Strokes and heart attacks kill some 12 million people a year worldwide - about 1 million of them in the U.S. And every 53 seconds, someone in the U.S. experiences a stroke - the blockage of blood flow to the brain. These blood clots cut off the delivery by red blood cells (RBCs) of oxygen and nutrients to the vital cerebral neurons.

The most striking and deadly example of thrombotic disease is atherosclerosis, in which unwanted clots build up in the coronary arteries that nourish the heart. Also, a clot fragment in a leg can break loose and hitchhike via the bloodstream until it lodges in a lung. That pulmonary embolism is often fatal.

"The only way to prevent thrombosis," observed cardiovascular researcher Vladimir Muzykantov at the University of Pennsylvania in Philadelphia, "is to intervene with blood thinners. The classical anticoagulants are heparin, aspirin and [Centocor Inc.'s ReoPro]. Those agents, by different mechanisms, all pursue the same goal, which is to prevent formation of pathological clots in the bloodstream. But they have their imperfections," he continued. "So even patients placed on heparin infusion or aspirin or ReoPro, still might suffer intravascular clots.

"ReoPro," Muzykantov summarized, "is a drug and monoclonal antibody against platelet aggregation. Aspirin inhibits platelet aggregation due to metabolic inhibition. Heparin blocks coagulation due to inhibition of enzymatic cascades of fibrin clot formation. So these three - and there are some other anticoagulants as well - mainly work on those three areas. But still none of them is 100 percent effective, and there are some clinical situations in which doctors have to either suspend anticoagulant therapy or minimize the dose, because of high propensity of bleeding."

Muzykantov explained: "For example, in or around surgery and immediately after the operation, surgeons usually try to minimize or even eliminate anticoagulants, because there is a very high risk of bleeding from the wound. The problem is that once these bad' pathological clots - as opposed to good' hemostatic clots - are formed, despite anticoagulation, they don't dissociate these clots, and the only way to do so is to inject into the blood tPA or fibrinolytics like tPA, which was developed two decades ago.

"One serious problem," Muzykantov added, "is that you cannot use blood tissue activators as a prophylactic." He reviewed the reasons why: "First, because tPAs are eliminated from the blood very readily; secondly, because they have the same deleterious side effects of indiscriminate dissolution of hemostatic clots as well as pathologic clots. Those plasminogen activators have three downsides: First, they don't last; all are eliminated within minutes from the bloodstream. Second, they dissolve hemostatic good clots because they diffuse inside these clots and lyse them. And third, they diffuse into tissues and cause collateral damage, especially in tissues that are hemorrhaging, as in the brain. So you cannot use plasminogen activators for prophylactic administration."

Targeting New Blood Clots Lethal To Patients

Muzykantov is senior author of an article in Nature Biotechnology, released online July 8, 2003. Its title: "Prophylactic fibrinolysis through selective dissolution of nascent clots by tPA-carrying erythrocytes."

"As we describe in this article," he told BioWorld Today, "we coupled plasminogen activators, in this case it was tPA, to autological red blood cells - erythrocytes. And after this we achieved a long circulation time of plasminogen activators by orders of magnitude. Because RBCs usually circulate for a very long time, we can couple tPA or other proteins in a biocompatible way with RBCs. They will circulate and target newly formed blood clots that are most lethal for patients.

"So if conventional anticoagulants like heparin don't work for any reason, and clots are formed, then we have tPA now incorporated in these clots from the inside," Muzykantov said. "They lyse newly formed pathological clots like a Trojan horse - from the inside. That is the concept we developed, thanks to two animal models. And those results of our animal studies show that indeed if we compare the action of a free tPA - which is the formulation currently used for therapies - with RBC-bound tPA, we see very effective dissolution of nascent but not pre-existing clots.

"From the general standpoint," Muzykantov noted, "this is the very first experimental study seen in animal models of pathological process relevant to human pathological process. That is, a drug carried by an RBC carrier could be used and is active in a very different way from a free drug. These Trojan horse fibrinolytics would have no access to pre-existing clots because they are bound to relatively large - a few micron-size - carriers, in this case RBC. This concept, converting these plasminogen activators from therapeutics to prophylactics, is totally new."

Inventing Technology Right In Bloodstream

"These RBCs were hitched by streptavidin-coupled biotinylated tPA to the surface of RBCs, and we injected them intravenously into the animals' tail veins," he said. "Theoretically, this technology could be used tomorrow for human practice, because in many clinical settings patients either donate their blood or just use a group of biocompatible blood from another donor to inject RBCs. In theory, you can soon do this without taking blood from a patient because we are now developing a new methodology for coupling tPA to RBCs directly in the human circulation.

"With both rats and mice we tested whether tPA circulates for a much longer time if injected as a complex with RBCs as free drug. In both animal species we found that after we conjugated tPA to RBC, we prolonged circulation time by orders of magnitude, similarly for rats and mice.

"One patent has already issued to the university, with myself as first inventor," Muzykantov noted. "And two or three applications are pending. The issued patent describes a very marked technique of biocompatible coupling of tPA and other RBCs. Continuing patents describe what this Nature Biotechnology paper reports.

"The goal of this particular paper was not to define potential side effects but to demonstrate proof of principle that the effects we were expecting are possible. We believe that ongoing and future animal studies will define potential applications and limitations of our strategy.

"The university now, I think," he concluded, "wants to find partners to translate this technology into the clinic."