Occlusive Arterial Thrombosis and Thrombolysis

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Kim, Dongjune A.
Ku, David N.
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Occlusive arterial thrombosis in a stenotic vessel can cause the cessation of blood flow to the brain or heart, which can result in a patient’s death. Following the incident, the patient may be given a thrombolytic agent or undergo a thrombectomy to recanalize the occluded vessel and to regain blood flow. The current standard for thrombolytic treatment involves the administration of tissue plasminogen activator (tPA), which has shown limited efficacy and creates a bleeding risk. An arterial thrombus differs from a venous thrombus in terms of both its mechanism of formation and composition. The von Willebrand Factor (VWF)-platelet-rich arterial thrombus forms by shear induced platelet aggregation (SIPA), and can occlude the vessel in harsh hemodynamic environments consisting of high shear rates and high blood pressure. It is critical to know what key factors affect the formation of occlusive SIPA clots, how these clots stabilize under arterial conditions, and which agents should be used for a lysis. The overall goal of this thesis is to investigate these three important aspects of arterial thrombosis formed under a pathological high shear. VWF is a protein that plays a critical role in forming SIPA clots under high shear conditions. Platelet ⍺-granules contain 50 times more concentrated VWF compared to plasma VWF. The role of ⍺-granules in forming occlusive arterial thrombi was studied using Nbeal2-/- mice that do not release ⍺-granules. Without ⍺-granule release, there was no rapid platelet accumulation and no subsequent channel occlusion occurred. Thus, ⍺-granules could be a potential new target for the prevention of arterial thrombosis. An occlusive SIPA clot formed with ⍺-granule release was VWF-platelet-rich. SIPA clots formed using arterial-like hemodynamic conditions in vitro were compared to coagulation clots generated under static conditions. SEM images of the SIPA clot revealed densely packed platelets, which can be converted into 1.23 billion platelets in the total clot. Histological images showed distinctive structural features that were different upstream, near the apex, and downstream of the stenosis. The upstream region showed mountain-like platelet aggregates protruding to the central lumen that left a large hole in the center, while RBCs trapped in valleys. Near the apex, a uniformly dense platelet mass formed throughout the lumen to fully occlude the channel. In the upstream part of the occlusive mass, platelets were amorphous consistent with activation. In the downstream part of the mass, spherical platelets dominated, consistent with no activation. The thrombus ended abruptly in the expansion past the throat of the stenosis and did not extend to the walls located in the flow separation zone. Measurements of pressure and flow could be used to characterize the SIPA clot as three orders of magnitude more permeable than a coagulation clot. However, although the SIPA clot was more porous and permeable, it was two times stiffer and seven times stronger than the coagulation clot, which would make it capable of occluding a stenosed coronary artery within a 2-mm clot length. Different types of clots may require different thrombolytic agents for lysis and vessel recanalization. Five different thrombolytic agents were tested on SIPA and coagulation clots in an in vitro model. A fibrinolytic agent, tPA lysed the coagulation clots but not the SIPA clots. Meanwhile, a novel thrombolytic agent, N,N’-Diacetyl-L-cystine (DiNAC) significantly lysed the SIPA clots without dissolving the coagulation clots. The results of this study may lead to the development of a new antithrombotic agent that inhibits formation or release of VWF from ⍺-granules of platelets, thrombectomy devices that account for SIPA clot permeability and strength, and a new thrombolytic treatment using DiNAC.
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