Microfluidic assay of antiplatelet agents for inhibition of shear-induced platelet adhesion and activation

Lab on a Chip ◽  
2021 ◽  
Vol 21 (1) ◽  
pp. 174-183
Author(s):  
Shekh Mojibur Rahman ◽  
Vladimir Hlady
Keyword(s):  
Whole Blood ◽  
Platelet Adhesion ◽  
Antiplatelet Agents ◽  
Microfluidic System ◽  
Flow Channel ◽  
Protein Capture ◽  
Capture Region

We have developed a microfluidic system to perfuse whole blood through a flow channel with an upstream stenotic region and a downstream protein capture region.

Carboxyl-Terminal Thrombospondin-Type 1 (TSP-1) Repeats of ADAMTS13 Inhibits Platelet Adhesion to Collagen-Coated Surface Under Shear Stress, Independent of Proteolytic Activity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 195-195 ◽  
Author(s):  
Juan (Jenny) Xiao ◽  
X. Long Zheng
Keyword(s):  
Shear Stress ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Surface Coverage ◽  
Thrombus Formation ◽  
Microfluidic System ◽  
Dependent Manner ◽  
C Terminus ◽  
Coated Surface

Abstract Abstract 195 ADAMTS13 contains multiple free thiols on its surface, which may form disulfide bonds with surface-exposed free thiols on plasma-derived von Willebrand factor (VWF). This interaction may prevent lateral association of apposed VWF under arterial shear stress. However, the functional consequence of ADAMTS13-VWF interaction without proteolysis is not known. We hypothesize that the interaction between the C-terminus of ADAMTS13 and the C-terminus of VWF inhibits thrombus formation under shear stress. Using a BioFlux microfluidic system, we showed that under arterial shear stress, 10 dyn/cm2, fluorescein-labeled platelets from PPACK (thrombin inhibitor) anti-coagulated human whole blood adhered to collagen (type I)-coated surface in a time-dependent manner. Addition of human recombinant full-length ADAMTS13 (10 nM) into whole blood dramatically reduced the surface coverage of fluorescein-labeled platelets. Conversely, addition of an inhibitory polyclonal anti-ADAMTS13 IgGs (150 ug/ml) to whole blood dramatically accelerated the accumulation of fluorescein-labeled platelets. These results suggest that this microfluidic system is highly sensitive for the assessment of anti-thrombotic function of ADAMTS13. Under the same conditions, we were able to further show that addition of recombinant C-terminal fragment of ADAMTS13 comprising of the 5th to 8th thrombospondin type 1 (TSP1) repeats and two CUB domains (T5C) or the 2nd to 8th TSP1 repeats and two CUB domains (T2C) into whole blood also inhibited the surface coverage of fluorescein-labeled platelets on collagen-coated surface in a concentration-dependent manner. In the presence of 0.1 μM and 0.5 μM of recombinant T2C or T5C, the surface coverage of fluorescein-labeled platelets was reduced by ∼40% and ∼60%, respectively. The inhibitory activity of these recombinant C-terminal fragments was nearly abolished if pre-treated with 40 mM of N-ethylmaleimide which blocked surface-exposed free thiols. Moreover, recombinant CUB domains at the highest concentration tested (1.0 μM) did not appear to alter the surface coverage of fluorecein-labeled platelets under the same conditions. These results suggest that the C-terminal TSP1 repeats of ADAMTS13 inhibit platelet adhesion and aggretion or thrombus formation through thiol-thiol interactions between ADAMTS13 and VWF (or other proteins). We conclude that the C-terminal TSP1 repeats may modulate thrombus formation independent of proteolytic activity. Disclosures: No relevant conflicts of interest to declare.

Fine Structure of Platelet Interaction with a New Microcrystalline Collagen Preparation

1974 ◽  
Vol 32 ◽  
pp. 58-59
Author(s):  
W. H. Zucker ◽  
R. G. Mason
Keyword(s):  
Fine Structure ◽  
Platelet Aggregation ◽  
Hydrochloric Acid ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Hemostatic Agent ◽  
Native Collagen ◽  
Fibrillar Morphology ◽  
Clinical Interest ◽  
New Form

Platelet adhesion initiates platelet aggregation and is an important component of the hemostatic process. Since the development of a new form of collagen as a topical hemostatic agent is of both basic and clinical interest, an ultrastructural and hematologic study of the interaction of platelets with the microcrystalline collagen preparation was undertaken.In this study, whole blood anticoagulated with EDTA was used in order to inhibit aggregation and permit study of platelet adhesion to collagen as an isolated event. The microcrystalline collagen was prepared from bovine dermal corium; milling was with sharp blades. The preparation consists of partial hydrochloric acid amine collagen salts and retains much of the fibrillar morphology of native collagen.

Inhibition of Platelet Adhesion to Fibrin(ogen) in Flowing Whole Blood by Arg-Gly-Asp and Fibrinogen γ-Chain Carboxy Terminal Peptides

1992 ◽  
Vol 68 (06) ◽  
pp. 694-700 ◽  
Author(s):  
Roy R Hantgan ◽  
Silvia C Endenburg ◽  
I Cavero ◽  
Gérard Marguerie ◽  
André Uzan ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Integrin Receptor ◽  
Shear Rates ◽  
Perfusion Chamber ◽  
Receptor Recognition ◽  
Adhesive Interactions ◽  
Coated Surfaces ◽  
Carboxy Terminal

SummaryWe have employed synthetic peptides with sequences corresponding to the integrin receptor-recognition regions of fibrinogen as inhibitors of platelet aggregation and adhesion to fibrinogen-and fibrin-coated surfaces in flowing whole blood, using a rectangular perfusion chamber at wall shear rates of 300 s–1 and 1,300 s–1. D-RGDW caused substantial inhibition of platelet aggregation and adhesion to fibrinogen and fibrin at both shear rates, although it was least effective at blocking platelet adhesion to fibrin at 300 s–1. RGDS was a weaker inhibitor, and produced a biphasic dose-response curve; SDRG was inactive. HHLGGAK-QAGDV partially inhibited platelet aggregation and adhesion to fibrin(ogen) at both shear rates. These results support the identification of an RGD-specific receptor, most likely the platelet integrin glycoprotein IIb: III a, as the primary receptor responsible for platelet: fibrin(ogen) adhesive interactions under flow conditions, and indicate that platelet adhesion to surface bound fibrin(ogen) is stabilized by multivalent receptor-ligand contacts.

An integrated microfluidic system for on-chip enrichment and quantification of circulating extracellular vesicles from whole blood

Lab on a Chip ◽  
2019 ◽  
Vol 19 (19) ◽  
pp. 3305-3315 ◽  
Author(s):  
Yi-Sin Chen ◽  
Yu-Dong Ma ◽  
Chihchen Chen ◽  
Shu-Chu Shiesh ◽  
Gwo-Bin Lee
Keyword(s):  
Extracellular Vesicles ◽  
Immunosorbent Assay ◽  
Whole Blood ◽  
Magnetic Beads ◽  
Extracellular Vesicle ◽  
Microfluidic System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Human Whole Blood ◽  
On Chip ◽  
Chip Enrichment

An integrated microfluidic system was developed for extracellular vesicle (EV) enrichment and quantification by using anti-CD63-coated magnetic beads and an on-chip enzyme-linked immunosorbent assay in human whole blood.

IS THE ENDOTHELIAL EXTRACELLULAR MATRIX THROMBOGENIC OR THROMBORESISTANT? EFFECT OF PREPARATION AND 13-HODE LEVELS

1987 ◽  
Author(s):  
M R Buchanan ◽  
E Bastida ◽  
J Aznar-Salatti ◽  
P de Groot
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cell ◽  
Cellulose Acetate ◽  
Surface Area ◽  
Whole Blood ◽  
Hplc Analysis ◽  
Platelet Adhesion ◽  
Flow Conditions ◽  
Hydroxyoctadecadienoic Acid ◽  
Static Conditions

It is generally thought that the extracellular matrix (ECM) is thrombogenic.However,one of us (MRB) has reported that the ECM is thromboresistant,and postulated that this was due to the release of endothelial cell (EC) 13-hydroxyoctadecadienoic acid (13-HODE) into the ECM. To test this possibility, we measured platelet adhesion (PLT ADH) onto cultured ECs and their ECMs exposed by 3 methods. We also extracted the ECMs for HPLC analysis of 13-HODE.PLT ADH was expressed as i)adhesion of 3H-adenine labelled platelets/mm2 of ECs or ECMs under static conditions, and ii) % surface^ area coverage measured morphometrically following 5"perfusion with citrated whole blood at 1300 sec-1 in the flat chamber.ECMs were prepared by removing the EC monolayers by freeze thawing , cellulose acetate stripping or NH4OH treatment. PLT ADH to ECs under static and flow conditions were 4700±240/mm2 and 0.1%, respectively, and were associated with 12,6± 1 pg of 13-HODE/mm2 of EC surface (M+SEM). Removal of the ECs by freeze thawing or stripping, resulted in a 18% and 25% increase in PLT ADH to the ECM,under static and flow conditions respectively, and a 80% decrease in ECM associated 13-HODE level. Removal of the EC by NH4OH resulted in a 380% and 770% increase in PLT ADH to the ECM in static and flow conditions. 13-HODE was undetectable.These data support the hypothesis that 13-HODE released from ECs influences the ECM thrombogenecity, and indicate that the residual amounts of components present in the ECMs following EC removal is influenced by the method of ECM preparation.

scholarly journals Fibulin-1 mediates platelet adhesion via a bridge of fibrinogen

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2569-2577 ◽  
Author(s):  
S Godyna ◽  
M Diaz-Ricart ◽  
WS Argraves
Keyword(s):  
Extracellular Matrix ◽  
Vascular Injury ◽  
Whole Blood ◽  
Platelet Adhesion ◽  
Solid Phase ◽  
Blood Perfusion ◽  
General Mechanism ◽  
Binding Assays ◽  
Solid Phase Binding ◽  
Coated Surfaces

Fibulin-1 is a component of the extracellular matrix that surrounds vascular smooth muscle. This observation, along with the recent finding that fibulin-1 can bind fibrinogen (J Biol Chem 270:19458, 1995), prompted investigation into the potential role of fibulin-1 as a thrombogenic agent. In perfusion chamber assays, platelets in whole blood under flow conditions attached and spread on surfaces coated with fibulin-1. This adhesion was completely blocked by fibulin-1 antibodies. Platelets free of plasma did not attach to fibulin-1 coated surfaces; however, with the addition of fibrinogen, platelet adhesion to fibulin-1 took place. When detergent extracts of platelets were subjected to fibulin-1-Sepharose affinity chromatography, the integrin alpha IIb beta 3 was selected. Solid phase binding assays using purified components showed that integrin alpha IIb beta 3 could not bind directly to fibulin-1 but in the presence of fibrinogen the integrin bound to fibulin-1-coated surfaces. Monoclonal alpha IIb beta 3 antibodies capable of blocking its interaction with fibrinogen completely blocked platelet adhesion to fibulin-1 in both whole blood perfusion and static adhesion assays. The results show that fibulin-1 can support platelet attachment via a bridge of fibrinogen to the platelet integrin alpha IIb beta 3. When fibroblast monolayers containing extracellular matrix-incorporated fibulin-1 were used as adhesion substrates, platelet adhesion in the presence of fibrinogen could be inhibited by 30% using antibodies to fibulin-1. Following vascular injury, fibulin-1 present in the extracellular matrix of the vessel wall may therefore interact with plasma fibrinogen and promote platelet adhesion, leading to the formation of a platelet plug. Thus, fibulin-1 joins the list of matrix proteins including collagens I and IV and fibronectin that mediate platelet adhesion via a plasma protein bridge. This bridging phenomenon may represent a general mechanism by which platelets interact with exposed subendothelial matrices following vascular injury.

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