You ought to protect the clot: allosteric inhibition of plasmin

Plasmin is a protein in the body that degrades blood clots, making regulation of plasmin expression very advantageous from a health perspective. In the case of a long, invasive surgery that involves lots of blood loss, an inhibitor of plasmin could be used to mitigate this blood loss and reduce amount of blood transfusions required. This paper talks about new plasmin inhibitors discovered by Al-Horani et al. with the goal of inhibiting plasmin in humans. 

But hold on… plasmin inhibitors already exist and are being used today, so why would we want new ones? Well, the inhibitors that exist now are mostly large molecules that bind to the active site of plasmin. The issues with the current molecules are that they have limited toxicity and selectivity, requiring a higher dose and yielding more side-effects. A smaller inhibitor molecule that is currently being used, tranexamic acid (TXA), requires a whopping dose of 1 to 20 grams to achieve the desired inhibition. The active site of plasmin is very similar to those of thrombin, factor Xa, and factor XIa. This means that that there is a potential for this drug to slow down the clotting cascade and increase blood loss; the opposite of the desired effect. Other side effects of the low specificity of the drug can include: seizures, renal dysfunction, and chest tube “drainage” (although I think they mean clogging).

So we could definitely use some new drugs that are more specific and also more toxic. The specificity would yield less side effects, and a more toxic drug requires a lower dosage to be effective. But how does one go about designing a new drug? Well, you start with what you know first.

Heparin is a sulphated glycosaminoglycan (carbohydrate monomer repeats with amide functional groups) which is known to allosterically control plasmin activity with an IC50 in the nanomolar range, among other interactions. Al-Horani et al. reasoned that small sulphated drugs would have interactions similar to the sulphur groups in heparin, while the specificity of the interaction would increase due to two things: 1. a smaller molecule will have fewer functional groups, leading to a statistically lower chance of having non-specific interactions; 2. the highly charged sulphur groups prevents the molecule from entering cells and crossing the blood brain barrier, keeping the molecule from ending up in places where it is not useful and can even cause undesirable reactions. Supported by the results of others that have been able to make active site plasmin inhibitors, they began synthesis of allosteric inhibitors in hopes of finding the next big plasmin inhibitor.

After using a variety of molecular backbones, a library of molecules was built. By using a chromogenic substrate hydrolysis assay they were able to identify 55 molecules with promise as inhibitors for heparin (the top four compounds being 31, 32, 52, and 54 from scheme 1 of the paper). Inhibition of plasmin with 31 and 32 yielded IC50 values of around 50 uM while 52 and 54 had IC50 values of around 75 uM. The compounds were tested for selectivity by comparing their ability to inhibit closely related serine proteases: thrombin and factor Xa. It was found that compared to plasmin, the compounds inhibited thrombin 7 to 10-fold less (IC50 >500uM) and factor Xa was inhibited about 5-fold less (IC50 >250 uM), indicating that selectivity was achieved with these compounds.

In summary, this paper is mostly a proof-of-concept that we can allosterically inhibit plasmin in order to reduce blood loss in people undergoing surgery. Allosteric inhibition will increase specificity and will have fewer side effects than drugs currently used to inhibit plasmin, like tranexamic acid. All this so that you can be more comfortable while you keep holding on to your life juice.

-Jeff