title: Thrombosis-associated antifibrinogen IgG1 κ impairs fibrin polymerization and enhances platelet activation.
abstract: The present study extends our previous investigation of circulating antibody/fibrinogen/C1q complexes (FgIgC) associated with thrombosis in a heterophenotypic AαR16C proband, by focusing on the molecular and functional characteristics of the FgIgC, isolated by cryoprecipitation, FgIgC components were demonstrated by SDS-PAGE and by rotary shadowing electron microscopy. Affinity chromatography was used to isolate IgG and fibrinogen from FgIgC. Thrombin-induced clots were examined by scanning electron microscopy and turbidity measurements. IgG/fibrinogen binding was measured by ELISA. Fibrinogen Aα1-19 peptides, cleaved by thrombin from fragment N-DSK, were examined by mass spectrometry. Clot stiffness, platelet release of P-selectin, and fibrinogen self-assembly were assessed by thromboelastography, flow cytometry, and atomic force microscopy, respectively. The FgIgC effects included the following: increased P-selectin release from gel-sieved platelets, finer fiber networks and decreased stiffness of its clots, and marked inhibition of fibrinogen self-assembly. The abnormal proband fibrinogen structure displayed phosphorylated AαR16C-AαR16C homodimers and AαR16C-glutathione heterodimers. ELISA measurements disclosed pronounced binding by proband fibrinogen to proband IgG, which was blocked by the IgG's Fab fragment and by proband, but not by normal plasmic fragment E1. There was appreciable, but much weaker, binding to normal fibrinogen, to its fragments E1, and D1, and to homodimeric AαR16C fibrinogen. The antibody's primary target epitope included heterodimeric AαR16C-glutathione; a secondary epitope resided in the D region. Moreover, both the enhanced platelet activation (i.e. increased P-selectin release induced by FgIgC) and the highly phosphorylated FpA (i.e. resulting in its accelerated release by thrombin) may have contributed to the thrombotic diathesis.
authors: aDepartment of Pathology Stony Brook University, Stony Brook, New York bDepartment of Pathology, West Virginia University, Health Science Center, Morgantown, West Virginia cDepartment of Cell Biology and Biochemistry, University of Pennsylvania, Philadelphia, Pennsylvania dDepartment of Material Science and Engineering, College of Engineering and Applied Science, Stony Brook University, Stony Brook, New York eDepartment of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
authors: Dennis K
authors: DK
authors: Galanakis
authors: Eric D
authors: ED
authors: Spitzer
authors: Peter
authors: P
authors: Perrotta
authors: Chandrasekaran
authors: C
authors: Nagaswami
authors: Rita
authors: R
authors: Marchi
authors: Silvia
authors: S
authors: Spitzer
authors: Miriam
authors: M
authors: Refailovich
authors: Roy E
authors: RE
authors: Smith
authors: Liudi
authors: L
authors: Zhang
authors: Clement
authors: C
authors: Marmorat
authors: John W
authors: JW
authors: Weisel
mesh_terms: D000328:Adult; D001792:Blood Platelets / Q000378:metabolism*; D005337:Fibrin / Q000378:metabolism*; D015241:Fibrinogens, Abnormal / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D008297:Male; D015539:Platelet Activation; D058105:Polymerization; D013927:Thrombosis / Q000378:metabolism*
chemical_list: D015241:Fibrinogens, Abnormal; D007074:Immunoglobulin G; D005337:Fibrin