Bovine thrombin

D, H W Hoeffken, D Crosse, J Stuerzebecher, P D Martin, B F P Edwards and W Bode 1992. Refined 2.3 Angstroms X-Ray Crystal Structure of Bovine Thrombin Complexes Formed witli he 3 Benzamidine and Arginine-Based Thrombin Inhibitors NAPAP, 4-TAPAP and MQPA A Starting Point for Improving Antithrombotics. Journal of Molecular Biology 226 1085-1099. 

Components/ mechanism of action Human plasma, fibrinogen and thrombin, virally inactivated, hemostat, sealant. Autologous fibrinogen -t-platelet-rich plasma, hemostatic gel. Bovine collagen, bovine thrombin, plus autologous human plasma, hemostatic agent. Bovine collagen and bovine thrombin. Expands 20% which aids in hemostatic effect. 

Prior to May of 1998 when the commercial product was approved by the FDA in the United States, surgeons in this country formed fibrin sealant by using topical bovine thrombin, which is a commercially available product, together with concentrated fibrinogen most frequently obtained from the blood bank. Standard blood bank cryoprecipitate is a good source of concentrated fibrinogen. Also... 

Daniels, T.M. and Fisher et al., PK., Antibodies to bovine thrombin and coagulation factor V associated with the use of topical bovine thrombin or fibrin glue a frequent finding. Blood, 82, 59a (1993). 

Prior, J., Wallace, D., Hamer, A. and Powers, N., A sprayable hemostat containing fibrillar collagen, bovine thrombin, and autologous plasma. Ann. Thome. Surg., 68, 479-485 (1999). 

Martin PD, Robertson W, Turke D, Bode W, Edwards BFP. The structure of residues 7-16 of the Aa-chain of human fibrinogen bound to bovine thrombin at 2.3 A resolution. J Biol Chem 1992 267 7911-7920. 

Martinelli, R. A., and Scheraga, H. A. (1980). Steady-state kinetic study of the bovine thrombin-fibrinogen interaction. Biochem. 19, 2343-2350. 

I. B. Baird and D, T, Elmore. The kinetics of inaction of bovine thrombin with p-nitrophenyl p -guanldinobenzDste and T-amlno-l-chloro-J-toluene-p-swlEbiianiwle-2-butanoiw. A new method for determining the operational molarity of thrombin solutions. FEBS Lett, 7 343 (196%. 

Zehnder JL, Leung LL. Development of antibodies to thrombin and factor V with recurrent bleeding in a patient exposed to topical bovine thrombin 1990 76 2011-6. 

Rapaport SI, Zivelin A, Minow RA, et al. Clinical significance of antibodies to bovine and human thrombin and factor V after surgical use of bovine thrombin. Am J Clin Pathol 1992 97 84-91. 

Lawson JH, Lynn KA, Vanmatre RM, et al. Antihuman factor V antibodies after use of relatively pure bovine thrombin. Ann Thorac Surg 2005 79 1037-8. 

Lundblad, R.L., A rapid method for the purification of bovine thrombin and the inhibition of the purified enzyme with phenylmethylsutfonyl fluoride. Biochemistry 10, 2501-2506, 1971. 

In three patients who underwent cardiovascular surgery subsequent abnormalities in hemostasis, characterized by increased activated partial thromboplastin time, prothrombin time, and bovine thrombin time, and by a markedly reduced concentration of factor V, developed between the seventh and eighth postoperative days after exposure to fibrin glue containing bovine thrombin (13). It was suggested that the glue also contains small amounts of factor V and that this may have caused the abnormalities. 

The use of preparations of fibrin glue containing bovine thrombin resulted in the development of antibovine thrombin antibodies. In a prospective study, 13 of 34 patients developed a thrombin inhibitor and reduced factor V activity (15,16). In another study a factor V inhibitor developed after cardiac surgery (17). 

Severe hypotension has been reported after the use of bovine fibrin glue for hemostasis in hepatic injury (18). In one there was cardiac arrest and death. These effects may have been the result of an anaphylactic reaction to one or more components of the glue. Of the three ingredients used to prepare fibrin glue, cryoprecipitate and bovine thrombin are antigenic and potentially the most likely causes of anaphylaxis. 

Berruyer M, Amiral J, Ffrench P, Belleville J, Bastien O, Clerc J, Kassir A, Estanove S, Dechavanne M. Immunization by bovine thrombin used with fibrin glue during cardiovascular operations. Development of thrombin and factor V inhibitors. J Thorac Cardiovasc Surg 1993 105(5) 892-7. 

Banninger H, Hardegger T, Tobler A, Barth A, Schupbach P, Reinhart W, Lammle B, Furlan M. Fibrin glue in surgery frequent development of inhibitors of bovine thrombin and human factor V. Br J Haematol 1993 85(3) 528-32. 

Heparin cofactor II may interfere, bovine thrombin minimizes this interference... 

When a suitable model of the unknown crystal structure is available, it can be used to solve the phase problem. Examples are the use of the stracture of human thrombin to solve the structure of bovine thrombin, the use of a known antibody fragment to solve the stracture of an unknown antibody, or the use of the stracture of an enzyme to solve the stracture of an inhibitor complex of the same enzyme in a different crystal form. The model is oriented and positioned in the unit cell of the unknown crystal with the use of rotation and translation functions, and the oriented model is subsequently used to calculate phases and an electron-density map. 

The amount of thrombin inhibited reflects the amount of AT-III in the sample. The addition of heparin reduces interference by other plasma inhibitors like a2-macroglobulin. It has been reported that the species of thrombin, bovine or human, used in the assay can influence abnormally low AT-III values. Friberger and co-workers in a 1982 report recommended bovine thrombin to insure better agreement with immunological AT-III values (F9). 

To demonstrate the effect of the active-site acylated enzyme, we prepared the benzoyl derivatives of bovine thrombin [22,23]. If benzoyl-thrombin is added to blood plasma, the clotting process is retarded in accordance with the deacylation rate (Fig. 7). 

McRae, B.J., Kurachi, K., Heimark, R. L, Fujikawa, K., Davie, E.W., Powers, J.C. 1981. Mapping the active sites of bovine thrombin, factor IXa, factor Xa, factor XIa, factor XI la, plasma kallikrein and trypsin with amino acid and peptide thioesters development of new sensitive substrates. Biochemistry 20, 7196-7206. 

Winterbottom N, Kuo J, Nguyen K, et al. Antigenic Responses to Bovine Thrombin Exposure During Surgery A Prospective Study of 309 Patients. J. Appl. Res. Clin. Exp. Then, 2002 2(1). 

The thrombin time was determined similarly by incubation of 2 IU of crude bovine thrombin (10 IU/mL, Miles Laboratories) with the beads for 5 min at room temperature in an albumin-coated glass tube, followed by 0.2 mL of citrated human plasma. The time to clot was noted by tilting the test tube gently every few seconds. PBS, after incubation with heparin-PVA beads for 5-60 min, was analyzed for the presence of heparin using both toluidine blue and the thrombin time test (PBS in place of gel beads). 

Note specific activities before labeling crude bovine thrombin—96 IU/mg (Parke-Davis), pure human antithrombin III—1000 IU/mg (M. Wick-erhauzer, American Red Cross, Bethesda, MD) open implies material used as eluent in conventional chromatographic mode and ratio of loaded protein does not include antithrombin III content of defibrinated plasma. 

Figure 2 shows that thrombin binds to both heparin-PVA beads and PVA beads without heparin. After a load of crude bovine thrombin (62 IU) followed by PBS and chromogen, color yields of 89% and 81% were obtained for the heparin-PVA and PVA gel, respectively, indicating the presence of active thrombin on the columns. Passing 15 mL of 20% (w/w) bovine albumin through the same columns followed by chromogen lowered the color yield for... 

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