Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Cryo-Electron Microscopy Structure of the αIIbβ3–Abciximab Complex

doi: 10.1161/ATVBAHA.119.313671

Figure Lengend Snippet: Cryo-EM structure of the αIIbβ3–abciximab complex. (a) Density map of the αIIbβ3–abciximab complex at 2.8-Å resolution, segmented and colored according to the individual polypeptide chains. (b) Left panel: Atomic model of the αIIbβ3–abciximab complex colored as in panel (a). The green spheres represent the metal ions in the MIDAS, ADMIDAS and SyMBS. VH: variable domain of the heavy chain; CH1: first constant domain of the heavy chain; VL: variable domain of the light chain; CL: constant domain of the light chain. Hinge region denotes the flexible linkers that connect the variable and constant domains in abciximab. Right panel: Interaction surfaces of abciximab (top) and αIIbβ3 (bottom). Side chains of residues that are within 4.5 Å from a side chain of the interacting protein are shown in stick representation. (c) Fibrinogen/RGD-binding pocket in the cryo-EM structure of the αIIbβ3–abciximab complex (colored as in panel (a)) and in the X-ray crystal structure of the unliganded αIIbβ3 integrin (light grey with metal ions shown as black spheres). The overlay shows that abciximab binding does not induce meaningful differences in the binding pocket. The boxed area is shown in panel d. (d) Conformation of the β3 SDL loop in the cryo-EM structure of the αIIbβ3–abciximab complex (dark grey with SDL in blue) and in the X-ray crystal structure of the unliganded αIIbβ3 integrin (light grey with SDL in cyan). Abciximab is shown as an orange transparent surface to illustrate that the SDL in the conformation seen in the X-ray crystal structure of the unliganded αIIbβ3 integrin (cyan) would clash with abciximab.

Article Snippet: Protein sequencing The primary amino-acid sequence of abciximab was determined by de novo peptide sequencing 23 , 24 using tandem mass spectrometry (MS) by Rapid Novor Inc.

Techniques: Cryo-EM Sample Prep, Binding Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Cryo-Electron Microscopy Structure of the αIIbβ3–Abciximab Complex

doi: 10.1161/ATVBAHA.119.313671

Figure Lengend Snippet: Abciximab prevents ligands from access to the integrin ligand-binding pocket. (a) Shown are superimpositions of our structure of the αIIbβ3–abciximab complex (αIIbβ3 in grey ribbon representation and abciximab as transparent grey surface) with other ligand–integrin complexes, which are αVβ6 (salmon) in complex with pro-TGF-β (red) (PDB: 5FFO)38, αVβ3 (light blue) in complex with the tenth domain of fibronectin (dark green) (PDB: 4MMX)37, and αIIbβ3 (light green) in complex with a peptide of the fibrinogen γ-chain (blue) (PDB: 2VDO)35. The overlays show that ligand binding would cause sterical clashes with the bound abciximab. (b) Ligand binding has little effect on the abciximab-binding region of αIIbβ3. Left panel: Superimposition of our αIIbβ3–abciximab complex structure (αIIbβ3 shown in light grey and abciximab in yellow and orange) with the crystal structure of unbound αIIbβ3 (light green) (PDB: 3FCS)22 shows that abciximab does not affect the ligand-binding pocket. Right panel: Superimposition of our αIIbβ3–abciximab complex structure (αIIbβ3 shown in light grey and abciximab in yellow and orange) with the crystal structure of αIIbβ3 (dark green) in complex with a peptide of the fibrinogen γ-chain (red) (PDB: 2VDO)35 shows that peptide binding induces only subtle changes to the abciximab-interacting region of αIIbβ3.

Article Snippet: Protein sequencing The primary amino-acid sequence of abciximab was determined by de novo peptide sequencing 23 , 24 using tandem mass spectrometry (MS) by Rapid Novor Inc.

Techniques: Ligand Binding Assay, Binding Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Cryo-Electron Microscopy Structure of the αIIbβ3–Abciximab Complex

doi: 10.1161/ATVBAHA.119.313671

Figure Lengend Snippet: Visualization of residues in (a) αIIbβ3 and (b) abciximab involved in inter-molecular contacts in at least 10% of the MD-simulation frames (see also Supplemental Figures VII and VIII). Insets in panel a refer to residues with calculated free-energy differences (ΔΔG(bind)) from wild type that are larger than 3.5 kcal/mol for top stabilizing (in green) and destabilizing (in purple) mutations of αIIbβ3 residues (see also Supplemental Figure IX).

Article Snippet: Protein sequencing The primary amino-acid sequence of abciximab was determined by de novo peptide sequencing 23 , 24 using tandem mass spectrometry (MS) by Rapid Novor Inc.

Techniques:

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Cryo-Electron Microscopy Structure of the αIIbβ3–Abciximab Complex

doi: 10.1161/ATVBAHA.119.313671

Figure Lengend Snippet: Residues of αIIbβ3 whose fluctuations are most affected by complex formation with abciximab are shown in stick representation and color-coded based on the change in RMSF. The RMSF changes are statistically significant in all of the residues depicted in stick representation (see also Supplemental Figure XI).

Article Snippet: Protein sequencing The primary amino-acid sequence of abciximab was determined by de novo peptide sequencing 23 , 24 using tandem mass spectrometry (MS) by Rapid Novor Inc.

Techniques:

Journal: Scandinavian Journal of Clinical and Laboratory Investigation

Article Title: Comparison of fibrin-based clot elasticity parameters measured by free oscillation rheometry (ReoRox ® ) versus thromboelastometry (ROTEM ® )

doi: 10.3109/00365513.2014.993698

Figure Lengend Snippet: ReoRox ® and ROTEM ® output traces. The ReoRox ® device displays information on viscosity (dashed curve) and elasticity (plain curve) during the coagulation process (A). Viscosity parameters include the time of the initial formation of fibrin strands (COT1) and the time to complete clot formation (COT2) before the clot starts to strengthen. Clot strength is represented by the maximum elasticity parameter G'max obtained either in the absence (Fibscreen1 G'max) or presence (Fibscreen2 G'max) of platelet inhibitor. Similar coagulation parameters are obtained with the ROTEM ® system (B), the equivalent of COT2 being the clotting time (CT, time from start to 2 mm amplitude). Maximum clot firmness (MCF) parameters, obtained either in the absence (EXTEM test) or presence (FIBTEM test) of platelet inhibitor, can be converted into maximum clot elasticity (MCE) parameters, using the following formula: MCE = (MCF × 100)/(100 − MCF).

Article Snippet: The Fibscreen2 test (MRX1917; Medirox AB, Nyköping, Sweden) was run on the ReoRox G2 ® apparatus (Medirox AB) according to the manufacturer's instructions.

Techniques: Viscosity, Coagulation, FIBTEM Assay

Journal: Scandinavian Journal of Clinical and Laboratory Investigation

Article Title: Comparison of fibrin-based clot elasticity parameters measured by free oscillation rheometry (ReoRox ® ) versus thromboelastometry (ROTEM ® )

doi: 10.3109/00365513.2014.993698

Figure Lengend Snippet: Stability analysis in re-calcified citrated blood samples. Stability of fibrin-based clot strength parameters of blood samples from two healthy volunteers was assessed (with a minimum of 12 measurements each) with the ROTEM ® FIBTEM and ReoRox ® Fibscreen2 tests.

Article Snippet: The Fibscreen2 test (MRX1917; Medirox AB, Nyköping, Sweden) was run on the ReoRox G2 ® apparatus (Medirox AB) according to the manufacturer's instructions.

Techniques: FIBTEM Assay

Journal: Scandinavian Journal of Clinical and Laboratory Investigation

Article Title: Comparison of fibrin-based clot elasticity parameters measured by free oscillation rheometry (ReoRox ® ) versus thromboelastometry (ROTEM ® )

doi: 10.3109/00365513.2014.993698

Figure Lengend Snippet: Comparison of functional fibrinogen in undiluted and diluted re-calcified citrated blood samples obtained with the ROTEM ® and ReoRox ® devices. Blood samples were diluted at 33% and 50% with saline (NaCl), gelatin (GEL) and hydroxyethyl starch (HES). Duplicates of each dilution of blood samples from six different volunteers were evaluated with (A) the derived FIBTEM maximum clot elasticity (MCE) and (B) Fibscreen2 maximum elasticity (G'max). For each group, the median is represented by a bar within a box (extending from the 25th to 75th percentiles). Whiskers extend from the smallest to the largest values.

Article Snippet: The Fibscreen2 test (MRX1917; Medirox AB, Nyköping, Sweden) was run on the ReoRox G2 ® apparatus (Medirox AB) according to the manufacturer's instructions.

Techniques: Comparison, Functional Assay, Saline, Starch, Derivative Assay, FIBTEM Assay

Journal: Scandinavian Journal of Clinical and Laboratory Investigation

Article Title: Comparison of fibrin-based clot elasticity parameters measured by free oscillation rheometry (ReoRox ® ) versus thromboelastometry (ROTEM ® )

doi: 10.3109/00365513.2014.993698

Figure Lengend Snippet: Fibrin-based clot strength parameters of citrated undiluted and hemodiluted blood samples. Undiluted and hemodiluted (33% and 50% with saline, gelatin or hydroxyethyl starch [HES]) blood of samples of healthy volunteers ( n = 6) were assessed with the ROTEM ® FIBTEM (A5, A10, A20, MCF, MCE) and ReoRox ® Fibscreen2 (G'max) tests. Values are presented as median [25th percentile − 75th percentile].

Article Snippet: The Fibscreen2 test (MRX1917; Medirox AB, Nyköping, Sweden) was run on the ReoRox G2 ® apparatus (Medirox AB) according to the manufacturer's instructions.

Techniques: Saline, Starch, FIBTEM Assay

Journal: Scandinavian Journal of Clinical and Laboratory Investigation

Article Title: Comparison of fibrin-based clot elasticity parameters measured by free oscillation rheometry (ReoRox ® ) versus thromboelastometry (ROTEM ® )

doi: 10.3109/00365513.2014.993698

Figure Lengend Snippet: Correlation of FIBTEM MCE and Fibscreen2 G'max. Maximum clot elasticity (MCE) derived from the ROTEM ® FIBTEM test of blood samples from six different volunteers were plotted against the maximum elasticity parameter (G'max) given by the ReoRox ® Fibscreen2 test. Citrated blood samples were run as duplicates in each test, undiluted or diluted (33% and 50%) with saline, gelatin and hydroxyethyl starch. Bold line: regression curve (y = 2.413 x + 10.19); dotted lines: 95% prediction/confidence band

Article Snippet: The Fibscreen2 test (MRX1917; Medirox AB, Nyköping, Sweden) was run on the ReoRox G2 ® apparatus (Medirox AB) according to the manufacturer's instructions.

Techniques: FIBTEM Assay, Derivative Assay, Saline, Starch