Platelet Membrane Receptors and Signalling Pathways.

Chapter 17 - Outside-in signaling by integrin αIIbβ3

In platelets, alpha(IIb)beta(3) exists in a form that cannot bind adhesive proteins in the plasma; although it can interact with immobilized fibrinogen it cannot interact with immobilized von Willebrand factor in the vessel wall. Soluble agonists such as thrombin convert alpha(IIb)beta(3) to a form that recognizes soluble and immobilized ligands. Attempts to reconstitute alpha(IIb)beta(3) activation in a non-hematopoietic, nucleated cell system have been unsuccessful. In the present study, we have developed a transfected Chinese hamster ovary cell model in which alpha(IIb)beta(3) activation is induced by signaling across glycoprotein (GP) Ib-IX by its ligand, von Willebrand factor. GPIb-IX activates not only the transfected alpha(IIb)beta(3) but also endogenous alpha(v)beta(3). Activation of the pathways leading to integrin activation occurred even in cells transfected with GPIb-IX lacking the domain on GPIbalpha that binds 14-3-3 or that which binds actin-binding protein. These studies demonstrate that signals induced by interaction of GPIb-IX with von Willebrand factor lead to alpha(IIb)beta(3) activation and suggest that the signaling pathways by which GPIb-IX induces alpha(IIb)beta(3) activation are different to those used by thrombin. Elucidation of these differences may provide insights into therapeutic ways in which to inhibit integrin activation in selective clinical settings.

It is a common understanding that the rupture of an atheroma in the coronary arteries is the initial event in the onset of arterial thrombosis resulting in myocardial infarction.1 Ex vivo perfusion experiments using human blood have clearly demonstrated that platelet accumulation occurs immediately when the subendothelial matrix, such as collagen, is exposed to the blood stream.2 However, initiation of platelet thrombus formation after endothelial disruption resulting in exposure of the subendothelial matrix does not directly represent the onset of symptomatic atherothrombotic diseases, such as myocardial infarction, which were caused by thrombotic arterial occlusion. For example, coronary intervention, while causing damage to the endothelium, does not, in most cases, result in any symptomatic myocardial ischemia. Moreover, recent advances in clinical imaging techniques, such as intracoronary ultrasonography, have revealed a much higher incidence of atheroma rupture than of symptomatic atherothrombotic coronary artery diseases, including myocardial infarction and unstable angina pectoris.3,4 These observations suggest the contribution of propagating factors for thrombus growth, besides exposure of the subendothelial matrix due to endothelial disruption, in the onset of symptomatic arterial thrombotic diseases. See …

PTP-1B Is the Protein Tyrosine Phosphatase That Initiates Integrin αIIbβ3 Outside-In Signaling in Platelets.

Integrin alphaIIbbeta3 is the major membrane protein and adhesion receptor at the surface of blood platelets, which after activation plays a key role in platelet plug formation in hemostasis and thrombosis. Small angle neutron scattering (SANS) and shape reconstruction algorithms allowed formation of a low resolution three-dimensional model of whole alphaIIb beta3 in Ca(2+)/detergent solutions. Model projections after 90 degrees rotation along its long axis show an elongated and "arched" form (135 degrees) not observed before and a "handgun" form. This 20-nm-long structure is well defined, despite alphaIIb beta3 multidomain nature and expected segmental flexibility, with the largest region at the top, followed by two narrower and smaller regions at the bottom. Docking of this SANS envelope into the high resolution structure of alphaIIb beta3, reconstructed from crystallographic and NMR data, shows that the solution structure is less constrained, allows tentative assignment of the disposition of the alphaIIb and beta3 subunits and their domains within the model, and points out the structural analogies and differences of the SANS model with the crystallographic models of the recombinant ectodomains of alphaIIb beta3 and alphaV beta3 and with the cryo-electron microscopy model of whole alphaIIb beta3. The ectodomain is in the bent configuration at the top of the model, where alphaIIb and beta3 occupy the concave and convex sides, respectively, at the arched projection, with their bent knees at its apex. It follows the narrower transmembrane region and the cytoplasmic domains at the bottom end. AlphaIIb beta3 aggregated in Mn(2+)/detergent solutions, which impeded to get its SANS model.

Understanding the pathology of acute coronary syndromes has led to significant improvements in patient outcome. Platelet adhesion to the arterial wall, especially under high shear forces, such as are present...

The small GTPase Rap1b regulates the cross talk between platelet integrin α2β1 and integrin αIIbβ3

Platelet aggregation and fibrin formation are essential for the maintenance of normal hemostasis, a system designed to act quickly and effectively to arrest hemorrhage. This system is also triggered by pathogenic events, such as the rupture of an atherosclerotic plaque, which can lead to thrombotic vaso-occlusion, ischemia, and infarction. Platelets are a major contributor to these damaging and life-threatening thrombotic phenomena because of their adhesive properties, which result in the release of soluble mediators, platelet aggregation, and enhancement of thrombin generation.1 The platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor is a key component in the pathway to platelet aggregation; consequently, this receptor has become the target for therapeutic intervention. A paradigm of this antiplatelet treatment modality is found naturally in the inherited disorder Glanzmann’s thrombasthenia. A key feature of this disease is that patients present with mucocutaneous bleeding but only rarely demonstrate spontaneous central nervous system hemorrhage,2 a feared complication of anticoagulant and antiplatelet therapy. All of the mutations that have been identified in patients with Glanzmann’s thrombasthenia result in a functional deficiency of GPIIb/IIIa receptors,2 3 and a hallmark of this disease is the absence of agonist-induced platelet aggregation. The molecular characterization of mutations causing Glanzmann’s thrombasthenia has provided a wealth of information on structure-function relations of the GPIIb/IIIa receptor. This review will briefly summarize those mutations that affect ligand-binding domains and receptor activation and present them in the context of predicted structures. More comprehensive coverage can be found in reviews discussing the structure and function of the GPIIb/IIIa receptor complex4 5 and the clinical and molecular basis of Glanzmann’s thrombasthenia.2 3 Platelets are the first line of defense in preventing blood loss from injured blood vessels via recognition and adhesion to components of the subendothelial matrix. This event is followed by formation of a platelet …

Platelet adhesion, activation, and aggregation at sites of vascular endothelial disruption caused by atherosclerosis are key events in arterial thrombus formation. Platelet tethering and adhesion to the arterial wall, particularly under high shear forces, are achieved through multiple high-affinity interactions between platelet membrane receptors (integrins) and ligands within the exposed subendothelium, most notably collagen and von Willebrand factor (vWF). Platelet adhesion to collagen occurs both indirectly, via binding of the platelet glycoprotein (GP) Ib-V-IX receptor to circulating vWF, which binds to exposed collagen, and directly, via interaction with the platelet receptors GP VI and GP Ia/IIb. Platelet activation, initiated by exposed collagen and locally generated soluble platelet agonists (primarily thrombin, ADP, and thromboxane A2), provides the stimulus for the release of platelet-derived growth factors, adhesion molecules and coagulation factors, activation of adjacent platelets, and conformational changes in the platelet alpha(IIb)beta3 integrin (GP IIb/IIIa receptor). Platelet aggregation, mediated primarily by interaction between the activated platelet GP IIb/IIIa receptor and its ligands, fibrinogen and vWF, results in the formation of a platelet-rich thrombus. Currently available antiplatelet drugs (aspirin [acetylsalicylic acid], dipyridamole, clopidogrel, ticlopidine, abciximab, eptifibatide, tirofiban) act on specific targets to inhibit platelet activation and aggregation. Elucidation of the multiple mechanisms involved in platelet thrombus formation provides opportunities for selectively inhibiting the pathways most relevant to the pathophysiology of atherothrombosis.

Blood platelets play an essential role in hemostasis. The primary physiological function of platelets is to form hemostatic thrombi that prevent blood loss and maintain vascular integrity, which requires a robust and precise response of platelets to vascular injury. On the other hand, platelet activation must be tightly controlled. Dysregulated thrombus formation (thrombosis) causes blockage of blood vessels, leading to ischemia. Platelets in normal circulation are in a non-adherent “resting” state. Upon vascular injury, platelets are activated following initial adhesion to adhesive proteins like von Willebrand factor (VWF) and collagen or by soluble platelet agonists like ADP, thrombin, and thromboxane A2 (TXA2). These adhesive proteins and soluble agonists induce signal transduction via their respective receptors and receptor-specific signaling pathways. The various receptor-specific platelet activation signaling pathways converge into common signaling events, such as activation of PKC and Ca2+ mobilization, which stimulate platelet shape change, granule secretion, and ultimately induce the “inside-out” signaling process leading to activation of the ligand binding function of integrin αIIbβ3. Ligand binding to integrin αIIbβ3 mediates platelet adhesion and aggregation and triggers “outside-in” signaling, resulting in platelet spreading, granule secretion, stabilization of platelet adhesion and aggregation, and clot retraction. Defects in platelet signal transduction may lead to bleeding disorder. This chapter briefly introduces normal signal transduction of platelet activation followed by detailed description of inherited defects in the signaling molecules in platelets including receptors and kinases, leading to impaired platelet function and bleeding disorders (Table 1).

In thrombus formation associated with hemostasis or thrombotic disease, blood platelets first undergo a rapid transition from a circulating state to an adherent state, followed by activation and aggregation. Under flow conditions in the bloodstream, this process potentially involves platelet-platelet, platelet-endothelium, platelet-subendothelial matrix, and platelet-leukocyte interactions. Specific adhesion receptors on platelets mediate these interactions, by engaging counter-receptors on other cells, or noncellular ligands in the plasma or matrix. The glycoprotein (GP) Ib-IX-V complex on platelets initiates adhesion at high shear stress by binding the adhesive ligand, von Willebrand Factor (vWF). GP Ib-IX-V may also mediate platelet-endothelium or platelet-leukocyte adhesion, by recognition of P-selectin or Mac-1, respectively. Other membrane glycoproteins, such as the collagen receptor GP VI, may trigger platelet activation at low shear rates. Engagement of GP Ib-IX-V or GP VI leads ultimately to platelet aggregation mediated by the integrin, alphaIIbbeta3 (GP IIb-IIIa). This review will focus on recent advances in understanding structure-activity relationships of GP Ib-IX-V, its role in initiating thrombus formation, and its emerging relationships with other vascular cell adhesion receptors.

von Willebrand factor mediates platelet spreading through glycoprotein Ib and alpha(IIb)beta3 in the presence of botrocetin and ristocetin, respectively.

JAM-A protects from thrombosis by suppressing integrin αIIbβ3-dependent outside-in signaling in platelets

alpha(1)-acid glycoprotein (AGP) is an acute-phase protein that contributes to inflammation processes. The role of AGP in platelet activation and thrombosis is, however, largely unknown. Therefore, we thoroughly investigated the effects of AGP on human platelets. Platelets were isolated from healthy volunteers and subsequently exposed to AGP. Platelet responses were monitored as change in light transmission, intracellular calcium concentration, light microscopy and protein phosphorylation by Western blot. We found that AGP induced platelet shape change independently of a second release of adenine nucleotides or thromboxane A(2), and that effect was abolished by endothelium-derived platelet inhibitors such as nitric oxide (NO) and adenosine. Furthermore, AGP triggered a minor calcium response and a pronounced Rho/Rho-kinase-dependent increase in Thr696 phosphorylation of myosin phosphatase target subunit 1 (MYPT1). Moreover, the Rho/Rho-kinase inhibitor Y-27632 significantly decreased the AGP-induced shape change. The results also showed that the AGP-elicited shape change was antagonised by pretreatment with low doses of collagen and thrombospondin-1. Our results describe a novel mechanism by which AGP stimulates platelet shape change via activation of the Rho/Rho-kinase signalling pathway. Physiological important platelet inhibitors, such as NO, completely counterbalance the effect of AGP. Hence, the present study indicates that AGP directly contributes to platelet activation, which in turn might have an impact in physiological haemostasis and/or pathological thrombosis.

90 - von Willebrand factor