Effect of the Pl^A2 alloantigen on the function of β₃-integrins in platelets

Platelet aggregation results from platelet cross-linking by fibrinogen or von Willebrand factor (or both) bound to the platelet-specific integrin α_IIbβ₃(GPIIb-IIIa).1 Because of critical roles played by platelets and α_IIbβ₃ in the pathogenesis of arterial thrombosis,2 it is noteworthy that a case-control study of patients with myocardial infarction and unstable angina suggested that the presence of at least one allele for the β₃ alloantigen Pl^A2 was associated with an odds ratio for a coronary event of 2.8; the odds ratio increased to 6.2 for patients less than 60 years old.3 Because nearly 20% of non-Asian populations are heterozygous for Pl^A2,4 its presence could put a substantial portion of these populations at increased risk for coronary thrombosis.

Pl^A2 results from a L→P (single-letter amino acid code) polymorphism at amino acid 33 in the extracellular portion of β₃.5 Although amino acid 33 is located at a substantial distance in the linear β₃ sequence from its putative ligand binding region, β₃ is a highly folded molecule and it is conceivable that amino acid 33 is in proximity to the ligand binding region of the mature protein.6 If Pl^A2 affects platelet function, it likely does so by altering the interaction of α_IIbβ₃ with ligands such as fibrinogen, von Willebrand factor, or fibronectin.6,7 β₃-Integrin is also a component of the platelet vitronectin (VN) receptor (α_vβ₃). Although platelets contain only a small amount of α_vβ₃, it is capable of supporting platelet adhesion to substrates such as osteopontin (OPN) and VN.8 Thus, Pl^A2 could also affect platelet α_vβ₃ function. To provide a biochemical basis for the suggestion that Pl^A2 is a risk factor for coronary thrombosis, we measured α_IIbβ₃ and α_vβ₃ function on platelets expressing Pl^A1 or Pl^A2 or both. Because the number of subjects homozygous for Pl^A2 is limited, many of our measurements were made using platelets of individuals who expressed one Pl^A2 allele. Nevertheless, we found that the presence of at least one Pl^A2 allele did not alter the function of either platelet α_IIbβ₃ and α_vβ₃ and conclude that factors unrelated to the function of these integrins likely account for the reported association of Pl^A2 with coronary thrombosis.

DNA was purified from the lymphocytes of 100 healthy volunteers. The polymerase chain reaction (PCR) was then used to amplify a 266-bp fragment from exon 2 of the β₃ gene that contains the polymorphism responsible for the Pl^Aphenotype.5 PCR was performed according to the manufacturer's instructions in a 25-μL reaction volume containing 6.5 pM of each primer, 1.25 U Amplitaq (Applied Biosystems, Foster City, CA), and 150 ng genomic DNA. The antisense primer was 5′-TCTCTCCCCATGGCAAAGAGT-3′ and the sense primer was 5′-TTCTGATTGCTGGACTTCT-3′. Following heating at 94°C for 7 minutes, 30 PCR cycles were performed using the following conditions: denaturation, 94°C for 10 seconds; annealing, 62°C for 30 seconds; extension, 69°C for 90 seconds. This was followed by an extension for 7 minutes at 70°C. Allele-specific hybridization was performed at 40°C using the probes 5′-CCTGCCTCTGGGCTCAC-3′ and 5′-CCTGCCTCCGGGCTCAC-3′. Genotyping was validated by sequencing the affected region of the β₃ gene in 5 Pl^A1/ Pl^A1 homozygotes and 5 Pl^A1/Pl^A2 heterozygotes.

Citrate-anticoagulated platelet-rich plasma (PRP) was obtained from 21 healthy normotensive Pl^A1/ Pl^A1homozygotes and Pl^A1/Pl^A2 heterozygotes whose ages were evenly distributed between 29 and 62. Seventeen subjects were women and 8 were African American. The subjects did not smoke tobacco products and were not taking aspirin or other medications known to affect platelet function. PRP was also obtained by apheresis at the Musser Blood Center of the Penn Jersey Region of the American Red Cross from 5 known white male Pl^A2 homozygotes whose ages ranged from 40 and 56 and was transported to the University of Pennsylvania for study. Platelets were isolated from PRP by gel filtration on Sepharose 2B (Pharmacia, Piscataway, NJ) as previously described.7 Fibrinogen binding to ADP-stimulated platelets was measured using ¹²⁵I-labeled human fibrinogen.7 Briefly, 0.5-mL aliquots of ≈5 × 10⁷ gel-filtered platelets (GFP) were mixed with¹²⁵I-fibrinogen, 0.5 mM CaCl₂, and 10 μM ADP. Following a 5-minute incubation at 37°C without stirring, the platelets were sedimented through silicone oil in an Eppendorf centrifuge (Brinkman Instruments, Westbury, NY). The tips of the centrifuge tubes containing the pelleted platelets were amputated and counted for ¹²⁵I. Nonspecific binding was determined by including a 15-fold excess of unlabeled fibrinogen in the assay. The effect of 2 concentrations of the tetrapeptide RGDS (Sigma) on fibrinogen binding was determined by adding the peptide to the platelet suspensions prior to the addition of ADP.9 

The dissociation constant (K_d) of α_IIbβ₃ for fibrinogen and maximum fibrinogen binding (B_max) were obtained by Scatchard analysis.7 The inhibitory constant (K_i) for RGDS was derived by plotting the slopes of double reciprocal plots of fibrinogen binding obtained in the presence or absence of RGDS against the RGDS concentration.10 

Overlap PCR was used to convert the codon for β₃L33 to P.11 pREP vectors containing α_IIb and β₃ complementary DNAs (cDNAs) were introduced into 7.5 × 10⁶ GM1500 B lymphocytes by electroporation (250 V and 960 μF). Stable cotransfectants were selected using G418 and hygromycin. The α_IIbβ₃ on the lymphocyte surface was quantified by flow cytometry using the monoclonal antibody (mAb) A2A9.12 The Pl^A allotype of each cell line was verified using polyclonal antisera that specifically recognize Pl^A1 or Pl^A2.13 Phorbol myristate acetate (PMA)–stimulated adhesion of³⁵S-methionine-labeled lymphocytes to fibrinogen was measured as previously described.11 Adherence data were normalized for the level of α_IIbβ₃expression by dividing the percent adherence by the mean fluorescence intensity (MFI) determined by flow cytometry.11 

Platelet-dependent hemostasis was assessed using the PFA-100 Platelet Function Analyzer (Dade-Behring, Deerfield, IL).14 Turbidometric platelet aggregation was measured using a Bio-Data PAP-4 aggregometer.15 Thrombin receptor-activating peptide (TRAP)–stimulated P-selectin expression was measured by flow cytometry using the ADIAflo Platelet Gp kit (American Diagnostica, Greenwich, CT) according to the manufacturer's instructions. Briefly, TRAP-stimulated platelets were incubated sequentially with the mAb CD62P (Biocytex, Marseilles, France) and fluorescein isothiocyanate (FITC)–labeled antimouse IgG, followed by flow cytometry. The measured platelet fluorescence was then compared to that obtained using 4 populations of 2 μm latex beads, each population coated with a known quantity of mouse IgG. The amount of P-selectin expressed per platelet was calculated from plots of bead fluorescence versus the amount of antibody bound per bead. The effect of the α_IIbβ₃ antagonist RWJ 53308 on TRAP-stimulated platelet aggregation was tested after determining whether Pl^A allotype affected RWJ 53308 binding. RWJ 53308 is a peptidomimetic whose structure is based on the K-Q-A-G-D sequence present at the carboxyl terminus of the fibrinogen γ chain.16 Platelets in citrated whole blood were stained with 2 anti-α_IIbβ₃ mAbs: Mab1, whose binding was not affected by RWJ 53308, and Mab2, whose binding to α_IIbβ₃ is inhibited by RWJ 53308. Flow cytometry measurements were converted to occupied α_IIbβ₃ complexes using microbeads coated with known amounts of FITC.

Platelet adhesion to OPN and VN was measured as previously described.8 Aliquots of GFP (100 μL) were added to the wells of microtiter plates coated with 5 μg/mL recombinant OPN or human VN (Sigma). Platelets were stimulated with 10 μM ADP. After a 30-minute incubation at 37°C, the number of adherent platelets was determined using a colorimetric assay.8 

Power calculations were based on the variability of our fibrinogen binding assay.7 Sample size was estimated as 10 per genotype to detect a log difference in K_d and K_i with 90% power. Statistical significance of differences was determined using a 2-tailed t test for unpaired samples.

To study the effect of Pl^A genotype on the affinity of α_IIbβ₃ for fibrinogen, we determined the Pl^A genotype of 100 healthy subjects. The frequency of the Pl^A2 allele among the 100 subjects was 16%, consistent with the frequency of the Pl^A2 allele in non-Asian populations.4 No Pl^A2 homozygotes were identified. We then measured both the K_d and B_max of fibrinogen binding to ADP-stimulated platelets of 10 subjects homozygous for Pl^A1 and 11 subjects heterozygous for Pl^A1/ Pl^A2. We also measured the K_d and B_max of fibrinogen binding to the platelets of 5 subjects homozygous for Pl^A2 obtained from the Musser Blood Center of the Penn Jersey Region of the American Red Cross. As shown in Figure 1A, there was overlap in the distribution of K_d values among the 3 groups of subjects. The means of these distributions were 1.36 ± 0.22 × 10⁻⁷ M, 1.28 ± 0.13 × 10⁻⁷ M, and 0.73 ± 0.08 × 10⁻⁷ M for the Pl^A1homozygotes, Pl^A1/Pl^A2 heterozygotes, and Pl^A2 homozygotes, respectively. Although the means for the Pl^A1 homozygotes and Pl^A1/Pl^A2heterozygotes were not statistically different (P = .74), the mean for the Pl^A2 homozygotes was significantly lower than that for the Pl^A1/Pl^A2 heterozygotes (P = .02), but not for the Pl^A1 homozygotes (P = .07). The distribution of values for B_maxis shown in Figure 1B. The means of these distributions ranged from 2.65 ± 0.15 × 10⁻¹⁹ mole/platelet for Pl^A1 homozygotes, 2.90 ± 0.14 × 10⁻¹⁹mole/platelet for Pl^A1/Pl^A2 heterozygotes, and 2.17 ± 0.11 × 10⁻¹⁹ mole/platelet for Pl^A2 homozygotes. Again, there was no significant difference between Pl^A1 homozygotes and Pl^A1/Pl^A2 heterozygotes (P = .24), whereas the mean B_max of the P^lA2 homozygotes was significantly less than that of the other 2 groups (P = .048 and .006, respectively). The data shown in Figure 1 indicate that the presence of a single Pl^A2 allele did not alter the ability of α_IIbβ₃ to interact with fibrinogen when the platelets were maximally stimulated by ADP. To address the possibility that a difference might be apparent at lower agonist concentrations, we measured fibrinogen binding to platelets of individuals homozygous for Pl^A1 and heterozygous for Pl^A1/Pl^A2 as a function of ADP concentration. As shown in Figure 2A, there was no difference in fibrinogen binding to the platelets of these individuals as the concentration of ADP was varied from 0.25 μM to 10 μM. Moreover, the median effective concentration (EC₅₀) values for ADP-stimulated fibrinogen binding (0.77 ± 0.18 μM and 0.82 ± 0.24 μM, respectively) were not statistically different (Figure 2B). RGD-containing peptides competitively inhibit fibrinogen binding to α_IIbβ₃.17 To determine if the inhibitory effect of the tetrapeptide RGDS is affected by the presence of Pl^A2, we measured the K_i for RGDS using platelets from 10 Pl^A1/Pl^A1 and 10 Pl^A1/Pl^A2 subjects. As shown in Figure3, the distribution of K_ivalues for the 20 subjects overlapped completely, nor was there a significant differences in the means of these distributions (33.9 ± 3.0 μg/mL and 35.0 ± 3.6 μg/mL, respectively,P = .82).

It is possible that sequence differences in β₃linked to Pl^A, but not Pl^A itself, could affect α_IIbβ₃ function. To address this possibility, we cotransfected the B-lymphocyte line GM1500 with an α_IIb cDNA and a β₃ cDNA encoding either Pl^A1 or Pl^A2 and measured unstimulated and PMA-stimulated lymphocyte adhesion to immobilized fibrinogen.11 As shown in Figure4, there was no significant difference in either the unstimulated or the PMA-stimulated adhesion of lymphocytes expressing α_IIbβ₃ containing either the Pl^A1 or Pl^A2 alloantigen (P = .45 and P = .65, respectively).

Postreceptor events, including platelet secretion, are thought to stabilize platelet aggregates.18 To determine whether macroscopic platelet aggregation is affected by the presence of a Pl^A2 allele, we compared the aggregation of Pl^A1/Pl^A1, Pl^A1/Pl^A2, and Pl^A2/Pl^A2 platelets induced by TRAP in a conventional platelet aggregometer. We found no significant differences in either the extent (Figure 5A) or rate (data not shown) of platelet aggregation as the TRAP concentration was increased from 0.4 to 1.0 μM. At low concentrations, even strong agonists like TRAP require α_IIbβ₃-mediated platelet aggregation to induce platelet secretion. To determine whether this is affected by Pl^A2, we measured P-selectin expression as an indication of platelet secretion on TRAP-stimulated platelets (Figure 5B). We found baseline P-selectin expression was slightly increased on unstimulated Pl^A2/Pl^A2 platelets. Nonetheless, when P-selectin expression was corrected for this difference in baseline expression, there were no differences in TRAP-stimulated P-selectin expression between Pl^A1/Pl^A1, Pl^A1/Pl^A2, and Pl^A2/Pl^A2 platelets. We next compared the rate of platelet thrombus formation in whole blood as a function of Pl^A allotype using the PFA-100 instrument.14As seen in Figure 6, there were no detectable differences in the time required to make an occlusive thrombus among the 3 groups of subjects. We also studied the effect of Pl^A allotype on the ability of the low-molecular-weight α_IIbβ₃ antagonist RWJ 53308 to inhibit macroscopic platelet aggregation.16 We first determined whether there were differences in binding of the compound to platelets according to their Pl^A allotype. As seen in Figure7A, RWJ 53308 bound equally well to platelets regardless of Pl^A allotype. We then measured TRAP-stimulated platelet aggregation in the presence of increasing concentrations of RWJ 53308. As seen in Figure 7B, there were no significant differences in the ability of RWJ 53308 to inhibit the aggregation of Pl^A1/Pl^A1, Pl^A1/Pl^A2, and Pl^A2/Pl^A2 platelets.

The integrin α_vβ₃ mediates platelet adhesion to OPN- and VN-coated surfaces.8 To determine if the presence of a Pl^A2 allele affects platelet adherence to OPN and VN, we compared platelet avidity for each substrate by determining the 50% inhibitory concentration (IC₅₀) for RGDS, reasoning that there is a direct relationship between the IC₅₀ and the avidity of platelet adhesion. There were no significant differences in the IC₅₀ for RGDS inhibition of platelet adhesion to OPN (35.0 ± 10.5 μM versus 19.1 ± 8.5 μ, P = .3) or VN (52.5 ± 15.6 μM and 62.1 ± 29.8 μM, P = .79) as a function of Pl^A allotype (Figure 8). These data suggest that platelet α_vβ₃, as well as α_IIbβ₃, is not affected by the presence of a Pl^A2 allele.

The formation of a coronary thrombus in vivo is a complex process that is influenced by a number of factors, including the nature of the vascular wall pathology, the ambient shear rate, and platelet reactivity. Therefore, it is not surprising that the contribution of a single factor, such as the polymorphism responsible for the Pl^A2 alloantigen on platelet α_IIbβ₃, to the risk for coronary artery thrombosis is uncertain. Moreover, it is noteworthy that of 37 epidemiologic studies addressing this question, only 15 implicated Pl^A2 in various aspects of arterial thrombosis.3,19-54 We reasoned that if the presence of Pl^A2 directly affects coronary thrombus formation, it should be possible to detect an effect of Pl^A2 on β₃-integrin function. Although our studies were limited by the small number of subjects who were homozygous for Pl^A2, heterozygous subjects were available in the expected numbers, and the presence of a single Pl^A2 allele in heterozygous individuals has been reported to confer significant thrombotic risk in all of the positive studies3,41-52,54but one.53 

Initially, we measured parameters of α_IIbβ₃function using radiolabeled fibrinogen. We found no differences in the affinity of fibrinogen binding or in maximal fibrinogen binding using platelets from 10 subjects homozygous for Pl^A1 and 11 subjects heterozygous for Pl^A1/Pl^A2. Although it is conceivable that differences might have become apparent if more subjects were studied, the extent of the overlap of these values within the groups suggests that this would be unlikely. On the other hand, the values for K_d and B_max of platelets from subjects homozygous for Pl^A2 were clustered tightly at the lower end of the distribution ranges of the other 2 groups, raising the possibility that the slightly increased affinity of these platelets for fibrinogen could enhance their ability to form thrombi. However, the K_d for fibrinogen binding of each allotype is well below the plasma concentration of fibrinogen,7 a disparity likely to be magnified within a thrombus because agonist-stimulated platelets secrete fibrinogen. Thus, under conditions of substantial fibrinogen excess, small differences in affinity are likely to have a negligible effect on fibrinogen binding and would not be expected to account for the reported effects of Pl^A2.10 

A more likely possibility is that the presence of Pl^A2enhances the ability of agonists to induce ligand binding to α_IIbβ₃ and a number of functional studies comparing Pl^A1 and Pl^A2 platelets have been reported. However, like the epidemiologic studies cited above, these reports have been contradictory. Thus, although Feng and colleagues55 reported that threshold concentrations for epinephrine-stimulated platelet aggregation were significantly lower when platelets expressed one or 2 copies of Pl^A2 and Michelson and coworkers concluded that Pl^A2-positive platelets displayed a lower threshold for activation,56Lasne and associates measured the threshold for biphasic aggregation induced by TRAP and ADP and found that platelets expressing Pl^A2 were actually less sensitive to either agonist.57 Similarly, Cooke and coworkers reported that the platelets of Pl^A1/Pl^A2 heterozygotes were more sensitive to the inhibitory effects of aspirin,58whereas Andrioli and colleagues reported that Pl^A2platelets were hyporesponsive to thromboxane A₂.39 In addition, Goodall and coworkers found increased fibrinogen binding to Pl^A2 platelets stimulated by ADP, but not when the platelets were stimulated by thrombin.59 On the other hand, Meiklejohn and colleagues reported that there was no difference in fibrinogen binding to ADP-stimulated Pl^A1 and Pl^A2platelets.60 We found that the presence of a single Pl^A2 allele had no effect when fibrinogen binding was measured as a function of ADP concentration. Moreover, we did not detect a difference in the TRAP-stimulated aggregation of homozygous or heterozygous Pl^A2 platelets compared to homozygous Pl^A1 platelets when platelet aggregation was measured as a function of TRAP concentration, nor did we detect differences in PMA-stimulated adhesion of B cells expressing either Pl^A1or Pl^A2 to fibrinogen-coated surfaces. Similarly, Wang and Newman did not detect functional differences between platelets expressing the Pen^a and Pen^b alloantigens that result from an R143Q polymorphism located in the RGD-binding domain of β₃.61 Thus, our measurements argue against the possibility that the presence of the Pl^A2 polymorphism facilitates the agonist-induced conversion of α_IIbβ₃ from a resting to an activated ligand-binding state.

α_IIbβ₃ function is associated with a number of “postreceptor” events including platelet aggregation, platelet secretion when platelets are stimulated by weak agonists such as ADP and epinephrine, clot retraction, and phosphorylation of focal adhesion kinase. It is worth noting that each of these events firstrequires ligand binding to α_IIbβ₃. Thus, each is either absent or perturbed in platelets from patients with Glanzmann thrombasthenia, a hereditary bleeding disorder due to the absence of sufficient numbers of functional α_IIbβ₃ complexes. Vijayan and associates addressed the influence of Pl^A2 on postreceptor events initiated through α_IIbβ₃ by expressing α_IIbβ₃ heterologously in Chinese hamster ovary (CHO) and 293 cells.62 Although they found no difference in soluble fibrinogen binding to transfected CHO cells expressing either Pl^A1 or Pl^A2, they observed increased adhesion of both CHO and 293 cells expressing Pl^A2 to immobilized fibrinogen. We performed similar experiments using transfected PMA-stimulated human B lymphocytes, but detected no significant difference in the adhesion of cells expressing either Pl^A1 or Pl^A2. We also measured agonist-stimulated platelet aggregation using both conventional aggregometry and the PFA-100 instrument and P-selectin expression as a measure of platelet secretion in patients who expressed 0, 1, or 2 Pl^A2 alleles. Again, we detected no differences. However, like Michelson and coworkers,56 we did find a slight increase in basal P-selectin expression on unstimulated platelets homozygous for Pl^A2. In our case, this was likely due to a slight degree of platelet activation during apheresis at the Red Cross or during the transport of blood from the Red Cross to the University of Pennsylvania because there was no difference in basal P-selectin on Pl^A1/Pl^A1 and Pl^A1/Pl^A2platelets obtained at the University of Pennsylvania.

β₃-integrin is a component of the VN receptor α_vβ₃. We have observed that despite the small number of α_vβ₃ complexes present on platelets, this integrin can mediate platelet adhesion to surfaces coated with OPN or VN.8 Thus, it is conceivable that perturbation of α_vβ₃ function could be responsible for the association of the Pl^A2 with myocardial infarction. Nonetheless, we found that the presence of Pl^A2did not effect the avidity of α_vβ₃-mediated adhesion of platelets to either OPN or VN, suggesting that the presence of a Pl^A2 allele does not modify the function of this integrin.

In summary, we have examined the effect of the Pl^A2polymorphism on β₃-integrin function in human platelets, focusing on agonist-stimulated ligand binding because this is central to the function of these integrins. Because we did not detect substantial differences in the ability of β₃-integrins to interact with ligands based on this polymorphism, our results suggest that factors unrelated to β₃-integrin function may account for the reported association of the Pl^A2 allele with coronary thrombosis.