Methylene Blue Enhances Lateral Association of Fibrin Resulting in Rapid Gelation and Thick Fiber Formation

Marcus E Carr Jr.; Patrick L Powers

doi:10.1055/s-0038-1646564

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1989; 61(02): 230-233
DOI: 10.1055/s-0038-1646564

Original Article

Schattauer GmbH Stuttgart

Methylene Blue Enhances Lateral Association of Fibrin Resulting in Rapid Gelation and Thick Fiber Formation

Marcus E Carr Jr.

The Division of Hematology/Oncology and Coagulation Special Studies Laboratory, Departments of Medicine and Pathology, Medical College of Virginia, USA, and McGuire V. A. Medical Center, Richmond, Mrginia, USA

,

Patrick L Powers

The Division of Hematology/Oncology and Coagulation Special Studies Laboratory, Departments of Medicine and Pathology, Medical College of Virginia, USA, and McGuire V. A. Medical Center, Richmond, Mrginia, USA

› Author Affiliations

Abstract

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Summary

Methylene blue (MB) has been suggested as a therapeutic alternative for heparin reversal in patients sensitive to protamine. We investigated the impact of MB on the assembly and structure of thrombin induced fibrin and plasma gels. MB (1,600 μg/ml) reduced the thrombin clotting time (TCT) of plasma by 30% and of purified fibrinogen by 46%. Above 1,600 μg/ml, TCTs were prolonged due to MB mediated fibrinogen precipitation. The presence of 5 mM CaCl₂ masked the effect of MB in both purified and plasma systems and lowered the threshold for MB-induced purified fibrinogen precipitation to 800 μg/ml. MB shortened the lag phase prior to thrombin-induced turbidity increase, and enhanced final gel turbidity. The fibrin fiber mass/length ratio increased from 5.2 to 13.1 × 10¹³ dalton/cm in purified fibrin gels and from 3.2 to 10.4 × 10¹³ dalton/cm in plasma gels as the MB concentration increased from 0 to 200 μglml. Due to the photooxidant effect of MB on fibrinogen, rapid time-dependent loss of fibrinogen clottability was obvious at low MB concentrations (50 to 400 μg/ml). At high MB concentrations, intense MB light absorption partially protected fibrinogen within the sample. Accurate measurements could only be made, however, when MB was added just prior to thrombin and the assays were performed in the dark. While erythrocytes may reduce the impact of MB photooxidation in whole blood, plasma samples must be shielded from light if reproducible results are to be obtained.

Keywords

Methylene blue - Fibrinogen - Fibrin

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References

References
1 Sloand EM, Kessler CM, McIntosh CL, Klein HG. Neutralization of heparin-induced anticoagulation with methylene blue. Blood 1987; 70 Suppl (Suppl. 01) 409 a
2 Carr ME, Gabriel DA, Herion JC, Roberts HR. Granulocyte lysosomal cationic protein alters fibrin assembly: a possible mechanism for granulocyte control of clot structure. J Lab Clin Med 1986; 107: 199-203
3 Carr ME, Gabriel DA, McDonagh J. Influence of calcium on the structure of reptilase and thrombin derived fibrin gels. Biochem J 1986; 239: 513-516
4 Carr ME, White GC, Gabriel DA. Platelet factor 4 enhances fibrin fiber polymerization. Thromb Res 1987; 45: 539-543
5 Carr ME, Cromartie HL, Gabriel DA. The effect of homo poly-L- amino acids on fibrin assembly the role of charge and molecular weight. Biochemistry 1989; 28: 1384-1388
6 Clauss A. A rapid physiological coagulation method for determination of fibrinogen. Acta Haematol 1957; 17: 237-246
7 Carr ME, Gabriel DA. Dextran-induced changes in fibrin fiber size and density based on wavelength dependence of gel turbidity. Macromolecules 1980; 13: 1473-1477
8 Carr ME, Gabriel DA. The effect of dextran 70 on the structure of plasma-derived fibrin gels. J Lab Clin Med 1980; 96: 985-993
9 Carr ME, Hermans J. Size and density of fibrin fibers from turbidity. Macromolecules 1978; 11: 46-50
10 Saito Y, Shimizu A, Matsushima A, Inada Y. Chemical modification of fibrinogen and the effect on fibrin formation. Ann NY Acad Sci 1983; 408: 288-300
11 Ferry JD, Morrison PR. Preparation and properties of serum and plasma proteins. VIII. The conversion of human fibrinogen to fibrin under various conditions. J Am Chem Soc 1947; 69: 388-399
12 Shulman S, Ferry JD, Tinoco I. The conversion of fibrinogen to fibrin. XII. Influence of pH, ionic strength and hexamethylene glycol concentration on the polymerization of fibrinogen. Arch Biochem Biophys 1953; 42: 245-256
13 Carr ME. Fibrin formed in plasma is composed of fibers more massive than those formed from purified fibrinogen. Thromb Haemostas 1988; 59: 535-539
14 Shen LL, Hermans J, McDonagh J, McDonagh RP, Carr ME. Effects of calcium ion and covalent crosslinking on formation and elasticity of fibrin gels. Thromb Res 1975; 6: 255-265
15 Marx G, Hopmeier P. Zinc inhibits FPA release and increases fibrin turbidity. Am J Hematol 1986; 22: 347-353
16 Bale MD, Westrick LG, Mosher DF. Incorporation of thrombospondin into fibrin clots. J Biol Chem 1985; 260: 7502-7508
17 Carr ME. The effect of hydroxyethyl starch on the structure of thrombin and reptilase induced fibrin gels. J Lab Clin Med 1986; 108: 556-563