Thrombosis and Haemostasis Research

AnniversaryIssueContribution

Thrombosis and haemostasis research:

1957–2007) Stimulating, hardwork andfun y( Margareta Blombäck Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital,Stockholm, Sweden

Anniversar odoresearch is to be inquisitive,tobeprepared to setout The first attemptatclinical useoffraction I-0 prepared from th in unexpected directions in response to newfindings –be humanplasma wasmade on apatient with acquired fibrinogen 50 T theynegativeorpositive. Research is likeanincurable in- deficiency; however, the patient went into averysevere fibrino- fectious disease –once you’ve got it, you’ve got it for life. A lytic state.Welater understood thatpyrogens cangiverise to fi- negative result or aresultthat contradicts what youpreviously brinolysis (wehad,for the preparation, used waterwhich wasnot believedoften leads to newideasand inventions.Research also pyrogen free).Weknew, however, thatCohn’sfraction Icon- givesyou friends all over the world, both collaboratorsand com- tained an antihaemophilic component, so working with Inga petitors. BelowImention just some colleagues with whom we Marie Nilsson from Malmö, we testedfraction I-0 for the pres- performed the basic and clinicalresearch,but rest assured: Ihave ence of factor VIII (FVIII). Thefirst sample testedgaveavery not forgotten anyone. high yield. Later it wasfound out that we had used haemophilia Bplasma in testing the activity of this sample.Wehad notyet characterized Stockholm’shaemophiliacs in termsofhaemophi- 1950s: Fibrinogen, factorVIII, vonWillebrand lia Aand B. However, it did indeed have 100% yield of the FVIII factor(VWF) activity of the original plasma when testedproperly. Dr.IkuoYamashina (3),watching me and Inga Marie sitting After afew years studying literatureand socialwork, respect- on eitherside of awaterbathtilting tubestostudy clot formation, ively, BirgerBlombäck and I, then24years old, started studying said “Maybe or maybe not”. Ihope he meant this as ajoke. We medicine at KarolinskaInstitutet, where we are still doing re- continuedtopurify fibrinogen and FVIII from fraction I-0(2–8). search.In1951 Birger Blombäck wasasked to join the research Birger also continuedwith structural analyses of fibrinogen (3). group of Erik Jorpes, the "heparin man". As Birger’swifeIwas At thetime we were fortunate enoughtohavegood financial also allowedtojoin the group (1).Jorpes had acommission from supportfrom ErikJorpes, whohad very high royalty incomes Vitrum (a private firm laterincorporated with Kabi/Pharmacia/ from Vitrum (donated tax-free to research). He alsoreceivedbig Pfizer) to test heparin activity and the analysis required purified grantsfrom the NationalInstitute of Health (NIH). Generous fibrinogen. This wasprepared from bovine blood,which we supportwas also providedbyJorpes’friends, the thoraxsurgeon fetched from the slaughterhouse, travelling by tram and laterby ClarenceCrafoord,and the headofStockholm Blood Centre, motorbike, lugging the blood around in large glass bottles. The ErikSköld,who provided the human blood free of charge. Cra- fibrinogen (preparedaccording to the methodsthen in use) was, foordneeded fraction I-0 for thosepatients whowere bleeding however, stable onlyfor afew hours. Birger,who has always read duetofibrinogendeficency(fibrinolysis due to pyrogens?) (3, the literaturemore carefullythan I, wasintrigued by Edward 4). In order to test the heparinactivity in the blood,wepartici- Cohn’sidea of plasma fractionation and demonstration of fibri- patedinthe second heart-lungoperation ever performed in

nogen in fraction I. We found thatwhen fraction Iwas extracted Stockholm, in 1955. This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. in the presence of high concentrations of glycine,the solubility In 1956 IngaMarie Nilssonhad apatient, Birgitta, with severe of fibrinogen wasreduced butmost of the impurities went into pseudohaemophilia (in1957 identified as being vonWillebrand ´s solution. The stable fibrinogen fraction thus obtained we called disease [VWD] type 3) whonolongertoleratedbloodtransfusions fraction I-0 (2, 3). Theclinical studiesdescribed in the following andwhosemenstrualbleedingwas life-threatening. We decided to would probablynot have beenallowedtoday, buttherewere no trytostop the bleedingwith fraction I-0 (containing FVIII). It had ethical committees at thattime. to be prepared in arush. Thetrains andaeroplanesenlisted to

Correspondence to: Received May12, 2007 MargaretaBlombäck Accepted May21, 2007 Dept. of MolecularMedicine and Surgery Karolinska Institutet Prepublished onlineJune 12, 2007 Clin. Chem. Build. L205 doi:10.1160/TH07–05–0343 Karolinska University Hospital Stockholm, 17177Sweden ThrombHaemost 2007; 98: 8–15 Te l.: +4685177 4437, Fax: +46 8312 438 E-mail: [email protected]

8 Blombäck:Thrombosisand haemostasis research transportthe freshly preparedfraction I-0 from Stockholm hadto of Åland,who had aformofpseudohemophilia, describedin wait until we got it to them. But it did have adramatic effect: bleed- 1926 by the Finnish doctor Erik vonWillebrand (13).Most of the ing stopped, the FVIII levelincreased andthe bleedingtime was population living in the archipelago of Åland,situatedverynear normalized (4, 8–12). In different studieswetriedtofigure out Stockholm, are of Swedish origin and speak Swedish. Prompted whythe bleedingtime wascorrected.Birger and ourteam pre- by Erik Jorpes, whowas born and raisedthere, we took atripto paredthe fractions,and IngaMarie wasthe one mainlyincharge these islands,tostudy the surviving members of the originalvon

of treating the patients. Purifiedfibrinogen and purified FVIII had Willebrand family(for results seebelow). Becauseofdifferences 1957–2007) no normalizingeffect on the bleedingtime, whereas fraction I-0 in voltage,the large centrifuge we had brought from Stockholm devoid of FVIII or prepared fromhaemophiliaAplasma corrected to spin down platelets did not work, forcing ayoung doctor to y( it. We also observedthatthe half-lives of FVIIIwerequitediffer- makedaily flights back and forthtoStockholm. Everyday he entinpatients with haemophilia Aversus thosewith VWD (4, 8) flewout with patient blood samples (and duty-free liquor) and (Fig. 1). In this wayweshowedthe existence of anew “anti-bleed- worked with Birgertoprepare the platelets for the assays (4,11). ing”factorabsentinVWD (byusfirst called the bleedingtime fac- The investigation showedthat the findings in the original family tor) andwenamed it “von Willebrand factor” (4, 12). were the same as thoseinthe patients in Sweden. Beforetravel- Anniversar Most of the fraction I-0 thatwas prepared wasgiven to pa- ling to Åland,ErikJorpes had met Prof. RudolfJürgens on the th tients with haemophilia AorVWD.For the first tenyears,itwas streetand revealed ourplans to him, so Jürgens, whowas on his 50 prepared aseptically (as we lost too muchofthe FVIII in filters waytothe islands to hunt seals, of course also brought ateam to used at thattime), by the devotedstaff in alaboratorywheredur- investigatethe same individuals. Theyfound adecreased levelof ing the first years insulin and peptideswere also prepared from FVIII, just as we did. We could confirm our suspicion that the animalintestines (3, 4). Swedish patients had the same disease and the same heredity as In 1956–1957 IngaMarieNilsson and I, accompaniedbya those from the Åland archipelago (11, 14). geneticist,travelledaround central Sweden in acar thatgaveus In 1958 IngaMarie, Birgerand Iwere going to present ourre- headaches. (Halfwaythrough the trip we discovered the exhaust sults at the VII th InternationalSociety of Hematology Congress system wasbroken.)Our mission wastostudy different families in Rome. We travelledinour car, whichhad alot of problems withpseudohaemophilia,aswecalleditthen. We visited high with its brakes. En route,wepassedthrough Vienna, where we and low, rich and poor alike (10).At thattime heredity could only visited anothermeeting. Laterwestayedinasmall place on the be analysedbyblood group determination; nonetheless, in two Italian east coast, happilydrinking the Italian wine there and in out of ten familieswefound a(healthy) child whose purported othersmall places.Arriving in Rome one daylate, we found that fatherwas notthe real father. IngaMarie’sboss (JanWaldenström) and ErikJorpes had given The clinicalfeatures,heredity and some laboratoryfindings us up for lost and were discussing whoshould present our find- in our patients were similartothose of patients from the islands ings. The congress participants were invitedtomeet the Pope

Figure1:Difference in sur- This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. vival time forfactorVIII in apatient withseverevWD type3(left)and apatient withmoderate haemophilia (right) in connection withhysterectomy and ap- pendectomy, respectively. Theadministered factorVIII is expressedasU/g protein. At the first occasion, thus3.9 x 300 U= 1,170 Uoffactor VIII were given.The broken arrow represents freshblood administration.(printed withper- mission from Haemostasis 1992; 22: 239.)

9 Blombäck:Thrombosisand haemostasis research

Pius XII, whotold us that he wasnot against abortions if the child In Pehr Edman’slaboratorywestudiedthe amino acid se- wasdiseased (a haemophiliac). quenceofthe human fibrinopeptides A(FPA) and B(FPB). Allthrough this timewehad to earnaliving, in addition to When we had returned to SwedenBirgerand his teamincluding studying medicine.Ihad ajob teaching medicalbiochemistryto Russ Doolittle continued to sequence the corresponding pep- medical students. It wasnot easy as Ihad almost no basic know- tides from manyspecies to study the evolutionarypattern.Ire- ledge except fortraining in the laboratory. Furthermore, we had memberlying on the floor at home trying to figure out (bypuzzl-

1957–2007) to write our theses. Birger’sthesis wasonfibrinogen. Mine was ing) the evolution patterns of cow, sheep, reindeer,mink, dog, on antihaemophilic factor (later named FVIII) butJorpes did not seal and even elephant fromthe sequencesfound. There were no y( allowmetocallitthat way. Ihad to callit“antihaemophilic glo- computers or computerprogramsavailablefor this job at that bulin”.Wepassedour doctorate examsinthe spring of 1958, time.All FPAsincluded an extremelywellpreservedsequence in Birger oneday earlierthan me (5, 8). The dayafter that, Birger the C-terminalpartofthe peptides close to the bond splitby had to give his lecture to qualify as an associate professor.These thrombin.Acomparison of the line of reindeer with thoseof tight timeschedules were orders from ErikJorpes. sheep and goat indicated thatthe latter branchedfrom adeer line Anniversar The 1950s were ahard time,but in manyrespectsavery re- rather thanfrom the main bovine line in classicalphylogeny (3, th warding period.Wehad alot of fun and learned to work in teams 20)(Fig. 2). Birger wasinvitedbyRobertG.Macfarlane to lec- 50 withboth laboratorystaff and clinicians. tureabout our findings in the ZoologicalSociety of London. Also in the beginning of the 1960s we started to work with Per Olssononantithrombin and its effect togetherwith heparin. 1960s andearly 1970s: Treatment of patients, Birgercontinued to work on the structureofthe N-terminal dis- fibrinogenstructure, fibrinogenDetroit, ulphide knot of fibrinogen togetherwith his colleagues men- tionedabove. thrombin inhibitorsand chromogenic substrates In 1966,with moreand more patients seeking diagnosis and Of course we hadthe idea that we could also purify afraction for advice, Imoved fromthe Karolinska Institutetothe hospital treatment of haemophilia Bfrom Cohnfraction II+III.We did so but (across the road), though Iwas still an assistant professor con- hadtowaitayear untilwecould tryitinapatient. Birger,who did nected with the Institute. The production of fraction I-0was the injection,reported that the haemostatic effect wasgood, butthe takenoverbyKabi,giving us aniceroyalty. blood pressure went down to lowlevelssoheinterrupted the injec- In May1967 we arranged afantastic meeting on dissemi- tion.Whennewly prepared,the fraction hadbeen tested for adverse natedintravascularcoagulation (DIC) in Grisslehamn, asmall effectsinarabbit without problems.Now when we trieditinadog village on the coastnorth of Stockholm. Manyscientists from all it showed the samenegativeeffect as in the patient. We suspected over the worldattendedthe conference. Theywere alittle shock- thatkallikrein activity haddevelopedand wastoblame for the ad- ed by the obligatorysauna, butaccepted this and other wild verseeffect.Two medical companieshavehad the sameexperience Swedish habits, including fermented Baltic herring. Afterthe when storing similarlyproduced material. When we wanted to start meeting some of the participants, very special friendsofours, the production againwecould not find our protocols. visited an island belonging to Birger and me.Bernadette Ver- While still engaged in production of fraction I-0 for our straete, aguest from Belgium,slippedinto the waterand Marc- Swedish patients, we designedanew fractionation plant (see Verstraete went in to rescue her.Theywere both savedbyPer [15]) whichwas inaugurated by KingGustaf Adolf Vand Queen Olsson. Louise (4).Inthe beginning of the 1960s Iworked mainlywith When Dr.ErwinDeutsch, thenthe editor of Thrombosis and clinical aspectsofhaemophilia (16)and on carriers of hae- Haemostasis,heard about the meeting he suggestedthat we mophilia in collaboration with IngaMarieNilsson,Olof should publish the presentations of the meeting in this prestig- Ramgren and others (17). One thing Iamveryproudofisthat we ious journal. But when we were going to submit the manuscripts, started prophylactic treatment as earlyas1958 in atwo-year-old we learned thatacleaner had thrownthe entire –veryheavy – boywho is alive to this day(18), though he has had aliver trans- boxwith allthe material into the trash (Birger and Iwere thenin

plant due to hepatitis C. Birger continuedtowork on the struc- NewYork butour collaboratorstried to find them in the Stock- This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. ture of fibrinogen, especiallyincollaboration withTorvard Laur- holm dump). Later we managedtofind allmanuscripts butthen ent,Birgit Hesseland Sadaki Iwanaga (3). it wastoo late to publishthem. From 1961 to 1962 we spent ayear in Australia at the invi- In 1967 we spent half ayear at the NewYork Blood Center. tation of Dr.Percival(Val) Baileyofthe Serum Laboratories in While there, we were contacted by Dr.EberhardMammenin Melbourne to startproduction of fraction I-0 there and of Dr. Detroit, whohad apatient, ablack girl,with severe menstrual Pehr Edman to do protein research(19). Whenwearrived,Dr. bleedings, whohethought must have an abnormalfibrinogen: Baileywas no longer in charge. Strugglingwith an overlyre- Herfibrinogenlevel wasnormal, butnoclot wasformed when stricted budget, he had publiclycriticized the government and thrombin wasadded (3).Asthe release of FPAand FPBseemed had therefore been fired (an English tradition).Wewere shock- to be normalweguessedthat the amino acid sequence following ed. In spite of this we had awonderful timeinAustralia and made the FPAsequence wasperturbed. We made atryptic digestion of manyfriends. We travelledaround the continent, lecturing, en- the A α chainofthe Nterminaldisulphide knot(calledN-DSK) joying ourselves and learning about Aborigine culture. We cher- and subjected the digest to two-dimensionalelectrophoresis- ish many memoriesfrom thattime.Fraction I-0 wasusedfor chromatographyoncellulosethin-layerplates(3). As canbede- long timetotreat the haemophiliacs in Australia. duced fromFigure3,the tripeptide Gly-Pro-Arg–which in nor-

10 Blombäck:Thrombosisand haemostasis research 1957–2007) y( Anniversar th 50

Figure2:Phylogeneticre- lationship of different ar- tiodactyls. Therelationship was obtainedbycomparisons of residues at the sameposi- tion in thedifferent species. (From ArkivKemi1966; 25: 418.) mal fibrinogen follows the thrombin-susceptible bond of the A α son and Lars Svendsen(theninMölndal nearGothenburg–the chainafter FPA–had moved. Subsequentlywecould showthat firm waslater takenoverbyKabi)the possibilityofusing this the arginine in position 19 of the A α chainhad been substituted knowledge formaking thrombin inhibitors. They synthesized by aserine residue.Wewroteapaper presenting our findings at both the full nona-peptide sequence and shorter variants. The alounge of the airportinDetroit (3, 21). On the waytoDetroit we maximum inhibitoryeffect of these peptidesonthrombin was stoppedatDr. Oscar Ratnoff’s laboratoryinCleveland to give found with the Phe-Val-Arg variant (3, 22). In 1967 the team ap- some lectures. We also presented dataonthe abnormal fibri- pliedfor apatent on the prototype tripeptide for useasinhibitor

nogen (fibrinogen Detroit) with the second mutation found in a of thrombin (23).Atthat timethe pharmaceuticalindustrywas This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. protein that could be coupledtoadefectivefunction (the first notmuch interested in thrombin inhibitors, though Göran Clae- wasamutation found for sicklecellhaemoglobin,afindingpres- son continuedtowork partlywith the inhibitorswhen he had ented in the Viennameeting mentioned above). Oscar Ratnoff movedtoStockholm and latertothe UK. kindlytook acolour photo of the gel (Fig. 3). At one of our meetings with Bofors-Kabi in Mölndal the Iloved working in Australia and NewYork, as in both these question wasput forward “Couldn’tweuse the tripeptide for places Ihad the time to work at the benchwith my ownhandsfor measuring thrombin activity?”This wasachieved by coupling a whichIhavehad almost no time sincethen. chromogenicgroup, paranitroanilide,tothe carboxylic partof Earlyon, we and Inga Marie Nilsson were asked by Bofors the arginine residue in the tripeptide.When the enzyme thrombin Company(Karlskoga, Värmland) to investigateexperimentally acted on this substrate,paranitroaniline wasreleased and the ac- the function of the snake venom from Bothrops Jarararaca (Rep- tivity (colour) could be measured in aspectrophotometer tilase)suggestedtobeusedfor treatment of bleedings. We then (24–26).Now we could also measureantithrombin activity.In also met some organic chemists there, Göran Claeson and others. similar ways, using knowledge of the cleavage sites forfactor However, as partofthe C-terminus of FPAseemed to be vitalfor Xa, plasmin, trypsin, kallikrein, and other proteases chromo- the thrombin action (seeabove), we discussed with Göran Clae- genic peptide substrates were constructed and in afew years

11 Blombäck:Thrombosisand haemostasis research 1957–2007) y( Anniversar th 50

Figure3:Two-dimensional electrophoresis ( ←→)-chromatography(↓↑)oftryptic digests of α -chain fragment. To pleft: normal fibrinogen. To pright: fibrinogenDetroit(homozygote). Bottom:fibrinogen Detroit (heterozygote). Photos takenbyOscar Ratnoff. Part of normal A α chain…..-PHE-LEU-ALA-GLU-GLY-GLY- GLY- VAL-ARG-GLY -PRO- ARG -VAL VAL….Thrombin splitsbetween ARG-GLY whenreleasingFPA.Infi- brinogen Detroit SER substituted ARG .(From NRFHématologie 1970; 5: 671-678).

thereappearedmore than 1,000 papers where differentsub- work togetherafter ourdivorcein1972, though until Iretired in stratesfor measurement of proenzymes, enzymes and their in- 1992, the clinical work as head of acoagulation laboratorytook hibitors had beenused. most of my time. Looking back, Icannot understandhow we could getso My laboratorydiagnosedhaemophilia,VWD,and other muchdone during the 1960s and early1970s. We owealot to the bleeding disorders, butalso did investigations in thrombophilic excellent collaborators(3, 4) mentioned above and to Agnes patients. We were on call to handle allsuch patients, including

Henschen and our foreign visitors IkuoYa mashina, Sadaki Iwan- patients bleeding in the intensive care (DIC etc).We did labora- This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. aga, Janeand Rick McDonagh and Gene Murano, Barbara Kow- toryanalyses and medicalcontrols of haemophilic patients from alska Lothtonameafew.While Birger continuedthe studies on alarge partofSweden and took care of them all when theyhad to fibrinogen and related topics, Ihad to devote most of my time to be hospitalized foroperations or similar problems. We took the clinical side becauseofmyposition at the hospital. Igot special care of the haemophilic children in Stockholm as well. manyyoung collaboratorsfrom differentclinics(6) whopro- Togetherwith Barbro Wiechel,who wasincharge of the out-pa- duced doctoral thesestogether with Birgerand me or me alone as tient care of adults, we introduced prophylactic treatment as well supervisor. as self-treatment for adult patients with severe and moderate haemophilia and with VWDtype 3in1976. Forthe patients the possibilityofself-treatment gave asenseofgreat freedom. Late 1970s, 1980s and1990s: Clinicalwork, My lab wasone of the first in Europetostart using instru- teaching,purifiedVWF, HIV,hepatitisC ments for clotting factor analyses;the laboratoryassistants told me Idid this just to save staff. Ialso introduced agood computer- After our sojourninNewYo rk, Birgerspent much timeasavisit- ized systemveryearlyfor the hospital accountants, butwhen my ing professor at the Blood Center,New Yo rk. We continuedto laboratorywas fusedwith Clinical Chemistry,the headofthe de-

12 Blombäck:Thrombosisand haemostasis research partment said we had to go backtodoitbyhand again. Idid afair VWDwhose health wasatstake.Some of them have died of liver amount of teaching especially trying to regularlyupgrade asmall damage before good treatment wasavailable. bookcalled“CoagulationNews” on clinical diagnosis, labora- toryanalyses and treatment of haemostatic disorders. VonWillebrand disease again Ihad ahighlydedicated staffand manygood medicalcollab- In the beginning of the 1980s, Birgerand Thorell designeda orators. Iespecially want to mention Nils Egberg from my lab- good method for preparation of VWF(28). This preparation

oratory, Hans Johnssonfrom the medical clinic,Dag Nyman(see showedbysequence analyses,for the first time,that VWFwas a 1957–2007) below),Margareta Hellgren and laterKatarina Bremme from the single chainprotein (29). We trieditontwo patients in whom it gynaecologyclinic,Julius Sorefffrom the orthopaedicclinic, indeedhad an extraordinarilybeneficialeffect (30).Weoffered y( and SixtenBredbacka from the intensive care unit, buttherewere the procedure to Kabi buttheyanswered too late.The Finish Red many others, among them several thoracicsurgeons (6).Inthe Cross then started to preparethe fraction. However, due to the beginning Ihad arather high royalty income from Kabi for our procedures for inactivating HIVitdid not retainactivity.Norbert inventions and Icould therefore supportthe researchwork in the Heimburger at the BehringwerkeinMarburg(Germany) early laboratory. However, afterwardsIregretted Ihad too little time to devicedapasteurization method for FVIII and other concen- Anniversar supportthe collaboratorsintellectuallyand Iwastedmuch trates in ordertoget ridofhepatitis virus (31). This resultedin th moneyunnecessarily, using it for routine work. thatmost of the haemophilic patients in Germanycould be 50 But my patients have also been very good collaborators. Ihad treatedwith aproduct free of HIV. oneverydevoted patient with severe haemophilia A, whom I Wayback when Iwas writing my thesis Iwas certain thatpa- could not persuade thatheneeded treatment with FVIII concen- tients with VWDtype 3were homozygous, though Iwas unable trate: He knew Ineeded hisplasma for analyses of FVIII. Nils to persuade either my opponent or IngaMarieNilsson thatIwas Egberg and Iwere working with the possibility of treating pa- right. However, Ihad asecretary, Elisabeth Söderlind,who had tients with thrombotic disorders with Defibrase or Arvin (An- kept track of allVWDfamilies, and,guided by this material, gen- crod) butwefirst wanted to find out if it gave rise to increased eticist MariaAnvret and Istarted investigating the polymor- bleeding tendencysowetried Defibraseinthis patient. The pa- phismsusing DNAlinkage analysis. We found thatpatients were tient reported onlyaslightlyincreased capillarybleeding. homozygous in five families, and were either compoundhet- erozygousorbore anew mutation in three families(32). After HIV and hepatitis C much discussion with an editor,who earlier had rejected our In the summerof1982, Frank Schnabel,the headand creator of papers on polymorphismsinhaemophilia Aand B, this paper the “World Federation of Hemophilia”,cametoStockholm for a waspublished in anotherjournal. Ioften wonder if editors pro- shortvisit. He told me,“Meta, there is aterriblediseasespread long the reviewprocesses becausetheywant to publish their own by blood products –Iamsure this will be acatastrophefor hae- results first.In1991, Zhiping Zhang ayoung Chinese,cameto mophilic patients.”In1983 at the XVth WorldFederation of He- our departments in KarolinskaInstitutet. He had stated that he mophilia Congress in Stockholm (27)therewere several lectures had read all my papers on VWD(laterheconfessed he had only onAIDSand the immunologicalstatus of haemophiliacs,though read arecent abstract). therewere notyet anytests for HIV.InSweden we had been forced to use FVIII products from the USA in ordertotreat our My triumph patients, as the supplyoffraction I-0 wasinsufficient (the plasma In 1992 at the XXth WorldFederation of Hemophilia Congress in wasfrom Sweden and Finland).Also the patients preferred the Athens,Ian Peakesaidinhis lecturethat it wasimpossible to find American product as it could be injected in asmall volume. anymutation in the VWFgene. In the discussionIpresentedpart Sadly, 100 of 136 patients with severe haemophilia Agot in- of what we had found in Stockholm (Fig. 4). The same year, fectedwith HIVand about 70% died,ofthose manyyoung boys. Zhiping Zhang published twoimportant papers (33,34), and his Whenwestarted treating haemophiliacs with fraction I-0 the findings were summarized in his thesis (35). By using PCR tech- mean age was16years,but before the HIV- era appeared the niquefollowedbydirect sequencing he found asingle cytosine

mean age wasnear thatofthe healthypopulation. In 1979 the deletion in exon 18, nonsense mutations in exons 28, 32 and 45 This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. Swedish Board forTechnical Innovation hadstarted aprogram to and nine other newmutations (33–35) (Fig. 4). The deletion in trytoprovide moreSwedish plasma, butthe resulting increase in exon 18 is the most common one found in the Swedish patients. plasma volume for fractionation didnot reachthe patients until Heredity investigations showedthat it canbetraced back several HIVwas afact.The HIVinfection wasclearlyadisasterfor the hundred years.Ibelieve it originatedfrom immigrantsorsailors patients butwas also extremelyhard forthe mothers whohad in- from the South. Thenonsense mutation in exon 28 wasfound in jected the FVIII concentrates into their children. OurVWD pa- patients whose parents came from Finland. tients did not have HIV as they were still treated exclusivelywith In the meantimeDag Nyman, whothen wasworking in my fraction I-0 produced from blood from Swedenand Finland laboratory, had shown that the familiesfrom Åland thatwere where HIV infection at thattime waslow. suggested to have VWD,could be divided into several categories Already in theearlydayswehad notedthat administration of (36). Thesurvivors of the originalfamilyhad the characteristics fraction I-0could give rise to hepatitis butasthe therapysaved of what Iprefer to callVWD type 1, similartothe relativesofthe livesIthink we did not bother so much about this infection. In Swedish patients with VWDtype 3, i.e. decreasedlevel of VWF later years we became aware what hepatitis Ccould do to our pa- and ristocetin cofactor activity in addition to normaland de- tients, and nowitwas notonlyhaemophiliacs butalso thosewith creased levels of FVIII; the plateletaggregation wasnormal. As

13 Blombäck:Thrombosisand haemostasis research

in the Swedish patients, one cytosinedeletion wasdetected in opedaverypromising oral thrombin inhibitor (41)that could be exon 18 in the surviving relatives. We investigated ahairfrom a used for short-termprophylactic treatment againstthromboem- small boywith VWDtype 3. He washomozygous for the dele- bolism. However, the administered drug ledtoliver damage in a tion (37). fewcases and waswithdrawn. During the past fewyears new At present Iwould liketostudy howthe mutants especially thrombin and factor Xa inhibitorshavebeen developedand thoseinexon 18 and in exon 28 have travelledinEurope,but this launched onto the market. Thehope is that theywill be good

1957–2007) is too much work for an oldlady and the moneyisnot available. enoughtoreplace both heparinoids and coumarinderivatives. My teamispresentlyexerting muchefforttoinvestigate y( Fibrinogen, fibrinogenstructure again, arteriosclerosis, some inhibitors with our newmethods (the fibrin network influence of acetylsalicylic acid(ASA) and global assay method and the global assay), hoping the resultswill help the of coagulation clinicians to find out about both the favourableand the unfavour- By studying the fibrin gel network in 1981 and 1989, Birger able effectsofthese newtreatment methods(42). showedwhat influenced the final fibrin network structureina Anniversar pure fibrinogen system (3) and also later in aplasma environ- th ment; he statedthat the clot structureisinfluencedbythe first Otherareas we worked in, missed 50 formednetwork (38). This could of coursebeofinterest for ar- opportunities, SSCwork teriosclerosisresearch,and,indeed, Anders Hamsten and I, examining this phenomenon,found that the initial network was In retrospect Isee Iwas ofteninterestedintoo manyaspects. On muchtighterinmales whohad experienced myocardialinfarc- one occasion Birgerand Ipurified coagulation factorV, probably tion at ayoung age (39).Sincethen much of my research work due to that we considereditanimportant factor in haemostasis. has been in this direction, especially togetherwith the groups of Birgerand Iwere always interested in antithrombin and its mech- Håkan Wallenand Gun Jörneskog and my postdoc Shu He.In anism and we did much interesting work with our great friend Per this respect the influenceofacetyl salicylic acid(ASA) has also I. Olsson whohas devotedthe last twodecades to work on hepa- attracted my attention.Ifound that lowdoses of ASA makethe rin-coated surfaces for clinical application. Averygood collab- network more porous and that it canthus be degraded more orator of mine in the antithrombin area is also my good friend Ul- easilybyplasmin (40). Ithink this finding is of much greater im- richAbildgaard,whom Iforgot to include in apaper on thatsub- portancethan plateletresearchers admit. ject. Apaper by PetterFriberger on heparin cofactorIIwas sent Formanyyears Ihavebeen frustrated by the belief–appar- to the chief editor of ThrombosisResearch –Birgerhimself – ently sharedbymany–that problems in the complexhaemosta- whoprobably thought it wasjust for comments from him person- sis systemcan be detected by measuring asingle factor or in- ally, as there wasnoansweruntil after reminding him, so we hibitor. Although Iamveryproudofmywork togetherwith missedthe opportunityofbeing oneofthe first writing about this Anders Hamsten and BjörnWimanonplasminogenactivator-1, topic in the literature. Ihavehad agreat interest in developingaglobalhaemostasis Agrave scientific mistake-information is difficultevento assay. Even though manyglobalassays are nowentering the mar- thosearound you. ket, Iamstill working on this subject togetherwith Shu He and Ihavemade many studiesonthe influenceofhormones on others. haemostasis. In astudy on the menstrualcycle Ifound agreat in- creaseinVWF and FVIII in the luteal phase, especially in one volunteer being my laboratoryassistant formanyyears.Though Thrombin inhibitorsagain–whatweare doing my conclusion in the paperwas correct thatfemalehormones at present caninfluence the levelofthese coagulation factorsitwas exag- gerated.Almost 10 years laterIfound out my laboratoryassistant After thefirst trials of thrombin inhibitors(seeabove)Astra- hadbeen running to the placeofblood sampling,allowingto Zeneca,together with one of BirgerBlombäck’sformer PhD stu- drawalternativeconclusions about the relation betweenexercise

dents Ann-Catrine Teger Nilsson, continued the work and devel- and haemostatic status. This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited. Formanyyears Ihavebeen interested in what happensduring storage of plasma for transfusion purposes or for fractionation of factor concentrates.Ienjoyedthe work with Joanna Chmielew- ska on this topic and recentlyAnna-Maija Suontaka from my group published athesis on the subject(43). Ithink the issue has beenlooked into fartoo little by blood transfusion specialists. Forseveral years aroundthe startofthe 1990s Ico-chaired and chairedthe Scientific and Standardization Subcommittee (SSC) of the InternationalSociety on Thrombosis and Haemos- tasis (ISTH). Ienjoyedthe work, though it wasnot easy to get 20 Figure4:StructureoftheVWFgeneand corresponding pro- subcommitteestoagree on newnomenclature issues (published tein domains. Mutations and polymorphisms found by Zhiping Zhang in 1994 butstill not usedbyall in full).Withoutthe dedication of areindicated.For details seereferences33–35, 37. (Printed withper- the Executive SecretaryCathy Cole and the supportfrom the mission from Zhiping Zhang.) ExecutiveDirector Harold RobertsofISTH, thework would not

14 Blombäck:Thrombosisand haemostasis research have beenpossible. In this regard an earlygreat mentor for me with and stimulate young doctorstodoresearch.Tobeableto and afriend wasRosemary Biggs, aformer editor-in-chief of apply basic researchfindings in the clinic,and to be able to solve Thrombosis and Haemostasis. questions from the clinicians, is aprerequisite for furtherdevel- opment of the area,and will be of benefitfor the patients. Concluding remarks Acknowledgement

The area of haemostasishas widened tremendouslysincethe Birger Blombäck has kindlyread the manuscript and corrected it in places 1957–2007) 1950s. It has givenmemuch pleasure to be allowedtotakepart

wherehethought Iwas wrong. y( both in the basic and the clinicalwork. Ialwaysliked to work

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