The HPA-1b (Pro33) Isoform of Platelet Integrin αIIbβ3 Is a Prothrombotic Variant: Characterization by Fluorescence Resonance Energy Transfer

Abstract

2028 Background and Objectives: The HPA-1 polymorphism of αIIbβ3 arises from a Leu→Pro exchange at residue 33 of the β3 subunit resulting in HPA-1a (Leu33) or HPA-1b (Pro33). We have documented that patients with coronary artery disease who are carriers of HPA-1b (Pro33) experience their myocardial infarction 5.2 years earlier than HPA-1a/1a (Leu33) patients (J Thromb Haemost 2005; 3: 1522). Based on these observations, it has been postulated that HPA-1b (Pro33) is a prothrombotic variant of αIIbβ3. We have now generated a model overexpressing fluorescent proteins fused with αIIbβ3 in transfected HEK293 cells. Methods: :A yellow protein (YFP) and a cyan fluorescent protein (CFP) were cloned to the C-termini of the β3 and αIIb subunits prior to transfection of HEK293 cells, subsequently expressing the fusion proteins of both HPA-1 isoforms. Using flow cytometry, Western blotting and specific antibodies directed against αIIb or β3, we identified 12 HPA-1a and 11 HPA-1b positive clones. For further experiments only those cell lines expressing equal amounts of fluorescent fusion proteins, i.e. a 140 kD αIIb-CFP and a 113 kD β3-YFP, were used. Results: Functional integrity of both integrin variants and proper membrane insertion were documented by intact activation of transfected HEK293 cells through G protein-coupled receptors with organic acid (1-stearoyl-2-arachidonoyl-sn-glycerol) or direct phorbol 12-myristate 13-acetate-induced stimulation of protein kinase C and by specific binding of Alexa488 fibrinogen to αIIbβ3 in response to inside-out signaling. In the presencence of pertussis toxin or abciximab, activation or ligand binding of αIIbβ3 were completely (>98%) inhibited in both isoforms. Activation of αIIbβ3 stimulates the tyrosine kinase Src, constitutively associated with the the β-subunit of the integrin. To determine whether αIIbβ3-dependent outside-in signaling is responsible for a polymorphism-related modulation, we performed adhesion experiments under static conditions with fibrinogen (50 μg/ml) in the absence or presence of Mn2+ (0.5 mM). Specific activation of the phosphotyrosine motif (Src-pY418), as determined by Western blotting and quantified by densitometry (ratio of Src-pY418/total Src), was 15 + 1.5% higher in HPA-1b than HPA-1a cells in the presence of Mn2+ (n=6 independent experiments, p<0.01). To explore the molecular nature of this difference in terms of putative changes in the allostery of integrin αIIbβ3 with regard to the HPA-1 polymorphism, dynamic measurements were performed using fluorescence resonance energy transfer (FRET). The relative decrease in FRET signal, indicating spatial separation of the cytoplasmic tails of the α- and β-subunit as a consequence of integrin activation, was recorded every minute over 0.5 hrs in transfected HEK293 cells adherent onto fibrinogen. At every time point, the kinetic measurements revealed a significantly faster and more distict (> 5%) decrease in HPA-1b than in HPA-1a cells under static adhesion (p<0.009). Upon exposure of adherent HEK293 cells to increasing shear rates (stepwise elevation from 50 to 1600 sec-1 by doubling the initial shear rate every minute), the spatial separation of the integrin subunits occurred significantly faster and more distinct (> 10%) in HPA-1b (Pro33) than HPA-1a (Leu33) cells in response to shear (p<0.0014). Under the same conditions, the rate of HPA-1b cells still adherent onto immobilized fibrinogen was 80%, while the relative number of residual HPA-1a cells decreased to 20% upon exposure to 1600 sec-1 (p<0.0001). These displacement experiments suggest that the HPA-1b (Pro33) variant is more resistant to biomechanical stress than the HPA-1a (Leu33) isoform. Conclusions: Our findings suggest that the HPA-1 polymorphism can have a significant impact on the activation of αIIbβ3. This is evident from a higher outside-in signaling and a higher resistance to biomechanical stress upon exposure to increasing shear of HPA-1b (Pro33) in comparison with HPA-1a (Leu33) transfectants. The difference in spatial separation of the cytoplasmic tails of the integrin in response to activation, as demonstrated by FRET analyses under static and flow dynamic conditions, reflects allosteric changes that may contribute to the prothrombotic phenotype of the HPA-1b (Pro33) variant. Disclosures: No relevant conflicts of interest to declare.

Platelet Signaling in Primary Haemostasis and Arterial Thrombus Formation: Part 2

“…43 These results suggest that the Leu!Pro exchange may modulate functional properties of αIIbβ3, resulting in a prothrombotic integrin variant. 44 Indeed, in subsequent experiments under flow-dynamic conditions, it was documented that Pro33 (HPA-1b) platelets and αIIbβ3 (Pro33)-transfected cells display a prothrombotic phenotype, as characterized by increased adhesion activity, 45,46 increased resistance of adherent transfectants to high shear, 47,48 increased aggregate/thrombus formation 49,50 and increased outside-in signaling. 51,52 Recently, the molecular nature of this prothrombotic integrin variant was elucidated.…”