- Email: [email protected]
Heparin-accelerated inhibition of activated factor X by its natural plasma inhibitor
387
SHORT COMMUNICATIONS
BBA 23559
Heparin-accelerated inhibition of activated Factor X by its natural plasma inhibitor The fluidity of the circulating blood is controlled, in part, b y the plasminogenplasmin system and by plasma antithrombins. It has been proposed that blood fluidity is also preserved through a clearance mechanism whereby coagulation intermediates are rapidly removed from the circulation by certain cells 1. Activated Factor X occupies a key position in both the intrinsic and extrinsic coagulation pathways, and like thrombin is also absent from serum. The potentially important role of a naturally occurring plasma inhibitor against activated Factor X in maintaining blood fluidity, has received very little attention since the observation by SEEGERS AND MARCINIAK2 of the neutralization of activated Factor X (autoprothrombin C) by plasma antithrombin I I I . We have recently described a method for the isolation and purification of a naturally occurring plasma inhibitor to activated blood clotting Factor X (ref. 3)Further studies on the physicochemical properties of this inhibitor, on its identity with antithrombin I I I and heparin co-factor are all in progress. This report was occasioned by the unexpected finding of a marked increase in the activated Factor X inhibitor activity in the plasma of a patient receiving heparin therapy for recurrent venous thrombosis. Moreover, at the time the activated Factor X inhibitor level was measured, the patient's .whole blood clotting time was reported to be within the normal range. This capacity of the patient's diluted plasma to inactivate an added amount of activated Factor X is compared to a normal plasma control in Fig. I. Further evidence in support of this observation is herewith presented. ]
i
,
i
,
,
,
,
,
3
3
6
9
12
15
Ig
21
t~
IncubatiOn time (mtn)
Fig. I. N e u t r a l i z a t i o n of a c t i v a t e d F a c t o r X b y n o r m a l a n d h e p a r i n i z e d (patient) p l a s m a . T h e r e a c t i o n m i x t u r e c o n s i s t e d of i n c u b a t i n g o.9 m l p l a s m a ( I : I O dilution) w i t h o.I m l a c t i v a t e d F a c t o r X g i v i n g a final a ~ t i v i t y of 5 u n i t s ] m l i n c u b a t i o n m i x t u r e . A t t h e i n d i c a t e d t i m e i n t e r v a l s , o . I - m l aliquots of t h e r e a c t i o n m i x t u r e were r e m o v e d a n d t e s t e d for residual a c t i v a t e d F a c t o r X a c t i v i t y . A io % solution of b o v i n e s e r u m a l b u m i n replaced p l a s m a in t h e control e x p e r i m e n t ( & ). O , n o r m a l p l a s m a , 1 : IO dilution; O , p a t i e n t p l a s m a , i : IO dilution.
Biochim. Biophys. Acta, 2Ol (197o) 387-39o
388
SHORT COMMUNICATIONS
Activated Factor X was prepared in our laboratory 4 and stabilized in Io % crystalline bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) 3. The activated Factor X activity for the incubation mixtures was utilized as previously described 3, except that the reagents were added in the following order in rapid succession: test fraction, CaC12, cephalin and Seitz plasma. Activated Factor X inhibitor was purified from plasma in our laboratory 3. Electrophoresis of 20o #g of this preparation on 7 % acrylamide gel at p H 9.5 (Ornstein) yielded only a single protein band. Sodium heparin-Liquaemin Sodium " i o " (IOOO U.S.P. units/ml) was obtained from Organon, West Orange, N.J. The concentrations of heparin used in these experiments were expressed in U.S.P. units, the heparin being appropriately diluted with o.85 %
iO o
• . . . . . . . . .
•
.
.
.
.
.
.
• .
.
. . . . . .
0 .
.
.
.
.
.
.
~) . . . . . . . . . .
•
D
_e
O-
'
I I
'
I 2
i
I 3
i
I
i
I 5
4
Incubatton
'
I
I
I
I
t
6
7
8
9
I0
ttme (mtt~)
Fig. 2. Neutralization of activated F a c t o r X b y n o r m a l p l a s m a in the presence and absence of heparin. The reaction m i x t u r e consisted of i n c u b a t i n g 0.8 ml plasma, o.i nil heparin or 0.85 % NaCI and o.I ml activated F a c t o r X (io units). At the indicated time intervals, o . I - m l aliquots were r e m o v e d from the incubation m i x t u r e and tested for residual activated F a c t o r X activity. Bovine s e r u m a l b u m i n replaced the p l a s m a in the control e x p e r i m e n t w i t h o.I u n i t heparin (Q). O , plasma, undiluted, + NaC1; O , plasma, i : io dilution, + NaC1; × , p l a s m a i : io dilution, + o.ooi u n i t h e p a r i n ; Z~, plasma, i : IO dilution, + o.oi u n i t h e p a r i n ; ~k, plasma, I : io dilution, + o.I u n i t heparin.
NaCI solution. In all experiments described any heparin carried over from the primary incubation mixture to the clotting assay system was found to be too small to influence the final clotting tests. All coagulation assays and reaction experiments were performed at 37°. The effect of heparin on the capacity of normal human plasma to neutralize added activated Factor X is shown in Fig. 2. In the absence of heparin a I : IO dilution of normal plasma inhibited IO units of added activated Factor X in Io rain. Upon addition of only o.oi unit of heparin to the same reaction mixture, inactivation of activated Factor X activity occurred in 2 m i n - a result comparable to that obtained without heparin when the plasma was used undiluted. When o.I unit of heparin was Biochim. Biophys. Acta, 2oi (197 o) 387-390
389
SHORT COMMUNICATIONS
added to the reaction mixture in the presence of a I : IO dilution of plasma, the inactivation of an added IO units of activated Factor X was complete in less than 3° sec. Heparin alone (o.I unit) had no discernible effect on activated Factor X activity (Fig. 2). To establish that heparin was indeed functioning together with the activated Factor X inhibitor rather than with another plasma component to cause the rapid inhibition of activated Factor X, an additional series of experiments were performed in which purified inhibitor replaced normal plasma. A low concentration of inhibitor and a high concentration of activated Factor X were selected to facilitate accurate recognition of slight changes in reaction rates (Fig. 3)- When IO #g of inhibitor I
10
8
6-
|
.
.
.
.
.
]
•
-O--
I
.
.
I
.
.
.
.
.
.
I
.
.
.
.
0•O
4"
P, 2-
OI
0
'
'
'
'1
I0
I
I
I
I
I
20
30
40
50
60
Incubation t/me (rain) Fig. 3. N e u t r a l i z a t i o n of a c t i v a t e d F a c t o r X b y purified i n h i b i t o r in t h e presence a n d absence of h e p a r i n . T h e r e a c t i o n m i x t u r e c o n s i s t e d of i n c u b a t i n g o.i rrd i n h i b i t o r (IO #g), o. 7 m l 1 % b o v i n e s e r u m a l b u m i n in o.oi M T r i s - m a l e a t e (pH 7-5), o.I m l h e p a r i n or 0.8 5 % NaC1 a n d o.i m l a c t i v a t e d F a c t o r X (io units), A t t h e i n d i c a t e d t i m e i n t e r v a l s , o . i - m l aliquots were r e m o v e d f r o m t h e i n c u b a t i o n m i x t u r e a n d t e s t e d for residual a c t i v a t e d F a c t o r X a c t i v i t y , o.8 5 ~o NaC1 replaced t h e i n h i b i t o r in t h e control e x p e r i m e n t w i t h 4- o.2 5 u n i t h e p a r i n (Q). O , IO /zg i n h i b i t o r + NaC1; O , i o / , g i n h i b i t o r + o.oi u n i t h e p a r i n ; × , IO/zg i n h i b i t o r + o.o 5 u n i t h e p a r i n ; A , IO/zg i n h i b i t o r + o.i u n i t h e p a r i n .
were incubated with IO units of activated Factor X, more than 95% of the activated Factor X was neutralized in 60 min. In striking contrast, when o.I unit of heparin was added to the same reaction mixture, the IO units of activated Factor X were neutralized in less than 30 sec. Finally, incubation of 0.25 unit of heparin alone with io units of activated Factor X resulted in no effect on the activated Factor X activity. SEEGERS AND MARCINIAK2 were the first to show in vitro that antithrombin I I I neutralized autoprothrombin C (activated Factor X) as well as thrombin. In the present study we have obtained, for the first time, in vitro data showing that, while heparin alone cannot inhibit activated Factor X activity, heparin does markedly Biochim. Biophys. Acta, 2oT (i97 o) .387-390
39 °
SHORT COMMUNICATIONS
p o t e n t i a t e the i n h i b i t o r y a c t i v i t y of the n a t u r a l p l a s m a i n h i b i t o r of a c t i v a t e d F a c t o r X. This t y p e of c a t a l y s i s is reminiscent of the effect of p h o s p h o l i p i d in p r o t h r o m b i n conversion at t h e level of p r o t h r o m b i n a c t i v a t o r formationS, 6. The d a t a p r e s e n t e d in Figs. 2 a n d 3 suggest t h a t the p r i m a r y role of heparin in r e t a r d i n g i n t r a v a s c u l a r coagulation m a y be in p o t e n t i a t i n g the n e u t r a l i z a t i o n of a c t i v a t e d F a c t o r X b y its n a t u r a l p l a s m a inhibitor. This thesis is s t r e n g t h e n e d b y the knowledge t h a t a c t i v a t e d F a c t o r X s t a n d s at the confluence of the intrinsic a n d extrinsic clotting p a t h w a y s , t h a t in the absence of a c t i v a t e d F a c t o r X no t h r o m b i n can be formed, a n d t h a t the i n t r a v e n o u s injection of 5 units of a c t i v a t e d F a c t o r X is more explosive in g e n e r a t i n g endogenous t h r o m b i n f o r m a t i o n in vivo t h a n is the injection of IOO units of purified t h r o m b i n itself 7. This work was s u p p o r t e d in p a r t b y a g r a n t from the Life Insurance Medical Research F u n d , b y U.S. Public H e a l t h Service G r a n t H E 1147o , a n d b y the Sig a n d Clara W o l f o r t Research F u n d . T h a n k s are due to Mrs. Virginia Kerschen a n d Mrs. S a n d y Bilyeu for technical assistance.
Department of Medicine, Washington University School of Medicine ; and The Jewish Hospital of St. Louis, St. Louis, Mo. 6311o (U.S.A.) i 2 3 4 5 6 7
E. THYE YIN STANFORD WESSLER
T. H. SPAET AND M. KROPATKIN,Proc. Soc. Exptl. Biol. ll/Ied., 95 (1957) 492SV. H. SEEGERS AND E. MARCINIAK, Nature, 193 (1962) 1188. E. T. YIN AND S. WESSLER, Thromb. Diath. Haemorrhag., 21 (1969) 398. E. T. YIN AND S. WESSLER, J. Biol. Chem., 243 (1968) 112. J- H. MILSTONE, Federation Proc., 23 (1964) 742. P. G. BARTON, C. IV[.JACKSONAND D. J. HANAHAN,Nature, 214 (1967) 923. E. T. YIN AND S. WESSLER, Thromb. Diath. Haemorrhag., 2o (1968) 465 .
R e c e i v e d S e p t e m b e r 25th, 1969
Biochim. Biophys. Acta, 2Ol (i97 o) 387-39 °
SHORT COMMUNICATIONS
BBA 23559
Heparin-accelerated inhibition of activated Factor X by its natural plasma inhibitor The fluidity of the circulating blood is controlled, in part, b y the plasminogenplasmin system and by plasma antithrombins. It has been proposed that blood fluidity is also preserved through a clearance mechanism whereby coagulation intermediates are rapidly removed from the circulation by certain cells 1. Activated Factor X occupies a key position in both the intrinsic and extrinsic coagulation pathways, and like thrombin is also absent from serum. The potentially important role of a naturally occurring plasma inhibitor against activated Factor X in maintaining blood fluidity, has received very little attention since the observation by SEEGERS AND MARCINIAK2 of the neutralization of activated Factor X (autoprothrombin C) by plasma antithrombin I I I . We have recently described a method for the isolation and purification of a naturally occurring plasma inhibitor to activated blood clotting Factor X (ref. 3)Further studies on the physicochemical properties of this inhibitor, on its identity with antithrombin I I I and heparin co-factor are all in progress. This report was occasioned by the unexpected finding of a marked increase in the activated Factor X inhibitor activity in the plasma of a patient receiving heparin therapy for recurrent venous thrombosis. Moreover, at the time the activated Factor X inhibitor level was measured, the patient's .whole blood clotting time was reported to be within the normal range. This capacity of the patient's diluted plasma to inactivate an added amount of activated Factor X is compared to a normal plasma control in Fig. I. Further evidence in support of this observation is herewith presented. ]
i
,
i
,
,
,
,
,
3
3
6
9
12
15
Ig
21
t~
IncubatiOn time (mtn)
Fig. I. N e u t r a l i z a t i o n of a c t i v a t e d F a c t o r X b y n o r m a l a n d h e p a r i n i z e d (patient) p l a s m a . T h e r e a c t i o n m i x t u r e c o n s i s t e d of i n c u b a t i n g o.9 m l p l a s m a ( I : I O dilution) w i t h o.I m l a c t i v a t e d F a c t o r X g i v i n g a final a ~ t i v i t y of 5 u n i t s ] m l i n c u b a t i o n m i x t u r e . A t t h e i n d i c a t e d t i m e i n t e r v a l s , o . I - m l aliquots of t h e r e a c t i o n m i x t u r e were r e m o v e d a n d t e s t e d for residual a c t i v a t e d F a c t o r X a c t i v i t y . A io % solution of b o v i n e s e r u m a l b u m i n replaced p l a s m a in t h e control e x p e r i m e n t ( & ). O , n o r m a l p l a s m a , 1 : IO dilution; O , p a t i e n t p l a s m a , i : IO dilution.
Biochim. Biophys. Acta, 2Ol (197o) 387-39o
388
SHORT COMMUNICATIONS
Activated Factor X was prepared in our laboratory 4 and stabilized in Io % crystalline bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) 3. The activated Factor X activity for the incubation mixtures was utilized as previously described 3, except that the reagents were added in the following order in rapid succession: test fraction, CaC12, cephalin and Seitz plasma. Activated Factor X inhibitor was purified from plasma in our laboratory 3. Electrophoresis of 20o #g of this preparation on 7 % acrylamide gel at p H 9.5 (Ornstein) yielded only a single protein band. Sodium heparin-Liquaemin Sodium " i o " (IOOO U.S.P. units/ml) was obtained from Organon, West Orange, N.J. The concentrations of heparin used in these experiments were expressed in U.S.P. units, the heparin being appropriately diluted with o.85 %
iO o
• . . . . . . . . .
•
.
.
.
.
.
.
• .
.
. . . . . .
0 .
.
.
.
.
.
.
~) . . . . . . . . . .
•
D
_e
O-
'
I I
'
I 2
i
I 3
i
I
i
I 5
4
Incubatton
'
I
I
I
I
t
6
7
8
9
I0
ttme (mtt~)
Fig. 2. Neutralization of activated F a c t o r X b y n o r m a l p l a s m a in the presence and absence of heparin. The reaction m i x t u r e consisted of i n c u b a t i n g 0.8 ml plasma, o.i nil heparin or 0.85 % NaCI and o.I ml activated F a c t o r X (io units). At the indicated time intervals, o . I - m l aliquots were r e m o v e d from the incubation m i x t u r e and tested for residual activated F a c t o r X activity. Bovine s e r u m a l b u m i n replaced the p l a s m a in the control e x p e r i m e n t w i t h o.I u n i t heparin (Q). O , plasma, undiluted, + NaC1; O , plasma, i : io dilution, + NaC1; × , p l a s m a i : io dilution, + o.ooi u n i t h e p a r i n ; Z~, plasma, i : IO dilution, + o.oi u n i t h e p a r i n ; ~k, plasma, I : io dilution, + o.I u n i t heparin.
NaCI solution. In all experiments described any heparin carried over from the primary incubation mixture to the clotting assay system was found to be too small to influence the final clotting tests. All coagulation assays and reaction experiments were performed at 37°. The effect of heparin on the capacity of normal human plasma to neutralize added activated Factor X is shown in Fig. 2. In the absence of heparin a I : IO dilution of normal plasma inhibited IO units of added activated Factor X in Io rain. Upon addition of only o.oi unit of heparin to the same reaction mixture, inactivation of activated Factor X activity occurred in 2 m i n - a result comparable to that obtained without heparin when the plasma was used undiluted. When o.I unit of heparin was Biochim. Biophys. Acta, 2oi (197 o) 387-390
389
SHORT COMMUNICATIONS
added to the reaction mixture in the presence of a I : IO dilution of plasma, the inactivation of an added IO units of activated Factor X was complete in less than 3° sec. Heparin alone (o.I unit) had no discernible effect on activated Factor X activity (Fig. 2). To establish that heparin was indeed functioning together with the activated Factor X inhibitor rather than with another plasma component to cause the rapid inhibition of activated Factor X, an additional series of experiments were performed in which purified inhibitor replaced normal plasma. A low concentration of inhibitor and a high concentration of activated Factor X were selected to facilitate accurate recognition of slight changes in reaction rates (Fig. 3)- When IO #g of inhibitor I
10
8
6-
|
.
.
.
.
.
]
•
-O--
I
.
.
I
.
.
.
.
.
.
I
.
.
.
.
0•O
4"
P, 2-
OI
0
'
'
'
'1
I0
I
I
I
I
I
20
30
40
50
60
Incubation t/me (rain) Fig. 3. N e u t r a l i z a t i o n of a c t i v a t e d F a c t o r X b y purified i n h i b i t o r in t h e presence a n d absence of h e p a r i n . T h e r e a c t i o n m i x t u r e c o n s i s t e d of i n c u b a t i n g o.i rrd i n h i b i t o r (IO #g), o. 7 m l 1 % b o v i n e s e r u m a l b u m i n in o.oi M T r i s - m a l e a t e (pH 7-5), o.I m l h e p a r i n or 0.8 5 % NaC1 a n d o.i m l a c t i v a t e d F a c t o r X (io units), A t t h e i n d i c a t e d t i m e i n t e r v a l s , o . i - m l aliquots were r e m o v e d f r o m t h e i n c u b a t i o n m i x t u r e a n d t e s t e d for residual a c t i v a t e d F a c t o r X a c t i v i t y , o.8 5 ~o NaC1 replaced t h e i n h i b i t o r in t h e control e x p e r i m e n t w i t h 4- o.2 5 u n i t h e p a r i n (Q). O , IO /zg i n h i b i t o r + NaC1; O , i o / , g i n h i b i t o r + o.oi u n i t h e p a r i n ; × , IO/zg i n h i b i t o r + o.o 5 u n i t h e p a r i n ; A , IO/zg i n h i b i t o r + o.i u n i t h e p a r i n .
were incubated with IO units of activated Factor X, more than 95% of the activated Factor X was neutralized in 60 min. In striking contrast, when o.I unit of heparin was added to the same reaction mixture, the IO units of activated Factor X were neutralized in less than 30 sec. Finally, incubation of 0.25 unit of heparin alone with io units of activated Factor X resulted in no effect on the activated Factor X activity. SEEGERS AND MARCINIAK2 were the first to show in vitro that antithrombin I I I neutralized autoprothrombin C (activated Factor X) as well as thrombin. In the present study we have obtained, for the first time, in vitro data showing that, while heparin alone cannot inhibit activated Factor X activity, heparin does markedly Biochim. Biophys. Acta, 2oT (i97 o) .387-390
39 °
SHORT COMMUNICATIONS
p o t e n t i a t e the i n h i b i t o r y a c t i v i t y of the n a t u r a l p l a s m a i n h i b i t o r of a c t i v a t e d F a c t o r X. This t y p e of c a t a l y s i s is reminiscent of the effect of p h o s p h o l i p i d in p r o t h r o m b i n conversion at t h e level of p r o t h r o m b i n a c t i v a t o r formationS, 6. The d a t a p r e s e n t e d in Figs. 2 a n d 3 suggest t h a t the p r i m a r y role of heparin in r e t a r d i n g i n t r a v a s c u l a r coagulation m a y be in p o t e n t i a t i n g the n e u t r a l i z a t i o n of a c t i v a t e d F a c t o r X b y its n a t u r a l p l a s m a inhibitor. This thesis is s t r e n g t h e n e d b y the knowledge t h a t a c t i v a t e d F a c t o r X s t a n d s at the confluence of the intrinsic a n d extrinsic clotting p a t h w a y s , t h a t in the absence of a c t i v a t e d F a c t o r X no t h r o m b i n can be formed, a n d t h a t the i n t r a v e n o u s injection of 5 units of a c t i v a t e d F a c t o r X is more explosive in g e n e r a t i n g endogenous t h r o m b i n f o r m a t i o n in vivo t h a n is the injection of IOO units of purified t h r o m b i n itself 7. This work was s u p p o r t e d in p a r t b y a g r a n t from the Life Insurance Medical Research F u n d , b y U.S. Public H e a l t h Service G r a n t H E 1147o , a n d b y the Sig a n d Clara W o l f o r t Research F u n d . T h a n k s are due to Mrs. Virginia Kerschen a n d Mrs. S a n d y Bilyeu for technical assistance.
Department of Medicine, Washington University School of Medicine ; and The Jewish Hospital of St. Louis, St. Louis, Mo. 6311o (U.S.A.) i 2 3 4 5 6 7
E. THYE YIN STANFORD WESSLER
T. H. SPAET AND M. KROPATKIN,Proc. Soc. Exptl. Biol. ll/Ied., 95 (1957) 492SV. H. SEEGERS AND E. MARCINIAK, Nature, 193 (1962) 1188. E. T. YIN AND S. WESSLER, Thromb. Diath. Haemorrhag., 21 (1969) 398. E. T. YIN AND S. WESSLER, J. Biol. Chem., 243 (1968) 112. J- H. MILSTONE, Federation Proc., 23 (1964) 742. P. G. BARTON, C. IV[.JACKSONAND D. J. HANAHAN,Nature, 214 (1967) 923. E. T. YIN AND S. WESSLER, Thromb. Diath. Haemorrhag., 2o (1968) 465 .
R e c e i v e d S e p t e m b e r 25th, 1969
Biochim. Biophys. Acta, 2Ol (i97 o) 387-39 °