John H. Ferguson

Studies on a Proteolytic Enzyme System of the Blood. IV. Activation of Profibrinolysin by Serum Fibrinolysokinase.

Conclusion Serum, independent of cellular elements, contains a fibrinolysokinase which is capable of converting profibrinolysin to active fibrinolysin.

Hemorrhagic Diathesis Due to PTC (Plasma Thromboplastin Component) Deficiency.

Summary Five patients suffering from hemorrhagic diatheses manifesting prolongation of the whole blood coagulation time are presented. On the basis of the experimental evidence, 3 of these patients have been classified as PTC deficient and 2 as hemophilic.

Crystalline Trypsin-Inhibitor and Blood-Clotting

Through the courtesy of Dr. T. E. Weich-selbaum, Washington University, St. Louis, we were supplied with 10 mg of pure crystalline trypsin-inhibitor (1 mg = 4.0 × 10-2 [T.U.]Hb), a polypeptide which was originally isolated from pancreas by Northrop and Kunitz. 1 The sample, dissolved in 10 cc physiological saline, was brought from pH = 5.1 to pH = 7.2 with a trace of n/10 NaOH (= inhib.). The other clotting reagents and test methods have been described in previous publications, 2 the dialyzed crude albumin (= alb.) being prepared from rabbit plasma. I. Inhibition of clotting of recalcified citrated plasma (rabbit): Whole plasma and several saline dilutions showed unequivocal retardation of clotting on recalcification in the presence of inhib., as compared to controls; e.g., clotting-time of plasma (1:2 dilution) on adding CaCl2: (a) control (with saline) = 3′ 20″; (b) with inhib. = 7′ 05″. 11. Inhibition of thrombic clotting of fibrinogen: −4 1: 100 dilution of Parfentjevs: 1 rabbit clottine globulin (Lederle Lab.) proved an excellent and stable thrombin (T). The following clotting-times (C.T.) were determined at room temperature (21 C) and pH = 7.2, the thrombin, in each case, being mixed with the cited agents for 10 min prior to the addition of the fibrinogen (F): No. 4 shows a definite antithrombic effect, as compared with the absence of any direct action of the inhibitor, alone, on the thrombin-fibrinogen interaction (No. 2).

Thromboplastic Action of Plasma Protease (Tryptase).

Summary The natural protease (tryptase) in several plasma protein fractions resembles a weak pancreatic trypsin in its fibrino-genolytic, fibrinolytic, and “thromboplastic” effects. These actions are inhibited by crystalline trypsin-inhibitors from pancreas and soybean. The clear demonstration of thromboplastic action in these quantitatively-controlled tests is direct proof of participation of plasma tryptase in the blood-clotting system.

Modus Operandi of Commercial Heparin

The full explanation of the clot-inhibiting action of heparin (Howell and Holts 1 anticoagulant liver extract) has awaited the reconciliation of the conflicting results of various investigators. The present experiments were designed to assist in the search for the experimental variables involved. Three technical points received special attention: (1) adequate controls 2 ; (2) the time factor in certain reactions; (3) the effective concentration zones of the chief reagents as measured by a dilution method. 0.5 cc. of a moderately weak thrombin mixture [prothrombin soln. (10) + 0.1 % cephalin (1) + N/10 CaCl2(1)] gave solid clots in 1.0 cc. of prothrombin-free dog fibrinogen in 15-60 sec. 2 Successive dilutions of a 1% stock solution of commercial (H. W. & D.s) heparin were used in the following tests (all performed at 38°C). A. A true antithrombic action could be demonstrated by adding 1-3 mg. heparin (1:1000 soln.) per one cc. of ‘ripe’ (i. e., maximally activated) thrombin and subsequently testing its clotting power. As long as the reagents were added simultaneously it was immaterial whether the heparin was added to the thrombin or to the fibrinogen first. In other words the inhibition was ‘immediate’. Nevertheless, if the heparin was incubated with the thrombin before the clotting test, the neutralization was definitely enhanced. The rapid and solid clots with control thrombins substantiate HoweHs (Cekada 3 ) claim that “antithrombin is absent from the solutions of prothrombin prepared by the acetone method.” There is nothing to suggest that this antithrombic action of heparin is other than direct. A flocculation of fibrinogen by the heparin proved troublesome in some of the tests but it was found possible to prevent this by controlled alkalinization with N/10 KOH.

Stability of Prothrombin in the Presence of Thrombin

In earlier numbers of these PROCEEDINGS, Mertz, Seegers and Smith 1 , 2 allege that thrombin can inactivate its precursor prothrombin. We have published evidence 3 that a trypsin-like enzyme is not only a thromboplastic agent 4 but also a progressive antithrombin 5 of major importance in natural thrombin destruction. It is present, to a varying extent, in serum and in most prothrombin and thrombin preparations. There is doubt whether such a factor was ruled out in the Iowa experiments. It should be pointed out that Smith, et al., use extremely potent thrombic agents and assay them by a dilution technic which yields values up to several thousands. In our experience, strong thrombins (and prothrombins) appear unusually stable but turn out to be much less so when diluted to, say, about 5–25 Iowa units. In Table I of another Iowa communication, 6 there is a 9% loss of activity of thrombin in the presence of prothrombin as compared with a much more startling 95% fall in the prothrombin titer during the same 4-hour period. Reagents and Methods. Our routine prothrombins, 7 when maximally activated with CaCl2 and brain thromboplastin, usually clot a test fibrinogen in some 5-20+ seconds (temp. = 37.5°C, pH = 7.5). A typical prothrombin solution, assayed through the courtesy of Dr. H. P. Smith (Iowa) gave 47 units per cc (38 units per mg N). Persisting in our unwillingness to use any system of thrombin “units” for comparing one preparation with another, without convincing proof of control of instability factors, we restrict the assay to a single prothrombin preparation and the thrombins freshly prepared from it. Under these narrowed conditions, thrombin concentrations are measured, with a 5–10% accuracy, in a relative manner from the clotting-times obtained with a series of dilutions tested under strictly comparable conditions.

Assay of Tryptases by Lysis of Fibrinogen

A recent method 1 , 2 for assay of tryptases by lysis of fibrin clot is sufficiently sensitive to give excellent results with normal dog, cat and human plasma tryptases, which, in a limited series of observations to date, fall within the range of 20-200 “units” (v. infra), in which the test is accurate to within 5-10%. The principle of the method is the comparison of the “lytic times” of activated tryptase ounknown” with a standard reference series of known trypsin concentrations. A convenient practical standard of 100 “units” is defined as the lytic activity of 1 mg per cc of a reliable commercial trypsin∗ (prepared by dilution of a “stock” 2% solution preserved in glycerol-borate buffer, according to Burdon 3 ). Since some species (e.g. rabbit 4 ) and certain human bloods yield plasma tryptase values under 5 “units” by the above test, a new method has been devised to operate with a sensitivity of ten times that of the original test. It has the same (5-10%) experimental error in the new range of 10-1 “units.” Like a number of older attempts (e.g. 5 ), the new method involves lysis of fibrinogen. This is timed by testing the residual clotting activity with a potent thrombin. As in the fibrinolytic method, the assay depends upon comparison of the “unknown” with a series of standard enzyme dilutions. Reagents. 1. Simplified borate bufler solution. 2.5% H3BO3 (45 parts), 0.5% NaCl (45 parts), 4% Na3B4O7,10H2O (10 parts): pH = 7.7 ± 0.1 (glass electrode). This solution is used as solvent and diluent for fibrinogen and thrombin. 2. Fibrinogen. A lyophilized preparationa is convenient and an especially pure preparation (about 50% coagulable protein) of human fibrinogen, prepared by Dr. R. M. Ferry and the staff of the Harvard Plasma Fractionation Laboratory and kindly supplied by Dr. E. J. Cohn for this work, is excellent.

Does Magnesium Sulphate Cause Catharsis by Being Absorbed and then Excreted into the Colon

Conclusion (1) Magnesium sulphate in the dog does not cause laxation or catharsis by being absorbed into the blood and then excreted into the colon. (2) The parenteral administration of magnesium sulphate does not augment the excretion of magnesium by the colonic mucosa to an appreciable extent.∗

Estimation of Trypsin by Fibrinolysis

It has been pointed out that the actions of crystalline trypsin on blood-clotting systems are: 1. thromboplastic, 1 2. prothrombinolytic 2 and thrombinolytic, 3 3. fibrinogenolytic and fibrinolytic. 4 Preliminary tests with varying amounts of crystalline trypsin (X.T. = preparation of Dr. T. E. Weichselbaum, Washington University, St. Louis) and commercial trypsin (F. T. = Fairchild Bros, and Foster) established the fact that fibrinogen and fibrin are more sensitive than thrombin to the lytic action of the protease. This is a serious handicap in attempts to assay trypsin by the thrombinolytic method previously suggested. 3 However, fibrinolysis offers an alternative and simpler technic for the enzyme assay, which may be made especially accurate if the fibrinolysis is followed by relative turbidimetry (cf. 4 ) using the Evelyn photoelectric colorimeter. Method. Essential reagents include a stable fibrinogen (F) 4 and a stable thrombin (e.g. TG = 1:100 dilution of “rabbit clotting globulin,” kindly supplied by Dr. I. A. Parfentjev, 5 Lederle Labs., N. Y.). In each of a series of enzyme dilutions, 1 cc trypsin is added to 5 cc F + 3 cc TG + 1 cc saline (= control for experiments, other than those cited, in which it is desired to test effects of inhibitors, etc.), a few sec prior to the onset of clotting. Control of clotting conditions must include temperature and pH. Relative turbidity is measured at intervals by the galvanometer scale deflection of the Evelyn apparatus. Data are plotted in the form of a series of curves corresponding to the various dilutions of trypsin used. It is not the shape of the curves which is significant but their relative positions along the time axis. Hence, conversion of the galvanometer reading (G) into “photometric density” (L), by means of the formula L = 2-log G, is an unnecessary refinement of the method described.

The Assay Recovery of Prothrombin Added to Plasma

In the promulgation of current methods of prothrombin assay 1 , 2 there has been some investigation of plasma dilutions but no direct experiments to show the ability to recover prothrombin added to plasma. This is important in connection with the possibility that clot-inhibitors 3 , 4 , 5 , 6 may complicate the interpretation of the assay. It is recognized, for instance, that heparin interferes with prothrombin determinations 2 and heparin can act both as an antiprothrombin 6 and as an immediate antithrombin, 5 in the presence of the respective plasma co-factors. The natural antithrombins also include a progressive thrombinolytic factor. 4 As an example of the kind of interference which may be expected, it has recently been found that a series of prothrombin dilutions, subsequently activated with Ca++ and brain thromboplastin, gives more than the theoretical thrombin yield, as determined from the clotting-times for purified fibrinogen, using a similar dilution series of the same (full-strength) thrombin as the standard of reference. 6 In the absence of equivalence it is, of course, impossible to assay prothrombin both in terms of an arbitrarily fixed clotting-time and a definite thrombin dilution value. Inherent in the clinical tests is a false assumption that inhibitors do not influence the results obtained. Actually, there must be a rôle for the natural inhibitors of the types mentioned. The Iowa workers 2 recognized only the progressive antithrombins and succeeded in controlling these by dilution. It is an unproved assumption that immediate antithrombins and antiprothrombins do not interfere with the prothrombin assay. In the following experiment, in which a prepared prothrombin is assayed in the presence of plasma, these problems are readily brought to test.