Volatile anesthetics, not intravenous anesthetic propofol bind to and attenuate the activation of platelet receptor integrin αIIbβ3

Abstract

Background: In clinical reports, the usage of isoflurane and sevoflurane was associated with more surgical field bleeding in endoscopic sinus surgeries as compared to propofol. The activation of platelet receptor αIIbβ3 is a crucial event for platelet aggregation and clot stability. Here we studied the effect of isoflurane, sevoflurane, and propofol on the activation of αIIbβ3.

Methods: The effect of anesthetics on the activation of αIIbβ3 was probed using the activation sensitive antibody PAC-1 in both cell-based (platelets and αIIbβ3 transfectants) and cell-free assays. The binding sites of isoflurane on αIIbβ3 were explored using photoactivatable isoflurane (azi-isoflurane). The functional implication of revealed isoflurane binding sites were studied using alanine-scanning mutagenesis.

Results: Isoflurane and sevoflurane diminished the binding of PAC-1 to wild-type αIIbβ3 transfectants, but not to the high-affinity mutant, β3-N305T. Both anesthetics also impaired PAC-1 binding in a cell-free assay. In contrast, propofol did not affect the activation of αIIbβ3. Residues adducted by azi-isoflurane were near the calcium binding site (an important regulatory site termed SyMBS) just outside of the ligand binding site. The mutagenesis experiments demonstrated that these adducted residues were important in regulating integrin activation.

Conclusions: Isoflurane and sevoflurane, but not propofol, impaired the activation of αIIbβ3. Azi-isoflurane binds to the regulatory site of integrin αIIbβ3, thereby suggesting that isoflurane blocks ligand binding of αIIbβ3 in not a competitive, but an allosteric manner.

Figure 1. Integrin structure and conformational change.

(A) αIIbβ3 consists of the α subunit (αIIb) and the βsubunit (β3). Domains within the primary structure of α- and β- subunits suggested by X ray crystal structures of αVβ3 and αIIbβ3 , are shown. The β-propeller and the thigh domains of the α subunit and the PSI, the hybrid and the I domains of the β subunit constitute the headpiece of αIIbβ3. (B) Schema of conformational change of the headpiece. The metal-ion dependent adhesion site (MIDAS) undergoes conformation change and interacts directly with ligands when it is in an active form. In a conformation where the hybrid domain faces inward toward the α subunit, the MIDAS is inactive. When the hybrid domain swings out, the conformational change of the MIDAS ensues with ligand or PAC-1 binding.

Figure 2. PAC-1 binding assays with anesthetics in platelets.

Flow cytometry based PAC-1 binding assays were performed using platelet-rich plasma stimulated with 20 µM adenosine 5′-diphosphate (ADP) in the presence of isoflurane (2%) or propofol (50 µM). Data is shown as mean +/− S.D. of mean fluorescence intensity (MFI) of six independent experiments. Data were analyzed using a one-way analysis of variance with Tukey post hoc pairwise comparisons. * denotes p<0.05 versus ADP-treated control sample.

Figure 3. PAC-1 binding assays using αIIbβ3 transfectants in the presence of anesthetics.

(A) Scheme of PAC-1 interaction with αIIbβ3 WT and β3-N305T mutant. While αIIbβ3 wild-type (WT) binds to PAC-1 only in an activating condition (1 mM Mn²⁺/0.4 mM Ca²⁺), activating β3-N305T mutant can bind to PAC-1 in a resting condition (1 mM Mg²⁺/Ca²⁺) due to its constitutive swing-out of the hybrid domain. (B–D) Flow cytometry based PAC-1 binding assays were performed using CHO cells stably transfected with wild type αIIbβ3 or N305T mutant in the presence of isoflurane (B) or sevoflurane (C) at various concentrations. For propofol, only wild type αIIbβ3 was tested (D). PAC-1 binding % was calculated as [(mean fluorescence intensity (MFI) at various concentrations of anesthetics) – (MFI of isotyoe control)]/[(MFI without anesthetics) – (MFI of isotype control)]×100 (%). Data is shown as mean +/− S.D. of three independent experiments. Binding experiment was done at 1 mM Mn²⁺/0.4 mM Ca²⁺. One-way analysis of variance with Tukey post hoc pairwise comparisons was used to compare the data at different anesthetic concentrations within wild-type or mutant transfectants. * denotes p<0.05 versus mock-treated sample (no anesthetic).

Figure 4. The effect of anesthetics on αIIbβ3 cell surface expression.

Surface expression of αIIbβ3 WT (A) or β3-N305T (B) was probed by AP3 antibody and expressed using mean fluorescence intensity (MFI). Data was shown as [(MFI of αIIbβ3 exposed to anesthetic)/(MFI of αIIbβ3 of sample not exposed to anesthetic)]×100%, and expressed as mean +/− S.D. of three independent experiments. Isoflurane, sevoflurane, and propofol used were 5%, 4%, and 100 µM, respectively.

Figure 5. Cell-free PAC-1 binding assays with anesthetics.

ELISA type PAC-1 binding assays were performed using full-length ectodomain or headpiece αIIbβ3 in the presence of isoflurane (A) or sevoflurane (B) at various concentrations. For propofol, experiments were performed using full-length αIIbβ3 (C). PAC-1 binding % was calculated as [(OD at various concentrations of anesthetics)- (OD of background)]/[(OD of mock treated sample)-(OD of background)]×100 (%). Data is shown as mean +/− S.D. of three independent experiments. Binding experiment was done at 1 mM Mn²⁺/0.4 mM Ca²⁺. One-way analysis of variance with Tukey post hoc pairwise comparisons was used to compare the data at different anesthetic concentrations within full-length or headpiece protein. * denotes p<0.05 versus mock-treated sample (no anesthetic).

Figure 6. Amino acid residues of the β I domain covered by mass spectrometry.

The amino acid residues of the β I domain are shown. Covered residues by mass spectrometry are shown in red. Adducted residues are shown in asterisk.

Figure 7. αIIbβ3 headpiece structure and adducted residues.

(A) The X ray crystal structure of αIIbβ3 headpiece was obtained from protein data bank (PDB; 3FCS). There are three metal binding sites in the I domain of the β subunit. Mg²⁺ in the MIDAS (this site is directly involved in ligands binding) is shown in yellow sphere, while Ca²⁺ in the SyMBS and ADMIDAS are shown in orange and light orange spheres, respectively. (B) The blowout of residues around metal binding sites from Figure 7 (A) is shown. The adducted residues of photolabeling experiments are shown in blue. Again, Mg²⁺ in the MIDAS is shown in yellow sphere, while Ca²⁺ in the SyMBS is shown in orange sphere. Both figures were created using PYMOL. (C) The structure of αIIbβ3 in the open conformation was obtained from Protein data bank (http://www.rcsb.org/pdb/home/home.do; PDB 3FCU). Residues shown as green spheres on αIIbβ3 are suggested PAC-1 binding sites by Puzon-McLaughlin et al. . This figure was created using PYMOL.

Figure 8. β3 mutants of adducted residues.

(A) PAC-1 binding to mock, αIIbβ3 wild type or mutant in 1 mM Mg²⁺/Ca²⁺ or 1 mM Mn²⁺/0.4 mM Ca²⁺. MFI; mean fluorescence intensity. One-way analysis of variance with Tukey post hoc analysis was performed to compare different groups (excluding mock group). * denotes p<0.05 versus wild type, 1 mM Mn²⁺/0.4 mM Ca²⁺ group. (B) Surface expression of mock, αIIbβ3 wild-type or mutants probed by AP3 is shown. Data is shown as mean +/− S.D. of three independent experiments. One-way analysis of variance with Tukey post hoc analysis was performed (excluding mock group). * denotes p<0.05 versus wild type.