrosine residues are important regulators of outside-in signaling; however, tyrosine phosphorylation is not required

Role of GPVI and alpha2beta1 in platelet collagen responses

In this thesis, we examine the functionality of two platelet collagen receptors, GPVI and α2β1, in their role in platelet collagen interactions as a result of vascular injury. Using rat basophilic leukemia cells transfected with the GPVI receptor, we show that GPVI is capable of mediating adhesion to collagen under shear stress in a density-dependent fashion that is independent of signaling. To extend these findings to a more physiological context, we used pharmacologic and genetic approaches to study human and mouse platelet adhesion to collagen under arterial shear rates. Our studies demonstrate that both GPVI and integrin α2β1 play significant roles for platelet adhesion to collagen under flow, and that the loss of both receptors completely ablates this response. Intracellular signaling mediated by the cytoplasmic adaptor SLP-76, but not by the transmembrane adaptor LAT, is critical for platelet adhesion to collagen under flow. In addition, reduced GPVI receptor density results in severe defects in platelet adhesion to collagen under flow. Defective adhesion to collagen under flow is associated with prolonged tail bleeding times in mice lacking one or both collagen receptors. These studies establish platelet collagen responses under physiologic flow as the consequence of a close partnership between two structurally distinct receptors and suggest that GPVI and α2β1 play significant hemostatic roles in vivo. With the establishment that integrin α2β1 is required for platelet collagen responses, we next address how it mediates bidirectional signaling in those responses. Specifically, we study whether or not the tyrosine residues of its β1 cytoplamic domain contribute to the response in outside-in signaling by inducing mutations, YF and YA, in the NPXY motifs. We find that YF mutant platelets maintain a near normal response in platelet adhesion and spreading on collagen but the YA results show a defective response. These results suggest that the tyrosine residues are important regulators of outside-in signaling; however, tyrosine phosphorylation is not required

GPVI and α2β1 play independent critical roles during platelet adhesion and aggregate formation to collagen under flow

The roles of the 2 major platelet-collagen receptors, glycoprotein VI (GPVI) and integrin α2β1, have been intensely investigated using a variety of methods over the past decade. In the present study, we have used pharmacologic and genetic approaches to study human and mouse platelet adhesion to collagen under flow conditions. Our studies demonstrate that both GPVI and integrin α2β1 play significant roles for platelet adhesion to collagen under flow and that the loss of both receptors completely ablates this response. Intracellular signaling mediated by the cytoplasmic adaptor Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) but not by the transmembrane adaptor linker for activation of T cells (LAT) is critical for platelet adhesion to collagen under flow. In addition, reduced GPVI receptor density results in severe defects in platelet adhesion to collagen under flow. Defective adhesion to collagen under flow is associated with prolonged tail-bleeding times in mice lacking one or both collagen receptors. These studies establish platelet-collagen responses under physiologic flow as the consequence of a close partnership between 2 structurally distinct receptors and suggest that both receptors play significant hemostatic roles in vivo