High-throughput measurement of human platelet aggregation under flow: application in hemostasis and beyond

Figures & data

Table I. Changed parameters of platelet thrombus formation on a collagen-I surface (no coagulation) observed for patients with genetically linked disorders.

Shear rates are indicated in 1000× (k). Color intensity reflects reported frequency (of effect). References: a (37,39); b (30); c (15,30); d (15); e (41); f (15,42); b (30); g (42); h (43,44); i (45,46); j (47); k (48,50); l (48–52); and m (49).

Table II. Changed parameters of platelet-fibrin thrombus formation on a collagen-I surface with(out) tissue factor observed for patients with coagulation deficiencies.

Shear rates are indicated in 1000× (k). Color intensity reflects reported frequency (of effect). References: a (48,54,55); b (53); c (53,54,56); d (48,54,55); e (53); f (56,57); and g (54).

Table III. Changed parameters of platelet-fibrin thrombus formation on collagen surface with(out) tissue factor observed for antithrombotic drugs.

* active metabolite, coinfusion with ADP

Shear rates are indicated in 1000× (k). Color intensity reflects reported frequency (of effect). References: a (60,64,65,76); b (59); c (60,63,65,67); d (59,61–63,66,76); e (66,68); f (56,70,71); and g (71).

Box 1. Useful protocols.

A. Construction of flow chambers for blood perfusion. Both commercial and home-made parallel-plate flow devices can be used for whole blood perfusion studies to measure platelet deposition, activation, and aggregation by the use of brightfield and fluorescence microscopy (73). Soft-material polydimethylsiloxane (PDMS) chambers though may contain irregularities in the flow channels, so that hard-plastic chambers with high-precision dimensions are preferred. The Maastricht flow chamber is made of polymethyl methacrylate with channel dimensions of 50 µm height, 3 mm width, and 20 mm length. At one side it is covered with a rectangular glass coverslip. The small channel proportions reduce the amount of blood needed per flow run to about 0.5 mL (27). Flow disturbances are prevented by tubular inlets entering the channel at low angle of 11 . Coverslips are usually coated with microspots of 1–2 mm Ø (0.5–2 μL applied per spot) for obtaining a consistent pattern of thrombi (15). A variety of platelet-adhesive ligands can be used, but most common are collagen type-I which binds VWF (73). Protocol details are given elsewhere (74).

B. Whole-blood thrombus formation without coagulation. Preferably, blood is collected on thrombin inhibitors. Alternatively citrate-anticoagulated blood is supplemented, prior to the experiment, with a CaCl2/MgCl2 mixture in the presence of thrombin inhibitors, in order to achieve physiological (millimolar) concentrations of free Ca^2+ and Mg^2+ (74). In our experience, dual thrombin inhibition is required to prevent “back-ground” clotting (e.g. PPACK/fragmin or hirudin/fragmin), certainly when using hyper-coagulant or mouse blood. We prefer to perfuse whole blood samples in a plastic syringe in push mode, thus limiting leakage artifacts. A large set of fluorescent labels is available for quantifying purposes (27). For most surfaces, thrombus formation tends to maximize in a time span of 5–8 min, meaning that end-stage images can be recorded only. Labeling for platelet activation then is done post hoc, i.e., while recording (enhanced-contrast) brightfield images. Additional recording of fluorescence images (e.g. using spectrally non-overlapping green fluorescent protein (GFP), red fluorescent protein (RFP), and Cy5 filter sets) ultimate provides multiparameter insight into the content, structure, and platelet-activation properties of a thrombus (15).

C. Whole-blood thrombus formation with coagulation. Samples of citrate-anticoagulated blood may be supplemented with CaCl2/MgCl2 mixture, and then perfused over a collagen/tissue factor surface. However, a disadvantage of this procedure is ongoing contact activation in the blood before reaching the tissue factor (this is partly prevented by addition of corn trypsin inhibitor). Our preferred way of operation is to continuously co-infuse citrated-anticoagulated whole blood with a CaCl2/MgCl2 mixture (two plastic syringes in push mode), with tissue factor either added to the mix (56) or present with collagen in microspots (57). On microspots with tissue factor alone, no fibrin is formed due to the absence of platelets (57). Since the formation of thrombin and fibrin is an ongoing process, preferably kinetic information is collected, i.e., by recording time series of microscopic images. Fluorescent probes can be added to the blood samples, e.g., detecting platelets, procoagulant activity and fibrin (using GFP, RFP, and Cy5 filter sets).

D. Microscopic image analysis. High-resolution digital microscopic (≥8-bit, brightfield and fluorescence) images can be scored for thrombus morphology, and analyzed for coverage of platelets and labeled activation markers. Image analysis using FIJI software (75) is convenient since the program allows to write scripts for different optics and image types. Scripts may include a correction for background illumination (fast Fourier transform bandpass filter), followed by (manual) adjustment of a threshold setting, and a measurement of the surface area coverage of supra-threshold pixels. For brightfield images, it is useful to include Gray morphology conversions (large-/medium-sized close, followed by a small dilate) in order to reduce striping and improve the detection accuracy.

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