The role of platelet adhesion receptor GPIbalpha far exceeds that of its main ligand, von Willebrand factor, in arterial thrombosis

Abstract

GPIbalpha binding to von Willebrand factor (VWF) exposed at a site of vascular injury is thought to be the first step in the formation of a hemostatic plug. However, our previous studies in VWF-deficient mice demonstrated delayed but not absent arterial thrombus formation, suggesting that, under these conditions, GPIbalpha may bind other ligands or that a receptor other than GPIbalpha can mediate platelet adhesion. Here, we studied thrombus formation in transgenic mice expressing GPIbalpha in which the extracellular domain was replaced by that of the human IL-4 receptor (IL4Ralpha/GPIbalpha-tg mice). Platelet adhesion to ferric chloride-treated mesenteric arterioles in IL4Ralpha/GPIbalpha-tg mice was virtually absent in contrast to avid adhesion in WT mice. As a consequence, arterial thrombus formation was inhibited completely in the mutant mice. Our studies further show that, when infused into WT recipient mice, IL4Ralpha/GPIbalpha-tg platelets or WT platelets lacking the 45-kDa N-terminal domain of GPIbalpha failed to incorporate into growing arterial thrombi, even if the platelets were activated before infusion. Surprisingly, platelets lacking beta3 integrins, which are unable to form thrombi on their own, incorporated efficiently into WT thrombi. Our studies provide in vivo evidence that GPIbalpha absolutely is required for recruitment of platelets to both exposed subendothelium and thrombi under arterial flow conditions. Thus, GPIbalpha contributes to arterial thrombosis by important adhesion mechanisms independent of the binding to VWF.

Fig. 1.

In vitro characterization of IL4Rα/GPIbα-tg platelets. (A) IL4Rα/GPIbα-tg platelets show normal surface expression of important adhesion receptors. WT or IL4Rα/GPIbα-tg platelets were stained with FITC-labeled antibodies against the indicated adhesion receptors and immediately were analyzed by flow cytometry. n = 4; ∗, P < 0.0001. (B) αIIbβ3 activation. Washed WT (squares) or IL4Rα/GPIbα-tg (diamonds) platelets were stimulated with the indicated concentrations of thrombin or ADP (in the presence of 5 μM concentration of the thromboxane A₂ mimetic U46619) for 5 min, stained with an antibody recognizing activated αIIbβ3 (JON/A-PE), and immediately analyzed by flow cytometry. n = 6. (C) Standard aggregometry traces of WT (black line) or IL4Rα/GPIbα-tg (gray line) platelets stimulated with 1 μM ADP, 10 μg/ml collagen, or thrombin (0.04 units/ml). Platelet aggregation (light transmission) was monitored over 12 min. Results are representative of three experiments. No defects in receptor expression and αIIbβ3 activation were observed in IL4Rα/GPIbα-tg platelets.

Fig. 2.

Platelet adhesion and thrombus formation in FeCl₃-injured arterioles. (A) WT (Left) or IL4Rα/GPIbα-tg (Right) mice were injected with calcein-green-labeled platelets of the respective genotype, and platelet adhesion was monitored in mesenteric arterioles upon application of FeCl₃. Images show platelet adhesion and thrombus formation in arterioles at the indicated times after application of FeCl₃. The direction of blood flow is from upper right to lower left. Note that adhesion in arterioles from IL4Rα/GPIbα-tg mice is virtually absent for the entire observation period (40 min). (B) Comparison of the time of the appearance of the first thrombus (>20 μm) and the time of occlusion in FeCl₃-treated mesenteric arterioles of WT and IL4Rα/GPIbα-tg mice. No thrombi formed in the mutant mice during the 40 min of observation. Movies showing platelet adhesion/thrombus formation in injured arterioles in real time can be found in supporting information.

Fig. 3.

IL4Rα/GPIbα-tg platelets show strongly impaired incorporation into a growing WT thrombus. Washed platelets were labeled with calcein-green (WT; Left) or calcein-red/orange (IL4Rα/GPIbα-tg; Right) and transfused into the same WT recipient mouse to reach a total of 2% labeled platelets for each genotype. Vascular injury was induced by application of FeCl₃, and thrombus growth was monitored until blood flow stopped (occlusion). Although WT platelets incorporated avidly into the thrombus, only a few entrapped IL4Rα/GPIbα-tg platelets could be seen. Results are representative of five independent experiments.

Fig. 4.

Platelets lacking the 45-kDa N-terminal domain of GPIbα are unable to incorporate into a growing WT thrombus. (A) Washed platelets were treated with O-sialoglycoprotein endopeptidase. Removal of the 45 kDa N-terminal domain of GPIbα was monitored by flow cytometry with antibodies against epitopes within (p0p4; Left) and outside (p0p5; Center) this domain of the receptor (8). Antibodies against GPV (Right) were used as a control. (B) Platelets were labeled with calcein-green (WT; Left) or calcein-red/orange (WT lacking 45-kDa N-terminal domain; Right) and transfused into the same WT recipient mouse. Vascular injury was induced by application of FeCl₃ and thrombus growth was monitored. Results are representative of five independent experiments. (C) WT mice were infused with WT platelets labeled in calcein-green (Left), and thrombosis was induced by FeCl₃. When the first thrombus <10 μm in diameter was observed, thrombin-activated IL4Rα/GPIbα-tg platelets labeled with calcein-red/orange (Right) were infused into the same mouse. Thrombus growth then was monitored until blood flow stopped (occlusion). Only a few activated IL4Rα/GPIbα-tg platelets were entrapped in the occlusive thrombus.

Fig. 5.

Incorporation of β3-deficient platelets into a growing WT thrombus requires the extracellular domain of GPIbα. (A) Platelets were labeled with calcein-green (WT; Left) or calcein-red/orange (β3^−/−; Right) and transfused into the same WT recipient mouse. Vascular injury was induced by application of FeCl₃, and thrombus growth was monitored until blood flow stopped (occlusion). Results are representative of five independent experiments. (B) Arterial thrombosis was studied in a WT recipient mouse infused with β3^−/− platelets (calcein-green; Left) and β3^−/− platelets lacking the 45-kDa N-terminal domain of GPIbα (calcein-red/orange; Right). Only platelets containing the N-terminal domain of GPIbα can incorporate successfully into thrombi.