Thrombotic disease is a common multiple disease that imperils people's life. The formation of thrombosis is a key event in the pathogenesis of myocardial infarction, stroke, and other ischemia conditions that comprise the most common and growing causes of morbidity and mortality (1). LK consists of a group of serine proteases with strong fibrinolytic activity (2,3), can dissolve fibrin either directly or indirectly by activating profibrinolysin. LK has been widely used as a novel, orally administered drug for thrombotic diseases and has caused growing interest in East Asia (4). Although some thrombolytic agents such as urokinase (uPA), streptokinase (SK) and tissue plasminogen activator (t-PA) are all typically used to dissolve clots, they are not specific to fibrin and have severe side effects such as heavy blood loss (5,6). In contrast, LK is very specific to fibrin as a substrate and does not cause excessive bleeding (7). Furthermore, some researches had shown that LK contained several active components (8,9) which could dissolve the fibrin directly or can convert plasminogen to plasmin by inducing endogenous t-PA activity to dissolve fibrin clots (10). However, The LK is an oral drug which belongs to biological products with very short half-life, it needs oral 2–3 times a day. And more importantly, given the fact that thrombotic disease is an acute illness, oral administration of the drug can not serve as first choice because of its low bioavailability and slow absorption in human body. In order to solve this problem, we have prepared it into intravenous agents by PEG modification which could improve its half-life, reduce dosing frequency. According to the results of the pharmacokinetics, the ideal result is that the drug can maintain for 12 to 24 h in the body after intravenous injection.

The attachment of proteins to polymers, especially to polyethylene glycol (PEG), is an established technique to enhance the therapeutic and biotechnological potential of numerous proteins, macromolecular carriers, oligonucleotides, and some biomolecules (11). PEGylation can increase the size and molecular weight of conjugated biomolecules and improve their pharmacokinetics. At the same time, it can protect the drug from enzymatic degradation, reduce renal clearance as well as inhance pharmacological activity in vivo (12–14). The covalent conjugation of PEG to therapeutic proteins is a clinically successful strategy for extending serum half-life to reduce administration frequency (15–17). Many biochemical agents such as tPA (18), SK (19), uPA (20), and staphylokinase (Sak) (21,22) have been modified with PEG as prodrugs for preparing injective formulation and for prolonging half-life. Currently, there are at least 10 PEGylated products with FDA marketing approval, and numerous other conjugates are entering clinical trials (23,24).

In our previous study, we investigated the optimal PEGylation condition with buffer pH of 8.0, reaction time of 5 h, reaction temperature of 0°C and molar ratio of 1:25. The primary amino group of LK and functional group of mPEG react under the buffer solution system, and finally generate PEGylated LK (Fig. 1). The in vitro fibrinolytic activity of both native and PEGylated LK was measured in comparison to that of uPA on fibrin plates. The results showed that mPEG₂₀₀₀₀-SC-LK was prepared successfully and had strong fibrinolytic activity in vitro. In this study, conjugation of LK with mPEG-SC₂₀₀₀₀ was prepared and analyzed as previously reported (25). Then, we studied the pharmacokinetics and in vivo anti-thrombosis efficacy of mPEG₂₀₀₀₀-SC-LK with rat carotid artery thrombosis model. This is the first systematic report to evaluate the anti-thrombotic effect of PEGylated injection form of LK in animal model.

Present study focused on development and evaluation of a long-acting anti-thrombosis agent through PEGylation of LK. PEGylated LK was successfully modified with mPEG₂₀₀₀₀-SC in optimal condition as we reported before (25). As the effective molecular weight of crude LK was around 24 kDa, the theoretical molecular weight of PEGylated LK was around 44 kDa. Both the SDS-PAGE and MALDI-TOF results were consistent with our expectation, suggesting that LK was successfully modified with mPEG₂₀₀₀₀-SC. The in vitro fibrinolytic activity of the PEGylated products was lower than that of purified LK although the calues were not significantly different. Despite the trend for PEGylation to decrease in vitro fibrinolytic activity, this may not influence in vivo activity because of the longer circulation half-life of PEG modified LK.

Multiple approaches have been devised to extend the half-life and increase activity of anti-thrombotic agents in the bloodstream, such as polymer coating and the use of drug carriers. The most commonly used approach to prolong the half-life of anti-thrombotic agents involves conjugation of drugs with PEG. PEG chains form a hydrated shell that enhances drug solubility and limits renal filtration, proteolytic degradation, and immunogenicity (1). In practice, it has been reported that PEG₂₀₀₀₀-IFN and PEG₄₀₀₀₀-IFN significantly prolonged serum half-life and lowered the renal clearance rate (14,23). In our study, the serum activity of mPEG₂₀₀₀₀-SC-LK showed 2.2-fold increase in t_1/2 than the native LK, and the mPEG₂₀₀₀₀-SC-LK displayed 3.7-fold decrease in CL. The reduction ranges of mPEG₂₀₀₀₀-SC-LK were much smaller than those of native LK. Seven h after the injection of native LK, the serum LK concentration was decreased below 10 IU/ml, while the serum mPEG₂₀₀₀₀-SC-LK concentration was remained above 170 IU/ml, indicating that mPEG₂₀₀₀₀-SC-LK had long-circulating effects and prolonged biological half-life and residence time in vivo. PEGylation increased the size and molecular weight of LK and improved its pharmacokinetics by protecting from enzymatic degradation, prolonging circulation time and reducing renal clearance. PEG polymer linked to LK, may creates a bulky hydrophilic shield which could efficiently mask the conjugated LK from enzymatic digestion.

LK extracted from the earthworm has been used to treat stroke and cardiovascular diseases (32,33). Recent studies (34) have shown that the fibrinolytic enzymes could dissolve blood fibrin clots and inhibit activation and aggregation of platelets. However, most studies mainly focused on the oral preparation of these enzymes. This is the first report to systematically evaluate the ant-thrombotic effect of PEGylated injection form of LK in animal model. Artery-vein bypass thrombosis animal model was widely used in evaluating properties of anti-thrombotic preparations. In our experiment, mPEG₂₀₀₀₀-SC-LK showed nearly 3.5-fold increase in the inhibition ratio of thrombosis effect compared with the native one in this rat model.

FeCl₃-induced carotid artery thrombosis model is another typical model to eveluate an anti-thombosis agent. To investigate changes of TXB₂, PGF_1α, t-PA and t-PA/PAI-1 ratio, thrombi were induced by topical ferric chloride in our experiment. TXB₂ as the end product of the metabolism of XA₂ (35,36), has an important role in the promotion of platelet aggregation and contraction of blood vessels. It has been reported that patients with atherosclerosis showed high plasma TXB₂ values and low PGF_1α content. After taking 2 weeks of LK, the plasma level of TXB₂ significantly decreased and PGF_1α level significantly increased in patients, suggesting that LK has a strong inhibitory effect on platelet aggregation. After the injection of LK and mPEG₂₀₀₀₀-SC-LK, we saw that the TXB₂ concentrations decreased compared to the model group, and the TXB₂ content in mPEG₂₀₀₀₀-SC-LK group decreased more than that of LK group. The content of PGF_1α was higher in both LK and mPEG₂₀₀₀₀-SC-LK groups when compared with the model group, and the PGF_1α content in mPEG₂₀₀₀₀-SC-LK group was increased greater than that in LK group.

Endogenously mediated fibrinolysis is initiated by t-PA and u-PA, the enzymes convert plasminogen to the fibrinolytic protease plasmin (37). T-PA and PAI are a pair of biological regulatory factors which are of great significance to the function of fibrinolytic system. T-PA can specifically combine with fibrin in plasma, possessing selective thrombolysis effect. PAI as a rapid inhibitor can inactivate t-PA by combining with it. It plays a decisive role in fibrinolytic system by inhibiting the role of anti-thrombosis effect. According to the literature (38), the t-PA/PAI ratio was often used as an index of anti-thrombosis. This study showed that in FeCl₃-indeced thrombosis rat model, the content of TXB₂ was increased, whereas PGF_1α content, t-PA content and t-PA/PAI ratio were all decreased in the model group. This is because as endothelial damage was occurred due to vascular endothelium, the platelet and coagulation system was activated and the anti-coagulant protein in the blood was consumed, leading to decreased anti-coagulant activity. Meanwhile, because of the formation of thrombus, the t-PA in the blood combined to the thrombus location decreased the activity of t-PA. As an anti-thrombosis agent, LK can directly dissolve fibrin or indirectly dissolve fibrin by activating profibrinolysin. Thus, LK can eventually reduce thrombotic diseases in patients with PAI and activate t-PA, resulting in increased fibrinolysis. After administration of the drugs, t-PA and t-PA/PAI ratio was increased, and the rising range of mPEG₂₀₀₀₀-SC-LK was greater than the native LK group. The lower in vitro potencies of the PEGylated LK relative to those of the native LK may be due to steric shielding of the protein by the PEG polymer with its comb-like structure (39). But when it comed to in vivo situation, PEGylated LK presented better anti-thrombosis efficacy than the native one. Because PEGylation is able to enhance drug efficacy by reducing its clearance either through glomeular filtration or through proteolytic degradation.

Vascular thrombotic disease is a serious threat to human health which is the main cause of human disability and death. According to the results of our research, it demonstrated that mPEG₂₀₀₀₀-SC-LK exhibited greater anti-thrombosis effect in both artery-vein bypass thrombosis and FeCl₃-induced carotid artery thrombosis models than native LK. It could effectively improve the clinical symptoms of patients, reduce morbidity and morbidity, and it is an ideal drug to treat vascular thrombotic disease in clinical, such as myocardial infarction combined high blood fibrinogen, high stick sluggish blood disease, retinal vein occlusion, cerebrovascular disease, infarction, and so on. Therefore, it is a potential anti-thrombotic agent for long-acting treatment of the thrombus diseases.

Present study showed that mPEG modified LK have prolonged half-life and strong fibrinolytic and thrombolytic activities in vivo. These results indicate that the PEGylated LK is a novel anti-thrombosis agent for the treatment of thrombosis diseases in clinic. These results can provide the foundation of the research of injective LK in the future study.