Blood coagulation research at Karolinska Institutet 1920-2004

PART I - Basic research

Margareta Blombäck Essäist

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

PART I - Basic research: on heparin and its derivatives, on thrombogenic and non-thrombogenic properties of the vessel wall; on purification of hemostatic factors: fibrinogen, prothrombin, Factor VIII and von Willebrand Factor and fibrinolytic fac-tors; 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 vari-ous ways to continue his education in Fin-land, 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 bet-ween 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 (Stock-holm).

With permission from the Swedish go-vernment, he resumed the study of medi-cine at Karolinska Institutet in 1920. It was said that he had to promise the finance minister, the leader of the Social Demo-crats, never to work with politics - a pro-mise 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 he-ralded 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 se-cretion 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 trea-ting 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 in-vested 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, Jor-pes 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 success-fully with American interests and obtained funds from them as well as from the Swe-dish authorities. Erik Jorpes and heparinWhy did Erik Jorpes start to work with he-parin? Clarence Crafoord in 1940 descri-bed how, on reading a paper by Howell on the effects of heparin in 1929, he was

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 ne-gative effects were due to contaminants. In August 1935 Crafoord started treating patients prophylactically.

According to Jorpes, the beds were sha-king hard when the patients received heparin (the importance of pyrogen con-tamination was unknown). Jorpes, alone or together with Sune Bergström and Olle Wilander in the Thirties and later in the Fif-ties with Harry Boström and Viktor Mutt, succeeded in unravelling the complex chemical structure of heparin and demon-strated its biosynthesis in the mast cells.

Crafoord, as well as many other collea-gues/friends of Jorpes, started working

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 procee-dings from 1940. He reported all that was known about heparin’s history, chemistry, mode of action, synthesis and standardi-zation; its clinical advantages and draw-backs, death rates among treated compa-red 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 he-parin was not only as an inhibitor of coa-gulation but also as a reducer of inflam-mation - a view that is coming into focus more and more.

As an individual, Erik was reserved and lived simply, according to strict principles. He was an excellent but feared teacher.

Fibrinogen

Basic studiesBirger Blombäck and I (Margareta Blombäck)In 1950, when Birger Blombäck and I (Margareta Blombäck) had begun to stu-dy 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 re-searchers, all very spoiled - thanks to Jor-pes’ royalties - by having the services of a woman who cleaned the glassware, an ef-ficient 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 fibrinogenOur first duty was to prepare fibrinogen to be used in a method for the standardiza-tion of heparin (one of Jorpes’ underta-kings with Vitrum). We did this from bovi-ne blood, acquired in 50-litre glass bottles

Erik Jorpes at work

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.

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 solu-tion while the fibrinogen remained as a precipitate which could easily be dis-solved. We called this fraction I-0 and it provided a foundation not only for our ba-sic research but also for much work by us and our collabora-tors in the clinical field. (Ref 2, Tree figure).

Fibrinogen Metamorpho-sis - a tree drawn by Birger Blombäck

Tree, drawn by Birger Blom-bäck. The tree illustrates the development ot the research by Birger and his team on fibrino-gen, its structure, funcion and evolution, on fibrinopeptide A, on the abnormal fibrinogen (fbg Detroit), on the development of chromogenic peptide substra-tes, and on thrombin inhibitors and on the coagulation factors VIII and Von Willebrand s fac-tor.

(ko=cow; hjort=deer; får=sheep; svin=pig; arters utveckling = evolution of species. Trombin hämmare= thrombin inhibitors; syntetiska substrat=synthetic chromoge-nic peptide substrates; kedjestruktur=chain structure; tätt nätverk=tight network; glest nätverk=porous network.)

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). Bir-ger Blombäck and Ikuo Yamashina then demonstrated that fibrinogen, a very lar-ge molecule, consists of three different chains and that each unit of fibrinogen contains two of each. Agnes Henschen isolated the chains and since the indivi-dual chains were obtained after reduc-tion (breaking the disulfide bonds), it was concluded that they were held together by disulfide bonds. Blombäck et al sho-

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 clea-ved off from fibrinogen when it was transfor-med to fibrin (the th-readlike substance being the basic sub-stance in a clot) by the enzyme thrombin. Bir-ger Blombäck and his team were particularly interested in mapping the structure not only of these peptides but also of the site of clea-vage by thrombin. By studying the release of the peptides by th-rombin, it was shown how polymerisation, gelation and aggrega-tion 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 struc-tural analyses were

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

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

Margareta and Birger Blombäck with a bottle of fraction I-O in the laboratory of Medical Chemistry. Press photo taken in connection with thier dispu-tations

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 frag-ment presented in the figure. T=bond split by thrombin; TRY=bond split by trypsin.

Having purified fibrinopeptides from mam-malian and other species, we determined their structure and found that the sequen-ce of nine amino acids (nonapeptide) near the binding split by thrombin, Arg-Gly, es-pecially the ninth position (phenylalanine), had been well preserved during evolution. We also demonstrated that the species dif-ferences and similarities (with the excep-tion 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 fasci-nating. (Ref 3)

Of great importance for understanding the initial polymerisation (fibrin forming) pro-cess was the discovery of a homo-zygous (inherited the same genetic defect from both parents) point mu-tation (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 prote-in, leading to a disease, had been demonstrated (the first was for he-moglobin in sickle-cell anemia). As the release of FPA was not preven-ted in this fibrinogen (called fibrino-gen Detroit), we suggested that the mutation had inactivated a polyme-risation site that was exposed on release of fibrinopeptides.

(Ref 4)

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 inhi-bitory 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 (la-ter Kabi) Göran 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 (para-nitroanilide =pNA), the compound (chro-mogenic 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 sub-strates 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-

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

Birger Blombäck and collaborators con-tinued their research on which parts of the fibrinogen molecule are of importan-ce for thrombin’s action. One of the very important studies was together with Ann-Catrine 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 ef-fect 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 (im-purity?). Together with Inga Marie Nils-son (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 he-mophilia 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 pri-mitive premises (now KI’s administrative building) where purification of compounds from animal intestines etc was also in pro-gress. Inga Marie Nilsson had a patient (a young girl) in Malmö with a severe blee-ding disorder known as pseudohemophi-lia (later shown to be identical to von Willebrand´s disease, VWD).

She had a low FVIII level (similar to a he-mophilia 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 frac-tion I-0 stopped the life-threatening men-strual bleeding, the level of FVIII in her blood increased and surprisingly the pro-longed bleeding time normalized. A few days later her uterus was removed under cover of fraction I-0 administration. Frac-tion I-0 was prepared, as mentioned, at KI in Stockholm and we delayed planes and trains for emergency deliveries to the hos-pital 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 hemophi-lia 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 in-creased for several hours even though the preparation did not contain any FVIII (nowadays this would probably not be al-lowed but at that time the transfer of he-patitis virus with blood products was unk-

nown).

This and the fact that FVIII could be re-moved from Fraction I-0 without elimina-ting the bleeding-time activity, were proof that the latter was caused by an indepen-dent factor.(Ref 8). The factor respon-

sible for this increase was then called von Willebrand s Factor (VWF). We suggested that the factor was involved in produc-tion or activation of FVIII. More or less the same conclusion was subsequently reached by French researchers. Later, se-veral 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 hea-ted 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 in-creased but there was very little effect on the bleeding time.

In the early Eighties, Lars Thorell and co-workers succeeded in preparing a fraction with a high FVIII content that also correc-ted 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 endothe-lium, this fraction could induce normali-zed 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 con-stituted 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 bet-ween VWF and FVIII, showing that VWF was a single-chain protein that polymeri-zes into a gigantic active polymer. (Ref 9).

Prothrombin

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

Birger Blombäck, Inga Marie Nilsson and Marga-reta 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 Walden-ström, in a heateddisscusion about which of them should present our findings

do protein research but from 1970 on-wards he held senior posts in Denmark. He devoted his life to the primary and se-condary 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 si-milar to Scandinavian ”kringles”).

Fibrinolysis

Basic researchPer Wallen and Kurt BergströmWhen Birger Blombäck and I started medi-cal school (Karolinska Institutet) in 1949, two of our colleagues were Per Wallen and Per Olsson. We all focused our re-search 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 app-ropriate ionic strength and pH conditions it was possible to prepare an almost homo-genous 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 perfor-med. The Edman technique was used to show how plasmin and thrombin digest fib-rinogen. Together with Sadaaki Iwanaga, a guest researcher of Birger Blombäck, further studies were carried out on the degradation products obtained from fib-rinogen by thrombin and trypsin. In 1970 Per Wallen moved to Umeå. There the re-search of Per and his group, especially his pupil Björn Wiman who also had moved to Umeå, led to a breakthrough for thrombo-lytic therapy with the tissue plasminogen activator ( tPA) when they showed that fi-bin 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- Ar-dalani, he also reported that a partially de-graded product of plasminogen (Lys-plas-minogen) adsorbed to fibrinogen gave, on clotting with thrombin, a more porous network than when non-degraded plasmi-nogen (Glu-plasminogen) was adsorbed.

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

Björn WimanBjörn Wiman returned to Stockholm in 1982 to join the coagulation laboratory at Karolinska Hospital and in 1992 he suc-ceeded 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 fib-rinolysis inhihibitor in plasma, the plas-minogen activator inhibitor-1, was a real break-through and since then most of his scientific work has centred on this inhibi-tor. Thus, he and his collaborators have developed PAI-1 methods for assays in different body mediums, he has charac-terized 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 PAI-1 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 Sten-Linder he has developed a method for the measurement of angiostatin (an angi-ogenis inhibitor consisting of fragments of plasminogen) in urine. He and collabo-rators have developed and applied in in-tensive 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).

Heparin, antithrombin and heparin coated surfaces

Per Olsson and his pupils Kjell Råde-gran, Jesper Swedenborg and Ulf HedinDuring our first years in medical school, Per Olsson (Pelle) and Birger Blombäck were having lively discussions about reli-gion, 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 hepa-rin assay. I remember how we participated by measuring heparin levels at Crafoord’s second and subsequent heart-lung ope-rations. Åke Senning was standing at the other operating table (their mutual langu-age reminded me of a couple of dockers). Pelle was also the first to point out the risk of hepatitis with blood products (fibrino-gen). Unlike us, however, Pelle in his fu-ture research focused on the mechanisms of how to keep the blood in a liquid state. Besides being about the influence of he-parin 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 disse-minated 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 endo-genous (vessel wall) surfaces and how to study the mechanism as well as protec-tion. 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 sur-faces had a sufficient density of heparin-binding sequences, there was no activa-tion of coagulation. Immobilized heparin activates coagulation factor XII (FXII) but as the activated FXII (FXIIa) is immedia-tely 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 hepa-rin (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 im-mediately inhibited by concomitantly ad-sorbed antithrombin. Thus, when the

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

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

Per Olsson and his pupils

Experimental research in athero-th-rombotic disorders

Jesper Swedenborg, Stefan Nydahl and Siw FrebeliusSince the time when Jesper Swedenborg started doing research with Per Olsson, he has devoted his interest to arterial ves-sel 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 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 antith-rombin on the vessel wall; in the absence of heparin the disappearance was unaf-fected 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 antith-rombin on the vessel wall; in the absence of heparin the disappearance was unaf-fected 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 throm-bomodulin. In a study together with Hedin, using vessel walls from balloon-injured rabbit aortas, they showed that after an injury, antithrombin inhibits the appearan-ce of thrombin on the vessel wall and the-reby 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 vascu-lar smooth-muscle cells.

Starting from the clinically derived hypo-thesis that the thrombus is of importance for growth and rupture of abdominal aor-tic aneurysms, they continued the studies with specimens from human aneurysms. Recent work together with Monsuur Kazi demonstrated that the aneurysm wall co-vered 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 doc-toral student of Ulf Hedin, demonstrated in transgenic deficient mice that the endo-genous heparin sulphate (HS) side-chains of the perlecans (the major HS proteogly-cans) contribute to the control of smooth muscle cell growth both in vitro and during hyperplasia, possibly by sequestering he-parin-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 fibrino-gen levels.

In 1981 Kjell Rådegran and collabora-tors found that if prostacyclin was infused during heart operations, the platelet count was better preserved and platelet aggre-gation was diminished.

Jan SvenssonJan Svensson´s interest in platelets star-ted with the break-through in prostaglan-din 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 biologi-cal systems, including human and animal platelets, and found to induce platelet ag-gregation and vasoconstriction in submi-cromolar concentrations.

Jan Svensson et al showed that the antith-rombotic effect of aspirin could now be ex-plained as inhibition of the cyclo-oxygena-se enzyme that converts arachidonic acid to these potent intermediates. Methods based on measurements of stable meta-bolites (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 ef-fects of aspirin. The results showed that platelet regeneration time is shortened in patients with stroke or myocardial infarc-tion and in painters exposed to organic solvents, reflecting a shortened survival of the platelets in vivo.

Paul HjemdahlPaul Hjemdahl has focused his groups in-terest 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 acti-vation, as well as increased platelet-leuko-cyte 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 per-forms pathophysiological studies in pa-tients with atherosclerotic disease and/or its risk factors, and studies of effects of treatment. Increased inflammatory activity is associated with platelet activation. Pla-telet-leukocyte ”cross-talk” and inflamma-tory activity are, for example, enhanced in diabetes mellitus. Antiplatelet treatment with aspirin (acetylsalicylic acid) or clopi-dogrel 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 com-plications via platelet activation; the exact mechanisms behind these links are the subject of further studies. (Ref 15)

Basic studies on the influence of ace-tylsalicylic acid See also above

Dog experiments by Kjell Rådegran in 1972 demonstrated that if ASA was injec-

Jan Svensson and Paul Hjemdahl

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 publis-hed 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 inhi-bits one mol of cyklooxygenase and that each individual has a genetically determi-ned level of cyklooxygenase; the large in-terindividual variation in this level makes it difficult to arrive at the appropriate dose of aspirin for protection against CHD.

However, besides acetylating a serine re-sidue in cyklooxygenase, leading to the effect on platelets, ASA acetylates lysine residues in several hemostasis proteins such as fibrinogen and coagulation fac-tor 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 prop-hylaxis at present.

Work on development of methods to assay hemostasis

Nils Egberg, Shu He, Alexsandra Anto-vic and Jovan AtovicAs is shown above our studies on fibrino-gen and its cleavage by thrombin led to the development of new principles using chromogenic peptide substrates for the assay of many hemostatic and other en-zymes, 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 Eg-berg. 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 proth-rombin time, which is heavily influenced by factor VII. Factor VII has a short half-life and varies more during treatment than fac-tor X; most likely only extremely low levels of FVII give an antithrombotic effect, while moderately low FX levels reduce the reac-tion rate in the coagulation cascade.

In a multicentre study, Nils Egberg and collaborators have presented an alterna-tive model for reliable and reproducible lo-cal 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 de-veloped a method, Overall Hemostatic Po-tential (OHP), to determine the hemostatic balance in plasma, i.e. whether the patient has a hyper- or a hypo-coagulation, in-cluding how the formed fibrin is degraded (fibrinolysis). Previously, only single fac-tors were determined and that did not re-flect 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 fibrinoly-sis, TAFI (thrombin activatable fibrinolytic inhibitor), which has attracted attention fairly recently, has also been investigated in the laboratory with regard to methodolo-gy and various clinical materials by Jovan Antovic.

References

The references have mostly been cho-sen by the researchers themselves

1. Mutt V, Blombäck M, Erik Jorpes-a prag-matic physiological clinical chemist. Selected papers in the Historyof Biochemistry: Perso-nal 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 phyloge-ny 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 hu-man 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 pep-tide substrates in the laboratory diagnosis of clotting disorders. Haemostasis and Th-rombosis. Ed A.Bloom, D.Thomas, Churchill Livingstone, Edinburgh, Vol 56, p 967-81, 1986/1987

7. Blombäck B. Fibrinogen and Fibrin-Prote-ins with complex roles in hemostasis and th-rombosis. Thromb Res 1996; 83:1-75.

8. Nilsson IM,Blombäck M,Blombäck B. v Willebrand´s disease in Sweden. Its pathoge-nesis and treatment. Acta Med Scand 1959; 164: 263-78,

9. Hessel, B., Jörnvall, H., Thorell, L. Söder-man, S., Larsson, U., Egberg, N., Blombäck, B. and Holmgren, A. Structure-function re-lationship of factor VIII complex studied by thioredoxin dependent disulfide reduction. Thromb. Res 1984; 35, 637- 51.10. Wiman B: The fibrinolytic system. Ba-sic principles and links to venous and arte-rial 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 sur-face. 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, Eriks-son P, Hedin U, Swedenborg, J. Influence of intraluminal thrombus on structural and cellu-lar 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 ac-tive compounds derived from prostaglandin endoperoxides, PNAS 1975;72: 2994-8

15. Li N, Wallén NH, Hjemdahl P: Evidence of prothrombotic effects of exercise and li-mited protection by aspirin. Circulation 1999;100:1374-9.

Read Margareta Blombäcks essay

- Part II Clinical research - Blood coagula-tion research at KI 1956-2004

Margareta Blombäck, Essäist

Margareta Blombäck - professor eme-rita - one of the leading figures wihin the Swedish research on Blood coagulation 1956 - 2004. Together with her husband, Birger Blombäck, she has reached inter-national fame. Here you will find a sum-mary of the Swedish reseach in this field.

Read Margareta Blombäck’s essay

- Part II Clinical research - Blood coagu-lation research at KI 1956-2004