Effect of Clopidogrel on Thrombus Formation in an Ex Vivo Parallel Plate Flow Chamber Model Cannot Be Reversed by Addition of Platelet Concentrates or vWF Concentrate

SummaryEpi-fluorescent video microscopy was used to evaluate the effect of ethanol on platelet mural thrombus formation. Whole blood, treated with ethanol, was perfused over collagen coated glass in a parallel-plate flow chamber at a shear rate of 1,000/s. Digital image processing and photodiode measurements were used to analyze the dynamics of thrombus growth on this surface. Ethanol concentrations as low as 0.02% v/v were found to inhibit 45 + 33% (± S.D.) of normal platelet accumulation on the slide while 0.2% v/v ethanol effected an 82 ± 15% inhibition of mural thrombus formation. While platelet adhesion to the collagen surface appeared unaffected by ethanol concentrations up to 0.1% v/v, 0.2% v/v ethanol had an effect on adhesion as well as aggregation. These results imply that low ethanol concentrations inhibit the formation of mural thrombi in a model of a damaged blood vessel at physiological shear rates. This inhibition would not be detected in systems which measure bulk aggregation, e.g. in aggregometric determinations.

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VCAM-1 is more effective than MAdCAM-1 in supporting eosinophil rolling under conditions of shear flow

Blood ◽

10.1182/blood.v95.2.592 ◽

2000 ◽

Vol 95 (2) ◽

pp. 592-601 ◽

Cited By ~ 41

Author(s):

P. Sriramarao ◽

Richard G. DiScipio ◽

Ronald R. Cobb ◽

Myron Cybulsky ◽

Greg Stachnick ◽

...

Keyword(s):

Monoclonal Antibody ◽

Parallel Plate ◽

Cell Adhesion Molecule ◽

Flow Chamber ◽

Β1 Integrin ◽

Vascular Cell Adhesion ◽

Parallel Plate Flow Chamber ◽

Vascular Cell

The ability of the 4 integrin counterligands vascular cell adhesion molecule (VCAM)-1 or mucosal addressin (MAd)CAM-1 to support eosinophil rolling or firm adhesion under conditions of physiologic flow has not been delineated. Using a parallel plate flow chamber in vitro and intravital microscopy in vivo, we demonstrate that eosinophil rolling and adhesion on VCAM-1 is mediated by both 4β1 and 4β7 integrins. Eosinophils rolled equally efficiently on both VCAM-1 2 domain and VCAM-1 7 domain, suggesting that the N-terminal 2 domains of VCAM-1 are sufficient to support eosinophil rolling under conditions of flow. Furthermore, activation of the eosinophil β1 integrin with monoclonal antibody (mAb) 8A2 resulted in both resistance to shear stress–induced detachment from VCAM-1 in vitro and in stable arrest of rolling eosinophils on interleukin (IL)-1β–stimulated venules in vivo. Eosinophils rolled less efficiently on MAdCAM-1– than on VCAM-1–coated coverslips under conditions of flow. However, eosinophils firmly adhered as efficiently to MAdCAM-1 as to VCAM-1. Overall, these results demonstrate that both VCAM-1 and MAdCAM-1 can support eosinophil firm adhesion under conditions of flow. In contrast, VCAM-1 is significantly more efficient than MAdCAM-1 in supporting eosinophil rolling under conditions of flow.

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Platelets, circulating tissue factor, and fibrin colocalize in ex vivo thrombi: real-time fluorescence images of thrombus formation and propagation under defined flow conditions

Blood ◽

10.1182/blood-2002-03-0902 ◽

2002 ◽

Vol 100 (8) ◽

pp. 2787-2792 ◽

Cited By ~ 95

Author(s):

Viji Balasubramanian ◽

Eric Grabowski ◽

Alessandra Bini ◽

Yale Nemerson

Keyword(s):

Real Time ◽

Tissue Factor ◽

Ex Vivo ◽

Thrombus Formation ◽

High Sensitivity ◽

Flow Chamber ◽

Flow Conditions ◽

Coated Glass ◽

Relative Contribution ◽

Fluorescent Markers

Although it is generally accepted that the initial event in coagulation and intravascular thrombus formation is the exposure of tissue factor (TF) to blood, there is still little agreement about the mechanisms of thrombus propagation and the identities of the molecular species participating in this process. In this study, we characterized the thrombotic process in real-time and under defined flow conditions to determine the relative contribution and spatial distribution of 3 components of the thrombi: circulating or blood-borne TF (cTF), fibrin, and platelets. For this purpose, we used high-sensitivity, multicolor immunofluorescence microscopy coupled with a laminar flow chamber. Freshly drawn blood, labeled with mepacrine (marker for platelets and white cells), anti-hTF1Alexa.568 (marker for tissue factor), and anti-T2G1Cy5 (marker for fibrin) was perfused over collagen-coated glass slides at wall shear rates of 100 and 650 s−1. A motorized filter cube selector facilitated imaging every 5 seconds at 1 of 3 different wavelengths, corresponding to optimal wavelengths for the 3 markers above. Real-time video recordings obtained during each of 10 discrete experiments show rapid deposition of platelets and fibrin onto collagen-coated glass. Overlay images of fluorescent markers corresponding to platelets, fibrin, and cTF clearly demonstrate colocalization of these 3 components in growing thrombi. These data further support our earlier observations that, in addition to TF present in the vessel wall, there is a pool of TF in circulating blood that contributes to the propagation of thrombosis at a site of vascular injury.

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Wall Shear Stress Determination in a Small-Scale Parallel Plate Flow Chamber Using Laser Doppler Velocimetry Under Laminar, Pulsatile and Low-Reynolds Number Turbulent Flows

Journal of Fluids Engineering ◽

10.1115/1.4039158 ◽

2018 ◽

Vol 140 (6) ◽

Cited By ~ 1

Author(s):

Hamed Avari ◽

Kem A. Rogers ◽

Eric Savory

Keyword(s):

Shear Stress ◽

Wall Shear Stress ◽

Laser Doppler Velocimetry ◽

Parallel Plate ◽

Flow Chamber ◽

Laser Doppler ◽

Small Scale ◽

Doppler Velocimetry ◽

Flow Conditions ◽

Parallel Plate Flow Chamber

The parallel plate flow chamber (PPFC) has gained popularity due to its applications in fields such as biological tissue engineering. However, most of the studies using PPFC refer to theoretical relations for estimating the wall shear stress (WSS) and, hence, the accuracy of such quantifications remains elusive for anything other than steady laminar flow. In the current study, a laser Doppler velocimetry (LDV) method was used to quantify the flow in a PPFC (H = 1.8 mm × W = 17.5 mm, Dh = 3.26 mm, aspect ratio = 9.72) under steady Re = 990, laminar pulsatile (carotid Re0-mean = 282 as well as a non-zero-mean sinusoidal Re0-mean = 45 pulse) and low-Re turbulent Re = 2750 flow conditions. A mini-LDV probe was applied, and the absolute location of the LDV measuring volume with the respect to the wall was determined using a signal monitoring technique with uncertainties being around ±27 μm. The uniformity of the flow across the span of the channel, as well as the WSS assessment for all the flow conditions, was measured with the uncertainties all being less than 16%. At least two points within the viscous sublayer of the low-Re turbulent flow were measured (with the y+ for the first point < 3) and the WSS was determined using two methods with the differences between the two methods being within 5%. This paper for the first time presents the experimental determination of WSS using LDV in a small-scale PPFC under various flow conditions, the challenges associated with each condition, and a comparison between the cases. The present data will be useful for those conducting biological or numerical modeling studies using such devices.

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Abstract 54: ML355 in Prevention of Thrombosis in vivo with Minimal Effects on Hemostasis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.54 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Reheman Adili ◽

Katherine Mast ◽

Michael Holinstat

Keyword(s):

Ex Vivo ◽

Platelet Reactivity ◽

Thrombus Formation ◽

Flow Chamber ◽

Human Platelets ◽

Mass Spectrometry Analysis ◽

Vessel Occlusion ◽

Spectrometry Analysis ◽

Thrombosis Model

12-lipoxygenase (12-LOX) has been demonstrated to regulate platelet function, hemostasis, and thrombosis ex vivo , supporting a key role for 12-LOX in regulation of in vivo thrombosis. While pharmacologically targeting 12-LOX in vivo has been a challenge to date, the recent development of the 12-LOX selective inhibitor, ML355, as an effective antiplatelet therapeutic in vivo was assessed. ML355 potently inhibited thrombin and other agonist-induced platelet aggregation ex vivo in washed human platelets and inhibited downstream oxylipin production of platelet 12-LOX as confirmed by Mass spectrometry analysis. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen was attenuated in human whole blood treated with ML355 to a greater extent compared to aspirin. In vivo , PK assessment of ML355 showed reasonable 12-LOX plasma levels 12 hours following administration of ML355. FeCl 3 -induced injury of the mesenteric arterioles resulted in less stable thrombi in 12-LOX -/- mice and ML355-treated WT mice resulting in impairment of vessel occlusion. Additionally, ML355 dose-dependently inhibited laser-induced thrombus formation in the cremaster arteriole thrombosis model in WT, but not in 12-LOX -/- mice. Importantly, hemostatic plug formation and bleeding following treatment with ML355 were not affected in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model. Our data strongly supports 12-LOX as a key determinant of platelet reactivity in vivo and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapeutics.

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