The effect of upstream platelet-fibrinogen interactions on downstream adhesion and activation

Abstract

Circulating activated platelets roll and make transient contacts before ultimately adhering to a substrate. However, despite the dynamic nature of platelet adhesion, most in vitro adhesion and activation studies have focused on establishing local cause and effect relationships. Here, we determined the effect of exposing platelets to immobilized upstream human fibrinogen on downstream adhesion and activation. Microcontact printing was used to prepare substrates that contained well defined fibrinogen priming regions. Washed platelets were perfused over the substrates and adhesion and activation in a downstream capture region were compared with samples that did not contain a fibrinogen priming region. It was found that samples containing an upstream priming region resulted in higher adhesion, platelet spreading areas and aggregation than samples that lacked the priming region. Also, when the priming region was selectively blocked with a polyclonal anti-fibrinogen antibody, the platelet response was attenuated. To characterize this phenomenon further, flow cytometry was used to assess bulk platelet activation following fibrinogen priming. The expression of two activation markers, PAC-1 and P-selectin were quantified. Expression of both activation markers was found to be higher after perfusion over fibrinogen versus albumin-coated substrates.

Copyright © 2011 Elsevier Ltd. All rights reserved.

Figures

Fig. 1

Sample preparation using μCP: (A) Image of a random pattern used to prepare PDMS stamps with an 85% relative coverage area. (B) Nexterion-H samples were prepared with covalently immobilized protein patterns using μCP. (C) A schematic representation of the μCP process. First PDMS stamps are cast and cured in patterned masks. The stamps are transferred to a protein solution where they are “inked” by allowing the protein to adsorb to the surface. The protein coated stamp is placed in contact with the reactive surface allowing the protein transfer to occur. On Nexterion-H substrates, the printed surface was incubated in an albumin solution to passivate the unpatterned regions.

Fig. 2

A representative fluorescence image of an 85% printed fibrinogen pattern where the grey represents Alexa Fluor 488 labeled fibrinogen. Images were acquired after a vigorous surfactant rinse.

Fig. 3

Effect of an upstream immobilized fibrinogen priming region on downstream (A) adhesion, (B) aggregation, and (C) spreading area on Nexterion-H substrates. Samples (n = 30) were acquired 10–15 mm downstream of the priming region. The error bars represent the standard error of the mean with a 95% confidence interval.

Fig. 4

Blocking fibrinogen priming region with an anti-fibrinogen polyclonal antibody attenuates the downstream adhesion response. The effect of priming platelets with immobilized upstream fibrinogen on downstream adhesion was compared with samples containing no priming region and a priming region blocked with an anti-fibrinogen polyclonal antibody. Samples (n = 30) in the downstream capture region were averaged and error bars represent the standard error of the mean with a 95% confidence interval.

Fig. 5

Flow cytometry analysis of bulk platelet activation. (A) Pac-1 and (B) P-selectin expression were quantified on washed platelets following perfusion over both an albumin and a fibrinogen substrate. These samples were also compared with unstimulated (negative control) and thrombin stimulated (positive control) samples collected prior to perfusion. Samples (n = 4) represent the average percent of platelets expressing each receptor out of out of 10,000 recorded events. The error bars represent the standard error of the mean calculated from a 95% confidence interval.