Binding of Erythrocyte ICAM–4 to the Platelet Activated Integrin αIIbβ3 leads to a Direct Erythrocyte-Platelet Adhesion Under Venous Flow Shear Rate
Authors: Du Vivian Xiaoyan , de Groot Philip G , van Wijk Richard , Ruggeri Zaverio M. , De Laat Bas 
Published: October 2019

Abstract
Abstract 105
Erythrocytes are the major cellular component of blood and they have been shown to contribute to primary hemostasis, predominantly due to their rheological properties. Direct platelet-erythrocyte interaction has been published but no information is available on the mechanism of interaction and the physiological function. Our aim was to characterize platelet-erythrocyte interactions under near physiological conditions in-vitro.
At first we studied whether erythrocytes are able to bind to platelets adhered to surfaces coated with different adhesive proteins at different flow-rates. For this purpose, an in vitro perfusion system connected to a light microscope and a digital camera was used. Erythrocytes bind to platelets both in buffer (washed platelets and erythrocytes) and in whole blood. Erythrocytes were attached to platelets with a sort of “focal adhesion point”, resulting in a tear-drop shape (Fig 1a, b, erythrocyte binding to platelets under flow). Erythrocyte-platelet adhesion was inversely correlated with flow rate and predominately occurred at shear rates lower than 300S−1. The addition of platelet agonists, i.e collagen related peptide (CRP), adenosine diphosphate (ADP), thrombin and arachidonic acid increased erythrocyte binding to platelets 3 to 6 folds indicating that platelet activation is involved in capturing erythrocytes from the circulation.
An Arg-Gly-Asp (RGD) containing peptide (d-RGDW), known to inhibit αIIbβ3 mediated platelet aggregation inhibited erythrocyte-platelet adhesion with 29% to 72%, depending on the agonist used (p<0.05, n=4). As erythrocyte ICAM-4 has been reported to be a ligand for platelet activated αIIbβ3(Hermand P. et al, J.Biol.Chem, 2003,), we tested whether ICAM-4 and platelet αIIbβ3 are the ligand/receptor pair responsible for the erythrocyte-platelet adhesion. Experiments with inhibitory antibodies revealed that the erythrocyte-platelet adhesion under conditions of flow was inhibited with both anti-ICAM-4 (40%, p<0.01, n=8) and anti- integrin β3 (CD61) (46%, p<0.001, n=8). In addition, an ICAM-4 peptide resembling the extracellular domain of human ICAM-4 demonstrated a significant inhibitory effect on erythrocyte-platelet adhesion.
To further characterize the binding between ICAM-4 and αIIbβ3, flow cytometry analysis was performed. We found a decreased fibrinogen binding to platelets (43% at ADP concentration of 125μM, p<0.05, n=5) and an increased P-selectin expression (60%, p<0.01, n=5) on platelets upon ADP stimulation in the presence of ICAM-4 peptide. This finding suggests that ICAM-4 peptide compete with fibrinogen for binding to activated αIIbβ3. The increase of P-selectin expression in the presence of ICAM-4 peptide suggests that binding of ICAM peptide to αIIbβ3 results in outside-in signalling and further platelet activation.
In conclusion, we found direct erythrocyte-platelet interaction under conditions of low shear stresses. This interaction is partly mediated via erythrocyte receptor ICAM-4 and platelet activated integrin αIIbβ3. In addition we found an indication that interaction with erythrocytes further enhances platelet activation. Direct erythrocyte-platelet adhesion seems to play a role in platelet depending thrombus formation.
Fig 1a: Erythrocyte binding to platelets under flow Fig 1a:. Erythrocyte binding to platelets under flow
Fig 1b: Scanning electron microscopy picture of erythrocyte-platelet interaction under flow Fig 1b:. Scanning electron microscopy picture of erythrocyte-platelet interaction under flow

Disclosures:
No relevant conflicts of interest to declare.