Microfluidic focal thrombosis model for measuring murine platelet deposition and stability: PAR4 signaling enhances shear-resistance of platelet aggregates

J Thromb Haemost. 2008 Dec;6(12):2193-201. doi: 10.1111/j.1538-7836.2008.03188.x. Epub 2008 Oct 7.


Background: Flow chambers allow the ex vivo study of platelet response to defined surfaces at controlled wall shear stresses. However, most assays require 1-10 mL of blood and are poorly suited for murine whole blood experiments.

Objective: To measure murine platelet deposition and stability in response to focal zones of prothrombotic stimuli using 100 microL of whole blood and controlled flow exposure.

Methods: Microfluidic methods were used for patterning acid-soluble collagen in 100 microm x 100 microm patches and creating flow channels with a volume of 150 nL. Within 1 min of collection into PPACK and fluorescent anti-mouse CD41 mAb, whole blood from normal mice or from mice deficient in the integrin alpha(2) subunit was perfused for 5 min over the patterned collagen. Platelet accumulation was measured at venous and arterial wall shear rates. After 5 min, thrombus stability was measured with a 'shear step-up' to 8000 s(-1).

Results: Wild-type murine platelets adhered and aggregated on collagen in a biphasic shear-dependent manner with increased deposition from 100 to 400 s(-1), but decreased deposition at 1000 s(-1). Adhesion to patterned collagen was severely diminished for platelets lacking a functional alpha(2)beta(1) integrin. Those integrin alpha(2)-deficient platelets that did adhere were removed from the surface when challenged to shear step-up. PAR4 agonist (AYPGKF) treatment of the thrombus at 5 min enhanced aggregate stability during the shear step-up.

Conclusions: PAR4 signaling enhances aggregate stability by mechanisms independent of other thrombin-dependent pathways such as fibrin formation.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Collagen / metabolism
  • Disease Models, Animal
  • Integrin alpha2beta1
  • Mice
  • Microfluidics*
  • Platelet Adhesiveness*
  • Platelet Aggregation
  • Receptors, Thrombin / agonists
  • Receptors, Thrombin / metabolism
  • Receptors, Thrombin / physiology*
  • Signal Transduction
  • Stress, Mechanical
  • Thrombosis / pathology*


  • Integrin alpha2beta1
  • Receptors, Thrombin
  • Collagen
  • protease-activated receptor 4