Dysregulated coagulation associated with hypofibrinogenaemia and plasma hypercoagulability: Implications for identifying coagulopathic mechanisms in humans

Rita Marchi; Bethany L. Walton; Colleen S. McGary; Feng-Chang Lin; Alice D. Ma; Rafal Pawlinski; Nigel Mackman; Robert A. Campbell; Jorge Di Paola; Alisa S. Wolberg

doi:10.1160/TH12-05-0355

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2012; 108(03): 516-526
DOI: 10.1160/TH12-05-0355

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

Dysregulated coagulation associated with hypofibrinogenaemia and plasma hypercoagulability: Implications for identifying coagulopathic mechanisms in humans

Rita Marchi

¹Laboratorio Biologia del Desarrollo de la Hemostasia, Instituto Venezolano de Investigaciones Cientificas (IVIC), Caracas, Venezuela

²Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

,

Bethany L. Walton

²Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

,

Colleen S. McGary

²Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

,

Feng-Chang Lin

³Department of Biostatistics and North Carolina Translational and Clinical Sciences Institute, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

,

Alice D. Ma

⁴Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

,

Rafal Pawlinski

⁴Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

,

Nigel Mackman

⁴Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

,

Robert A. Campbell

⁵Program in Molecular Medicine, University of Utah, Salt Lake City, Utah, USA

,

Jorge Di Paola

⁶Department of Pediatrics and Human Medical Genetics and Genomics Program, University of Colorado, Denver, Colorado, USA

,

Alisa S. Wolberg

²Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

› Author Affiliations

Abstract

Summary

Identifying coagulation abnormalities in patients with combined bleeding and thrombosis history is clinically challenging. Our goal was to probe the complexity of dysregulated coagulation in humans by characterizing pathophysiologic mechanisms in a patient with both bleeding and thrombosis. The patient is a 56-year-old female with a history of haematomas, poor wound healing, and thrombosis (retinal artery occlusion and transient cerebral ischaemia). She had a normal activated partial thromboplastin time, prolonged thrombin and reptilase times, and decreased functional and antigenic fibrinogen levels, and was initially diagnosed with hypodysfibrinogenaemia. This diagnosis was supported by DNA analysis revealing a novel FGB mutation (c.656A>G) predicting a Q189R mutation in the mature chain that was present in the heterozygote state. However, turbidity analysis showed that purified fibrinogen polymerisation and degradation were indistinguishable from normal, and Bβ chain subpopulations appeared normal by two-dimensional difference in-gel electrophoresis, indicating the mutated chain was not secreted. Interestingly, plasma thrombin generation testing revealed the patient’s thrombin generation was higher than normal and could be attributed to elevated levels of factor VIII (FVIII, 163–225%). Accordingly, in an arterial injury model, hypofibrinogenaemic mice (Fgn^+/−) infused with factor VIII demonstrated significantly shorter vessel occlusion times than saline-infused Fgn^+/− mice. Together, these data associate the complex bleeding and thrombotic presentation with combined hypofibrinogenaemia plus plasma hypercoagulability. These findings suggest previous cases in which fibrinogen abnormalities have been associated with thrombosis may also be complicated by co-existing plasma hypercoagulability and illustrate the importance of “global” coagulation testing in patients with compound presentations.

Keywords

Thrombin generation - factor VIII - hypofibrinogenaemia - thrombosis - bleeding

Full Text

References

References
1 Allen GA, Wolberg AS, Oliver JA. et al. Impact of procoagulant concentration on rate, peak and total thrombin generation in a model system. J Thromb Haemost 2004; 02: 402-413.
2 Butenas S, van't Veer C, Mann KG. ″Normal″ thrombin generation. Blood 1999; 94: 2169-2178.
3 Machlus KR, Colby EA, Wu JR. et al. Effect of tissue factor, thrombomodulin, and elevated clotting factor level son thrombingeneration in th ecalibrated automated thrombogram. Thromb Haemost 2009; 102: 936-944.
4 Kyrle PA, Minar E, Hirschl M. et al. High plasma levels of factor VIII and the risk of recurrent venous thromboembolism. N Engl J Med 2000; 343: 457-462.
5 Tracy RP, Arnold AM, Ettinger W. et al. The relationship of fibrinogen and factors VII and VIII to incident cardiovascular disease and death in the elderly: results from the cardiovascular health study. Arterioscler Thromb Vasc Biol 1999; 19: 1776-1783.
6 Kraaijenhagen RA, in't Anker PS, Koopman MM. et al. High plasma concentration of factor VIIIc is a major risk factor for venous thromboembolism. Thromb Haemost 2000; 83: 5-9.
7 Bank I, Libourel EJ, Middeldorp S. et al. Elevated levels of FVIII:C within families are associated with an increased risk for venous and arterial thrombosis. J Thromb Haemost 2005; 03: 79-84.
8 Machlus KR, Lin F-C, Wolberg AS. Procoagulant activity induced by vascular injury determines contribution of elevated factor VIII to thrombosis and thrombus stability in mice. Blood 2011; 118: 390-398.
9 Kant JA, Fornace Jr AJ, Saxe D. et al. Evolution and organization of the fibrinogen locus on chromosome 4: gene duplication accompanied by transposition and inversion. Proc Natl Acad Sci USA 1985; 82: 2344-2348.
10 Henschen A, Lottspeich F, Kehl M. et al. Covalent structure of fibrinogen. Ann NY Acad Sci 1983; 408: 28-43.
11 Redman CM, Xia H. Fibrinogen biosynthesis. Assembly, intracellular degradation, and association with lipid synthesis and secretion. Ann NY Acad Sci 2001; 936: 480-495.
12 Weisel JW. Fibrinogen and fibrin. Adv Protein Chem 2005; 70: 247-299.
13 Mosesson MW. Update on antithrombin I (fibrin). Thromb Haemost 2007; 98: 105-108.
14 Niwa K, Yaginuma A, Nakanishi M. et al. Fibrinogen Mitaka II: a hereditary dysfibrinogen with defective thrombin binding caused by an A alpha Glu-11 to Gly substitution. Blood 1993; 82: 3658-3663.
15 Sugo T, Nakamikawa C, Yoshida N. et al. End-linked homodimers in fibrinogen Osaka VI with a B beta-chain extension lead to fragile clot structure. Blood 2000; 96: 3779-3785.
16 Bornikova L, Peyvandi F, Allen G. et al. Fibrinogen replacement therapy for congenital fibrinogen deficiency. J Thromb Haemost 2011; 09: 1687-1704.
17 Carrell N, Gabriel DA, Blatt PM. et al. Hereditary dysfibrinogenemia in a patient with thrombotic disease. Blood 1983; 62: 439-447.
18 Collet J-P, Soria J, Mirshahi M. et al. Dusart syndrome: a new concept of the relationship between fibrin clot architecture and fibrin clot degradability: hypofibrinolysis related to an abnormal clot structure. Blood 1993; 82: 2462-2469.
19 Tarumi T, Martincic D, Thomas A. et al. Familial thrombophilia associated with fibrinogen Paris V: Dusart syndrome. Blood 2000; 96: 1191-1193.
20 Wada Y, Lord ST. A correlation between thrombotic disease and a specific fibrinogen abnormality (A alpha 554 Arg-->Cys) in two unrelated kindred, Dusart and Chapel Hill III. Blood 1994; 84: 3709-3714.
21 Koopman J, Haverkate F, Briet E. et al. A congenitally abnormal fibrinogen (Vlissingen) with a 6-base deletion in the gamma-chain gene, causing defective calcium binding and impaired fibrin polymerization. J Biol Chem 1991; 266: 13456-13461.
22 Marchi R, Lundberg U, Grimbergen J. et al. Fibrinogen Caracas V, an abnormal fibrinogen with an Aalpha 532 Ser-->Cys substitution associated with thrombosis. Thromb Haemost 2000; 84: 263-270.
23 Haverkate F, Samama M. Familial dysfibrinogenemia and thrombophilia. Report on a study of the SSC Subcommittee on Fibrinogen. Thromb Haemost 1995; 73: 151-161.
24 Dargaud Y, Luddington R, Baglin TP. Elimination of contact factor activation improves measurement of platelet-dependent thrombin generation by calibrated automated thrombography at low-concentration tissue factor. J Thromb Hae-most 2006; 04: 1160-1161.
25 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74: 5463-5467.
26 Kazal LA, Amsel S, Miller OP. et al. The preparation and some properties of fibrinogen precipitated from human plasma by glycine. Proc Soc Exp Biol Med 1963; 113: 989-994.
27 Osorio C, Sullivan PM, He DN. et al. Mortalin is regulated by APOE in hippocampus of AD patients and by human APOE in TR mice. Neurobiol Aging 2007; 28: 1853-1862.
28 Pratt CW, Monroe DM. Microplate coagulation assays. Biotechniques 1992; 13: 430-433.
29 Ploplis VA, Wilberding J, McLennan L. et al. A total fibrinogen deficiency is compatible with the development of pulmonary fibrosis in mice. Am J Pathol 2000; 157: 703-708.
30 Machlus KR, Cardenas JC, Church FC. et al. Causal relationship between hyperfibrinogenemia, thrombosis, and resistance to thrombolysis in mice. Blood 2011; 117: 4953-4963.
31 Emeis JJ, Jirouskova M, Muchitsch EM. et al. A guide to murine coagulation factor structure, function, assays, and genetic alterations. J Thromb Haemost 2007; 05: 670-679.
32 Kawasaki T, Kaida T, Arnout J. et al. A new animal model of thrombophilia confirms that high plasma factor VIII levels are thrombogenic. Thromb Haemost 1999; 81: 306-311.
33 Neyman M, Gewirtz J, Poncz M. Analysis of the spatial and temporal characteristics of platelet-delivered factor VIII-based clots. Blood 2008; 112: 1101-1108.
34 Shi Q, Fahs SA, Kuether EL. et al. Targeting FVIII expression to endothelial cells regenerates a releasable pool of FVIII and restores hemostasis in a mouse model of hemophilia A. Blood 2010; 116: 3049-3057.
35 Moller F, Tranholm M. A ferric chloride induced arterial injury model used as haemostatic effect model. Haemophilia 2010; 16: e216-222.
36 Baumgartner B, Jaki T, Wolfsegger MJ. et al. Optimization, refinement and reduction of murine in vivo experiments to assess therapeutic approaches for haemophilia A. Lab Anim 2010; 44: 211-217.
37 Swiss Institute of Bioinformatics. ExPASy Proteomics Server. Available at: http://ca.expasy.org/swiss-2dpage?combined=fibrinogen Accessed June 2011.
38 de Bosch NB, Mosesson MW, Ruiz-Saez A. et al. Inhibition of thrombin generation in plasma by fibrin formation (antithrombin 1). Thromb Haemost 2002; 88: 253-258.
39 Asselta R, Duga S, Tenchini ML. The molecular basis of quantitative fibrinogen disorders. J Thromb Haemost 2006; 04: 2115-2129.
40 Vu D, Neerman-Arbez M. Molecular mechanisms accounting for fibrinogen deficiency: from large deletions to intracellular retention of misfolded proteins. J Thromb Haemost 2007; 05 (Suppl. 01) 125-131.
41 Hanss M, Biot F. A database for human fibrinogen variants. Ann NY Acad Sci 2001; 936: 89-90.
42 Doolittle RF, Goldbaum DM, Doolittle LR. Designation of sequences involved in the ″coiled-coil″ interdomainal connections in fibrinogen: constructions of an atomic scale model. J Mol Biol 1978; 120: 311-325.
43 Zhang JZ, Redman CM. Role of interchain disulfide bonds on the assembly and secretion of human fibrinogen. J Biol Chem 1994; 269: 652-658.
44 Xu W, Chung DW, Davie EW. The assembly of human fibrinogen. The role of the amino-terminal and coiled-coil regions of the three chains in the formation of the alphagamma and betagamma heterodimers and alphabetagamma half-molecules. J Biol Chem 1996; 271: 27948-27953.
45 Asselta R, Duga S, Spena S. et al. Missense or splicing mutation? The case of a fibrinogen Bbeta-chain mutation causing severe hypofibrinogenemia. Blood 2004; 103: 3051-3054.
46 Suh TT, Holmback K, Jensen NJ. et al. Resolution of spontaneous bleeding events but failure of pregnancy in fibrinogen-deficient mice. Genes Develop 1995; 09: 2020-2033.
47 Homer VM, Brennan SO, George PM. Novel fibrinogen Bbeta gene mutation causing hypofibrinogenaemia. Thromb Haemost 2002; 88: 1066-1067.
48 Homer VM, Brennan SO, Ockelford P. et al. Novel fibrinogen truncation with deletion of Bbeta chain residues 440-461 causes hypofibrinogenaemia. Thromb Haemost 2002; 88: 427-431.
49 Mimuro J, Hamano A, Tanaka T. et al. Hypofibrinogenemia caused by a nonsense mutation in the fibrinogen Bbeta chain gene. J Thromb Haemost 2003; 01: 2356-2359.
50 Xu X, Wu J, Zhai Z. et al. A novel fibrinogen Bbeta chain frameshift mutation in a patient with severe congenital hypofibrinogenaemia. Thromb Haemost 2006; 95: 931-935.
51 Brennan SO, Fellowes AP, Faed JM. et al. Hypofibrinogenemia in an individual with 2 coding (gamma82 A-->G and Bbeta235 P-->L) and 2 noncoding mutations. Blood 2000; 95: 1709-1713.
52 Wolberg AS, Aleman MM, Leiderman K. et al. Procoagulant activity in hemostasis and thrombosis: Virchow's triad revisited. Anesth Analg 2012; 114: 275-285.
53 Madeira CL, Layman ME, de Vera RE. et al. Extrahepatic factor VIII production in transplant recipient of hemophilia donor liver. Blood 2009; 113: 5364-5365.
54 Shovlin CL, Angus G, Manning RA. et al. Endothelial cell processing and alternatively spliced transcripts of factor VIII: potential implications for coagulation cascades and pulmonary hypertension. PLoS One 2010; 05: e9154.
55 Ingram IC. The determination of plasma fibrinogen by the clot-weight method. Biochem J 1952; 51: 583-585.
56 Weitz JI, Hudoba M, Massel D. et al. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest 1990; 86: 385-391.
57 Hemker HC, Giesen P, Al Dieri R. et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003; 33: 4-15.