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Influence of heparin;of different heparin fractions and of a low molecular weight heparin-like substance on the mechanism of fibrinolysis
THROMBOSIS RESEARCH 27; 341-352, 1982 0049-3838/82/150341-12$03.00/O Printed in the USA. Copyright (c) 1982 Pergamon Press Ltd. All rights reserved.
INFLUENCE OF HEPARIN;OF OIFFERENT HEPARIN FRACTIONS AND OF A LOW MOLECULAR WEIGHT HEPARIN-LIKE SUBSTANCE ON THE MECHANISM OF FIBRINOLYSIS
H.Vinazzer, A.Stemberger,
S.Haas and G.BlUmel
Blood Coagulation Laboratory,Linz,Austria Surgery, Technical University, Munich,FRG
and Institute for Experimental
(Received 23.4.1982; in revised form 9.5.1982. Accepted by Editor H. Stormorken)
ABSTRACT The influence of different heparin fractions and of a synthetic polysulfated polysaccharide (SP54) on the fibrinolytic mechanism was examined. In vitro,a significant shortening of the euglobulin lysis time (ELT) was found after addition of standard mucosa heparin, of high MW heparin and of SP 54 respectively. Low MW heparin fractions had no influence on the ELT. Simultaneously with the shortening of the ELT an activation of factor XII and of kallikrein was observed. A similar effect was found in groups of volunteers after i.v. or S.C. injections and even after oral administration of SP 54. The effect of venous occlusion on the ELT and on the activation of factor XII was considerably increased when heparin or SP 54 was njected '2 h prior to the test. When a comparable concentration of the test substances was added to plasma samples before and af t er venous occlusion,the effect on the ELT was much less pronounced than after injection whilst the effect on factor XII was comparable in both tests. From these results the conclusion was drawn that activation of fibrinolysis by polysulfated polysaccharides is achieved by an endogenous pathway as well as by an increased availability of the vascular activator. The magnitude of the activation of fibrinolysis partly depends on the MW of the substance but apparently also on the degree of sulfatation since a low MW substance with a high number of sulfate bonds such as SP 54 was considerably more active than the low MW fraction of standard heparin.
Key words: Heparin, Heparin fractions,
Polyanion
341
SP 54, Fibrinolysis
342
HEPARIN A&D FIBRINOLYSIS
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INTRODUCTION In contrast to the well known influence of heparin and of heparin analogues on coagulation a possible activation of the fibrinolytic mechanism by heparin was less thoroughly examined. Though an overall activation of fibrinolysis by heparin was demonstrated long ago (1,2,3,4), a detailed analysis of this mechanism was not carried out and interest was apparently lost when highly effective activators of fibrinolysis such as streptokinase and urokinase were introduced. For this reason the effect of heparin on fibrinolysis was reexamined. Additional tests were carried out with high molecular weight (HMW) and low molecular weight (LMW) heparin fractions and with a synthetic LMW polysulfated polysaccharide (Polyanion SP 54). MATERIALS
AND METHODS
Materials Heparin was pig mucosa sodium heparin with an average MW of 11,000 D and an activity of 200 I.U./mg from AB KABI, Stockholm,Sweden. LMW and HMW heparin fractions were prepared from heparin of the same source by KABI laboratories, Stockholm. A LMW fraction with a MW of 3,300 D and a HMW fraction with a MW of 18,300 D were obtained. The synthetic polysulfated polysaccharide was Polyanion SP 54 from Bene-Chemie, Munich, FRG. This substance was obtained in 1 ml ampouls containing 100 mg SP 54 and in capsules containing 150 mg and 250 mg respectively. The MW of SP 54 was 3,000 D. Reagents for assays of plasminogen and of antiplasmins were contained in the Coatest Antiplasmin kit from AB KABI Diagnostica, Stockholm. The reagent for kallikrein was from the same source and was the chromogenic substrate S-2302. Human prekallikrein was a gift from Cutter Laboratories, Berkeley, Calif. batch no. 1704-42-l-1937-4. The substance had the following characteristics: It was free from kallikrein,plasmin,plasminogen,thrombin and factor Xa. 1 mg of the lyophilized powder corresponded to the amount of prekallikrein contained in 170,ul normal human plasma. For tests,a solution of 6 mg of the substance per ml distilled water was made. Thrombin was from AB KABI Diagnostica,Stockholm. One vial contained 15 NIH units highly purified lyophilized human thrombin. Photometer: Model S-100 by Labtronic, Zurich,Switzerland. Clot Lysis Time Recorder: by Medicon,Glasgow,Scotland. This instrument was connected to a Rikadenki linear recorder which allowed paper speeds between 1 cm/h and 60 cm/min. Methods Euglobulin lysis time: Milstone (5); Plasminogen: Soria et al. (6); Alpha-2antiplasmin and total antiplasmin: Teger-Nilsson et al. (7). Factor XIIf was assayed by its ability to convert prekallikrein into kallikrein. 20,ul plasma was pipetted into a semimicro cuvette at 37OC. 20~1 of the test substance or of Tris-HCl buffer pH 7.4 was added which was immediately followed by 160,ul of the same buffer and by lOO,ul prekallikrein. After an incubation time of 60 set the chromogenic reaction was started by addition of 200,ul S-2302 ( 4 mmol/l). The difference in optical density per min was corresponding to the amount of active kallikrein formed. The blank containing buffer instead of the test substance was subtracted from the result. Since prekallikrein was in abundance and inhibitors were strongly diluted, XIIf was the limiting factor for kallikrein formation. Kallikrein in plasma was assayed in a similar way but for these tests the addition of prekallikrein was omitted. Both methods were described elsewhere in this journal (8).
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Fibrin assays: In some test series the diminution of a fibrin clot during incubation was quantitated. For this purpose 200,ul plasma before and after injections of the test substances was pipetted into 9 glass tubes and was diluted with 800,ul isotonic saline solution pH 7.35. After addition of 200,ul 20 mmol/l calcium chloride solution in which 10 NIH thrombin per ml was dissolved,the tubes were left at 37OC. After various intervals of time between 30 min and 12 h the fibrin clots which had formed where wound on glass rods and were repeatedly washed and squeezed between the glass rod and the wall of the tube in order to remove traces of soluble plasma proteins. The tyrosine equivalent of the clots was determined by the method of Ratnoff and Menzie (3). Plasma samples For in vitro tests citrate blood ( 1 part 0.15 mol/l trisodium citrate and 9 parts of venous blood) was collected from 10 healthy individuals. Blood was centrifuged for 20 min at 2,000 x g and the platelet poor citrate plasma was used. For tests on volunteers venous blood was obtained from different groups of healthy male students between 21 and 28 years of age. These blood samples were collected before and at different intervals after administration of the test substances. Comparison
of the test substances
The different fractions were compared by their concentrations based on the weight of the dry substances. This was felt to be the best way for the following reasons: Comparison on a molar base was impossible since standard heparin is not a homogenous substance but consists of subunits of different MW. Comparison by activities in I.U. was also impracticable because the LMW heparin fraction and the synthetic substance Polyanion SP 54 had a considerably lower thrombin inhibiting activitythan standard heparin or HMW heparin. Statistical
analysis
This was carried out by Student's
t-tests for paired groups.
RESULTS Heparin fractions could only be tested in vitro since the available quantities were not sufficient for in vivo trials. The influence of different concentrations of LMW and HMW heparin in comparison to standard heparin on the ELT was tested on 10 plasma samples from healthy individuals (fig. 1). Whilst there was no significant influence of LMW heparin on the ELT,a dose dependent shortening of the ELT was observed when euglobulin fractions were prepared from plasma containing standard heparin and HMW heparin respectively. There was no alteration of the plasminogen levels in the plasma samples containing LMW heparin. The highest concentrations of standard heparin and HMW heparin used in these tests resulted in a maximum diminution of plasminogen of 6%. This difference from plasma without heparin just reached the level of significance ( p 0.04). Alpha-2-antiplasmin remained at the same level in all plasma samples and the activity of total antiplasmin showed a maximum diminution of 5% which was not significant. A possible endogenous activation was examined by tests for the presence of fractioned factor XII (XII f) and of kallikrein (KK) (fig. 2). The highest concentration of LMW heparin in the plasma samples resulted in a trace of XII f and inasomewhathigher activity when standard heparin was used. HMW heparin produced a distinct and dose dependent activity of XII f in plasma. Tests for active kallikrein gave rather similar results and also showed a distinct and dose dependent activity in the HMW heparin samples.
HEPARIN AND
344
FIBRXNOLYSlS
ELT,min 2507
0
2
5
10
FIG.
20yg/ml
1
Influence of Standard Heparin and of LMW and HMW Heparin Euglobulin Lysis Time in vitro n = 10, M and 1 SD
Significances:
+ p below 0.05
Fractions
on the
; +’ p below 0.01
K K ,‘I. 1
30-
HMW
;.’
,
,:’
, HMW
1
20 -
IOHEP
HEP LMW
O,. 02
0
I
5
10
Oii
20pgtml
FIG.
io
Zbrglml
2
Influence of Standard Heparin, LMW and HMW Heparin Fractions ion of Factor XII and of Kallikrein
on the Activat-
n = 10, M and 1 SD
These in vitro experiments showed a dose dependent shortening of the ELT by heparin for which the HMW fraction of heparin is mainly responsible.Anactivation of factor XII and of kallikrein points to a possible endogenous path-
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345
way of the fibrinolytic mechanism. A possible influence on the exogenous pathway of fibrinolysis was examined by the following experiment. Venous blood was collected from 10 healthy male volunteers and citrate plasma was prepared. After blood collection the venous blood flow of the opposite arm was occluded by a blood pressure cuff inflated to a pressure of 90 mm Hg. After an occlusion time of 15 min,blood was collected from the occluded arm. Standard heparin in a dose of 50 mg was then injected subcutaneously. Two hours later,blood before and after venous occlusion was again collected. Heparin in a dose of Syg/ml was also added to fractions of the blood samples obtained prior to the injection of heparin. ELT's and factor XIIf levels were measured in all samples. The results are shown in fig. 3.
Xllf,% 2,
ELT,min 300
T
I
jooi h
1
T
T
100
0’
r
B
I
OtB
0 L/J(
I
B
0
FIG.
OtB
0
3
ELT and Factor XIIf Before and After Venous Occlusion. Influence of Heparin after S.C. Injection and after Addition in vitro. n = 10, M and 1 SD B= basic, 0= occlusion, arrows: 50 mg heparin S.C. 2 h prior to occlusion. Squares: heparin added in vitro to plasma samples before and after occlusion
The test showed the well known shortening of the ELT after venous occlusion which is a consequence of the release of a vascular activator of plasminogen. After subcutaneous heparin,the basic ELI was significantly shortened and further shortening was found after venous occlusion. A similar concentration of heparin added in vitro to plasma samples before and after venous occlusion had a considerably weaker effect on the ELT. There was no detectable factor XIIf in the basic blood samples but a trace could be found after occlusion. After heparin,some XIIf was found in the basic blood sample and a further increase was seen after occlusion. The samples which had heparin added in vitro showed a rather similar pattern.
346
HEPARIN
AtiD
FIBRINOLYSIS
Vol.2i,
so.3
A series of tests was also carried out with Polyanion SP 54. This substance was of special interest since it had a MW comparable to the LMW fraction of heparin. The influence of SP 54 on the ELT in vitro and on the activation of factor XII is shown in fig. 4.
ELT,min zoo-
Xllf,% 14 -
12 IO -
ISO-
06h2-
100^
0-’
025
10
2Opg /ml
02
5
10
20pglml
FIG. 4 Influence of SP 54 on the ELT and on the Activation n = 10, M and 1 SD, Significances:
of Factor
XII in vitro
+ p below 0.05, +' p below 0.01
In spite of considerable scattering of the ELT's of the plasma samples of these test persons the shortening of the ELT by SP 54 was comparable with the results obtained with standard heparin. The activation of factor XII was even as strong as the results obtained with HMW heparin. There was no significant influence on plasminogen nor on the antiplasmin activities in this series. Further test series were carried out in vivo on healthy volunteers. Single i.v. doses of 5, 10 and 50 mg respectively were given to groups of 5 individuals and blood samples were collected prior to the injections and 10, 30, 60 and 120 min thereafter. Other groups of 5 volunteers received subcutaneous doses of 50 and 100 mg SP 54 respectively. In a third series of groups SP 54 was given as a single oral dose of 150, 250 and 500 mg respectively. In these groups blood samples were collected before and between 30 min and 6 h after the administration of SP 54. Furthermore, in a group of 8 volunteers the experiment with venous occlusion described above was also made with a subcutaneous dose of 50 mg. The influence on the ELT of different intravenous,subcutaneous and oral doses is shown in fig. 5. For better comparability the ELT's which were slightly different in different groups were converted into percentages. The average ELT prior to SP 54 is therefore,given as a 100% level. Placebo groups were also included into these series. After intravenous injections a maximum decrease of the ELT was found in the samples obtained at 45 min. The effect was diminishing after 2 h and there was a distinct dose dependence. A similar effect was found after subcutaneous injections. In these series,shortening of the ELT was evident after 2 h and
347
HEPARIN AND FIBRINOLYSIS
Vo1.27, No.3
was still detectable after 6 h. Considerably higher oral doses were required to obtain a significant effect on the ELT. After 500 mg however,the shortening of the ELT was comparable with the effect. of the 50 mg subcutaneous dose.
i.v. mg
EL1.V.
mg
I
0
t im
I
1
1
012
2h
I
oral mg
S.C.
at
e
L te
r
6h
0
2
4
6h
application
FIG. 5 Influence of Different
i.v., S.C. and oral Doses of SP 54 on the ELT
n= 5 per dose, M and 1 SD, significances
as above
Fibrin mg/dl 3LQ-
. %
300-
%$
??
P\
I..>\ * p 260-
T;,\, ..>, * IOmg i.v. ...\. ..... v yT;... .. ...... . .., .__._. f ..?* SOOmg oral -------___________ * 100mg S.C.
220-
P
180I,, 0 2
I , 4 6
I1
I 12 Incubation
1 1 2Lh
FIG. 6 Kinetics of Clot Lysis in Plasma samples after Administration n= 5 per dose, M and 1 SD, significances
of SP 54
as above
In some of these series,quantitative assays of the fibrin clot were also carried out. The kinetics of this reaction were examined in 3 groups of 5 individuals. Blood samples were obtained 15 min after 10 mg SP 54 i.v.,
HEPARIN AND FIBRINOLYSIS
348
Vo1.27, X0.3
90 min after 100 mg SP 54 S.C. and 120 min after an oral dose of 500 mg SP 54. From every plasma sample 9 identical fibrin clots were obtained as described above. After incubation times between 30 min and 24 h the remaining fibrin was assayed (fig. 6). In these series,there was no detectable clot lysis within the first hour of incubation. Thereafter fibrin diminished until a minimum was reached after an incubation time of 12 h. No further clot lysis occurred thereafter. Clot lysis tests carried out in the other groups are therefore only shown as the difference of the fibrin assay between the samples incubated for 45 min and for 12 h. The results of these tests are demonstrated in fig. 7.
Fibrin diminution
i.v.
S.C.
oral
%
mg
mg
m9
1
0 30 time
I..,,.
120min
012
af
L 6h application
tar FIG.
0
I
2
A
6h
7
InfluenceofDifferent DosesandApplications of SP 540nClot Lysis in Plasma n= 5 per dose, M and 1 SD, significances as above
The influence of SP 54 on the ELT and on the activation of factor XII is shown in fig. 8. In these tests SP 54 in a concentration of 5,ug/ml was also added to fractions of the blood samples taken before and after occlusion but prior to the injection of SP 54. The results were rather similar to the ones obtained with heparin which are shown in fig. 3. There was also a more pronounced influence on the ELT before and after venous occlusion when SP 54 was injected but only a slight shortening of the ELT when SP 54 was added in vitro. No significant difference between the in vitro and the in vivo samples was detected when the activities of factor XIIf were measured. Finally a daily oral dose of 500 mg SP 54 was given for one week to 9 patients suffering from postthrombotic syndrome but without acute signs of thrombosis. 2 of these patients were male and 7 were female. The average age was 52.6 + 12.3 years and the average weight was 71.5 !: 13.3 kg. Blood samples were collected prior to SP 54, after 24 h and after one week. Samples 2 and 3 were taken between 2 h and 3 h after ingestion of SP 54. The test results are shown in table I. A diminution of the ELT after 24 h as well as after 1 week could be demonstrated in these cases. Furthermore,there was a slight but significant diminution of plasminogen and of alpha-2-antiplasmin after 24 h but not after one week.
HEPARIN AND FIBRINOLYSIS
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349
ELT.min 250 b
200
‘\ _ Xllf,%
11 150
IUU
.nn
SO
31
;
1 1’ 1
*
2-
*
1
*
,r-d' 1 1
+
0
,
r
B
OTB
B = basic, = 5,uglml
??
t = 100mg in vitro
O= occlusion, SP 5-iadded
1
BOtB
0
SP 54 i.m. 2 h before +p (0.05. * ~(0.01
0
occlusion
FIG. 8 ELT and Factor XIIf Before and After Venous Occlusion. Influence of SP 54 in vitro.n=8, M and 1 SO 2 h after SO mg S.C. and afteradditionofj;,ug/ml
TABLE I Administration of Daily Oral Doses of 500 mg SP 54 for One Week Influence on ELT,Plasminogen and Alpha-Z-Antiplasmin. n=9 Test ELT, min M 1 SD Plasminogen,% Fl 1 SD Alpha-2-Antiplasmin,% I1 1 SD
Basic
after 24 h
after 1 week
127++ 51
150+ 64
97 10
87+ 12
96 12
101 10
87+ 11
99 10
234 95
DISCUSSION Previous investigations on the influence of heparin on fibrinolysis were a source of considerable controversy. There were reports on an enhancement of fibrinolysis induced by heparin in vitro (1,2) and after intravenous injection (10). On the other hand heparin was found to have a dual effect on fibrinolysis in vitro. Concentrations of over 125,ug/ml inhibited fibrinolysis whilst it was enhanced by heparin up to 5,ug/ml (11). Another group of investigators found no influence of heparin in vitro on the ELT nor on the diluted clot lysis time (12). Since natural heparin is a mixture of polysulfated polysaccharides of different MW,different heparin fractions were examined in the
350
HEPARIN AND FIBRINOLYSIS
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present study. In vitro, a clear difference was found between LFl!U' heparin which had no influence on fibrinolysis and HMW heparin which showed a dose dependent enhancement of the ELT in vitro. Since the ELT is influenced by the quality of fibrin,by the amount of plasminogen and by the activity of endogenous and exogenous activators,detailed examinations were carried out. An influence on the quality of fibrin was ruled out by addition of heparin to plasma prior to the precipitation of euglobulin. Heparin was thereby discarded with the supernatant and the absence of heparin was controlled by thrombin clotting times of the euglobulin fraction. Also the amount of plasminogen remained constant in most tests and only showed a minimal diminution at the highest concentration of HMW heparin. It is known that the endogenous activation Of fibrinolysis is influenced by activated factor XII and by kallikrein (I3,14). An activation of these proteins by heparin was already known (15). In the present study this activation was depending on the MW of the heparin fraction and on the dose and was running parallel to the enhancement of the ELT in vitro. A possible influence on the vascular activator of fibrinolysis was investigated in vivo by blood sampling before and after venous occlusion. This test was carried out prior to heparin and after a heparin injection. A comparable quantity of heparin was also added to fractions of the blood samples obtained prior to the heparin injection. There was a similar activation of factor XII in vitro and in vivo which indicated that endogenous activation was about the same when heparin was either added to plasma or injected. The enhancement of the ELT was however,considerably more pronounced in the samples obtained from heparinized patients. This speaks in favour of an additional influence of heparin on the exogenous activation of fibrinolysis. This assumption is further backed by the results of experiments in pigs which showed that a vascular activator of fibrinolysis is released when heparin or Polyanion SP 54 is administered by intravenous infusion (16). Though the effect on the ELT was highly significant the degree of fibrinolysis was rather mild in comparison to strong activators such as streptokinase or urokinase. This was also shown by the influence on plasminogen and on the antiplasmins. These proteins either remained at a constant level during the experiments or only showed a minimal diminution. When natural heparin fractions were examined there was a clear dependence on the MW. Nevertheless a synthetic heparin analogue with a MW comparable to the LMW heparin fraction also had an influence on fibrinolysis comparable to the action of HMW heparin. The MW is therefore,not the only limiting factor on the fibrinolytic action. Most natural heparins contain one sulfate group per hexose unit whilst Polyanion SP 54 has a higher degree of sulfatation (14). This can serve as a possible explanation for the different influence on fibrinolysis. Whilst heparin is known to be ineffective when given orally the smaller molecule of Polyanion SP 54 is absorbed from the intestinal tract (17). An enhancement of fibrinolysis was therefore,also found after ingestion of this drug though the dose had to be considerably higher than the amount given parenterally. The duration of the fibrinolytic action depended on the route of administration. It was shortest after intravenous injection and considerably longer when given subcutaneously or when ingested. In addition to the ELT,overall fibrinolysis was also examined by quantitative assays of fibrin clots which were obtained by addition of thrombin to diluted plasma and were incubated. This method is similar to the plasma clot lysis time but instead of complete lysis the degree of partial lysis after a constant incubation time is measured. The method was found to be more sensitive than the ELT and gave a better reflection of overall fibrinolysis since the plasmin inhibitors were not removed from this test system. A possible disadvantage was the presence in the system of the heparin-like substance which had a chance to alter the quality of the fibrin clot and thereby to influence the result. Nevertheless the same trend was found when partial lysis and ELT were
Vo1.27, No.3
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351
compared. The result of this series of experiments is an argument in favour of a fibrinolytic action of the HMW fraction of heparin and of a polysulfated heparin-like polysaccharide. No comment can yet be'made on the possibility of a clinical importance of this mechanism.
REFERENCES
1. HALSE,Th. Heparin und Heparinoide,
Dicumarol.
2. VINAZZER,H. Untersuchungen Wien.Z.Inn.
S. Hirzel,ZUrich
Uber die fibrinolytische Med. -23, 167-173, 1951
1950
Wirkung des Heparins
3. HALSE,Th. Aktivierung der Fibrinolyse und Thrombolyse durch Polysaccharidschwefelsaureester. Arzneimittelforsch. -12, 574-582, 1962 4. HAl_SE,Th. Polyelectrolytes as activators lysis. ___Sangre 9, 149-152, 1964
of fibrinolysis
and thrombo-
5. MILSTONE,H. A factor in normal human blood which participates coccal fibrinolysis. J. Immunol. 42, 109-116, 1941
in strepto-
6. SORIA,J., SORIA,C. and SAMAMA,M. Dosage du plasminogene a l'aide d'un substrat chromogene tripeptidique. Pathol. Biol. -24, 725-732, 1976 7. TEGER-NILSSON,A.C., FRIBERGER,P. and GYZANDER,E. Determination of a new rapid plasmin inhibitor in human blood by means of a plasmin specific tripeptide substrate. Scand.J.Clin.Lab.Invest. -37, 403-414, 1977 8. VINAZZER,H. Assay of total factor XII and of activated factor XII in plasma with a chromogenic substrate. Thromb. Res. 14, 155-166, 1979 9. RATNOFF,O.D. and MENZIE,C. A new method for the determination ogen in small samples of plasma. J.Lab.clin.Med. -37, 316-325,
of fibrin1951
10. LACKNER,H. and MERSKEY,C. Variation in fibrinolytic activity after myocardial infarction and after the administration of oral anticoagulant drugs and after intravenous heparin. Brit.J.Haemat. -6, 402-410,196O 11. VON KAULLA,K.N. and McDONALD,T.S. The effect of heparin on components the human fibrinolytic system. -Blood 13, 811-821, 1958
of
12. HOLEMANS,R., ADAMIS,D. and HORACE,J.F. Interaction of heparin with fibrinolysis. Thrombos. Diathes. haemorrh. -9, 447-458, 1963 13. KLUFT,C., TRUMPI-KALSHOVEN,M.,JIE,A. and VELDHUYZEN-STOLK,E. Factor XII dependent fibrinolysis:A double function of plasma kallikrein and the occurrence of a previously undescribed factor XII- and kallikrein dependent plasminogen activity. Thrombos. Haemostas. -41, 756-773, 1979 14. MANDLE,R. and KAPLAN,A. Hageman factor dependent fibrinolysis: generation of fibrinolytic activity by the interaction of human activated factor XI and plasminogen. -Blood 54, 850-862, 1979
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15. MOSKOWITZ,R.W., SCHWARTZ,H,J., MICHEL,B., RATNOFF,O.D. and ASTRUP !T. Generation of kinin-like agents by chondroitin sulfate,heparin,ch i tin sulfate and human articular cartilage. Possible pathophysiologic implications. J.Lab.clin.Med. -76, 790-798. 1970 16. MARKWARDT,F. and KLUCKING,H.P. Heparin induced release of plasminogen activator. Haemostasis -6, 370-374, 1977 17. SUE,T.K., JAQUES,L.B. and YUEN,E. Effects of acidity, cations and alcoholic fractionation on absorption of heparin from gastrointestinal tract. Can.J.Physiol.Pharmacol. -54, 613-617, 1976
INFLUENCE OF HEPARIN;OF OIFFERENT HEPARIN FRACTIONS AND OF A LOW MOLECULAR WEIGHT HEPARIN-LIKE SUBSTANCE ON THE MECHANISM OF FIBRINOLYSIS
H.Vinazzer, A.Stemberger,
S.Haas and G.BlUmel
Blood Coagulation Laboratory,Linz,Austria Surgery, Technical University, Munich,FRG
and Institute for Experimental
(Received 23.4.1982; in revised form 9.5.1982. Accepted by Editor H. Stormorken)
ABSTRACT The influence of different heparin fractions and of a synthetic polysulfated polysaccharide (SP54) on the fibrinolytic mechanism was examined. In vitro,a significant shortening of the euglobulin lysis time (ELT) was found after addition of standard mucosa heparin, of high MW heparin and of SP 54 respectively. Low MW heparin fractions had no influence on the ELT. Simultaneously with the shortening of the ELT an activation of factor XII and of kallikrein was observed. A similar effect was found in groups of volunteers after i.v. or S.C. injections and even after oral administration of SP 54. The effect of venous occlusion on the ELT and on the activation of factor XII was considerably increased when heparin or SP 54 was njected '2 h prior to the test. When a comparable concentration of the test substances was added to plasma samples before and af t er venous occlusion,the effect on the ELT was much less pronounced than after injection whilst the effect on factor XII was comparable in both tests. From these results the conclusion was drawn that activation of fibrinolysis by polysulfated polysaccharides is achieved by an endogenous pathway as well as by an increased availability of the vascular activator. The magnitude of the activation of fibrinolysis partly depends on the MW of the substance but apparently also on the degree of sulfatation since a low MW substance with a high number of sulfate bonds such as SP 54 was considerably more active than the low MW fraction of standard heparin.
Key words: Heparin, Heparin fractions,
Polyanion
341
SP 54, Fibrinolysis
342
HEPARIN A&D FIBRINOLYSIS
vo1.27, No.3
INTRODUCTION In contrast to the well known influence of heparin and of heparin analogues on coagulation a possible activation of the fibrinolytic mechanism by heparin was less thoroughly examined. Though an overall activation of fibrinolysis by heparin was demonstrated long ago (1,2,3,4), a detailed analysis of this mechanism was not carried out and interest was apparently lost when highly effective activators of fibrinolysis such as streptokinase and urokinase were introduced. For this reason the effect of heparin on fibrinolysis was reexamined. Additional tests were carried out with high molecular weight (HMW) and low molecular weight (LMW) heparin fractions and with a synthetic LMW polysulfated polysaccharide (Polyanion SP 54). MATERIALS
AND METHODS
Materials Heparin was pig mucosa sodium heparin with an average MW of 11,000 D and an activity of 200 I.U./mg from AB KABI, Stockholm,Sweden. LMW and HMW heparin fractions were prepared from heparin of the same source by KABI laboratories, Stockholm. A LMW fraction with a MW of 3,300 D and a HMW fraction with a MW of 18,300 D were obtained. The synthetic polysulfated polysaccharide was Polyanion SP 54 from Bene-Chemie, Munich, FRG. This substance was obtained in 1 ml ampouls containing 100 mg SP 54 and in capsules containing 150 mg and 250 mg respectively. The MW of SP 54 was 3,000 D. Reagents for assays of plasminogen and of antiplasmins were contained in the Coatest Antiplasmin kit from AB KABI Diagnostica, Stockholm. The reagent for kallikrein was from the same source and was the chromogenic substrate S-2302. Human prekallikrein was a gift from Cutter Laboratories, Berkeley, Calif. batch no. 1704-42-l-1937-4. The substance had the following characteristics: It was free from kallikrein,plasmin,plasminogen,thrombin and factor Xa. 1 mg of the lyophilized powder corresponded to the amount of prekallikrein contained in 170,ul normal human plasma. For tests,a solution of 6 mg of the substance per ml distilled water was made. Thrombin was from AB KABI Diagnostica,Stockholm. One vial contained 15 NIH units highly purified lyophilized human thrombin. Photometer: Model S-100 by Labtronic, Zurich,Switzerland. Clot Lysis Time Recorder: by Medicon,Glasgow,Scotland. This instrument was connected to a Rikadenki linear recorder which allowed paper speeds between 1 cm/h and 60 cm/min. Methods Euglobulin lysis time: Milstone (5); Plasminogen: Soria et al. (6); Alpha-2antiplasmin and total antiplasmin: Teger-Nilsson et al. (7). Factor XIIf was assayed by its ability to convert prekallikrein into kallikrein. 20,ul plasma was pipetted into a semimicro cuvette at 37OC. 20~1 of the test substance or of Tris-HCl buffer pH 7.4 was added which was immediately followed by 160,ul of the same buffer and by lOO,ul prekallikrein. After an incubation time of 60 set the chromogenic reaction was started by addition of 200,ul S-2302 ( 4 mmol/l). The difference in optical density per min was corresponding to the amount of active kallikrein formed. The blank containing buffer instead of the test substance was subtracted from the result. Since prekallikrein was in abundance and inhibitors were strongly diluted, XIIf was the limiting factor for kallikrein formation. Kallikrein in plasma was assayed in a similar way but for these tests the addition of prekallikrein was omitted. Both methods were described elsewhere in this journal (8).
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Fibrin assays: In some test series the diminution of a fibrin clot during incubation was quantitated. For this purpose 200,ul plasma before and after injections of the test substances was pipetted into 9 glass tubes and was diluted with 800,ul isotonic saline solution pH 7.35. After addition of 200,ul 20 mmol/l calcium chloride solution in which 10 NIH thrombin per ml was dissolved,the tubes were left at 37OC. After various intervals of time between 30 min and 12 h the fibrin clots which had formed where wound on glass rods and were repeatedly washed and squeezed between the glass rod and the wall of the tube in order to remove traces of soluble plasma proteins. The tyrosine equivalent of the clots was determined by the method of Ratnoff and Menzie (3). Plasma samples For in vitro tests citrate blood ( 1 part 0.15 mol/l trisodium citrate and 9 parts of venous blood) was collected from 10 healthy individuals. Blood was centrifuged for 20 min at 2,000 x g and the platelet poor citrate plasma was used. For tests on volunteers venous blood was obtained from different groups of healthy male students between 21 and 28 years of age. These blood samples were collected before and at different intervals after administration of the test substances. Comparison
of the test substances
The different fractions were compared by their concentrations based on the weight of the dry substances. This was felt to be the best way for the following reasons: Comparison on a molar base was impossible since standard heparin is not a homogenous substance but consists of subunits of different MW. Comparison by activities in I.U. was also impracticable because the LMW heparin fraction and the synthetic substance Polyanion SP 54 had a considerably lower thrombin inhibiting activitythan standard heparin or HMW heparin. Statistical
analysis
This was carried out by Student's
t-tests for paired groups.
RESULTS Heparin fractions could only be tested in vitro since the available quantities were not sufficient for in vivo trials. The influence of different concentrations of LMW and HMW heparin in comparison to standard heparin on the ELT was tested on 10 plasma samples from healthy individuals (fig. 1). Whilst there was no significant influence of LMW heparin on the ELT,a dose dependent shortening of the ELT was observed when euglobulin fractions were prepared from plasma containing standard heparin and HMW heparin respectively. There was no alteration of the plasminogen levels in the plasma samples containing LMW heparin. The highest concentrations of standard heparin and HMW heparin used in these tests resulted in a maximum diminution of plasminogen of 6%. This difference from plasma without heparin just reached the level of significance ( p 0.04). Alpha-2-antiplasmin remained at the same level in all plasma samples and the activity of total antiplasmin showed a maximum diminution of 5% which was not significant. A possible endogenous activation was examined by tests for the presence of fractioned factor XII (XII f) and of kallikrein (KK) (fig. 2). The highest concentration of LMW heparin in the plasma samples resulted in a trace of XII f and inasomewhathigher activity when standard heparin was used. HMW heparin produced a distinct and dose dependent activity of XII f in plasma. Tests for active kallikrein gave rather similar results and also showed a distinct and dose dependent activity in the HMW heparin samples.
HEPARIN AND
344
FIBRXNOLYSlS
ELT,min 2507
0
2
5
10
FIG.
20yg/ml
1
Influence of Standard Heparin and of LMW and HMW Heparin Euglobulin Lysis Time in vitro n = 10, M and 1 SD
Significances:
+ p below 0.05
Fractions
on the
; +’ p below 0.01
K K ,‘I. 1
30-
HMW
;.’
,
,:’
, HMW
1
20 -
IOHEP
HEP LMW
O,. 02
0
I
5
10
Oii
20pgtml
FIG.
io
Zbrglml
2
Influence of Standard Heparin, LMW and HMW Heparin Fractions ion of Factor XII and of Kallikrein
on the Activat-
n = 10, M and 1 SD
These in vitro experiments showed a dose dependent shortening of the ELT by heparin for which the HMW fraction of heparin is mainly responsible.Anactivation of factor XII and of kallikrein points to a possible endogenous path-
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345
way of the fibrinolytic mechanism. A possible influence on the exogenous pathway of fibrinolysis was examined by the following experiment. Venous blood was collected from 10 healthy male volunteers and citrate plasma was prepared. After blood collection the venous blood flow of the opposite arm was occluded by a blood pressure cuff inflated to a pressure of 90 mm Hg. After an occlusion time of 15 min,blood was collected from the occluded arm. Standard heparin in a dose of 50 mg was then injected subcutaneously. Two hours later,blood before and after venous occlusion was again collected. Heparin in a dose of Syg/ml was also added to fractions of the blood samples obtained prior to the injection of heparin. ELT's and factor XIIf levels were measured in all samples. The results are shown in fig. 3.
Xllf,% 2,
ELT,min 300
T
I
jooi h
1
T
T
100
0’
r
B
I
OtB
0 L/J(
I
B
0
FIG.
OtB
0
3
ELT and Factor XIIf Before and After Venous Occlusion. Influence of Heparin after S.C. Injection and after Addition in vitro. n = 10, M and 1 SD B= basic, 0= occlusion, arrows: 50 mg heparin S.C. 2 h prior to occlusion. Squares: heparin added in vitro to plasma samples before and after occlusion
The test showed the well known shortening of the ELT after venous occlusion which is a consequence of the release of a vascular activator of plasminogen. After subcutaneous heparin,the basic ELI was significantly shortened and further shortening was found after venous occlusion. A similar concentration of heparin added in vitro to plasma samples before and after venous occlusion had a considerably weaker effect on the ELT. There was no detectable factor XIIf in the basic blood samples but a trace could be found after occlusion. After heparin,some XIIf was found in the basic blood sample and a further increase was seen after occlusion. The samples which had heparin added in vitro showed a rather similar pattern.
346
HEPARIN
AtiD
FIBRINOLYSIS
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A series of tests was also carried out with Polyanion SP 54. This substance was of special interest since it had a MW comparable to the LMW fraction of heparin. The influence of SP 54 on the ELT in vitro and on the activation of factor XII is shown in fig. 4.
ELT,min zoo-
Xllf,% 14 -
12 IO -
ISO-
06h2-
100^
0-’
025
10
2Opg /ml
02
5
10
20pglml
FIG. 4 Influence of SP 54 on the ELT and on the Activation n = 10, M and 1 SD, Significances:
of Factor
XII in vitro
+ p below 0.05, +' p below 0.01
In spite of considerable scattering of the ELT's of the plasma samples of these test persons the shortening of the ELT by SP 54 was comparable with the results obtained with standard heparin. The activation of factor XII was even as strong as the results obtained with HMW heparin. There was no significant influence on plasminogen nor on the antiplasmin activities in this series. Further test series were carried out in vivo on healthy volunteers. Single i.v. doses of 5, 10 and 50 mg respectively were given to groups of 5 individuals and blood samples were collected prior to the injections and 10, 30, 60 and 120 min thereafter. Other groups of 5 volunteers received subcutaneous doses of 50 and 100 mg SP 54 respectively. In a third series of groups SP 54 was given as a single oral dose of 150, 250 and 500 mg respectively. In these groups blood samples were collected before and between 30 min and 6 h after the administration of SP 54. Furthermore, in a group of 8 volunteers the experiment with venous occlusion described above was also made with a subcutaneous dose of 50 mg. The influence on the ELT of different intravenous,subcutaneous and oral doses is shown in fig. 5. For better comparability the ELT's which were slightly different in different groups were converted into percentages. The average ELT prior to SP 54 is therefore,given as a 100% level. Placebo groups were also included into these series. After intravenous injections a maximum decrease of the ELT was found in the samples obtained at 45 min. The effect was diminishing after 2 h and there was a distinct dose dependence. A similar effect was found after subcutaneous injections. In these series,shortening of the ELT was evident after 2 h and
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HEPARIN AND FIBRINOLYSIS
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was still detectable after 6 h. Considerably higher oral doses were required to obtain a significant effect on the ELT. After 500 mg however,the shortening of the ELT was comparable with the effect. of the 50 mg subcutaneous dose.
i.v. mg
EL1.V.
mg
I
0
t im
I
1
1
012
2h
I
oral mg
S.C.
at
e
L te
r
6h
0
2
4
6h
application
FIG. 5 Influence of Different
i.v., S.C. and oral Doses of SP 54 on the ELT
n= 5 per dose, M and 1 SD, significances
as above
Fibrin mg/dl 3LQ-
. %
300-
%$
??
P\
I..>\ * p 260-
T;,\, ..>, * IOmg i.v. ...\. ..... v yT;... .. ...... . .., .__._. f ..?* SOOmg oral -------___________ * 100mg S.C.
220-
P
180I,, 0 2
I , 4 6
I1
I 12 Incubation
1 1 2Lh
FIG. 6 Kinetics of Clot Lysis in Plasma samples after Administration n= 5 per dose, M and 1 SD, significances
of SP 54
as above
In some of these series,quantitative assays of the fibrin clot were also carried out. The kinetics of this reaction were examined in 3 groups of 5 individuals. Blood samples were obtained 15 min after 10 mg SP 54 i.v.,
HEPARIN AND FIBRINOLYSIS
348
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90 min after 100 mg SP 54 S.C. and 120 min after an oral dose of 500 mg SP 54. From every plasma sample 9 identical fibrin clots were obtained as described above. After incubation times between 30 min and 24 h the remaining fibrin was assayed (fig. 6). In these series,there was no detectable clot lysis within the first hour of incubation. Thereafter fibrin diminished until a minimum was reached after an incubation time of 12 h. No further clot lysis occurred thereafter. Clot lysis tests carried out in the other groups are therefore only shown as the difference of the fibrin assay between the samples incubated for 45 min and for 12 h. The results of these tests are demonstrated in fig. 7.
Fibrin diminution
i.v.
S.C.
oral
%
mg
mg
m9
1
0 30 time
I..,,.
120min
012
af
L 6h application
tar FIG.
0
I
2
A
6h
7
InfluenceofDifferent DosesandApplications of SP 540nClot Lysis in Plasma n= 5 per dose, M and 1 SD, significances as above
The influence of SP 54 on the ELT and on the activation of factor XII is shown in fig. 8. In these tests SP 54 in a concentration of 5,ug/ml was also added to fractions of the blood samples taken before and after occlusion but prior to the injection of SP 54. The results were rather similar to the ones obtained with heparin which are shown in fig. 3. There was also a more pronounced influence on the ELT before and after venous occlusion when SP 54 was injected but only a slight shortening of the ELT when SP 54 was added in vitro. No significant difference between the in vitro and the in vivo samples was detected when the activities of factor XIIf were measured. Finally a daily oral dose of 500 mg SP 54 was given for one week to 9 patients suffering from postthrombotic syndrome but without acute signs of thrombosis. 2 of these patients were male and 7 were female. The average age was 52.6 + 12.3 years and the average weight was 71.5 !: 13.3 kg. Blood samples were collected prior to SP 54, after 24 h and after one week. Samples 2 and 3 were taken between 2 h and 3 h after ingestion of SP 54. The test results are shown in table I. A diminution of the ELT after 24 h as well as after 1 week could be demonstrated in these cases. Furthermore,there was a slight but significant diminution of plasminogen and of alpha-2-antiplasmin after 24 h but not after one week.
HEPARIN AND FIBRINOLYSIS
Vo1.77, No.3
349
ELT.min 250 b
200
‘\ _ Xllf,%
11 150
IUU
.nn
SO
31
;
1 1’ 1
*
2-
*
1
*
,r-d' 1 1
+
0
,
r
B
OTB
B = basic, = 5,uglml
??
t = 100mg in vitro
O= occlusion, SP 5-iadded
1
BOtB
0
SP 54 i.m. 2 h before +p (0.05. * ~(0.01
0
occlusion
FIG. 8 ELT and Factor XIIf Before and After Venous Occlusion. Influence of SP 54 in vitro.n=8, M and 1 SO 2 h after SO mg S.C. and afteradditionofj;,ug/ml
TABLE I Administration of Daily Oral Doses of 500 mg SP 54 for One Week Influence on ELT,Plasminogen and Alpha-Z-Antiplasmin. n=9 Test ELT, min M 1 SD Plasminogen,% Fl 1 SD Alpha-2-Antiplasmin,% I1 1 SD
Basic
after 24 h
after 1 week
127++ 51
150+ 64
97 10
87+ 12
96 12
101 10
87+ 11
99 10
234 95
DISCUSSION Previous investigations on the influence of heparin on fibrinolysis were a source of considerable controversy. There were reports on an enhancement of fibrinolysis induced by heparin in vitro (1,2) and after intravenous injection (10). On the other hand heparin was found to have a dual effect on fibrinolysis in vitro. Concentrations of over 125,ug/ml inhibited fibrinolysis whilst it was enhanced by heparin up to 5,ug/ml (11). Another group of investigators found no influence of heparin in vitro on the ELT nor on the diluted clot lysis time (12). Since natural heparin is a mixture of polysulfated polysaccharides of different MW,different heparin fractions were examined in the
350
HEPARIN AND FIBRINOLYSIS
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present study. In vitro, a clear difference was found between LFl!U' heparin which had no influence on fibrinolysis and HMW heparin which showed a dose dependent enhancement of the ELT in vitro. Since the ELT is influenced by the quality of fibrin,by the amount of plasminogen and by the activity of endogenous and exogenous activators,detailed examinations were carried out. An influence on the quality of fibrin was ruled out by addition of heparin to plasma prior to the precipitation of euglobulin. Heparin was thereby discarded with the supernatant and the absence of heparin was controlled by thrombin clotting times of the euglobulin fraction. Also the amount of plasminogen remained constant in most tests and only showed a minimal diminution at the highest concentration of HMW heparin. It is known that the endogenous activation Of fibrinolysis is influenced by activated factor XII and by kallikrein (I3,14). An activation of these proteins by heparin was already known (15). In the present study this activation was depending on the MW of the heparin fraction and on the dose and was running parallel to the enhancement of the ELT in vitro. A possible influence on the vascular activator of fibrinolysis was investigated in vivo by blood sampling before and after venous occlusion. This test was carried out prior to heparin and after a heparin injection. A comparable quantity of heparin was also added to fractions of the blood samples obtained prior to the heparin injection. There was a similar activation of factor XII in vitro and in vivo which indicated that endogenous activation was about the same when heparin was either added to plasma or injected. The enhancement of the ELT was however,considerably more pronounced in the samples obtained from heparinized patients. This speaks in favour of an additional influence of heparin on the exogenous activation of fibrinolysis. This assumption is further backed by the results of experiments in pigs which showed that a vascular activator of fibrinolysis is released when heparin or Polyanion SP 54 is administered by intravenous infusion (16). Though the effect on the ELT was highly significant the degree of fibrinolysis was rather mild in comparison to strong activators such as streptokinase or urokinase. This was also shown by the influence on plasminogen and on the antiplasmins. These proteins either remained at a constant level during the experiments or only showed a minimal diminution. When natural heparin fractions were examined there was a clear dependence on the MW. Nevertheless a synthetic heparin analogue with a MW comparable to the LMW heparin fraction also had an influence on fibrinolysis comparable to the action of HMW heparin. The MW is therefore,not the only limiting factor on the fibrinolytic action. Most natural heparins contain one sulfate group per hexose unit whilst Polyanion SP 54 has a higher degree of sulfatation (14). This can serve as a possible explanation for the different influence on fibrinolysis. Whilst heparin is known to be ineffective when given orally the smaller molecule of Polyanion SP 54 is absorbed from the intestinal tract (17). An enhancement of fibrinolysis was therefore,also found after ingestion of this drug though the dose had to be considerably higher than the amount given parenterally. The duration of the fibrinolytic action depended on the route of administration. It was shortest after intravenous injection and considerably longer when given subcutaneously or when ingested. In addition to the ELT,overall fibrinolysis was also examined by quantitative assays of fibrin clots which were obtained by addition of thrombin to diluted plasma and were incubated. This method is similar to the plasma clot lysis time but instead of complete lysis the degree of partial lysis after a constant incubation time is measured. The method was found to be more sensitive than the ELT and gave a better reflection of overall fibrinolysis since the plasmin inhibitors were not removed from this test system. A possible disadvantage was the presence in the system of the heparin-like substance which had a chance to alter the quality of the fibrin clot and thereby to influence the result. Nevertheless the same trend was found when partial lysis and ELT were
Vo1.27, No.3
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compared. The result of this series of experiments is an argument in favour of a fibrinolytic action of the HMW fraction of heparin and of a polysulfated heparin-like polysaccharide. No comment can yet be'made on the possibility of a clinical importance of this mechanism.
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Dicumarol.
2. VINAZZER,H. Untersuchungen Wien.Z.Inn.
S. Hirzel,ZUrich
Uber die fibrinolytische Med. -23, 167-173, 1951
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Wirkung des Heparins
3. HALSE,Th. Aktivierung der Fibrinolyse und Thrombolyse durch Polysaccharidschwefelsaureester. Arzneimittelforsch. -12, 574-582, 1962 4. HAl_SE,Th. Polyelectrolytes as activators lysis. ___Sangre 9, 149-152, 1964
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5. MILSTONE,H. A factor in normal human blood which participates coccal fibrinolysis. J. Immunol. 42, 109-116, 1941
in strepto-
6. SORIA,J., SORIA,C. and SAMAMA,M. Dosage du plasminogene a l'aide d'un substrat chromogene tripeptidique. Pathol. Biol. -24, 725-732, 1976 7. TEGER-NILSSON,A.C., FRIBERGER,P. and GYZANDER,E. Determination of a new rapid plasmin inhibitor in human blood by means of a plasmin specific tripeptide substrate. Scand.J.Clin.Lab.Invest. -37, 403-414, 1977 8. VINAZZER,H. Assay of total factor XII and of activated factor XII in plasma with a chromogenic substrate. Thromb. Res. 14, 155-166, 1979 9. RATNOFF,O.D. and MENZIE,C. A new method for the determination ogen in small samples of plasma. J.Lab.clin.Med. -37, 316-325,
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12. HOLEMANS,R., ADAMIS,D. and HORACE,J.F. Interaction of heparin with fibrinolysis. Thrombos. Diathes. haemorrh. -9, 447-458, 1963 13. KLUFT,C., TRUMPI-KALSHOVEN,M.,JIE,A. and VELDHUYZEN-STOLK,E. Factor XII dependent fibrinolysis:A double function of plasma kallikrein and the occurrence of a previously undescribed factor XII- and kallikrein dependent plasminogen activity. Thrombos. Haemostas. -41, 756-773, 1979 14. MANDLE,R. and KAPLAN,A. Hageman factor dependent fibrinolysis: generation of fibrinolytic activity by the interaction of human activated factor XI and plasminogen. -Blood 54, 850-862, 1979
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15. MOSKOWITZ,R.W., SCHWARTZ,H,J., MICHEL,B., RATNOFF,O.D. and ASTRUP !T. Generation of kinin-like agents by chondroitin sulfate,heparin,ch i tin sulfate and human articular cartilage. Possible pathophysiologic implications. J.Lab.clin.Med. -76, 790-798. 1970 16. MARKWARDT,F. and KLUCKING,H.P. Heparin induced release of plasminogen activator. Haemostasis -6, 370-374, 1977 17. SUE,T.K., JAQUES,L.B. and YUEN,E. Effects of acidity, cations and alcoholic fractionation on absorption of heparin from gastrointestinal tract. Can.J.Physiol.Pharmacol. -54, 613-617, 1976