Blood coagulation research atKarolinska Institutet 1920-2004PART I - Basic research

JUBILEUMS E S S Ä E R

Blood coagulation research at Karolinska Institutet 1920-2004 PART I - Basic research

Margareta Blombäck Essäist

Blood coagulation research at Karolinska Institutet 1920-2004 PART I - Basic research PART I - Basic research: on heparin and its derivatives, on thrombogenic and nonthrombogenic properties of the vessel wall; on purification of hemostatic factors: fibrinogen, prothrombin, Factor VIII and von Willebrand Factor and fibrinolytic factors; and on platelets.

It all began with Erik Jorpes...

Erik Jorpes was born in 1894 as the eldest son of a poor fisherman on Kökar, a small island in the Åland archipelago between Sweden and Finland. Since then, about a hundred theses and thousands of papers in this field have been published from KI. (Ref 1).

Erik’s high intelligence was recognised by a schoolteacher who helped him in various ways to continue his education in Finland, where he studied medicine and took his Bachelor’s degree with the top mark in medical chemistry.

This was the time of the vicious war between the ”Whites” and the ”Reds” in Finland, which in December 1917 had declared its independence from Russia.

Although he lacked clinical experience, Erik Jorpes started to treat the wounded and sick on the Red side. The Reds were forced to retire to Russia and during his stay there Erik was among those who started the first Finnish communist party.

In 1919 he had to flee to Sweden (Stockholm).

With permission from the Swedish government, he resumed the study of medicine at Karolinska Institutet in 1920. It was said that he had to promise the finance minister, the leader of the Social Democrats, never to work with politics - a promise he kept.

Erik soon got in touch with the nucleic acid expert Einar Hammarsten at the Department of Chemistry and defended his thesis on pancreatic nucleic acids in 1928 - seen in retrospect, this thesis heralded the downfall of the then prevalent tetranucleotide theory of the structure of nucleic acids. Through Hammarsten, Erik obtained a one-year fellowship at the Rockefeller Institute for Medical Research (1928-29), where he continued to study nucleic acids and carbohydrates. He also became interested in how pancreatic secretion was regulated and this ultimately led to his work on the isolation of secretin.

Erik never practised medicine but was very concerned to find new ways of treating patients. Back in Sweden, he worked on a method for the preparation of insulin and found a small Swedish firm, Vitrum, whose owner became interested in his work and in 1930 launched a technique based on Jorpes’ method. In that Jorpes obtained very generous royalties and invested the greater part in his research (he had an agreement with the state about not paying taxes), the collaboration with Vitrum came to be very beneficial for his research and that of all his pupils. If a collaborator had financial problems, Jorpes willingly provided funds from his own pocket. He passed on his knowledge to Vitrum and does not seem to have had any patents. He also cooperated successfully with American interests and obtained funds from them as well as from the Swedish authorities.

Erik Jorpes and heparin

Why did Erik Jorpes start to work with heparin? Clarence Crafoord in 1940 described how, on reading a paper by Howell on the effects of heparin in 1929, he was

on heparin. Jorpes summarized the early research in a fascinating book Heparin in the Treatment of Thrombosis in 1939 (2nd edition 1946) and in symposium proceedings from 1940. He reported all that was known about heparin’s history, chemistry, mode of action, synthesis and standardization; its clinical advantages and drawbacks, death rates among treated compared with untreated patients. Jorpes must have been very encouraged by all the clinical uses his purification work had led to. He also suggested that the effect of heparin was not only as an inhibitor of coagulation but also as a reducer of inflammation - a view that is coming into focus more and more.

Erik Jorpes at work

struck by the idea that heparin, besides being of value in the treatment of venous thrombosis, might be a prophylactic agent against postoperative thromboembolism.

When he discussed this with Jorpes, the latter considered that there was a rebound after the anticoagulant effect had worn off and that heparin had toxic properties.

However, in 1935 (the year in which his only son was born), Jorpes managed to prepare absolutely pure heparin - the negative effects were due to contaminants.

In August 1935 Crafoord started treating patients prophylactically.

According to Jorpes, the beds were shaking hard when the patients received heparin (the importance of pyrogen contamination was unknown). Jorpes, alone or together with Sune Bergström and Olle Wilander in the Thirties and later in the Fifties with Harry Boström and Viktor Mutt, succeeded in unravelling the complex chemical structure of heparin and demonstrated its biosynthesis in the mast cells.

Crafoord, as well as many other colleagues/friends of Jorpes, started working

As an individual, Erik was reserved and lived simply, according to strict principles.

He was an excellent but feared teacher.

Fibrinogen Basic studies Birger Blombäck and I (Margareta Blombäck) In 1950, when Birger Blombäck and I (Margareta Blombäck) had begun to study medicine, Jorpes invited Birger to join the research group at the department and I accompanied him. We came to a place of hard work, full of glad and energetic researchers, all very spoiled - thanks to Jorpes’ royalties - by having the services of a woman who cleaned the glassware, an efficient secretary, a librarian and later also personnel for preparing blood fractions.

There was a complete set of machinery and a glass-blower funded with money that Hammarsten had acquired from the state.

Birger Blombäck and fibrinogen

Our first duty was to prepare fibrinogen to be used in a method for the standardization of heparin (one of Jorpes’ undertakings with Vitrum). We did this from bovine blood, acquired in 50-litre glass bottles

from the slaughterhouse (in exchange, the butchers received bottles of alcohol bought at the Swedish Monopoly) and transported at first in the tram, later in our Volvo. However, the fibrinogen prepared by precipitation remained stable for just a few hours.

(ko=cow; hjort=deer; får=sheep; svin=pig; arters utveckling = evolution of species.

Trombin hämmare= thrombin inhibitors; syntetiska substrat=synthetic chromogenic peptide substrates; kedjestruktur=chain structure; tätt nätverk=tight network; glest nätverk=porous network.)

Birger Blombäck studied Edvin Cohn’s procedure for the purification of plasma proteins (developed for rational use of blood proteins under war conditions) and suggested that perhaps we could further purify the first fraction in that procedure (fraction I) by washing it with a solution containing optimal concentrations of the amino acid glycine, ethanol and salts etc so that the contaminants went into solution while the fibrinogen remained as a precipitate which could easily be dissolved. We called this fraction I-0 and it provided a foundation not only for our basic research but also for much work by us and our collaborators in the clinical field. (Ref 2, Tree figure).

However, as fraction I-0 still contained small amounts of contaminating proteins, we further purified it until we obtained a very pure fibrinogen (fraction I-4). Birger Blombäck and Ikuo Yamashina then demonstrated that fibrinogen, a very large molecule, consists of three different chains and that each unit of fibrinogen contains two of each. Agnes Henschen isolated the chains and since the individual chains were obtained after reduction (breaking the disulfide bonds), it was concluded that they were held together by disulfide bonds. Blombäck et al sho-

Fibrinogen Metamorphosis - a tree drawn by Birger Blombäck Tree, drawn by Birger Blombäck. The tree illustrates the development ot the research by Birger and his team on fibrinogen, its structure, funcion and evolution, on fibrinopeptide A, on the abnormal fibrinogen (fbg Detroit), on the development of chromogenic peptide substrates, and on thrombin inhibitors and on the coagulation factors VIII and Von Willebrand s factor.

made with the well-known protein sequencing method designed by Pehr Edman in the Chemistry Department in the Forties.

Margareta and Birger Blomb채ck with a bottle of fraction I-O in the laboratory of Medical Chemistry.

Press photo taken in connection with thier disputations

wed that each half-molecule was linked to the other by disulfide bonds. It had been shown that two peptides (fibrinopeptides A and B, that is, FPA and FPB) were cleaved off from fibrinogen when it was transformed to fibrin (the threadlike substance being the basic substance in a clot) by the enzyme thrombin. Birger Blomb채ck and his team were particularly interested in mapping the structure not only of these peptides but also of the site of cleavage by thrombin. By studying the release of the peptides by thrombin, it was shown how polymerisation, gelation and aggregation led to the fibrin in the clot. The action of thrombin was located in the N-terminal part (see page 3) of the peptides. The structural analyses were

The sequence of fibrinopeptide A and part of the aminoterminal of the Aalpha chain

The sequence of fibrinopeptide A and part of the aminoterminal of the Aalpha chain of normal human fibrinogen and of Detroit fibrinogen (within parenthesis).

The figure above the horizontal arrows refer to the peptide spots observed in the fingerprint of normal Aalpha-chain fragment presented in the figure. T=bond split by thrombin; TRY=bond split by trypsin.

Having purified fibrinopeptides from mammalian and other species, we determined their structure and found that the sequence of nine amino acids (nonapeptide) near the binding split by thrombin, Arg-Gly, especially the ninth position (phenylalanine), had been well preserved during evolution.

We also demonstrated that the species differences and similarities (with the exception of reindeer) agreed with the course of evolution suggested by fossil findings and anatomical details. With no computering techniques available at that time, the work of fitting all the pieces together was fascinating. (Ref 3) Of great importance for understanding the initial polymerisation (fibrin forming) process was the discovery of a homozygous (inherited the same genetic defect from both parents) point mutation (exchage of one amino acid to another) in the fibrinogen of a girl who suffered from life-threatening menstrual bleedings; this was the second time a mutation in a protein, leading to a disease, had been demonstrated (the first was for hemoglobin in sickle-cell anemia). As the release of FPA was not prevented in this fibrinogen (called fibrinogen Detroit), we suggested that the mutation had inactivated a polymerisation site that was exposed on release of fibrinopeptides.

(Ref 4)

Text????

We considered that the sequence of the nonapeptide was of great importance for the action of thrombin. However, as the nonapeptide as such had just a slight inhibitory effect on thrombin, we argued that in the natural substrate there might be a structure that brings phenylalanine (Phe) closer to the arginine (Arg) Together with Per Olsson and researchers at Bofors (later Kabi) Gรถran Clesson et al, we showed that synthetic peptides in which Phe was placed in position 3 from the C-terminal Arg (see figure above) had a pronounced inhibitory effect on thrombin (Ref 5) and that, when coupled to a substance (paranitroanilide =pNA), the compound (chromogenic peptide substrate) could be used for the determination of thrombin activity (when split by thrombin, pNA develops a colour). (Ref 6).Soon other researchers together with the Kabi group were able to show that the sequence adjoining the cleavage site for other enzymes could be used for constructing chromogenic substrates for many other enzymes, not only for those of coagulation and fibrinolysis but also for kallikrein, elastase, trypsin etc. The substrates could also be used for measuring proenzymes, enzyme acti-

vators and inhibitors of proteolytic enzymes. More than ten thousand publications involving such chromogenic and similar substrates have been published.

Birger Blombäck and collaborators continued their research on which parts of the fibrinogen molecule are of importance for thrombin’s action. One of the very important studies was together with AnnCatrine Teger-Nilsson in 1974 on the rate of thrombin-fibrinogen reaction in some mammalian species. Birger and several of his collaborators also investigated how the polymerisation actually occurs. This led to studies of the network structure formed by fibrin, which he determined by measuring optical properties and the flow through the fibrin gel that formed when thrombin was added to the patient s blood (plasma) or to purified fibrinogen; the final results were obtained with three-dimensional confocal microscopy. See part II. A review about the fibrinogen work is found in Ref 7.

Coagulation FVIII (AHG,AHF) and hemophilia A; von Willebrand Factor (VWF) and von Willebrands disease (VWD) Basic research For genetic research se part II. In the mid Forties there were indications that Cohn’s fraction I had a hemostatic effect in hemophiliacs but the picture was not clear-cut. We (Birger Blombäck and I) therefore wanted to investigate whether fraction I-0 contained such an activity (impurity?). Together with Inga Marie Nilsson (from Malmö), we found that fraction I-0 had a very high activity of coagulation factor FVIII (lacking in hemophilia A), while fraction I-4 had almost no activity. Could we use the fraction for treatment of hemophilia A patients? Thanks to Jorpes’

friends Clarence Crafoord and Erik Sköld, an expert in the treatment of hemophilia and head of the blood centre we obtained fresh human blood to this end. As sterile filtration eliminated the activity, we had to develop a new aseptic technique for the preparation of I-0 to be carried out in primitive premises (now KI’s administrative building) where purification of compounds from animal intestines etc was also in progress. Inga Marie Nilsson had a patient (a young girl) in Malmö with a severe bleeding disorder known as pseudohemophilia (later shown to be identical to von Willebrand´s disease, VWD).

She had a low FVIII level (similar to a hemophilia A patient) but also a prolonged bleeding time she had life-threatening menstrual bleedings and could no longer be treated with blood because of severe side reactions. Administration of our fraction I-0 stopped the life-threatening menstrual bleeding, the level of FVIII in her blood increased and surprisingly the prolonged bleeding time normalized. A few days later her uterus was removed under cover of fraction I-0 administration. Fraction I-0 was prepared, as mentioned, at KI in Stockholm and we delayed planes and trains for emergency deliveries to the hospital in Malmö.

Early on in our research we had noticed that after intravenous administration of fraction I-0 (containing FVIII) to patients, the plasma level of FVIII disappeared quickly after reaching a peak in hemophilia A patients, while in VWD patients FVIII continued to increase and disappeared slowly.This was confirmed when fraction I-0 prepared from plasma from patients with hemophilia A was administered to VWD patients - their FVIII level was increased for several hours even though the preparation did not contain any FVIII (nowadays this would probably not be allowed but at that time the transfer of hepatitis virus with blood products was unk-

nown).

This and the fact that FVIII could be removed from Fraction I-0 without eliminating the bleeding-time activity, were proof that the latter was caused by an independent factor.(Ref 8). The factor respon-

Birger Blombäck, Inga Marie Nilsson and Margareta Blombäck in Rome in 1958.

We enjoyed our trip to the congress so much that we arrived late, to find Birger and Margaretas boss, erik Jorpes, and Inga Maries, Jan Waldenström, in a heated disscusion about which of them should present our findings

sible for this increase was then called von Willebrand s Factor (VWF). We suggested that the factor was involved in production or activation of FVIII. More or less the same conclusion was subsequently reached by French researchers. Later, several international research groups made it clear that the VWF simply carries and protects the FVIII in the circulating blood.

We enjoyed our trip to the congress so much that we arrived late, to find Birger and Margareta s boss, Erik Jorpes, and Inga Marie s, Jan Waldenström, in a heated discussion about which of them should present our findings.

Our basic research was then directed to further purification of FVIII and VWF. We were lucky to be able to separate FVIII (fraction I-1A) from fraction I-0 in a one-step procedure. When fraction I-1A was tried on patients with VWD, FVIII increased but there was very little effect on the bleeding time.

In the early Eighties, Lars Thorell and coworkers succeeded in preparing a fraction with a high FVIII content that also corrected the bleeding time, as shown in two patients with severe VWD . Unfortunately, this concentrate was never produced on an industrial scale. The effect was also demonstrated in an in vitro model by Kjell Sakariassen et al, who showed that when tested on vessels with damaged endothelium, this fraction could induce normalized adhesion of platelets to the damaged vessel wall but that this was not the case when plasma from patients with severe VWD was tested. Since FVIII protein constituted only a few per cent of the purified FVIII-VWF complex, it was obvious that the VWF was the main component in the complex.

In 1984 Birgit Hessel and coworkers were the first to report the N-terminal amino acid sequence for the VWF in a complex between VWF and FVIII, showing that VWF was a single-chain protein that polymerizes into a gigantic active polymer. (Ref 9).

Prothrombin Staffan Magnusson Another coagulation protein, prothrombin, was investigated by Staffan Magnusson, a medical student who joined Jorpes’ department in 1952. After much hard work on the purification of prothrombin, he managed to study the conversion of prothrombin to thrombin by using the Edman method to show where the split in prothrombin occurs. Soon after his dissertation in 1965 he left for America and Britain to

do protein research but from 1970 onwards he held senior posts in Denmark.

He devoted his life to the primary and secondary structures and functions of many hemostatic and other proteins, In several of them he found disulfide-bonded loops which he called kringles (as they were similar to Scandinavian ”kringles”).

Fibrinolysis Basic research Per Wallen and Kurt Bergström When Birger Blombäck and I started medical school (Karolinska Institutet) in 1949, two of our colleagues were Per Wallen and Per Olsson. We all focused our research on the hemostatic mechanism but did so from different angles. Per Wallén studied the fibrinolytic process rather than coagulation itself. His first goal was the purification of plasminogen, the precursor of plasmin which degrades the fibrin clot.

His thesis on this subject was presented in 1962. He and his pupil Kurt Bergström showed that plasminogen in the presence of the amino acid lysine was not adsorbed to anionic surfaces. By choosing the appropriate ionic strength and pH conditions it was possible to prepare an almost homogenous plasminogen fraction as well as a purified fibrinogen with no contaminating plasminogen. Studies on urokinase, the plasminogen activator from urine, and its effect on plasminogen were also performed. The Edman technique was used to show how plasmin and thrombin digest fibrinogen. Together with Sadaaki Iwanaga, a guest researcher of Birger Blombäck, further studies were carried out on the degradation products obtained from fibrinogen by thrombin and trypsin. In 1970 Per Wallen moved to Umeå. There the research of Per and his group, especially his pupil Björn Wiman who also had moved to Umeå, led to a breakthrough for thrombolytic therapy with the tissue plasminogen activator ( tPA) when they showed that fibin potentiates the effect of tPA. After his

retirement in 1993, Per returned to KI and continued his research in the fibrinolytic field, trying to find endothelial receptors for tPA. Together with Kamaran Fatah- Ardalani, he also reported that a partially degraded product of plasminogen (Lys-plasminogen) adsorbed to fibrinogen gave, on clotting with thrombin, a more porous network than when non-degraded plasminogen (Glu-plasminogen) was adsorbed.

Per was an excellent researcher with an international reputation, besides being a good musician.

Björn Wiman Björn Wiman returned to Stockholm in 1982 to join the coagulation laboratory at Karolinska Hospital and in 1992 he succeeded Birger Blombäck as professor of coagulation research at KI. He has made many very important contributions to the field of fibrinolysis. His discovery of a fibrinolysis inhihibitor in plasma, the plasminogen activator inhibitor-1, was a real break-through and since then most of his scientific work has centred on this inhibitor. Thus, he and his collaborators have developed PAI-1 methods for assays in different body mediums, he has characterized PAI-1 especially with regard to its interaction with the activator of fibrinolysis (tissue plasminogen activator-tPA) and he has investigated the stability of PAI-1.

Björn has also identified a carrier for PAI1 in plasma (vitronectin) and studied the interactions between the two proteins.

He and his collaborators (Göran Kronvall et al) studied plasminogen receptors on microorganisms. With Margareta StenLinder he has developed a method for the measurement of angiostatin (an angiogenis inhibitor consisting of fragments of plasminogen) in urine. He and collaborators have developed and applied in intensive care an important method for the measurement of soluble fibrin in plasma.

Björn’s recent functional studies of the plasmin inhibitor (antiplasmin) have yiel-

ded a better insight into the regulation of fibrinolysis. (Ref 10).

Per Wallen and Björn Wiman, who likes to fish and won the competition that year

Heparin, antithrombin and heparin coated surfaces Per Olsson and his pupils Kjell Rådegran, Jesper Swedenborg and Ulf Hedin During our first years in medical school, Per Olsson (Pelle) and Birger Blombäck were having lively discussions about religion, life, poetry and politics - Pelle more on the democratic side, Birger more on the conservative. Pelle, like Birger and me, is still working at KI. It was he, with an early interest in thoracic surgery, who introduced us to Clarence Crafoord and his team and together we worked on methods for heparin assay. I remember how we participated by measuring heparin levels at Crafoord’s second and subsequent heart-lung operations. Åke Senning was standing at the other operating table (their mutual language reminded me of a couple of dockers).

Pelle was also the first to point out the risk of hepatitis with blood products (fibrinogen). Unlike us, however, Pelle in his future research focused on the mechanisms of how to keep the blood in a liquid state.

Besides being about the influence of heparin and its elimination, his thesis in 1963 was the first to describe the importance of antithrombin for activation of the heparin effect.

He applied his knowledge to the treatment of patients, especially those with disseminated intravascular coagulation. In the late Sixties he was joined by two pupils, Kjell Rådegran and Jesper Swedenborg, still working at KI, who were interested in the effects of platelet aggregation - its influence on pulmonary obstruction - and in the importance of non-thrombogenic surfaces of catheters, for example, for avoiding undue site coagulation. Jesper Swedenborg and his foremost pupil Ulf Hedin have turned their interest to endogenous (vessel wall) surfaces and how to study the mechanism as well as protection. In keeping with his main concern of keeping the blood running, Pelle has for many years worked on obtaining artificial surfaces that do not activate coagulation.

Together with his colleagues Rolf Larsson and Olle Larm, he showed that heparin can bind (covalently) to artificial surfaces, rendering them thromboresistant, and in various ways prevent clotting. If the surfaces had a sufficient density of heparinbinding sequences, there was no activation of coagulation. Immobilized heparin activates coagulation factor XII (FXII) but as the activated FXII (FXIIa) is immediately inactivated by antithrombin (which is not the case with free heparin in the blood), the surface will be compatible with blood. Given such surfaces, patients can be treated in extracorporeal machines without being anticoagulated with heparin (ECMO or ExtraCorporeal Membrane Oxygenation treatment). (Ref 11-13).

Two of Pelle s pupils, Javier Sanchez and Graciela Elgue, have subsequently moved to Uppsala University. Pelle and Javier found that when the endothelium bound heparin sulphate (with its specific heparin-binding sequences like artificial heparin-coated surfaces), adsorbed FXII and activated it to FXIIa, the latter was immediately inhibited by concomitantly adsorbed antithrombin. Thus, when the

antithrombin level in the patient s blood reaches a critically low level, the endothelium will turn to become an activator of the contact activation coagulation and fibrinolytic systems as well as of the kallikrein and complement systems.

Per Olsson and his pupils

Experimental research in athero-thrombotic disorders Jesper Swedenborg, Stefan Nydahl and Siw Frebelius Since the time when Jesper Swedenborg started doing research with Per Olsson, he has devoted his interest to arterial vessel surgery and been the leader here in research as well as clinical work.

Swedenborg, together with Stefan Nydahl and Siw Frebelius, found in 1993 that recirculation through the microvasculature in a rat heart preparation led to increased disappearance of thrombin when it was performed together with heparin, which probably reacts with endogenous antithrombin on the vessel wall; in the absence of heparin the disappearance was unaffected by antithrombin.

Swedenborg, together with Stefan Nydahl and Siw Frebelius, found in 1993 that re-

circulation through the microvasculature in a rat heart preparation led to increased disappearance of thrombin when it was performed together with heparin, which probably reacts with endogenous antithrombin on the vessel wall; in the absence of heparin the disappearance was unaffected by antithrombin.

This is compatible with the theory that antithrombin/glycosaminoglycans plays a minor role for inhibition of thrombin activity and that thrombin binds mainly to thrombomodulin. In a study together with Hedin, using vessel walls from balloon-injured rabbit aortas, they showed that after an injury, antithrombin inhibits the appearance of thrombin on the vessel wall and thereby reduces the risk of thrombosis. This effect was later attributed to the ?-isoform of antithrombin. They also showed that the mitogenic activity of thrombin on smooth muscle cells is regulated by antithrombin, indicating that it may serve dual functions, inhibiting both thrombin coagulant activity and its growth-promoting effect on vascular smooth-muscle cells.

Starting from the clinically derived hypothesis that the thrombus is of importance for growth and rupture of abdominal aortic aneurysms, they continued the studies with specimens from human aneurysms.

Recent work together with Monsuur Kazi demonstrated that the aneurysm wall covered by thrombus is thinner and has more signs of inflammation, apoptosis of smooth muscle cells and a degraded extracellular matrix, indicating that these factors could increase the risk of aneurysm rupture and that the thrombus-covered wall may be the site of rupture.

In a recent paper, Phan-Kiet Tran, a doctoral student of Ulf Hedin, demonstrated in transgenic deficient mice that the endogenous heparin sulphate (HS) side-chains of the perlecans (the major HS proteoglycans) contribute to the control of smooth muscle cell growth both in vitro and during hyperplasia, possibly by sequestering heparin-binding mitogens such as fibroblast growth factor-2.

Platelets and acetylsalicylic acid (aspirin) Basic research

Hans Johnsson showed as early as in 1975 that circulating fibrinogen is present not only in plasma but also in platelets, with no exchange between media. Even so, the platelet fibrinogen is of importance for hemostasis, especially at low fibrinogen levels.

In 1981 Kjell RĂĽdegran and collaborators found that if prostacyclin was infused during heart operations, the platelet count was better preserved and platelet aggregation was diminished.

Jan Svensson Jan Svensson´s interest in platelets started with the break-through in prostaglandin research in 1974-75 in Nobel laureate Bengt Samuelsson’s group at KI with the isolation of prostaglandin endoperoxides and the discovery of thromboxane A2 (Ref 14).The effects of these unstable but highly biologically potent compounds were then investigated in several biological systems, including human and animal platelets, and found to induce platelet aggregation and vasoconstriction in submicromolar concentrations.

Jan Svensson et al showed that the antithrombotic effect of aspirin could now be explained as inhibition of the cyclo-oxygenase enzyme that converts arachidonic acid to these potent intermediates. Methods based on measurements of stable metabolites (e.g. thromboxane B2) have been used to study platelet regeneration time in patients with athero-thrombotic diseases as well as in studies on the inhibiting effects of aspirin. The results showed that platelet regeneration time is shortened in patients with stroke or myocardial infarction and in painters exposed to organic solvents, reflecting a shortened survival of the platelets in vivo.

Paul Hjemdahl Paul Hjemdahl has focused his groups interest on the dynamic regulation of hemo-

stasis in vivo, especially platelet function and interactions with white blood cells (”platelet-leukocyte cross-talk”). His group has therefore put efforts into reducing in vitro artifacts influencing platelet function measurements and developing methods for in vivo studies. Prothrombotic effects of stress, with platelet and leukocyte activation, as well as increased platelet-leukocyte aggregation and thrombin generation have been shown. Platelet activation in vivo during stress is parallelled by reduced platelet aggregation stimulated by ADP in vitro, which illustrates the complexity of platelet function studies. The group performs pathophysiological studies in patients with atherosclerotic disease and/or its risk factors, and studies of effects of treatment. Increased inflammatory activity is associated with platelet activation. Platelet-leukocyte ”cross-talk” and inflammatory activity are, for example, enhanced in diabetes mellitus. Antiplatelet treatment with aspirin (acetylsalicylic acid) or clopidogrel reduces some measures of platelet activity at rest, but does not protect against the prothrombotic effects of stress. Stress and increased inflammatory activity may thus contribute to atherothrombotic complications via platelet activation; the exact mechanisms behind these links are the subject of further studies. (Ref 15)

Jan Svensson and Paul Hjemdahl

Basic studies on the influence of acetylsalicylic acid See also above Dog experiments by Kjell Rådegran in 1972 demonstrated that if ASA was injec-

ted before thrombin infusion, most of the thrombin-induced increase in pulmonary arterial pressure and tracheal insufflation pressure could be inhibited.

In the early Eighties, Per Olsson published reports, especially together with R. Malmgren and H.Beving, on the extent to which platelets are influenced by ASA, serotonin and various toxic substances.

They showed that one mol of aspirin inhibits one mol of cyklooxygenase and that each individual has a genetically determined level of cyklooxygenase; the large interindividual variation in this level makes it difficult to arrive at the appropriate dose of aspirin for protection against CHD. However, besides acetylating a serine residue in cyklooxygenase, leading to the effect on platelets, ASA acetylates lysine residues in several hemostasis proteins such as fibrinogen and coagulation factor XIII, leading to a porous fibrin network (see above fibrinogen and part II) with thicker fibres, especially at low doses of ASA similar to those used in stroke prophylaxis at present.

Work on development of methods to assay hemostasis Nils Egberg, Shu He, Alexsandra Antovic and Jovan Atovic As is shown above our studies on fibrinogen and its cleavage by thrombin led to the development of new principles using chromogenic peptide substrates for the assay of many hemostatic and other enzymes, their proenzymes , and inhibitors Such methods but also immunological procedures, used in the research at KI have been developed at the coagulation laboratory, led for many years by Nils Egberg. Together with Kurt Bergström, Nils investigated the possibility of monitoring anti-vitamin-K (AVK) treatment (coumarin/ warfarin treatment) by measuring just one of the vitamin K dependent coagulation

factors, factor X. The advantage would be that factor X reflects the antithrombotic effect of the treatment better than prothrombin time, which is heavily influenced by factor VII. Factor VII has a short half-life and varies more during treatment than factor X; most likely only extremely low levels of FVII give an antithrombotic effect, while moderately low FX levels reduce the reaction rate in the coagulation cascade.

In a multicentre study, Nils Egberg and collaborators have presented an alternative model for reliable and reproducible local calibration of prothrombin time assays, using combined thromboplastin reagents (Owren-type reagents). This has been introduced in Sweden and has promoted inter-laboratory uniformity and reduced the costs of calibration.

In recent years Shu He, post doc, has developed a method, Overall Hemostatic Potential (OHP), to determine the hemostatic balance in plasma, i.e. whether the patient has a hyper- or a hypo-coagulation, including how the formed fibrin is degraded (fibrinolysis). Previously, only single factors were determined and that did not reflect what actually happens. Together with Aleksandra Antovic in particular, she has further developed and applied this method to several clinical materials, especially pregnancy and its complications.

A link between coagulation and fibrinolysis, TAFI (thrombin activatable fibrinolytic inhibitor), which has attracted attention fairly recently, has also been investigated in the laboratory with regard to methodology and various clinical materials by Jovan Antovic.

References The references have mostly been chosen by the researchers themselves

1. Mutt V, Blombäck M, Erik Jorpes-a pragmatic physiological clinical chemist. Selected papers in the Historyof Biochemistry: Personal Recollections VI. Eds G Semenza and R. Jaenicke Comprehensive Biochemistry 2000;41:263-389,.

2. Blombäck B, Blombäck M. Purification of human and bovine fibrinogen. Arkiv Kemi 1956; 10; 415-43.

3. Blombäck B, Blombäck M, Gröndahl NJ & Holmberg E. Structure of fibrinopeptides its relation to enzyme specificity and phylogeny and classification of species. Arkiv Kemi.

1966; 25; 411-28 4.Blombäck M, Blombäck B, Mammen EF, Prasad AS. Fibrinogen Detroit a molecular defect in the N-terminal disulphide knot of human fibrinogen? Nature. 1968; 218; 134-7.

5. Blombäck B, Blombäck M, Olsson P. Svendsen L, Åberg G. Synthetic peptides with anticoagulant and vasodilating activity. Scand Clin Lab Invest 1969, Suppl 107: 59-64.

6. Blombäck M, Egberg N: Chromogenic peptide substrates in the laboratory diagnosis of clotting disorders. Haemostasis and Thrombosis. Ed A.Bloom, D.Thomas, Churchill Livingstone, Edinburgh, Vol 56, p 967-81, 1986/1987 7. Blombäck B. Fibrinogen and Fibrin-Proteins with complex roles in hemostasis and thrombosis. Thromb Res 1996; 83:1-75.

8. Nilsson IM,Blombäck M,Blombäck B. v Willebrand´s disease in Sweden. Its pathogenesis and treatment. Acta Med Scand 1959; 164: 263-78, 9. Hessel, B., Jörnvall, H., Thorell, L. Söderman, S., Larsson, U., Egberg, N., Blombäck, B. and Holmgren, A. Structure-function relationship of factor VIII complex studied by thioredoxin dependent disulfide reduction.

Thromb. Res 1984; 35, 637- 51.

10. Wiman B: The fibrinolytic system. Basic principles and links to venous and arterial thrombosis. Blood stasis and Thrombosis 2000;14, 325-38.

11.Sanchez J, Olsson P. On the control of the plasma contact activation system on human endothelium: comparisons with heparin surface. Thromb Res 1999; 93:27-34 12.Rådegran K. The effect of acetylsalicylic acid on the peripheral and pulmonary vascular responses to thrombin . Acta Anaesth Scand 1972; 16: 140-6.

13.Kazi M, Thyberg J, Religa P, Roy J, Eriksson P, Hedin U, Swedenborg, J. Influence of intraluminal thrombus on structural and cellular composition of abdominal aortic aneurysm wall. J Vasc Surg 2003;38:1283-92.

14. Hamberg M, Svensson J, Samuelsson B. Thromboxanes: a new group of biological active compounds derived from prostaglandin endoperoxides, PNAS 1975;72: 2994-8 15. Li N, Wallén NH, Hjemdahl P: Evidence of prothrombotic effects of exercise and limited protection by aspirin. Circulation 1999;100:1374-9.

Read Margareta Blombäcks essay - Part II Clinical research - Blood coagulation research at KI 1956-2004

Margareta Blombäck, Essäist Margareta Blombäck - professor emerita - one of the leading figures wihin the Swedish research on Blood coagulation 1956 - 2004. Together with her husband, Birger Blombäck, she has reached international fame. Here you will find a summary of the Swedish reseach in this field.

Read Margareta Blombäck’s essay - Part II Clinical research - Blood coagulation research at KI 1956-2004