Abstract
Background
The pathogenesis and the impact of therapy on thrombin activation in children with acute lymphoblastic leukemia (ALL) are unknown. Steroids may contribute to ALL-associated thrombosis. We explored the hemostatic effects of methylprednisolone monotherapy (MpMT) (32 mg/m2/day IV × 3 days) in children with newly diagnosed ALL.
Methods
Children (>1 to ≤18 years of age) enrolled on DFCI ALL05-01 protocol (n = 30; mean age 6.3 years), without prior steroid therapy, were eligible for study. Overnight fasting pre- and post-MpMT samples were analyzed for coagulation factors [FVIII:C, von Willebrand factor antigen (vWF:Ag) and fibrinogen] and parameters of thrombin generation [prothrombin fragments 1.2 (F1.2), thrombin–antithrombin complex (TAT), and D-dimer].
Results
At diagnosis F1.2 (1.5 nmol/L), TAT (10.9 µg/L), and D-dimers (2,766 ng/ml) levels were increased indicating endogenous thrombin activation. Patients with peripheral blasts (n = 17) had higher levels of vWF:Ag (1.89 vs. 1.14 P = 0.001), TAT (15.39 vs. 5.02 P = 0.038), and D-dimer (3,640 vs. 1,623 P = 0.019) compared to those without peripheral blasts. Following MpMT the blast count decreased significantly from 24% to 3.5% (P < 0.001) with reduction in level of vWF:Ag (1.5, P < 0.01), TAT (8.9, P = 0.42), and D-dimer (P = 0.018) despite 30% increase in FVIII:C levels (P = 0.005). However, patients without peripheral blasts had no significant change in vWF:Ag levels (1.14 vs. 1.25; P = 0.142) and had an increase in thrombin generation parameters.
Conclusions
We postulate that peripheral blasts through endothelial activation stimulate vWF:Ag production/secretion causing coagulation activation. Methylprednisolone therapy reduces the blast count and indirectly suppresses the coagulation activation. Future studies are required to confirm these findings. Pediatr Blood Cancer 2010;54:963–969
Uma Athale MD, MSc1,2,*, Albert Moghrabi MD3, Trishana Nayiager BScH, CCRP2, Yves-Line Delva RN3, Lehana Thabane PhD4,5,6, Anthony K.C. Chan MBBS1,2
Pediatric Blood & Cancer
Volume 54, Issue 7, pages 963–969, 1 July 2010
Wednesday
Thursday
Bone marrow-derived progenitor cells prevent thrombin-induced increase in lung vascular permeability
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
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
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)
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