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Blood, Vol. 109, Issue 9, 3725-3732, May 1, 2007
Mapping early conformational changes in  IIb and ß3 during biogenesis reveals a potential mechanism for IIbß3 adopting its bent conformation
Blood Beau Mitchell et al.
109: 3725
Supplemental materials for: Mitchell et al, Vol 109, Issue 9, 3725-3732
Files in this Data Supplement:
- Figure S1. αIIb-specific LIBS mAbs recognize only small amounts of free pro-αIIb (JPG, 75 KB)
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(A) Epitopes of the mAb anti-V5 and the  IIb-specific LIBS mAbs are indicated by spheres or bars on the bent, unactivated, and the proposed extended structure of ligand-bound IIb 3. (B) PMI-1 binding to IIb3 receptors on the same cells as in panel C increased more than 5-fold in the presence of EDTA, indicating that the PMI-1 epitope is available on these receptors under conditions which increase PMI-1 binding to platelets. (C) IIb-specific LIBS mAbs B1B5 and PMI-1 reacted with only small subpopulations of free pro-IIb, pro-IIb3, and mature IIb3. In addition, the polyclonal antibody anti-IIb (H-160), which was raised against the amino acid sequence 847 to 1006, thus encompassing both the B1B5 and PMI-1 epitopes, precipitated only trace amounts of free pro-IIb. The 90 minute time point is depicted from 1 of more than 5 experiments. Equivalent amounts of protein were loaded in each lane. Nonreduced Western blots of platelet lysate (Plt) and of lysate from the same HEK293 cells used in panel B (293) show that each of the antibodies is reactive with IIb.
- Figure S2. Free pro-αIIb, like dissociated mature αIIb, is readily cleaved by thrombin (JPG, 92.6 KB)
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Previous studies have shown that IIb in complex with 3 is not cleaved by thrombin and that IIb becomes a thrombin substrate when dissociated from 3 with EDTA.1,2 To determine whether free pro-IIb is protease-resistant during biogenesis, we assessed thrombin cleavage of pro-IIb. (A) A ribbon model of IIb is shown with and without a space-filling model of 3. The -carbons of amino acids R41 and R386 at the consensus thrombin cleavage sites on the -propeller of IIb are indicated in yellow. (B) HEK293 cells expressing IIb3 were pulse-labeled for 15 minutes and then harvested after 90 minutes. Samples were immunoprecipitated with mAb anti-V5, and the precipitates were incubated with 40 U/mL thrombin with or without 2 mM EDTA for 1 hour at 37°C. Mature IIb subunits were protected from cleavage in the absence of EDTA but were cleaved in the presence of EDTA. In the absence of EDTA, the mature IIb was protected from thrombin cleavage, but pro-IIb was almost completely digested, indicating that the thrombin cleavage sites are readily available on pro-IIb subunits as well as on dissociated mature IIb subunits.
REFERENCES
1. Fujimura K, Phillips DR. Calcium cation regulation of glycoprotein IIb-IIIa complex formation in platelet plasma membranes. J Biol Chem. 1983;258:10247-10252.
2. Phillips DR, Fujimura K, Jennings LK, Parise L, Fitzgerald L, Fox JE. Calcium regulation of glycoproteins IIb and IIIa in human platelet membranes. Ann N Y Acad Sci. 1983;416:166-175.
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