Human fibrinogen 1 is homodimeric with respect to its ␥ chains ('␥ A -␥ A '), whereas fibrinogen 2 molecules each contain one ␥ A (␥ A 1-411V) and one ␥ chain, which differ by containing a unique C-terminal sequence from ␥408 to 427L that binds thrombin and factor XIII. We investigated the structural and functional features of these fibrins and made several observations. First, thrombin-treated fibrinogen 2 produced finer, more branched clot networks than did fibrin 1. These known differences in network structure were attributable to delayed release of fibrinopeptide (FP) A from fibrinogen 2 by thrombin, which in turn was likely caused by allosteric changes at the thrombin catalytic site induced by thrombin exosite 2 binding to the ␥ chains. Second, cross-linking of fibrin ␥ chains was virtually the same for both types of fibrin. Third, the acceleratory effect of fibrin on thrombin-mediated XIII activation was more prominent with fibrin 1 than with fibrin 2, and this was also attributable to allosteric changes at the catalytic site induced by thrombin binding to ␥ chains. Fourth, fibrinolysis of fibrin 2 was delayed compared with fibrin 1. Altogether, differences between the structure and function of fibrins 1 and 2 are attributable to the effects of thrombin binding to ␥ chains. IntroductionFibrinogen is a multidomain disulfide-linked protein composed of symmetric halves, each consisting of 3 polypeptide chains termed A␣, B␤, and ␥. 1 Human fibrinogen can be separated by ion exchange chromatography into 2 major fractions, fibrinogen 1 (peak 1 fibrinogen) and fibrinogen 2 (peak 2 fibrinogen). 2,3 Plasma fibrinogen contains approximately 15% fibrinogen 2. Structurally, the 2 fibrinogens differ from each other with respect to the composition of their ␥ chains. Fibrinogen 1 contains 2 ␥ A chains, each composed of 411 amino acids, whereas heterodimeric fibrinogen 2 molecules each contain one ␥ A and one ␥Ј chain. 3,4 The variant ␥Ј chain is longer (427 residues) and has a more anionic, carboxyl terminal sequence than the ␥ A chain beyond position 408. 4 Alternative mRNA splicing at the exon 9-exon 10 boundaries gives rise to the variant ␥Ј chain. 5 Thrombin binds to fibrinogen at the substrate site through its exosite 1, 6-8 thereby mediating cleavage of fibrinopeptide A 9-12 and slower cleavage of fibrinopeptide B. 13,14 Fibrin assembly commences with the formation of double-stranded twisting fibrils in which fibrin molecules are arranged in a staggered, overlapping manner. 15 Subsequently, lateral fibril associations occur, resulting in thicker fibrils and fibers. Concomitant with converting fibrinogen to fibrin, thrombin activates factor XIII to factor XIIIa. [16][17][18][19][20][21] In the presence of factor XIIIa and Ca 2ϩ , fibrin undergoes intermolecular covalent cross-linking by the formation of ⑀-amino(␥-glutamyl) lysine isopeptide bonds. 22 Generally speaking, intermolecular cross-linking occurs rapidly between ␥ chains to form ␥-dimers and more slowly among ␣ chains to create oligomers and larger ␣ chain...

SummaryFibrinogen plays a central role in surface-induced thrombosis. However, the interactions of fibrinogen with different substrata remain poorly understood because of the difficulties involved in imaging globular proteins under aqueous conditions. We present detailed three dimensional molecular scale images of fibrinogen molecules on a hydrophobic surface under aqueous conditions obtained by atomic force microscopy. Hydrated fibrinogen monomers are visualized as overlapping ellipsoids; dimers and trimers have linear conformations predominantly, and increased affinity for the hydrophobic surface compared with monomeric fibrinogen. The results demonstrate the importance of hydration on protein structure and properties that affect surface-dependent interactions.

A cold precipitable fibrin-precursor, designated cryoprofibrin, was separated from the plasma of endotoxin-treated rabbits, and shown on the basis of its peptide composition to correspond to a product of limited action of thrombin on fibrinogen (1). The present communication is concerned with the stability and the mechanism of formation of cryoprofibrin. The studies were undertaken principally to evaluate the proposed utility of cryoprofibrin in determining intravascular deposition of fibrin.