Identification of a 2-stage platelet aggregation process mediating shear-dependent thrombus formation

Abstract

Disturbances of blood flow at sites of atherosclerotic plaque rupture are one of the key pathogenic events promoting platelet activation and arterial thrombus formation. Shear effects of platelets have been extensively investigated in vitro; however, the mechanisms by which shear promotes platelet aggregation in vivo remain incompletely understood. By employing high-resolution imaging techniques to in vitro and in vivo thrombosis models, we demonstrate a unique mechanism initiating shear-dependent platelet aggregation involving aggregate formation between discoid platelets. These discoid platelet aggregates are initially unstable and result from the development of membrane tethers between coadhering platelets. Tether formation involves the adhesive function of GPIb/V/IX and integrin ␣ IIb ␤ 3 , and conversion of discoid platelet aggregates into stable aggregates requires released ADP. The efficiency of this process is regulated by 3 independent variables, including the reactivity of the adhesive substrate, the level of shear flow, and the platelet density at the adhesive surface. These studies identify a new mechanism initiating platelet aggregation that is critically influenced by shear, physical proximity between translocating platelets, and membrane tether formation. Moreover, they provide a model to explain how the discoid morphology of platelets facilitates the maintenance of adhesive interactions with thrombogenic surfaces under high shear stress conditions. IntroductionExcessive accumulation of platelets at sites of atherosclerotic plaque rupture is one of the key pathogenic events precipitating arterial thrombus formation, leading to acute myocardial infarction, sudden death, and ischemic stroke. This pathological process is responsible for more morbidity and mortality than any other disease process and, as a consequence, the platelet represents a major target for therapeutic intervention. Several factors contribute to the potent platelet-activating properties of ruptured plaques, including the high content of fibrillar collagens in the lesion, 1-3 the presence of tissue factor, 4 as well as the direct platelet-activating effects of high shear stress caused by arterial narrowing. [5][6][7][8] Rheologic disturbances at sites of arterial stenosis are dynamic and complex and are highly influenced by the level of narrowing and altered geometry of the vascular lumen. Nonetheless, high shear is an inevitable consequence of progressive vascular occlusion, establishing a potentially hazardous cycle of further platelet activation and thrombus growth.The mechanisms underlying platelet aggregation and thrombus formation have been extensively investigated and are highly influenced by the prevailing blood flow conditions. Under conditions of relatively low shear (0 to 1000 s Ϫ1 ), platelet aggregation is primarily mediated by soluble fibrinogen, which physically crosslinks platelets through engagement of integrin ␣ IIb ␤ 3 . 9-11 At progressively higher wall shear rates (1000 to 10 000 s Ϫ1 ) aggregation becomes mor...

Platelets lacking PIP5KIγ have normal integrin activation but impaired cytoskeletal-membrane integrity and adhesion

“…Our previous studies indicated that megakaryocytes derived from PIP5KIg-null yolk sac progenitor cells have a defect in anchoring their integrins to the underlying cytoskeleton. 35 We also observed that this defect was dependent on the lipid kinase activity of PIP5KIg. Although our prior work suggested that PIP5KIg was important for anchoring integrins, this study did not analyze membrane dynamics in a cell that required integrins for their principal function.…”

“…In addition, it has been postulated that platelets regulate the elasticity of their cell membranes during the initial phases of platelet adhesion. 35 Therefore, we reasoned that PIP5KIg might contribute to the regulation of integrins and cell elasticity in platelets. We observed by using an optical trap that PIP5KIg E17 2/2 PF4 Cre 1 platelets had a defect in anchoring their integrins to the underlying actin cytoskeleton ( Figure 3B).…”

Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity

“…59 ADP released from dense granules regulates a-granule secretion, [60][61][62] forming dense regions that stabilize the platelet aggregate. 63 Concomitant release of polyP with ADP from dense granules suggests that it may be retained in these low-solute transport areas. a-Granule release occurs at lower agonist concentrations than dense granule release, 64,65 suggesting that platelets at the edge of the core and within the shell may release fibrinogen and bind fibrin before polyP secretion, thereby providing an anchor to retain polyP in the locale of the activated platelet aggregates.…”

Apelin: an antithrombotic factor that inhibits platelet function

“…Differences in the chronological implication of these platelet activators and their intracellular signaling may be contributing factors, because platelet aggregation is a sequential process, with reversible and irreversible steps. [42][43][44] Therefore, inhibition of thrombin by apelin may affect all potential thrombin downstream effectors involved in platelet activation. Furthermore, the inability of apelin to inhibit platelet activation in the presence of TXA 2 or ADP may be related to ADP/TXA 2 intracellular signaling pathways that are not regulated by apelin or to those that may interfere with the apelin signaling.…”