Platelet aggregation at sites of vascular injury is essential for hemostasis and arterial thrombosis. It has long been assumed that platelet aggregation and thrombus growth are initiated by soluble agonists generated at sites of vascular injury. By using high-resolution intravital imaging techniques and hydrodynamic analyses, we show that platelet aggregation is primarily driven by changes in blood flow parameters (rheology), with soluble agonists having a secondary role, stabilizing formed aggregates. We find that in response to vascular injury, thrombi initially develop through the progressive stabilization of discoid platelet aggregates. Analysis of blood flow dynamics revealed that discoid platelets preferentially adhere in low-shear zones at the downstream face of forming thrombi, with stabilization of aggregates dependent on the dynamic restructuring of membrane tethers. These findings provide insight into the prothrombotic effects of disturbed blood flow parameters and suggest a fundamental reinterpretation of the mechanisms driving platelet aggregation and thrombus growth.

Platelet aggregation, the process by which platelets adhere to each other at sites of vascular injury, has long been recognized as critical for hemostatic plug formation and thrombosis. Until relatively recently, platelet aggregation was considered a straightforward process involving the noncovalent bridging of integrin ␣ IIb ␤ 3 receptors on the platelet surface by the dimeric adhesive protein fibrinogen. However, with recent technical advances enabling real-time analysis of platelet aggregation in vivo, it has become apparent that this process is much more complex and dynamic than previously anticipated. Over the last decade, it has become clear that platelet aggregation represents a multistep adhesion process involving distinct receptors and adhesive ligands, with the contribution of individual receptorligand interactions to the aggregation process dependent on the prevailing blood flow conditions. It now appears that at least 3 distinct mechanisms can initiate platelet aggregation, with each of these mechanisms operating over a specific shear range in vivo. The identification of shear-dependent mechanisms of platelet aggregation has raised the possibility that vascular-bed-specific inhibitors of platelet aggregation may be developed in the future that are safer and more effective than existing antiplatelet agents. IntroductionThe propensity of platelets to clump together at sites of vascular injury was first recognized more than 100 years ago. [1][2][3][4] This phenomenon, most accurately described as platelet cohesion although more commonly referred to as platelet aggregation, was quickly identified as important for hemostatic plug formation. 5 It was also recognized at the time that platelets played a key role in the development of thrombosis, 6 but, it was not until almost a century later that it became widely accepted that platelets played a pivotal role in development of cardiovascular disease. 7 As a consequence, inhibitors of platelet aggregation have become increasingly important parts of the armamentarium for the prevention and treatment of many atherothrombotic disorders. 8,9 For more than 3 decades, the factors mediating platelet aggregation appeared conceptually straightforward, requiring a platelet stimulus (agonist), a soluble adhesive protein (fibrinogen), and a membrane-bound platelet receptor (integrin ␣ IIb ␤ 3 or GPIIb-IIIa), leading to a simple unified model of platelet aggregation ( Figure 1). Although these core elements remain fundamental, recent technical advances allowing real-time analysis of platelet aggregation in vivo have demonstrated a much more complex and dynamic process than previously anticipated. It is now widely accepted that one of the key elements influencing the platelet aggregation process is blood flow, with evidence that distinct aggregation mechanisms operate under different shear conditions. [10][11][12][13] This has raised the interesting possibility that vascular bed-specific inhibitors of platelet aggregation may be developed in the future and has stimulated a...

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The formation of blood clots--thrombosis--at sites of atherosclerotic plaque rupture is a major clinical problem despite ongoing improvements in antithrombotic therapy. Progress in identifying the pathogenic mechanisms regulating arterial thrombosis has led to the development of newer therapeutics, and there is general anticipation that these treatments will have greater efficacy and improved safety. However, major advances in this field require the identification of specific risk factors for arterial thrombosis in affected individuals and a rethink of the 'one size fits all' approach to antithrombotic therapy.

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It is well established that preexposure of human neutrophils to proinflammatory cytokines markedly augments the production of reactive oxygen species (ROS) to subsequent stimuli. This priming event is thought to be critical for localizing ROS to the vicinity of the inflammation, maximizing their role in the resolution of the inflammation, and minimizing the damage to surrounding tissue. We have used a new generation of isoform-selective phosphoinositide 3-kinase (PI3K) inhibitors to show that ROS production under these circumstances is regulated by temporal control of class I PI3K activity. Stimulation of tumor necrosis factor-␣ (TNF-␣)-primed human neutrophils with N-formylmethionyl-leucyl-phenylalanine (fMLP) results in biphasic activation of PI3K; the first phase is largely dependent on PI3K␥, and the second phase is largely dependent on PI3K␦. The second phase of PI3K activation requires the first phase; it is this second phase that is augmented by TNF-␣ priming and that regulates parallel activation of ROS production. Surprisingly, although TNF-␣-primed mouse bone marrow-derived neutrophils exhibit superficially similar patterns of PI3K activation and ROS production in response to fMLP, these responses are substantially lower and largely dependent on PI3K␥ alone. These results start to define which PI3K isoforms are responsible for modulating neutrophil responsiveness to infection and inflammation. IntroductionNeutrophils are critical components of the immune system and have a vital role in combating bacterial and fungal infections. 1 A key weapon in the neutrophil armory is the so-called "respiratory burst," the generation of reactive oxygen species (ROS) by a multicomponent oxidase complex. 2,3 Patients with chronic granulomatous disease (CGD) caused by defective expression of active oxidase components experience recurrent, life-threatening infections. 4 The role of ROS in fighting infections is complex. ROS are involved in the killing process directly through the damaging actions of oxygen radicals and their halogenated derivatives and indirectly through the activation of phagosomal proteases. [5][6][7] It is also becoming apparent that ROS may regulate the neutrophil lifespan, modify the extracellular matrix through which the neutrophils migrate, and modulate the function of other cells participating in the inflammatory response. [8][9][10][11][12] Given the potential for self-damage, a key feature of the inflammatory response is to confine ROS generation in time and space to areas where it is required. One way in which this is thought to occur is through a form of signal integration in which prior exposure to local proinflammatory factors is necessary for maximal activation by subsequent oxidase-triggering signals. [13][14][15][16][17] One of best studied examples of this "priming" phenomenon is the ability of tumor necrosis factor-␣ (TNF-␣), a cytokine released primarily by macrophages, to dramatically augment the oxidase response to bacterially derived peptides (eg, N-formyl-methionyl-leucylphenylal...

Summary. Recent in vivo studies have highlighted the dynamic and complex nature of platelet thrombus growth and the requirement for multiple adhesive receptor-ligand interactions in this process. In particular, the importance of von Willebrand factor (VWF) in promoting both primary adhesion and aggregation under high shear conditions is now well established. In general, the efficiency with which platelets adhere and aggregate at sites of vessel wall injury is dependent on the synergistic action of various adhesive and soluble agonist receptors, with the contribution of each of the individual receptors dependent on the prevailing blood flow conditions. In this review, we will discuss the major platelet adhesive interactions regulating platelet thrombus formation under high shear, with specific focus on the VWF (GPIb and integrin a IIb b 3 ) and collagen receptors (GPVI and integrin a 2 b 1 ). We will also discuss the signaling mechanisms utilized by these receptors to induce platelet activation with specific emphasis on the role of cytosolic calcium flux in regulating platelet adhesion dynamics. The role of soluble agonists in promoting thrombus growth will be highlighted and a model to explain the synergistic requirement for adhesive and soluble stimuli for efficient platelet aggregation will be discussed.

IntroductionPlatelet aggregation at sites of vascular injury is essential for the formation of the primary hemostatic plug and also for the development of pathological thrombi at sites of atherosclerotic plaque rupture. The initial contact of platelets with the injured vessel wall (platelet adhesion) is a complex process involving multiple adhesive substrates (von Willebrand factor [vWf], collagen) and receptors on the platelet surface (GPIb/V/IX, integrins α IIb β 3 and α 2 β 1 ) (1). The interaction between matrix-bound vWf and GPIbα on the platelet surface serves primarily to tether platelets to the area of vascular injury (2, 3), particularly under conditions of high shear stress, as a prerequisite step for integrin-mediated cell arrest (4). Whereas the molecular events underlying platelet adhesion under different shear conditions have been well delineated, the mechanism(s) by which platelets in freeflowing blood subsequently adhere to the initial layer of adherent platelets (platelet cohesion or aggregation) under flow have been less clearly defined.The traditional model of platelet aggregation, in which integrin α IIb β 3 was thought to have an exclusive role in mediating platelet-platelet adhesion contacts, has been largely determined from studies using a platelet aggregometer (5). With this method, the addition of a soluble agonist to a stirred platelet suspension induces activation of integrin α IIb β 3, converting it from a low-to a high-affinity receptor capable of binding soluble fibrinogen. The dimeric nature of fibrinogen enables it to cross-link adjacent activated platelets leading to stable platelet aggregation. Studies of platelet aggregation under high shear conditions, using a coneplate viscometer, have demonstrated that plasma vWf becomes the relevant ligand responsible for platelet activation (6). Shear-induced binding of soluble vWf to GPIbα initiates platelet activation independent of the addition of exogenous stimuli. Whereas the vWf-GPIbα interaction is indispensable for the initiation of platelet-platelet adhesion contacts under high shear, irreversible platelet aggregation requires a second adhesive interaction between vWf and integrin α IIb β 3 (7).The molecular events governing the formation of stable adhesion contacts between platelets in suspension have been well delineated; however, the mechanism by In this study we have examined the mechanism of platelet aggregation under physiological flow conditions using an in vitro flow-based platelet aggregation assay and an in vivo rat thrombosis model. Our studies demonstrate an unexpected complexity to the platelet aggregation process in which platelets in flowing blood continuously tether, translocate, and/or detach from the luminal surface of a growing platelet thrombus at both arterial and venous shear rates. Studies of platelets congenitally deficient in von Willebrand factor (vWf) or integrin α IIb β 3 demonstrated a key role for platelet vWf in mediating platelet tethering and translocation, whereas integrin α IIb β 3 mediated cell ...