Abstract
ICAM-4 (LW blood group glycoprotein) is an erythroid-specific membrane component that belongs to the family of intercellular adhesion molecules and interacts in vitro with different members of the integrin family, suggesting a potential role in adhesion or cell interaction events, including hemostasis and thrombosis. To evaluate the capacity of ICAM-4 to interact with platelets, we have immobilized red blood cells (RBCs), platelets, and ICAM-Fc fusion proteins to a plastic surface and analyzed their interaction in cell adhesion assays with RBCs and platelets from normal individuals and patients, as well as with cell transfectants expressing the alpha(IIb)beta(3) integrin. The platelet fibrinogen receptor alpha(IIb)beta(3) (platelet GPIIb-IIIa) in a high affinity state following GRGDSP peptide activation was identified for the first time as the receptor for RBC ICAM-4. The specificity of the interaction was demonstrated by showing that: (i) activated platelets adhered less efficiently to immobilized ICAM-4-negative than to ICAM-4-positive RBCs, (ii) monoclonal antibodies specific for the beta(3)-chain alone and for a complex-specific epitope of the alpha(IIb)beta(3) integrin, and specific for ICAM-4 to a lesser extent, inhibited platelet adhesion, whereas monoclonal antibodies to GPIb, CD36, and CD47 did not, (iii) activated platelets from two unrelated type-I glanzmann's thrombasthenia patients did not bind to coated ICAM-4. Further support to RBC-platelet interaction was provided by showing that dithiothreitol-activated alpha(IIb)beta(3)-Chinese hamster ovary transfectants strongly adhere to coated ICAM-4-Fc protein but not to ICAM-1-Fc and was inhibitable by specific antibodies. Deletion of individual Ig domains of ICAM-4 and inhibition by synthetic peptides showed that the alpha(IIb)beta(3) integrin binding site encompassed the first and second Ig domains and that the G65-V74 sequence of domain D1 might play a role in this interaction. Although normal RBCs are considered passively entrapped in fibrin polymers during thrombus, these studies identify ICAM-4 as the first RBC protein ligand of platelets that may have relevant physiological significance.
Highlights
The main physiological function of red blood cells (RBCs),1 which encapsulate hemoglobin, is to ensure the respiratory
RBCs Interact with Activated Platelets—To analyze molecular events occurring during RBC-platelet interaction, in vitro cell adhesion assays were developed using RBCs from donors of common and rare phenotypes immobilized to plastic surface via anti-GPA binding and platelets from normal healthy donors, pretreated or not with the synthetic GRGDSP peptide in the presence of inhibitors of platelet activation, resulting in specific ␣IIb3 integrin activation and the acquisition of high affinity Fg-binding state without addition of a cellular agonist [37]
We found that the ␣IIb3 integrin had to be in its high affinity state to bind ICAM-4, as the interaction occurred only after synthetic GRGDSP peptide activation, but not with untreated resting platelets
Summary
The main physiological function of red blood cells (RBCs), which encapsulate hemoglobin, is to ensure the respiratory. It is interesting that many RBC adhesion molecules contain protein domains characteristic of the immunoglobulin superfamily, suggesting some recognition function These molecules might participate in the normal RBC physiology by playing a role during erythropoiesis (differentiation, maturation, enucleation, release), self-recognition mechanisms, red cell turnover, and cell aging through cellular interactions with counter receptors present on macrophages from bone marrow or reticuloendothelial system in spleen and liver (1, 4 –9). The conversion of non-adherent RBCs to adherent state arises in several diseases In such circumstances, adhesion molecules might be involved in the pathophysiology of malaria [12, 13], sickle cell disease (14 –17), and diabetes [18, 19], mainly through an abnormal adhesion to the vascular endothelium [1, 20]. The purpose of this report was to examine the potential role of ICAM-4 in RBC-platelet interaction and to demonstrate that this protein interacts in vitro with the high affinity state of activated platelet ␣IIb3 integrin
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