THROMEJOSIS RESEARCH 16; 147-158 Pergamon Press Ltd.1979. Printed
in Great
Britain
ESTIMATION OF TZBROMBUS FORMATIOK BY LABELING OF PLATELETS, RED CELLS AND FIBRIKOGEN IN EXPERIMENTAL TBROMBOSIS
R.Zimmermann, C.Zeltsch and
D.Lange *
Medizinische Universitdtsklinik (Ludolf-Krehl-Klinik) and Universitbts-Strahlenklinik (Czerny-Krankenhaus) ?? Universitat Heidelberg, 69 Heidelberg 1,Bergheimerstr. 58 GFR (Received
62.1979; in revised form 17.5.1979. Accepted by Editor S. Witte)
ABSTRACT Thrombus analyses were derived from thrombus-to-blood ratios of radiolabeled platelets (51Cr1, red cells csgFe or 51Cr) and fibrinogen (1*5I) in different thrombogenic conditions in 130 rabbits. While the composition of stasis thrombi nearly resembled that of blood the specific thrombus/blood ratio of platelets, red cells and fibrinogen was estimated at 51, 0.6 and 4 in typical arterial and 12, 2 and 7 in venous thrombi, respectively. These data suggest an increased thrombus content and significance of platelets with increasing blood flow velocities or shear rates. Red cells and fibrinogen demonstrated a different behaviour with the maximal thrombus uptake at lower blood flow velocity. Antithrombotic therapy did not give rise to any substantial alteration of the thrombus composition. Comparing the radiometric methods and the thrombus weight in estimating thrombus formation, the correlation analysis showed reliable correlation coefficients rs of more than 0.8 in the venous system. rs values about 0.6 supposed that gravimetry should be the primary method in estimating fomation of arterial thrombosis.
IN'TRODUCTION The formation of experimental thrombi can be established by their rate of incidence (1,2),subjective grading (3,4),morphometry (51,determination of the thrombus weight (61 or the flow rate of the thrombosed vessels (7).After development of techniques for radioisotope labeling of blood components especially radiolabeled fibrinogen has been used as a thrombus localizing agent in experimental (8,9,10) and clinical investigations (11,12,13).
Key
words:
Thrornbus formation, radioactive labeling, I 125 fibrinogen, radiolabeled platelets, antithrombotic therapy and thrombus composition. 147
Eo-rmerly,the experimentally induced throirbi '&ere chara.cteriaed str,~-,~. _CII-L rally by microscopy :!4,15) or on the basis of the thrombogenic conditions of the employed experimental model (16).1n the present study the simultaneous thrombus uptake of radiolabeled platelets i5iCrJ,red celis (5'Fe or 51Cr) and fibrinogen (1251) was investigated in rabbits and qcalitatlve ~anc! quantitative analyses of venous and arterial thrombi performed.In addition the incorporation of blood elements was studied iz different thrombogenic conditions and in antithrombotic therapy with antiplatelet drugs and anticoagulants.
MATERIALS AND METHODS Radioisotopic procedures. In three different series blood elements were labeled in 130 rabbits as follows: series
I (70 rabbits):
platelets 51Cr red cells 59Fe fibrinogen 1251
series
II (50 rabbits):
red cells 51Cr fibrinogen 1251
series
III (10 rabbits):
platelets 51Cr red cells 5gFe fibrinogen 125~
Autologous platelets were labeled with 51Cr according to Aas and Gardner (17) with slight modifications.Blood (8 ml) was collected 1:5 with an acidcitrate-dextrose solution (Biotest Laboratories,Frankfurt) from the central ear artery of each rabbit.The suspension of platelets was labeled by incubation for 1 hour at room temperature with 0.3 ml of 50 WCi 51Cr (230 mCi per mg of sodium chromate,Amersham Laboratories,Buckinghamshire).Thereafter platelets were resuspended and washed twice with previously saved platelet-poor rabbit plasma and reinjected intravenously within 3 hours after blood collection. Red cells were labeled in vivo by intravenous injection of 4 ICi 5gFe seven to eight days prior to the experiments (18).In series 11 red cells were labeled with S*Cr by a standard technique (19),with some modifications. 2 ml of blood were taken from the marginal ear vein 1:5 with acid-citratedextrose solution.Some of the supernatant citrate plasma was removed before incubation with 30 BCi 'lCr (sodium chromate,from Amersham Laboratories, B::ckinghamshire) for 30 min at 37 O C.The red cells were then washed three times with 0.9 % NaCl solution and reinjected intravenously. Freshly prepared,heterologous human 1251-fibrinogen from Amersham Laboratories,Buckinghamshire was administered because of its high in vivo clottability of nearly 90 % when used in the first week after iodination (20) and the nearly identical turnover of human fibrinogen in rabbits during the first days after application (21).The isotopic clottable activity was controlled according to Regoeczi (22). 4 PCi of 1251-fibrinogen were injected intravenously one hour before producing thrombi. Thrombus formation. In the experimental series I thrombosis was produced by endothelial lesions in 70 rabbits.The animals were anesthetized with sodium pentobarbital (Nembutal R) (30 mg/kg body weight) via an ear vein cannula in the ipsilateral carotid artery for radioactivity determinations
vo1.16,so.1/2
ON THROMBUS
COMPOSITIOS
z-7 coagulation analyses.Thereafter the contralateral jugular vein and the abdominalis were prepared.In the jugular veins slight endothelial lesions were induced according to the method of Schmidt (23),with slight modifications.The vessels (1 cm in length) were frozen at -2C' C for 4 min.In the aortic segment thrombus formation was induced by perivascular application of a 20 % solution of silver nitrate,as described by Jansen (24).The artery was kept in a plastiv groove (1.5 cm length) in order to achieve a standardized lesion.Thrombus formation was induced not earlier than 1 hour after application of labeled platelets and fibrinogen.After 3 hours the thrombi were carefully dissected out from the vessel,immediately (within a half minute) weighed and the radioactivity determined.
aorta
In the series II rapidly growing venous thrombi were induced in 50 rabbits and the effect of antithrombotic drugs on quantitative and qualitative thrombus formation investigated-The jugular vein was treated with 20 % silver nitrate solution,inducing a stiffening of the vessel wall with a slightly reduced blood flow.Thrombosis developed frequently within 20 min. Arterial thrombosis was induced as in the first model.In this study red cells were labeled with SICr. In the series III development of stasis thrombi was induced in 10 rabbits in the jugular vein and in the aorta by injection of contact activation product as described earlier (25).Thrombi formed within 10 min. Labeling of platelets,red cells and fibrinogen was performed as reported in the first series. Antithrombotic therapy. The 50 rabbits of series II were randomized into five groups of ten animals.The first group received 0.9 % sodium chloride solution.The second group was treated with acetylsalicylic acid (ASA) 100 mg/kg body weight per OS on the evening before starting the experiments and in addition 90 min before.A further group was given a low dose of heparin of 80 IV/3 hours continuously by an infusion pump.Ten animals received a combination of low dose heparin and ASA.The last group of ten animals were injected with heparin in a dose of 460 IV/3 hours.The antithrombotic effect was evident in all groups before onset of thrombus formation. Radioactivity determinations. The measurement of radioactivity of the three radioisotopes in the samples was carried out in a scintillation counter with a pulse hight analyser in the usual manner.The total count rate in each of the windows was corrected by background radiation,the photoeffect and the interference of the Compton effect of high energy gamma rays in lower windows.Correction factors were evaluated by counting pure standards. Calculations and definitions. For the quantitative and qualitative analyses of the experimentally induced thrombi the following definitions were chosen and computed as follows: For the three blood components,platelets,red cells and fibrinogen,the isotopically estimated thrombus size was called the 'radiometric thrombus size' and calculated from the equation: radiometric thrombus size
=
cpm (thrombus) cpm / ml blood
For characterizing the content and composition of the thrombi of radiolabeled blood elements the term 'specific thrombus/blood ratio' was chosen and computed from the equation: specific thrombus / blood ratio
=
cpm / g thrombus cpm / g blood
150
OS THRO.XEKJS COXPOSITION
vo1.16,so.1/2
In addition the platelet,red c ell and fibrinogen content was expressed count or concentration per mg thrombus weight.
as
Statistical analvsis. The correlation of the radiometric thrombus size and the thrombus weight was performed by the Spearman rank correlation, because the distribution of the original values was far from normal.The effect of the antithrombotic therapy was controlled by a one way analysis of variance in the usual way.The original data xi were transformed according tO Ti = 100 log [ 100 ( Xi + I ) ].As level of significance p = 0.05 was chosen.
RESULTS
Thrombus composition. The specific thrombus/blood ratios of the labeled blood elements are represented in the Table I.In series I in the arterial system the uptake rate-of slCr labeled platelets was 33.This indicates that the platelet content of the thrombi was 33 times that of an equivalent weight of blood.In the ten largest and occlusive thrombi the specific thrombus/blood ratio was estimated at 25,while the ten smallest and non-occluding thrombi showed the highest platelet uptake of 50.0. The 5gFe labeled red cells were incorporated at only half the concentration of blood. 1251-fibrinogen uptake was 4.5 times higher,in comparison to normal blood and amounted at 7.6 in occlusive and 3.7 in the smallest thrombi.In the venous system (series I) the incorporation of platelets was smaller than in the arterial thrombi,while specific thrombus/blood ratio of red cells and fibrinogen was measured at higher values.In the thrombosis model II ( Table I) the incorporation of 5ICr labeled red cells and 1251-fibrinogen was investigated in rapidly growing venous thrombi.The specific thrombus/blood ratios of stasis thrombi (series III) are shown in the bottom of Table I. The computed thrombus content of platelets,red cells and fibrinogen are given in the Table II as count or concentration for a defined thrombus weight. Experimental conditions and thrombus composition. Figure I illustrates the incorporation of the three measured blood elements dependent on the thrombogenic conditions of the three experimental models in relation to the supposed blood flow velocity.The four experimental conditions used represent different shear rates,increasing from the model III to maximal values in the model I. Because of the induced by silver nitrate slight stiffening of the vein wall the blood flow can be assumed to be slower in the model II and stagnant in areas of stasis as in the model III.The incorporation of blood platelets increased from the model III to a maximum in the aorta (model I), while the uptake of fibrinogen and red cells decreased under these conditions. Antithrombotic therapy and thrombus formation. The antithrombotic effect of antiplatelet drugs and anticoagulants on tbrombus formation was measured by gravimetry and radiometrically from the equation cpm cpm
( thrombus f / ml blood
.
In the venous system a significant reduction of the thrombus growth was found only under treatment with sodium heparin (Liquemin R) (p' 0.051 (Table III). Under antithrombotic therapy a slight but not statisticaly significant alteration of the specific thrombus blood ratio could be seen (Table III). Especially high and low doses of heparin did not change the incorporation of 1251-fibrinogen. The measurement of 51Cr labeled red cells did not show a significant reduction of the incorporation under therapy with antitbrombotic drugs.
03 THROHBGS
VOl.l6,XO.l/2
1-1
COMPOSITIOS
Specific thrombus/blood ratios of the labeled blood elements in the arterial and venous thrombi of the series I,11 and III.The values are given in group medians and (means).In addition to the total the ratios of the ten smallest and ten largest thrombi were presented.All of the greatest venous and nearly all (9 of 10) of the greatest arterial thrombi were occluding.
series I
arterial thrombi
platelets
(51Cr)
red cells
CsgFe)
fibrinogen (1251) weight
venous thrombi
(mg)
platelets
cSICr)
red cells
tsgFe)
fibrinogen (l-251) weight
(rng)
series II red cells venous thrombi
fibrinogen weight
c5kr) (1251) (mgl
arterial thrombi
platelets
(51Cr)
red cells
t59Fe)
fibrinogen (1251) weight
venous thrombi
(mg)
platelets
(5kr)
red cells
tsgFe)
fibrinogen (1*5I) weight
total
50.8 (48.9: 0.6 (0.6) 3.7 (6.5) 3.2 (6.6)
33.0 (5C.2) 0.6 il.71 :. 5 (6.1) lC.5 (18.3)
24.7 (28.7) 0.9 (4.7) 7.6 (6.6) 60.2 (52.7)
20.1 (32.2) 2.8 (5.5) 4.4 (8.02 4.5 15.61
12.2 (19.0)
5.7 (6.7) 2.2 (2.3) 3.4 (5.4) 41.6 (65.3)
small
total
1.3 (1.1) 3.1 (4.3) 3.1 (4.3)
,f:Z, 6.8 (12.7) 8.9 (23.5)
1.5 (1.5) 4.9 (5.5) 5.1 (5.7) total
serifs III
(mg)
occluding
small
2.7 (3.0) 1.3 (1.2) 2.8 (2.3) 58.4 (60.3) 2.7 (2.9) 1.5 (1.7) 2.6 (2.2) 64 5 (72:31
occluding 1.8 (1.8) 5.2 (4.9) 13.3 (15.0)
VO1.16,XO.1/2
Platelet, red cell and fibrinogen content of thrombi.
series I
arterial thrombi
venous thrombi
venous thrombi
total
24.0 (23.0)
15.6 (23.7)
(5lcr)
red cells 106/mg
(59Fe)
3.2 (3.4)
3.5 (9.5)
5.1 (3.2)
fibrinogen (1251) m9/g
8.2 (14.3)
9.8 (13.5)
16.7 (14.6)
~$~~$e'"
(5lCr)
9.5 115.2)
5.8 (9.0)
2.7 (3.2)
~$,~~lls
(59Fe)
15.5 (30.4)
12.1 (28.0)
12.0 (12.7)
fibrinogen (1251j mg/9
9.8 (11.9)
15.1 (28.11
7.4 (17.1)
series II
small
total
red cells 106/mg
11.7 (13.6)
occluding
(slCr)
7.3 (6.2)
8.4 (8.4)
10.1 (10.1)
fibrinogen (12~~) 106/mg
6.8 (9.5)
10.8 (12.1)
11.4 (10.8)
total
y;;;etets
(5iCr)
1.3 (1.4)
red cells l@/mg
(5gFej
7.1 (6.6)
fibrinogen (125,) mg/9
6.2 (5.1)
1.3 (1.0) venous thrombi
occluding
y$;;;ets
series III
arterial thrombi
small
tsgFef
8.5 (9.4)
fibrinogen (12511 mg/g
5.8 (4.9)
red cells 106/mg
~
Vo1.16,No.1/2
ON THROMBUS
4-
-c-
COMPOSITION
,----_
153
---_ ---_
/
/
--a
/
/
d’ *.....-**
_*...... *I’- .*a..
. . ...* .-*...*
O................‘Q“
*...
....
*...
-...
*...
a...
.. . .
....
.*..
a...
-...
“0
L
‘It
1
P-
I
I-
I-
inncasingblood flowmtt FIG. I or Thrombus incorporation of 5lCr labeled platelets (A---&,5gFe 51Cr labeled red cells (O.*.O) and 1251-fibrinogen (w-+ in the series I - III. TABLE III Thrombus weight and specific thrombus/blood ratios of venous thrombi in antithrombotic therapy (series II).
Control
ASA
Heparin low
Heparin low •t ASA
Heparin
weight (mg)
5.1 (5.7)
4.0 (4.5)
1.6 (2.5)
1.5 (2-O)
0.9 (1.2)
fibrinogen
5.0 (5.2)
5.0 (6.4)
5.4 (6.6)
3.3 (4.5)
4.5 (4.9)
red cells
1.6 (1.8)
1.6 (I.71
1.5 (1.3)
1.4 (1.3)
1.2 (1.1)
OS THFtO,MXJS COHPOSITION
TABLE The
results by
of the correlation analysis the correlation coefficient
series
arterial thrombi
venous thrombi
I
are presented (rs).
rs
n
platelets
tSICr)
0.62
57
red
cells
csgFe)
0.64
60
fibrinogen
(1251)
0.55
60
platelets
(51Cr)
0.8
35
red
cells
(5gFe)
0.83
39
fibrinogen
(1251)
0.76
43
series venous thrombi
IV
red
II
rs
n
cells
(51Cr)
0.92
43
fibrinogen
(1251)
0.84
43
Correlation analysis of radiometrically and gravimetrically measured thrombus size. The reliability of isotopic methods and gravimetry in estimating thrombus formation was controlled by means of the Speannan rank correlation. The results of the statistical calculations are represented by Table IV by the correlation coefficient (rs). rs ranged from-O.5 - 0.6 in the arterial system.In the venous system a rs of more than 0.8 could be observed.
DISCUSSION Analysis of the thrombus structure by radioisotopic methods provides a reliable labeling procedure without separation of radioactivity and uneffected functions of the labeled blood elements.In the present study platelets were labeled using the slightly modified technique of Aas and Gardner (17) with addition of ascorbic acid and reinjected already 3 hours after the labeling procedure;thus an intact platelet function could be preserved 126, 27).Investigating of the radioactivity of the platelet sediment,platelte Cr rich plasma and whole blood confirmed that only minimal quantities of (< 3 %) separated from the labeled platelets.Radioactive iron is rapidly cleared from plasma,incorporated in the hemoglobin molecule and then labels selectively red cells (18).The used 125I-fibrinogen demonstrates a high clottability of more than 80 %,when employed in the first days of radioiodination as described by Bradley (ZO).Thus ,it can be supposed that the measured radioactivities nearly represent only these three blood elements. In the arterial system the specific thrombus/blood ratio for 51Cr labeled platelets was measured at 51 in the small non-occluding thrombi,while a specific thrombus/blood ratio of 33 was estimated as group median in this first series.Likewise,in the venous system the highest uptake rate was calculated at 20 in the small thrombi,whereas it decreased when the thrombi
VO1.16,NO.1/2
ON THROMBUS
COMPOSITIOX
155
became occlusive.These results confirm the significance of platelets in the formation of arterial thrombi as known from morphological investigations (14,15,28),in vitro experiments (5,29,30) and platelet turnover studies in man (31).In the initial stages of venous thrombus growth the platelets are still the predominant blood elements if thrombi are forming at the site of endothelial lesions.The highest specific thrombus/blood ratios of platelets were found in the smallest thrombi,representing the initial stages of thrombus formation or the structure of small but not further grown thrombi. In the stasis thrombi the specific thrombus/blood ratio for platelets nearly resembled that of blood and represents structurally the so called 'whole blood clot' (14,321. The specific thrombus/blood ratio of 59Fe or 51Cr labeled red cells was calculated in arterial thrombi at nearly half the concentration of that found in the blood.In the venous system the uptake rates of the red cells ranged from 1 to 2 in the thrombosis model I and II and were nearly identically to that of the stasis thrombi as shown in the model III. The specific thrombus/blood ratios of lz51-fibrinogen amounted to nearly similar values of 6.8 and 4.9 in the venous system and 4.5 in the aorta, respectively.The mode of thrombus incorporation of radiolabeled fibrinogen is up to now an unsolved problem.Radiofibrinogen will be incorporated as well into a forming as into a recently formed thrombus, 18 to 72 hours after onset of the thrombus formation (33,34,35).The highest uptake rates of 17 to 23 were reported by Coleman (33) and Knight (35) when radiofibrinogen was applicated 4 to 6 hours after starting thrombus production.More than 22 hours old thrombi showed radioactivity ratios of only 2-3 (34,35).In view of these data and our findings we can suggest that not only growth but also an alteration in structure occurs in the first min as well as in the first three hours of thrombus formation. The wall shear rates of the vessel areas used for inducing experimental thrombi cannot be calculated accurately in vivo.In our study the presented thrombosis models or vessel areas suggest different blood flow velocities or shear rates,respectively.According to Baumgartner (361,Copley (37) and Schmid-Schonbein (38) the wall shear rates can be estimated at values of about 800-1000 see-1 in the rabbit aorta abdominalis,at lower values in the venous system and at a shear rate of 0 sec- 1 in areas of complete stasis. Our results obtained in rabbits in vivo demonstrated an increasing specific thrombus/blood ratio for 5lCr labeled platelets in thrombogenic conditions with increasing blood flow velocities or shear rates.The highest flow rate canbe assumed in the model I in the arterial system-The presented data are consistent with the findings of other authors,who described a shear rate dependent platelet deposition on subendothelium or growth rate of platelet thrombi (29,39,40).According to Richardson (29) the platelet deposition rate will again decrease if theoretical very high shear rates at higher blood flow velocities were reached.Such conditions were not attained in our experimental models in vivo.In analogy to the refered behaviour of platelets,red cells and fibrinogen demonstrated a similar blood flow dependent incorporation-The maximal thrombus uptake or deposition was seen at lower shear rates in the venous system and decreased at higher blood flow velocity as demonstrated i..the arterial system. On the basis of the heparin-induced inhibition of the fibrinogen-fibrin converslon,a reduced uptake or a decrease of the specific thrombus/blood ratio of lz51-fibrinogen Igould be conceivable.The thrombus formation could
have been quantitativeiy inhibited by heparin,but without any change in the 1251-fibrinogen content.Antit;hrombotic therapy with antiplatelet drugs caused no substantial alteration of the thrombus compositon. Evaluation of gravimetry and the radiometric methods in estimating quantitative thrombus formation by the correlation analysis in the venous system demonstrated a reliable agreement with a correlation coefficient rs of more than 0.8 in all models.In the arterial system the thrombus composition differed more obviously from that of blood and showed a rs of about 0.6. In view of these results we conclude,that venous thrombus formation can be reliably estimated by labeling of blood elements.In the arterial system gravimetry should be the primary method in controling thrombus growth.
ACKNOWLEDGEMENTS We thank Dr.G.Weckesser,Institut fur Dokumentation und Statistik der Universitat Heidelberg,for valuable assistance in statistical analyses.
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OX THROMBUS
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ijS
O?i THRO?BUS
COXPOSITIOS
VOl.l6,SO.l/~~
31. HARKER,L.A.,SLICHTER,S.J. Platelet and fibrinogen consumption in zaa. N.Eng.J.Med. 287, 999, 1972. 32. SPAET,T.H.,TSAO,C.H. Vascular endothelium and thrornbogenesis.In: Thrombosis. Sherry,S.,BrinWlous,K.M.,Genton,E.,Stengle,J.M.(Eds.) Washington,D.C.,National Academy of Sciences, 1969, p-416. 33. COLEMAN,R.E.,AARWIG,S.S.L.,RARWIG,J.F.,SIEGEL,B.A,,WELCH,M.J. Fibrinogen uptake by thrombi:Effect of thrombus age. J.Nucl.Med. 16, 370, 1975. 34. MOSCHOS,C.B.,OLDEWURTEL,H.A.,HAIDER,B.,REGAN,T.J. Effect of coronary thrombus age on fibrinogen uptake. Circ. 54, 653, 1976. 35. i(NIGHT,L.C.,PRIMEAU,J.L.,SIEGEL,B.A.,WEI.CH,M.J.Comparison of In-llllabeled platelets and iodinated fibrinogen for the detection of deep vein thrombosis. J.Nucl.Med. 19, 391, 1978. 36. BAUMGARTNER,H.R. Platelet interaction with collagen fibrils in flowing blood.I.Reaction of human platelets with chymotrypsin-digested subendothelium. Thrombos.Haemostas. 37, 1, 1977. 37. COPLEY,A.L.,HUANG,C.R.,KING,R.G. Rheogoniometric studies of whole human blood at shear rates from 1000 to 0.0009 set-1 .Part I.Experimental findings. Biorheology 10, 17, 1973. 38. SCHMID-SCH&JBEIN,H. Microrheology of erythrocytes and thrombocytes,blood viscosity and the distribution of blood flow in the microcirculation.In: Handbuch der Allgemeinen PathologierIII.7.Teil,Mikrozirkulation. Springer_Verlag,Berlin,l977, p.289. 39. BAUMGARTNER,H.R. The role of blood flow in platelet adhesion,fibrin deposition and formation of mural thrombi. Microvasc.Res. 5, 167, 1973. 40. BEGENT,N.,BORN,G.V.R. Growth rate in vivo of platelet thrombi,produced by Iontophoresis of ADP,as a function of mean blood flow velocity. Nature 227, 926, 1970.
in Great
Britain
ESTIMATION OF TZBROMBUS FORMATIOK BY LABELING OF PLATELETS, RED CELLS AND FIBRIKOGEN IN EXPERIMENTAL TBROMBOSIS
R.Zimmermann, C.Zeltsch and
D.Lange *
Medizinische Universitdtsklinik (Ludolf-Krehl-Klinik) and Universitbts-Strahlenklinik (Czerny-Krankenhaus) ?? Universitat Heidelberg, 69 Heidelberg 1,Bergheimerstr. 58 GFR (Received
62.1979; in revised form 17.5.1979. Accepted by Editor S. Witte)
ABSTRACT Thrombus analyses were derived from thrombus-to-blood ratios of radiolabeled platelets (51Cr1, red cells csgFe or 51Cr) and fibrinogen (1*5I) in different thrombogenic conditions in 130 rabbits. While the composition of stasis thrombi nearly resembled that of blood the specific thrombus/blood ratio of platelets, red cells and fibrinogen was estimated at 51, 0.6 and 4 in typical arterial and 12, 2 and 7 in venous thrombi, respectively. These data suggest an increased thrombus content and significance of platelets with increasing blood flow velocities or shear rates. Red cells and fibrinogen demonstrated a different behaviour with the maximal thrombus uptake at lower blood flow velocity. Antithrombotic therapy did not give rise to any substantial alteration of the thrombus composition. Comparing the radiometric methods and the thrombus weight in estimating thrombus formation, the correlation analysis showed reliable correlation coefficients rs of more than 0.8 in the venous system. rs values about 0.6 supposed that gravimetry should be the primary method in estimating fomation of arterial thrombosis.
IN'TRODUCTION The formation of experimental thrombi can be established by their rate of incidence (1,2),subjective grading (3,4),morphometry (51,determination of the thrombus weight (61 or the flow rate of the thrombosed vessels (7).After development of techniques for radioisotope labeling of blood components especially radiolabeled fibrinogen has been used as a thrombus localizing agent in experimental (8,9,10) and clinical investigations (11,12,13).
Key
words:
Thrornbus formation, radioactive labeling, I 125 fibrinogen, radiolabeled platelets, antithrombotic therapy and thrombus composition. 147
Eo-rmerly,the experimentally induced throirbi '&ere chara.cteriaed str,~-,~. _CII-L rally by microscopy :!4,15) or on the basis of the thrombogenic conditions of the employed experimental model (16).1n the present study the simultaneous thrombus uptake of radiolabeled platelets i5iCrJ,red celis (5'Fe or 51Cr) and fibrinogen (1251) was investigated in rabbits and qcalitatlve ~anc! quantitative analyses of venous and arterial thrombi performed.In addition the incorporation of blood elements was studied iz different thrombogenic conditions and in antithrombotic therapy with antiplatelet drugs and anticoagulants.
MATERIALS AND METHODS Radioisotopic procedures. In three different series blood elements were labeled in 130 rabbits as follows: series
I (70 rabbits):
platelets 51Cr red cells 59Fe fibrinogen 1251
series
II (50 rabbits):
red cells 51Cr fibrinogen 1251
series
III (10 rabbits):
platelets 51Cr red cells 5gFe fibrinogen 125~
Autologous platelets were labeled with 51Cr according to Aas and Gardner (17) with slight modifications.Blood (8 ml) was collected 1:5 with an acidcitrate-dextrose solution (Biotest Laboratories,Frankfurt) from the central ear artery of each rabbit.The suspension of platelets was labeled by incubation for 1 hour at room temperature with 0.3 ml of 50 WCi 51Cr (230 mCi per mg of sodium chromate,Amersham Laboratories,Buckinghamshire).Thereafter platelets were resuspended and washed twice with previously saved platelet-poor rabbit plasma and reinjected intravenously within 3 hours after blood collection. Red cells were labeled in vivo by intravenous injection of 4 ICi 5gFe seven to eight days prior to the experiments (18).In series 11 red cells were labeled with S*Cr by a standard technique (19),with some modifications. 2 ml of blood were taken from the marginal ear vein 1:5 with acid-citratedextrose solution.Some of the supernatant citrate plasma was removed before incubation with 30 BCi 'lCr (sodium chromate,from Amersham Laboratories, B::ckinghamshire) for 30 min at 37 O C.The red cells were then washed three times with 0.9 % NaCl solution and reinjected intravenously. Freshly prepared,heterologous human 1251-fibrinogen from Amersham Laboratories,Buckinghamshire was administered because of its high in vivo clottability of nearly 90 % when used in the first week after iodination (20) and the nearly identical turnover of human fibrinogen in rabbits during the first days after application (21).The isotopic clottable activity was controlled according to Regoeczi (22). 4 PCi of 1251-fibrinogen were injected intravenously one hour before producing thrombi. Thrombus formation. In the experimental series I thrombosis was produced by endothelial lesions in 70 rabbits.The animals were anesthetized with sodium pentobarbital (Nembutal R) (30 mg/kg body weight) via an ear vein cannula in the ipsilateral carotid artery for radioactivity determinations
vo1.16,so.1/2
ON THROMBUS
COMPOSITIOS
z-7 coagulation analyses.Thereafter the contralateral jugular vein and the abdominalis were prepared.In the jugular veins slight endothelial lesions were induced according to the method of Schmidt (23),with slight modifications.The vessels (1 cm in length) were frozen at -2C' C for 4 min.In the aortic segment thrombus formation was induced by perivascular application of a 20 % solution of silver nitrate,as described by Jansen (24).The artery was kept in a plastiv groove (1.5 cm length) in order to achieve a standardized lesion.Thrombus formation was induced not earlier than 1 hour after application of labeled platelets and fibrinogen.After 3 hours the thrombi were carefully dissected out from the vessel,immediately (within a half minute) weighed and the radioactivity determined.
aorta
In the series II rapidly growing venous thrombi were induced in 50 rabbits and the effect of antithrombotic drugs on quantitative and qualitative thrombus formation investigated-The jugular vein was treated with 20 % silver nitrate solution,inducing a stiffening of the vessel wall with a slightly reduced blood flow.Thrombosis developed frequently within 20 min. Arterial thrombosis was induced as in the first model.In this study red cells were labeled with SICr. In the series III development of stasis thrombi was induced in 10 rabbits in the jugular vein and in the aorta by injection of contact activation product as described earlier (25).Thrombi formed within 10 min. Labeling of platelets,red cells and fibrinogen was performed as reported in the first series. Antithrombotic therapy. The 50 rabbits of series II were randomized into five groups of ten animals.The first group received 0.9 % sodium chloride solution.The second group was treated with acetylsalicylic acid (ASA) 100 mg/kg body weight per OS on the evening before starting the experiments and in addition 90 min before.A further group was given a low dose of heparin of 80 IV/3 hours continuously by an infusion pump.Ten animals received a combination of low dose heparin and ASA.The last group of ten animals were injected with heparin in a dose of 460 IV/3 hours.The antithrombotic effect was evident in all groups before onset of thrombus formation. Radioactivity determinations. The measurement of radioactivity of the three radioisotopes in the samples was carried out in a scintillation counter with a pulse hight analyser in the usual manner.The total count rate in each of the windows was corrected by background radiation,the photoeffect and the interference of the Compton effect of high energy gamma rays in lower windows.Correction factors were evaluated by counting pure standards. Calculations and definitions. For the quantitative and qualitative analyses of the experimentally induced thrombi the following definitions were chosen and computed as follows: For the three blood components,platelets,red cells and fibrinogen,the isotopically estimated thrombus size was called the 'radiometric thrombus size' and calculated from the equation: radiometric thrombus size
=
cpm (thrombus) cpm / ml blood
For characterizing the content and composition of the thrombi of radiolabeled blood elements the term 'specific thrombus/blood ratio' was chosen and computed from the equation: specific thrombus / blood ratio
=
cpm / g thrombus cpm / g blood
150
OS THRO.XEKJS COXPOSITION
vo1.16,so.1/2
In addition the platelet,red c ell and fibrinogen content was expressed count or concentration per mg thrombus weight.
as
Statistical analvsis. The correlation of the radiometric thrombus size and the thrombus weight was performed by the Spearman rank correlation, because the distribution of the original values was far from normal.The effect of the antithrombotic therapy was controlled by a one way analysis of variance in the usual way.The original data xi were transformed according tO Ti = 100 log [ 100 ( Xi + I ) ].As level of significance p = 0.05 was chosen.
RESULTS
Thrombus composition. The specific thrombus/blood ratios of the labeled blood elements are represented in the Table I.In series I in the arterial system the uptake rate-of slCr labeled platelets was 33.This indicates that the platelet content of the thrombi was 33 times that of an equivalent weight of blood.In the ten largest and occlusive thrombi the specific thrombus/blood ratio was estimated at 25,while the ten smallest and non-occluding thrombi showed the highest platelet uptake of 50.0. The 5gFe labeled red cells were incorporated at only half the concentration of blood. 1251-fibrinogen uptake was 4.5 times higher,in comparison to normal blood and amounted at 7.6 in occlusive and 3.7 in the smallest thrombi.In the venous system (series I) the incorporation of platelets was smaller than in the arterial thrombi,while specific thrombus/blood ratio of red cells and fibrinogen was measured at higher values.In the thrombosis model II ( Table I) the incorporation of 5ICr labeled red cells and 1251-fibrinogen was investigated in rapidly growing venous thrombi.The specific thrombus/blood ratios of stasis thrombi (series III) are shown in the bottom of Table I. The computed thrombus content of platelets,red cells and fibrinogen are given in the Table II as count or concentration for a defined thrombus weight. Experimental conditions and thrombus composition. Figure I illustrates the incorporation of the three measured blood elements dependent on the thrombogenic conditions of the three experimental models in relation to the supposed blood flow velocity.The four experimental conditions used represent different shear rates,increasing from the model III to maximal values in the model I. Because of the induced by silver nitrate slight stiffening of the vein wall the blood flow can be assumed to be slower in the model II and stagnant in areas of stasis as in the model III.The incorporation of blood platelets increased from the model III to a maximum in the aorta (model I), while the uptake of fibrinogen and red cells decreased under these conditions. Antithrombotic therapy and thrombus formation. The antithrombotic effect of antiplatelet drugs and anticoagulants on tbrombus formation was measured by gravimetry and radiometrically from the equation cpm cpm
( thrombus f / ml blood
.
In the venous system a significant reduction of the thrombus growth was found only under treatment with sodium heparin (Liquemin R) (p' 0.051 (Table III). Under antithrombotic therapy a slight but not statisticaly significant alteration of the specific thrombus blood ratio could be seen (Table III). Especially high and low doses of heparin did not change the incorporation of 1251-fibrinogen. The measurement of 51Cr labeled red cells did not show a significant reduction of the incorporation under therapy with antitbrombotic drugs.
03 THROHBGS
VOl.l6,XO.l/2
1-1
COMPOSITIOS
Specific thrombus/blood ratios of the labeled blood elements in the arterial and venous thrombi of the series I,11 and III.The values are given in group medians and (means).In addition to the total the ratios of the ten smallest and ten largest thrombi were presented.All of the greatest venous and nearly all (9 of 10) of the greatest arterial thrombi were occluding.
series I
arterial thrombi
platelets
(51Cr)
red cells
CsgFe)
fibrinogen (1251) weight
venous thrombi
(mg)
platelets
cSICr)
red cells
tsgFe)
fibrinogen (l-251) weight
(rng)
series II red cells venous thrombi
fibrinogen weight
c5kr) (1251) (mgl
arterial thrombi
platelets
(51Cr)
red cells
t59Fe)
fibrinogen (1251) weight
venous thrombi
(mg)
platelets
(5kr)
red cells
tsgFe)
fibrinogen (1*5I) weight
total
50.8 (48.9: 0.6 (0.6) 3.7 (6.5) 3.2 (6.6)
33.0 (5C.2) 0.6 il.71 :. 5 (6.1) lC.5 (18.3)
24.7 (28.7) 0.9 (4.7) 7.6 (6.6) 60.2 (52.7)
20.1 (32.2) 2.8 (5.5) 4.4 (8.02 4.5 15.61
12.2 (19.0)
5.7 (6.7) 2.2 (2.3) 3.4 (5.4) 41.6 (65.3)
small
total
1.3 (1.1) 3.1 (4.3) 3.1 (4.3)
,f:Z, 6.8 (12.7) 8.9 (23.5)
1.5 (1.5) 4.9 (5.5) 5.1 (5.7) total
serifs III
(mg)
occluding
small
2.7 (3.0) 1.3 (1.2) 2.8 (2.3) 58.4 (60.3) 2.7 (2.9) 1.5 (1.7) 2.6 (2.2) 64 5 (72:31
occluding 1.8 (1.8) 5.2 (4.9) 13.3 (15.0)
VO1.16,XO.1/2
Platelet, red cell and fibrinogen content of thrombi.
series I
arterial thrombi
venous thrombi
venous thrombi
total
24.0 (23.0)
15.6 (23.7)
(5lcr)
red cells 106/mg
(59Fe)
3.2 (3.4)
3.5 (9.5)
5.1 (3.2)
fibrinogen (1251) m9/g
8.2 (14.3)
9.8 (13.5)
16.7 (14.6)
~$~~$e'"
(5lCr)
9.5 115.2)
5.8 (9.0)
2.7 (3.2)
~$,~~lls
(59Fe)
15.5 (30.4)
12.1 (28.0)
12.0 (12.7)
fibrinogen (1251j mg/9
9.8 (11.9)
15.1 (28.11
7.4 (17.1)
series II
small
total
red cells 106/mg
11.7 (13.6)
occluding
(slCr)
7.3 (6.2)
8.4 (8.4)
10.1 (10.1)
fibrinogen (12~~) 106/mg
6.8 (9.5)
10.8 (12.1)
11.4 (10.8)
total
y;;;etets
(5iCr)
1.3 (1.4)
red cells l@/mg
(5gFej
7.1 (6.6)
fibrinogen (125,) mg/9
6.2 (5.1)
1.3 (1.0) venous thrombi
occluding
y$;;;ets
series III
arterial thrombi
small
tsgFef
8.5 (9.4)
fibrinogen (12511 mg/g
5.8 (4.9)
red cells 106/mg
~
Vo1.16,No.1/2
ON THROMBUS
4-
-c-
COMPOSITION
,----_
153
---_ ---_
/
/
--a
/
/
d’ *.....-**
_*...... *I’- .*a..
. . ...* .-*...*
O................‘Q“
*...
....
*...
-...
*...
a...
.. . .
....
.*..
a...
-...
“0
L
‘It
1
P-
I
I-
I-
inncasingblood flowmtt FIG. I or Thrombus incorporation of 5lCr labeled platelets (A---&,5gFe 51Cr labeled red cells (O.*.O) and 1251-fibrinogen (w-+ in the series I - III. TABLE III Thrombus weight and specific thrombus/blood ratios of venous thrombi in antithrombotic therapy (series II).
Control
ASA
Heparin low
Heparin low •t ASA
Heparin
weight (mg)
5.1 (5.7)
4.0 (4.5)
1.6 (2.5)
1.5 (2-O)
0.9 (1.2)
fibrinogen
5.0 (5.2)
5.0 (6.4)
5.4 (6.6)
3.3 (4.5)
4.5 (4.9)
red cells
1.6 (1.8)
1.6 (I.71
1.5 (1.3)
1.4 (1.3)
1.2 (1.1)
OS THFtO,MXJS COHPOSITION
TABLE The
results by
of the correlation analysis the correlation coefficient
series
arterial thrombi
venous thrombi
I
are presented (rs).
rs
n
platelets
tSICr)
0.62
57
red
cells
csgFe)
0.64
60
fibrinogen
(1251)
0.55
60
platelets
(51Cr)
0.8
35
red
cells
(5gFe)
0.83
39
fibrinogen
(1251)
0.76
43
series venous thrombi
IV
red
II
rs
n
cells
(51Cr)
0.92
43
fibrinogen
(1251)
0.84
43
Correlation analysis of radiometrically and gravimetrically measured thrombus size. The reliability of isotopic methods and gravimetry in estimating thrombus formation was controlled by means of the Speannan rank correlation. The results of the statistical calculations are represented by Table IV by the correlation coefficient (rs). rs ranged from-O.5 - 0.6 in the arterial system.In the venous system a rs of more than 0.8 could be observed.
DISCUSSION Analysis of the thrombus structure by radioisotopic methods provides a reliable labeling procedure without separation of radioactivity and uneffected functions of the labeled blood elements.In the present study platelets were labeled using the slightly modified technique of Aas and Gardner (17) with addition of ascorbic acid and reinjected already 3 hours after the labeling procedure;thus an intact platelet function could be preserved 126, 27).Investigating of the radioactivity of the platelet sediment,platelte Cr rich plasma and whole blood confirmed that only minimal quantities of (< 3 %) separated from the labeled platelets.Radioactive iron is rapidly cleared from plasma,incorporated in the hemoglobin molecule and then labels selectively red cells (18).The used 125I-fibrinogen demonstrates a high clottability of more than 80 %,when employed in the first days of radioiodination as described by Bradley (ZO).Thus ,it can be supposed that the measured radioactivities nearly represent only these three blood elements. In the arterial system the specific thrombus/blood ratio for 51Cr labeled platelets was measured at 51 in the small non-occluding thrombi,while a specific thrombus/blood ratio of 33 was estimated as group median in this first series.Likewise,in the venous system the highest uptake rate was calculated at 20 in the small thrombi,whereas it decreased when the thrombi
VO1.16,NO.1/2
ON THROMBUS
COMPOSITIOX
155
became occlusive.These results confirm the significance of platelets in the formation of arterial thrombi as known from morphological investigations (14,15,28),in vitro experiments (5,29,30) and platelet turnover studies in man (31).In the initial stages of venous thrombus growth the platelets are still the predominant blood elements if thrombi are forming at the site of endothelial lesions.The highest specific thrombus/blood ratios of platelets were found in the smallest thrombi,representing the initial stages of thrombus formation or the structure of small but not further grown thrombi. In the stasis thrombi the specific thrombus/blood ratio for platelets nearly resembled that of blood and represents structurally the so called 'whole blood clot' (14,321. The specific thrombus/blood ratio of 59Fe or 51Cr labeled red cells was calculated in arterial thrombi at nearly half the concentration of that found in the blood.In the venous system the uptake rates of the red cells ranged from 1 to 2 in the thrombosis model I and II and were nearly identically to that of the stasis thrombi as shown in the model III. The specific thrombus/blood ratios of lz51-fibrinogen amounted to nearly similar values of 6.8 and 4.9 in the venous system and 4.5 in the aorta, respectively.The mode of thrombus incorporation of radiolabeled fibrinogen is up to now an unsolved problem.Radiofibrinogen will be incorporated as well into a forming as into a recently formed thrombus, 18 to 72 hours after onset of the thrombus formation (33,34,35).The highest uptake rates of 17 to 23 were reported by Coleman (33) and Knight (35) when radiofibrinogen was applicated 4 to 6 hours after starting thrombus production.More than 22 hours old thrombi showed radioactivity ratios of only 2-3 (34,35).In view of these data and our findings we can suggest that not only growth but also an alteration in structure occurs in the first min as well as in the first three hours of thrombus formation. The wall shear rates of the vessel areas used for inducing experimental thrombi cannot be calculated accurately in vivo.In our study the presented thrombosis models or vessel areas suggest different blood flow velocities or shear rates,respectively.According to Baumgartner (361,Copley (37) and Schmid-Schonbein (38) the wall shear rates can be estimated at values of about 800-1000 see-1 in the rabbit aorta abdominalis,at lower values in the venous system and at a shear rate of 0 sec- 1 in areas of complete stasis. Our results obtained in rabbits in vivo demonstrated an increasing specific thrombus/blood ratio for 5lCr labeled platelets in thrombogenic conditions with increasing blood flow velocities or shear rates.The highest flow rate canbe assumed in the model I in the arterial system-The presented data are consistent with the findings of other authors,who described a shear rate dependent platelet deposition on subendothelium or growth rate of platelet thrombi (29,39,40).According to Richardson (29) the platelet deposition rate will again decrease if theoretical very high shear rates at higher blood flow velocities were reached.Such conditions were not attained in our experimental models in vivo.In analogy to the refered behaviour of platelets,red cells and fibrinogen demonstrated a similar blood flow dependent incorporation-The maximal thrombus uptake or deposition was seen at lower shear rates in the venous system and decreased at higher blood flow velocity as demonstrated i..the arterial system. On the basis of the heparin-induced inhibition of the fibrinogen-fibrin converslon,a reduced uptake or a decrease of the specific thrombus/blood ratio of lz51-fibrinogen Igould be conceivable.The thrombus formation could
have been quantitativeiy inhibited by heparin,but without any change in the 1251-fibrinogen content.Antit;hrombotic therapy with antiplatelet drugs caused no substantial alteration of the thrombus compositon. Evaluation of gravimetry and the radiometric methods in estimating quantitative thrombus formation by the correlation analysis in the venous system demonstrated a reliable agreement with a correlation coefficient rs of more than 0.8 in all models.In the arterial system the thrombus composition differed more obviously from that of blood and showed a rs of about 0.6. In view of these results we conclude,that venous thrombus formation can be reliably estimated by labeling of blood elements.In the arterial system gravimetry should be the primary method in controling thrombus growth.
ACKNOWLEDGEMENTS We thank Dr.G.Weckesser,Institut fur Dokumentation und Statistik der Universitat Heidelberg,for valuable assistance in statistical analyses.
REFERENCES 1.
WESSlER,S.,MORRIS,L.E.Studies in intravascular coagulation.IV.The effect of heparin and dicumarol on serum-induced venous thrombosis.Circ. 12, 553, 1955.
2.
MARBET,G.A.,DUCKERT,F. Action of coagulation factors on experimental thrombogenesis and their changes after thrombotiis.Thrombos.Diathes. Haemorrh. 29, 619, 1973.
3.
KRICHEFF,I.I.,ZUCKER,M.B.,TSCHOPP,T.B.,KOLODJIEZ,A. Inhibition of thrombosis on vascularcatheters in cats. Diagn.Radiol. 106, 49, 1973.
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ZIMMERMANN,R.,BARTH,P.,EHLERS,W. Effects of acetylsalicylic acid,heparin and phenprocoumon on formation of experimental red thrombi.In:Platelets, recent advances in basic research and clinical aspects.Ulutin,O.N.(ed.), Exc.Med.,Amsterdam, 1974, p.378.
5.
BAUMGARTNER,H.R.,MUGGLI,R.,TSCH~PP,T.B.,TURITTO,V.T. Platelet adhesion, release and aggregation in flowing blood.Effects of surface properties and platelet function. Thrombos.Di.athes.Haemorrh. 35, 124, 1976.
6.
ZWEIFLER,A.J. Standardized technique for the study of thrombus growth: Effect of heparin therapy. J.Lab.Clin.Med. 60, 254, 1962.
7.
DANESE,C.A.,VOLETI,C.D.,WEISS,H.J. Protection by aspirin againstexperimentally induced arterial thrombosis in dogs. Thrombos.Diathes.Haemorrh. 25, 288, 1971.
8.
HOBBS,J.T.,DAVIES,J.W.L. Detection of venous thrombosis with 1311-labeled fibrinogen in the rabbit. Lancet 2, 134, 1960.
9.
CHIU,H.M.,HIRSH,J.,YUNG,W.L.,REGOECZI,E.,GENT,M. Relationship between the anticoagulant and antithrombotic effects of heparin in experimental venous thrombosis. Blood 49, 171, 1977.
10. KAUFMANN,H.H.,WOO,J.,ANDERSON,J.H.,CANNON,D.C.,BANDEL,S.F.,REVEZI,J.M. Radioiodinated fibrinogen for clot detection in a canine model of cervical carotid thrombosis. J.Nucl.Med. 19, 370, 1978. 11. MEDICAL RESEARCH COUNCIL (Report of the Steering Commitee).Effect of aspirin on postoperative venous thrombosis. Lancct 2, 441, 1972.
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COMPOSITIOS
15:
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