A leucine to proline substitution at amino acid 33 defines the PlA1 (Leu33, HPA-1a) and PlA2 (Pro33, HPA-1b) isoforms of integrin β3. The Pro33 isoform has received a great deal of attention since it was first reported to be associated with acute coronary artery syndromes . A correlation between the Pro33 polymorphism of integrin αIIbβ3 and increased platelet activation  and aggregation  has also been reported. However, the few other studies that have evaluated platelet functional differences based on the Pro33 polymorphism have observed inconsistent and, in several cases, possibly conflicting results [4,5]. The interpretation of studies designed to assess the functional consequences of the Pro33 polymorphism is limited by interindividual differences in platelet functional assays and by variables that affect platelet functions like donor age, gender, cigarette smoking and medications. To overcome such concerns, we purified αIIbβ3 integrins from all three Leu33Pro genotypes and examined the in vitro binding of αIIbβ3 to two prothrombotic ligands fibrinogen and prothrombin.
Outdated platelets from approximately 19 Leu33/Leu33, 14 Pro33/Leu33 and two Pro33/Pro33 subjects were pooled and αIIbβ3 purified as described by Shock et al. . The protein preparation was >95%αIIbβ3 based on silver staining and immunoblotting with anti-αIIb and anti-β3 antibodies (Fig. 1A,B). αIIbβ3 receptors purified by this protocol largely remained in an inactive state with regard to binding adhesive proteins  and therefore were exogenously activated with 0.5 mmol L−1 MnCl2. Immobilized fibrinogen and prothrombin supported the binding of the activated integrins demonstrating that the purified integrins are functional. Compared with Leu33/Leu33 integrin, Pro33/Leu33 integrin demonstrated approximately 30% increased binding (P = 0.01) to fibrinogen and approximately 90% increased binding (P = 0.02) to prothrombin. Furthermore, compared with Leu33/Leu33 integrin, Pro33/Pro33 integrin demonstrated a approximately 35% greater binding (P = 0.04) to fibrinogen and approximately 250% increased binding to prothrombin (P = 0.04) (Fig. 1C). The increased binding of the purified Pro33-positive integrins to fibrinogen is consistent with previous adhesion studies using heterologous cells expressing both isoforms of αIIbβ3 . Because binding of prothrombin to αIIbβ3 in the presence of factor (F) Va and FXa accelerates the conversion of prothrombin to thrombin , our findings are consistent with the data of Undas et al. who demonstrated increased thrombin generation in subjects with Pro33-positive platelets .
Although several studies [2–5,7] have previously assessed integrin function based on the PlA status, none have utilized the purified integrins. The use of purified receptors devoid of other cell membrane constituents eliminate several potential mechanisms that could account for the differences in the thrombotic tendency between Leu33-positive and Pro33-positive platelets, including the possibilities that: (i) the preferential association of Leu33-expressing integrins with an inhibitory factor; (ii) the preferential association of Pro33-expressing integrins with an activating factor; (iii) greater expression levels of αIIbβ3 in Pro33-expressing platelets; and (iv) increased avidity changes in the Pro33-positive integrins. In light of these considerations, our findings are most consistent with the possibility that the Pro33 substitution induces an alteration in αIIbβ3 structure that favors a more stable association with ligands than that observed with the Leu33 isoform. A limitation of this study is the possible contamination up to 5% with other platelet integrins like αVβ3, α2β1, α5β1 and α6β1 . However, except αVβ3 no other platelet integrins bind fibrinogen and prothrombin. Taken together, the increased fibrinogen and prothrombin binding by the purified human Pro33-positive integrins provides further support to the prothrombotic feature for the Pro33 isoform of integrin β3.