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Bob Lazarus and Kevin Judice
The Genentech teaching team
February 1, 1995

Using Leeches as Bait to Go Fishing for New Anticlotting Drugs

It was hot and humid. Bzzzzzzzzz-Zap! While on vacation in August in northern Minnesota several years ago visiting my wife's family, I had just been bitten by a huge mosquito, also known as the state bird. I struck at the little bugger (no pun intended) and nailed him (or was it a her?). I observed the relatively large amount of blood the little beast had managed to suck out of me. I continued to bleed for a bit but soon I returned to 'normal' other than having that obnoxious little bump to deal with.

Undaunted, I decided to take a hike. After I got back to the house, I noticed a little hard 'thing' on my leg - great! I'd been bitten by a tick. After disposing of the tick, I noticed more bleeding at my wound. Despite all of this, I later went swimming in the local lake to cool off and get away from the mosquitoes, ticks, gnats, and other fun loving creatures of the state. After I got out I noticed a little worm-like organism on my leg - I didn't even realize I'd been bitten by a leech! More blood sucking creatures! Again I noticed that I continued to bleed somewhat after I'd removed the leech.

I sat out for a while watching the sunset and listened to the loons. As a molecular biochemist studying various aspects of blood clotting, I began to wonder... The ability to form blood clots is critical to our survival. When you cut yourself, you need to clot so that you don't bleed to death. However, sometimes too much clotting leads to serious trouble. For instance, heart attacks are caused by blood clots in the heart and strokes are caused by blood clots in the brain.

Triggering clotting in humans is very complex, making sure our blood clots when it should, but not when it shouldn't. Blood clots are basically made up of two components --platelets and fibrin. Upon injury, platelets are the first cells on the scene. They adhere to the vessel wall and then aggregate to form what's known as a hemostatic plug. This plug is often strengthened by fibrin - the end product of the coagulation cascade. This cascade involves many zymogens (inactive serine proteases, or enzymes which degrade proteins) which become activated upon injury. The activated zymogens ultimately cause the formation of fibrin from a precursor protein molecule called fibrinogen. The fibrin molecules then polymerize to produce a more stable network of fibrin and platelets. It is this network that we call a blood clot. Although all blood clots involve both fibrin and platelets, blood clots that form on the arterial side of the blood supply are usually more platelet-rich, whereas venous thrombosis is usually more fibrin mediated.

There are lots of strategies for dealing with blood clots. One strategy involves the use of thrombolytic therapy with drugs such as t-PA (tissue plasminogen activator), a human protein which can dissolve blood clots after they have been formed. t-PA works by activating plasmin, a serine protease that can cleave fibrin, one of the major components of blood clots, into smaller fragments. Treatment with t-PA, or other thrombolytic agents, is becoming a standard therapy for patients with heart attacks. Despite the existence of successful thrombolytic therapy, many of the diseases associated with thrombosis are perhaps better treated by preventing blood clots from forming in the first place. This is the rationale for the current use of low dose daily aspirin, which is known to affect platelets, another key component in the formation of blood clots.

Back to Minnesota... I thought we might be able to address this problem of how to prevent formation of blood clots by looking into how nature accomplishes this goal. The blood sucking creatures such as leeches, ticks, vampire bats, mosquitoes, snakes, and others have successfully adapted to feeding on mammals by shutting down the clotting process of the "victim." I thought, wouldn't it be nice to know that mosquitoes, ticks, and leeches could save lives? It seemed reasonable that these highly successful species might have a variety of pharmacologically active substances that overcome the clotting mechanisms of the host, i.e. me!.

Well, this is in fact the case! Within the past several years potent agents that act as fibrinolytics (clot-busters), anticoagulants (often mistakenly referred to as blood thinners - agents which prevent fibrin formation), and antiplatelets (which prevent platelet aggregation) have been discovered in all of these species, isolated and characterized at the molecular level. By understanding how Mother Nature deals with blood clots, we hoped to be able to use either the specific molecules that she has provided or the details of the molecular interactions of these molecules with their respective targets to be able to design new compounds useful as drugs for thrombotic disease.

Perhaps the best understood of all the bloodsucking animals is the leech. Therefore, for practical purposes I'll limit most of this discussion to leeches although the other blood sucking animals are equally fascinating and we can "talk" about them during the seminar.

Leeches have had a long standing place in medicine; the medicinal leech, Hirudo medicinalis, was used extensively in the 19th century for a wide variety of indications. In fact, leeches are increasingly being used today as surgical tools in tissue grafts and reattachment surgery because of their ability to prevent blood clots from forming and to help keep tissues healthy. In 1884, John B. Haycraft, Professor of Physiology at the University of Wales, discovered that blood within the gut of the leech did not coagulate and that blood continued to flow from leech wounds for abnormally long times. It was not until the 1950's in Germany that Fritz Marquardt isolated a protein from H. medicinalis termed hirudin, which contained 65 amino acids, and which very potently and specifically inhibited the procoagulant protease thrombin. Since thrombin both makes fibrin from fibrinogen and activates platelets, its inhibition by hirudin decreases blood clot formation. Subsequently, the biochemical, structural, biological, and pharmacological activities of hirudin, which is now made by recombinant DNA technology, have been well studied. Today, hirudin is in human clinical trials for the treatment of thrombotic disease and may someday become an approved drug.

At Genentech, our own ventures -- started in the late 1980's --had a different direction. We decided to try to prevent platelets from aggregating by inhibiting binding of fibrinogen (Fg) to its receptor (termed GP IIb-IIIa) on the activated platelet surface. Fibrinogen is a dimer, having 2 binding sites for GP IIb-IIIa, and can thus serve as a cross-link between activated platelets. (Platelets normally circulate in an unactivated state which do not bind to fibrinogen - otherwise we would continually form clots.) The Fg/GP IIb-IIIa complex represents the basis by which platelets aggregate. Using our purified fibrinogen and activated platelet receptors, and antibodies that we had generated against these two types of molecules, we set out to develop an assay which would detect binding of fibrinogen to the activated receptor. We would, in turn, use this assay to detect and measure compounds which inhibited fibrinogen/receptor binding. Remember, we wanted to identify any leech molecules that had lots of inhibitory activity.

The assay that we developed was a type of ELISA (enzyme linked immunosorbant assay) which involved coating plastic 96-well microtiter plates with fibrinogen molecules. The coated plates were then treated with purified receptor molecules. Some of the plates were also treated with potential binding inhibitors. After allowing sufficient time for any possible binding to occur between fibrinogen, receptor, and/or inhibitor molecules, the treated plates were washed. This washing removed any molecules which had not bound to the fibrinogen which, you will recall, is physically attached to the plastic plates. We now wanted to measure how much receptor bound to the fibrinogen. This measurement made use of antibodies to the receptor, along with an organic dye. The result of this was that any microtiter wells that had no inhibitors appeared yellow, while wells that contained inhibitors remained clear.

With a method for measuring levels of fibrinogen/receptor binding , we were now prepared to look for antagonists that might be present in our natural blood sucking friends. We bought several species of leeches from various suppliers, mostly in Minnesota and Louisiana. Each type of leech looked different, ranging from the worm-like, agile swimming H. medicinalis to the small, flat, almond shaped and well-camouflaged turtle leech, Placobdella ornata. But they shared the common trait we wanted to mimic -- the ability to inhibit formation of blood clots.

Searching for a particular substance in the leeches can be both time-consuming and costly. As a first attempt we decided to look at "the whole system." We put each species into a waring blender containing some buffer- a mixture of salts and water- and turned it on. Leechomatic! After centrifuging the 'leechshake', we put the clear supernatant into the binding assay and voila - we saw no color. That indicated, for the first time, that leeches have an antagonist which interrupts binding of fibrinogen to the activated platelet receptor. We subsequently purified the active compound and found that it was a protein of only 39 amino acid residues and that it potently inhibited platelet aggregation. So far - so good.

However, it was difficult to isolate this protein from all of the other undesired components of the ground-up leeches, so we set out to devise a simpler procedure. We reasoned that the protein was likely secreted by glands in the mouth of the leech so we set out to collect leech saliva. We reasoned that if the protein really does exist in the saliva, we might be able to purify the protein more easily. Well, it just doesn't work to ask them to spit (We tried!).

To get around this leech limitation, we made a rather ingenuous use of natural lambskin condoms. We filled up the condoms with a warm arginine-salt solution, tied them off, and put them into a tank with 'hungry' leeches. The leeches swam over, attached themselves to the condom, and slowly filled up with fluid. After about 20 minutes they were full and dropped to the bottom of the tank, no longer swimming, and appearing quite bloated. We then picked up the leeches and squeezed their contents (something we referred to as leech ingestate!) into a test tube - this was pretty analogous to squeezing out the contents of a tube of toothpaste. After doing this the leeches went back to swimming and could feed again. The 'leech ingestate' we collected was put over a chromatography column and the protein which had the potent antiplatelet activity was isolated. This is undoubtedly the most unusual purification scheme we've ever done, as well as the most novel use of a condom that I've ever heard of!

Upon obtaining the amino acid sequence of this protein, which we named decorsin, from the leech Macrobdella decora, we synthesized a gene which would encode the protein and inserted it into E. coli bacteria using recombinant DNA techniques. The engineered E. coli cells then synthesized the leech protein which we purified by conventional methods for use in further studies. Interestingly, we had also isolated related proteins from the snake venoms of various pit vipers. These were also very potent inhibitors of platelet aggregation. It turned out that the one common thread among all of these proteins was the fact that they contained the amino acid sequence 'Arg-Gly-Asp', a recognition sequence that is thought to be important in many cell/matrix and cell/cell interactions.

We then set out on a number of paths. The three dimensional structural analysis of the leech protein, decorsin, was carried out using a technique called 2-dimensional nuclear magnetic resonance (2D-NMR). In addition we changed the amino acid sequences of decorsin and related proteins using a technique called site-directed mutagenesis. In so doing, we could replace any specific amino acid residue with a different one. The purpose of these studies was to ascertain which amino acid residues in these proteins were important for binding to the activated platelet receptor, thereby inhibiting platelet aggregation. These experiments confirmed that only the sequence 'Arg-Gly-Asp' was important for binding, thus defining the 'binding epitope'.

The proteins were evaluated in vivo in animal models of thrombosis in order to prove our initial concept - that inhibiting formation of the Fg/GP IIb-IIIa complex would, in fact, inhibit formation of blood clots. The data looked good.

At this point we could have chosen to develop these inhibitors further, however these proteins would only be useful as injectable drugs in acute situations. Furthermore, they would likely be antigenic since they are foreign proteins. We really wanted to develop an orally active agent - a pill that could be taken once or twice a day. This is where the peptide and medicinal chemists and molecular modelers took over - literally synthesizing thousands of compounds that were designed to contain the essential elements of the 'binding epitope' - the 'Arg-Gly-Asp' sequence - and turn it into something that had the desired properties to make an orally active drug which inhibits clot formation. These properties include such things as efficacy, nontoxicity, bioavailability, appropriate half-life, ease of synthesis, and many others. Once the compounds looked good in the Fg/GP IIb-IIIa platelet aggregation and other in vitro assays, they were tested in various animal models of thrombosis to evaluate their efficacy and side effects, i.e. how good do they work and what problems do they cause?

As you can imagine, it's difficult trying to inhibit this very complex process of blood clots formation without creating the potential for excessive bleeding. Compounds that inhibit platelet aggregation by antagonizing the Fg/GP IIb-IIIa interaction are at various developmental stages at Genentech and a number of other companies. These range from basic research to evaluation in human clinical trials - time (and a lot of money!) will tell whether this approach will be successful. A great deal of progress has been made in this area and I am confident much will be accomplished in the future, ultimately resulting in new drugs that will be very useful in saving lives as well as improving the quality of lives well beyond what currently available drugs can accomplish.

We can thank Mother Nature for providing us with some clues as to how to better our lives. Sometimes we just need to keep our eyes open... anybody wanna go fishin'?

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