Profibrinolysin, Antifibrinolysin, Fibrinogen and Urine Fibrinolytic Factors in the Human Subject
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Profibrinolysin, Antifibrinolysin, Fibrinogen and Urine Fibrinolytic Factors in the Human Subject M. Mason Guest J Clin Invest. 1954;33(11):1553-1559. https://doi.org/10.1172/JCI103033. Research Article Find the latest version: https://jci.me/103033/pdf PROFIBRINOLYSIN, ANTIFIBRINOLYSIN, FIBRINOGEN AND URINE FIBRINOLYTIC FACTORS IN THE HUMAN SUBJECT 1 By M. MASON GUEST (From the Department of Physiology, Wayne University College of Medicine, Detroit, Michi- gan, and the Carter Physiology Laboratory, University of Texas Medical Branch, Galveston, Texas) (Submitted for publication March 30, 1954; accepted July 22, 1954) This report describes methods for the quanti- in 90 ml. of 0.1 M HCI. The pH was adjusted to 7.25 tative assay of human plasma profibrinolysin with HO or NaOH, and the volume brought to 100 (plasminogen) and for the semi-quantitation of ml. with distilled, or demineralized, water. Streptokinase (VaridaseD) 8 was used to activate the fibrinolytic factors in urine. The concentrations profibrinolysin to fibrinolysin. The streptokinase-strep- of plasma profibrinolysin, antifibrinolysin, fibrino- todornase preparation was dissolved in 0.9 per cent NaCl gen and lytic factors in urine of patients with an solution to produce a streptokinase solution containing active neoplasm and in pregnant women are com- 15,000 units per ml. pared with their concentrations in normal indi- The fibrinogen used for preparation of the fibrin sub- strate was obtained from bovine plasma by the freeze- viduals. thawing method (6). A 1.5 to 3.0 per cent fibrinogen Although several methods have been described solution containing 1.5 per cent NaCi and 5 per cent of for the separation of human profibrinolysin from the imidazole buffer solution was stored at -20° C. other constituents of plasma (1-5), the method Prior to use, a test tube containing the stored fibrinogen described here appears to have the advantages solution was thawed in a 37.5° C. water bath. The fibrin- ogen was then diluted with 0.9 per cent NaCl and dis- that: (a) It can be utilized for quantitation; (b) tilled water to a fibrinogen concentration of 0.2 per cent it can be performed on a relatively small aliquot of and a final salinity equivalent to 0.9 per cent NaCl. plasma; (c) it is not influenced by the antifibrin- These fibrinogen solutions were stable for five hours or olysin titer of the plasma; (d) it is highly re- longer when maintained at 37.5° C. producible; and (e) it does not require complex Thrombin, Topical,4 was employed to clot the fibrino- gen. The solution, containing 2500 Iowa units per ml., procedures or reagents which are difficult to was prepared by dissolving the thrombin in 0.9 per cent obtain. NaCl and adding 50 per cent glycerol for stabilization The blood and urine of approximately 200 hu- during storage under refrigeration. man subjects were analyzed. Significant mean Bovine fibrinolysin5 was dissolved and standardized increases in plasma profibrinolysin, antifibrinoly- for the antifibrinolysin assay as described in previous sin, and reports (7, 8). fibrinogen were found during pregnancy The phenol reagent of Folin and Ciocalteu (9) was and in patients with neoplastic disease when com- used to determine tyrosine in the assay of fibrinogen. pared with normal subjects. The fibrinolytic ac- Collection and preparation of plasma. Nine parts of tivity of urine was slightly but not significantly human blood, obtained from the antecubital vein with- higher in patients with neoplasms than in normal out stasis, was immediately mixed with one part of 3.2 per cent sodium citrate. The plasma was removed by individuals. pipetting, after centrifugation and reading of the hemato- crit. All fibrinogen determinations were completed dur- MATERIALS AND METHODS ing the day on which the blood was drawn. The plasma was stored at C. whenever or 2 -20° profibrinolysin anti- Reagents. Protamine sulfate powder (Salmine) was fibrinolysin assays could not be completed on the same dissolved in 0.9 per cent NaCl to a final concentration of day. 0.8 per cent. Imidazole, employed to buffer the fibrin clots at pH 8 Generously supplied by Dr. J. M. Ruegsegger of the 7.25, was prepared by dissolving 1.72 g. of the free base Lederle Laboratories Division, the American Cyanamide Co. 1 This work was supported by a Cancer Control Grant 'Generously supplied through the courtesy of E. C. from the United States Public Health Service. Loomis of Parke, Davis and Co. 2 Supplied through the kind cooperation of Dr. W. R. 5Generously supplied through the courtesy of E. C. Kirtley of Eli Lilly and Co. Loomis of Parke, Davis and Co. 1553 1554 M. MASON GUEST Profibrinolysin isolation and assay. The separation of In determining the fibrinolytic activity, aliquots of the profibrinolysin from antifibrinolysin and other inter- activated re-dissolved precipitate were diluted with sa- fering substances was accomplished by treatment with line to adjust the lysis times of 0.1 per cent fibrin clots to protamine sulfate, alteration in pH, dilution, centrifuga- between 200 and 400 seconds.6 Dilutions of the fibrinoly- tion, and digestion of any remaining fibrinogen follow- sin in the clot between 1 to 16 and 1 to 64 usually pro- ing activation of the proenzyme with streptokinase. Stud- duced lysis within the prescribed time range. In mak- ies have indicated that this procedure results in the quan- ing this assay, 0.2 ml. of the diluted, activated fibrinoly- titative precipitation of profibrinolysin. Four mg. of sin solution to be tested was pipetted into a test tube, 50 protamine sulfate were added to a 2 ml. aliquot of the mm. X 8 mm. I.D. As 0.2 ml. of 0.2 per cent fibrinogen plasma by pipetting in 0.5 ml. of the protamine sulfate was pipetted into the tube a stop watch was started. solution. The pH of the mixture was brought to 9.0 Thrombin was immediately added by stirring the mixture (glass electrode) by adding 0.1 M NaOH dropwise with with a 2 mm. diameter rod which had been wetted with stirring. After allowing the solution to remain at pH the thrombin described above. The tube was placed in 9.0 for one hour at room temperature, it was diluted a 37.5° water bath and allowed to remain there except with distilled or demineralized water to a volume of 40 when examined for the lytic end-point. A solid clot ml. and the pH adjusted to 7.0 (glass electrode) with 0.1 formed within 15 to 20 seconds after the thrombin was M HC1. With dilution and lowering the pH a fairly added. The progress of the lysis was followed by tip- heavy precipitate, which contained fibrinogen and pro- ping the tube at intervals. Just prior to the end-point the fibrinolysin, appeared. The precipitate was separated clot acquired a semifluid consistency and slid down the in 50 ml. containers by centrifugation for 30 minutes at tube with a humped appearance. The end-point was 3000 r.p.m. (2000 RCF). The supernatant, containing reached when the "hump" disappeared. Occasional de- antifibrinolysin, was decanted and discarded. No meas- bris was disregarded. Lysis times were determined in urable fibrinolytic activity following treatment with strep- triplicate for each 2 ml. sample of plasma and in most tokinase has ever been found in numerous tests of the cases duplicate samples of plasma were analyzed. supernatant. Traces of antifibrinolysin occasionally re- The 37.5° C. assay curve in Figure 1 was based upon mained with the precipitate, but the amount present was dilutions of the activated plasma profibrinolysin (pro- barely detectable by the most sensitive assay methods. cedure described above) from approximately 200 indi- The trace contamination with antifibrinolysin was re- viduals. The average values for each dilution of all de- moved by repeated washing with distilled water and re- terminations were utilized in constructing this curve. centrifugation in several experiments but this procedure This 37.5° curve coincided with the curve obtained by did not improve the accuracy or precision of the method. dilution of bovine fibrinolysin (Loomis) with assay at When added to the test plasma, profibrinolysin prepa- 37.5° C. and the units are the same as those which have rations prepared by the method of Kline (10), may be previously been described except that the centiunit is recovered as fibrinolysin. However, if an appreciable 1/100 of the conventional unit. The 280 C. curve is in- quantity of profibrinolysin is added to plasma, the amount cluded since the assay for fibrinolysin was originally of streptokinase utilized for activation must also be in- described at this temperature and repeated checks have creased, since the reaction between streptokinase and indicated that the human profibrinolysin assay also falls profibrinolysin appears to be stoichiometric (11). In the on this curve when the lysis is carried out at 28° C. analyses reported here, additional streptokinase was added The identical slopes of the human profibrinolysin assay to the precipitates from a number of the native plasmas curves and the bovine fibrinolysin assay curves would in which the fibrinolytic activity, as measured in the con- indicate that there is no more inhibitor in our profibrin- ventional assay, was high. No additional fibrinolysin olysin preparations than in the best bovine fibrinolysin was recovered when the higher concentrations of strep- available. tokinase were used, indicating that the streptokinase Antifibrinolysin assay. This assay has been described preparations in use for the assays reported here were in detail (7, 8); in essence it involves measuring the adequate for complete activation. Furthermore, the sta- amount of residual bovine fibrinolysin, following incu- tistical increase obtained in profibrinolysin in malignancy bation under controlled conditions of a known quantity and pregnancy is indicative of adequate activation of the of fibrinolysin with diluted defibrinated plasma.