Journal of Clinical InvestigationVol. 42, No. 6, 1963

MASSIVE THROMBOSISPRODUCEDBY FATTY ACIDINFUSION *

BY WILLIAM E. CONNOR,JOHN C. HOAK,t AND EMORYD. WARNER

(From the Cardiovascular Research Laboratories and Departments of Internal Medicine andPathology, State University of Iowa College of Medicine, Iowa City, Iowa)

(Submitted for publication November 19, 1962; accepted February 21, 1963)

Previous investigations have shown that fattyacids affect the coagulation of blood (2-4). Inparticular, certain fatty acids greatly acceleratedthe formation of thrombi from rat or human bloodin the Chandler apparatus 1 (6, 7). Long-chain,saturated fatty acids, such as stearic and palmitic,shortened the time of thrombus formation, whereasthe unsaturated fatty acids of similar chain length(oleic, linoleic, linolenic, and arachidonic) did notinfluence the thrombus formation time. Short-chain, saturated fatty acids of chain length lessthan C12 were likewise without effect. Themechanism whereby thrombus formation was ac-celerated by long-chain, saturated fatty acids prob-ably resulted from the activation of the Hagemanor contact factor of plasma (7).

These observations relating fatty acids to throm-bus formation in vitro have prompted additionalinvestigations designed to test the effects of differ-ent fatty acids given intravenously to anesthetizeddogs. Many previous workers have infused fattyacids (or their sodium salts) into animals. Asearly as 1889, Munk had noted that dogs de-veloped hypotension and asystole after the intra-venous injection of oleate or palmitate (8).Others have documented that fatty acids or theirsodium salts, when given to animals intravenously,produced hemolysis (9, 10), capillary damage(11), and death (10-13). In none of these re-

* Presented in part at the annual meeting of The Fed-eration of American Societies for Experimental Biology,Atlantic City, N. J., April 15, 1962 (1). Supported byU. S. Public Health Service research grants H-4621 andH-7239, by U. S. Public Health Service research careerprogram award HE-K3-18,406 from the National HeartInstitute, and by the Iowa Heart Association.

t Research Fellow, American and Iowa Heart Asso-ciations.

1 The thrombi produced in this system form in amoving stream of blood and are similar in structure tothrombi occurring in human thromboembolic disease(5).

ports has thrombosis been alluded to as a patho-logical consequence of fatty acid infusion, norhas any differentiation been made between effectsfrom different fatty acids.

In the present study, extensive thrombosis anddeath occurred after the intravenous infusion oflong-chain, saturated fatty acids into dogs. Theseeffects did not occur when unsaturated fatty acidsor short-chain, saturated fatty acids were given.These more innocuous fatty acids did, however,induce some hypercoagulability of the blood asindicated by thrombus formation in a jugular veinsegment isolated after the completion of the infu-sion.

MATERIALS AND METHODS

The sodium salts of different fatty acids were pre-pared in a 0.1%o concentration (6). Control solutionsconsisted of a 0.9% NaCl solution and a hypotonic NaClsolution of the same molarity (0.097 M) as that used inthe preparation of sodium stearate. A 0.1% suspension ofbentonite and a 2%o suspension of soybean phosphatidewere prepared in 0.9%o NaCl. The pH of all solutionswas adjusted to 8.0. The preparations of long-chainfatty acids, both saturated and unsaturated, and of ben-tonite and soybean phosphatide appeared cloudy at roomtemperature. The physical state of the fatty-acid prepa-rations associated with turbidity was important in theproduction of thrombosis and other effects. As has beenpreviously discussed, the acceleration of thrombus for-mation in vitro was thought to be caused by the ionicmicelle, a particle consisting of a negatively chargedaggregation of fatty acid ions (6). Loss of activityresulted when the fatty acid concentration was reducedsufficiently for the solution to appear clear, or whenthe preparation was filtered through a bacterial filterthat removed the particles responsible for turbidity andgreatly lowered the fatty acid concentration (to about3.5%o of the original concentration).

Plasma FFA were determined by the method of Doleand Meinertz (14). The whole blood clotting time insilicone-coated test tubes was measured in 16 dogs (15).Determinations of the thrombus formation time of wholeblood (6), one-stage prothrombin (16), two-stage pro-thrombin (17), accelerators (factors V and VII) (18),and fibrinogen were performed in 9 dogs before and 3

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MASSIVE THROMBOSISPRODUCEDBY FATTY ACID INFUSION

to 8 minutes after the start of the infusion of a long-chain, saturated fatty acid. Blood for these determina-tions was obtained from a vein not previously used andin a limb different from the infusion site. A silicone-coated needle was used, and the blood was allowed toflow freely through plastic tubing into collecting testtubes. Plasma fibrinogen was measured by the deter-mination of the total nitrogen in the clot formed afterthe addition of thrombin. The micro-Kjeldahl techniquewas used. Plasma levels of free hemoglobin were meas-ured before and after fatty acid infusions in some ex-periments (19). The pH of arterial blood was deter-mined with a Radiometer pH-meter, and it did not changeafter the fatty acid infusions.

Healthy mongrel dogs were anesthetized by intrave-nous sodium pentobarbital. An external jugular veinwas exposed and a 3.4-cm segment isolated without dis-turbance of blood flow. The 0.1% fatty acid salt prepa-ration, in an amount equal to 10 ml per kg of bodyweight, was infused into a foreleg vein over a 5-minuteperiod through a blood transfusion filter set. Thisamount was selected on the basis of calculations thatthe plasma FFA levels of most dogs would approxi-mately double if the infused substance remained in theplasma compartment for the duration of the infusion.Such an increase in FFA occurs physiologically after avariety of stimuli such as starvation, stress, or the in-jection of adrenaline (20). In a few dogs, the time ofinfusion was lengthened, or the quantity of fatty acid in-fused was reduced. Bentonite, 0.1%, and soybean phos-phatide, 2%o, were given in amounts equal to 10 ml perkg of weight over a 5-minute period.

After completion of the infusion, the isolated seg-ment of the jugular vein was clamped off, as describedby Wessler (21). Fifteen minutes after the initial clamp-ing, the vein segment was tied off, opened up longitudi-nally, and examined for the presence of a thrombus. Ifthe animal did not survive the infusion, the vein seg-ment was clamped at time of death and inspected simi-larly 15 minutes later for the presence of a thrombus.With this particular technique, the two factors importantin thrombus formation are stasis and hypercoagulabilityof the blood confined in the isolated vein segment.

In the event of death, the chambers of the heart andthe great vessels were opened and examined for thepresence of thrombi immediately after breathing stopped.In most instances, the examination was made whencardiac contractions were still occurring. Specimens ofappropriate vessels, lung, heart, liver, spleen, kidney,and brain were taken for subsequent histologic exami-nation.

RESULTS

The long-chain, saturated fatty acids almost in-varibly caused the death of the animal within 3 to5 minutes after the beginning of the infusion.Electrocardiographic monitoring revealed an ini-tial increase in the heart rate, followed by pro-found depression of the ST segment, and later by

an irregular cardiac rhythm and periods of asys-tole. A loud systolic murmur could be auscultatedat about the same time that the electrocardio-graphic changes appeared.

At post-mortem examination, these dogs hadlarge thrombi 2 in the inferior and superior venacavae and in the right heart extending into thepulmonary artery. The chambers of the heartwere opened immediately after breathing stoppedand while the cardiac muscle was still irregularlycontracting.3 Occasionally the left atrium and leftventricle contained thrombi. Thrombi in theaorta and peripheral arteries occurred at times.Microscopic sections of the viscera usually werenot abnormal. In some instances, however, mi-croscopic thrombi did occur and demonstratedlayering of fibrin and cellular elements, thus sug-gesting that they formed in moving blood.

In contrast, the infusion of either long-chain,unsaturated fatty acids or the short-chain, satu-rated fatty acids did not kill any animal, nor werethere overt signs of toxicity. Table I sum-marizes the results from the infusion of variousfatty acids. Nineteen dogs were given long-chain,saturated fatty acids; 16 died, and 15 of these hadlarge thrombi in the heart and great vessels.When fatty acids of shorter chain length wereused, the toxic effects of their infusion disap-peared. The critical change occurred from C14: 0to C12: 0. Thus, 13 dogs infused with short-chain, saturated fatty acids sustained no demon-strable ill effects. Infusions of five, different, un-saturated fatty acids were given to 16 dogs. Theseacids included oleic and erucic, both with onedouble bond, and the polyunsaturated acids lino-leic, linolenic, and arachidonic. None of thesefatty acids was lethal.

Ten dogs were infused with a sodium chloridesolution of the same molarity and pH as the solu-tions of fatty acid salts. This control solution

2 The term "thrombus" is used to indicate intravas-cular clotting during the life of an animal. Such thrombioften will not have the classic structure of clots formedfrom moving blood. The structural pattern of layeringof blood elements will not develop if the clotting is rapid,or if, like a propagated thrombus, the thrombus de-velops in a static blood column.

3 In this connection, it should be noted that dogskilled with sodium pentobarbital and immediately autop-sied did not have thrombi; the blood in the cardiacchambers was still fluid.

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WILLIAM E. CONNOR,JOHN C. HOAK, ANDEMORYD. WARNER

TABLE I

Results of fatty acid infusions

No. of No. of MassiveSubstance infused dogs deaths thrombosis

Saturated fatty acids

Long-chainC22:0 2 2 2C18:0 9 8 7C16:0 6 4 4C14:0 2 2 2

Total 19 16 15

Short-chainC12:0 3 0 0C10:0 4 0 0C6 :0 6 0 0

Total 13 0 0

Unsaturated fatty acidsC18:1 3 0 0C18:2 4 0 0C18:3 3 0 0C20:4 3 0 0C22:1 3 0 0

Total 16 0 0

Bentonite 2 2 2

Soybean phosphatide 2 0 0

Control - NaCl 10 0 0

produced no observable systemic toxic effects.The infusion of the opalescent soybean phospha-tide suspension was likewise without toxicity. A0.1%o suspension of bentonite, a substance acti-vating the Hageman factor and thereby initiatingblood clotting, produced death and massive throm-bosis in the 2 dogs to which it was given.

A reduction in the amount of stearic acid in-fused, from 0.1 to 0.01%, prevented the usualthrombosis and death. When the time of theusual 0.1% infusion of stearic acid was increasedfrom 5 minutes to 30 or 40 minutes, no toxic ef-fects appeared. When the concentration was givenin a 10-minute infusion, however, massive throm-bosis and death resulted.

Thrombus formation in the isolated jugularvein segment occurred in most dogs receivinglong-chain fatty acids, whether saturated or un-

saturated. In Table II, the jugular segmentthrombi are classified as absent, small, or as largeif over 1.5 cm long. Long-chain, saturated fattyacids caused jugular thrombosis in 9 of 11 dogs.Short-chain fatty acids produced thrombi less of-ten, in 5 of 13 instances. The infusion of unsatu-rated fatty acids, in contrast to their lack of ef-

TABLE II

Thrombi in isolated jugular vein segments afterfatty acid infusions

Jugular vein thrombiNo. of

Substance infused dogs Absent Small Large*

Saturated fatty acidsLong-chain 11 2 2 7Short-chain 13 8 2 3

Unsaturated fatty acids 16 2 5 9

Soybean phosphatide 2 2 0 0

Control - hypotonic 10 8 1 1NaCI

Control - 0.9% NaCl 5 5 0 0

*Over 1.5 cm long.

fect in causing systemic thrombosis, caused jugu-lar thrombi in 14 of 16 animals. The injection ofthe hypotonic control NaCl solution resulted injugular thrombi in only 2 of 10 dogs. A 0.9%NaCl solution produced no jugular thrombi in 5dogs.

At this point, it should be indicated that previ-ous reports over the past 30 years have stressedespecially the hemolysis resulting from the infu-sion of fatty acids into animals (9). Becausehemolyzed erythrocytes have been incriminatedin the causation of hypercoagulability of the bloodand thrombosis, it was considered important todetermine whether there was a relationship be-tween the extent of hemolysis and the productionof thrombosis. Accordingly, the plasma hemo-globin was measured before and after the variousinfusions (Table III). Since these solutionswere hypotonic, all resulted in some hemolysis.The amount of hemolysis was highly variable andcould not be correlated with intravascular clotting.In fact, infusion of the long-chain, saturated fattyacids usually was associated with the least hemoly-sis. Thus hemolysis was not a major factor inthrombosis or death.

TABLE III

Hemolysis of dog blood before and after infusion of fatty acids*

After

Saturated fatty acidsUnsaturated

Before Long-chain Short-chain fatty acids

32.34117.5 (SD) 84 152.2 148.2 153.4 104.64±75.9

* Mean values ISD in milligrams per 100 ml free hemoglobin.

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MASSIVE THROMBOSISPRODUCEDBY FATTY ACID INFUSION

Blood coagulation tests were carried out invitro before and after the infusion of long-chain,saturated fatty acids (either C18: 0 or C22: 0).An attempt was made to obtain blood before thedeath of the animal and while blood was stillfluid and circulating. The results of blood clottingtests are given in Table IV. The two-stage pro-thrombin, fibrinogen, and thrombus formationtimes were slightly and variably reduced afterfatty acid infusion. The one-stage prothrombintime and accelerators were not changed. Thewhole blood clotting time in silicone-coated tubeswas decreased, from an average of 32 minutes to17 minutes. These changes were variable, prob-ably due to rapid death and poor mixing of thefatty acid with the blood generally. To indicatethe extremes, in 2 dogs the silicone clotting timedecreased from 20 to 2 minutes; in another dog, itactually increased from 33 to 44 minutes. Thissuggests the possibility of an inhibitor.

Many investigators have shown that the plasmaFFA have a rapid turnover (20). This may oc-cur because of their prompt metabolism by mus-cle and liver, or possibly from rapid diffusibility.In 25 of our experiments, the plasma FFA con-centrations were measured before the fatty acidinfusion and exactly at the time of completion ofthe infusion. They did not change significantly,despite the administration of an amount designedto raise the plasma concentration by 100%o. Thislack of change was observed with all three groupsof fatty acids infused. The plasma FFA concen-tration was 517 ± 218 uM before the infusionsof long-chain, saturated fatty acids and 490 4

170 ,uM immediately afterward (p > 0.5). With

TABLE IV

Blood clotting tests after the infusion of long-chain,saturated fatty acids

Before After p

One-stage prothrombin 12.2 4z 2.3 12.7 d 2.8 >0.5time, seconds

Two-stage prothrombin, 206 41 29 185 4 39 >0.05units

Test for accelerators, 107 4: 21 108 A 21 >0.8factors V and VII. %

Fibrinogen, mg per 100 ml 348 4 100 320 i 100 >0.05Thrombus formation time, 4.7 4 1.2 3.7 4- 1.6 >0.1

minutesClotting time of whole 32 i 11 17 :i: 11 <0.001

blood in silicone-coatedtubes, minutes

the short-chain, saturated fatty acid infusions, thevalues were 604 + 192 juM before and 584 ± 166MMafterward (p > 0.5). With the long-chain,unsaturated fatty acids, the values were 446 +

130 uM before and 483 + 141 MMafterward (p> 0.2).

DISCUSSION

It is not clear why dogs tolerate the infusion ofunsaturated fatty acids, whereas they die fromsaturated fatty acids of similar chain length. Adifference in thrombotic effect was anticipatedfrom previous work in vitro (5, 6). The toxiceffect on the myocardium, likewise limited to thelong-chain, saturated fatty acids, was distinct fromthe induced intravascular clotting. Significantly,one dog died from stearic acid infusion, but didnot have any thrombosis upon immediate autopsy.Wesuppose that myocardial failure in this animaloccurred before there was time for intravascularclotting. Furthermore, when clotting was pre-vented in animals by pretreatment with heparin,these fatty acids still produced electrocardio-graphic abnormalities and impaired cardiac con-tractility (22). Neither the thrombotic nor themyocardial effects were manifested if the dura-tion of the infusion was prolonged, or the concen-tration of the infused fatty acid was considerablyreduced.

Although fatty acids constitute a major energysource for the working cells of the body, it haslong been appreciated that in plasma-free sys-tems they are highly toxic to cellular function andintegrity (23-25). This was found to be trueof both saturated and unsaturated fatty acids.In a recent study, Helinski and Cooper demon-strated an enzymatic disturbance that might ac-count for the toxicity of fatty acids (26). Fattyacids blocked metabolic activity in liver mito-chondria by uncoupling oxidative phosphoryla-tion. When infused into dogs under the condi-tions of our experiments, the long-chain, saturatedfatty acid group, as represented by stearic acid,is highly toxic. The other group, of which oleicacid is a prototype, however, is almost completelyharmless. A possible explanation for the greatdifference in action might be that unsaturatedfatty acids are rapidly bound and so renderednontoxic with respect to both thrombosis and theeffect of the myocardium.

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WILLIAM E. CONNOR,JOHN C. HOAK, AND EMORYD. WARNER

Under physiological conditions, most of thecirculating FFA is complexed with the plasma al-bumin, and smaller quantities are complexedwith lipoproteins and erythrocytes (27-29). Inour study, unbound fatty acids were infused intothe blood. In all probability, as a fatty acid andblood mixed intravascularly, there was a strongtendency for the fatty acid to be bound to theplasma albumin and other blood elements. Per-haps oleic acid, given intravenously, may be rap-

idly bound to the albumin of dog blood and there-fore exerts little toxic effect. Fredrickson andGordon indicated that the binding of sodium oleatewith albumin was very rapid, occurring as soon

as mixing of the two substances occurred (20).They made no mention of the rate of binding ofstearate to albumin. Goodman has studied thenumber of serum albumin binding sites for stear-ate, oleate, and linoleate, as well as for other fattyacids (27). His data do not suggest that thereare fewer binding sites of the serum albumin forsaturated than for unsaturated fatty acids, andtherefore do not provide any explanation for a

probability of more rapid binding of unsaturatedfatty acids. In fact, lipoproteins had more bind-ing sites for stearate than for linoleate (28).Since all incubations of fatty acids with albuminwere carried out for 48 hours, the rate of bindingfor different fatty acids was not reported.

If stearic acid is more slowly bound, it mightexert its toxic effects before binding occurs.

Studies are now in progress to test the validity ofthis hypothesis. It has already been demonstratedthat the incubation of stearic acid and bovine al-bumin for some time prevented the usual shorten-ing of thrombus formation time (6). Further-more, the mixing of stearic acid with bovine albu-min under special conditions of temperature hasbeen shown to prevent the expected thrombosisafter the intravenous infusion (22).

Although long-chain, unsaturated fatty acidsdid not produce generalized thrombosis or evi-dence of toxic damage to the heart, they did in-duce some hypercoagulability of the blood as

demonstrated by the thrombi formed in the iso-lated jugular vein segments. Similarly, short-chain, saturated fatty acids caused jugular thrombiin 5 of the 13 attempts. The infusion of hypo-tonic saline also led to jugular thrombi in 2 of 10dogs. This suggests that with both the unsatu-

rated and the short-chain fatty acids, hemolysismay have contributed to a degree in the forma-tion of jugular vein thrombi in some animals.Since the infusion of the long-chain, unsaturatedfatty acids usually caused jugular vein thrombi(in 14 of 16 experiments), it appears that theyexerted some effect upon coagulation beyond thecontribution from hemolysis. Perhaps some ac-tivation of coagulation mechanisms occurred asthese fatty acids entered the blood and beforethey became bound to the plasma proteins. Thiseffect was not sufficient to cause intravascularclotting in moving blood.

Activation of the Hageman factor has been con-sidered a possible mechanism in the causation ofthrombotic disease (30, 31). This factor nor-mally circulates in an inactive form. Any agentcapable of activating it might well produce throm-bosis, since the Hageman factor initiates the earlyphases of blood clotting. In this study, two sub-stances believed to activate the Hageman factordid, in fact, cause thrombosis when given intra-venously. These were the long-chain, saturatedfatty acids and bentonite (7).

If Hageman factor activation was the mecha-nism of the thrombotic effect from the long-chain,saturated fatty acid, it would be suspected that theblood clotting tests reflecting active Hagemanfactor would be altered. Shortening of the sili-cone clotting time reflects well the extent of invivo Hageman factor activation. In this study,there was shortening of the silicone clotting timeof blood in almost all dogs. It seems likely thatactivation of the Hageman factor with subsequentin vivo plasma thromboplastin generation was themechanism of the intravascular clotting inducedby the fatty acid infusion. After plasma throm-boplastin had formed, the massive intravascularclotting was very rapid and probably did not al-low time for either uniform mixing or the pro-gressive depletion of clotting factors in the circu-lating blood. Undoubtedly, the concurrent de-cline of cardiac function was also a factor in thismatter. The intravenous infusion of particulatematter and thromboplastins ordinarily produces adeficiency in certain clotting factors (32), or evenincoagulable blood, the defibrination syndrome.Fatty acid infusion, on the other hand, induced no,or only slight, reduction of clotting factors.

The recent demonstrations that diets high in

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MASSIVE THROMBOSISPRODUCEDBY FATTY ACID INFUSION

saturated fat may produce thrombosis and organ

infarction may have some relationship with thethrombosis resulting from long-chain, saturatedfatty acids in our study. Whereas Thomas andHartroft found that butter fat and lard fed to ratscaused coronary thrombosis, they noted no throm-bosis after corn oil feeding (33). Corn oil was ap-

parently not thrombotic because of its predomi-nantly unsaturated fatty acid composition. Theseexperiments in rats have been confirmed byGresham and Howard (34, 35). Saturated fat(butter, beef fat, or synthetic stearates) causedthrombosis and infarction. Rats fed linoleate or

oleate did not develop thrombi. Extending thesefindings to another species, Hartroft, Suzuki, andO'Neal found that the thrombosis-producing dietof butter fat induced both thrombosis and athero-sclerosis in the dog (36). In addition, hyperco-agulability of the blood was reported in rats fedsuch a diet (37).

The relationship of stress to episodes of throm-bosis and to sudden death in man has long in-trigued investigators. The early work of Cannonand Gray suggested that adrenaline secretionmight be a mediating factor in causing hyperco-agulability of the blood (38). Since both stressand adrenaline stimulate a prompt increase in theplasma FFA, Poole reasonably speculated thatFFA might be involved in the initiating of throm-bosis (39). Saturated fatty acids make up 37.8%oof the FFA composition of plasma (40). DeLong, Uhley, and Friedman found that stress inrats accelerated the blood clotting times (41).Rats under stress also had a rise in FFA (42).The stress of repeated breeding, unilateral ne-

phrectomy, and administration of ACTH pro-

duced thrombosis in female rats (43). Obviously,these interrelationships require much further workbefore any conclusions are possible.

SUMMARY

1. Long-chain, saturated fatty acids producedextensive thrombosis and death when given in-travenously to dogs. Thrombi were found in theveins and chambers of the heart and occasionallyin the peripheral arteries. Thrombosis and deathdid not occur if the fatty acids were infused more

slowly or in lower concentration.2. This fatty-acid-induced thrombosis probably

represented an example of intravascular clotting

occurring from rapid activation of the Hagemanfactor. The clotting time of whole blood in sili-cone-coated tubes was shortened by fatty acidinfusion. Thus a hypercoagulable state of thecirculating blood was demonstrated before the oc-currence of massive thrombosis.

3. Similar infusions of long-chain, unsaturatedfatty acids and of saturated fatty acids of chainlength C12 and below did not cause massivethrombosis or death. They did, however, inducesome hypercoagulability of the blood as indicatedby the formation of thrombi in the jugular veinsegments isolated after the completion of the fattyacid infusions.

ACKNOWLEDGMENT

We thank Dr. Raymond Sheets for analyzing plasmasamples for free hemoglobin.

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