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Keywords:

  • heparin;
  • warfarin;
  • discovery

Summary

  1. Top of page
  2. Summary
  3. The discovery of heparin
  4. The discovery of warfarin
  5. Moving into the modern era of clinical trials
  6. References

Heparin and coumarins have been the mainstay of anticoagulant therapy throughout our working lives. As we stand on the threshold of a new era of anticoagulants it is timely to look back upon their discovery and development. Both have fascinating stories to tell. Jay McLean claimed to have discovered heparin whilst a medical student, although this is disputed. The story of warfarin leads us from a mysterious haemorrhagic disease of cattle to the development of a rat poison which became one of the most commonly prescribed drugs in history. Many people were involved in both stories and we owe them all a debt of gratitude.

Heparin and vitamin K antagonists have been the main anticoagulant drugs for decades. We are now approaching an era of many new anticoagulants as reviewed in this journal recently (Bates & Weitz, 2006). It is thus timely to look back at the discovery of heparin and warfarin in the first part of the 20th century.

The discovery of heparin

  1. Top of page
  2. Summary
  3. The discovery of heparin
  4. The discovery of warfarin
  5. Moving into the modern era of clinical trials
  6. References

Heparin, one of the oldest drugs still in widespread clinical use, is a naturally occurring glycosaminoglycan whose main function is to inhibit the coagulation of blood. It was discovered almost a century ago and took many years to move from the laboratory to the bedside. There has been significant controversy regarding the distribution of credit its discovery. James Marcum has chronicled both the discovery and development (Marcum, 1990, 1997), and the dispute (Marcum, 2000).

Initial discoveries: from fat-soluble phosphatides to water-soluble heparin

In 1916, at Johns Hopkins Medical School, Baltimore, USA, a second year medical student, Jay McLean, was working under the physiologist William Henry Howell (Fig 1). Howell’s main interests were the substances controlling blood clotting; he thought there was a balance between a clotting inhibitor (termed antithrombin) and a procoagulant (termed thromboplastin). He believed that the release of cephalin (so called because it was first isolated from canine brain) from platelets and leucocytes neutralised antithrombin, permitting activation of prothrombin by calcium (Howell, 1912). McLean had come up to Baltimore the previous year and was assigned by Howell to examine the chemical purity of cephalin preparations, and to demonstrate that it was cephalin and not a contaminant in the preparation that accounted for the procoagulant activity. After finishing this work early, McLean extracted phosphatides (fat soluble compounds) from canine liver that appeared to demonstrate anticoagulant, properties in vitro and subsequently led to excessive bleeding in experimental animals. McLean then moved to the University of Pennsylvania to research cephalins further under Richard Mills Pearce. In October 1917, he returned to Baltimore but did no further research on the phosphatides he had isolated the previous year. Instead, he continued research on cephalin, feeling that work on a procoagulant rather than an anticoagulant, was better for the ongoing efforts in The Great War.

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Figure 1.  William Henry Howell (courtesy of USA National Library of Medicine).

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Back in Howell’s laboratories, work on anticoagulants continued. Alongside another medical student L. Emmett Holt Jr, Howell had isolated another fat soluble anticoagulant apparently distinct from that isolated by McLean 2 years previously (Howell & Holt, 1918). The term ‘heparin’ was coined by Howell from the Greek ‘hepar’, or liver, the tissue from which it was first isolated. At an annual meeting of the American Physiological Society in 1922, Howell introduced an aqueous extraction protocol for isolating heparin and, at The 12th International Physiological Congress in 1926, he presented refinements to this protocol and identified the water-soluble carbohydrate as glucuronic acid. This he correctly claimed was a compound distinct both to the entity isolated by himself and Holt in 1918 and by McLean in 1916.

This water soluble heparin began to be produced commercially by a local pharmaceutical company in Baltimore, Hynson, Westcott, and Dunning, but studies conducted at Mayo Clinic, Minnesota, by Edward Mason demonstrated that this preparation caused side effects including headaches, fevers and nausea (Mason, 1924). Howell was concerned production would cease as its toxic effects might preclude widespread use (Howell & MacDonald, 1930). However, heparin did continue to be available commercially despite these fears, although the pharmaceutical company did not advance its isolation beyond Howell’s original protocol. In 1931, Howell retired from his post at Johns Hopkins and did no further research on heparin.

Bringing heparin to the bedside

Investigators in other parts of the world were working on a way to better purify heparin and thus avoid its side effects. In 1928, the physician and physiologist Charles Best (Fig 2), most famous for his work on insulin at the Connaught Laboratories, Toronto, began to assemble a team of biochemists, physiologists and clinicians at the city’s university. He had spent the last few years in the National Institute of Medical Research (established in London in 1913 as the first institute of the Medical Research Council) and decided to develop heparin into something useful for research and clinical purposes. In 1929 he became Professor and Head of Physiology at the university and it was at this time that he and his graduate student, Arthur Charles, began work in earnest. Their aims were twofold; to further purify heparin to reduce or eliminate its side effects and to demonstrate its effects in the prevention of thrombus formation. In 1929, Erik Jorpes, a Swedish physiologist visited Best to observe the production of insulin at Toronto. Jorpes was shown around the Connaught Laboratories and introduced to the work on heparin. He subsequently returned to Stockholm and began his own attempt to isolate and characterise the substance.

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Figure 2.  Charles Best (courtesy of USA National Library of Medicine).

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It was not until 1933, four years after Best’s team had begun serious work on the project, that Charles, and his more experienced colleague David Scott, who had served as an assistant director at the Connaught Laboratories, published a series of papers on their work thus far (Charles & Scott, 1933a,b,c). In the first paper (Charles & Scott, 1933a) they outlined a protocol for isolating a crude heparin preparation from bovine liver. To increase the amount of heparin yielded, the tissue had to be autolysed but the smell of the decaying tissue was so bad that the production had to be moved from the laboratories in the city to the local Connaught Farm! Their next paper outlined a survey of extra-hepatic tissues where heparin could be identified, partly because of the high cost of liver (Charles & Scott, 1933b). They concluded that liver, muscle, and lung tissues were where heparin was most abundant. The only tissue found to have little or none isolated was the tissue considered most integral to its mode of action; blood. Almost a decade earlier Howell had claimed that heparin was responsible for the fluidity of blood, he believed that although the amount of heparin in the circulation was probably small, its overall potency was greater there (Howell, 1925). The third and final paper in the series presented a purification protocol for heparin (Charles & Scott, 1933c). As Best later clarified, the preparations were not pure in the chemical sense but rather free from toxic components. It was hoped that the preparations would be of uniform potency but, although Charles and Scott were finally able to produce a crystalline form of heparin, there were problems getting consistent results from batch to batch. This not only hampered clinical progress but also sparked complaints from researchers who had bought heparin from Connaught laboratories for their own research purposes. One such investigator was Robert Cornish who was experimenting with dogs. In 1934, he wrote a letter to the Connaught laboratories complaining that the heparin he had bought ‘caused failure of two of [their] experiments as well as damage to blood’. He went on ‘…if you intend to sell much more heparin you should avoid such serious misbranding in the future!’.

To address the clinical and physiological aspects the Canadians worked in two teams; Murray led a team at the Toronto General Hospital while Best’s team continued to work in the Department of Physiology and School of Hygiene. Their initial publication was on the application of heparin for thrombus prevention in dogs (Murray et al, 1937), where they showed that heparin prevented thrombus formation in veins traumatised by mechanical or chemical means. The first use of this newer, purer, form of the anticoagulant in a human was 16 April 1937 – a solution of heparin in saline was passed into the brachial artery resulting in a significantly increased clotting time during the 2-h infusion. There were no toxic side effects. Murray tentatively claimed during a Hunterian lecture in the same year that although its development was still in its early days, its use could potentially extend to embolectomy, splenectomy, venous grafts and pulmonary embolism.

In 1939 Jay McLean, the medical student who first isolated the anti-coagulant fat-soluble phosphatides over 20 years previously, had began to investigate the clinical efficacy of heparin (along with sulfapyridine) in patients with endocarditis (McLean et al, 1941). Unfortunately, both patients succumbed to the disease despite the treatment. More promisingly, in 1943, heparin was used to preserve a gangrenous leg from full amputation (McLean & Johnson, 1946). Following discussions with McLean, Kurt Lange from New York Medical Center reported that gangrene resulting from frostbite, and military trauma such as paratroopers landing injury might be treated with heparin (Lange et al, 1945).

After the war, Connaught Laboratories continued to produce heparin and attempted to increase its potency. However, by 1949, Drs Peter Moloney and Edith Taylor had patented a method that obtained a greater yield of heparin at lower cost, making heparin cheaper to produced elsewhere. By the early 1950s, Connaught had stopped producing the drug it had pioneered altogether (Rutty, 2000).

The distribution of credit

Before the 1940s, most researchers in the biomedical community regarded Howell as the discoverer of heparin (Murray, 1940). According to James Marcum’s account of the origin of the dispute over heparin, Jay McLean, unhappy that he had not received appropriate recognition for heparin (something he saw as his own discovery) began a campaign in the 1940s to re-dress the balance of distribution of credit (Marcum, 2000). McLean gave several national lectures and wrote a number of letters to Best claiming that he, and not Howell, was the discoverer of heparin. However, McLean kept his campaign relatively discreet until Howell’s death in 1945, largely because he wanted to avoid controversy because of his long-standing relationship with Howell. It is important to note, as Marcum points out (Marcum, 2000), that it was not the intention of McLean to deceive but rather ‘correct the perception of the biomedical community that Howell was responsible for the discovery of the anticoagulant’. Louis Jaques was irritated by McLean’s claims to be the discoverer of heparin and claimed that McLean’s contribution was part of ‘American medical folk-lore’ (Jaques, 1978) and in a letter written in 1987 he stated ‘the key person for heparin was Charles Best as he had the novel combined role of top academician and director of production of biological products’ (Marcum, 2000). McLean’s efforts in persuading the biomedical community of his discovery were largely fruitful. After his death in 1959, his obituary credited him with the accolade ‘discoverer of heparin’ and did not mention Howell at all (Marcum, 2000). In 1963, a plaque was unveiled in Johns Hopkins to commemorate the ‘major contribution [of McLean] to the discovery of heparin in 1916 in collaboration with Professor William Henry Howell’ (Ulin & Gollub, 1964).

The discovery of warfarin

  1. Top of page
  2. Summary
  3. The discovery of heparin
  4. The discovery of warfarin
  5. Moving into the modern era of clinical trials
  6. References

The sweet clover problem

Warfarin is the most widely used anticoagulant in the world. In the UK it is estimated that at least 1% of the population and 8% of the over-80s are taking it regularly (Pirmohamed, 2006). The fascinating story of its discovery begins on the prairies of Canada and the Northern Plains of America in the 1920s. Previously healthy cattle in these areas began dying of internal bleeding with no obvious precipitating cause. Given that livestock was one of the most important industries in these areas and because most North Americans were already severely economically wounded by The Great Depression, this was a devastating blow for the farmers’ livelihoods. As there was an apparent lack of a recognisable pathogenic organism or nutritional deficiency responsible for the haemorrhage, the diet of the livestock was questioned. The cattle and sheep had grazed on sweet clover hay (Melilotus alba and Melilotus officinalis) and the incidence of bleeding occurred most frequently when the climate, and therefore the hay, in these areas were damp. Such damp hay became infected by moulds such as Penicillium nigricans and Penicillium jensi which appeared to be integral in the disease process occurring in the cattle (Schofield, 1924; Roderick, 1929, 1931). As Duxbury and Poller point out in their review article on warfarin, such hay would normally have been discarded if it spoiled in storage but in the financial hardship of the 1920s few farmers could afford to buy supplementary fodder for their cattle and thus the mouldy hay was used to feed them (Duxbury & Poller, 2001). The resultant haemorrhagic disease, which became known as ‘sweet clover disease’, became manifest within 15 d of ingestion and killed the animal within 30–50 d (Duxbury & Poller, 2001). Schofield and Roderick, two local veterinary surgeons, had demonstrated sweet clover disease to be potentially reversible if the offending mouldy hay was removed, or if fresh blood was transfused into the bleeding animals (Schofield, 1924; Roderick, 1929). The recommendations to local farmers were that they should avoid feeding their cattle with the mouldy sweet clover hay. Roderick showed that the acquired coagulation disorder was caused by what he called a ‘plasma prothrombin defect’ (Roderick, 1929).

Isolation of the oral anticoagulant

Ten years after the original outbreak of sweet clover disease, a young Wisconsin farmer, Ed Carlson, was at his wits’ end from losing so many of his prized cows and bulls from internal bleeding. Like so many local farmers he had no faith in the theory of sweet clover disease. After all, they had fed the cows with the hay for generations and to no ill effect. One winter’s day he travelled 200 miles in a blizzard, with a dead cow in the back of his truck, to the local agricultural experimental station where investigators Karl Link and his senior student Wilhelm Schoeffel were working. As the story goes, Carlson entered Link’s office (the only one he found open in the building) carrying a milk can of unclotted blood. Although clearly sympathetic, all that Link could recommend was the avoidance of the mouldy hay and the possibility of a transfusion of blood, as Schofield and Roderick had demonstrated some years previously. Karl Link (Fig 3) had only become interested in the sweet clover problem a month earlier. Nonetheless it would appear that Carlson had indeed come to the right place. Schofield experimented with the blood that evening and, as Duxbury and Poller comment, ‘The can of uncoagulated blood lying on the floor of Link’s laboratory was to change the course of history, and little did Link know what the long-term implications would be’ (Duxbury & Poller, 2001). Although the cause of the haemorrhagic malady had been found, in the sweet clover, the actual active compound had yet to be identified or even isolated. Link and colleagues got to work on finding the active substance from the spoiled hay.

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Figure 3.  Karl Link in his laboratory (courtesy of University of Wisconsin).

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A new in vitro clotting assay using plasma from rabbits was developed to guide chemical fractionation of compounds found in the hay. After some 6 years of intensive work, Link’s laboratory was finally able to crystallise the substance. It proved to be 3,3′-methylene-bis[4-hyfroxycoumarin] (Campbell et al, 1940, 1941; Campbell & Link, 1941; Stahmann et al, 1941). They found that that natural coumarin became oxidised in mouldy hay, to form the substance that would become better known as dicoumarol. Large scale isolation of dicoumarol was accomplished by graduate student Mark Stahmann who went on to become a professor of the biochemistry department at Wisconsin (Last, 2002). The work was funded by the Wisconsin Alumni Research Foundation (WARF), and patent rights for dicoumarol were given to WARF in 1941.

From test tube to rat poison

In 1945, whilst recovering in a sanatorium from ‘wet pleurisy’, Link got the idea of using a coumarin derivative as a rodenticide, the rodents dying of internal haemorrhage. He reviewed all the bioassay data to select the best variations of dicoumarol to create what he called ‘better mousetraps’. He thought dicoumarol a poor rodenticide because it acted too slowly (Last, 2002). Link and colleagues, still funded by WARF began working on several variations of the naturally occurring coumarin. Number 42 of a list of 150 appeared to be particularly active; warfarin (named after the authority with the financial muscle that funded the research) was born. It was promoted in 1948 as a rodenticide (Fig 4).

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Figure 4.  Karl Link promoting warfarin as a rodenticide (courtesy of University of Wisconsin).

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From rat poison to clinical application

After its success as a rodenticide, the transition of warfarin to clinical application was made under the name ‘Coumadin’. Other anticoagulants were available; but of these heparin required parenteral administration and dicoumarol had a long latent period before onset of therapeutic effect. The principal advantages of warfarin were its high water solubility and high oral bioavailability (Last, 2002). It was more potent than dicoumarol but retained the ability to have its effect reversed by vitamin K (Overman et al, 1942).

In 1955, Warfarin was given to President Dwight Eisenhower following a myocardial infarction As Duxbury and Poller point out; ‘What was good for a war hero and the President of the United States must be good for all, despite being a rat poison!’ (Duxbury & Poller, 2001).

A major problem in the widespread clinical application of warfarin proved to be the laboratory method used for dosage control; the prothrombin time (PT). In the 1950s, commercial sources of thromboplastin became available but they varied markedly in their responsiveness to the defect induced by vitamin K antagonists. As a result the PT varied greatly depending on the thromboplastin used. Expressing the results as a prothrombin ratio (PTR) did not solve the problem. If less responsive commercial thromboplastins were used larger doses were given to meet the target PTR. This led to overdosage and widespread reports of bleeding. In the UK more sensitive thromboplastin was used than in the US and it became clear that the UK had fewer bleeding complications. It was later realised that if samples are taken from patients on vitamin K antagonists and the PTs or PTRs obtained with two different thromboplastins plotted against each other in a log-log plot the points lay on a straight line. Based on this fact the World Health Organisation (WHO) in 1982 adopted a model to convert the PTR obtained with any reagent to an International Normalised Ratio (INR), that is the PTR that would have been obtained if an International Reference Preparation (IRP) had been used (Kirkwood, 1983; WHO Expert Committee on Biological Standardization, 1983). If the PTRs using the IRP are plotted on the y-axis and the PTRs using the local laboratory reagent on the x-axis the slope of the line is known as the International Sensitivity Index (ISI). The INR is then the PTR found in the local laboratory raised to the power of the ISI. This system standardised anticoagulant control worldwide.

Moving into the modern era of clinical trials

  1. Top of page
  2. Summary
  3. The discovery of heparin
  4. The discovery of warfarin
  5. Moving into the modern era of clinical trials
  6. References

The first randomised trial of these anticoagulants was performed in 1960 (Barritt & Jordan, 1960). Patients with pulmonary embolism were randomised to anticoagulation with heparin and nicoumalone or to no anticoagulation. Of the 16 patients randomised to anticoagulation none died from pulmonary embolism and there were no non-fatal recurrences. Of the 19 patients randomised to no treatment, five died from pulmonary embolism and there were also five non-fatal recurrences. The importance of the initial treatment with heparin was confirmed much more recently when heparin plus acencoumorol was compared with acencoumorol alone for the initial treatment of proximal deep vein thrombosis (Brandjes et al, 1992). In the 60 patients treated with heparin and acencoumorol there were no extensions of the thrombus and there were two pulmonary emboli and two recurrences. In the 60 patients treated with acencoumorol alone there were two extensions of the thrombus, two pulmonary emboli and eight recurrences. The importance of heparin was also illustrated by studies that looked at the adequacy of the dose (Hull et al, 1986; Raschke et al, 1993). Hull et al (1986) compared subcutaneous and intravenous heparin for the treatment of proximal DVT. It was noticed that, irrespective of mode of delivery, inadequate heparinisation in the first 24 h resulted in a 25% recurrence rate whilst it was only 1·6% in those adequately anticoagulated. Raschke et al (1993) compared two intravenous heparin regimens. The fixed dose regimen resulted in an inadequate anticoagulation in 68% at 6 h and 23% at 24 h, the figures for the weight based regimen were 14 and 3% respectively. The risk of recurrent thromboembolism was fivefold greater in the former group. From these studies it seemed that an inadequate activated partial thromboplastin time response to unfractionated heparin in the first 24 h increased the risk of recurrence. However, a meta-analysis has suggested that this does not seem to be critical if the starting rate is at least 1250 U/h (Anand et al, 1996).

The dosing of warfarin was also controversial (see above). Was dosing in the UK too low, increasing the incidence of thromboses, or was the dosing in the USA excessive, leading to unacceptable bleeding risks? The matter was resolved in a randomised trial in which patients with venous thromboembolism were assigned to a target PTR of 2·0–3·0 using either the British thromboplastin (supplied by Dr Leon Poller) or the less sensitive thromboplastin used at McMaster Hospital. The incidence of recurrent thrombosis was 2% in both groups but bleeding rates were five times higher if the North American thromboplastin was used (22% vs. 4%) (Hull et al, 1982). As the British reagent had an ISI of 1·0 this lent to the widespread adoption of an INR target of 2·0–3·0.

Low molecular weight heparins have now largely replaced unfractionated heparin. The key reason is that they produce a much more predictable anticoagulant response. This, combined with the fact that they have very high bioavailability after subcutaneous injection, means that the dose can be calculated by body weight and be given subcutaneously without any monitoring or dose adjustment. They are at least as effective and at least as safe as unfractionated heparin even when given once a day and this made out-patient treatment possible (Koopman et al, 1996; Levine et al, 1996). We now await the results of phase III clinical trial of direct oral thrombin inhibitors and direct oral anti-factor Xa inhibitors. These drugs will surely be available in the next few years. Will they be suitable for all patients and for all indications (no trials are taking place in patients with prosthetic heart valves)? We are left to speculate whether heparin and warfarin will still be used 100 years after their discovery.

References

  1. Top of page
  2. Summary
  3. The discovery of heparin
  4. The discovery of warfarin
  5. Moving into the modern era of clinical trials
  6. References
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  • Brandjes, D.P., Heijboer, H., Buller, H.R., De Rijk, M., Jagt, H. & Ten Cate, J.W. (1992) Acenocoumarol and heparin compared with acenocoumarol alone in the initial treatment of proximal-vein thrombosis. New England Journal of Medicine, 327, 14851489.
  • Campbell, H.A. & Link, K.P. (1941) Studies on the hemorrhagic sweet clover disease. IV. The isolation and crystallization of the hemorrhagic agent. Journal of Biological Chemistry, 138, 2133.
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  • Raschke, R.A., Reilly, B.M., Guidry, J.R., Fontana, J.R. & Srinivas, S. (1993) The weight-based heparin dosing nomogram compared with a ‘standard care’ nomogram. A randomized controlled trial [see comments]. Annals of Internal Medicine, 119, 874881.
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