Thrombin Applications
Production of fibrin clot in plasma:
Typicallty one to two units of Thrombin will clot one mL of plasma.
Cleavage of Fusion Proteins:
Thrombin can be used for the cleavage of many peptides at the Thrombin recognition site using concentrations of 0.5 NIH units thrombin per one nanomole polypeptide in 20 microliters of 50 mM ammonium bicarbonate, pH 8.0.
Thrombin cleavage of fusion proteins can be carried out at a Thrombin to fusion protein ratio of 1:500.
Fusion proteins may be cleaved in Thrombin cleavage buffer consisting of 50 mM Tris, pH 8.0, 150 mM NaCl, 2.5 mM CaCl2 and 0.1% 2-mercaptoethanol. 2 mg of fusion protein was incubated with 4 µg of thrombin for 20 minutes at RT in the cleavage buffer.
Several conventions are used in Thrombin literature:
1 IOWA unit= 0.83 NIH unit
1 WHO unit = 0.56 NIH unit
1 NIH unit = 0.324 +/- 0.073 µg
1 NIH unit = 1 USP unit
Thrombin (human and bovine) will catalyze the hydrolysis of several peptide p-nitroanilides, tosyl-arg-nitrobenzyl ester, and a thiobenzyl ester synthetic substrates
thrombin references
1.Enzyme Nomenclature: EC 3.4.21.5
2.Chang, J.Y., Eur. J. Biochem., 151, 217?224 (1985).
3.The Plasma Proteins, 2nd ed., 2, Putnam, F. W., ed, p. 148.
4.Machovich, R., The Thrombin, 1, 63-66 (1984)
5.Machovich, R., The Thrombin, 1, 111 (1984)
6.Prasad, S., J. Biol. Chem. 279, 10103-10108 (2004)
7.Kisiel, W., Human plasma protein C: isolation, characterization, and mechanism of activation by alpha-thrombin. J. Clin. Invest. 64, 761-769, (1979)
8.The Plasma Proteins, 2nd ed., 2, Putnam, F. W., ed: Table 2. See also: The Enzyme Explorer: Plasma and Blood Protein Resource
9.Qian, W.J., et al., J. Proteome Res., 4, 2070-2080 (2005).
10.Nilsson, B., et al., Arch. Biochem. Biophys., 224, 127-133 (1983)
11.Boyer, P.D., The Enzymes, Academic Press (New York), 3rd ed., Vol. III, p. 277-321 (1971).
12.Expasy/SwissProt: P00743
13.Boissel, J.P., et al., J. Biol. Chem., 259, 5691-5697 1984).
14.Righetti, P.G., and Tudor, G., Isoelectric points and molecular weights of proteins, a new table. Journal of Chromatography, 220, 115-194 (1981).
15.Butkowski, R.J. et al., J. Biol. Chem., 252, 4942 (1977).
16.Winzor, D. J. and Scheraga, H. A., Arch. Biochem. Biophys. 104, 202-207 (1964)
17.Human Blood Coagulation, Haemostasis and Thrombosis, 2nd ed., R. Biggs, ed., p. 722 (1976).
18.The Handbook of Synthetic Substrates, Hemker, H. C., Martinus Nijhoff publisher (1983).
19.Lottenberg, R., et al., Assay of Coagulation Proteases Using Peptide Chromogenic and Fluorogenic Substrates. Meth. Enzymol., 80-C, 341-361 (1981).
20.Chang, Y., Thrombin specificity. Requirement for apolar amino acids adjacent to the thrombin cleavage site of polypeptide substrate. Eur. J. Biochem., 151(2), 217-224 (1985).
21.Hakes, D.J. and Dixon, J.E., Anal. Biochem., 202, 293 (1992).
22.Gaun, KL and Dixon, JE,, Anal. Biochem., 192, 262, 1991
23.De Cristofaro, R. and De Candia, E., J. Thromb. Thrombolysis, 15, 151-163 (2003)
24.Sherwood, J.A., Mol. Biochem. Parisitol., 40, 173-181 (1990)
25.Berg, D.T., et al., Science, 273, 1389-1391 (1996)
26.Lundblad, R.L. et al., Methods Enzymol., 45, 156 (1976)
27.Matsuoka, S., et al., JP. J. Pharmacol., 51, 455-463 (1989)
28.Wimen, B., Meth. Enzymol., 80, 395-408 (1981)
29.Magnusson, S. The Enzymes, 3rd ed., III, pp. 277-321, Boyer, P.D., ed., Academic Press (1971)
Wednesday
Thursday
Thrombin generation in trauma patients.
BACKGROUND: Trauma patients are at risk of developing an acute coagulopathy of trauma (ACT) related to tissue injury, shock, and hemodilution. ACT is incompletely understood, but is similar to disseminated intravascular coagulation (DIC) and is associated with poor outcome.
STUDY DESIGN AND METHODS: Thrombin generation assays were used to evaluate plasma hemostasis in 42 trauma patients, 25 normal subjects, and 45 patients on warfarin and in laboratory-prepared factor reduced plasma. RESULTS: Prolonged prothrombin time (PT), more than 18 seconds, or an international normalized ratio of greater than 1.5 was present in 15 trauma patients indicating possible ACT. Native thrombin generation (no activator added, contact activation blocked) showed that Trauma with ACT patients had lag times 68% shorter and peak thrombin generation threefold higher than normal patients indicating the presence of circulating procoagulants capable of initiating coagulation systemically. Trauma patients had lower platelet counts and fibrinogen and Factor (F)II levels putting them at increased risk of bleeding. In laboratory-prepared isolated factor-reduced samples and in patients with vitamin K-dependent factor deficiency due to warfarin, thrombin generation decreased in direct proportion to FII levels. In contrast, in diluted plasma and in trauma patients with reduced factor levels, thrombin generation was increased and associated with slower inhibition of thrombin generation (prolonged termination time) and decreased antithrombin levels (43% of normal in Trauma with ACT).
CONCLUSIONS: Thrombin generation studies indicate that Trauma with ACT patients show dysregulated hemostasis characterized by excessive non-wound-related thrombin generation due to a combination of circulating procoagulants capable of activating coagulation systemically and reduced inhibitor levels allowing systemic thrombin generation to continue once started.
Dunbar NM, Chandler WL.
From the Department of Laboratory Medicine, University of Washington, Seattle, Washington
STUDY DESIGN AND METHODS: Thrombin generation assays were used to evaluate plasma hemostasis in 42 trauma patients, 25 normal subjects, and 45 patients on warfarin and in laboratory-prepared factor reduced plasma. RESULTS: Prolonged prothrombin time (PT), more than 18 seconds, or an international normalized ratio of greater than 1.5 was present in 15 trauma patients indicating possible ACT. Native thrombin generation (no activator added, contact activation blocked) showed that Trauma with ACT patients had lag times 68% shorter and peak thrombin generation threefold higher than normal patients indicating the presence of circulating procoagulants capable of initiating coagulation systemically. Trauma patients had lower platelet counts and fibrinogen and Factor (F)II levels putting them at increased risk of bleeding. In laboratory-prepared isolated factor-reduced samples and in patients with vitamin K-dependent factor deficiency due to warfarin, thrombin generation decreased in direct proportion to FII levels. In contrast, in diluted plasma and in trauma patients with reduced factor levels, thrombin generation was increased and associated with slower inhibition of thrombin generation (prolonged termination time) and decreased antithrombin levels (43% of normal in Trauma with ACT).
CONCLUSIONS: Thrombin generation studies indicate that Trauma with ACT patients show dysregulated hemostasis characterized by excessive non-wound-related thrombin generation due to a combination of circulating procoagulants capable of activating coagulation systemically and reduced inhibitor levels allowing systemic thrombin generation to continue once started.
Dunbar NM, Chandler WL.
From the Department of Laboratory Medicine, University of Washington, Seattle, Washington
Wednesday
Assessment of thrombin generation: useful or hype?
School of Human Life Sciences, University of Tasmania, Tasmania, Australia. Murray.
Thrombin plays an important role in hemostasis through its multiple functions across blood coagulation, platelet activation, and fibrinolysis. The measurement of thrombin generation is therefore viewed as a potentially useful test that could be applied to the screening, monitoring, and/or diagnosis of hemostatic abnormalities. Indeed, advances in thrombin generation assays have created significant interest and debate as to whether they may provide a more physiologically relevant testing system than do traditional coagulation tests.
A variety of thrombin generation assays, including commercially available systems, have been investigated for their correlation with hypocoagulable and hypercoagulable states. Although there is an extensive body of literature that has investigated the application of thrombin generation assays, some limitations remain. These include poor standardization of reagents and methods and a lack of large prospective studies that demonstrate clear relationships between thrombin generation with bleeding and thrombosis phenotypes, as well as with monitoring anticoagulation.
Whether thrombin generation assays become more "useful" than "hype" will require well-designed, large, prospective multicenter trials using standardized methods
Thrombin plays an important role in hemostasis through its multiple functions across blood coagulation, platelet activation, and fibrinolysis. The measurement of thrombin generation is therefore viewed as a potentially useful test that could be applied to the screening, monitoring, and/or diagnosis of hemostatic abnormalities. Indeed, advances in thrombin generation assays have created significant interest and debate as to whether they may provide a more physiologically relevant testing system than do traditional coagulation tests.
A variety of thrombin generation assays, including commercially available systems, have been investigated for their correlation with hypocoagulable and hypercoagulable states. Although there is an extensive body of literature that has investigated the application of thrombin generation assays, some limitations remain. These include poor standardization of reagents and methods and a lack of large prospective studies that demonstrate clear relationships between thrombin generation with bleeding and thrombosis phenotypes, as well as with monitoring anticoagulation.
Whether thrombin generation assays become more "useful" than "hype" will require well-designed, large, prospective multicenter trials using standardized methods
Friday
What is Thrombin
THROMBIN(activated Factor II [IIa]) is a coagulation protein that has many effects in the coagulation cascade. It is a serine protease (EC 3.4.21.5) that converts soluble fibrinogen into insoluble strands of fibrin, as well as catalyzing many other coagulation-related reactions.
Activation of prothrombin is crucial in physiological and pathological coagulation. Various rare diseases involving prothrombin have been described (e.g., hypoprothrombinemia). Anti-thrombin antibodies in autoimmune disease may be a factor in the formation of the lupus anticoagulant also known as ( antiphospholipid syndrome ).
In addition to its activity in the coagulation cascades, thrombin also promotes platelet activation, via activation of protease-activated receptors on the platelet.
Thrombin, a potent vasoconstrictor and mitogen, is implicated as a major factor in vasospasm following subarachnoid hemorrhage. Blood from a ruptured cerebral aneurysm clots around a cerebral artery, releasing thrombin. This can induce an acute and prolonged narrowing of the blood vessel, potentially resulting in cerebral ischemia and infarction (stroke).
Due to its high proteolytic specificity, thrombin is a valuable biochemical tool. The thrombin cleavage site (Leu-Val-Pro-Arg-Gly-Ser) is commonly included in linker regions of recombinant fusion protein constructs. Following purification of the fusion protein, thrombin can be used to selectively cleave between the Arginine and Glycine residues of the cleavage site, effectively removing the purification tag from the protein of interest with a high degree of specificity.
References
^ McMillen, S.I. (1984), None of These Diseases (Old Tappan, NJ: Revell)
^ Schmidt A (1872). "Neue Untersuchungen ueber die Fasserstoffesgerinnung". Pflüger's Archiv für die gesamte Physiologie 6: 413–538. doi:10.1007/BF01612263.
Recommended Reading
Further reading
Esmon CT (1995). "Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface". Faseb J 9 (10): 946–55. PMID 7615164.
Lenting PJ, van Mourik JA, Mertens K (1999). "The life cycle of coagulation factor VIII in view of its structure and function". Blood 92 (11): 3983–96. PMID 9834200.
Plow EF, Cierniewski CS, Xiao Z, et al. (2002). "AlphaIIbbeta3 and its antagonism at the new millennium". Thromb. Haemost 86 (1): 34–40. PMID 11487023.
Maragoudakis ME, Tsopanoglou NE, Andriopoulou P (2002). "Mechanism of thrombin-induced angiogenesis". Biochem. Soc. Trans 30 (2): 173–7. doi:10.1042/ (inactive 28 June 2008). PMID 12023846.
Howell DC, Laurent GJ, Chambers RC (2002). "Role of thrombin and its major cellular receptor, protease-activated receptor-1, in pulmonary fibrosis". Biochem. Soc. Trans 30 (2): 211–6. doi:10.1042/ (inactive 28 June 2008). PMID 12023853.
Firth SM, Baxter RC (2003). "Cellular actions of the insulin-like growth factor binding proteins". Endocr. Rev 23 (6): 824–54. doi:10.1210/er.2001-0033. PMID 12466191.
Minami T, Sugiyama A, Wu SQ, et al. (2004). "Thrombin and phenotypic modulation of the endothelium". Arterioscler. Thromb. Vasc. Biol 24 (1): 41–53. doi:10.1161/01.ATV.0000099880.09014.7D. PMID 14551154.
De Cristofaro R, De Candia E (2004). "Thrombin domains: structure, function and interaction with platelet receptors". J. Thromb. Thrombolysis 15 (3): 151–63. doi:10.1023/B:THRO.0000011370.80989.7b. PMID 14739624.
Tsopanoglou NE, Maragoudakis ME (2004). "Role of thrombin in angiogenesis and tumor progression". Semin. Thromb. Hemost 30 (1): 63–9. doi:10.1055/s-2004-822971. PMID 15034798.
Bode W (2007). "Structure and interaction modes of thrombin". Blood Cells Mol. Dis 36 (2): 122–30. doi:10.1016/j.bcmd.2005.12.027. PMID 16480903.
Wolberg AS (2007). "Thrombin generation and fibrin clot structure". Blood Rev 21 (3): 131–42. doi:10.1016/j.blre.2006.11.001. PMID 17208341.
Degen S: Prothrombin. In: High K, Roberts H, eds. Molecular Basis of Thrombosis and Hemostasis. New York, NY: Marcel Dekker; 1995:75.
[show]v • d • eProteins: coagulation
Activation of prothrombin is crucial in physiological and pathological coagulation. Various rare diseases involving prothrombin have been described (e.g., hypoprothrombinemia). Anti-thrombin antibodies in autoimmune disease may be a factor in the formation of the lupus anticoagulant also known as ( antiphospholipid syndrome ).
In addition to its activity in the coagulation cascades, thrombin also promotes platelet activation, via activation of protease-activated receptors on the platelet.
Thrombin, a potent vasoconstrictor and mitogen, is implicated as a major factor in vasospasm following subarachnoid hemorrhage. Blood from a ruptured cerebral aneurysm clots around a cerebral artery, releasing thrombin. This can induce an acute and prolonged narrowing of the blood vessel, potentially resulting in cerebral ischemia and infarction (stroke).
Due to its high proteolytic specificity, thrombin is a valuable biochemical tool. The thrombin cleavage site (Leu-Val-Pro-Arg-Gly-Ser) is commonly included in linker regions of recombinant fusion protein constructs. Following purification of the fusion protein, thrombin can be used to selectively cleave between the Arginine and Glycine residues of the cleavage site, effectively removing the purification tag from the protein of interest with a high degree of specificity.
References
^ McMillen, S.I. (1984), None of These Diseases (Old Tappan, NJ: Revell)
^ Schmidt A (1872). "Neue Untersuchungen ueber die Fasserstoffesgerinnung". Pflüger's Archiv für die gesamte Physiologie 6: 413–538. doi:10.1007/BF01612263.
Recommended Reading
Further reading
Esmon CT (1995). "Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface". Faseb J 9 (10): 946–55. PMID 7615164.
Lenting PJ, van Mourik JA, Mertens K (1999). "The life cycle of coagulation factor VIII in view of its structure and function". Blood 92 (11): 3983–96. PMID 9834200.
Plow EF, Cierniewski CS, Xiao Z, et al. (2002). "AlphaIIbbeta3 and its antagonism at the new millennium". Thromb. Haemost 86 (1): 34–40. PMID 11487023.
Maragoudakis ME, Tsopanoglou NE, Andriopoulou P (2002). "Mechanism of thrombin-induced angiogenesis". Biochem. Soc. Trans 30 (2): 173–7. doi:10.1042/ (inactive 28 June 2008). PMID 12023846.
Howell DC, Laurent GJ, Chambers RC (2002). "Role of thrombin and its major cellular receptor, protease-activated receptor-1, in pulmonary fibrosis". Biochem. Soc. Trans 30 (2): 211–6. doi:10.1042/ (inactive 28 June 2008). PMID 12023853.
Firth SM, Baxter RC (2003). "Cellular actions of the insulin-like growth factor binding proteins". Endocr. Rev 23 (6): 824–54. doi:10.1210/er.2001-0033. PMID 12466191.
Minami T, Sugiyama A, Wu SQ, et al. (2004). "Thrombin and phenotypic modulation of the endothelium". Arterioscler. Thromb. Vasc. Biol 24 (1): 41–53. doi:10.1161/01.ATV.0000099880.09014.7D. PMID 14551154.
De Cristofaro R, De Candia E (2004). "Thrombin domains: structure, function and interaction with platelet receptors". J. Thromb. Thrombolysis 15 (3): 151–63. doi:10.1023/B:THRO.0000011370.80989.7b. PMID 14739624.
Tsopanoglou NE, Maragoudakis ME (2004). "Role of thrombin in angiogenesis and tumor progression". Semin. Thromb. Hemost 30 (1): 63–9. doi:10.1055/s-2004-822971. PMID 15034798.
Bode W (2007). "Structure and interaction modes of thrombin". Blood Cells Mol. Dis 36 (2): 122–30. doi:10.1016/j.bcmd.2005.12.027. PMID 16480903.
Wolberg AS (2007). "Thrombin generation and fibrin clot structure". Blood Rev 21 (3): 131–42. doi:10.1016/j.blre.2006.11.001. PMID 17208341.
Degen S: Prothrombin. In: High K, Roberts H, eds. Molecular Basis of Thrombosis and Hemostasis. New York, NY: Marcel Dekker; 1995:75.
[show]v • d • eProteins: coagulation
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