A Plasma Clot Lysis Assay Based on the Release of Fibrin Degradation Products: Application to the Diagnosis of Hypofibrinolytic States

Summary

Using a monoclonal antibody-based assay, we measured the fibrin degradation product release in the supernatant of plasma clots obtained before and after venous occlusion (VO) in 30 patients with definite or suspected vascular thrombosis (19 definite and 2 suspected deep vein thrombosis, 6 recurrent superficial thrombophlebitis, 3 arterial occlusions of lower limbs). tPA and PAI-1 concentrations were determined using ELISA assays; the post-occlusion values were corrected for haemoconcentration. The increase in tPA during VO was correlated with haemoconcentration (r = 0.74), but 3 patients had ineffective VO (<2% increase in proteins). The fibrinolytic response to VO was evaluated using the shortening of the time necessary for the release of 200 μg of fibrin degradation products per mg of fibrinogen (Δ T 200). Two among the 27 patients with effective VO were bad responders with a Δ T 200 <3 h (whereas all the others had Δ T 200 >10 h). These patients had respectively a deficient tPA release (Δ tPA = 1 ng/ml) and an elevated PAI-1 level at rest (33 ng/ml). Several other patients were bad responders in terms of tPA release or of shortening of the euglobulin clot lysis time but they had a normal Δ T 200. This plasma clot test reflects the ability of free tPA to bind to fibrin (the amount of which depends on the level of tPA and PAI-1), and may be useful in the diagnosis of a hypofibrinolytic state.

Keywords

Thrombosis - Plasma clot lysis - Fibrin degradation products

• References

• 1 Bachmann F. Fibrinolysis. In: Thrombosis and Haemostasis Ver-straete M, Vermylen J, Lijnen HR, Arnout J. (eds). International Society on Thrombosis and Haemostasis and Leuven University Press; Leuven: 1987: 227-265
• 2 Prowse CV, Farrugia A, Boulton FE, Tucker J, Ludlam CA, MacLaren M, Belch JJ F, Prentice CR M, Dawes J, MacGregor IR. A comparative study using immunological and biological assays of the haemostatic responses to DDAVP infusion, venous occlusion and exercise in normal men. Thromb Haemostas 1984; 51: 110-114
• 3 Wun TC, Capuano A. Initiation and regulation of fibrinolysis in human plasma at the plasminogen activator level. Blood 1987; 69: 1354-1362
• 4 Sanzo MA, Marasa JC, Wittwer AJ, Siegel NR, Harakas NK, Feder J. Purification and characterization of a tissue plasminogen activator-inhibitor complex from human umbilical vein endothelial cell conditioned medium. Biochemistry 1987; 26: 7443-7449
• 5 Masson C, Anglès-Cano E. Kinetic analysis of the interaction between plasminogen activator inhibitor-1 and tissue-type plasminogen activator. Biochem J 1988; 256: 237-244
• 6 Wun TC, Capuano A. Spontaneous fibrinolysis in whole human plasma. Identification of a tissue activator-related protein as the major plasminogen activator causing spontaneous activity in vitro. J Biol Chem 1985; 260: 5061-5066
• 7 Koppert PW, Hoegee de NobelE, Nieuwenhuizen W. A monoclonal antibody-based enzyme immunoassay for fibrin degradation products in plasma. Thromb Ilacmuslas 1988; 59: 310-315
• 8 Kluft C. Studies on the fibrinolytic system in human plasma: quantitative determination of plasminogen activators and proactivators. Thromb Haemostas 1979; 41: 365-383
• 9 Francis RB, Seyfert U. Tissue plasminogen activator antigen and activity in disseminated intravascular coagulation: clinicopathologic correlations. J Lab Clin Med 1987; 110: 541-547
• 10 Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogen. Acta Haematologica 1957; 17: 237-246
• 11 Nilsson IM, Ljungnér H, Tengborn L. Two different mechanisms in patients with venous thrombosis and defective fibrinolysis: low concentration of plasminogen activator or increased concentration of plasminogen activator inhibitor. Br Med J 1985; 290: 1453-1456
• 12 Wiman B, Ljungberg B, Chmielewska J, Urdén G, Blombäck M, Johnsson H. The role of the fibrinolytic system in deep vein thrombosis. J Lab Clin Med 1985; 105: 265-270
• 13 Juhan-Vague I, Valadier J, Alessi MC, Aillaud MF, Ansaldi J, Philip-Joet C, Holvoet P, Serradimigni A, Collen D. Deficient t-PA release and elevated PA inhibitor levels in patients with spontaneous or recurrent deep venous thrombosis. Thromb Haemostas 1987; 57: 67-72
• 14 Nguyen G, Horellou MH, Kruithof EK O, Conard J, Samama MM. Residual plasminogen activator inhibitor activity after venous stasis as a criterion for hypofibrinolysis: a study in 83 patients with confirmed deep vein thrombosis. Blood 1988; 72: 601-605
• 15 Hamsten A, Wiman B, De Faire U, Blombäck M. Increased plasma levels of a rapid inhibitor of tissue plasminogen activator in young survivors of myocardial infarction. N Eng J Med 1985; 313: 1557-1563
• 16 Paramo JA, Colucci M, Collen D. Plasminogen activator inhibitor in the blood of patients with coronary artery disease. Br Med J 1985; 291: 573-574
• 17 Nicoloso G, Hauert J, Kruithof EK O, Van Melle G, Bachmann F. Fibrinolysis in normal subjects – Comparison between plasminogen activator inhibitor and other components of the fibrinolytic system. Thromb Haemostas 1988; 59: 299-303
• 18 Juhan-Vague I, Alessi MC, Fossat C, Valadier J, Aillaud MF, Serradimigni A. Clinical relevance of reduced t-PA release upon venous occlusion in patients with idiopathic/recurrent deep venous thrombosis. Fibrinolysis 1988; 2 (Suppl. 02) 112-113
• 19 Brommer EJ P, Barrett-Bergshoeff MM, Allen RA, Schicht I, Bertina RM, Schalekamp MA D H. The use of desmopressin acetate (DDAVP) as a test of the fibrinolytic capacity of patients – Analysis of responders and non-responders. Thromb Haemostas 1982; 48: 156-61
• 20 Sultan Y, Harris A, Strauch G, Venot A, De Lauture D. A dynamic test to investigate potential tissue plasminogen activator activity. Comparison of deamino-8-D-arginine vasopressin with venous occlusion in normal subjects and patients. J Lab Clin Med 1988 6. 645-653