M i n i m a l
H e p a r i n
Cofactor
D i s s e m i n a t e d
Activity
in
Intravascular a n d
C o a g u l a t i o n
Cirrhosis
KENNETH M. BRAUNSTEIN, B.A., AND KARL EURENIUS, M.D.
Department of Medicine, Division of Hematology, Medical University ofSouth Carolina, Charleston, South Ca ABSTRACT
With a modification of the technic of Egeberg, 6 we have measured heparin-activated antithrombin activity, or plasma heparin cofactor activity (HCA), in blood from four patients with disseminated intravascular coagulation (DIC) and ten patients with cirrhosis, two clinical conditions associated with coagulation disorders and low antithrombin III activity.5,9'18 A causal relationship between low antithrombin III activity and thromboembolic phenomena has been demonstrated in various kindreds. 6,10,17 Low antithrombin activity in DIC may be the result of intravascular thrombin inactivation of the antithrombin molecule.6,14 HCA has not been assessed in patients with cirrhosis, but would be exReceived September 30, 1975; received revised pected to be low since antithrombin III manuscript. November 7, 1975; accepted for publicalevels in such patients are reported detion November 7, 1975. Address reprint requests to Dr. Eurenius: Veterans creased and thought to reflect reduced synAdministration Hospital, 109 Bee Street, Charleston, thesis.5 If heparin is to be considered South Carolina 29403.
THROMBIN INACTIVATION in vitro is regu-
lated by mechanisms that have been outlined in a recent review. 3 Antithrombin II, better known as heparin cofactor, and antithrombin III are perhaps the most important of these, and in addition, they have recently been shown to have anti-factor X a , 20 ' 21 anti-factor XI a , 4 and antiplasmin 8 activity. Recent biochemical fractionation studies 2,15,21 indicate that antithrombin III and heparin cofactor reside on the same molecule. Heparin would appear to be a coenzyme for antithrombin III, markedly accelerating the rate of binding of antithrombin III to thrombin, X a , XI a , and plasmin.
488
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Braunstein, Kenneth M., and Eurenius, Karl: Minimal heparin cofactor activity in disseminated intravascular coagulation and cirrhosis. Am J Clin Pathol 66: 488-494, 1976. An assay technic for measuring heparin cofactor activity in which antithrombin activity can be assessed without plasma attenuation even in the presence of therapeutic levels of heparin is presented. Heparin-activated anti-thrombin activity was markedly depressed in plasmas of four patients with disseminated intravascular coagulation and in ten patients with cirrhosis. Residual activity in those plasmas appeared qualitatively normal, and no inhibitor (platelet factor IV activity) was observed. Plasmas from patients with disseminated intravascular coagulation and cirrhosis required more heparin to obtain in vitro clotting time prolongation equivalent to normal. (Key words: Antithrombin; Antithrombin II; Heparin; Heparin cofactor; Hemorrhagic disorders.)
September 1976 Material Fibrinogen 500 agZ Heparin 10,000 U/al Buffer and Plasma
HEPARIN COFACTOR ACTIVITY Volume In ml 0.20 0.023 0.15
489
Final Concentration* 210 mgl 526 U/ml 0 - 30Z
+ (no preincubation time) Thrombin 50 U/ml
0.10
37*C 10.5 U/ml
] clotting time
*Baaed upon final volume of 0.475 ml FIG. 1. Heparin cofactor assay procedure.
Materials and Methods Blood was collected from ten VA research employees who served as normal controls, from ten patients who had biopsyproven cirrhosis, and from four patients diagnosed as having disseminated intravascular coagulation (DIC). The four patients with DIC had an acquired disseminated bleeding disorder characterized by hypofibrinogenemia (4/4), erythrocyte fragmentation (2/4), acute thrombocytopenia (4/4), and acquired elevation of clotting times and fibrin—fibrinogen split product titers (4/4). Blood was collected with plastic syringes, anticoagulated with 3.2% trisodium citrate (9/l:V/V) and kept on ice in plastic tubes until centrifuged (1,000 Xg, 10 min, 4 C) to yield plateletpoor plasma. Blood in glass tubes was allowed to clot (10 min, room temperature), the clot was rung out with a wood stick and discarded, and the remaining blood centrifuged (1,000 X g, 10 min, 4 C) to yield serum. Plasma and serum were stored at —70 C. No difference in heparinactivated anti-thrombin activities of samples frozen at - 7 0 C and - 1 5 C and unfrozen fresh samples was found.
Fibrinogen standard was prepared by reconstituting 1 g fibronogen (Cutter Laboratories, Berkeley, California, Lot K8439) with 200 ml of 0.05 M sodium phosphate buffer, pH 7.40, and stored in 4-ml amounts at —15 C. Heparin (sodium heparin, Riker Laboratories, 10,000 U/ml, Lot 45179) was stored at 4 C. Thrombin was prepared by reconstitution of thrombin (Parke, Davis and Company, 10,000 U/vial, Lot 903461B) in physiologic saline solution to yield a concentration of 50 U/ml. This was stored in 5-ml amounts at - 7 0 C. T h e 0.05 M sodium phosphate buffer used to prepare fibrinogen was stored at —15 C. Assessment of heparin cofactor, a modification of the technic of Egeberg, 6 is described in Figure 1. To a mixture containing 0.2 ml fibrinogen solution (final concentration 2.1 mg/ml (.6 /u.mol/1)), and heparin 0.025 ml (final concentration 526 U/ml) was added 0.15 ml of either test plasma or serum serially diluted in buffer. Immediately following this, 0.1 ml of thrombin solution (final concentration 10.5 U/ml) was added, and the stopwatch was started. T h e watch was stopped when the first signs of a fibrin clot were found with a platinum wire loop. Fibrin formation was considered to have occurred when a fibrin strand was seen. Test plasma or serum was serially diluted to produce 30, 25, 20, 15, 10, and 5% final concentrations of the test material. A control for heparin activity independent of plasma or
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
optimal therapy for defibrination and thromboembolic syndromes, then heparinactivated antithrombin activity should be assessed, since this may influence the dosage and efficacy of the anticoagulant.
490
A.J.C.P. —Vol. 66
BRAUNSTEIN AND EURENIUS
no demonstrable cofactor activity, suggesting that what we are measuring is heparin cofactor, which is heat- and acid-labile, and not platelet factor IV, which is heat8 100 and acid-stable. Furthermore, when plateS let-rich plasma and platelet-poor plasma, o LU (/) or sera obtained by recalcification of plateUJ let-rich and platelet-poor plasma, were Icompared, clotting time curves were identio z cal. H c5 Figure 2 describes the exponential rela_J o tionship obtained when serially diluted normal plasma was plotted against clotting time. This relationship contrasts with the apparent linear relationship seen when normal serum was tested (Fig. 3). HCA was found to be markedly reduced in 10 15 20 samples from four patients with disTEST PLASMA, % seminated intravascular coagulation (Fig. FIG. 2. Clotting time in seconds versus 7c test 4). Note that in both Figure 4 and plasma (normal plasma). Data are presented as the means ± 1 standard deviation for ten normal Figure 5, the ordinates are linear, and only volunteers. Hatched areas represent standard devia- a portion of the normal plasma curve tion of the assay with 07c plasma. is shown. In samples from ten patients with cirrhosis (Fig. 5), plasma HCA was serum heparin-activated antithrombin, again markedly reduced. When we inconsisting of 0.15 ml buffer in the ab- creased the concentration of the plasma of sence of test plasma or serum, is shown in one cirrhotic patient to 65% of the total all studies as 0% test plasma. 200 -
The following controls were performed better to characterize heparin cofactor activity in our assay system. First, we demonstrated that imidazole-buffered saline solution could be used to replace phosphate buffer without any change in resultant data. When saline solution was substituted for either buffer, decreased heparin cofactor activity was observed. When plasma was replaced in the assay system with saline solution, clotting times were constant at all phosphate buffer concentrations. Second, the possibility of platelet factor IV activity was examined by either heating serum from recalcified platelet-rich plasma to 75 C for 1 hour or acidifying it to pH 5.5 for 1 hour. Both of these experiments yielded a serum that had
10
15
20
25
30
TEST PLASMA, SERUM, %
FIG. 3. Clotting time in seconds versus 7c normal plasma and normal serum. Mean values ± 1 standard deviation are depicted
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Results
September 1976
491
HEPARIN COFACTOR ACTIVITY
Discussion Plasma antithrombin III activity is usually determined with heat-or thrombindefibrinated plasma. 3 While such attenuation of plasma is claimed to have no influence upon antithrombin III activity, Yin has recently demonstrated that heatingplasma to 56 C destroys both the antiXa and antithrombin activity of antithrombin III. 21 T h e present heparin cofactor activity assay, a modification of Egeberg's technic, 6 does not require such attenuation and is a variant of the assay used by others to determine purified cofactor activity.1,13 One feature of this procedure is FIG. 4. Clotting lime in seconds versus 7c test plasma from four patients with disseminated intra- the concentration of heparin (526 U/ml), vascular coagulation. All values arc presented as which would allow the determination of means ± 1 standard deviation. Hatched area represents clotting with 09? plasma ± 1 standard devia- HCA in blood samples from patients receiving therapeutic heparin anticoagulation.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
assay mixture (Fig. 6), some minimal cofactor was present. While it is not clear from these studies whether the curve of this patient's cofactor activity was exponential, like that in normal plasma, or linear, like that of normal serum, we obtained a delayed but clearly exponential relationship by decreasing the thrombin concentration in the mixture by half and by increasing plasma concentration. Low heparin-activated antithrombin activity in the blood of this patient prevented sufficient inactivation of thrombin to prolong the clotting time. However, when the thrombin concentration in this assay was reduced to half standard levels (5.2 U/ml), an exponential dose-response curve was again obtained. Also, by increasing heparin concentration TEST PLASMA, % in this assay (Fig. 7), we were able to extend the clotting times in both normal FIG. 5. Clotting time in seconds versus 7c test plasma from ten patients with cirrhosis. All values and cirrhotic plasma without interfering are presented as means ± 1 standard deviation. with the basic characteristics of either nor- Hatched area represents clotting with 07c plasma ± 1 mal or cirrhotic slopes. When cirrhotic standard deviation. or DIC plasma containing minimal HCA was preincubated (20 min, 37 C) with an equal amount of normal plasma, the mixture was as active as the normal control alone, thereby excluding a plasma inhibitor of HCA.
492
A.J.C.P. —Vol. 66
BRAUNSTEIN AND EURENIUS
200
/ NORMAL f PLASMA (Thrombin = 10.5 U/ml)
/ ,X / /
C.G. (CIRRHOSIS) PLASMA (Thrombin • 5.2 U/ml)
15 20 25 30 40 50 60 70 Percent Test Plasma
tion, since this concentration greatly exceeds and minimizesin-vivo concentrations, In addition, this high level of heparin minimizes the effects of binding of heparin
by inert carriers or substrates such as fibrinogen, which have been shown to lower the potency of heparin as an anticoagulant. 7 Excess heparin does not appear to alter
200 HEPARIN 1578 U/ml 052 U/ml
100 -
NORMAL PLASMA
O u V
CO
50 1578 U/ml
I0S2 U/ml 20
526 U/ml
10
10
15
20
Percent Test Plasma
25
30
C.G. PLASMA (Cirrhosis)
FIG. 7. Clotting time in seconds versus changes in heparin concentration. Pooled normal plasma (•) and cirrhotic plasma (D) assessed for heparin cofactor activity with 526 u heparin/ml '(•), 1,052 u/ml (O), and 1.578 u/ml (X).
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
C.G. (CIRRHOSIS) PLASMA (Thrombin • 10. 5 U/ml )
FIG. 6. Cloning time in seconds versus changes in % lest plasma concentration and thrombin concentration. Clotting times were evaluated in one cirrhotic palient (•) at concentrations to as much as 66% of the final assay volume, and compared with controls (O). The clotting time curve generated when thrombin concentration was decreased from 10.5 to 5.2 U/ml is shown by the dolled lines (x).
September 1976
HEPARIN COFACTOR ACTIVITY
more quantitative manner. The combination of HCA and the factor Xa assay for heparin concentration would appear to us to be the logical approach to heparin anticoagulation. With our assay, we found both blood samples from cirrhotic and DIC patients to have significantly lower HCA than did controls, and these required significantly more heparin than control plasma to obtain the equivalent in-vit.ro thrombin time anticoagulation. Heparin cofactor deficiency may present an additional mechanism of heparin resistance. While this assay is a measure of a phenomenon and is not quantitative, it reflects a phenomenon that is significant, since heparin exerts its anticoagulant activity by interacting with heparin cofactor. Acknowledgment. Mrs. Linda S. Todd assisted in the preparation of this manuscript.
1.
2. 3. 4. 5. 6. 7. 8.
9. 10. 11.
References Abildgaard U: Highly purified antithrombin 3 with heparin cofactor activity prepared by disc electrophoresis. Scand J Clin Lab Invest 21: 89-91, 1968 Abildgaard U, Gravem K, Godal HC: Assay of progressive antithrombin in plasma. Thromb Diath Haemorrh 24:224-229, 1970 Biggs R: Human Blood Coagulation, Haemoslasis and Thrombosis. Oxford, Blackwell Scientific Publications, 1972 Damns PS, Hicks M, Rosenberg RD: Anticoagulent action of heparin. Nature 246:355-357, 1973 Duckert F: Behaviour of antithrombin 111 in liver disease. Scand J Gastroenterol (suppl 19) 8:109-112, 1973 Egeberg O: Inherited antithrombin deficiency causing thrombophilia. Thromb Diath Haemorrh 13:516-530, 1965 Estes JW, Poulin PF: Pharmacokinetics of heparin. Thromb Diath Haemorrh 33:26-37, 1975 Highsmith RK, Rosenberg RD: The inhibition of human plasmin by human antithrombin-heparin cofactor. J Biol Chem 249:4335-4338, 1974 Mannucci L, Dioguardi N, Del Ninno E, et al: Value of normotest and antithrombin III in the assessment of liver function. Scand J Gastroenterol (suppl 19) 8:103-107, 1973 Marciniak E, Farley CM, DeSimone PA: Familial thrombosis due to antithrombin III deficiency. Blood 43:219-231, 1974 Mihalyi E: Observations on thrombin inactivation by human serum. J Gen Physiol 37:139156, 1954
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
the heparin-activated antithrombin activity in plasma, since doubling or tripling this concentration (Fig. 7) alters only the baseline of the assay, and not the slope. It has been shown by others 11,13,16 that antithrombin III activity is altered by clotting. We have noticed similar changes with HCA. While plasma HCA measured by the thrombin time increased exponentially with plasma concentration, this relationship was linear with serum. When plasma with low HCA activity was studied using reduced thrombin concentrations (Fig. 6), this exponential relationship persisted. It appears either that the conversion of plasma to serum alters the cofactor molecule or that a second heparin cofactor that is either consumed or attenuated by coagulation exists. Low HCA activity in both DIC and cirrhosis could be due to an acquired heparin resistance related to the release of platelet factor IV or heparin-neutralizing activity. Since relatively greater heparin concentrations are needed for equivalent levels of anticoagulation in blood from patients with increased heparin neutralizing activity6,12 and similar results were obtained in our studies (Fig. 7), this possibility was considered. T h e presence of such inhibitor activity seems unlikely since the progressive addition of plasma or serum to the assay continues to prolong the clotting time, mixtures of deficient (cirrhotic, DIC) and normal plasmas indicate no inhibitory activity, and HCA values in samples from our cirrhotic and DIC patients were clearly lower than control serum values, where platelet factor IV and heparin-neutralizing activity are thought to be maximal. Finally, heat and acid lability observed in our assay system are characteristic of heparin cofactor and are not characteristic of platelet factor IV. T h e recent introduction of the activated factor X a assay19 has permitted the measurement of plasma heparin activity and the regulation of heparin anticoagulation in a
493
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12. O'Brien JR: Antithrombin III and heparin clotting time in thrombosis and atherosclerosis. Thromb Diath Haemorrh 32:116-123, 1974 13. Owen CA, Bollman JL: Serum and plasma antithrombin. Proc Soc Exp Biol Med 67: 367369, 1948 14. Rosenberg RD: Actions and interactions of antithrombin and heparin. N Engl J Med 292: 146-151, 1975 15. Rosenberg RD, Damus PS: The purification and mechanism of action of human thrombinheparin cofactor. J Biol Chem 248:6490-6505, 1973 16. Seegers WH, Miller KD, Andrews EB, et al: Fundamental interactions and effects of storage, ether, absorbants, and blood clotting on plasma antithrombin activity. Am J Physiol 169:700-711, 1952
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66
17. Van Der Meer J, Stoepman-Van Daleni EA, Jansen JMS: Antithrombin III deficiency in a Dutch family. J Clin Pathol 26:532-538, 1973 18. vonKaulla E, vonKaulla KN: Antithrombin 111 and diseases. Am ) Clin Pathol 48:69-80, 1967 19. Yin ET, Wessler S, Butler JU: Plasma heparin: A unique, practical submicrogram sensitivity assay. J Lab Clin Med 81:298-310, 1973 20. Yin ET, Wessler S, Stoll PJ: Biological properties of the naturally occurring plasma inhibitor to activated factor X. J Biol Chem 246:37033711, 1971 21. Yin ET, Wessler S, Stoll PJ: Identity of plasmaactivated factor X inhibitor with antithrombin III and heparin cofactor. J Biol Chem 246: 3712-3719, 1971 Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
H e p a r i n
Cofactor
D i s s e m i n a t e d
Activity
in
Intravascular a n d
C o a g u l a t i o n
Cirrhosis
KENNETH M. BRAUNSTEIN, B.A., AND KARL EURENIUS, M.D.
Department of Medicine, Division of Hematology, Medical University ofSouth Carolina, Charleston, South Ca ABSTRACT
With a modification of the technic of Egeberg, 6 we have measured heparin-activated antithrombin activity, or plasma heparin cofactor activity (HCA), in blood from four patients with disseminated intravascular coagulation (DIC) and ten patients with cirrhosis, two clinical conditions associated with coagulation disorders and low antithrombin III activity.5,9'18 A causal relationship between low antithrombin III activity and thromboembolic phenomena has been demonstrated in various kindreds. 6,10,17 Low antithrombin activity in DIC may be the result of intravascular thrombin inactivation of the antithrombin molecule.6,14 HCA has not been assessed in patients with cirrhosis, but would be exReceived September 30, 1975; received revised pected to be low since antithrombin III manuscript. November 7, 1975; accepted for publicalevels in such patients are reported detion November 7, 1975. Address reprint requests to Dr. Eurenius: Veterans creased and thought to reflect reduced synAdministration Hospital, 109 Bee Street, Charleston, thesis.5 If heparin is to be considered South Carolina 29403.
THROMBIN INACTIVATION in vitro is regu-
lated by mechanisms that have been outlined in a recent review. 3 Antithrombin II, better known as heparin cofactor, and antithrombin III are perhaps the most important of these, and in addition, they have recently been shown to have anti-factor X a , 20 ' 21 anti-factor XI a , 4 and antiplasmin 8 activity. Recent biochemical fractionation studies 2,15,21 indicate that antithrombin III and heparin cofactor reside on the same molecule. Heparin would appear to be a coenzyme for antithrombin III, markedly accelerating the rate of binding of antithrombin III to thrombin, X a , XI a , and plasmin.
488
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Braunstein, Kenneth M., and Eurenius, Karl: Minimal heparin cofactor activity in disseminated intravascular coagulation and cirrhosis. Am J Clin Pathol 66: 488-494, 1976. An assay technic for measuring heparin cofactor activity in which antithrombin activity can be assessed without plasma attenuation even in the presence of therapeutic levels of heparin is presented. Heparin-activated anti-thrombin activity was markedly depressed in plasmas of four patients with disseminated intravascular coagulation and in ten patients with cirrhosis. Residual activity in those plasmas appeared qualitatively normal, and no inhibitor (platelet factor IV activity) was observed. Plasmas from patients with disseminated intravascular coagulation and cirrhosis required more heparin to obtain in vitro clotting time prolongation equivalent to normal. (Key words: Antithrombin; Antithrombin II; Heparin; Heparin cofactor; Hemorrhagic disorders.)
September 1976 Material Fibrinogen 500 agZ Heparin 10,000 U/al Buffer and Plasma
HEPARIN COFACTOR ACTIVITY Volume In ml 0.20 0.023 0.15
489
Final Concentration* 210 mgl 526 U/ml 0 - 30Z
+ (no preincubation time) Thrombin 50 U/ml
0.10
37*C 10.5 U/ml
] clotting time
*Baaed upon final volume of 0.475 ml FIG. 1. Heparin cofactor assay procedure.
Materials and Methods Blood was collected from ten VA research employees who served as normal controls, from ten patients who had biopsyproven cirrhosis, and from four patients diagnosed as having disseminated intravascular coagulation (DIC). The four patients with DIC had an acquired disseminated bleeding disorder characterized by hypofibrinogenemia (4/4), erythrocyte fragmentation (2/4), acute thrombocytopenia (4/4), and acquired elevation of clotting times and fibrin—fibrinogen split product titers (4/4). Blood was collected with plastic syringes, anticoagulated with 3.2% trisodium citrate (9/l:V/V) and kept on ice in plastic tubes until centrifuged (1,000 Xg, 10 min, 4 C) to yield plateletpoor plasma. Blood in glass tubes was allowed to clot (10 min, room temperature), the clot was rung out with a wood stick and discarded, and the remaining blood centrifuged (1,000 X g, 10 min, 4 C) to yield serum. Plasma and serum were stored at —70 C. No difference in heparinactivated anti-thrombin activities of samples frozen at - 7 0 C and - 1 5 C and unfrozen fresh samples was found.
Fibrinogen standard was prepared by reconstituting 1 g fibronogen (Cutter Laboratories, Berkeley, California, Lot K8439) with 200 ml of 0.05 M sodium phosphate buffer, pH 7.40, and stored in 4-ml amounts at —15 C. Heparin (sodium heparin, Riker Laboratories, 10,000 U/ml, Lot 45179) was stored at 4 C. Thrombin was prepared by reconstitution of thrombin (Parke, Davis and Company, 10,000 U/vial, Lot 903461B) in physiologic saline solution to yield a concentration of 50 U/ml. This was stored in 5-ml amounts at - 7 0 C. T h e 0.05 M sodium phosphate buffer used to prepare fibrinogen was stored at —15 C. Assessment of heparin cofactor, a modification of the technic of Egeberg, 6 is described in Figure 1. To a mixture containing 0.2 ml fibrinogen solution (final concentration 2.1 mg/ml (.6 /u.mol/1)), and heparin 0.025 ml (final concentration 526 U/ml) was added 0.15 ml of either test plasma or serum serially diluted in buffer. Immediately following this, 0.1 ml of thrombin solution (final concentration 10.5 U/ml) was added, and the stopwatch was started. T h e watch was stopped when the first signs of a fibrin clot were found with a platinum wire loop. Fibrin formation was considered to have occurred when a fibrin strand was seen. Test plasma or serum was serially diluted to produce 30, 25, 20, 15, 10, and 5% final concentrations of the test material. A control for heparin activity independent of plasma or
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
optimal therapy for defibrination and thromboembolic syndromes, then heparinactivated antithrombin activity should be assessed, since this may influence the dosage and efficacy of the anticoagulant.
490
A.J.C.P. —Vol. 66
BRAUNSTEIN AND EURENIUS
no demonstrable cofactor activity, suggesting that what we are measuring is heparin cofactor, which is heat- and acid-labile, and not platelet factor IV, which is heat8 100 and acid-stable. Furthermore, when plateS let-rich plasma and platelet-poor plasma, o LU (/) or sera obtained by recalcification of plateUJ let-rich and platelet-poor plasma, were Icompared, clotting time curves were identio z cal. H c5 Figure 2 describes the exponential rela_J o tionship obtained when serially diluted normal plasma was plotted against clotting time. This relationship contrasts with the apparent linear relationship seen when normal serum was tested (Fig. 3). HCA was found to be markedly reduced in 10 15 20 samples from four patients with disTEST PLASMA, % seminated intravascular coagulation (Fig. FIG. 2. Clotting time in seconds versus 7c test 4). Note that in both Figure 4 and plasma (normal plasma). Data are presented as the means ± 1 standard deviation for ten normal Figure 5, the ordinates are linear, and only volunteers. Hatched areas represent standard devia- a portion of the normal plasma curve tion of the assay with 07c plasma. is shown. In samples from ten patients with cirrhosis (Fig. 5), plasma HCA was serum heparin-activated antithrombin, again markedly reduced. When we inconsisting of 0.15 ml buffer in the ab- creased the concentration of the plasma of sence of test plasma or serum, is shown in one cirrhotic patient to 65% of the total all studies as 0% test plasma. 200 -
The following controls were performed better to characterize heparin cofactor activity in our assay system. First, we demonstrated that imidazole-buffered saline solution could be used to replace phosphate buffer without any change in resultant data. When saline solution was substituted for either buffer, decreased heparin cofactor activity was observed. When plasma was replaced in the assay system with saline solution, clotting times were constant at all phosphate buffer concentrations. Second, the possibility of platelet factor IV activity was examined by either heating serum from recalcified platelet-rich plasma to 75 C for 1 hour or acidifying it to pH 5.5 for 1 hour. Both of these experiments yielded a serum that had
10
15
20
25
30
TEST PLASMA, SERUM, %
FIG. 3. Clotting time in seconds versus 7c normal plasma and normal serum. Mean values ± 1 standard deviation are depicted
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
Results
September 1976
491
HEPARIN COFACTOR ACTIVITY
Discussion Plasma antithrombin III activity is usually determined with heat-or thrombindefibrinated plasma. 3 While such attenuation of plasma is claimed to have no influence upon antithrombin III activity, Yin has recently demonstrated that heatingplasma to 56 C destroys both the antiXa and antithrombin activity of antithrombin III. 21 T h e present heparin cofactor activity assay, a modification of Egeberg's technic, 6 does not require such attenuation and is a variant of the assay used by others to determine purified cofactor activity.1,13 One feature of this procedure is FIG. 4. Clotting lime in seconds versus 7c test plasma from four patients with disseminated intra- the concentration of heparin (526 U/ml), vascular coagulation. All values arc presented as which would allow the determination of means ± 1 standard deviation. Hatched area represents clotting with 09? plasma ± 1 standard devia- HCA in blood samples from patients receiving therapeutic heparin anticoagulation.
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
assay mixture (Fig. 6), some minimal cofactor was present. While it is not clear from these studies whether the curve of this patient's cofactor activity was exponential, like that in normal plasma, or linear, like that of normal serum, we obtained a delayed but clearly exponential relationship by decreasing the thrombin concentration in the mixture by half and by increasing plasma concentration. Low heparin-activated antithrombin activity in the blood of this patient prevented sufficient inactivation of thrombin to prolong the clotting time. However, when the thrombin concentration in this assay was reduced to half standard levels (5.2 U/ml), an exponential dose-response curve was again obtained. Also, by increasing heparin concentration TEST PLASMA, % in this assay (Fig. 7), we were able to extend the clotting times in both normal FIG. 5. Clotting time in seconds versus 7c test plasma from ten patients with cirrhosis. All values and cirrhotic plasma without interfering are presented as means ± 1 standard deviation. with the basic characteristics of either nor- Hatched area represents clotting with 07c plasma ± 1 mal or cirrhotic slopes. When cirrhotic standard deviation. or DIC plasma containing minimal HCA was preincubated (20 min, 37 C) with an equal amount of normal plasma, the mixture was as active as the normal control alone, thereby excluding a plasma inhibitor of HCA.
492
A.J.C.P. —Vol. 66
BRAUNSTEIN AND EURENIUS
200
/ NORMAL f PLASMA (Thrombin = 10.5 U/ml)
/ ,X / /
C.G. (CIRRHOSIS) PLASMA (Thrombin • 5.2 U/ml)
15 20 25 30 40 50 60 70 Percent Test Plasma
tion, since this concentration greatly exceeds and minimizesin-vivo concentrations, In addition, this high level of heparin minimizes the effects of binding of heparin
by inert carriers or substrates such as fibrinogen, which have been shown to lower the potency of heparin as an anticoagulant. 7 Excess heparin does not appear to alter
200 HEPARIN 1578 U/ml 052 U/ml
100 -
NORMAL PLASMA
O u V
CO
50 1578 U/ml
I0S2 U/ml 20
526 U/ml
10
10
15
20
Percent Test Plasma
25
30
C.G. PLASMA (Cirrhosis)
FIG. 7. Clotting time in seconds versus changes in heparin concentration. Pooled normal plasma (•) and cirrhotic plasma (D) assessed for heparin cofactor activity with 526 u heparin/ml '(•), 1,052 u/ml (O), and 1.578 u/ml (X).
Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
C.G. (CIRRHOSIS) PLASMA (Thrombin • 10. 5 U/ml )
FIG. 6. Cloning time in seconds versus changes in % lest plasma concentration and thrombin concentration. Clotting times were evaluated in one cirrhotic palient (•) at concentrations to as much as 66% of the final assay volume, and compared with controls (O). The clotting time curve generated when thrombin concentration was decreased from 10.5 to 5.2 U/ml is shown by the dolled lines (x).
September 1976
HEPARIN COFACTOR ACTIVITY
more quantitative manner. The combination of HCA and the factor Xa assay for heparin concentration would appear to us to be the logical approach to heparin anticoagulation. With our assay, we found both blood samples from cirrhotic and DIC patients to have significantly lower HCA than did controls, and these required significantly more heparin than control plasma to obtain the equivalent in-vit.ro thrombin time anticoagulation. Heparin cofactor deficiency may present an additional mechanism of heparin resistance. While this assay is a measure of a phenomenon and is not quantitative, it reflects a phenomenon that is significant, since heparin exerts its anticoagulant activity by interacting with heparin cofactor. Acknowledgment. Mrs. Linda S. Todd assisted in the preparation of this manuscript.
1.
2. 3. 4. 5. 6. 7. 8.
9. 10. 11.
References Abildgaard U: Highly purified antithrombin 3 with heparin cofactor activity prepared by disc electrophoresis. Scand J Clin Lab Invest 21: 89-91, 1968 Abildgaard U, Gravem K, Godal HC: Assay of progressive antithrombin in plasma. Thromb Diath Haemorrh 24:224-229, 1970 Biggs R: Human Blood Coagulation, Haemoslasis and Thrombosis. Oxford, Blackwell Scientific Publications, 1972 Damns PS, Hicks M, Rosenberg RD: Anticoagulent action of heparin. Nature 246:355-357, 1973 Duckert F: Behaviour of antithrombin 111 in liver disease. Scand J Gastroenterol (suppl 19) 8:109-112, 1973 Egeberg O: Inherited antithrombin deficiency causing thrombophilia. Thromb Diath Haemorrh 13:516-530, 1965 Estes JW, Poulin PF: Pharmacokinetics of heparin. Thromb Diath Haemorrh 33:26-37, 1975 Highsmith RK, Rosenberg RD: The inhibition of human plasmin by human antithrombin-heparin cofactor. J Biol Chem 249:4335-4338, 1974 Mannucci L, Dioguardi N, Del Ninno E, et al: Value of normotest and antithrombin III in the assessment of liver function. Scand J Gastroenterol (suppl 19) 8:103-107, 1973 Marciniak E, Farley CM, DeSimone PA: Familial thrombosis due to antithrombin III deficiency. Blood 43:219-231, 1974 Mihalyi E: Observations on thrombin inactivation by human serum. J Gen Physiol 37:139156, 1954
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the heparin-activated antithrombin activity in plasma, since doubling or tripling this concentration (Fig. 7) alters only the baseline of the assay, and not the slope. It has been shown by others 11,13,16 that antithrombin III activity is altered by clotting. We have noticed similar changes with HCA. While plasma HCA measured by the thrombin time increased exponentially with plasma concentration, this relationship was linear with serum. When plasma with low HCA activity was studied using reduced thrombin concentrations (Fig. 6), this exponential relationship persisted. It appears either that the conversion of plasma to serum alters the cofactor molecule or that a second heparin cofactor that is either consumed or attenuated by coagulation exists. Low HCA activity in both DIC and cirrhosis could be due to an acquired heparin resistance related to the release of platelet factor IV or heparin-neutralizing activity. Since relatively greater heparin concentrations are needed for equivalent levels of anticoagulation in blood from patients with increased heparin neutralizing activity6,12 and similar results were obtained in our studies (Fig. 7), this possibility was considered. T h e presence of such inhibitor activity seems unlikely since the progressive addition of plasma or serum to the assay continues to prolong the clotting time, mixtures of deficient (cirrhotic, DIC) and normal plasmas indicate no inhibitory activity, and HCA values in samples from our cirrhotic and DIC patients were clearly lower than control serum values, where platelet factor IV and heparin-neutralizing activity are thought to be maximal. Finally, heat and acid lability observed in our assay system are characteristic of heparin cofactor and are not characteristic of platelet factor IV. T h e recent introduction of the activated factor X a assay19 has permitted the measurement of plasma heparin activity and the regulation of heparin anticoagulation in a
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12. O'Brien JR: Antithrombin III and heparin clotting time in thrombosis and atherosclerosis. Thromb Diath Haemorrh 32:116-123, 1974 13. Owen CA, Bollman JL: Serum and plasma antithrombin. Proc Soc Exp Biol Med 67: 367369, 1948 14. Rosenberg RD: Actions and interactions of antithrombin and heparin. N Engl J Med 292: 146-151, 1975 15. Rosenberg RD, Damus PS: The purification and mechanism of action of human thrombinheparin cofactor. J Biol Chem 248:6490-6505, 1973 16. Seegers WH, Miller KD, Andrews EB, et al: Fundamental interactions and effects of storage, ether, absorbants, and blood clotting on plasma antithrombin activity. Am J Physiol 169:700-711, 1952
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17. Van Der Meer J, Stoepman-Van Daleni EA, Jansen JMS: Antithrombin III deficiency in a Dutch family. J Clin Pathol 26:532-538, 1973 18. vonKaulla E, vonKaulla KN: Antithrombin 111 and diseases. Am ) Clin Pathol 48:69-80, 1967 19. Yin ET, Wessler S, Butler JU: Plasma heparin: A unique, practical submicrogram sensitivity assay. J Lab Clin Med 81:298-310, 1973 20. Yin ET, Wessler S, Stoll PJ: Biological properties of the naturally occurring plasma inhibitor to activated factor X. J Biol Chem 246:37033711, 1971 21. Yin ET, Wessler S, Stoll PJ: Identity of plasmaactivated factor X inhibitor with antithrombin III and heparin cofactor. J Biol Chem 246: 3712-3719, 1971 Downloaded from http://ajcp.oxfordjournals.org/ by guest on June 6, 2016
