Shear stress and platelet-induced tensile forces regulate ADAMTS13-localization within the platelet thrombus

Abstract

Background: The multimeric glycoprotein von Willebrand factor (VWF) mediates platelet adhesion and aggregation at the site of vessel injury. The adhesive activity of VWF is influenced by its multimer length which is regulated by the metalloprotease ADAMTS13. The ability of ADAMTS13 to regulate platelet thrombus growth in a shear-dependent manner has been described, however, the mechanistic basis of this action has not been well characterized.

Methods: We developed an mCherry-tagged murine ADAMTS13 protein and utilized an ex vivo flow chamber system to visualize the localization of ADAMTS13 within the platelet thrombus under different conditions of shear. Using this system, we also assessed the influence of platelet-mediated tensile force on ADAMTS13 localization within the thrombus using gain-of-function GPIb binding and loss-of-function GPIIbIIIa binding mutants in VWF/ADAMTS13 DKO mice.

Results: ADAMTS13 was visualized on the growing platelet thrombus under very high shear using ADAMTS13-mcherry. ADAMTS13-mCherry localized particularly at the top portion of the thrombus and reduced thrombus size as it grew to occlusion. At the pathological high shear of 7500 s^-1, platelet-mediated tensile force, involving GPIb but not GPIIbIIIa receptors, influenced localization of ADAMTS13 to the thrombus under conditions of shear.

Conclusions: Tensile force applied on VWF produced by shear stress and platelet GPIb binding has a crucial role in ADAMTS13 activity at the site of thrombus formation. These results suggest that ADAMTS13 activity at the site of platelet thrombus formation is regulated by a shear stress and platelet-dependent feedback mechanism to prevent vessel occlusion and pathological thrombosis.

Keywords: ADAMTS13; GPIb; platelet; shear stress; von Willebrand factor.

Figure 1

Murine A Disintegrin And Metalloproteinase with Thrombospondin type 1 motif, 13 (ADAMTS13)‐mCherry reduces murine von Willebrand factor (VWF) multimer size and decreases platelet thrombus formation in a flow chamber model. (A) ADAMTS13‐mCherry reduces the high molecular weight content of VWF multimers. Recombinant mouse VWF was treated with ADAMTS13‐mCherry (0, 0.5, 2 U/mL) in the presence of 0.75 mol/L urea and the reactions were terminated by the addition of Ethylenediaminetetraacetic acid (EDTA, 10 mmol/L). The multimer profiles of these treated proteins were confirmed on the 1% sodium dodecyl sulfate agarose gel electrophoresis. ADAMTS13‐mCherry was added to whole blood from ADAMTS13 KO mice and perfused across the collagen‐coated flow chamber. Platelets were labeled with 3,3'‐dihexyloxacarbocyanine iodide (DiOC₆, green). Influence of ADAMTS13‐mCherry on (B) surface area coverage and (C) platelet thrombus volume after 9 min under 500 s⁻¹, 2500 s⁻¹, or 7500 s⁻¹ shear conditions. (D) Visualization of ADAMTS13‐mCherry at the sites of platelet accumulation. Platelets were labeled with DiOC₆ (green). The signal from ADAMTS13‐mCherry was enhanced with an anti‐cherry antibody (red). Scale bar = 25 μm . *P < 0.05, **P < 0.01

Figure 2

Association between von Willebrand factor (VWF), A Disintegrin And Metalloproteinase with Thrombospondin type 1 motif, 13 (ADAMTS13), and platelets under high and very high shear stress. 2 U/mL of ADAMTS13‐mCherry was added to the whole blood obtained from ADAMTS13 KO mice and perfused into the collagen coated flow chamber at 7500 s⁻¹. After 9 min of perfusion, the thrombus was fixed and immunostaining was performed. Platelet, VWF and ADAMTS13 are shown in blue, green and red pseudo‐color, respectively. Merged images are also shown and white pseudo‐color indicates the three color co‐localization. Representative images after 9 min of perfusion from seven separate experiments are shown. (A) Longitudinal view of a representative thrombus formed under 7500 s⁻¹. (B) Longitudinal view of a representative thrombus formed under 2500 s⁻¹. (C) Total fluorescent intensity of platelet, VWF, and ADAMTS13 under 7500 s⁻¹, 2500 s⁻¹, and 500 s⁻¹ are shown. (D) The difference of ADAMTS13 fluorescent intensity in the top, middle, and bottom portion of thrombus under 7500 s⁻¹, 2500 s⁻¹, and 500 s⁻¹ are shown. Each data point represents the means of 21 areas examined (three areas randomly selected in seven independent perfusions). Data was expressed in arbitrary units (AU). (E) Ratio of ADAMTS13 to VWF staining in incorporated thrombus. *P < 0.05, **^** P < 0.0001

Figure 3

Influence of Glycoprotein Ib (GPIb), and Glycoprotein IIbIIIa (GPIIbIIIa), on the association between von Willebrand factor (VWF), A Disintegrin And Metalloproteinase with Thrombospondin type 1 motif, 13 (ADAMTS13), and platelets. (A) Wild type murine recombinant (r) VWF, (B) murine V1316M rVWF, (C) murine RGG rVWF were added to whole blood from VWF/ADAMTS13 double knockout (DKO) mice and perfused into the flow chamber, respectively. Longitudinal views of a representative thrombus after 9 min of perfusion from four separate experiments are shown. Platelet, VWF, ADAMTS13, and merged images are shown. (D) The total fluorescent intensity of platelets, VWF and ADAMTS13 are shown. Data was expressed in arbitrary units (AU). Each data point represents the means of 8 areas examined (two to three areas randomly selected in three to four independent perfusions). (E) Ratio of ADAMTS13 to VWF staining in incorporated thrombus. (F) Overlap coefficient between ADAMTS13 and VWF as determined by Coloc 2 analysis. **P < 0.01, **^* P < 0.001, **^** P < 0.0001