Fibrinogen and prothrombin binding is enhanced to the Pro33 isoform of purified integrin αIIbβ3

Authors


Paul F. Bray, Thrombosis Research Section, Baylor College of Medicine, One Baylor Plaza, BCM 286, N1319, Houston, TX 77030, USA.
Tel.: +1 713 798 3480; fax: +1 713 798 3415; e-mail: pbray@bcm.tmc.edu

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 [1]. A correlation between the Pro33 polymorphism of integrin αIIbβ3 and increased platelet activation [2] and aggregation [3] 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Outdated platelets from approximately 19 Leu33/Leu33, 14 Pro33/Leu33 and two Pro33/Pro33 subjects were pooled and αIIbβ3 purified as described by Shock et al. [6]. 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 [6] 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 [7]. Because binding of prothrombin to αIIbβ3 in the presence of factor (F) Va and FXa accelerates the conversion of prothrombin to thrombin [8], our findings are consistent with the data of Undas et al. who demonstrated increased thrombin generation in subjects with Pro33-positive platelets [9].

Figure 1.

Purification of integrin αIIbβ3. Twenty micrograms of total platelet lysates and 0.5 μg of purified integrins were separated by 10% SDS-PAGE, and proteins detected by silver staining (A) or transferred to a nitrocellulose membrane and immunoblotted with either anti-αIIb (132.1), anti-Leu33 specific (SZ21) [10] and anti-β3 (AP3) antibodies (B). (C) Integrin-ligand binding assays: 4.5 μm polystyrene beads were coated with bovine serum albumin (BSA), 12.5 μg mL−1 fibrinogen or 50 μg mL−1 of prothrombin, washed and blocked with BSA. Beads were incubated with 1 μg of integrin and Mn+2 and the integrin binding to the beads was detected with FITC-P2 antibody using flow cytometry. Results are expressed as MFI±SE of five (Leu33/Leu33 and Pro33/Leu33 integrin) and three (Pro33/Pro33 integrin) experiments. For fibrinogen binding, *P = 0.01 for Leu33/Leu33 vs. Pro33/Leu33 and †P = 0.04 for Leu33/Leu33 vs. Pro33/Pro33. For prothrombin binding, ‡P = 0.02 for Leu33/Leu33 vs. Pro33/Leu33 and †P = 0.04 for Leu33/Leu33 vs. Pro33/Pro33. For binding to either fibrinogen or prothrombin there was no significant difference between the Pro33/Leu33 and Pro33/Pro33 integrin.

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 [6]. 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.

Acknowledgements

This work was supported by grants HL65967 and HL65229 from the National Institute of Health. K. V. V. is supported by a Scientist Development Grant 0435017N from the National American Heart Association.

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