Fifty Years of Fibrinogen Structure and Function

Comment on: The Molecular Structure of Fibrinogen Semin Thromb Hemost 2024; 50(01): 131-147 DOI: 10.1055/s-0043-1776311 Seminars in Thrombosis and Hemostasis Reissuing historical scientific papers from the Seminars in Thrombosis and Hemostasis “vault” or treasure trove to celebrate 50 years of publishing is a fantastic idea, bringing important original studies or reviews such as “The Molecular Structure of Fibrinogen” by Murano[ 1 ] back into the light for new generations of scientists to discover them or more senior colleagues to rediscover. It is very appropriate that the first article published in Seminars in Thrombosis and Hemostasis focused on fibrinogen, the final substrate of the coagulation cascade and without much contest (but maybe a little bias!) the molecule which is the most central for maintaining a healthy hemostatic balance between clot formation and clot lysis. Indeed, abnormal fibrinogen levels or abnormal fibrin(ogen) structures can perturb the balance, leading to bleeding manifestations, thrombosis, or both. Why was it so important to understand the molecular structure of fibrinogen in 1974 and why is research in this field still so active today? It is worth mentioning that, to our knowledge, fibrinogen is the only coagulation protein that has its own society, the International Fibrinogen Research Society, founded in 1990 in Rouen, France, which organizes an International Workshop every 2 years to bring together basic and clinical research on fibrinogen, in a dynamic and friendly atmosphere. With fibrin, produced by thrombin-mediated cleavage, fibrinogen plays important roles in many physiological processes that are essential for human health.[ 2 ] Polymerized and cross-linked fibrin, present in the stable blood clot, is essential to prevent blood loss and initiate wound healing. The complete absence of fibrinogen in patients with congenital afibrinogenemia causes a life-threatening bleeding disorder, which can also include thrombotic complications. Even reduced fibrinogen levels can lead to severe bleeding or thrombosis under challenging circumstances, such as pregnancy or surgery. On the contrary, elevated levels of fibrinogen resulting from inflammation can lead to hypercoagulability, a risk factor for cardiovascular disease. Through its immunomodulatory effects, fibrin(ogen) has been associated with metabolic diseases such as obesity and diabetes but also cancer development and progression. When fibrin(ogen) is found outside the bloodstream, fibrin deposits in the central nervous system can cause stroke while fibrin aggregates in the kidney can cause renal amyloidosis. The review published in 1974 by Genesio Murano focuses mostly on the physiochemical properties of fibrinogen, based on studies of chemically (e.g., sulfite or cyanogen bromide) or proteolytically (e.g., thrombin, plasmin, and trypsin) cleaved fibrinogen fragments as well as single chain molecules.[ 1 ] Many of the findings described in this review, stemming from research performed in the late 1960s and early 70s by scientists including Birger Blombäck and Margaret Blombäck, Michael Mosesson, Russell Doolittle, John Ferry, and Agnes Henschen, have held the test of time and have been replicated in later studies using more advanced technology. For example, the primary structure of fibrinogen, a hexamer containing two copies of three polypeptide chains (AαBbγ) 2 with a molecular weight of 340 kDa, based on diffusion and sedimentation characteristics of the hexamer and the sum of the values obtained for the individual chains migrating on SDS-PAGE has been confirmed to be correct, as indeed have the general chemical composition of amino acids, disulfide bonds, and carbohydrate content. The conversion of fibrinogen to fibrin by thrombin is already described in the 1974 review.[ 1 ] Cleavage of an arginyl–glycine bond in A–α (at Arg16–Gly17) allows the rapid release of fibrinopeptide A which induces a conformational change and exposes a “dormant” polymerization domain (aA, now known as “knob A”), which is then free to interact with a second site in the first step of fibrin polymerization. This second, apparently always active, site was correctly thought to be in the C-terminal “D domain” at the end of the molecule, although it was not known in which chain. This site has since been located in the C-terminus of the fibrinogen γ chain and is now known as “hole a.”[ 3 ] While the exposure of “knob A” is sufficient to initiate fibrin polymerization, the conformational change following fibrinopeptide A release also allows thrombin access to the Arginyl-Glycine bond bond in B–β. Thus, cleavage at Arg14–Gly15 occurs later, removing fibrinopeptide B and exposing domain bB (now known as “knob B”) which also interacts with a second site in the D domain (in the C-terminus of the β-chain of another molecule; i.e., “hole b”). The sequences of Fibrinopeptides A and B obtained by thrombin digestion and Edman degradation analyses are shown for numerous species, allowing taxonomic classifications which are in agreement with morphological taxonomy. Reading this paper now, it was rather intriguing to find a discussion of a second thrombin cleavage site in A–α, located just three residues C-terminally to the Arg16–Gly17 (i.e., Arg19–Val20; see Fig. 6 in Genesio Murano's review).[ 1 ] Indeed, studies by Blombäck et al[ 4 ] [ 5 ] showed that in N-DSK as in native fibrinogen, the Arg16–Gly17 bond is cleaved at a fast rate releasing fibrinopeptide A and the Arg19–Val20 is cleaved at a slower rate, releasing the tripeptide Gly17–Pro18–Arg19 (GPR). If this is the case, what would be the impact on fibrin polymerization, since as mentioned above the GPR residues of knob A are essential for the first steps of fibrin polymerization by binding to the complementary pocket “hole a”? What could be the biological significance of this second site in case of mutations affecting the thrombin cleavage site at Arg16–Gly17? Would cleavage at the second site be possible, and if so, would the remaining “Knob” Val Glu Arg be of any use for fibrin polymerization? Here the analogy with a well-known television quiz show came to mind. The “Call-a-Friend” lifeline can be used if a contestant does not know the answer to a question. We contacted John Weisel at the University of Pennsylvania, an expert on fibrinogen and fibrin structure and function[ 6 ] with our (million dollar?) questions who answered: “Fibrinogen missing GPR would likely not polymerize, although it might polymerize via GHR from the beta chain. Regarding the Blomback JBC 1972 paper, fibrinogen was digested with 35 NIH Units/ml of thrombin for 2 hr at pH 8.5. yielding 0.15 mole of GPR per mole of FpA. That is a whopping amount of thrombin and a long digestion for a low yield of GPR peptide, so I think we can say that the other cleavage is unlikely under normal circumstances.” How has the field evolved since 1974? Many major advances have been made in the field in 50 years: X-ray crystal structures have been obtained for most of the fibrinogen domains, and these studies in combination with computational modeling of missing areas, high-resolution electron microscopy, and atomic force microscopy have allowed us to confirm a great number of findings reviewed by Murano[ 1 ] and have resolved some issues which were subject to debate at the time (e.g., the more or less linear structure of native fibrinogen (yes!) versus a “swollen spheroidal one” (no!). An important line of research on fibrinogen which is not developed in the review by Genesio Murano is how the study of fibrinogen variants, due to mutations or posttranslational modifications, both naturally occurring in human individuals or experimentally designed, has helped to better understand how the structure of fibrinogen impacts on function. While it was already observed that circulating fibrinogen could be found in various forms, described by Murano as “a population of slightly different molecules,” with variable solubility, variable N termini, phosphorylation, and variable proteolytic outcomes, the underlying molecular basis of these differences and their possible impact on function was not well understood at that time. Of course, this is largely because, in 1974, the nucleotide sequences of the three human fibrinogen encoding genes were unknown. Partial coding sequences and the genomic organization of the fibrinogen cluster were determined by Crabtree and coworkers,[ 7 ] [ 8 ] and the gene sequences were published in 1990 by Chung and coworkers.[ 9 ] The availability of these sequences allowed, among other findings, the identification of different splicing isoforms for fibrinogen α (i.e., the A-α-E extended isoform present as a homodimer in 1–2% of fibrinogen in the circulation of human adults) and for fibrinogen γ (i.e., the γ prime isoform which is present in approximately 8–15% circulating fibrinogen). Also, while in 1974 only one pathogenic variant (fibrinogen Detroit) had been identified accounting for abnormal clot polymerization and dysfibrinogenaemia,[ 10 ] sequencing the fibrinogen genes has since allowed the identification of numerous normal polymorphisms and disease-causing pathological variants. Indeed, to date, more than 350 distinct causative mutations have been characterized both in quantitative disorders (afibrinogenemia and hypofibrinogenemia) and qualitative disorders (dysfibrinogenemia and hypodysfibrinogenemia) allowing a more definitive diagnosis for patients with congenital fibrinogen disorders and facilitating patient management.[ 11 ] Unsurprisingly, one of the two “hot-spot” mutation sites in dysfibrinogenemia is the Arg16 residue at the thrombin cleavage site of A–α (Arg35 in the nascent peptide containing the signal peptide). The other “hot-spot” site is found in the γ chain, at residue Arg275 (Arg301 with the signal peptide), which is important for end-to-end connection of adjacent monomers and elongation of fibrin strands.[ 6 ] Another vast body of new knowledge has been gained from the study of fibrin clot properties. As introduced in the review of Murano,[ 1 ] with the description of Fibrinogen Detroit, studies of hereditary dysfibrinogenemia have provided a great deal of information on fibrin polymerization mechanisms, fibrinolysis, and fibrin network structures.[ 12 ] Similarly, these investigations have given valuable clues for correlating fibrinogen function and clinical phenotypes.[ 11 ] [ 13 ] For instance, some fibrinogen variants lead to altered fibrin networks with resistance to fibrinolysis[ 14 ] and eventually to a very strong thrombotic phenotype. Others affect fibrin polymerization with the production of thicker fibrin fibers, reduced fiber branching, and large pores, resulting in defective clot ability and a bleeding tendency.[ 15 ] Overall, assessment of fibrin clot properties in such patients can help in tailoring their management.[ 16 ] More generally, it is now well established that fibrin clot properties and fibrin biomechanical characteristics play a determining role in many bleeding or thrombotic diseases.[ 17 ] [ 18 ] For instance, impaired fibrin clot properties have been reported as factors associated with poor prognosis in coronary artery disease, stroke, peripheral arterial disease, and aortic aneurysm (reviewed in[ 19 ]). Altered fibrin clot structure has recently been highlighted in COVID-19 acute infection which, combined with dysregulated fibrinolysis, could contribute to the high thrombotic risk.[ 20 ] In long COVID, the presence of fibrin amyloid microclots resisting fibrinolysis may play a role in the underlying coagulopathy although their specific contribution to the disease is subject to debate and requires further investigation.[ 21 ] [ 22 ] The role of splice variants, post-translational modifications, environmental factors, and interactions with complement, neutrophil extracellular traps, red-blood cells, platelets, and leucocytes are only some characteristics of the fibrinogen molecule that have been unraveled since the publication of Genesio Murano's review.[ 1 ] There is no doubt that we still have many other pieces to look forward to that will contribute to solving the fascinating puzzle of fibrin(ogen)'s role in human physiology and disease. * This is a commentary on: Murano G. The molecular structure of Fibrinogen. Semin Thromb Hemost 1974;1:1–31 Publication History Article published online: 09 October 2023 © 2023. Thieme. All rights reserved. Thieme Medical Publishers, Inc. 333 Seventh Avenue, 18th Floor, New York, NY 10001, USA