Effect of Glycosaminoglycans on Thrombin- and Atroxin-Induced Fibrin Assembly and Structure

Summary

This study was performed to quantitate the impact of several glycosaminoglycans (GAG) on fibrin assembly and structure. Gel formation was monitored as the increase in optical density at 633 nm subsequent to thrombin (2 NIH u/ml) or atroxin (0.10 mg/ml) addition to solutions of buffered fibrinogen (1 mg/ml) or plasma. Gel absorbance was measured as a function of wavelength (400 to 800 nm) and gel fiber diameter and mass/length ratio (μ) were calculated. Chondroitin sulfate A (CSA)shortened the lag phase, enhanced the maximal rate of turbidity increase, and increased the final gel turbidity of fibrin gels formed by thrombin or atroxin. CSA (16 mg/ml) increased fiber μ from 1.3 to 3.1 × 10¹³ dalton/cm and fiber radius from 6.0 to 8.6 × 10^-6 cm in thrombin-induced gels. μ increased from 0.7 to 2.7 × 10¹³ dalton/cm and fiber radius from 4 to 7.8 × 10^-6 cm for atroxin-induced gels. Above 16 mg/ml, CSA caused fibrinogen precipitation in purified solutions but not in plasma. CSA inhibited thrombin-induced plasma clotting of plasma but effects in atroxin-mediated plasma gels paralleled those seen in purified solutions. Chondroitin sulfate B (CSB)-induced changes in fibrin were similar but slightly less dramatic than those seen with CSA. μ increased from 0.9 to 2.0 × 10¹³ dalton/cm for thrombin-induced fibrin gels and from 0.8 to 2.3 × 10¹³ dalton/cm for atroxininduced gels. Low molecular weight heparin (M_r = 5100) slowed fibrin assembly and reduced fiber size by 50% in thrombininduced gels. Changes in μ of atroxin-induced gels were much less pronounced (<20%). This study documents pronounced GAGinduced changes in fibrin structure which vary with GAG species and may mediate significant physiologic functions.

Keywords

Chondroitin sulfate A - Chondroitin sulfate B - Heparin

• References

• 1 Bettelheim FR, Bailey K. The products of the action of thrombin on fibrinogen. Biochim Biophys Acta 1952; 09: 578-579
• 2 Lorand L. Fibrinopeptide. Biochem J 1952; 52: 200-203
• 3 Hantgan RR, Hermans J. Assembly of fibrin: a light scattering study. J Biol Chem 1979; 254: 1127-1181
• 4 Hantgan R, McDonagh J, Hermans J. Fibrin assembly. Ann NY Acad Sci 1983; 408: 344-366
• 5 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
• 6 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
• 7 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
• 8 Shen LL, Hermans J, McDonagh JP, McDonagh R, Carr ME. Effects of calcium ion and covalent crosslinking on formation and elasticity of fibrin gels. Thromb Res 1975; 06: 255-265
• 9 Marx G. Divalent cations induce protofibril gelation. Am J Hematol 1988; 27: 104-109
• 10 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
• 11 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
• 12 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
• 13 Carr ME, Gabriel DA, 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
• 14 Carr ME, White GC, Gabriel DA. Platelet factor 4 enhances fibrin fiber polymerization. Thromb Res 1987; 45: 539-543
• 15 Gabriel DA, Smith LA, Folds JD, Davis L, Cancelosi SE. The influence of immunoglobulin (IgG) on the assembly of fibrin gels. J Lab Clin Med 1983; 101: 545-552
• 16 Bale MD, Westrick LG, Mosher DF. Incorporation of thrombospondin into fibrin clots. J Biol Chem 1985; 260: 7502-7508
• 17 Carr ME. Turbidimetric evaluation of albumin’s impact on the structure of thrombin mediated fibrin gels. Haemostasis 1987; 17: 189-194
• 18 Carr ME, Gabriel DA, McDonagh J. Influence of factor XIII and fibronectin on fiber size in thrombin-induced fibrin gels. J Lab Clin Med 1987; 110: 747-752
• 19 Carr ME, Cromartie HL, Gabriel DA. The effect of homo poly-Lamino acids on fibrin assembly and the role of charge and molecular weight. Biochemistry 1989; 28: 1384-1388
• 20 Carr ME. Fibrin formed in plasma is composed of fibers more massive than those formed from purified fibrinogen. Thromb Haemostas 1988; 59 (03) 535-539
• 21 LeBoeuf BD, Raja RH, Fuller GM, Weigel PH. Human fibrinogen specifically binds hyaluronic acid. J Biol Chem 1986; 261: 12586-12592
• 22 LeBoeuf RD, Gregg RR, Weigel PH, Fuller GM. Effects of hyaluronic acid and other glycosaminoglycans on fibrin polymer formation. Biochemistry 1987; 26: 6052-6057
• 23 Jones M, Gabriel DA. Influence of the subendothelial basement membrane components on fibrin assembly. Evidence for a fibrin binding site.on Type IV collagen J Biol Chem 1988; 263: 7043-7048
• 24 Laki K. The polymerization of proteins: the action of thrombin on fibrinogen. Arch Biochem Biophys 1951; 32: 317-324
• 25 Clauss A. A rapid physiological coagulation method for determination of fibrinogen. Acta Haematol 1957; 17: 237-246
• 26 Carr ME, Hermans J. Size and density of fibrin fibers from turbidity. Macromolecules 1978; 11: 46-50
• 27 Stathakis NE, Mosesson MW. Interactions among heparin, coldinsoluble globulin, and fibrinogen in formation of the heparinprecipitable fraction of plasma. J Clin Invest 1977; 60: 855-865
• 28 Carr ME, Qureshi GD. The impact of delayed fibrinopeptide-A release on fibrin structure, studies of an abnormal fibrinogen. J Biol Chem 1987; 262: 15568-15574
• 29 Carr ME. coagulation events have minimal impact on plasma fibrin structure. Am J Med Sci 1988; 295: 433-437
• 30 Carr ME, Hardin CL. Fibrin has larger pores when formed in the presence of erythrocytes. Am J Physiol 1987; 253: Hl069-Hl073
• 31 Carr ME, Shen LL, Hermans J. Mass-length ratio of fibrin fibers from gel permeation and light scattering. Biopolymers 1977; 16: 1-15
• 32 Stewart U, Bradley M, Johnson Jr CS, Gabriel DA. Measurement of the diffusion of macromolecules through fibrin gels using holographic relaxation spectroscopy. Biopolymers 1988; 27: 173-185
• 33 Carr ME, Hardin CL. Large fibrin fibers enhance urokinase-induced plasmin digestion of plasma clots. Blood 1987; 70 Suppl (Suppl. 01) 400 a
• 34 Ciano pS, Colvin RB, Dvorak AM, McDonagh J, Dvorak HF. Macrophage migration in fibrin gel matrices. Lab Invest 1986; 54: 62-70
• 35 Carrell N, Gabriel DA, Blatt PM, Carr ME, McDonagh J. Hereditary dysfibrinogenemia in a patient with thrombotic disease. Blood 1983; 62: 439-447