Development of a method to quantify platelet adhesion and aggregation under static conditions

Abstract

Platelets are important players in hemostasis and thrombosis. Thus, accurate assessment of platelet function is crucial for identifying platelet function disorders and measuring the efficacy of antiplatelet therapies. We have developed a novel platelet aggregation technique that utilizes the physical parameter of platelet concentration in conjunction with volume and mass measurements to evaluate platelet adhesion and aggregation. Platelet aggregates were formed by incubating purified platelets on fibrinogen- or fibrillar collagen-coated surfaces at platelet concentrations ranging from 20,000 to 500,000 platelets/ L. Platelets formed aggregates under static conditions in a platelet concentration-dependent manner, with significantly greater mean volume and mass at higher platelet concentrations ( 400,000 platelets/ L). We show that a platelet glycoprotein IIb/IIIa inhibitor abrogated platelet-platelet aggregation, which significantly reduced the volume and mass of the platelets on the collagen surface. This static platelet aggregation technique is amenable to standardization and represents a useful tool to investigate the mechanism of platelet activation and aggregation under static conditions.

Keywords: Aggregation; Blood; Microscopy; Platelets.

FIGURE 1. Platelet aggregation on fibrinogen-coated surfaces

(a,d,g,j) XY and XZ DIC projections of platelet aggregates formed on fibrinogen-coated coverslips at 20,000, 100,000, 400,000, or 500,000 platelets/ L, respectively. Representative image from 3 independent experiments. (b,e,h,k) Magnified image of region within the white box in (a,d,g,j). (c,f,i,l) Projected density of the white box in (a,d,g,j) determined with the NIQPM technique.

FIGURE 2. Platelet aggregation on collagen-coated surfaces

(a,d,g,j) XY and XZ DIC projections of platelet aggregates formed on fibrillar collagen-coated coverslips at 20,000, 100,000, 400,000, or 500,000 platelets/ L. Representative image from 3 independent experiments. (b,e,h,k) Magnified image of region within the white box in (a,d,g,j). (c,f,i,l) Projected density of the white box in (a,d,g,j) determined with the NIQPM technique.

FIGURE 3. Platelet aggregate formation under static conditions as a function of platelet count

Mean platelet aggregate (a) volume and (b) mass for a 32 μm by 32 μm region collected over three trials. Error bars are ± standard error of the mean.

FIGURE 4. Platelets treated with a GPIIb/IIIa inhibitor form platelet monolayers on collagen-coated surfaces

Platelets were incubated with the GPIIb/IIIa inhibitor, eptifibatide (20 μg/mL; 10 min at 25°C), followed by incubation on fibrillar collagen-coated glass coverslips at platelet concentrations of (a,b,c) 400,000 or (d,e,f) 500,000 platelets/ L. (a,d) XY and XZ DIC projections of GPIIb/IIIa-treated platelet aggregates. Representative image from 3 independent experiments. (b,e) Magnified image of region within the white box in (a,d). (c,f) Projected density of the white box in (a,d) determined with the NIQPM technique. Mean platelet aggregate (g) volume and (h) mass for a 32 μm by 32 μm region collected over three trials. Error bars are ± standard error of the mean.