Acta physiol. scand. 1972. 85. 523-531 From the Institute of Physiology and the Institute of Forensic Medicine University of Oslo,

Norway

Effects on the Pulmonary Circulation of Suddenly Induced Intravascular Aggregation of Blood Platelets

BY G. BPI and J. HOGNESTAD

Received 18 January 1972

Abstract

Ba, G. and J. HOGNESTAD. Effects on the pulmonary circulation of suddenly induced intravascular aggregation of blood platelets. Acia physiol. scand. 1972. 85. 523-53 1.

When intravascular platelet aggregation was induced by intravenous infusion of collagen extract, a temporary and marked increase in pulmonary vascular resistance (PVR) was observed. The number of circulating platelets was gradually reduced after the first few infusions of collagen. After 4-6 infusions of collagen the vascular response vanished, and the platelet count in arterial blood remained fairly stable. When a vasodilator was infused simultaneously with the collagen, the pressure response could be abolished. Histological examinations of lungs at the peak of a pressor response to collagen revealed aggregated platelets in small arterial vessels and capillaries. After many infusions larger vessels were found occluded by aggregated platelets. It is concluded that platelet aggregation in the blood arriving in the lung creates a strong vasoconstriction in the pulmonary vascular bed. This response is related to release of material from the platelets.

I t has been shown in previous investigations on cats that blood platelets are a prerequisite for the development of high pulmonary vascular resistance during hemorrhagic hypotension. Thrombocytopenic animals were protected against the progressive rise in pulmonary vascular resistance (PVR) which developed in animals with a normal number of blood platelets (B0 and Hognestad 1971). From the investigations of Bergentz, Lewis and Ljungquist ( 1971) it is furthermore known that labelled platelets are trapped in the lungs during shock and trauma. I t has therefore been suggested that the pulmonary hypertension seen subsequent to trauma and hypotension may in some way be caused by aggregated blood platelets and/or released material.

The aim of the present work has been to study the effect on pulmonary vascular resistance of induced intravascular platelet aggregation. Platelet aggregation was achieved by intravenous injections of tendon extract. Collagen is known to cause platelet aggregation both in vivo and in vitro. Platelets will adhere to collagen fibrils, and this will again give rise to a secondary aggregation of other platelets

523

524 G . BB AND J. HOGNESTAD

(Hovig 1963). During aggregation a platelet release reaction will also take place. Collagen itself has no effect on smooth muscle cells, and the fibrils of the extract are so small that they should not occlude the pulmonary vascular bed.

A considerable but transient increase in pulmonary vascular resistance was observed upon injections of collagen. The results of the experiments indicate that this response was not caused by physical obstruction of the vascular bed, but by contraction of smooth muscle cells in the pulmonary resistance vessels. This contrac- tion was apparently directly or reflexly induced by materials released from the platelets.

Methods Animals: Cats, weighing 2.5-4 kg were used. They were anesthetized by intraperitoneal injections (30 mg/ kg) of sodium pentobarbitone (Nembutala, Abbott).

Ventilation: After tracheostomy a muscle relaxant, Alloferine (1 /2 mg/kg) was given and positive pressure ventilation started with a piston pump respirator (The Ideal Respiration Pump, C. F. Palmer Ltd., London), The respiration frequency was 14 per min. With the use of water seals the end tidal pressures were usually kept a t 6-43 and 2 cm of water, respectively. T h e mpiratcft’s tidal volume was adjusted so as to keep the arterial Pcoz at 30 mm Hg, which is the normal level for cats. Standardized hypkrinflations were carried out carefully at regular intervals and between each test. The ratio of the respirator’s tidal volume to the inspiratory peak pressure was calculated during and between each test, and was used as an expression of the pulmonary compliance.

Surgical procedures. Pressure and flow recordings: The thorax was opened widely by a sternum-splitting incision. Catheters of polyethylene were introduced into the femoral artery, the pulmonary artery and left atrium for recordings of the femoral arterial pressure (PFA, with a Statham P23Gb transducer), the pulmonary arterial pressure (PPA, with a Statham P23Db transducer) and the left atrial pressure (PLA, with a Statham P23De transducer), respectively. Infusions were carried out through catheters placed in the femoral veins and with their tips in the caval vein.

A flowprobe was placed around the ascending aorta and flow was recorded by a Nycotron square wave flowmeter (type 372, Nycotron A/S, Norway). The pressure and flow transducers were connected to a six-channel Sanborn recorder (model 320, Sanborn Co., California).

The animals were placed on a heated table. Postoperatively they were covered by a poly- ethylene tent into which was led warm moist air. The animals’ body temperature was thereby maintained at the normal level.

Mean pulmonary vascular resistance, PVR, was calculated according to the formula:

PPA-PLA (mm Hg) Mean flow (ml/mh) PVR =

Percentage changes are given when PVR alterations in different animals are compared. Thrombocyte counts in arterial blood samples were carried out according to the method of

Brecher and Cronkite (1950). pH-measurements were done using Radiometer equipment (pH-meter 22 equipped with a

pH-electrode type G 297/G and a gas mixing apparatus). The arterial Pcoz was calculated by the equilibration method and according to Siggaard-Andersen’s nomogram. A polyethylene catheter was inserted into a femoral vein with the tip placed in the caval vein.

Collagen and bradykinin infusions: 0.5 ml of collagen suspension (prepared as described by Holmsen 1969) was infused intravenously in the course of 1 min. When the subsequent pressure response had vanished (usually after 10 min) a new infusion was given. The infusions were usually repeated until there was no pressure response to a collagen dose.

In some experiments bradykinin (Sandoz) was also infused in doses of 80 pglmin. For this infusion a Harvard Infusion Pump (model 947, Harvard Apparatus Co., Mass.) was used. Bradykinin infusions were either carried out before or simultaneously with the collagen infusions.

Histological examination of the lungs: The animals were killed by an overdose of Nembutal. The fixative (Zenker’s solution) was then poured into the bronchial tree so as to prevent collapse of the lungs.

PULMONARY CIRCULATION AND PLATELETS 525

Fig. 1. Effect in a cat of 0.5 ml of collagen adminstered intravenously on mean aortic blood flow, mean pulmonary arterial pressure ( PPA) , mean left atrial pressure (Pt.4) and mean femoral arterial pressure ( P F . ~ ) .

The lungs were then immediately removed and immersed into the fixative. Specimens from all lobes of the lung were embedded and sections 4 ,M thick were cut.

In most animals lung tissue sections were taken when several (6-7) infusions of collagen had been carried out. In 3 cats, however, only one infusion of collagen was given. The lungs were then removed at the peak of the subsequent response, and sections were taken from the hilus region as well as from peripheral parts of the lung lobes.

Results Intravenous infusion of 0.5 ml of the collagen suspension caused a sharp rise in Ppz,, usually from the mean value of about 15 mm Hg and up to about 35 mm Hg. Aortic flow simultaneously decreased by 25 per cent of the resting value. This pressure/flow response lasted for 5-7 min. Both PpA and flow returned to near initial values within 10 min (Fig. 1 ) . The-calculated pulmonary vascular resistance (PVR) consequently rose tremendously subsequent to such an infusion, the mean rise in 5 animals being almost 500 per cent. Great variations were, however, seen between the individual animals (range 1 5 7 4 5 9 ) . PL, showed a minute and transient reduction, whereas PFA fell more markedly (Fig. 1 ) .

TABLE I. Percentage rise in PVR in 5 animals upon repeated infusion of collagen.

lnfusionsno. A B C D E

1 2 3

- 4 5 6 7

157 500 859 208 669 143 416 769 2 00 669 114 942 1102 183 717 107 447 663 166 669

- - 596 526

- - - - - -

- c--o---o

s - - E 18 - E - - - -

6 - 2 - -

Fig. 2. Pulmonary vasopressor responses to repeated infusions of collagen in the same cat. Interval between infusions 10-1 5 min.

I I

300 6 I

E E

200 f - z d

100 LL.

Subsequent to the first infusion the number of circulating thrombocytes in arterial blood was found to be reduced by about 20 per cent. In some experiments platelet counts in arterial blood were taken both at the top of an infusion response and afterwards. A consistently higher count was always obtained after the response rather than during its peak.

The ratio of the respirator’s tidal volume to the respiratory peak pressure fell during the response to a mean of 60 per cent. This ratio returned to its preinfusion level simultaneously with the normalization of flow and pressure.

The following trend was then observed (Table I and Fig. 2 ) : In 5 animals the first infusion of collagen was followed by 6-7 subsequent infusions. The first 4 infusions caused a marked rise in PVR in all the 5 animals. Four of the animals did

lnfu sions i n m i v v v r v u

Fig. 3. Platelet count in 5 animals during a period of repeated intravenous infusions of collagen.

PULMONARY CIRCULATION AND PLATELETS 527

Fig. 4. Influence of simultaneous bradykinin infusion on the pulmonary vasopressor response to collagen infusions. Collagen response exhausted at infusion no. 5.

not respond to further infusions. One animal (E) responded also to the 5th and the 6th infusions, but there was no response to the 7th. The platelet count in arterial blood (Fig. 3) decreased in all animals up to the 3rd infusion. In 4 animals it then seemed to stabilize or even rise moderately. In one animal (E) it decreased further.

Bradykinin, which has been previously found to temporarily reduce the high pulmonary vascular resistance in hypotensive animals, were given in connection with the collagen infusions in 2 of the present experiments. Bradykinin infusions were either started somewhat before or simultaneously with the collagen infusions (Fig. 4). Following one collagen response two subsequent pressor responses to such combined infusion were weak or absent. "Pure" collagen infusion carried out before or just after the combined ones did, however, give the usual pressure response.

In 1 expt. the vagal nerve was cut bilaterally between the first and the subsequent infusions of collagen. This did not alter the pressure response to collagen in any way.

Morphology The histological picture differed in the two animal groups examined. Macroscopially the lungs from both groups showed an almost normal appearance. In the first group of animals, which had only one infusion of collagen, and where the lungs were removed at the peak of the pressor response, aggregated platelets were found in small arterioles and capillaries (Fig. 5). The aggregates were unevenly distributed in the lung. The diameter of the occluded vessels was usually less than 100 p. Some larger arteries contained freely floating clusters of aggregated platelets (Fig. 6) .. Large vessels, including the main pulmonary artery, showed no aggregates.

528 G. B 0 AND J . H3GNESTAD

Fig. 5 Fig. 6

Fig. 5. Platelet aggregates in small arterioles and capillaries (arrows) from lung cut out on top of the pressure response.

Fig. 6. Clusters of aggregates in large vessel from lung cut out on top of thc prcssure response (arrow).

In the second group of animals, which had received sevcral infusions of collagen, a different picture was seen. Several of the larger vessels with a diameter greater than 100 ,u were occluded with apparently densely packed platelets. Few smaller vessels were occluded. The aggregated platelets showed a varying degree of viscous metamorphosis (Fig. 7) .

Discussion

Acute pulmonary hypertension m a y be provoked in a variety of ways. One is to cause partial occlusion of the pulmonary vasculature. When part of the vascular bed becomes occluded during unchanged cardiac output, then flow must @ease in

PULMONARY CIRCULATION AND PLATELETS 529

Fig. 7. Densely packed platelets occluding large vessel from lung where several infusions of collagen was given. Note the varying stages of metamorphosis.

the remaining open vessels, and there is a tendency towards increase in pressure. However, the vascular compartment of the lung may be distended to a large extent. At constant flow conditions it has been shown that a large fraction of the vasculature must be closed off before there is much rise in the pulmonary arterial pressure. Our own experiments on the cat show that when we double the flow through the right lung by clamping the left pulmonary artery, PVR in the right lung will decrease by more than 20 per cent.

The usual method used to occlude pulmonary vessels is to introduce emboli of a certain size into the pulmonary artery. The size of the emboli is of great importance for the pressure/flow response in the lung (Price, Hata and Smith 1955). The reason is apparently that a vasoconstrictor reflex mechanism operates when small arteries and arterioles are triggered. When only larger arteries are occluded no reflex vasoconstriction will occur. Smaller sized particles will cause a pressor response even when moderate quantities are injected. (Dexter 1965.)

The usual response to collagen infusions in the present experiment was a rise in P,, and a fall in aortic flow. Since PLA was fairly constant, PVR must have risen considerably. The collagen particles themselves were too small to occlude the vessels. This was also born out by the responses to late collagen infusions. When the pressor

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530 G. B0 AND J. HOGNESTAD

response was abolished, great amounts of the collagen suspension could be infused without any influence on flow and pressure. However, in the experiments where the lungs were removed at the peak of the first collagen response occluding platelet aggregates were found in small arterial vessels and in capillaries. Judging from the accompanying pressor response, aggregates in such positions are apparently connected with an element of strong vasoconstriction. The effect of physical blockade of some such vessels cannot in itself explain the large increase in PVR.

The experiments with bradykinin infusion give additional support to the presence of a strong vasoconstriction element in the pressor response to collagen. Bradykinin, which acts on the smooth muscle cells, will in animals with normal PVR cause a rise in PPA and also a rise in PLA. At the same time the cardiac output shows some increase, copsequently PVR is not much changed. During pulmonary hypertension, however, bradykinin reduce PVR markedly (Bra, Hauge and Hognestad, in prepara- tion). When bradykinin was infused simultaneously with collagen, the pressor response of the latter substance was very much reduced. Two components then probably A p e t e on the level of smooth muscle cells, namely one or more vaso- constrictor effects from aggregated platelets and the vasodilating effect of bradykinin. The number of platelets trapped in the lungs was the same during a combined infusion of bradykinin and collagen. We assume, therefore, that the physical vessel blockade caused by the aggregates was in the same in the 2 series.

Aggregated platelets will more or less markedly release their content of serotonin, histamine, catecholamines and nucleotides. These substances may exert a vaso- constrictor influence directly on the smooth muscle cells in the lung vessels. More- over, they may stimulate receptive sites in the lungs, and cause some reflex vaso- constriction.

The present experiments do not allow a differentiation between direct and reflex vasoconstriction effects of released platelet material.

During one individual pressure response the lowest platelet count was obtained at, the peak of the response, Afterwards the count increased somewhat. This indicates that some of the platelets are only temporarily trapped in the pulmonary vascular bed. Ultrastructural studies on platelets in arterial blood after collagen infusion have indicated that some of the platelets have been involved in a reversible aggregation reaction (Nicolaysen et al. in preparation).

In the lungs where several collagen infusions had been carried out, vessels of larger size were occluded, and the aggregates were more densely packed. In some of the vessels it appeared that several layers of aggregates were superimposed on each other.

A remarkable observation was that the pressor response in the lungs seemed to be exhausted when several collagen infusions had been given. The number of platelets in arterial blood did not show any alteration at this point. The exhaustion of the response can be explained in two ways: 1. The remaining number of platelets may be too mall to provoke the response. 2. The remaining circulating platelets do not react with collagen.

,

’ PULMONARY CIRCULATION AND PLATELETS 53 1

From Table I it appears that the pressure response is rather suddenly abolished. This points to the platelets becoming non-reactive to collagen which might be a consequence of the repeated stimulations towards platelet release. When using different embolizing materials the platelets and their reactions should be taken into account. Many of the foreign substances which have been used may attract platelets, and thereby an aggregation with subsequent release of platelets may contribute to the observed effects on the pulmonary vasculature.

References BERGENTZ, S.-E., D. H. LEWIS and U. LJUNGQUIST, Trapping of platelets in the lung after

experimental injury. In: Ditzel, J. and D. H. Lewis (eds.), Microcirculatory approaches to current therapeutic problems. S . Karger, Basel 1971. 35-40.

BRECHER, G. and E. P. CRONKITE, Morphology and enumeration of human blood platelets. J . appl. Physiol. 1950. 3. 365-377.

B0, G., A. HAUGE and J. HOGNESTAD, The nature of the increased pulmonary vascular resistance during hemorrhagic hypotension. In preparation.

B0, G. and J. HOGNESTAD, Thrombocytes and pulmonary vascular resistance during hemorrhagic hypotension. Acta physiol. scand. 1971. 82. 218-228.

DEXTER, L., Cardiovascular responses to experimental pulmonary embolism. In: SASAHARA, A. A. and M. STEIN (eds.), Pulmonary embolic disease. Grune and Stratton New York 1965. 101 -109.

HOLMSEN, H., Adenine nucleotide metabolism of blood platelets. Thesis. Universitetsforlaget, Oslo 1969.

HOVIG, T., Release of a platelet-aggregating substance (Adenosine ‘D$hosphate) from rabbit blood platelets induced by saline “extract” of tendons. Thrombos. Diathes. haemorrh. (Stuttgt.) 1963. 9. 264-278.

NICOLAYSRN, ANNE, T. HOVIG, G. BPI and J. HOGNESTAD, Ultrastructural studies of platelets stimulated by collagen in vivo. In preparation.

PRICE, K. S., D. HATA and J. R. SMITH, Pulmonary vasomotion resulting from miliary embolism of the lungs. Amer. 1. Physiol. 1955. 182. 183-190.