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Keywords:

  • acute coronary syndromes;
  • arterial thrombosis;
  • platelet aggregation;
  • rheology

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Dynamic thrombus formation and the acute coronary syndromes
  5. Insights from experimental thrombosis models
  6. Impact of blood flow on platelet aggregation and thrombus formation
  7. Contribution of soluble agonist and rheology-dependent platelet aggregation mechanisms to thrombus development
  8. Conclusions
  9. Disclosure of Conflict of Interests
  10. References

Summary.  Platelet aggregation and thrombus formation at sites of atherosclerotic plaque rupture is a dynamic process that can lead to intermittent or permanent obstruction to blood flow, resulting in ischemic tissue injury and organ dysfunction. There is a growing body of evidence suggesting that the dynamics of platelet aggregation and initial thrombus development are regulated by two distinct, complementary processes, involving: (i) rheological (biomechanical) and (ii) soluble-agonist-dependent mechanisms. Rheological-dependent platelet aggregation occurs between discoid platelets and requires the biomechanical adhesive and signaling function (mechanotransduction) of the major platelet adhesion receptors, GPIb and integrin αIIbβ3. Soluble agonists further potentiate platelet activation, stimulating global platelet shape change and degranulation, and play a major role in stabilizing formed aggregates. Unraveling the dynamics of platelet aggregation and thrombus formation in vivo requires consideration of the cooperative interplay between rheological- and soluble agonist-dependent platelet aggregation mechanisms.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Dynamic thrombus formation and the acute coronary syndromes
  5. Insights from experimental thrombosis models
  6. Impact of blood flow on platelet aggregation and thrombus formation
  7. Contribution of soluble agonist and rheology-dependent platelet aggregation mechanisms to thrombus development
  8. Conclusions
  9. Disclosure of Conflict of Interests
  10. References

Considerable progress has been made defining the key molecular events underlying platelet aggregation and thrombus development at sites of vascular injury. Technological developments in the generation and study of knock-out mouse models have helped refine our understanding of the processes initiating, propagating and stabilizing platelet thrombi [1]. Most of the major adhesive ligands, soluble agonists and receptors involved in supporting the hemostatic and prothrombotic function of platelets have been identified, and in large part, the major signaling pathways utilized by these receptors to regulate platelet activation have been identified. What is less clearly understood is the spatial and temporal relationship between specific platelet adhesion and activating events, and how these processes are coordinated in the rapidly changing shear environment that characterizes thrombus development. In this brief review, we will discuss (i) the clinical evidence supporting an important role for dynamic thrombus development in the pathogenesis of the acute coronary syndromes; (ii) the importance of hemodynamic influences on the initiation of platelet aggregation and thrombus development; and (iii) highlight the cooperative contribution of rheological and soluble agonist-dependent platelet aggregation mechanisms to thrombus growth.

Dynamic thrombus formation and the acute coronary syndromes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Dynamic thrombus formation and the acute coronary syndromes
  5. Insights from experimental thrombosis models
  6. Impact of blood flow on platelet aggregation and thrombus formation
  7. Contribution of soluble agonist and rheology-dependent platelet aggregation mechanisms to thrombus development
  8. Conclusions
  9. Disclosure of Conflict of Interests
  10. References

The acute coronary syndromes, manifest as unstable angina, acute myocardial infarction and sudden death are typically caused by intimal rupture or erosion of atherosclerotic plaques, leading to the formation of platelet-rich thrombi [2]. Post-mortem analysis of the coronary vasculature and myocardium of patients dying from the acute coronary syndromes have highlighted the dynamic nature of thrombus development, with evidence that subclinical thrombus development can occur over many hours or days prior to the onset of symptoms. Most thrombi forming in the coronary circulation have a layered structure with thrombus material of different ages, indicating periods of episodic growth by repeated platelet and fibrin mural deposits [3]. Moreover, microemboli and microinfarcts are frequently found in the myocardium downstream from the site of the coronary thrombus, indicating intermittent thrombus fragmentation and peripheral embolization [3,4].

Angiographic findings have revealed the presence of loosely attached platelet-thrombi that typically form in the post-stenotic region of atherosclerotic plaques [3]. In experimental models, these regions appear to offer ideal fluid-dynamic conditions for the progressive growth of platelet aggregates [5]. Nonetheless, these aggregates remain unstable, leading to the persistent embolism of thrombus fragments into the distal microcirculation [6]. Thus, platelet thrombus formation occurring within and downstream of the ruptured plaque appears to be a dynamic process in which the thrombus size waxes and wanes over hours and even days, explaining the intermittent and unstable clinical characteristics of the acute coronary syndromes.

Insights from experimental thrombosis models

  1. Top of page
  2. Abstract
  3. Introduction
  4. Dynamic thrombus formation and the acute coronary syndromes
  5. Insights from experimental thrombosis models
  6. Impact of blood flow on platelet aggregation and thrombus formation
  7. Contribution of soluble agonist and rheology-dependent platelet aggregation mechanisms to thrombus development
  8. Conclusions
  9. Disclosure of Conflict of Interests
  10. References

The dynamic growth, fragmentation and embolization of thrombi, followed by reformation of fresh thrombi, was first recognized more than 150 years ago following the application of intravital microscopy to in vivo thrombosis models [7]. Whilst being most prominent during the first few hours of thrombus development, continuous thrombus formation and embolization may occur over days and weeks [8]. Recent technical developments in intravital microscopy that have enabled concomitant real-time analysis of platelet adhesion and activation have highlighted the dynamic nature of platelet adhesive interactions in vivo [9–12]. Most initial platelet tethering interactions are rapidly reversible, with initial thrombus development characterized by the formation of unstable loose aggregates, particularly in the superficial layers of forming thrombi. Activation of platelets within a developing thrombus occurs in stages, both spatially and temporally [11,12] with recent in vivo evidence demonstrating that discoid platelets are capable of forming loose aggregates [11] and undergoing minimal intracellular calcium flux [12]. As discussed below, these initial layers of discoid platelet aggregates are primarily driven by changes in blood rheology, with stabilization of the aggregation process dependent on the subsequent generation of soluble agonists.

Impact of blood flow on platelet aggregation and thrombus formation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Dynamic thrombus formation and the acute coronary syndromes
  5. Insights from experimental thrombosis models
  6. Impact of blood flow on platelet aggregation and thrombus formation
  7. Contribution of soluble agonist and rheology-dependent platelet aggregation mechanisms to thrombus development
  8. Conclusions
  9. Disclosure of Conflict of Interests
  10. References

It has long been recognized that a key factor regulating the dynamics of thrombus development is blood rheology, with alterations in the local hemodynamic environment being one of the most important factors regulating platelet deposition and thrombus growth [13]. Platelets are unique in their ability to form stable adhesive interactions under conditions of rapid blood flow, and in general, increasing shear stress leads to increased deposition of platelets onto thrombogenic surfaces and increased rates of thrombus growth [14].

In healthy arteries away from branching sites, blood flow can be considered laminar, with adjacent layers of fluid travelling parallel to one another. As a consequence, platelets at the vessel wall are typically exposed to relatively uniform hemodynamic forces during initial hemostatic plug formation. Much of the current information on shear effects on platelets has been gained from the use of experimental flow chambers and viscometers which expose platelets to relatively uniform levels of shear stress. Based on recent findings from in vitro perfusion systems [10,11,15–17] and in vivo thrombosis models [11,17,18], at least three distinct shear-dependent platelet aggregation mechanisms have been identified [19].

  • 1
    Low-intermediate shear (<1000 s−1) – these wall shear rates are typically found in veins and larger arteries and the platelet aggregation process under these conditions is predominantly mediated by integrin αIIbβ3. Under lower shear, αIIbβ3 on the surface of free-flowing platelets can engage fibrinogen adsorbed onto the surface of thrombi. Subsequent stimulation of platelets by locally generated soluble agonists stimulates an increase in integrin αIIbβ3 affinity, thereby stabilizing integrin αIIbβ3-fibrinogen bonds.
  • 2
    High shear (1000–10 000 s−1) – these shear rates are typically found in the arterial microcirculation or in regions of moderate arterial stenosis. Under high shear conditions, platelet-platelet interactions become progressively more VWF-dependent with an important role for both GPIb and integrin αIIbβ3 in promoting the initial formation of discoid platelet aggregates (Fig. 1).
  • 3
    Pathological shear (>10 000 s−1) – at sites of severe vessel narrowing, as may occur at sites of atherothrombosis, wall shear rates may increase dramatically up to 40 000 s−1. At these shear rates, platelet aggregation does not require platelet activation or the adhesive function of integrin αIIbβ3 and is exclusively mediated by VWF-GPIb adhesive bonds [17].
image

Figure 1.  Model of platelet aggregation under laminar and disturbed flow conditions. (A) Under laminar shear conditions (typically > 1000 s−1), both GPIb and integrin αIIbβ3 promote the initial formation of membrane-tethered discoid platelet aggregates. Subsequent soluble agonist generation induces sustained cytosolic calcium flux, platelet shape change and degranulation, ultimately leading to the formation of tightly packed stable aggregates. (B) Aggregation under conditions of localized alterations in blood flow typically occurs between discoid platelets. Flow vortices create shear gradients resulting in stable discoid platelet aggregation mediated by membrane tether restructuring [22] and increases the accumulation of platelet agonists in post - stenotic zones thereby enhancing platelet accummulation and activation downstream from the site of vascular injury. (C) Characteristic features of the traditional model of platelet aggregate formation and the rheology-dependent aggregation model.

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It should be emphasized that these aggregation mechanisms operate under simplified laminar shear conditions and thus may vary significantly from the complex shear conditions experienced by platelets at sites of atherosclerotic plaque rupture [20,21]. For example, blood flowing through a stenosed vessel may experience deceleration at the entry of the stenosis, rapid acceleration across the stenosis and flow separation and reversal at the stenosis outlet. During thrombus development, hemodynamic perturbations leading to flow separation, temporal shear gradients and turbulent flow become more prevalent resulting in platelet exposure to a rapidly changing and complex rheological environment, with recent experimental evidence demonstrating that the degree of flow perturbations correlates directly with the magnitude of platelet aggregation [22]. In addition, in regions where platelets experience marked alterations in flow such as eddies and flow vortices, platelet agonists accumulate in post-stenotic zones, thereby enhancing platelet accumulation and activation downstream from the site of vascular injury (Fig. 1) [23].

Contribution of soluble agonist and rheology-dependent platelet aggregation mechanisms to thrombus development

  1. Top of page
  2. Abstract
  3. Introduction
  4. Dynamic thrombus formation and the acute coronary syndromes
  5. Insights from experimental thrombosis models
  6. Impact of blood flow on platelet aggregation and thrombus formation
  7. Contribution of soluble agonist and rheology-dependent platelet aggregation mechanisms to thrombus development
  8. Conclusions
  9. Disclosure of Conflict of Interests
  10. References

Rheology-dependent platelet aggregation typically occurs between discoid platelets and play an important role in initiating and propagating thrombus development, particularly under high shear conditions or in regions of disturbed blood flow (Fig. 1B) [11,22]. During the initial phases of aggregate development, discoid platelets are in a low activation state, as evidenced by minimal cytosolic calcium flux, granule release or morphological change, and as a consequence, adhesion contacts between discoid platelets are inherently unstable. Rheology-dependent platelet aggregation mechanisms are largely dependent on adhesion-induced activation pathways with signals operating downstream of GPIb and integrin αIIbβ3 inducing weak platelet stimulation in the absence of soluble agonists [24,25].

A key feature of rheology-dependent platelet aggregation is the development of membrane tethers. Membrane tethers are smooth cylinders of lipid membrane that are pulled from the surface of discoid platelets under the influence of hemodynamic drag forces [11,26]. Membrane tethers play an important role in sustaining the adhesive interaction of discoid platelets with matrix proteins and other platelets, by reducing the force exerted on adhesive bonds [11,26]. Recent studies demonstrate that membrane tethers respond to rapid changes in the shear environment, by undergoing physical restructuring [22]. Restructured tethers further enhance the stability of discoid platelet adhesive interactions, providing a potential mechanistic explanation for the stabilization of discoid platelet aggregates observed in vivo [22]. A key function of membrane tethers is to enable discoid platelets to maintain close physical contact with one another, thereby facilitating stimulation by locally generated agonists. Such a process may help limit the ‘wash-out’ effects of blood flow on soluble agonists, maintaining a high local concentration of activating signals within the confines of a developing aggregate.

Thus soluble agonists, long considered the primary initiators of platelet aggregation, may in fact play a secondary role stabilizing formed aggregates, particularly under high shear conditions. Soluble-agonist-dependent platelet aggregation on thrombogenic surfaces is typically associated with robust cytosolic calcium flux, platelet shape change and degranulation, leading to the formation of tightly packed stable aggregates (Fig. 1A) [19]. Granule release of ADP and TXA2 generation enhance platelet activation and the stability of thrombi; however, under the influence of hemodynamic drag forces, these thrombi remain unstable and prone to distal embolization in the absence of significant thrombin generation and fibrin polymerization.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Dynamic thrombus formation and the acute coronary syndromes
  5. Insights from experimental thrombosis models
  6. Impact of blood flow on platelet aggregation and thrombus formation
  7. Contribution of soluble agonist and rheology-dependent platelet aggregation mechanisms to thrombus development
  8. Conclusions
  9. Disclosure of Conflict of Interests
  10. References

It has long been recognized that platelets sense and respond to local changes in their hemodynamic environment. There is growing evidence that this mechanosensory function of platelets is central to the initiation and propagation of platelet thrombi, with soluble agonists playing a key role in stabilizing formed aggregates. Much remains to be learned about the complex, dynamic interplay between rheological and soluble-agonist-dependent platelet aggregation mechanisms, and their contribution to initial thrombus growth, stability and embolization. Unravelling this dynamic relationship will be important for a clearer understanding of the thromboembolic events that occur under conditions of disturbed blood flow and for the future development of more effective antithrombotic approaches.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Dynamic thrombus formation and the acute coronary syndromes
  5. Insights from experimental thrombosis models
  6. Impact of blood flow on platelet aggregation and thrombus formation
  7. Contribution of soluble agonist and rheology-dependent platelet aggregation mechanisms to thrombus development
  8. Conclusions
  9. Disclosure of Conflict of Interests
  10. References
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