Vascular sequestration of heparin

Vol.

IXROMBOSIS RESEARCH Printed in Great Britain

12, PP. 79-90, Pergamon Press,

1977 Ltd.

VASCULAR SEQUESTRATIONOF HSPAIUN

J. Nahadoo, L. Hiebert and L.B. Jagues Hemostasis - Thrombosis Hasearch Unit, Departmentof Physiology, Universityof Saskatchewan, Saskatoon, Saskatchewan,Canada S7N OWO.

(Received

10.10.1977; Accepted

by

in revised Editor

M.I.

form 24.10.1977. Barnhart)

ABSTRACT

Heparin was administeredintravenouslyand by intratrachealinstillation to rats. With %I-heparin,radioactivityaccumulatedin endothelium and vessel wall of aorta and vena cava. These vessels were also removed from rats receivinghigher doses of cold heparin, extracted for mucopolysaccharidesand heparin identified and measured by microelectrophoresis. This procedure revealed the presence of more heparin on the endotheliumof the aorta than of the vena cava. Despite the presence of high radioactivityin the vessel wall denuded of endothelium,no heparin was obtained by the chemical extraction, suggesting a heparin metabolizingactivity in the endothelium and/ or vessel wall. Haparin was also administeredintravenouslyto rats and by intratrachealinstillationto mice. The drug was identified by light and electron microscopy in the endothelialtissue of rat aorta and of mouse pulmonary vessels.

INTIUDDUCTION The main factors promoting thrombosisare described by Virchow's triad: (i) changes in the blood composition, (ii) slowing of the blood stream and (iii) changes in the vessel wall. It is now generally accepted that thrcnnbosis is of multiple etiology and that more than one of the factors mentioned above are necessary for intravascularclotting. Over 40 years ago, heparin was introduced in clinical medicine as an antithromboticagent. It is generally accepted that the antithromboticeffect of heparin resides in its action on blood composition. Heparin in conjunctionwith its co-factor Antithrombin III inhibits Factor Xa and thrombin (11 thus preventing the formation of a thrombus. Heparin may also lower the viscosity of blood thereby facilitating blood flow (2). Heparin increases the negative change on blood cells 79

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and the vessel wall, thereby having an antiaggregatingand antithromboticeffect (3). It is known that a rough surface (uncoatedglass, damaged endothelial lining, etc.) promotes clot formation and that coating plastic prostheses with heparin prevents formation of platelet deposits and thrombi. Hiebert and Jaques (4) have reported that endothelium accumulatesheparin after injection. The electronegativityimparted to the endothelium (5) will then prevent platelet or red cell deposit on vessel wall. Thus heparin inhibits all three aspects of Virchow's triad. However, least studied is its interactianwith the third component of the triad - the vessel wall. It has recently been shown that intrapulmonaryadministrationof heparin produces a moderate degree of hypocoagulabilitysustained over a period of days (6). The lung is easily isolated and examined. Previous studies have shown that most of the heparin is cleared from the lung within three hours (6). This suggests that the heparin may be stored in a cellular pool. We have now examined the heparin content of plasma and vascular tissue after intravenousand intrapulmonaryadministrationof the drug in order to find the extent of vascular sequestrationof heparin. We have also examined the vascular tissue by light and electron microscopy to obtain qualitativeevidence of this sequestration.

MATERIALS

ANDMETHODS

Heparin: Sodium heparin used for histologicalstudies in mice was from pig intestinalmucosa, Lot #1102-12, USP activity 157 u/mg. This was a gift from Fine Chemicals Division, Canada Packers Ltd. 3H-Heparinwas a gift from Mr. G. Barlow, Abbott Laboratories,North Chicago, U.S.A. It had a specific activity of 18.9 @/mg and a stated USP activity of 117 u/mg. In a dialysis test none of the radioactivitywas dialysable. Panheparin used for heparin determinationin blood and vasculaturewas of bovine mucosal origin. This was a gift from Abbott LaboratoriesLtd. Montreal. Wistar Albino rats weighing 300 to 400 grams each were divided into four groups. In the first group four rats received each 2000 units of Panheparin intravenously. These rats were used for the determinationof heparin in blood vessels by chemical extraction as well as by histologicaltechniques. In the second group four rats received 2000 units of Panheparin by intratracheal instillation. These rats were used for the determinationof heparin in plasma and in blood vessels. In the third group eight rats received 50 units of %-heparin: seven by intravenousinjection and one by intratracheal instillation. In the fourth group eight rats were used as controls and each received 0.1 ml of physiologicalsaline: four by intravenousinjecticn and four by intratrachealinstillation. Swiss Albino mice weighing about 30 grams each were divided into two groups. One group of four mice received 500 units of sodium heparin by intratrachealinstillation. The second group of four mice received 0.1 ml of physiologicalsaline also by intratracheal instillation. These mice were used for histologicalexamination of the lung.

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In all cases the volume of solution injected intravenously or instilled intratracheally was 0.1 ml. Fifteen minutes after administration of heparin or saline to rats, a mid-line incision was made to expose the aorta and inferior vena cava. Samples of arterial and venous blood were obtained from these vessels within 30 seconds of each other. The aorta and superior vena cava were then removed and their heparin content determined by chemical means when cold heparin or saline was injected and by radioisotope counting when 3H-heparin was injected. Samples of lung were taken for histological observation. The albino mice were also killed fifteen minutes after administration of heparin or saline. Samples of lung tissue were taken for histological examination. Determination of heparin in vascular tissue: Vessels were dissected out, washed thoroughly in Locke's solution and placed on a sheet of clean dental wax. The vessel was slit open and pinned down flat. The endothelium was removed by cellulose acetate paper which was then dissolved in cold acetone according to the method of Hiebert and Jaques (4) to produce a dry endothelial powder. A polysaccharide fraction was prepared from the vessel wall remaining after endothelial stripping with cellulose acetate paper by the method of Jaques and Debnath (7). The heparin content of these extracts was determined by the microelectrophoresis technique on agarose gel (8). For radioactivity determinations, the blood vessels were removed and washed as above and endothelium removed by cellulose acetate paper. The cellulose acetate paper with the endothelium on it was put in a vial containing 10 ml of Aquasol. The remaining vessel wall was digested with 0.1 ml of a 10% perchloric acid and decolorized with 0.2 ml of 30% (W/V) hydrogen peroxide. This colorless digest was mixed with 10 ml of Aquasol in a vial. The samples thus prepared were counted in an Isocap 300 liquid scintillation counter. Determination of heparin in blood: The samples of blood obtained from the rats were centrifuged at 3000 g to obtain plasma. This plasma was then centrifuged at 20,000 g to obtain platelet poor plasma which was used for the determination of heparin content by the coagulation test of Yin et al (9). For radioactivity determination procedure, 0.1 ml of the platelet poor plasma was mixed in 10 ml of Aquasol in a vial and counted in the Isocap 300 counter. Histological Preparation: Samples of rat aorta, vena cava and mouse lung from control and experimental animals were fixed in gluteraldehyde, cacodylate buffer and ruthenium red (1:l:l) for 1 hour. After a 5 minute rinse in cacodylate buffer they were further fixed in osminum tetroxide (4%), cacodylate buffer and ruthenium red (1:l:l) for another hour. Gradual dehydration was then carried out in 50%, lo%, 80%, 95% and absolute alcohol. After soaking in propylene oxide for 15 minutes, the tissue samples were gradually embedded in epon. One micron sections of these tissues were cut and stained with 1% Toluidine blue in borax for examination at the light microscopy level. Ultrathin sections were cut and stained in uranT:l acetate and lead citrate for electron microscn~~-,.

RESULTS

Results obtained when heparin content of aorta and superior vena cava were detenrined by extraction and chemical determination are shown in Table

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I. Heparin was found on aortic endothelium of all rats 15 minutes after intravenous injection and on two out of four rats 15 minutes after intratracheal instillation.

Heparin was found on vena caval endothelium of two

rats after intravenousinjection but no heparin was found on the vena caval endotheliumof rats after intratrachealinstillation. The heparin found on endothelium reflects the heparin sequestered from blood. The heparin concentrationin plasma is higher 15 minutes after intravenousas compared to intratrachealinstillation. Heparin could not be identifiedon the remainder of the vessel wall (containingthe media and adventitia)of the aorta or superior vena cava when heparin was given either intravenously or by tracheal instillation. TABLE

I

Heparin concentrationin vascular tissue after administration of 2000 units

I-tat

Heparin concentrationin vascular tissue units/&

IO.

Remaining

Indothelium

Tissue

1

I.V.

0.035

0

2

I.V.

0.042

0

3

I.V.

0.052

0

4

I.V.

0.039

0

0

5 Intratracheal

I

0.080

0

0

0

0.020

0

0

0

0

0

0

0

0

O

0

6

"

0.035

0

7

"

trace

0

8

"

0

0

I

1

IA

A01

Route

I

I I

0

n

In Figure 1 is shown a diagram of microelectrophoresisslides of endothelium and the remainder of vena cava and aorta after intravenousinjection of heparin. A band with migration similar to heparin can be observed when the polysaccharidefraction of aortic or vena caval endothelium is applied to the slide. When the remainder of the aorta or of vena cava is applied.to the slide some mucopolysaccharidecan be observed but none of these bands are comparable to that obtained when referenceheparin is applied or that found when endotheliumis applied. These bands are similar to that seen when the polysaccharidefraction of aorta or vena cava from control rats are applied.

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AE 0-

R

VCE

83

Ul'TAKS OF HEPARIN

R

A -0

00

R

VC

R

-e

+ FIG. 1 Electropherogram of polysaccharide fraction of blood vessels. A: Aorta; AR: Aortic endothelium; VC: Vena Cava; VCR: Vena caval endothelium; R: Reference heparin. 50 units/ml. (Lot #4439, Leo Pharmacutische Production N.V. Emmex, The Netherlands. Note:

The absence of polysaccharide of migration rate similar to heparin from vessel wall extract. In Table II are shown results for the concentration of heparin in the

plasma of aortic and vena caval blood 15 minutes after intratracheal instillation.

It can be seen that the arterial heparin concentration is consis-

tently higher than the venous heparin concentration for all rats examined. The arterio-venous difference is probably party due to uptake by the endothelium.

As shown in Table I heparin does accumulate in the endothelial tis-

sue following intravenous and intratracheal administration of the drug.

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TABLE

OF HEPAFUN

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II

Heparin concentration in plasma after intratracheal instillation of hepars

Pat No.

Heparin Concentration Arterial venous Plasma Plasma

A-V difference

A-V Diff. Circ. time

Amount cleared Circulation time

Plasma Volume

1.

0.72 u/ml

0.58 u/ml

0.14 u/m

0.02 u/ml

12.46 ml

0.25 unit

2.

1.50

w

1.10

"

0.40

"

0.05

n

13.50 *

0.67

"

3.

0.36

u

0.18

Im

0.18

Ia

0.02

"

11.76 w

0.23

"

1.00

It

0.88

"

0.12

w

0.02

"

0.27

"

4. 5.

17,384 B 11,614 cpm 5,770 z ml. ml. ml. ,

769 CJIJ ml.

13.69 " 12.85 "

9,882 cpm

Dose of Sodium heparin administered = 2,000 units. Circ. time = The vena cava - aorta circulation time, which in our preparation = 4 sets. In Table III are shown radioactive counts for plasma, aortic and vena caval endothelium, and the remainder of these vessels after administration of heparin.

In agreement with results in Table II, the concentration in

arterial plasma is usually higher than in venous plasma. TABLE III Distribution of radioactivity in aorta and vena cava after administration of H3-Heparin to rats. ROUtIS

lOSe lnitr

rimee

VENA C

AORTA

a

IA

wall c&cm:

Lz?!&!L

256

117,840

65

Plasma

endotheliuu Wall 2 lxn/cm2 * CPm/cm

50

15

125,110

65

I.V.

50

15

138,820

25

921

127,760

144

5373

I.V.

50

15

182,790

43

1003

196,700

128

10,936

I.V.

50

15

146,330

28

3308

123,760

0

0

1-v.

50

15

20,673

371

0

20,964

435

0

I.V.

50

15

12,303

159

0

11,758

88

0

I.V.

50

5

175,420

20

455

151,750

24

50

15

17,384

43

228

11,614

0

I.V.

Intratra

~cheal

188

414' 995

Table IIIC inlso shows accumulation of radioactivity in high amounts in the vessel wall (media and adventitia).

This is in sharp contrast'with the

observation in Figure 1 and Tab19 I where chemical extraction shows the presence of heparin only in the endothelium.

No heparin was recovered by

chemical extraction to correspond with the presence of high radioactivity in the remainder of the vessel wall.

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Heparin can be detected histologically in the endothelial tissue after both intravenous and intratracheal administration of the drug.

Figure

2

shows control and experimental endothelial tissue following intravenous injection.

Figure 2a is an endothelial cell taken from the aorta of a control

Figure 2b is an endothelial cell taken from the aorta of a rat 15

rat.

minutes after an intravenous injection of heparin.

There is an increase in

ruthenium red staining on the surface of the endothelial cell as compared to controls.

a

Endo-endothelium

FIG. 2 Aortic endothelium from rats. Magnification x 24,000.

"a" is endothelium from a control rat.

"b" is en-

dothelium from a rat 15 minutes after an intravenous injection of heparin. Note:

The thicker endo-endothelial layer in "5".

Figure 3 shows control and experimental pulmonary vessels 15 minutes after intrapulmonary administration of saline and heparin respectively. Figure 3a and 3b show light photomicrographs of 1 u sections stained with toluidine blue.

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Figure 3: Pulmonary Vessels from mouse Magnification x 6000.

(a) Pulmonary vessel from control mouse.

ary vessel from experimental mouse.

(b) Pulmon-

Note the presence of heparin-toluidine

blue particles only in the experimental vessel.

Figure 4 is an electron photomicrograph of a pulmonary vessel dissected 15 minutes after intratracheal administration of 5 mg of heparin.

The hep-

arin can be detected as electron dense material accumulated in the endothelium.

Ruthenium red was used as a marker and it was introduced early in the

fixation of the tissue blocks.

Since heparin appears in vesicles and ruth-

enium red does not cross the cell membrane it appears, that these vesicles are continuous with the cell membrane.

This photomicrograph also illustrates

the vesicular mode of transport of heparin across the vessel wall from the alveolar space.

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Figure 4: Pulmonary blood vessel from mouse. Magnification x 54,000. Note:

Cluster of electron dense particles (Heparin-Ruthenium red complexes) indicated by arrow.

DISCUSSION From our results it is obvious that heparin after intratracheal administration enters the blood stream and is dealt with in the same way as intravenous heparin.

During the phase of absorption from the lungs there is

a continuous addition of heparin to the blood on the arterial side of the systemic circulation.

Our results show that this heparin is taken up from

the blood by the endothelium of the blood vessels in a similar manner as after intravenous heparin.

The magnitude of endothelial uptake depends on

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the heparin concentrationin the blood (4). This is why, after intravenous injection of 2000 units of heparin we observe an amount of the drug in the endothelium larger than is found after intrapulmonaryinstillationof the same dose. The data reported in the Tables II and III allow calculationsto determine the quantitativesignificanceof this endothelialuptake. Ffsnalclearance of heparin occurs with plasma levels at and above 14 units per ml. Hence this is not a significantfactor in these experiments,as also indicated in (6). For samples used for the following calculationsthe plasma concentrationwas 0.4 to 1.5 units/ml. Table III indicates the approximate extent of endothelialuptake of heparin after intravenousinjection of 50 units of 31i-Iieparin in rats. Since all the heparin is already in the blood volume, the plasma arterio-venousdifference is mainly due to clearance by endothelialuptake. The arterio-venousdifferencewas 26,046 cpm/ml/min. Our calculationsindicate that at the 15 minute mark 12.9% of the administered dose was cleared per minute. The net plasma clearance was 65% after 15 minutes. Out of that, 0.01% was found on the endothelium of the aorta. As it would appear that the circulatingheparin comes in cantact with a much larger amount of endothelium than is present in the aorta, this could account for the sequestrationof the large amount of heparin that has been cleared from the plasma. The arterio-venousdifference in heparin concentrationin plasma after intratrachealinstillationof the drug shown in Table II is due to (1) the endothelialuptake of the drug and (ii) the additicn of heparin from the lungs to the arterial side of the systemic circulation. The mean concentrationof plasma heparin was 10.4 units (plasma volume x mixed venous concentrationof heparin). From data previously obtained (6) the amount of heparin that would leave the lung in 15 minutes is about 6.5% of the dose administered. This is equivalent to 130 units. Therefore the amount of heparin cleared from plasma would be the amount that has left the lung minus the amount present in the plasma. This calculationgives a clearance of 5.9% of administereddose at 15 minutes or a clearance rate of 0.62 unit/ml/min. 0.2 unit or 0.01% of the heparin that has left the lung was recovered from the* total aortic endothelium alone. It would appear that the remaining 99.99% could be easily sequestratedby the thousands - fold larger endothelialsurface that is in contact with the circulatingheparin. COlumns 5 and 8 of Table III show the amount of radioactivityfound in

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each square centimeterof endothelialsurface, and columns 3 and 5 of Table I show the amount of extractableheparin from an equal area of endothelium. Hence we can compare observationsobtained simultaneouslyof arterio-venpus difference in heparin concentrationin plasma, and the endothelialaccumulation of heparin as detected by isotope technique,by chemical means and by histologicaltechnique. The heparin found in the endotheliumafter intravenous or intrapulmonaryadministrationof the drug appears to be identical to the administeredheparin. The presence of radioactivityin the remainingwall of the vessels without any extractableheparin suggests that some metabolism of the drug may occur in the endotheliumand/ or the vessel well.

ACKNOWLEDGEMENT This study was supportedby grants-in-aidfrom the SaskatchewanHeart Foundation and Canada Packers Ltd., Toronto, Ontario. We are greatly indebted to Miss Sharon Steckler for skilled technical assistance. REFERENCES

1. WESSLER, S. and YIN, E.T. On the antithromboticaction of heparin. Thrombos. Diathes. Haemorrh. (Stuttg.)32, 71, 1974. 2. SRINIVASAN,S., DUIC, L., RANASAMY,N. and SAWYER, P.N. Electrochemical reactions of blood coagulationfactors - their role in thrombosis. Eerichte der Bunsen-GesellschafffUr PhysikalischeChemie Ba. 77, #lo/ 11, 799, 1973. 3. DINTENFASS,L. ted.) Blood microrheology. Butterworth and Co. (Publishers)Ltd. Trowbridge and London. 1971, p. 217-218. 4. HIEBERT, L.M. and JAQUES, L.B. Heparin uptake on endothelium.Artery 2 (1) 26, 1976. 5. SRINIVASAN,S., AARON, R., CXOPRA, P.S., LUCASE, T. and SAWYER, P.N. Effect of thromboticand antithromboticdrugs on the surface charge characteristicsof canine blood vessels: in vivo and in vitro studies Surgery. 64, #4, 827, 1968. 6. JAQUES, L.B., MAHADDC, J. and KAVANAGH, L.W. Intrapulmonaryheparin: A new procedure for anticoagulanttherapy. The Lancet 1157, 27th Nov. 1976. 7. JAQUES, L.B. and IEBNATH, A.K. Simultaneousevaluationof tissue heparin and mast cells in small tissue samples. Am. J. Physiol. 219, 1155, 1970.

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8. JAQUES, L.B. and WOLLIN, A. Identificationand quantitationof heparin by microelectrophoresison agarose gel. Anal. Biochem. 52, 219, 1973. 9. YIN, E-T., WBSSLER, S. and BUTLER, J.V. Plasma heparin: unique practical, submicrogram- sensitive assay. J. of Lab. and Clin. Med. 81, 298, 1973.