The methods currently used to measure plasminogen activator (PA) in tissue are the potassium thiocyanate extraction technique¹⁾ and the histochemical fibrin slide method, first described by Todd²⁾ and modified by Pandolfi³⁾. Many investigators have studied the relation between disease and tissue PA^4–8) using these methods, But despite various scoring systems, they remain semiquantitative ones. Also, it is not easy to obtain a human blood vessel in a physiologic state, so most studies dealing with vascular PA activity have been based on postmortem specimens or major resectional surgery specimens. There is no controversy over the fact that the main factor of fibrinolytic activity, t-PA, is from vascular endothelial cells, especially of the vein. But it is not known whether any correlation exists between the plasminogen activator in the arteries and/or veins and systemic fibrinolytic activity, because the PA activity of the vessels varies from one anatomic region to another^4–9,13). To assay the physiological and clinical significance of vascular PA activity, it is necessary to obtain artery and vein specimens in a homogenized condition from the given anatomic location. A/V fistular or shunt operation in chronic renal failure for hemodialysis gives us a good opportunity to obtainan artery or vein from the same anatomical area conveniently. In many cases, especially side-to-side anastomosis, small pieces of artery or vein can be obtained. Unfortunately, the quantity of the material is too small to extract fibrinolytic activity by the previously described methods, so we attempted a new method of extracting fibrinolytic activity from a small piece of vessel. With this new method, we could measure the fibrinolytic activity of a piece of vessel as small as 3 mg of wet weight. We compared vascular fibrinolytic activity with systemic fibrinolytic activity of basal and response to venous occlusion.

This study was designed to evaluate the relationship between the fibrinolytic activity and the systemic fibrinolytic activity. Kluft^15,16) reports that total fibrinolytic activity in dextran sulfate euglobulin fraction prepared from normal morning plasma mainly consist of factor XII dependent and independent intrinsic fibrinolytic activity, while an additional amount of approximately 1 – 2% is contributed by the extrinsic or vascular activator. However, the physiologic importance and the nature of the intrinsic fibrinolytic activity is uncertain. Our results showing the correlation between the fibrinolytic activity in dextran sulfate euglobulin in the morning, and the fibrinolytic activity of the artery and vein suggest that the intrinsic fibrinolytic activity is a reflection of the fibrinolytic activity of the artery and vein. Vascular fibrinolytic activity is released from endothelial cells by several stimuli^17,20), and the amount released is determined by the fibrinolytic capacity or reserve of the endothelial cells^21–24).

The fibrinolytic activity of the artery and vein is different²⁴⁾ and varies from one anatomic region to another^4,9–13). So it is not easy to characterize the fibrinolytic activity of one branch of vein and/or artery in total fibrinolytic activity of plasma. It was known that tissue plasminogen activator is insoluble under usual conditions. The inability to extract the fibrinolytic agent from tissues with saline was observed by Fleisher²⁵⁾, and Permin^26,27) tried unsuccessfully to extract the activator with the following solution: Phosphate buffer (N/15 pH 5.6 – 8.3), acetic acid (1%, 5%), HCl (0.01 N), ammounium hydroxide (0.01 N), sodium hydroxide (0.01 N), and glycerol (87%) containing 0.5% acetic acid. Based on the observation that the tissue fibrinolytic activator extracted from tissue with a strong solution of potassium thiocyanate and with an improved standandized fibrin plate method, a method for the quantitative extraction and assay of the tissue activator was developed by Astrup and Albrechtzen¹⁾. Using the plasminogen inactivated fibrin plates, it was demonstrated that the activity present in the extract of tissue was caused by an activator of plasminogen and not by a protease²⁸⁾. But the wide range of activity in the different organs and the large individual variations discouraged consideration of the origin of activator and its correlation with systemic fibrinolytic activity. In our method, fibinolytic activity could be extracted by incubating a small piece of vessel in distilled water dropped on a plasminogen-rich fibrin plate. This method was very simple, sensitive enough to measure a quantity of material as small as 1 mg, and did not require any equipment or reagent except a controlled fibrin plate and distilled water. But the material should be more than 3 mg for two reasons. First, material less than 3 mg is not on a dose-dependent curve, and second, the weight is not reproducible if the weighing material (wet vessel) is less than 3 mg. We do not understand the exact mechanism of how distilled water extracts t-PA. In our preliminary study, the lysed zone was negligible when we used normal saline, half saline, DMEM, or HANK solution instead of distilled water. We examined the vessels microscopically after incubation and found that the endothelium was disrupted and lysed out with distilled water but not with normal saline, half saline, DMEM or HANK solution. So the osmotic lysis of endothelial cells and the release of t-PA might be assumed. Also we speculated that distilled water may change the environment of the released t-PA and its inhibitor. For example, the change of ionic strength might be a factor in separating t-PA from its inhibitor. In conclusion, we could extract vascular fibrinolytic activity by distilled water on a fibrin plate. Basal fibrinolytic activity was linked to artery and venous fibrinolytic activity. The increment of fibrinolytic activity after venous occlusion was linked to venous fibrinolytic activity but not to arterial fibrinolytic activity.