Figure 1.
The αIIb C490-C545 disulfide bond is missing in approximately one-third of integrin molecules on the resting and activated human platelet surface. (A) Blood from healthy volunteers was drawn by venipuncture into 0.4% citrate as an anticoagulant. Platelet-rich plasma was prepared by centrifugation and transferred into a new tube with 20% (v/v) acid citrate dextrose (ACD) solution. After a 30-minute rest at 37°C, 1 µM prostaglandin E1 (PgE1) was added and platelets pelleted by centrifugation. The platelet pellet was resuspended in warm HEPES Tyrode’s buffer and unpaired cysteine thiols alkylated by the addition of 4 mM 12C-IPA, in some cases after the addition of 1 U/mL thrombin and 5 µg/mL collagen to activate the platelets. Labeled platelets were pelleted and resuspended in lysis buffer. The lysate was diluted in HEPES-buffered saline and incubated with anti-β3 mAb; the antibody-labeled integrin was then collected on protein A/G agarose, washed, labeled for a second time with 4 mM 12C-IPA, eluted in LDS buffer, and resolved on SDS-PAGE. The αIIb band was excised and washed before disulfide-bonded cysteines were reduced with dithiothreitol (DTT), and the resulting new cysteine thiols labeled with 5 mM 13C-IPA. The protein was deglycosylated by using PNGase F and digested with chymotrypsin and trypsin. Twenty-three peptides reporting on 8 of the 9 αIIb disulfide bonds were analyzed by using mass spectrometry (Table 1; supplemental Figure 1). The redox state of disulfide bonds was quantified as the percentage of 12C-IPA labeled Cys in which the total was the sum of 12C-IPA– and 13C-IPA–labeled Cys. Example of a Sypro Ruby–stained gel of 12C-IPA–labeled αIIbβ3 resolved on SDS-PAGE is shown at right. Molecular mass standards are shown in the left lane. (B) Positions of the αIIb integrin disulfide bonds in a modeled open structure4 of the complete αIIbβ3 integrin ectodomain (αIIb subunit in wheat ribbon and β3 subunit in gray ribbon). The cysteine residues comprising the nine αIIb disulfide bonds are shown as cyan, blue, and black spheres and the residue numbers indicated. The redox state of the cysteines in cyan and blue was measured by using differential alkylation and by mass spectrometry. The cysteines in black (C880 and C885) were not determined. Domain names are indicated. (C) Redox states of eight αIIb disulfide bonds in 13 healthy human donor platelets (6 male subjects, 7 female subjects; 21-48 years old). The bars and errors are mean ± standard deviation. (D) Percentage of the αIIb C490-C545 disulfide bond that is missing in total (6 male subjects, 7 female subjects; 21-48 years old) and platelet surface (2 female subjects; 22 and 23 years old, biological replicates) integrin in healthy human donor platelets. The bars and errors are mean ± standard deviation.

The αIIb C490-C545 disulfide bond is missing in approximately one-third of integrin molecules on the resting and activated human platelet surface. (A) Blood from healthy volunteers was drawn by venipuncture into 0.4% citrate as an anticoagulant. Platelet-rich plasma was prepared by centrifugation and transferred into a new tube with 20% (v/v) acid citrate dextrose (ACD) solution. After a 30-minute rest at 37°C, 1 µM prostaglandin E1 (PgE1) was added and platelets pelleted by centrifugation. The platelet pellet was resuspended in warm HEPES Tyrode’s buffer and unpaired cysteine thiols alkylated by the addition of 4 mM 12C-IPA, in some cases after the addition of 1 U/mL thrombin and 5 µg/mL collagen to activate the platelets. Labeled platelets were pelleted and resuspended in lysis buffer. The lysate was diluted in HEPES-buffered saline and incubated with anti-β3 mAb; the antibody-labeled integrin was then collected on protein A/G agarose, washed, labeled for a second time with 4 mM 12C-IPA, eluted in LDS buffer, and resolved on SDS-PAGE. The αIIb band was excised and washed before disulfide-bonded cysteines were reduced with dithiothreitol (DTT), and the resulting new cysteine thiols labeled with 5 mM 13C-IPA. The protein was deglycosylated by using PNGase F and digested with chymotrypsin and trypsin. Twenty-three peptides reporting on 8 of the 9 αIIb disulfide bonds were analyzed by using mass spectrometry (Table 1; supplemental Figure 1). The redox state of disulfide bonds was quantified as the percentage of 12C-IPA labeled Cys in which the total was the sum of 12C-IPA– and 13C-IPA–labeled Cys. Example of a Sypro Ruby–stained gel of 12C-IPA–labeled αIIbβ3 resolved on SDS-PAGE is shown at right. Molecular mass standards are shown in the left lane. (B) Positions of the αIIb integrin disulfide bonds in a modeled open structure4 of the complete αIIbβ3 integrin ectodomain (αIIb subunit in wheat ribbon and β3 subunit in gray ribbon). The cysteine residues comprising the nine αIIb disulfide bonds are shown as cyan, blue, and black spheres and the residue numbers indicated. The redox state of the cysteines in cyan and blue was measured by using differential alkylation and by mass spectrometry. The cysteines in black (C880 and C885) were not determined. Domain names are indicated. (C) Redox states of eight αIIb disulfide bonds in 13 healthy human donor platelets (6 male subjects, 7 female subjects; 21-48 years old). The bars and errors are mean ± standard deviation. (D) Percentage of the αIIb C490-C545 disulfide bond that is missing in total (6 male subjects, 7 female subjects; 21-48 years old) and platelet surface (2 female subjects; 22 and 23 years old, biological replicates) integrin in healthy human donor platelets. The bars and errors are mean ± standard deviation.

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