A systems approach to hemostasis: 1. The interdependence of thrombus architecture and agonist movements in the gaps between platelets

Abstract

Key Points• Thrombus formation and contraction alters local molecular transport, which regulates agonist distribution and platelet activation.• Semaphorin 4D contactdependent signaling increases platelet activation, but does not affect platelet packing or agonist transport.Hemostatic thrombi develop a characteristic architecture in which a core of highly activated platelets is covered by a shell of less-activated platelets. Here we have used a systems biology approach to examine the interrelationship of this architecture with transport rates and agonist distribution in the gaps between platelets. Studies were performed in mice using probes for platelet accumulation, packing density, and activation plus recently developed transport and thrombin activity probes. The results show that intrathrombus transport within the core is much slower than within the shell. The region of slowest transport coincides with the region of greatest packing density and thrombin activity, and appears prior to full platelet activation. Deleting the contact-dependent signaling molecule, Sema4D, delays platelet activation, but not the emergence of the low transport region. Collectively, these results suggest a timeline in which initial platelet accumulation and the narrowing gaps between platelets create a region of reduced transport that facilitates local thrombin accumulation and greater platelet activation, whereas faster transport rates within the shell help to limit thrombin accumulation and growth of the core. Thus, from a systems perspective, platelet accumulation produces an altered microenvironment that shapes thrombus architecture, which in turn affects agonist distribution and subsequent thrombus growth. (Blood. 2014;124(11):1808-1815 IntroductionThe hemostatic response balances the need to halt bleeding with the need to avoid inappropriate vascular occlusion. Recent reports of hemostatic thrombi formed in vivo have demonstrated that the extent of platelet activation within a growing thrombus is heterogeneous [1][2][3][4][5] and can result in a characteristic core-and-shell architecture. We have shown that the core region develops adjacent to the injury site and consists of fully activated, closely packed platelets that have undergone a-granule exocytosis, which allows them to be recognized by the appearance of the a-granule membrane protein, P-selectin, on their surface. 3 The shell is a less stable region that coats the core and consists of loosely packed, less activated platelets. 3Regional differences in the extent of platelet activation can potentially be driven by multiple factors. Here we have adopted a systems biology perspective, looking beyond the events in any one platelet to test the idea that the emerging architecture of the hemostatic response serves as both a driver and a reflection of differences in intrathrombus molecular transport rates and consequent differences in agonist distribution. Numerous platelet agonists are present during vascular injury, including collagen, thrombin, adenosine 59-diphosphate (ADP)...

Defective release of α granule and lysosome contents from platelets in mouse Hermansky-Pudlak syndrome models

“…Thrombin activity in this model extends as a gradient from the site of vascular injury [73][74][75][76] and is necessary for full platelet activation and a granule secretion. 51,77 Our results indicate that ADP signaling reinforces platelet activation at the edge of this gradient where thrombin activity starts to decline.…”

Platelets and hemostasis: a new perspective on an old subject

“…Second, they suggest that the strengths and limitations of some commonly used antiplatelet agents reflect not only their half-lives, affinities, and off-rates, but also where they work on the platelet signaling network and how well they penetrate thrombus structure. For example, observational studies performed in vivo suggest that widely prescribed P2Y 12 antagonists impair hemostasis and reduce recurrent thrombotic events by destabilizing the thrombus shell with comparatively little impact on the thrombus core, at least in the microcirculation where these studies were performed. Finally, the data suggest that tests of on-treatment platelet function in Figure 2.…”

“…PAR1 produces a quick burst of signaling; PAR4 a more sustained response. ADP activates P2Y 1 and P2Y 12 , the latter coupled to G i2 and the former to G q . Signals mediated by G q activate phospholipase Cb, leading to increased cytosolic Ca 21 , activation of Rap1b, and, ultimately, to the activation of a IIb b 3 .…”

“…As packing density increases, transport becomes dominated by diffusion rather than convection, slowing movement to an even greater extent ( Figure 1C). [10][11][12] Regional differences in packing density also affect the distribution of platelet agonists. The result is the appearance of concentration gradients in which the distribution of each agonist is also affected by its physical properties and binding to other molecules.…”

Platelets stop us leaking

“…A consequence of the tightly packed core is restricted permeation of plasma-borne molecules through the core, and therefore greater local thrombin activity resulting in fibrin formation and increased platelet activation. [6][7][8][9] Indeed, inhibition of thrombin disrupts thrombus core structure, while the size of the thrombus shell is reduced in the presence of adenosine 59-diphosphate (ADP) receptor antagonists. 6 In this issue, Welsh et al have explored which features of the heterogeneous architecture of platelet thrombi explain their ability to seal blood vessels to achieve "plasma stasis," and determined whether the use of antiplatelet and anticoagulant agents which impair thrombus structure interfere with the formation of a vascular seal.…”