4.1. Mechanisms of Platelet Aggregation

Aggregation involves platelet-to-platelet adhesion, and is necessary for effective hemostasis following the initial adhesion of platelets to the site of injury, described above in Chapter 3. Following adhesion, platelets are activated by a number of agonists such as adenosine diphosphate (ADP) and collagen present at the sites of vascular injury. These agonists activate platelets by binding to specific receptors on the platelet surface discussed earlier. Occupancy of these receptors leads to a series of downstream events that ultimately increases the intracytoplasmic concentration of calcium ions. The increase in platelet intracellular calcium occurs through release from intracellular stores and calcium influx through the plasma membrane [156]. Receptors coupled to G-proteins such as those to ADP, thromboxane A₂ (TXA₂) and thrombin, activate phospholipase Cβ (PLCβ), whereas receptors acting via the non-receptor tyrosine kinase pathways such as collagen receptor GpVI preferentially activate phospholipase Cγ (PLCγ) [83]. Activation of PLCβ or PLCγ results in the production of two second messengers: diacylglycerol (DAG) and inositol trisphosphate (IP₃). DAG mediates calcium influx while IP₃ liberates calcium from intracellular stores. In addition, calcium influx may be induced directly by certain agonists, such as ATP binding to the ligand-gated ion channel receptor, P2X1 [74].

Increased platelet free calcium concentration results in a number of structural and functional changes of the platelet. Morphologically, the platelet changes dramatically from a disc to a spiny sphere (a process called shape change). The granules in the platelet are centralized and their contents are discharged into the lumen of the open canalicular system, from which they are then released to the exterior (the release reaction). The increase in platelet calcium stimulates membrane phospholipase A₂ activity, which liberates arachidonic acid from membrane phospholipids. Arachidonic acid is converted to an intermediate product prostaglandin H₂ (PGH₂) by the enzyme cyclooxygenase 1 (COX-1). PGH₂ is further metabolized to TXA₂ by thromboxane synthase [117]. TXA₂ is a potent activator of platelets. The long membrane projections brought about by shape-change reaction allow the platelets to interact with one another to form aggregates. Shape change is mediated by the platelet cytoskeleton, composed by an organized network of microtubules and actin filaments and a number of associated proteins, linked to a variety of platelet signaling molecules [157]. Platelet shape change results in extensive reorganization of the cytoskeleton network, polymerization of actin, and myosin light chain phosphorylation [157–160]; these responses vary in a time- and stimulus-dependent manner. Examples of changes in platelet shape during activation and aggregation are depicted in Figure 4.1.

Figure 4.1

Transmission Electron microscopy images of mouse platelets illustrating various stages of activation, associated with microvascular thrombosis induced by photochemical injury. A) discoid-shape platelet revealing a dense granule with a characteristic “bull’s (more...)

A main adhesion molecule involved in platelet aggregation is the membrane protein, GPIIb/IIIa complex. GPIIb/IIIa is an integrin receptor present at high density on platelets, both on the plasma membrane and on α-granules [52]. It exists as an inactive form in resting platelets. Platelet activation by almost all agonists induces conformational changes (‘inside-out signaling’) of GPIIb/IIIa, which becomes competent to bind soluble plasma fibrinogen. In turn, ligand binding of GPIIb/IIIa results in conformational changes directed to the cytoplasm (‘outside-in signaling’). The precise sequence of events leading to these signaling events has not been fully elucidated [53,161]. The roles of receptor clustering, phosphorylation and association with cytoskeletal and other cytoplasmic molecules in inducing GPIIb/IIIa conformational changes are not totally delineated. Nevertheless, the receptor-bound fibrinogen acts as a bridge between two GPIIb/IIIa molecules on adjacent platelets [83]. This is the final common pathway of platelet aggregation induced by platelet chemical agonists. However, vWF substitutes for fibrinogen as a bridge molecule between GPIIb/IIIa for platelet aggregation induced by high shear conditions, although platelet aggregation under lower shear is mediated by fibrinogen binding to GPIIb/IIIa [162].

Although GPIIb/IIIa is the most widely studied mediator of bridging platelets to each other and stabilizing thrombi, other molecules have been proposed recently to mediate these responses. These include junctional adhesion molecules (JAMs), SLAM (signaling lymphocyte activation molecule) family proteins, and CD40 ligand [163–165]. The relative roles of these mechanisms in platelet aggregation are yet to be defined clearly.

Activated platelets recruit additional platelets to the growing hemostatic plug by several feedback amplification loops: they release platelet agonists (such as ADP and serotonin stored in the α-granules) and they synthesize de novo proaggregatory TXA₂. Release of ADP and TXA₂ synthesis consolidate the initial hemostatic plug by promoting the participation of other platelets in the hemostatic plug formed at sites of vascular injury. Finally, platelets also play a dominant role in secondary hemostasis by providing a highly effective catalytic surface for activation of the coagulation cascade, as discussed below in Chapter 5. When platelets are activated, negatively charged phospholipids move from the inner leaflet of the membrane bilayer to the to the outer leaflet. The transbilayer movement of anionic phospholipids is associated with blebbing and release of procoagulant vesicles rich in anionic phospholipids. Both activated platelets and the micro-vesicles provide binding sites for enzymes and cofactors of the coagulation system, which then efficiently generate thrombin, itself a potent platelet agonist.

4.2. Monitoring of Platelet Aggregation

4.2.1. Ex Vivo Monitoring: Platelet Aggregometry

Traditional approaches to monitor platelet aggregation involve exposure of platelets in suspension to a variety of stimuli ex vivo, a technique known as platelet aggregometry. Agonists used commonly include adenosine diphosphate (ADP), collagen, thrombin, and thromboxane A₂, among others [166]. Studies are performed on either platelet-rich plasma or whole blood; platelets are maintained in suspension by stirring. Following exposure to an agonist, formation of platelet aggregates results in an increase in light transmission through the sample; the kinetics of the responses and maximal aggregation provide quantitative assessment of platelet aggregation. Figure 4.2 illustrates aggregation response of platelets ex vivo in response to ADP. Platelet aggregometry may provide relevant information of platelet function abnormalities in some selected clinical conditions, though this technique is used more frequently experimentally than clinically.

Figure 4.2

Platelet aggregometry tracing of human platelets in response to ADP. Low-dose exposure results in reversible aggregation, evident as a transient increase in light transmission. Intermediate dose results in two “waves” of aggregation, whereas (more...)

4.2.2. In Vivo Monitoring of Platelet Aggregation

While platelet aggregometry allows for assessment of platelet aggregation kinetics under carefully controlled experimental conditions (agonist dose, platelet counts, temperature, etc.), it may not reflect the complexities involved in platelet aggregation in vivo. A variety of intravital video microscopy approaches have been used to monitor platelet aggregation during thrombus formation in vivo, these are reviewed in detail elsewhere [134]. These techniques typically involve an injury to microvascular walls, for example by micropuncture, electrical stimulation, laser, chemical, or photochemical injury [86,134,167–169]. The nature of vascular injury, as well as the vessel type may determine the molecular mechanisms responsible for platelet recruitment in the individual models. In some models (e.g., micropuncture, chemical stimulation) platelet adhesion occurs at sites of endothelial denudation and reflects the mechanisms of platelet adhesion to the subendothelial matrix outlined earlier. In others, platelet adhesion is evident without overt endothelial denudation [86,170]. Vascular injury results in platelet adhesion, which may progress to formation of an occlusive thrombus, as illustrated in Figure 4.3. The kinetics of platelet adhesion and aggregation may be monitored in real-time with these techniques, and they have provided important novel observations of the mechanisms mediating platelet recruitment in vivo. These approaches also illustrate the redundancy of many mechanisms responsible for platelet recruitment. For example, mice lacking both vWF and fibrinogen are able to form occlusive thrombi following injury, albeit with marked delay in responses [171]. While intravital microscopy to visualize thrombus formation is not a new technique (e.g., Bizzozero used it in the late 19^th century [2]), advances in image acquisition and processing techniques, molecular biology and genetic models of disease have expanded the recent interest in this approach to study interactions of platelets with vascular walls.

Figure 4.3

Platelet adhesion and aggregation in vivo in a mouse model of endothelial injury induced by photochemical stimulation. Sequential intravital microscopy images reveal progressive increase in platelet aggregates (arrows) within an injured cremaster venule; (more...)