Thrombin Adsorption to Fibrin.

The adsorption of thrombin to fibrin has been a subject of study for some time.   The work by Liu and coworkers (1) can be considered seminal for the subsequent work on the binding of thrombin to fibrin. Briefly, there are two classes of binding sites, the binding is reversible, and binding does not require the enzyme active site.   In addition, the bound thrombin was partially protected from inactivation by antithrombin and bound thrombin could be obtained from the fibrin clot by treatment with plasmin. The effect of plasmin was consistent with earlier work by Bloom (2).   Francis and workers (3) showed that most of the thrombin bound to fibrin was released by incubation in buffer or manual compression with 10-15% remaining bound to the fibrin. These investigators also established the bound thrombin retained catalytic activity.  Consistent with earlier studies, the bound thrombin could be solubilized with digestion by plasmin.  Advances in the knowledge of the structure of thrombin has permitted a more sophisticated understanding of the binding of thrombin to fibrin (4-6). Thrombin is thought to bind to the α and β chains via exosite I while binding to γ-chain involves exosite II (4). This is consistent with the earlier work from the late Hymie Nossel's laboratory describing two classes of binding sites on fibrin for thrombin.  The binding of thrombin to the gamma chain of fibrin via exosite II modulates the activity of thrombin (7) much in the same way that thrombin binding to platelet GPIbα modulates the activity of thrombin (8,9).* 

 It is of interest that the adsorption of thrombin to fibrin has been a subject of study since the observations of Buchanan in 1845 (10). Buchanan was able to purify a "fibrin ferment" from fibrin obtained from the dilution of plasma into water.  The clotting principle was insoluble in water but was eluted with 8% NaCl. Buchanan noted that the clotting principle had the properties of a globulin.  Buchanan's work was followed by Gamgee in 1878 (11) who was able to reproduce Buchanan's findings suggesting also that thrombin was a "ferment." However, a few years later in 1883, Lea and Green(12) , while reproducing the apparent adsorption and elution of a clotting material from fibrin, argued that this material was not a protein (proteid) or a ferment.  Some years later in 1910, Howell in1910 (13) was able to reproduce these early studies purifying thrombin by elution from fibrin with sodium chloride (8% assumed W/V; ca. 1.4 M).  It could be argued that these early studies might be the first examples of affinity purification of an enzyme.  It is also of interest that Howell's studies were prompted by another study (14), also from the Physiology Laboratory at Johns Hopkins.  Rettger (14), as with Lea and Green(12) argued that thrombin was not a ferment (enzyme).  The protein/enzyme controversy was not settled until Sumner's work on urease in 1927 (15,16) and even then the battle continued but that is another story.  Suffice to say, the protein nature of my old friend, factor VIII was still in question as late at 1966 (17).  Regardless of Rettger's mistaken (in hindsight) interpretation of the data, his paper is  a good review of the state of the art in coagulation at that time. Rettger also recognized the existence of "the thrombogen" which he suggested was activated by calcium ions.  Howell (13) first attempted to obtain thrombin from canine fibrin placed in 8% NaCl/40oC/24 hours.  Under these conditions, the fibrin clot was dissolved resulting in a large amount of protein in solution with thrombin.  A more satisfactory product was obtained from fibrin obtained from porcine blood (8% NaCl/40C/48-72 hrs).  Under these conditions a viscous solution is obtained which was processed by repeated chloroform extraction to obtain thrombin.  It is clear that the fibrin obtained from canine blood and porcine blood contained plasmin/plasminogen which permitted "solubilization" of the fibrin clot with release of thrombin.   

One consequence of the binding of thrombin by fibrin is the "inactivation" of thrombin by removal of thrombin from bulk solution.   It is of interest that fibrin was known as antithrombin I (7,18); antithrombin II is known as heparin cofactor II and antithrombin III is now known as antithrombin.   Platelets were also demonstrated to have antithrombin activity based on the binding of thrombin (19).  With fibrin and platelets, the bound thrombin retained some activity (20).

Another potential effect of the adsorption of thrombin by fibrin is the inhibition of earlier stages of coagulation although the data supporting this is weak and significance questionable. Quick and Favre-Gully (21) observed that little prothrombin is consumed during the process of blood coagulation and that, consistent with the earlier observations of Howell (13), thrombin was removed by fibrin.  These investigators also suggested that the removal of thrombin by fibrin decreased prothrombin consumption.  It is not clear that this observation was prescient of later observations by Sandy Schiffman and Sam Rapaport on the enhancement of factor V and factor VIII activity (22).   However, more recent work by Kremers and coworkers (23) suggests that the removal of fibrinogen by defibrination (Ancrod) decreased thrombin formation; the defect could be removed by addition of fibrinogen. 

I started thinking about this problem again while working on a review on the presence of thrombin in the interstitial space (24).   I was not so much interested in the role of thrombin in promoting interstitial fibrosis through activation of fibroblasts or fibrin formation but whether thrombin bound to fibrin(ogen) in the extracellular matrix had attenuated activity such as described above for interaction with the γ-chain of fibrin (or platelet GPIbα).   Fibrinogen has been shown to be deposited into the extracellular matrix without the action of thrombin (25).   As with many proteins, fibrinogen undergoes a conformational change on binding to a surface and can adopt characteristics of fibrin (26).  However the binding of fibrinogen to a surface depends on the quality of the surface (27) and the bound fibrinogen can bind in different orientations (28) which likely influences conformational change.  While acknowledging that there is considerable literature on the interaction of thrombin with fibrinogen and other extracellular matrix proteins not cited, it is possible to speculate that the binding of thrombin in the extracellular matrix (29) resulting in the modulation of thrombin activity in a manner similar to binding to fibrin as discussed above.  Thrombin bound to the extracellular matrix does possess mitogenic activity toward  vascular smooth muscle cells (30).   It is my sense that the action of thrombin in the interstitial space is poorly understood and is likely of considerable importance beyond a role in the development of fibrosis.
I have learned a lot from this exercise and did restrain myself from further discussion of the history. I do believe that as I think Winston Churchill said, "the further backward you can looks, the further forward you are able to see."

* This publication of these works (and others) gave me a great amount of personal satisfaction as it provided a basis for early work from my laboratory on the binding of thrombin to platelets (Workman, E.F. Jr., White, G.C.,II., and Lundblad, R.L., Structure-function relationships in the interaction of α-thrombin with blood platelets, J.Biol.Chem. 252, 7118-7123, 1977).   See also Lundblad, R.L. and White, G.C.,II, The interaction of thrombin with blood platelets, Platelets 16, 373-385, 2005.

1.  Liu, C.Y., Nossel, H.L., and Kaplan, K.L., The binding of thrombin by fibrin, J.Biol.Chem. 254, 10421-10425, 1979.
2.   Bloom, A.L., The release of thrombin from fibrin by fibrinolysin, Br.J.Haematol. 8, 129-133, 1962.
3.  Francis, C.W., Markham, R.E., Jr., Barrow, G.H., et al., Thrombin activity of fibrin thrombi and soluble plasmic derivatives, J.Lab.Clin.Med. 102, 220-230, 1983.
4.  Fredenburgh, J.C., Stafford, A.R., Popisil, C.H., and Weitz, J.I., Modes and consequences of thrombin's interaction with fibirn, Biophys.Chem. 112, 277-284, 2004.
5.  Vu, T.T., Stafford, A.R., Leslie, B.A., et al., Histidine-rich glycoprotein binds fibrin(ogen) with high affinity and competes with thrombin for binding to the γ'-chain, J.Biol.Chem. 286, 30314-30323, 2011.
6.  Chan, H.H., Leslie, B.A., Stafford, A.R., et al., By increasing the affinity of heparin for fibrin, Zn2=promotes the formation of a ternary heparin-thrombin-fibrin complex that protects thrombin from inhibition by antithrombin, Biochemistry 51, 7964-7973, 2012.
7. Mosesson, M.W., Update on antithrombin I (fibrin), Thromb.Haemost. 98, 105-108, 2007.
8.  Li, C.Q., Vindigni, A., Sadler, J.E., and Wardell, M.R., Platelet glycoprotein Ibα binds to thrombin anion-exosite II inducing allosteric changes in the activity of thrombin, J.Biol.Chem. 276, 6161-6168, 2001.
9.  Sabo, T.M. and Mauer, M.C., Biophysical investigation of GpIbα binding to thrombin anion binding exosite II, Biochemistry 48, 7110-7122, 2009.
10.  Buchanan, A.,On the coagulation of the blood and other fibriniferous liquids, London Med.Gazette  Vol. 1, 617-621, 1845.
11. Gamgee, A., Some old and new experiments on the fibrin ferment, J.Physiol. 2, 145-163, 1878-1879.
12.  Lea, S. and Green, J.R., Some notes on the fibrin ferment, J.Physiol. 4, 380-386, 1883.
13.  Howell, W.H., The preparation and properties of thrombin together with observations on antithrombin and prothrombin, Am.J.Physiol. 26, 453-473, 1910.
14.  Rettger, L.J., The coagulation of blood, Am.J.Physiol. 24, 406-435, 1909.
15.  Simoni, R.D., Hill, R.H., and Vaughn, M., Urease, the first crystalline enzyme and the proof that enzymes are proteins:  the work of James B. Sumner, J.B., J.Biol.Chem. 277, 23e, 2002.
16.  Manchester, K.L., The crystallization of enzymes and virus proteins: laying to rest the colloidal concept of living systems, Endeavor 28, 25-29, 2004.
17.  Veder, H.A., Is the antihaemophilic globulin a protein?, Nature  209, 202, 1966.
18.  Biggs, R. and Denson, K.W.,E., Natural and pathological inhibitors of blood coagulation, in Blood Coagulation, Haemostasis and Thrombosis,  2nd edn., ed. R. Biggs, Chapter 7, pps. 133-158, Blackwell Scientific, Oxford, United Kingdom, 1972.
19.  Watanabe, K., Chao, F.C., and Tullis, J.L., Antithrombin activity of intact human platelets, Thromb.Diath.Haemorrh. 34, 115-126, 1975.
20.  Wu, H.F., White, G.C., 2nd,  Workman, E.F., Jr., Jenzano, J.W., and Lundblad, R.L.., Affinity chromatography of platelets on immobilized thrombin: retention of catalytic activity by platelet-bound thrombin, Thromb.Res. 67, 419-427, 1992.
21.  Quick, A.J. and Favre-Gilly, J.E.,Fibrin, a factor influencing the consumption of prothrombin on coagulation, Am.J.Physiol. 158, 387-395, 1949.
22.  Rapaport, S.I., Schiffman,S., Patch, M.J., and Ames, S.B., The importance of activation of antihemophilic globulin and proaccelerin by traces of thrombin in the generation of intrinsic prothrombinase activity, Blood 21, 221-236, 1963.
23. Kremers, R.M., Wagenvoord, R.J., and Hemker, H.C., The effect of fibrin(ogen) on thrombin generation and decay, Thromb.Haemost. 112, 486-494, 2014.
24.  de Ridder, G.G., Lundblad, R.L., and Pizzo, S.V., Actions of thrombin in the interstitium, J.Thromb.Haemost., in press, 2016.
25.  Guadiz, G., Sporn, L.A., and Simpson-Haidaris, P.J., Thrombin cleavage-independent deposition of fibrinogen in extracellular matrixes, Blood 90, 2644-2653, 1997.
26.  Zamarron, C., Ginsberg, M.H., and Plow, E.F., Monoclonal antibodies specific for a conformationally altered state of fibrinogen, Thromb.Haemost. 64, 41-46, 1990.
27.  Geer, C.B., Rus, I.A., Lord, S.T., and Schoenfisch, M.H., Surface-dependent fibrinopeptide A accessibility to thrombin, Acta Biomater. 3, 663-668, 2007.
28.  Dyr, J.E., Tichý, I., Juroušková, M., et al., Molecular arrangement of adsorbed fibrinogen molecules characterized by specific monoclonal antibodies and a surface plasmon resonance sensor, Sensors Actuators  B 51, 268-272, 1998.
 29.  Bar-Shavit, R., Eldor, A., and Vlodavsky, I., Binding of thrombin to subendothelial extracellular matrix. Protection and expression of functional properties, J.Clin.Invest. 84, 1096-1104, 1989.
30.   Bar-Shavit, R., Benezra, M., Eldor, A., et al., Thrombin immobilized to extracellular matrix is a potent mitogen for vascular smooth muscle cells: nonezymatic mode of action, Cell Regul. 1, 453-463, 1990.

Roger L Lundblad
Chapel Hill, North Carolina,
January 16, 2016

© Roger L Lundblad
January 16, 2016