Residual Serum Thrombin

Transcription

1 Residual Serum Thrombin Activity Murray Weiner N RECENT YEARS a remarkable number of blood and tissue components have been found to play a role in the formation of thrombin, the enzyme ultimately responsible for clotting. Recently, there has been renewed interest in factors which destroy or inactivate thrombin. These factors may be of equal importance with those responsible for the speed and amount of thrombin formation. Thrombin in blood is believed to be inactivated by several different mechanisms such as adsorption on a fibrin clot; by heparin combined with a co-factor; and by so-called circulating anti-thrombin (1). There is as yet no simple method for estimating the over-all antithrombin activity of a plasma specimen analogous to the simple one stage Quick test for activity of the prothrombin complex. Most coagulation laboratories measure antithrombin by a multitube titration which estimates the inhibiting influence of serial dilutions of the test plasma on standard mixtures of fibrinogen and thrombin. This paper describes a method for estimating antithrombin activity as reflected by the residual thrombin activity after a standard period of incubation of the serum residue of the usual Quick prothrombin test. It is based on the fact that fresh serum, which contains antithrombin, rapidly loses its thrombin activity on incubation, although preparations of thrombin in equivalent concentrations in simple buffer solutions remain active for days. Some of the factors influencing the te8t are described below. PROCEDURE Figure 1 illustrates the method by which residual thrombin was estimated. Prothrombin times were determined at 37#{176} with the aid Prom the Research Service (Third Medical Division), Goldwater Memorial Hospital, New York, N. Y. and the Department of Medicine, New York University College of Medicine, New York Cit7. This work was aided by a grant from the New York Heart Association. Received for publication May 2,

2 272 WEINER Clinical Chemistry P1a C4im opiaau.s #{149}t.g.Pmth,ota U.. :: 0,... el.t..inst..rur eottlm * LI 0.2 ul. MS ra tr.. t,5, i.inta.ft.r c1,tti.,i. Ti....o..u, t.r fibrinog.. to olot us.. is tho Aoti-th,o Fig. 1. Method of estimating residual thrombin. Antithrombin time (residual thrombin time) is the residual thrombin activity 1 minute after clotting. of a nichrome wire ioop by the usual one-stage technic using 0.1 ml. citrated plasma to 0.2 mg. calcium-thromboplastin reagent (Simplastin, Warner-Chilcott Co.). Exactly 30 seconds after clot formation, the loop is lifted with the clot, and the clot gently pressed against the tube wall to extract the serum. The clot and loop are then removed, and exactly 1 minute after clotting, 0.1 ml. of this serum is added to 0.2 ml. of a standard fibrinogen solution (Warner-Chilcott Co.). The time necessary for the serum to clot the fibrinogen solution is a reflection of the activity of thrombin which has not yet been inactivated by antithrombin, and may be referred to as antithrombin time or residual thrombin time. The 1-minute incubation time was chosen for convenience. RESUITS Figure 2 illustrates the relationship of time of serum incubation to residual thrombin. One minute after clot formation, 107 normal human specimens showed an average residual thrombin time (antithrombin time) of 14.3 sec., with an average deviation of 2.2 sec. (Fig. 3). Paired determinations of antithrombin time after 1-minute incubation deviated from each other by an average of 0.5 seconds. After 3 minutes incubation, end points were more difficult to detect, and paired determinations differed on the average by 2.5 seconds. Values below 11 seconds after 1-minute incubation are considered to indicate abnormally low antithrombin activity. Values above 19 seconds reflect increased antithrombin if the plasma prothrombin

3 Vol. 4, No. 4 RESIDUAL SERUM THROMBIN ACTIVITY 273 I I 2& L 7 HE L U LJ U.t LUJ U UJ tge of S.r.-outa#{149} Fig. 2. Effect of incubation on the ability of fresh serum to clot a standard fibrinogen solution. S 12 to so so or I. a U Pt a,e FIg. 3. Antithrombin time of 107 subjects without frank clotflag abnormalities. activity is normal. There was no significant difference in the results obtained with untreated glassware as compared to silicone-coated tubes. Variations in prothrombin time of these 107 normal specimens did not correlate with the antithrombin time. This is consistent with the concept that variations in residual thrombin activity (i.e., in antithrombin time) is not a reflection of relatively minor variations in the amount of thrombin produced. However, where initial thrombin is definitely reduced as reflected by a prolonged prothrombin time, the residual thrombin after 1 minute will also be less than normal, i.e. with prolonged prothrombin time, a comparable prolonged antithrombin time is generally noted, and does not mean increased anti-

4 274 WEINER Clinical Chemistry Table 1. COMPARISON OF THE EFFECT OF SERUM INCUBATION IN THE PRESENCE oa ABSENCE OF THE Cior ON RESIDUAL THROMBIN AcTlvrvy &Uquot Seconds inoubatd Wtth clot Clot r.,ncv.d Ciotng time (Sec.) (residual thrombn) A B C thrombin activity. Scattergrams of the antithrombin time vs. recal. cification time or prothrombin consumption (i.e. serum prothrombin time) of the same blood specimens from 29 subjects failed to reveal any significant correlation. Influence oi Serum Incubation in Presence of Clot The influence of incubation of serum in contact with the clot, as compared to incubation with clot removed, on the residual thrombin was studied by the following experiment: The residual thrombin time after 3 minutes incubation was determined for several aliquots of the same plasma with different portions of the incubation time in contact with the clot. The results (Table 1) indicate that incubation in the presence of the clot leads to more residual thrombin than the same time of incubation in the absence of the clot. Influence of Fib4n on Residual Thrombin The influence of the presence of fibrin on residual thrombin was also studied in the following manner: Partially defibrinated plasma was obtained by adding 1 unit of thrombin (Ortho Pharmaceutical Co.) in 0.01 ml. saline to 1.0 ml. citrated plasma. The resulting clot was removed 10 minutes later. The residuum and an untreated aliquot of plasma were then incubated at 37#{176} for 10 minutes and the prothrombin and residual thrombin times determined (Table 2). The partially defibrinated specimen contained considerably more residual thrombin activity. APPLICATION Aside from prothrombin deficiency, residual thrombin has been found to be decreased in 2 cases of acute pancreatitis, a condition in 1The fact that a thin but definite clot was obtained with the thrombin-treated plasma. on the addition of thromboplastin indicated that it had been partially but not completely deflbrinated.

5 Vol. 4, No. 4 RESIDUAL SERUM THROMBIN ACTIVITY 275 Table 2. COMPARISON OF RESIDUAL THROMBIN Aovivrrv op Paam&u.y DEFIBRINATED PLASMA WITH UNTEx&!rgI PLASMA Plasma specimen Residual thrombin time (see.) Prothromnbin time (see.) After I,nin. incubation After S mist. incubation Untreated Partially defirinated which antithrombin activity by titration methods has been reported to be elevated (2). Decreased residual thrombin was also noted in some patients given relatively small doses of Varidase (streptokinasestreptodornase) intramuscularly. Tillett (3) found that intravenous doses of the order of 100,000 units may influence several of the proteins involved in coagulation. In our studies, intramuscular doses of 5000 units twice daily were found not to influence prothrombin time, recalcification time, lysis, or the pattern of the thrombelastograph (4). However, there was an increase in the average antithrombin activity in 9 out of 10 subjects while under the influence of these small doses of intramuscular Varidase as compared to control periods (Fig. 4). Since fever is a frequent reaction to intramuscular Vandase, the nonspecific effect of fever on residual thrombin time was studied in 2 subjects by inducing fever for several days with typhoid vaccine. No significant change in residual thrombin time was noted (Fig. 5). D Period of?r.st.uot - Period. 11Co.troi irtitiei 1 Fig. 4. Average antithrombin time of 10 patients during control period as compared to period of Varidase therapy (5000 unite twice daily intramuscularly).

6 276 WEINER Clinical Chemstry JOT. OH. 1*. F. FIg. 5. Effect of intramuscular Varidase (5000 units twice daily) and typhoid vaccine on antithrombin time. One patient with marked hypofibrinogenemia (between 45 and 65 mg./100 ml.) repeatedly showed greater than normal residual thrombin activity, i.e. a 1-minute antithrombin time of from 7 to 10 seconds. Since this patient also had a deficiency of other plasma proteins, including prothrombin, it is difficult to attribute the high residual thrombin solely to the hypofibrinogenemia. A true reduction of circulating antithrombin may have existed. Reconstituted lyophilized plasma specimens2 with normal prothrombin activity show high residual thrombin in the presence of normal fibrinogen levels, suggesting that antithrombins have been destroyed by the lyophilization process. Incubation of plasma at 37#{176} for from 4 to 8 hours does not alter antithrombin activity. Further incubation causes first a reduction in residual thrombin which roughly parallels reduced prothrombin complex activity (prolonged one-stage prothrombin time), and then increased residual thrombin, presumably a reflection of decreased antithrombin. DISCUSSION The role of the clot as a site of thrombin adsorption is not completely clear. Partially defibrinated plasma does show more residual 2Warner-Chilcott s lyophilized plasma, prepared as a standard for determining thromboplastin activity was used in this study and yielded a one-minute residual thrombin time of 8 seconds.

7 Vol. 4, No. 4 RESIDUAL SERUM THROMBIN ACTIVITY 277 thrombin activity than unaltered plasma, which is consistent with the concept that the presence of less fibrinogen, forming less clot, results in less thrombin adsorbed. However, if one compares the residual thrombin of serum left in contact with its clot, vs. serum from which the clot was promptly removed, the latter shows less residual thrombin. This is contrary to the idea that thrombin is adsorbed by the clot. It would fit the idea that thrombin is contained in the formed clot, and that it is released into serum on standing, perhaps with retraction. SUMMARY A simple test for residual thrombin activity of serum is described. This test is believed to reflect the over-all antithrombin activity of the specimen. These results are not altered by silicone vs. glass handling. The test is influenced by the presence or absence of contact with a fresh clot; by deficiencies in prothrombin or fibrinogen; by aging of plasma specimens and possibly by streptokinase administration in vivo, and by pancreatic disease. The full significance of alteration in this test remains to be determined. REFERENCES 1. Beegers, W. H., Miller, K. D., Andrews, E. B., and Murphy, B. C., Am. J. Physiol. 169, 700 (1952). 2. Innerfield, I., Angrist, A., and Benjamin, J. W. Am. J. Med. 12, 24 (1952). 3. Tillett, W. S., Johnson, A. J., and McCarty, W. B., J. GUn. Invest. 34, 169 (1955). 4. von Kaulla, K. N., and Weiner, M., Blood 10, 362 (1955).