Quantitative analysis of plasminogen activator in small arteries by modified fibrin plates

Abstract

The assay of plasminogen activator at the intimal surfaces of arteries less than 2 mm in diameter is possible using streptokinase-treated fibrin plates. Femoral arteries obtained from rats showed a reproducible fibrinolysis at their intimal surfaces that could be quantitated for the analysis of plasminogen activator. Utilization of the assay adjusted to the experimental situation would give a method of evaluating changes in the concentration of plasminogen activator at the intimal surface of small arteries when studied under various research conditions. A n awareness of the dynamic hemostatic balance that exists between coagulation and fibrin degradation in blood vessels is important for the microsurgeon. The lysis of accumulated fibrin is a contributing factor to the maintenance of circulation. Intravascular fibrinolysis is initiated by a plasminogen activator released from the intimal surface of blood vessels. The activator converts plasminogen to plasmin, the enzyme that actually lyses fibrin. The fibrinolytic activity of large vessels and tissue biopsies has been extensively studied. The fibrin-plate method of Permin' and Astrup and Mullertz2 has generally been used. This fibrinplate method produces quantitative and reproducible results with large vessels, but has not been sensitive enough to measure fibrinolysis in vessels less than 2 mm in diameter. Small arteries (0.5 to 2.0 mm in diameter) have consistently shown no demonstrable activity on standard fibrin plates. Smaller vessels have been evaluated by Todd? Astrup? Pandolfi et al.,5 Hobby: and others using the histochemical "fibrinolysis autograph" slide technique of Todd. The fibrin slide technique, while applicable to small vessels, relies on a system of grading fibrinolysis that we have found difficult to quantitate. The "fibrin slide sandwich" technique of Noordhoek-Hegt ,'I the "focalysis time" technique of Kwaan and Astrup; and the "Hauchen slide" technique of Warrens are also difficult to quantitate. Many of the above techniques interpret the total fibrinolytic activity of a vessel or tissue rather than fibrinolysis at the intimal surface. This article describes a method for direct and reproducible assaying of the fibrinolysis initiated by the plasminogen activator found on the intimal surfaces of arteries. This technique is sensitive enough to accurately quantitate fibrinolytic activity in vessels as small as 0.5 mm in diameter. JOURNAL OF MICROSURGERY MATERIALS AND METHODSOur method of measuring the fibrinolytic activity of vessels less than 2 mm in diameter uses the original formula of Astrup modified in two ways. First, a thinner layer of fibrin clot is prepared by using 6 ml rather than 9 ml of the fibrinogen solution on 100 x 15 mm Petri dishes. Second, streptokinase is added before the formation of the fibrin clot.Two sets of fibrin plates were prepared in the following manner. Either plasminogen-rich or plasminogen-free bovine fibrinogen (Miles Laboratories, Inc., Elkhart, IN) was assayed for potency, and a quantity sufficient to give a co...

Assessment of the Patency of Microvascular Venous Anastomosis

“…[9][10][11][12] These factors are constantly balanced by intravascular fibrinolysis, initiated by plasminogen activator released by the intima of the blood vessel. [13][14][15] Any imbalance in this mode of dynamic homeostasis can start the cascade toward thrombosis.…”

“…This could be attributed to the animals' strong fibrinolytic activity. 13,14,20 It has also been reported that a newlyformed blood channel based on the adventitious and perivascular networks can bridge the gap between the ligated venous stumps. The new blood channel is then differentiated from the bridging capillary networks.…”