The main goal of this study is to develop a better thrombolytic therapeutic agent for thrombosis diseases, such as deep vein thrombosis and peripheral arterial occlusion (PAO). PAO occurs when a clot blocks artery blood flow to a distant part of the body such as the legs, arms, feet, or hands. PAO is the result of peripheral arterial disease (PAD), in which atherosclerotic plaque build-up on the artery walls leads to obstructed blood flow, leading to ischemia in blood starved limbs of the body .
Current treatments of PAO include angioplasty, stents, and thrombolytic intervention with Activase® (tissue-type plasminogen activator, tPA) or Abbokinase® (urokinase-type plasminongen activator, uPA). Thrombolytic therapeutics are not currently approved by the FDA for PAO because they require infusions that last a day or more and are associated with high risk of serious bleeding including stroke . In humans, thrombolytic therapy with tPA has been shown to cause potentially fatal intracranial hemorrhage (ICH) in approximately 1% of patients receiving it for acute myocardial infarction . The patient risk for ICH is even higher at 2.9% after prolonged infusion treatment for PAO . Thrombolytic intervention against PAO has advanced concurrently with significant technical advances in catheter design and delivery, permitting local drug delivery directly into the clot. However, even under these circumstances, plasminogen activator (PA) mediated clot dissolution can be slow, cumbersome, and only partially effective, requiring 1-2 days to take effect . Furthermore, because the effectiveness of PAs is dependent on local plasminogen (Plg) levels, Plg depletion problems occur due to long, retracted clots and poor circulation. Consequently, this renders PA therapy only partially effective for PAO, and carries with it potential serious side effects . Therefore, alternative therapeutic options that are safer and more efficient are desired. One such alternative strategy is to directly use the activated form of Plg, Plasmin (Plm), which digests fibrin in vivo. In practical consideration, current advances in local drug delivery make Plm or its des-kringle derivatives attractive for PAO treatment. In particular, the issue of its very short serum half-life has been addressed through catheter infusion directly at the clot site as an alternative to IV administration. At the same time, the very short half-life of Plm offers the additional advantage of decreasing circulation of the active enzyme to non-specific sites, thereby reducing the risk of hemorrhaging and ICH.
As described above, breakdown of a fibrin clot (thrombi) into soluble components depends on Plm, a serine protease that is derived from the freely circulating proenzyme Plg . Plg binds to both fibrin and fibrinogen, thereby being incorporated into a clot as it is formed. In vivo, Plg is activated by tPA trapped in the blood clot . The resulting Plm is transformed into two separate subunits interconnected by 2 disulfide bridges. The A chain of the Plm molecule consists of 5 triple-loop disulfide kringle (Kr) domains (approximately 78-80 amino acids each), while the B chain contains a “linker” region of 20 amino acids and a serine protease domain (approximately 228 amino acids) . Through laboratory manipulations, 2 des-kringle variants of Plg with potential pharmacological application have been created. One of these, miniplasminogen (mPlg), consists of Kr5, the linker, and the serine protease domain. The other, microplasminogen (μPlg), consists of only the linker and serine protease domain itself. mPlg and μPlg are also activated to miniplasmin (mPlm) and microplasmin (μPlm), respectively, by digestion at the peptide bond between R561 and V562 (amino acid number adapted from reference ). As in Plg, activation of mPlg and μPlg by tPA, uPA , or other PAs forms two separate subunits interconnected by two disulfide bridges.
In vitro studies have identified several interesting functional differences between Plm, mPlm, and μPlm which may have potential clinical significance in developing a therapeutic drug. Functionally, μPlm is distinguished from mPlm and Plm by its inability to specifically bind to fibrin; μPlm lacks the fibrin binding resides in Kr1-Kr3 and Kr5 domains [9–11]. While Plm and mPlm have similar catalytic rates in digesting fibrin, μPlm is 6-fold slower than mPlm and 12-fold slower than Plm . Once fibrin bound Plm dissociates from the blood clot, it becomes immediately accessible to its principal inactivator, α2-antiplasmin (α2-AP). α2-AP first binds to specific lysine residues located in Kr5 and other kringle domains before binding to the catalytic domain, inactivating Plm for a resulting plasma half-life of only 0.2 seconds [9, 12, 13]. Apart from the half-life issue, the desirability for pharmaceutical thrombolysis development is Plm>mPlm>μPlm because of the fibrin binding specificity and the more rapid kinetics in digesting fibrin .
During the preclinical drug development stage and animal testing, we observed that recombinant mPlm has better pharmacological properties than μPlm, and decided to develop mPlm as a thrombolytic therapeutic candidate. However, during process development and scale up production, we faced a non-specific cleavage problem during activation of mPlg, hindering the development of this promising drug candidate. In order to solve this problem, we designed and screened mPlm mutants to select for those that retain the desired catalytic properties, but have much reduced tendency to be cleaved non-specifically.