von Willebrand factor (VWF) is essential for the induction of arterial thrombosis. In this study, we investigated the critical role of platelet VWF in occlusive thrombosis formation at high shear in mice that do not express platelet VWF (Nbeal2−/−). Using in silico modeling, in vitro high-shear microfluidics, and an in vivo Folts model of arterial thrombosis we reproduced the platelet dynamics that occur under pathological flow in a stenosed vessel. Computational fluid dynamics (CFDs) simulated local hemodynamics in a stenosis based on arterial geometries. The model predicted shear rates, time course of platelet adhesion, and time to occlusion. These predictions were validated in vitro and in vivo. Occlusive thrombosis developed in wild-type control mice that had normal levels of plasma VWF and platelet VWF in vitro and in vivo. Occlusive thrombosis did not form in the Nbeal2−/− mice that had normal plasma VWF and an absence of platelet VWF. Occlusive thrombosis was corrected in Nbeal2−/− microfluidic assays by the addition of exogenous normal platelets with VWF. Combining model and experimental data, we demonstrated the necessary requirement of platelet VWF in α-granules in forming an occlusive thrombus under high shear. These results could inspire new pharmacological targets specific to pathological conditions and prevent arterial thrombosis.
Thrombus formation in major arteries is life threatening. In this review article, we discuss how an arterial thrombus can form under pathologically high shear stresses, with bonding rates estimated to be the fastest Kon values in biochemistry. During occlusive thrombosis in arteries, the growth rate of the thrombus explodes to capture a billion platelets in about 10 min. Close to 100% of all platelets passing the thrombus are captured by long von Willebrand factor (vWF) strands that quickly form tethered nets. The nets grow in patches where shear stress is high, and the local concentration of vWF is elevated due to α-granule release by previously captured platelets. This rapidly formed thrombus has few red blood cells and so has a white appearance and is much stronger and more porous than clots formed through coagulation. Understanding and modeling the biophysics of this event can predict totally new approaches to prevent and treat heart attacks and strokes.
Atherothrombosis leads to complications of myocardial infarction and stroke as a result of shear-induced platelet aggregation (SIPA). Clinicians and researchers may benefit from diagnostic and benchtop microfluidic assays that assess the thrombotic activity of an individual. Currently, there are several different proposed point-of-care diagnostics and microfluidic thrombosis assays with different design parameters and end points. The microfluidic geometry, surface coatings, and anticoagulation may strongly influence the precision of these assays. Variability in selected end points also persists, leading to ambiguous results. This study aims to assess the effects of three physiologically relevant extrinsic design factors on the variability of a single end point to provide a quantified rationale for design parameter and end-point standardization. Using a design of experiments approach, we show that the methods of channel fabrication and collagen surface coating significantly impact the variability of occlusion time from porcine whole blood, while anticoagulant selection between heparin and citrate did not significantly impact the variability. No factor was determined to significantly impact the mean occlusion time within the assay. Occlusive thrombus was found to consistently form in the first third (333 μm) of the high shear zone and not in the shear gradient regions. The selection of these factors in the design of point-of-care diagnostics and experimental SIPA assays may lead to increased precision and specificity in high shear thrombosis studies.
The search persists for a safe and effective agent to lyse arterial thrombi in the event of acute heart attacks or strokes due to thrombotic occlusion. The culpable thrombi are composed either primarily of platelets and von Willebrand Factor (VWF), or polymerized fibrin, depending on the mechanism of formation. Current thrombolytics were designed to target red fibrin-rich clots, but may be not be efficacious on white VWF-platelet-rich arterial thrombi. We have developed an in vitro system to study the efficacy of known and proposed thrombolytic agents on white clots formed from whole blood in a stenosis with arterial conditions. The agents and adjuncts tested were tPA, ADAMTS-13, abciximab, N-acetyl cysteine, and N,N’-Diacetyl-L-cystine (DiNAC). Most of the agents, including tPA, had little thrombolytic effect on the white clots. In contrast, perfusion of DiNAC lysed thrombi as quickly as 1.5 min, which ranged up to 30 min at lower concentrations, and resulted in an average reduction in surface area of 71 ± 20%. The clot burden was significantly reduced compared to both tPA and a saline control (p<0.0001). We also tested the efficacy of all agents on red fibrinous clots formed in stagnant conditions. DiNAC did not lyse red clots, whereas tPA significantly lysed red clot over 48 h (p<0.01). These results lead to a novel use for DiNAC as a possible thrombolytic agent against acute arterial occlusions that could mitigate the risk of hyper-fibrinolytic bleeding.
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