By David N. Leff

Two of the 20th century's most celebrated inventors were the syndicated cartoonists Rube Goldberg (1883-1970) and Heath Robinson (1872-1944). Both - the American, Goldberg, and Britain's Robinson - fantasized intricate working drawings of absurdly complicated devices, which tweaked their readers' love of gadgets.

One example: Goldberg penned a contraption designed to wipe a diner's chin with a napkin. Its steps included lifting a spoon that pulled a string that jerked a ladle that threw a biscuit past a parrot, which jumped and tilted its perch - and so on, until a pendulum attached to a napkin swung past the chin.

A universal mammalian bodily function, the blood's coagulation cascade, could certainly qualify as a Rube Goldberg or Heath Robinson wound-healing modus operandi. To wit:

¿ When external trauma or internal abrasion damages the wall of a blood vessel, circulating factor VIIa binds swiftly and tightly to membrane-bound tissue factor (TF), thus triggering the clotting cascade.

¿ This complex then activates factor X, which releases thrombin from its precursor protein, prothrombin.

¿ Thrombin in turn then converts fibrinogen - a component of blood plasma - to fibrin, a long, rod-like molecule, which acts like the metal rebars in concrete to fabricate the actual clot, which closes the hole in the dyke of the vessel wall.

This simplified account of clot formation has one more vital step: stopping the coagulation process when it's gone far enough. If it's not turned off in time, all the veins, arteries and capillaries in the body's vascular network would suddenly solidify into a single rigid clot. The shutoff valve is a glyoprotein called thrombomodulin, which is ever-present in the endothelial cells lining the blood vessel wall. It binds thrombin, and turns it into an anticoagulant. To do so, it hooks up with two other proteins, which inactivate factors Va and VIIIa.

Hemophilia and thrombosis are two sides of the clotting cascade coin. Hemophiliacs lack factors VIII or IX, so they bleed uncontrollably at the slightest scratch, unless those proteins can be replaced.

Wanted: Antithrombotic Clot-Busting Control

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

Hence, for years researchers have been hot on the track of medicinal molecules that can inhibit excess clots from forming. The key targets on which they zero in are the serine proteases - protein-cleaving enzymes that regulate clot formation by binding substrate molecules in active-site pockets on their surface. That regulation often takes place through so-called "exosites," on the enzyme surface some distance away from their active site.

A recent entry in this sweepstakes is a paper in Nature dated March 30, 2000, bearing the title: "Peptide exosite inhibitors of factor VIIa as anticoagulants." Its senior author is biochemist Robert Lazarus, a senior scientist in the protein engineering department of Genentech Inc., of South San Francisco.

"I think the innovation in this work," Lazarus told BioWorld Today, "is the fact that we found peptides that bind to a new site on factor VIIa, and inhibit its function. Instead of blocking serine proteases by going after their active site, it permits having a pathway that goes after those inhibitors, using exosites.

"The one real advantage of these peptides with respect to other types of inhibitors," he observed, "is their selectivity. That's the one thing that's very hard to get with inhibitors at the active site. Whereas, as our paper reported, we were clearly able to get it at the exosite - potentially a distinct advantage that this mode of inhibition may have as opposed to other modes."

The co-authors used phage display technology to single out promising inhibitor peptides. "What we did," Lazarus recounted, "was make naove peptide libraries - peptides that contained many different possibilities, and displayed those on the surface of bacteriophage. We selected for phage-bearing peptide sequences that bound to factor VIIa in the presence of tissue factor.

"And I'd say," he observed, "that there was a certain amount of serendipity here. We were hoping to find things that might inhibit, but that wasn't a given at the beginning of this effort." He explained: "It wasn't surprising that we found peptides that bound, but that we discovered peptides that bound with high affinity, and in a functionally relevant way."

Putative Therapies Way Off In Offing

"The utility of that finding," Lazarus pointed out, "is an antithrombotic or anticoagulant, able to prevent blood clots from forming. Inhibitors of factor VIIa in general," he added, "are going to be used more as anticoagulants, which may supplant blood-thinning drugs, such as heparin and coumarin."

One of Genentech's lead products is Activase, the tissue plasminogen activator (t-PA) administered to patients in heart attack from clot-blocked coronary arteries.

"T-PA is also a serine protease," Lazarus observed, "but more involved in dissolving clots, whereas inhibitors of factor VIIa will aim at preventing clots from forming in the first place. The way in which such an inhibitor would work would be to hit the top of the coagulation cascade. Tissue factor," he went on, "is an essential trigger for factor VIIa. It then can catalyze the reaction of X to Xa or IX to IXa, which ultimately can go on to cleave thrombin from prothrombin. Thrombin in turn cleaves fibrinogen to fibrin, which can then polymerize and form a blood clot.

"There are many possible medical indications," Lazarus continued. "But one thing to take into account is that at this stage of the game it's still a fairly early piece of research. The inhibiting peptide in and of itself does not represent a drug. What we can do is turn that peptide into a drug - which we're now in the process of addressing. We would certainly re-engineer the peptide itself, or find small molecules that somehow mimic what the peptide can do in the therapeutic area. But," he concluded, "it's early."

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