i was excited to see the news that ATryn, a recombinant human antithrombin III protein (aka AT) produced in goats, is being discussed tomorrow for possible acceptance by the FDA. it has already been approved for use in Europe.

ATryn is produced by transgenic goats and purified for human use. this is a pretty exciting venture in molecular biology, and an important first in drug production. let’s discuss, shall we?

role of antithrombin in the clotting cascade

first we should catch up with the clotting cascade, yes?

when your circulatory system experiences damage, several things happen. first and foremost, you’ve got blood vessel constriction and recruitment of platelets. then the platelet aggregation leads to inflammatory processes and activation of the clotting cascade.

the clotting cascade is set up in a very cool way. it’s always on the ready, and can spring to action instantly. basically, clotting factors are inactive enzymes (aka zymogens) waiting to be enzymatically cleaved and activated. once activated, they cleave the next zymogen into the activated form, and so on and so forth until you reach the end. the last activated enzyme is thrombin, which cleaves fibrinogen into fibrin, which then becomes cross-liked among the platelets to form the clot.

because thrombin is such a critical protein, given that it’s the final enzyme in this critically important chain reaction, it has its own self-regulation pathways as well. it can activate cofactors  that lead to increased prothrombin cleavage. but it can also bind to thrombomodulin, which activates a pathway that degrades those same cofactors and leads to decreased activated thrombin. positive and negative feedback mechanisms at their finest.

as you might guess from the name, antithrombin is an inhibitor of thrombin. (you may also hear of it referred to as a “serpin”, which is short for serine protease inhibitor. and then you may deduce, thrombin is a type of enzyme known as a serine protease.)

but AT is also very involved in regulating clotting via thrombin. in its most basic move, it binds to thrombin and inhibits the formation of fibrin. (heparin increases the binding affinity by leaps and bounds, which is why it it also very important in anticoagulation.) but it also can inhibit the thrombin-thrombomodulin complex, which in effect keeps thrombin production up. this complex interplay of activation and inhibition in the clotting pathway make it terribly important to have enough antithrombin around to keep up the interactions as needed.

deficiency

unfortuantely, AT deficiency is an inhertited disorder in which blood clotting is not properly regulated via AT. there are several types, but the end result is that AT activity is not sufficient to properly regulate coagulation, and thrombosis ensues. of course, rogue blood clots are never welcome in the body. stroke, pulmonary embolism, and myocardial infaction are among the unwanted consequences.

so we really, REALLY want to treat these people. and so far, they are treated with heparin and warfarin.

but have you ever had a surgery, and they ask you to stop taking blood thinners for x hours prior to surgery to prevent excessive bleeding? well, then what? that would be the indication of ATryn, should it be approved.

and now onto the goats.

molecular biology is freakin’ AWESOME

as glad as i am that i steered more toward what i do now, i still have a special place in my heart for molecular biology. so this is more ooh-that’s-so-cool geekery from me.

so we have this protein we want to supplement in people, and we can’t give it orally because proteins don’t absorb from the GI tract. also, stomach acid chews them up. (after all, it spends all day digesting the proteins we put into our stomachs for nutrition.) and it’s got some complex post-translational modifications that keep us from producing it in simple organisms. so. what’s an easy-to-harvest mammalian fluid that does not harm your very expensive transgenic animals?

the correct answer is milk. (and for those of you wondering about animal health, i can guarantee the pricetag on the heads of these animals alone gets them high quality treatment.)

and here is where goats come in. goats can produce a lot of milk. we can genetically modify them. we have the gene in question. now to put them all together. we know that transcription information is specified by promoters, and that promoters are gene-specific. so take a promoter for a gene associated with milk- say, a casein protein- and stick the AT gene in there as the gene that gets expressed instead. now, when mammary cells go to make casein for the milk, the endogenous casein protein is made by the copy of the gene the goat already has AND AT is made because it’s got its own casein promoter. ta-da! milk with AT.

i can guarantee this took many, many, MANY trials to get right. i make it sound like it’s simple, but anyone who has done DNA work knows there are lots of attempts involved.

concerns?

this is a pretty big, pretty new way to manufacture drugs. it’s understandable people will be concerned. is the protein going to work? is there any goat stuff in there that can make me sick? what about animal diseases? aren’t farm animals dirty? i can understand being a little bit concerned about this. after all, it’s critical that the producer takes proper precautions, purifies the protein to a very high extent, ensures there are no pathogens, and etc.

they describe their purification process as an affinity column- they use the high binding affinity to heparin to actually remove the AT protein from the milk, while letting all the other stuff that’s in the milk pass on through. the final product is passed through a virus filter. (i didn’t know they had these!) they also address prions, but the specifics weren’t mentioned in the FDA briefing.

once they have the purified protein, they test it for activity against published numbers. i presume they have a rigorous quality assurance program in place to maintain the quality of their product and to protect the patients who will receive the drug.

the FDA will make the final call, but i think there are many advantages to a mass-produced recombinant AT. also, the major precedent of drugs from transgenic animals might change some thinking in the industry. i bet there will be some molecular biologist positions opening up when the economy comes around.

we will have to maintain constant vigilance to ensure the purity and safety of the drugs, and we will have to subject this and all future products to rigorous review to ensure that the activity of each protein produced in animals for human use is good enough. but i feel pretty optimistic about this (after due caution), and it’s a very cool story to write about too.