Microfluidic devices for studies of shear-dependent platelet adhesion - PubMed (original) (raw)

Microfluidic devices for studies of shear-dependent platelet adhesion

Edgar Gutierrez et al. Lab Chip. 2008 Sep.

Abstract

Adhesion of platelets to blood vessel walls is a shear stress dependent process that promotes arrest of bleeding and is mediated by the interaction of receptors expressed on platelets with various extracellular matrix (ECM) proteins that may become exposed upon vascular injury. Studies of dynamic platelet adhesion to ECM-coated substrates in conventional flow chambers require substantial fluid volumes and are difficult to perform with blood samples from a single laboratory mouse. Here we report dynamic platelet adhesion assays in two new microfluidic devices made of PDMS. Small cross-sections of the flow chambers in the devices reduce the blood volume requirements to <100 microl per assay, making the assays compatible with samples of whole blood obtained from a single mouse. One device has an array of 8 flow chambers with shear stress varying by a factor of 1.93 between adjacent chambers, covering a 100-fold range from low venous to arterial. The other device allows simultaneous high-resolution fluorescence imaging of dynamic adhesion of platelets from two different blood samples. Adhesion of platelets in the devices to three common ECM substrate coatings was verified to conform with published results. The devices were subsequently used to study the roles of extracellular and intracellular domains of integrin alphaIIbbeta3, a platelet receptor that is a central mediator of platelet aggregation and thrombus formation. The study involved wild-type mice and two genetically modified mouse strains and showed that the absence of the integrin impaired adhesion at all shear stresses, whereas a mutation in its intracellular domain reduced the adhesion only at moderate and high stresses. Because of small sample volumes required, the devices could be employed in research with genetically-modified model organisms and for adhesion tests in clinical settings with blood from neonates.

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Figures

Figure 1

Drawings of microchannel networks of the two microfluidic devices used in the study: A. device 1 and B. device 2. 24 µm and 250 µm deep channels are shown in dark and light grey, respectively. The 250 µm deep feeder channels minimize the shear stress on the way from the inlet to the test chamber and, just as the collector channels, have low flow resistance and nearly uniform pressure in them. Flow rate through the 24 µm test chambers is set by the flow resistance of the resistance channels connected in series with the test chambers. The flow rate is highest for the test chamber 1 and lowest for the test chamber 8. The bypass channels help synchronize the injection of blood into different test chambers.

Figure 2

Validation of platelet adhesion to physiologic matrices in microfluidic devices. A. Representative trajectories of individual platelets on VWF without inhibitor (black symbols; a and b) and with 20 µM integrilin, an anti-αIIbβ3 antagonist (grey symbols; c and d). Long plateaus correspond to prolonged periods of rest (a) that may lead to stable attachment (b) and are only observed for untreated cells. All platelet surface interactions are abolished in the presence of 10 µg/mL AP-1, an anti-GPIbα antibody (not shown). B. Histogram of the fraction of untreated cells and cells treated with integrilin that become stably attached to VWF at 3.4 and 50 dynes/cm2 after 1 min of flow. C. Adhesion to fibrinogen is αIIbβ3 dependent. Whole blood was incubated with or without 15 µg/mL Ib5, an anti-murine αIIbβ3 monoclonal antibody, and the platelet adhesion to fibrinogen was determined after 1 min of flow at τ = 3.4 and 13.4 dynes/cm2. Average values of 3 separate experiments ± SEM are shown. D. Fluorescence micrographs of a test chamber with collagen matrix in the device 1 at 13.4 dynes/cm2 showing normal thrombus growth from whole blood. After 1 minute of flow (left panel), there is a monolayer of adherent platelets serving as nucleation centers that develop into thrombi after 4 min of flow (right panel).

Figure 3

Dynamic adhesion of platelets to fibrinogen depends on the presence of αIIbβ3 and its cytoplasmic tail associations. A. Fluorescence micrographs showing adhesion of β3+/+ (left panel) and β3−/− platelets (right panel) to fibrinogen at 3.4 dynes/cm2, after 1 min of flow. B.C. Histograms of numbers of β3+/+ and β3Y747A platelets attached to fibrinogen within the field of view at different shear stresses in the device 1. B. Attachment to fibrinogen after 1 and 2 min of flow (average of 4 separate experiments). C. Whole blood from β3+/+ or β3Y747A mice was pre-incubated with PGE1 or ethanol vehicle control and then perfused over fibrinogen. Average values of 3 separate experiments ± SEM after 1 minute of flow are shown.

Figure 4

Dynamic adhesion of platelets to collagen depends on the presence of αIIbβ3 and its cytoplasmic tail associations. A. Fluorescence micrographs of test chambers with β3+/+ (left panel) and β3−/− (right panel) blood after 2 minutes of flow at 3.4 dynes/cm2. β3+/+ but not β3−/− platelets exhibit characteristic thrombus growth. B. Adhesion of β3+/+ and β3−/− platelets to collagen in the two adjacent test chambers of the device 2 after 2 min of flow at 50 dynes/cm2 as evaluated from a fluorescence micrograph. Surface coverage by mepacrine-labeled platelets is represented by color-coded fluorescence intensity (red corresponds to highest platelet density). Data shown is representative of at least 3 separate experiments. C. Histogram of the levels of attachment of β3+/+ and β3Y747A platelets to collagen after 1 minute of flow. Data are depicted as the total integrated fluorescence intensity for the field in arbitrary units (a.u.), and are an average of 3 separate experiments ± SEM.

Figure 5

Platelet-granulocyte interactions. A. Fluorescence micrographs of a test chamber with whole blood from wild-type (left panel) and β3−/− mice (right panel) labeled with PE-anti-Gr-1 antibody (granulocytes: red) and mepacrine (platelets: green) after 10 minutes of flow over collagen matrix at 3.4 dynes/cm2. Note extended β3−/− granulocyte tethers (arrowhead). (B-D). Quantification of interactions between granulocytes and collagen-adherent β3−/− platelets. B. Average translocation velocities of granulocytes on platelets. C. Percentage of stably attached granulocytes. D. Inhibition of granulocyte-platelet interactions. Whole blood from wild type and β3−/− mice was incubated with antibodies against P-selectin (a-P-sel), PSGL-1 (a-PSGL1) or with control IgG for 30 min, prior to flow. Histogram shows the percentage (± SEM) of granulocytes rolling on platelets from blood treated with each inhibitor versus IgG control antibody.

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Microfluidic devices for studies of shear-dependent platelet adhesion - PubMed (original) (raw)