developed by the federation of american societies …breakthroughs in bioscience 2 waiting for a...

Developed by the Federation of American Societies for Experimental Biology (FASEB) to educate the general public about the benefits of fundamental biomedical research.

INSIDEthis issueCClloott BBuusstteerrss!!!! ��

DDiissccoovveerryy ooffTThhrroommbboollyyttiicc

TThheerraappyy ffoorr HHeeaarrttAAttttaacckk && SSttrrookkee

A Convergence of Pathways

1In the Olden Days

1Changing Therapeutic

Directions

2The Road to Thrombolytics

4The Raison d’Etre for Natural Clot Busters

5Discovering t�PA

6The Life & Death of a Clot

8How Streptokinase &

t�PA Work

9Door�to�Needle Time

10Coming Attractions

14Out of Basic Research:

A Life�Saving Drug15

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AcknowledgmentsCLOT BUSTERS!! - DISCOVERY OF THROMBOLYTIC THERAPY FOR HEART ATTACK& STROKE

Author, Cathryn Delude

Scientific Advisor, Kenneth Mann, Ph.D.,

University of Vermont

Scientific Reviewers:, Craig Jackson, Ph.D.,

Hemosaga Diagnostics Corporation

Burton Sobel, M.D., University of Vermont

BREAKTHROUGHS IN BIOSCIENCE COMMITTEE

Fred R. Naider, Ph.D., Chair, College of Staten Island,

CUNY

David L. Brautigan, Ph.D., University of Virginia School of

Medicine

John Grossman, M.D., Ph.D., MPH, George Washington

University

Tony T. Hugli, Ph.D., California Toxicology Research

Institute

Richard G. Lynch, M.D., University of Iowa College of

Medicine

J. Leslie Redpath, Ph.D., University of California, Irvine

BREAKTHROUGHS IN BIOSCIENCE PRODUCTION STAFF

Science Policy Committee Chair: Leo T. Furcht, M.D.,

University of Minnesota Medical School

Managing Editor: Carrie D. Wolinetz, Ph.D., Associate

Director for Communications, FASEB Office of Public

Affairs

COVER IMAGE: The development of thrombolytics

(“clot�busters”) to treat heart attack and stroke followed a

complex pathway of basic research and clinical observation�

Natural clot busting agents� from human blood vessels� leeches�

vampire bat saliva� and bacteria all played a role in helping sci�

entists understand how to harness the power of thrombolytics

to save lives� Cover design by Corporate Press� photos courtesy

of Science Photo Library� Photo Researchers� Inc� and the

Organization for Bat Conservation�

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Breakthroughs in Bioscience 1Breakthroughs in Bioscience 1

THROMBOLYTICSClot�Busters!! � Discovery of ThrombolyticTherapy for Treating Heart Attack & Stroke

A Convergence ofPathwaysWhat do the search for antibi-

otics in the 1930s, the discoveryof the double helix in the 1950s,and an extremely aggressive caseof melanoma skin cancer in the1970s have in common? They allcontributed a piece of the puzzleto a breakthrough treatment forheart attacks and the most com-mon form of stroke.

That treatment, a class of “clotbusting drugs” called thrombolyt-ics, has saved untold lives. Yet asin so much of science, the intro-duction of thrombolytics took along, circuitous route, with bothhead-scratching bewildermentand “aha!” moments along theway. The emerging, disparatepieces did not appear to belong tothe same puzzle, much less toone pertaining to the heart orbrain. A proliferation of newresearch specialties (moleculargenetics, cancer biology, and bio-chemistry) co-evolved and turnedup more pieces to contemplate.Budding technologies (surgicalpathology, radiology, in vitro cellgrowth, and recombinant DNA)provided new ways to explorepast and present discoveries,including clues about naturallyoccurring thrombolytic agents.Unrelated areas of research unex-

ment of thrombolytics stated,“Physicians trained after theadvent of thrombolytic therapymay find it difficult to believethat this concept was one of themost hotly debated pathophysio-logical tenets of the 20th century.Yet, the development of throm-bolytic agents for treatment ofAMI remained a tortuous andstuttering process until this concept achieved widespread acceptance.”1

In The Olden DaysWhile playing tag in the back

yard of our Oklahoma home in1959, my 53-year old grandfather,John Alonzo McCullough, feltchest pain, went to lie down, andwaited for the house call from ourfamily doctor, Charles Johnson. A slightly overweight Texanpostal inspector and smoker,McCullough was not an unusuallyyoung heart attack victim in thosedays (Figure 2). At the dawn ofthe 1960s, when few people knewmuch about heart disease preven-tion and the average hospital hadfew cardiac procedures to offer,an even higher percentage ofAmericans who suffered heartattacks died from them than do so today.

In 1959, lying down was proba-bly the best thing to do while

1Maroo A and Topol EJ. (2004) Journal of Thrombosis and Haemostasis. 2:1-4.

pectedly converged, and unsuc-cessful experiments in one arealed to sudden advances in another.

Without any one of these sepa-rate advances, from microbiologyto DNA technology, the puzzlewould have remained incomplete.Instead, like the image on aPolaroid photo coming into focus,a new conceptual model of thenature of heart attacks (and thenstrokes) developed. That modelshowed that clots are the culpritsbehind acute heart attacks, knownas acute myocardial infarction(AMI), as well as most strokes,and it vindicated a previously dis-carded notion of treating a heartattack by using clot-dissolvingthrombolytics to open up theclogged arteries.

Because of decades of basicresearch and clinical studies, anew type of thrombolytic drug,known as t-PA for tissue plas-minogen activator, appeared at the bedside in record time.Meanwhile, acceptance of the“open artery” theory of treatingheart attacks led to still more life-saving techniques, includingangioplasty, and preventive thera-pies, such as a daily dose ofaspirin.

As a 2004 review article sum-marizing the history and develop-

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Breakthroughs in Bioscience 2

waiting for a doctor or ambu-lance. Dr. Johnson did not yetknow to give my grandfather afew aspirin to prevent furthergrowth of the clot that—unbeknownst to either man—wasblocking the flow of blood in acoronary artery and depriving the heart muscle of oxygen. Thelonger the heart muscle wentwithout oxygen, the more damageit sustained. Resting the heartmuscle reduced its oxygendemand and limited the damage.Yet Dr. Johnson probably had thisprognosis for my grandfather:either he dies, or he does not.(Luckily, he did not, and he livedanother non-smoking 35 years ona lean diet and long daily walks.)

Changing TherapeuticDirections Strangely enough, had this heart

attack happened in 1899, beforemy grandfather was born, his doc-tor might have read the just-pub-lished article, “Infarction of theHeart.” It proposed that acuteheart attacks are frequentlycaused by thrombosis, or clotting,in the coronary arteries. Otherscientists had observed clots inthe blood vessels for several cen-turies. One scientist called theclots seen in arteries at autopsy“chicken fat,” because that waswhat they resembled. Their paleyellow color stems from the factthat post-mortem arterial clotscontain few red blood cells, con-sisting mainly of coagulatedblood plasma. Another scientistcalled the clots in veins, which docontain red blood cells, “currant

FFiigguurree ��:: A lucky heart attack survivor c� ��: The longer a heart muscle goes withoutoxygen� the more damage it sustains� Resting the heart limits the damage and beforethe discovery of thrombolytics was the recommended therapy� Photo courtesy of JohnMcCullough and Cathryn Delude�

FFiigguurree ��:: The Toll of Clots� As leading causes of death in the United States� heartattack ranks first and stroke third (second� world�wide)� The total number ofheart disease deaths per year increased throughout the ��th century� along withthe growing population and the long term toll of smoking and a more sedentarylifestyle� Fewer and fewer heart attacks are fatal� in part due to thrombolytics�resulting in a dramatically declining death rate over thirty years (from �� to)� Sources: CDC/NCHS� American Heart Association �� Update� andNational Hospital Discharge Survey� NHLBI�



jelly” for their red, gelatinousappearance. Had they been ableto observe the blood vessels priorto death at this time, researchersmight have described white clots,as well, made up of clumps ofplatelets.

While doctors began to acceptthe theory that clots cause heartattacks at the turn of the 20th cen-tury, they assumed that AMIswere inevitably fatal. With thatpessimistic assumption, there waslittle impetus to develop thera-pies. During the 1910s, however,some researchers challenged theexpectation that heart attackswere always fatal, reporting thatmany patients did survive andrecover. That realization spawneda new therapeutic model: the“open artery hypothesis” pro-posed that restoring the bloodflow to the heart muscle couldimprove the survival and recov-ery rate for heart attack victims.

The field changed directionsagain during the 1940s.Prominent medical experts,including some at the NationalInstitutes of Health (NIH), heldthat coronary thrombosis was the result, not the cause of heart muscle damage.Electrocardiograms (EKGs)enabled doctors to observe thatpatients undergoing heart attackshad irregular EKG readings, indi-cating heart muscle spasms. Onthe other hand, autopsies revealedarterial clots in only about a thirdof heart attack victims. Thus,many experts concluded thatheart spasms could cause the

clotting, but that the reverse wasnot true. The drive to developthrombolytic drugs to treat heartattacks slackened.

Undaunted, Dr. Sol Sherry wasconvinced that a thrombolyticagent called streptokinase (SK),produced by streptococcal bacteria, could treat unwelcomeclots. Without knowing exactlyhow SK worked, Sherry used iton a heart attack patient (whosurvived) in 1947 and laterdemonstrated that it dissolvedclots in rabbit ear veins. In the1950s, he and collaborators treat-ed more heart attack patients withSK, using massive doses thatwere much higher than the thera-peutic dose given today.2

Looking back, the results ofSherry’s studies showed what hassince become the cardinal rule ofheart attack therapy: the soonerthe better. Patients who receivedSK treatment within 14 hours oftheir heart attack had a lowermortality rate in the hospital.Those who received delayedtreatment fared no better thanuntreated patients.

At the time, no one consideredthe potential importance of earlytreatment with thrombolytics. Ifthey had analyzed the dataaccording to how early patientsreceived treatment, they wouldhave noted clear-cut benefits forpatients who received SK in thefirst few hours. As it was, pool-ing all the results showed aninsignificant overall benefit com-pared to the elevated risk ofbleeding as a side effect of theintervention in the body’s blood-clotting system. Thus, manyresearchers reached erroneousconclusions, dismissed the bene-fits of thrombolytics, and ques-tioned the clot theory of the disease.

“We were dissolving clots inpeople whose heart muscles werealready dead. We didn’t realizewe had to treat early to receivebenefits,” recalls Dr. BurtonSobel of his days as a youngresearcher investigating throm-bolytic therapy for heart attacks.“We were all confused and wethought either something waswrong with our hypothesis thatheart attacks were caused by clotsor something else was wrong. Itturned out to be something else,the need for very early treatment.Our lack of subsidiary knowledgediscouraged progress and thefield dried up.”

The situation changed again in1980, when new technologiesbegan to converge with clinicalresearch and Dr. Marcus DeWoodfinally caught clots at the scene of the crime. He used

The “open artery” model forheart attacks proposed thatrestoring blood flow to the

heart muscles could preventdamage and improve survival

chances. But this theory took several decades to

become widely accepted.

2Tillet WS and Sherry S. (1949) Journal of Clinical Investigation 28:172-190; Fletcher AP, Alkjersig N, Smyrniotis FE and Sherry S. (1958) Trans.Assoc. Am. Phys. 71:287-296.


FFiigguurree ��:: Colored scanning electron micrograph (SEM) of Streptococcus� Streptococcalbacteria produce a molecule� streptokinase� to gain access to the human blood streamand to penetrate tissues� Scientists isolated SK to use as the first thrombolytic drug totreat acute heart attack� Science Photo Library/Photo Researchers� Inc�


angiography, the technique ofinserting a catheter for an X-rayexamination of the blood vesselsconnected to the heart, on 126patients within 24 hours of theonset of symptoms. Of thosepatients, almost 50% showed evi-dence of thrombi. DeWood couldeven retrieve the thrombi from 52patients, an amazing feat at thattime. During the next 24 hours,many fewer clots were observed.The decrease in thrombi overtime resulted from the body’s nat-ural clot-dissolving process (see“Fibrinolysis,” page 11), whichcontinues post-mortem andaccounts for the absence of clotsin the autopsies of many heartattack victims.

DeWood’s finding helped reaffirmthe open-artery hypothesis fortreating acute myocardial infarc-tion. Now the medical community

had an acute need for a thrombolyt-ic drug that could immediatelybegin dissolving the clot and quick-ly restore blood flow to endangeredheart muscle. “The hope was we

could have a therapy that was likethe additive you put in car radia-tors that plugs up only the holes,”recalls FASEB Executive Directorand medical researcher Dr.Frederick R. Rickles. “Onlythrombolytics would work inreverse. It would open up theplugs, and only the plugs, so thatit would not cause bleeding. But inpractice that’s difficult to do.”

The Road toThrombolyticsLuckily, a virtually ready-made

drug was already on the shelf:streptokinase. Sherry and his protégés had accumulated evi-dence showing that SK indeeddid save the lives of heart attackvictims. Other researchers were forging ahead with anothernaturally occurring thrombolytic,urokinase, which is produced bykidney cells and is used mainly for treatment of pulmonaryembolisms [blockages in thelungs]. The introduction of strep-tokinase into medical practice foracute heart attacks in the 1970sbegan to make its mark on thedeclining death rates from acuteheart attack. (Figure 5)

Just as SK was gaining glory, anew and glamorous kid arrivedon the block: recombinant-PA.While some researchers wereinvestigating SK as a therapy forheart attack, others, followingindependent paths in the basicresearch arena, had already dis-covered a naturally occurringhuman thrombolytic agent, calledtissue plasminogen activator or t-PA, that circulates in the humanbloodstream. Newly available

It took new medical technology to finally establishscientifically the true cause of

heart attacks: thrombi, alsoknown as blood clots, in

the coronary arteries.



molecular techniques for explor-ing the biochemistry of t-PA’sinteraction with various bloodproteins coincided with newlyminted DNA technologies. Thesetechnologies enabled scientists toidentify the genetic sequencesand then clone important bloodproteins and enzymes for furtherstudy or therapeutic applications.Proteins produced by this methodare called recombinant proteins.In just seven years, researcherswould use t-PA for therapy andintroduce the first recombinantdrug, rt-PA, to the pharmacyshelves.

The Raison d’Etre forNatural Clot BustersWhy are there naturally occur-

ring thrombolytic agents? Thehuman blood system is an

secrete molecules that breakdown clots or prevent them fromforming.

Streptococcal bacteria, orStreptococci, for instance, inflictnumerous types of infection onhumans, ranging from sore throatto rheumatic fever, and they double as the notorious “flesh-eat-ing bacteria.” Doctors had noticedthat some patients with strepto-coccal infections developed clotsthat then spontaneously dissolved.In 1933, around the time of grow-ing skepticism about the openartery model of treating heartattacks, bacterial infections were ahuge menace to public health. Noantibiotics were yet available,although a fervent search wasunderway, a search that laterturned out to be fortuitous yetcritical for the development ofthrombolytic therapy. In pursuingantibiotics, researchers werestudying Streptococci to under-stand the disease process and findways to subdue infections. In theprocess, they isolated the agentthat dissolves clots and called itstreptokinase.

Eventually, scientists came tounderstand that Streptococci pro-duce streptokinase as part of anevolutionary one-upmanship withthe human immune system.Immune cells recognize whenStreptococci are trying to penetratevarious tissues. In defense, theimmune cells instruct the body tobuild roadblocks, in the form ofclots, which also help stop thespread of infection to other partsof the body. In a counter attack,

FFiigguurree ��:: When the lining of the coronary artery is clear and healthy� blood flows easily to the heart� bringing it the oxygen it needs to function� Atherosclerosis� or hard�ening of the arteries� is caused by a build up of plaque (fatty material) along the walls ofthe artery� and can narrow the passageway bringing blood to the heart� It is thought thatatherosclerosis is caused by a response to damage to the artery walls from high cholesterol� high blood pressure� and cigarette smoking� A blood clot can plug the open�ing narrowed by the atherosclerosis� ceasing all blood flow to the heart muscle� When thishappens� the heart muscle does not receive oxygen� is damaged� stops working� and even�tually dies� This is a heart attack� or myocardial infarction� Designed by Corporate Press�

Normal artery

Atherosclerosis

Atherosclerosis and blood clot

blood clot

intricately complicated field ofchecks and balances, defensesand attacks. Take, for instance, a finger prick. A dot of bloodappears but then, normally, stopsflowing. The slight break in theblood vessel wall sets in motion acascade of events that coagulateblood and prevent uncontrolledbleeding, a natural clottingprocess called hemostasis. Theexcessive and sometimes life-threatening bleeding in hemo-philia, for example, results froma deficiency in just one of manyblood proteins involved in theclotting cascade. Bacteria, bats,leeches, and snakes long ago figured out the benefits of over-riding the human body’s bloodclotting system. To gain access to the blood stream or to invadehuman tissue, these organisms


the nutrients in human tissue andto spread infection.

Discovering t�PA andits SurprisingApplication�

In 1947, researchers reportedfinding an anti-clotting agent thathuman cells naturally produce to clear up clotting, later calledtissue plasminogen activator or t-PA. Meanwhile, cancerresearchers were studying anunidentified agent’s suspectedrole in cancer-forming cells. Theyknew the agent degraded a fiber-like substance called fibrin thathelps build blood clots, and themore of it a cell made, the morecancerous the cell looked.Researchers speculated that thesame agent responsible for chew-ing up fibrin also helped the cancer cell chew through theextra-cellular matrix of tissues,recalls one of those researchers,the Belgian biochemist, Dr.Desire Collen. His team waslooking for an inhibitor to thatagent in order to interfere withthe cancer cells’ advance.

In 1979, Collen received a lineof aggressive melanoma cellsderived from a patient namedBowes. These melanoma cellsshowed an unusual amount offibrinolytic activity, and Collenand colleagues worked to purifythe fibrin-digesting agent. Withinthe year, he demonstrated that theagent was t-PA. It turned out thatthe Bowes cell line produced 100to 1000 times more t-PA than anyother known source at the time.“We realized we had a cell linethat everyone had been lookingfor,” he says. He began to turn

the Streptococci secrete streptoki-nase, which dissolves the clotsand enables the bacteria to enjoy

Open-Artery Model for Treating AcuteHeart Attack• Blood clots (thrombi) block the blood supply to the heart

muscles.• Without oxygen from the blood, the heart muscles

suffer damage.• The longer a muscle goes without oxygen, the more

severe the damage.• Restoring the blood flow reduces damage to heart muscles.• There are three main ways to restore blood flow:

1. Clot-busting drugs (thrombolytics) can dissolve clots immediately.

2. Angioplasty, a surgical procedure, can open the artery with a balloon and then, if indicated, install a stent (a scaffold-like device) that helps keep the vessel open.

3. Bypass surgery provides an alternate route around the blockage, using a blood vessel from the calf or else-where in the body.

Figure 4: In-Hospital Death Rates from Heart Attack and

Stroke: 1970 -2000

0

5

10

15

20

25

30

1970 1975 1980 1985 1990 1995 2000

Year

Pe

rce

nt

De

ad

Heart Attack

Stroke

FFiigguurree ��:: The in�hospital death rate for acute myocardial infarction fell from �� in �� (when early thrombolytics were becoming marginally accepted) to �� in �� (whenthrombolytic use had doubled and a new recombinant form of tissue plasminogen acti�vator [t�PA] was introduced) to in �� (after angioplasty was more widely used)�for a total decline of �� National Hospital Discharge Survey Annual Reports; NationalHeart� Lung and Blood Institute�

In-Hospital Death Rates from Heart Attack and Stroke: 1970 – 2000

Year


3Collen D and Lijnen HR. (2004) Tissue-type plasminogen activator: a historical perspective and personal account. Journal of Thrombosis andHaemostasis. 2(4): 541-546.


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his attention to using t-PA to dissolve clots.

In the first half of 1980, Collenand colleagues used t-PA to dissolve clots in rabbits with pul-monary embolus and later report-ed their results in Nature4. Beforepublication, Collen presented hisfindings at a conference on June12, 1980. “I still remember it,” he says. “After the presentation a young lady, Dr. Diane Pennica,came up to me and said she wasinterested in cloning t-PA andthen to make a recombinantform.”

Although the Bowes cell linewas prolific compared to othercell lines, it could not produceenough t-PA for a marketable newdrug. Making larger quantities of

a cellular product required coax-ing another type of cell, grown intissue cultures, to churn out theprotein like a mini factory. Thenew biotechnology industry envi-sioned using recombinant DNAtechnology to identify, clone, andsometimes fine-tune genes forspecific products, and then insertthe genes in the DNA of cells thatcould mass-produce those prod-ucts. In this case, they wouldinsert the gene for t-PA in a cellline that would then “manufac-ture” the t-PA as a recombinantprotein.

As Collen explains, Pennica hadjust joined the very small biotechfirm Genentech, which had fewerthan 100 employees. When heagreed to collaborate with thecompany to work on a recombi-

FFiigguurree ��:: The widely practiced technique of bloodletting throughout history unwittinglyrelied on the potential of a leech protein to override the human body’s natural blood clot�ting system� This woodcut is from Historia Medica by W� van den Bossche� which waspublished in �� Science Photo Library/Photo Researchers� Inc�

nant t-PA (rt-PA), they embarkedon a new model for drug develop-ment: the academic and biotechcollaboration that is routine today.Within a year, working 80 to 90hour-weeks, Pennica had clonedthe gene for human t-PA and wasproducing rt-PA in a tissue cultureof Chinese hamster ovary cells.That was a speedy accomplish-ment in the early 1980s, but withtoday’s technology, “a graduatestudent should be able to do thiswork in a week,” Collen says.

Collen and U.S. collaborators,including Sobel, next demonstrat-ed in animal experiments that t-PAcould dissolve coronary clotswithout inducing systemic bleed-ing, followed by a similar “proofof concept” study involving sevenpatients with coronary thrombo-sis. After testing the recombinantform, rt-PA, in experimental ani-mals, promising results from asmall, placebo-controlled triallater in 1984 set the stage forlarge-scale randomized multi-cen-ter clinical trial.

In just three more years, rt-PA had received FDA approvalfor treating acute myocardial


4Matsuo O, Rijken DC, and Collen D. (1981) Thrombolysis by human tissue plasminogen activator and urokinase in rabbits with experimental pul-monary embolus. Nature. 291 (5816):590-591.

A serendipitous discovery by cancer researchers hadunanticipated benefits in

improved thrombolytic therapyfor heart attack and stroke. “We realized we had a cell line that everyone had been

looking for.”



infarction and soon hit the mar-ket. Seven years from bench tobedside (1980-1987) still standsas one of the fastest drug devel-opment processes in history, inspite of its occurrence duringbiotechnology’s infancy.

The Life and Death of a Clot�

The blood clotting system is agood example of how the bodyusually maintains a delicate bal-ance between opposing actions.In this case, the forces leading toa blood clot are opposed by thosethat dissolve the clot, which arecountered by another process thatslows down the dissolution of theclot to give time for the healingprocess to work. When thoseforces get out of balance, or whenone of the players is missing ordefective, diseases and disordersrear their ugly heads.

The key players in clot-buildingsystem are the proteins thrombinand fibrin, and special bloodcells, the platelets. Think back onthe finger prick and its tiny ves-sel break. Numerous round, whiteplatelets gather and then adhereto the tissue below the vessel surface. The clump of plateletsforms a plug that begins to pre-vent blood loss.

Throughout the body, cells areconstantly “talking” to eachother, sending molecular signalsnear and far that trigger varioustypes of activity. At the fingerprick, such signals trigger the for-mation of thrombin, which sum-mons up more clot-buildingactivity.

Meanwhile, an inactive proteincalled fibrinogen circulates in thebloodstream, but does not interactwith other blood proteins. At thesite of the finger prick, however,fibrinogen encounters thrombin,which activates the fibrinogen,transforming it into fibrin. Fibrinproteins coalesce into long, ropey

fibers that settle around theplatelets, reinforcing and anchor-ing the plug to prevent blood losswhile other systems kick into gearto repair the damage. Fibrin fibersfunction in a clot like the support-ing meshwork holding bouldersalong a highway or the rocks of adike. This meshwork stops bloodloss, but allows some blood topass through to the tissues, pro-viding nourishment while thewound is under repair.

At the molecular level, the sys-tem works essentially the samewhether a clot forms in responseto an injury to prevent blood loss(hemostasis) or in a misguidedresponse (thrombosis) to a misin-terpreted threat, such as arterialplaque. Thrombosis is any unwar-ranted response, in the absence ofimminent blood loss, that blocks ablood vessel.

Even while a clot forms, othermolecules begin to dissolve it, a counter-process called fibri-

nolysis. The cast of characters forthis biological drama includesplasmin, its precursor plasmino-gen, and plasminogen activators,such as t-PA.

Plasminogen flows ubiquitouslythrough the circulatory systembut, like fibrinogen, does notinteract with other molecules untilactivated. In fibrinolysis, the pres-ence of fibrin in a clot alerts theplasminogen activator protein t-PA to its upcoming role. Whenplasminogen encounters t-PA nearthe clot, the plasminogen is con-verted into plasmin. Plasmin snipsapart the fibrin mesh supportingthe clot. As fibrin degrades, theplatelets are removed and the clotdissolves.

To prevent the natural fibrinolyt-ic activity from happening tooquickly, molecular inhibitors nor-mally slow down the activation ofplasmin, allowing the clot tolinger as long as it is needed.When the inhibitors, for eithergenetic or environmental

FFiigguurree :: The molecular structure of tissue plasminogen activator (t�PA)�

Fibrin is a key player in building a blood clot. Plasmin

and tissue plasminogen activator (t-PA) are the keyplayers in dissolving a clot.

5Jackson CM. (2002) Biochemistry of Haemostasis, in Bhagavan, N.V. Medical Biochemistry, 4th Edition. Academy Press. San Diego. 840-872.



(lifestyle) reasons, get out of bal-ance and overpower the plasminactivation, fibrinolysis cannotfunction normally. Such animbalance can lead to the throm-bi that cause heart attacks andstrokes.

When renegade clots form inthe coronary arteries, they over-whelm the natural fibrinolyticprocess. Thrombolytic drugs arebasically pharmaceutical inter-ventions that provide a souped-upversion of fibrinolysis.

How Streptokinaseand t�PA WorkWith the refined understanding

of how plasmin becomes active at the site of a clot, researchersgained new insights into howstreptokinase works as a throm-bolytic. Scientists realized thatSK bypasses the intermediaryplasminogen activator (t-PA) andbinds directly to plasminogen in away that activates it. In addition,SK is unaffected by the naturalinhibitors of plasmin, so it worksquickly when applied as a throm-bolytic drug.

However, SK activates not onlythe plasminogen that is bound toa fibrin clot. It also activates thenormally inactive plasminogenthat courses throughout thebloodstream, forming plasminthroughout the body. This wide-spread plasmin can degradediverse molecules involved incoagulation. As a result, SK notonly dissolves clots, it also pre-disposes bleeding in other organsand tissues.

Building a ClotHemostasis:A Defensive Maneuver• Platelets gather around an injured

vessel and form a plug. • The injury triggers the creation of

thrombin at the site of the wound.• Thrombin activates fibrinogen

at that site, transforming it into fibrin.

• Fibrin fibers cast a reinforcing netaround the platelets, creating a clot that prevents blood loss.

• The fibrin clot prevents blood loss,while other systems in the body repairthe wound.

Thrombosis:A Biological Mistake• Damage inside a blood vessel

tricks the blood system into creating thrombin.

• Thrombin sets in motion the same process, forming a fibrin clot.

• The clot blocks the artery and shuts off the supply of blood and oxygen to the heart.

Clots Gone WildLike many of the body’s intricate biologi-

cal systems, what can help in one contextcan harm under other conditions. The normal blood clotting system is defensive,intended to prevent blood loss, a normalprocess called hemostasis. At other times,the body aggressively over-responds whenthere is not really a danger of blood loss, adisease process called thrombosis. In heartattacks and strokes, damage inside a blood vessel, often caused by cholesteroldeposits, can trigger the same molecularreactions and produce an undesirable clot,called a thrombus. Unlike a normal clot,thrombi cut off essential blood flow tovital organs.

Colorized scanning electron micro-graph of whole clot, showing fibrin(blue), platelets (purple) and redblood cells (red). Photo courtesy ofDr. John Weisel, published in Nature,Vol. 413 (6855), used with permission.(Y. Veklich/J.Weisel)

Colorized scanning electron micro-graph of platelets. Photo courtesy ofDr. John Weisel, published in Journalof Biological Chemistry, Vol. 278 (51)used with permission.)

Gross specimen of tissue from aheart, showing a blood clot (throm-bus, dark red, center) in one of thecoronary arteries. These are the arter-ies that supply blood to the heart mus-cle itself. A blood clot in one of thesecan block or reduce the blood supply,cutting the oxygen supply that keepsthe muscle functioning, which cancause a heart attack. CNRI/PhotoResearchers, Inc.


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Streptokinase’s systemic activityis responsible for its major draw-back as a thrombolytic drug:unwanted and potentially lethalbleeding. The risk of bleedingstems from the fact that heartattack victims generally have vas-cular damage elsewhere in thebody in addition to the coronaryarteries, and those damaged ves-sels are susceptible to bleeding.

Another drawback of SK relatesto the human body’s ability torecognize it as the product of abacterial invader. The body pro-duces antibodies to SK, and theantibodies can thwart the drug insubsequent doses. Additionally, asmall minority of people canhave a severe allergic reaction.

Like SK, t-PA activates plas-minogen, which generates thefibrin-degrading plasmin. UnlikeSK, however, human t-PA actsalmost exclusively on the plas-minogen that is bound to a fibrinclot. Thus, t-PA’s activity is con-sidered relatively “fibrin specif-ic,” meaning it only dissolvesclots and carries a lower risk ofsystemic bleeding. Moreover, rt-PA is engineered to jump intohigh gear when sitting on astrand of fibrin. Because rt-PAderives from a human protein, itdoes not arouse the suspicion ofthe immune system, instigate anallergic reaction, or lose its effec-tiveness with a repeated dose.

Door�to�Needle TimePracticing physicians wanted to

see rigorous proof of clear-cutclinical benefits to patientsbefore adopting thrombolytics asa treatment practice on a morewidespread basis. Early promis-ing evidence of SK’s benefitscame from studies using dogswith coronary thrombosis duringthe 1970s. After Dr. MarcusDeWood (mentioned previously)advanced his results establishingthe causal link between clots andheart attacks, the NIH beganplanning a large-scale clinicaltrial for the early 1980s to testthe safety and efficacy of SK foracute myocardial infarction.When researchers learned aboutt-PA, they added it, and later rt-PA, to the trials. Based on numer-ous studies that showed rt-PA’sgenerally higher efficiency andits lower risk of bleeding oradverse immune response, itcame to be viewed by many inthe United States as the drug ofchoice, despite its high cost.

The clinical trial phase clearlyestablished that time is of theessence when treating acute heartattack victims. The concept“door-to-needle time” emerged,indicating the time between whena patient first has symptoms andwhen he or she receives the firstdose of the thrombolytic.

One trial showed that patientswho receive thrombolytics—andsuccessfully had their arteriesopened—within one hour haveonly a 1% mortality rate during

the next 30 days. Waiting anotherhalf hour raises the 30-day mor-tality rate to 6.4%. After 3 hours,80% of the benefits were lost,and after 4 hours thrombolyticshave virtually no effect in reduc-ing mortality.6 Thus, the practicalclinical goal is to administer t-PAor SK within 90 (plus or minus30) minutes of the onset of symp-toms. A similar temporal windowof opportunity was establishedfor stroke in later trials.

Achieving the rapid door-to-needle time, however, requiresthat the patient recognize theearly onset of symptoms and callfor help immediately. Even withrapid treatment with thrombolyt-ics, about 60% of heart attackpatients still need more interven-tion to completely restore bloodflow. Many of these are candi-dates for angioplasty.

In angioplasty, a catheter carry-ing a deflated balloon is insertedinto the closed artery. The catheterinjects a dye that enables real-timeX-ray imaging of the heart’s arter-ies. Depending on what theseimages reveal, the balloon may beinflated to open the artery and/or astent placed to keep the vesselopen. Less frequently, the clot canbe removed or otherwise cut apart.The term percutaneous coronaryintervention (PCI) covers all theseintervention options. Like throm-bolytics, PCI’s success requires arapid “door to balloon” time: 90(± 30) minutes. If the surgery isdelayed three hours, 80% of thebenefit is lost.

6Bergman SR, Lerch RA, Fox KA, Ludbrook PA, Welch MJ, Ter-Pogossian MM and Sobel BE. (1982) Temporal dependence of beneficial effects ofcoronary thrombolysis characterized by positron tomography. American Journal of Medicine. 73:573-581.



In a typical community, howev-er, there are practical barriers tousing PCI.7 Only 20-30% of U.S. hospitals (and fewer still inEurope) have the capacity (emergency cardiac units andcatheterzion laboratories) to per-form emergency PCIs in the nar-row window of time required forbest results. Despite increased

Most researchers came to see recombinant t-PA as

the thrombolytic of choicebecause of its higher efficiency

and generally lower risk ofadverse side effects.

ThrombolyticTreatment forHeart AttackBenefits:

• Thrombolytic agentsalone reduce early mor-tality 30%, and they areeven more effective(40%) when adminis-tered with the anticoagu-lant, heparin.

• Thrombolytics providelonger-term benefits forsurvivors, who have justa 5% mortality rate atone year.

Risk:

• The risk of a majorbleed or brain hemor-rhage generally affects2-3% of heart attack victims. Minor bleeds,such as nosebleeds, aremore common.

• The risk particularlyaffects patients withother health complica-tions, those over the ageof 80, and those beingtreated for strokes (whorun, on average, a 6% risk).

Source: Fibrinolytic TherapyTrialists’ (FTT) Collaboration;Fuster and Verstraete, “Thrombolysisin the Treatment of AcuteMyocardial Infarction,” 1992, p. 299.

Blood vessel walls

PlasminogenCirculating TPA

Fibrin

Fibrin

Fibrin fragments

Fibrin signals TPA to activateplasminogen into plasmin

Plasmin breaks apartthe fibrin

Clot

Clot

Clot

Plasmin

A.

B.

C.

D.

Fibrinolysis

FFiigguurree :: ““SSwwoorrdd iinn tthhee ssttoonnee”” aannaallooggyy ooff ffiibbrriinnoollyyssiiss�� The inactive substance� plasmino�gen� binds to fibrin (shown as yellow fibers within the clot) becoming incorporated intothe clot as it forms� Tissue plasminogen activator (t�PA) circulates freely in the blood� (AA) Fibrin within the clot sends our signals which activates t�PA� causing it to bind toplasminogen� (BB) In turn� t�PA activates plasminogen� converting it to the enzyme� plas�min� (CC) Plasmin cleaves through the fibrin fibers in the clot� The clot dissolves� (DD)Figure designed by Dr� Carrie Wolinetz and Corporate Press�

7Pharmacoinvasive Therapy in Acute Myocardial Infarction. Edited by Harold L. Dauerman, M.D. and Burton E. Sobel, M.D., Marcel Dekker, Inc.,New York, January, 2005. (Pre-publication)


emphasis on the need for speed,hospitals reduced the door-to-bal-loon time by an average of onlyeight minutes during the 1990s.Thus, even though PCI can gen-erally open up the blood vesselseven more effectively than throm-bolytics when all other things areequal, in most communities PCIcomes too late to fully benefit.

The American College ofCardiology’s guidelines now

recommend that if transferringpatients to an angioplasty facilitydelays treatment by one hour,doctors should consider firstoffering thrombolytics to open thearteries immediately, at least par-tially. Then, those patients whoneed further intervention canreceive angioplasty or other pro-cedures. That way, patients willbenefit from both t-PA’s rapid res-cue of blood flow and from PCI’scomplete reopening of the artery.

Stroke Therapy LeapsForward

While thrombolytic therapy forheart attacks was advancing, sotoo was interest in using throm-bolytics to treat strokes. One doc-tor calls the arrival of t-PA a“leap forward” in treatingischemic strokes.

It was previously thought thatthere was nothing doctors coulddo to stop a stroke in process.That view began to change duringthe second half of the 20th centu-ry, in part because scientists bet-ter understood the nature of thisviolent event deep in the inacces-sible brain. As with heart attackresearchers, that better under-standing also raised the possibili-ty that strokes could be treated.

Strokes come in two types.More than 80% are ischemicstrokes caused by clots blockingblood flow in a vessel in thebrain. The remainder, hemorrhag-ic strokes, result from bursting ofa blood vessel that causes bleed-ing into the brain.

Research showed that quicklyrestoring the blood flow to thearea of the brain blocked by aclot could salvage the tissue andavert brain damage. However, themedical decision to give a strokepatient a thrombolytic drug ismore complicated than it is forcardiologists. Since some strokesare hemorrhagic, doctors mustuse a brain scan, CAT scan, orMRI to clearly exclude the possi-bility that the stroke is the bleeding type.

Recombinant t-PA receivedapproval for treatment ofischemic stroke in 1996, follow-ing an NIH trial indicating thattreatment with rt-PA could reducelong-term disability by 33% overuntreated patients. The drug iscurrently being used in the U.S.,Canada, Australia, and someEuropean countries, and approvalfor use throughout Europe isexpected soon.

In spite of the potential of rt-PAto reduce death and disability,only about 3% – 5% of strokepatients receive it. Door-to-nee-dle time is equally critical instroke as in acute myocardialinfarction. Treatment with rt-PAis most effective when used assoon as possible after the strokehappens, preferably within thefirst 3 hours after stroke onset.


Early Recognition of Heart Attack & Stroke Saves Lives Heart Attack

• Pain, discomfort, pressureor numbness in chest orupper body.

• Pain may feel like indiges-tion or heartburn.

• Shortness of breath.• Cold sweat, nausea, vomit-

ing or feeling light-headed.

Stroke (all symptoms areSUDDEN!)

• Numbness or weakness,especially on one side of the body.

• Confusion or troubledspeech, or trouble understanding speech.

• Trouble seeing with one or both eyes.

• Dizziness, difficulty walking, loss of balance or coordination.

• Severe headache with noknown cause.

8Bousser, MG. (2004) Antithrombic Strategy in Stroke. Journal of Thrombosis and Haemostasis. 86:1-7.; Silliman S. (1998) Acute stroke therapywith intravenous tissue plasminogen activator. Jacksonville Medicine. 492-494.

Stroke treatment is most effec-tive when started within three

hours of the onset of the stroke.



Many patients do not seek treat-ment within this time to receivemaximum benefits from throm-bolytics, largely because of a lowpublic awareness (17%) about thewarning signs and symptoms ofstroke. There also remains thefear of bleeding complicationsassociated with thrombolytics,which discourages some doctorsfrom prescribing them.

Good news may be on the hori-zon. Some early evidence sug-gests that some stroke patientsmay benefit from treatment withthrombolytics beyond three hoursfrom stroke onset. Doctors areusing new brain imaging tech-niques to identify and selectthose patients. Studies are alsoongoing to examine new throm-bolytic products that may havefewer risky side effects.

In other patients, however, stud-ies indicate that after three hoursof stroke onset, the blood vesselsbelow the clot become so weak-ened that the blood-brain barrieris easily breached. Administeringt-PA after that time frame canallow t-PA to seep through thevessel wall into the brain andincrease the damage being doneto the already-stressed neurons.Very recent research conductedby neuroscientists found thatanother drug, Activated Protein C(APC), protects neurons from thesame cell-death programs that t-PA accelerates. APC is approvedfor use in sepsis, in which thetoxins produced by infectiousmicroorganisms poison cells.Depending on the outcome ofsafety and efficacy trials inhumans, this combination could

potentially extend the window oftime for delivering t-PA safelyand make t-PA beneficial effectsavailable for many more patients.

With more patients survivingacute heart attacks and strokes,there is also more focus on howto maintain the survivor’s long-term cardiovascular health.Breaking up the clot can resolvethe immediate, acute problem,but not the underlying problem ofvascular disease. Along withtreatment by thrombolytics orangioplasty, all heart attackpatients receive an anti-coagulantthat prevents new clots fromforming, a combination termedadjuvant therapy. In addition, aheart attack or a stroke is not aone-time event; they often recur.To prevent recurrences, survivorscontinue taking anti-coagulantsand/or anti-platelets long-term.Anti-coagulants target and inhibitthrombin, thus preventing the formation of the fibrin that reinforces a clot. Anti-platelets

ThrombolyticBenefits for Stroke• Early treatment with

t-PA reduces long-termdisability by 33% to 55%and saves an estimated $4 million for every 1,000patients treated.

• Patient delay in seekingtreatment prevents widespread use of thrombolytics.

Clogged artery Balloon Stent

FFiigguurree ��:: Percutaneous Transluminal Coronary Angioplasty (PTCA)� often referred to assimply angioplasty� is used to widen narrowed or blocked arteries� It involves inserting acatheter through the patient’s leg� arm or groin and moving it to the blocked artery� Aballoon at the tip of the catheter is inflated� compressing the plaque on the walls of theblood vessel so that blood can flow more easily� Then the balloon is deflated and thecatheter is removed� Many of these procedures also involve placing a stent� Stents arewire mesh tubes used to permanently hold the artery open to improve blood flow�Designed by Corporate Press�


prevent platelets from clumpingtogether and forming the initialplug for a clot.

Coming AttractionsThree quarters of the world can-

not afford rt-PA, comments rt-PA’s co-creator Dr. Desire Collen,so most heart attack and strokepatients receive the less desirablestreptokinase, if they receive ther-apy. Collen and others are nowworking on a microbial relative ofstreptokinase, called staphyloki-nase, that elicits a much weakerand slower immune response thanSK. Produced by the staphylococ-cal bacterium, staphylokinase isfibrin specific, like t-PA, so itdoes not pose as great a risk ofbleeding as SK. Because it is thenatural product of bacteria, likeSK, it should not be as expensiveas rt-PA. As Collen explains,staphylokinase could have thesafety and efficiency of rt-PA,with the low cost of SK.

In addition to staphylokinase,several new modifications of rt-PA have entered the market or arein the drug pipeline. One drug,known as TNK-rt-PA, a modifi-cation of the initial recombinantdrug, remains in the bloodstream

longer and is more specific in itsactivity. Researchers continueusing molecular genetics and bio-chemistry to develop new throm-bolytics that activate plasmin

more efficiently with fewer nega-tive side effects (such as bleedingand immune reactions) that cur-rently exclude many heart attackand stroke patients.

On another front, researcherscontinue to study naturally pro-duced thrombolytics. One newdrug candidate derives from anenzyme in vampire bat saliva andmay be effective as long as 9hours after the onset of symp-toms. This drug, desmoteplase(DSPA), is in clinical trails for

stroke in Europe, Asia, andAustralia. Extending the narrowwindow of time allotted to mostexisting thrombolytics wouldallow more patients benefit fromthe treatment.

As mentioned earlier, basicresearch in neuroscience suggeststhat combining t-PA with analready approved drug for sepsis,APC, may one day increase thebenefits of thrombolytic therapyfor a broader segment of strokepatients while decreasing therisks. This apparent coalescenceof different lines of research isquite fitting for the field ofthrombolytics, with its long andrich tradition of the intertwiningof interdisciplinary fields.

Developing new and betterdrugs for adjuvant therapy is alsoa “hot field,” focusing on drugs toprevent clots from forming in thefirst place, or from recurring aftertreatment for a heart attack orstroke. Currently, much of theresearch on anti-coagulants isstudying how snake venomenzymes that dissolve clots work.That approach seems appropriate,since one widely used anti-coagu-lant, hirudin, is also derived froma naturally occuring protein from


FFiigguurree ��:: The saliva from vampire batsserves as the basis for a new candidate for a thrombolytic therapeutic� Photocourtesy of the Organization for BatConservation�

Timeline: �� Years of Thrombolytics Development

Theory that thrombosis

causes acute heart attack

(acute myocardialinfarction, AMI).

Open ArteryHypothesis fortreating AMI.

Conflicting hypothesis proposes

thrombosis as result, not cause,

of AMI.

Discovery thatstreptokinase has

thrombolytic activity.

Streptokinase (SK)used as thrombolyticfor AMI. Awareness that animal tissues

contain agent that activates plasminogen.

Streptokinase used as first

therapy available for

ischemic stroke.

Biochemical studies elucidate

molecular interventions regulating fibrinolysis.

Interest in t-PA as alternative to streptokinase.

1899 1910s 1930s 1933 1947 1960s 1970s


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t-PA purified from Bowes Melanoma

cell line (a fortuitous

circumstance).

DeWood reaffirms open-artery theory for treating AMI.

Collen presents his findings on t-PA andbegins collaboration

with Genentech. First animal experiments

using rt-PA in rabbitswith pulmonary

embolus.

Successful experimentswith t-PA on dogs withcoronary thrombosis.Extended studies with

rt-PA demonstrated clot specificity.

First AMIpatientsreceive

t-PA in a US/

European pilot trial.

First 50

patient trial of rt-PA in US.

rt-PAapproved

by FDA for

AMI.

rt-PAapproved

forischemic

stroke.

New thrombolytics,

anti-platelets, and anti-coagulants under

development.

1979 1980 1981 1983 1984 1987 1996 2000s


a leech. Researchers are alsointroducing thrombolytics andrelated agents for the treatment ofdeep vein thrombosis and othercardiovascular conditions.

Before new drugs are introduced,a great deal of basic research stillneeds to be done, and then eachdrug candidate needs to pass thehurdles of clinical trials.Introducing a new drug that willbe used by large numbers of peo-ple requires large trials, whichhave become prohibitively expen-sive. Dr. Desire Collen suggeststhat thrombolytics have become avictim of their own success; theyhave lowered mortality rates sosignificantly that it is difficult tomeasure incremental improve-ments in new modifications.

Out of Basic Research�A Life�Saving Drug Over the past century, basic

physiological studies led to a newunderstanding of the disease

processes involved in heartattacks and strokes. Yet the stepsfrom research to therapy did notfollow a straightforward path.

Science and medicine firstneeded to establish the cause andeffect relationship between bloodclots and disease. Only thenwould they realize the need forthrombolytic drugs and other

interventions that could removethe endangering clots. Haltingly,the outcomes of basic researchand clinical trials overturned onemodel of disease, and replaced itwith a model that links the bio-chemical reactions of blood clot-ting to heart attack and stroke.

In the end, the contributions of medical researchers, microbiol-ogists, cancer researchers, biochemists, biotech scientists,and cardiac specialists gave rise tothe world’s first widely used drugmade from recombinant proteins,rt-PA. That work is not complete,however. There are still manyrefinements needed to reduce sideeffects, increase speed and effec-tiveness, decrease cost, and makethe benefits available worldwide.

All together, curiosity, passion,and scientific and medical rigorbrought about fundamentalimprovement in therapy for heartattack and stroke victims.Millions of lives are saved andprolonged by these develop-ments. Serendipity and luckplayed a role in redirecting basicresearch towards thrombolytics,says Desire Collen, a principalfigure in the clot busting story.“But the rest was blood, sweat,and tears.”

Half a century of basic researchled to unanticipated new therapies and significant

improvements in human health.

FFiigguurree ��:: Researcher milks venom from aMalaysian pit viper (Calloselasma rho�dostoma)� The venom contains an antico�agulant drug which is being studied foruse in treating stroke patients� This drugimproves blood flow by reducing theamount of fibrinogen (a clotting protein)in the blood plasma� Strokes occur whenthe blood supply to part of the brain iscut off by a blockage or bleeding in thebrain� They can lead to brain damage andare a leading cause of death� VolkerSteger / Photo Researchers� Inc�


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Biographies Cathryn Delude writes about science, medicine, biotech-nology, water, and energy from Andover, Massachusetts. She has written for FASEB in the past, and also for ScientificAmerican, The Scientist, the Harvard School of Public HealthReview, Howard Hughes Medical Institute, the Boston Globe,and other publications, and she develops science educationmaterials with The Writing Company.

Kenneth Mann, Ph.D., professor and chair of bio-chemistry at the University of Vermont, College of Medicine,is an international leader in the field of blood clottingresearch and holds several patents related to the diagnosis andstudy of functions that are critical in normal blood coagula-tion. He is chair of the National Hemophilia FoundationResearch Working Group, a past vice-chair of medicine at theMayo Clinic, past associate editor of Blood and presentlyassociate editor of The Journal of Thrombosis and Hemostasisand The Journal of Atherosclerosis and Thrombosis. Mann hasheld volunteer positions with the National Institutes ofHealth, the American Heart Association, the AmericanSociety of Hematology, the International Society onThrombosis and Hemostasis and the American Society ofBiochemistry and Molecular Biology. He is the recipient ofnumerous national and international awards.

Selected ReferencesBousser, M-G. (2004) Antithrombic Strategy in Stroke.Journal of Thrombosis and Homeostasis. 86: 1.

Collen, D., Lijnen, H. R. (2004) Tissue-type plasminogenactivator: a historical perspective and personal account.Journal of Thrombosis and Homeostasis. 2(4):541.

Jackson, C.M. (2002) Biochemistry of Hemostasis inBhagavan, N.V. Medical Biochemistry, 4th Edition,Academic Press, San Diego 840-872.

Maroo A. and Topol E. J. (2004) The early history and devel-opment of thrombolysis in acute myocardial infarction.Journal of Thrombosis and Haemostasis. 2:1.

Additional ResourcesAmerican Heart Association7272 Greenville AvenueDallas, TX 75231800-242-8721http://www.heart.org

National Heart, Lung, and Blood Institute (NHLBI)National Institutes of HealthP.O. Box 30105 Bethesda, MD 20824-0105301 592 8573http://www.nhlbi.nih.gov/index.htm

Stroke: Hope through Research. National of Neurological Disorders and Stroke (NINDS)National Institutes of HealthP.O. Box 5801 Bethesda, MD 20824 http://www.ninds.gov800-352-9424

MayoClinic.com Heart Center/Brain & Nervous System Centerhttp://www.mayoclinic.com/

Special thanks to the following individuals for their invaluableexpertise and contributions:• Desire Collen, M.D., Ph.D., University of Leuven, Belgium• Victor J. Marder, M.D., University of California, Los Angeles• Merle Myerson, M.D, Ed.D., National Heart, Lung, and Blood

Institute • Magdy Selim, M.D., Beth Israel Deaconess Medical Center

Stroke Center• Thomas Thom, National Heart, Lung & Blood Institute• Scott Cooper, Ph.D., University of Wisconsin-LaCrosse• John Weisel, Ph.D., University of Pennsylvania School of

Medicine

The Breakthroughs in Bioscience

series is a collection of illustrat�

ed articles that explain recent

developments in basic biomed�

ical research and how they are

important to society� Electronic

versions of the articles are avail�

able in html and pdf format at

the Breakthroughs in Bioscience

website at:

www�faseb�org/opar/break/�


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