Development of a perfusion chamber assay to study in real time the kinetics of thrombosis and the antithrombotic characteristics of antiplatelet drugs

Abstract

BackgroundArterial thrombosis triggered by vascular injury is a balance between thrombus growth and thrombus fragmentation (dethrombosis). Unbalance towards thrombus growth can lead to vascular occlusion, downstream ischemia and tissue damage.Here we describe the development of a simple methodology that allows for continuous real time monitoring and quantification of both processes during perfusion of human blood under arterial shear rate conditions. Using this methodology, we have studied the effects of antiplatelet agents targeting COX-1 (aspirin), P2Y12 (2-MeSAMP, clopidogrel), GP IIb-IIIa (eptifibatide) and their combinations on the kinetics of thrombosis over time.ResultsUntreated samples of blood perfused over type III collagen at arterial rates of shear promoted the growth of stable thrombi. Modulation by eptifibatide affected thrombus growth, while that mediated by 2-MeSAMP and aspirin affected thrombus stability. Using this technique, we confirmed the primacy of continuous signaling by the ADP autocrine loop acting on P2Y12 in the maintenance of thrombus stability. Analysis of the kinetics of thrombosis revealed that continuous and prolonged analysis of thrombosis is required to capture the role of platelet signaling pathways in their entirety. Furthermore, studies evaluating the thrombotic profiles of 20 healthy volunteers treated with aspirin, clopidogrel or their combination indicated that while three individuals did not benefits from either aspirin or clopidogrel treatments, all individuals displayed marked destabilization profiles when treated with the combination regimen.ConclusionsThese results show the utility of a simple perfusion chamber technology to assess in real time the activity of antiplatelet drugs and their combinations. It offers the opportunity to perform pharmacodynamic monitoring of arterial thrombosis in clinical trials and to investigate novel strategies directed at inhibiting thrombus stability in the management of cardiovascular disease.

Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk

“…Partial disaggregation was achieved with physiologically relevant concentrations of adenosine. Meanwhile, others have shown that platelet aggregation stability is inversely proportional to platelet ADP receptor occupancy (44), and partial disaggregation can be caused by the P2Y 12 antagonist 2-MeSAMP (45). Hence, the immediate and complete reversal of human platelet aggregation by APT102 (Fig.…”

Microfluidic Assay of Platelet Deposition on Collagen by Perfusion of Whole Blood from Healthy Individuals Taking Aspirin

“…VerifyNow Aspirin is an assay based on light transmittance but lacks a hemodynamic flow field. Most recently, development of a perfusion chamber allows for study of ASA-dependent changes in platelet deposition (24). Our methodology is distinct from that of Stephens et al (24), where whole blood was perfused through an entire glass capillary coated with human type III fibrillar collagen at 1000 or 1500 s −1 .…”

“…Most recently, development of a perfusion chamber allows for study of ASA-dependent changes in platelet deposition (24). Our methodology is distinct from that of Stephens et al (24), where whole blood was perfused through an entire glass capillary coated with human type III fibrillar collagen at 1000 or 1500 s −1 . A collagen-coated capillary creates a distance of many centimeters of collagen for platelets to activate, adhere, embolize, and recapture on the surface.…”

A shear gradient-activated microfluidic device for automated monitoring of whole blood haemostasis and platelet function

“…2b ). Based on the previous studies showing that the size of a growing thrombus measured in vitro correlates linearly with measured light intensity 41 42 , we can approximate that , where A (t) is the cross-sectional area available for blood flow through the occluding channel at a given time, A max is the initial cross-sectional area of the microchannel and Sig( t ) is a sigmoidal function. Further, since the hydraulic resistance ( R h ) of the occluding microchannel approximately scales as (ref.…”