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 4.1Transmission 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 he 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.