The interaction of integrin αIIbβ3 with fibrin occurs through multiple binding sites in the αIIb β-propeller domain

Abstract

The currently available antithrombotic agents target the interaction of platelet integrin αIIbβ3 (GPIIb-IIIa) with fibrinogen during platelet aggregation. Platelets also bind fibrin formed early during thrombus growth. It was proposed that inhibition of platelet-fibrin interactions may be a necessary and important property of αIIbβ3 antagonists; however, the mechanisms by which αIIbβ3 binds fibrin are uncertain. We have previously identified the γ370-381 sequence (P3) in the γC domain of fibrinogen as the fibrin-specific binding site for αIIbβ3 involved in platelet adhesion and platelet-mediated fibrin clot retraction. In the present study, we have demonstrated that P3 can bind to several discontinuous segments within the αIIb β-propeller domain of αIIbβ3 enriched with negatively charged and aromatic residues. By screening peptide libraries spanning the sequence of the αIIb β-propeller, several sequences were identified as candidate contact sites for P3. Synthetic peptides duplicating these segments inhibited platelet adhesion and clot retraction but not platelet aggregation, supporting the role of these regions in fibrin recognition. Mutant αIIbβ3 receptors in which residues identified as critical for P3 binding were substituted for homologous residues in the I-less integrin αMβ2 exhibited reduced cell adhesion and clot retraction. These residues are different from those that are involved in the coordination of the fibrinogen γ404-411 sequence and from auxiliary sites implicated in binding of soluble fibrinogen. These results map the binding of fibrin to multiple sites in the αIIb β-propeller and further indicate that recognition specificity of αIIbβ3 for fibrin differs from that for soluble fibrinogen.

Keywords: Adhesion; Clot Retraction; Fibrin; Fibrinogen; Integrin; Platelets.

FIGURE 1.

The interaction of α_IIbβ₃ with the P3 peptide analyzed by SPR. A, representative profiles of the SPR responses for P3 peptide concentrations ranging from 0 to 45 μm binding to purified α_IIbβ₃ coupled onto a CM5 sensor chip. RU, response units. B, saturable binding curve and Scatchard plot (inset) of P3 binding to α_IIbβ₃. Req is the response at equilibrium. The abscissa in the Scatchard plot is the ratio of the number of P3 molecules bound per molecule of α_IIbβ₃. The ratio of bound to free peptide is given on the ordinate.

FIGURE 2.

The binding of P3 to the peptide library spanning the sequence of the α_IIb β-propeller. A, the amino acid sequence of the of α_IIb β-propeller (residues 1–451). The β-strands from the seven blades are marked and underlined. The numbering of residues is shown with a dot, which marks every 10th residue. B, a peptide library consisting of 9-mer peptides derived from the α_IIb β-propeller (residues 1–451) was screened for P3 binding. The membrane with covalently attached peptides was incubated with ¹²⁵I-labeled P3 and subjected to autoradiography. C, clusters of the overlapping sequences selected based on the highest P3 binding activity are shown. The numbers of peptides correspond to the numbering of spots in B.

FIGURE 3.

Effect of the α_IIb 241–255 peptide on platelet-mediated fibrin clot retraction. A, platelets were mixed with 0.25 mg/ml fibrinogen in isotonic HEPES buffer containing 1 mm CaCl₂ and different concentrations of the α_IIb 241–255 peptide (0–200 μm), and fibrin clots were formed by adding 1 unit/ml thrombin at 22 °C. Clot retraction was observed by taking photographs at different times (0–85 min). The left lane of tubes (0) shows clot retraction in the absence of peptide. A representative experiment is shown. B, clot areas in each tube were measured from images in A, and a percentage of clot retraction was calculated. Kinetic curves of retraction in the absence (●) or presence of 50 (○), 100 (■), and 200 (□) μm α_IIb 241–255 were generated by plotting clot areas versus time. C, dose-dependent inhibition of clot retraction by α_IIb 241–255 is shown. Clot retraction in the presence of selected concentrations of the peptide was determined at 85 min. At this time, clot retraction in the absence of peptide was complete (100% retraction), and clot retraction in the presence of each concentration of peptide was at different stages of completion. At 200 μm, no clot retraction was observed. The data shown are means and S.D. (error bars) from three experiments.

FIGURE 4.

Effect of the α_IIb 241–255 peptide on platelet-mediated retraction of clots formed from mutant γ407 and normal recombinant fibrinogens. Platelets (2 × 10⁸/ml) were mixed with 0.25 mg/ml normal fibrinogen (A) and Fg γ407 (B) in isotonic HEPES buffer containing 1 mm CaCl₂ and 60 μm (○) or 150 μm (▾) α_IIb 241–255 (final volume, 0.25 ml). Fibrin clots were formed by adding 1 unit/ml thrombin at 22 °C, and clot retraction was observed by taking photographs at different times. Kinetic curves of retraction of clots formed from normal fibrinogen in the absence (●) or presence of α_IIb 241–255 were generated by plotting clot areas versus time.

FIGURE 5.

Effect of the α_IIb β-propeller-derived peptides on platelet adhesion. Microtiter wells were coated with 10 μg/ml D98 fragment and postcoated with 1% BSA. 50-μl aliquots containing different concentrations of the α_IIb-derived peptides in isotonic HEPES buffer were added to the wells for 15 min at 37 °C followed by suspensions of calcein-labeled platelets (1 × 10⁷/50 μl). After 30 min at 37 °C, nonadherent cells were removed, and adhesion was determined. A, a dose-dependent inhibition of adhesion by α_IIb 94–108 is shown. B, inhibition of platelet adhesion by each peptide used at 250 μm is shown. The data are expressed as the percentage of adhesion in the absence of peptides. Error bars represent S.D. The results shown are the average of triplicate measurements at each experimental point and are representative of three to five experiments.

FIGURE 6.

Adhesion of HEK293 cells expressing wild-type and mutant α_IIbβ₃ receptors. A, adhesion of HEK293 cells expressing the α_IIb β-propeller double point mutant W100S/D102P is shown. Microtiter wells were coated with different concentrations of the D98 fragment (0–10 μg/ml). Calcein-labeled HEK293 cells expressing WT (●) or mutant (W100S/D102P) integrin α_IIbβ₃ (▾) were added to the wells (5 × 10⁴ cells/well) and allowed to adhere for 30 min at 37 °C. After washing of non-adherent cells, cell adhesion was assessed by measuring fluorescence. Adhesion on WT HEK293 cells (○) was also determined. B, adhesion of each mutant to different concentrations of D98 was performed as described in A. Adhesion of each mutant reached the maximal level at 10 μg/ml D98 and is shown as a percentage of maximal adhesion attained with HEK293 cells expressing WT α_IIbβ₃. The data are means ± S.D. (error bars) of triplicate determinations at experimental data point and are representative of 3–5 experiments.

FIGURE 7.

Clot retraction mediated by HEK293 cells expressing mutant α_IIbβ₃. A, aliquots (2 × 10⁶ cells/ml) of HEK293 cells stably expressing WT (left panel) or mutant α_IIbβ₃ (right panel)in Tyrode's/HEPES buffer were incubated with 10 mm tranexamic acid, 0.25 mg/ml fibrinogen, and 2 mm CaCl₂ in siliconized glass tubes for 5 min at 37 °C. mAb LM609 (10 μg/ml) was added to block α_vβ₃-mediated clot retraction. Fibrin polymerization and clot retraction were initiated by adding 1 unit/ml thrombin to cell suspensions, and fibrin gels were incubated at 37 °C for the next 220 min. Clot retraction was monitored by taking digital photographs. Representative photographs of retracted gels are shown. B, kinetic curves of retraction by WT α_IIbβ₃ (●) or mutant receptor carrying 10 point mutations (○) were generated by plotting clot areas versus time. HEK293 cells (▾) do not support clot retraction. The data are expressed as percentage of clot retraction as described under “Experimental Procedures.” Results are representative of three separate experiments.

FIGURE 8.

The ribbon model of the α_IIbβ₃ headpiece based on the crystal structure (Protein Data Bank code 2VDO). The α_IIb subunit is shown in gray, and β₃ is shown in tan. Amino acid residues identified as critical for the binding of fibrin-specific peptide P3 in the α_IIb β-propeller are shown in magenta (selected residues are labeled). Two views (A and B) are rotated relative to each other by 180° about the vertical axis.