Am J Physiol Lung Cell Mol Physiol 298: L36-L44, 2010
Since thrombin activation of endothelial cells (ECs) is well-known to increase endothelial permeability by disassembly of adherens junctions (AJs) and actinomyosin contractility mechanism involving myosin light chain (MLC) phosphorylation, we investigated the effects of bone marrow-derived progenitor cells (BMPCs) on the thrombin-induced endothelial permeability response.
We observed that addition of BMPCs to endothelial monolayers at a fixed ratio prevented the thrombin-induced decrease in transendothelial electrical resistance, a measure of AJ integrity, and increased mouse pulmonary microvessel filtration coefficient, a measure of transvascular liquid permeability. The barrier protection was coupled to increased vascular endothelial cadherin expression and increased Cdc42 activity in ECs.
Using small interfering RNA (siRNA) to deplete Cdc42 in ECs, we demonstrated a key role of Cdc42 in signaling the BMPC-induced endothelial barrier protection. Endothelial integrity induced by BMPCs was also secondary to inhibition of MLC phosphorylation in ECs. Thus BMPCs interacting with ECs prevent thrombin-induced endothelial hyperpermeability by a mechanism involving AJ barrier annealing, inhibition of MLC phosphorylation, and activation of Cdc42.
Yidan D. Zhao,* Hiroshi Ohkawara,* Stephen M. Vogel, Asrar B. Malik, and You-Yang Zhao
Department of Pharmacology and Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, Illinois
Thursday
Wednesday
Thrombin
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)
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)
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
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