Abstract
Abstract 1143
Background and Objectives:
Activation of αIIbβ3 is the final step leading to platelet aggregation. The polymorphism of the β3 gene of αIIbβ3 results in an amino acid exchange with a leucin (HPA-1a) or a prolin (HPA-1b) at residue 33. We have shown in patients with coronary artery disease that the HPA-1b variant of αIIbβ3 is associated with premature manifestation of acute myocardial infarction (MI) (JTH 2005). Autopsy studies have confirmed that HPA-1b is strongly associated with coronary thrombi after MI. Recently, we have documented that HPA-1b platelets display increased adhesion, increased thrombus stability, and increased outside-in signaling corresponding to their prothrombotic character. To explore the nature of this phenotype in further detail, we have now generated a model overexpressing fluorescent proteins fused with αIIbβ3 in transfected cells to assess the postulated differences between both HPA-1 isoforms with regard to αIIbβ3 activation and its concomitant conformational changes.
Materials and Methods:
Transfected and fluorescently tagged HEK293 cells stably expressing either HPA-1a or HPA-1b of αIIbβ3 were generated. The cyan (CFP) and yellow fluorescent protein (YFP) were cloned to the C-termini of the β3 and αIIb subunits prior to cell transfection. To explore conformational changes in the cytoplasmic tails and the activation of integrin, dynamic measurements were performed by fluorescence resonance energy transfer (FRET) under static and dynamic conditions (shear rates of 100 to 1600 s−1) using an established flow model.
Results:
Functional integrity of both integrin variants and correct membrane insertion were examined by flow cytometry and documented intact activation of αIIbβ3 in the transfected cells upon phorbol 12-myristate 13-acetate-induced (PMA)-induced stimulation of protein kinase C and specific binding of alexa647 fibrinogen (Fg) to αIIbβ3 upon inside-out activation. In the presence of abciximab, binding of alexa647 Fg to αIIbβ3 was completely blocked in both isoforms (PMA, mean: 119 units vs. abciximab 10 units). Upon cell adhesion under static condition and subsequent outside-in signaling, the HPA-1b isoform displayed a quantitatively different activation pattern than HPA-1a, as detected by FRET analyses. Thus, FRET signals, defined by the ratio of acceptor to donor fluoresecence intensity, revealed a faster and more distinct decrease in HPA-b (67+5) than in HPA-1a (75+6) (HPA-1b vs. HPA-1a, p=0.0053). This finding corresponds to are more pronounced spatial separation of the cytoplasmic tails in the HPA-1b variant of αIIbβ3. By contrast, stimulation with PMA (1 μM) caused an opposite effect in HPA-1a transfectants, as compared to HPA-1b (p<0.001), indicative of a distinct property upon inside-out activation. Upon interaction of αIIbβ3 with immobilized fibrinogen to induce outside-in signaling, HPA-1b exhibited an increased activation of the phosphotyrosine motif at residue Y418 (pY418) of the αIIbβ3-associated Src kinase, as compared with HPA-1a (p<0.05). No such difference was observed in Src phosphorylation upon stimulation with PMA (inside-out signaling). These findings suggest that the HPA-1 polymorphism of αIIbβ3 can modulate indeed outside-in signaling upon interaction of the transfectants with immobilized fibrinogen. When αIIbβ3-transfected HEK293 cells adherent onto immobilized fibrinogen were exposed to increasing arterial shear rates (800 to 1600 s−1), FRET analyses and digital imaging confirmed a more pronounced spatial separation of the cytoplasmic tails (αIIb-CFP; β3-YFP) in HPA-1b than in HPA-1a cells (p=0.0029). Likewise, the rate of residual adherent cells was significantly higher with HPA-1b than HPA-1a cell clones (p<0.0001). Interestingly, only arterial (> 800 s−1) but not venous shear rates increased the observed conformational changes in adherent HPA-1b cells.
Conclusions:
Our findings suggest that the prothrombotic phenotype of the HPA-1b variant is caused by increased outside-in signaling. The observed quantitative differences between both HPA-1 isoforms of αIIbβ3 are related to distinct conformational changes in their C-terminal cytoplasmic tails. These findings are in agreement with the contention that the HPA-1 polymorphism of αIIbβ3 can have a major impact on platelet adhesion, aggregation and thrombus formation under pathological conditions resulting from abnormally high shear stress.
Disclosures:
No relevant conflicts of interest to declare.
Structural rearrangement of the intracellular domains during AMPA receptor activation
