Spacer effect on heparin-triggered release of a thrombolytic drug from a camouflaged construct

Objective: The purpose of this study is to determine the effect of the polyethylene glycol (PEG) spacer between serum albumin (a camouflaging molecule) and protamine (a binding domain), on the amount of heparin required to release tPA from its inactive construct. Background: Human recombinant tissue plasminogen activator (tPA) is a major thrombolytic drug used for various thrombotic conditions, including myocardial infarction and ischemic stroke. Since the drug may lead to fatal hemorrhagic conditions by depleting clotting factors in blood, targeted prodrug constructs have been proposed to prevent excessive nonspecific plasmin generation. In these constructs, a reversible binding between the drug and a carrier molecule is created, which can be undone by an external trigger. The key to success of this type of strategy is to optimize the strength of the reversible binding, so that the construct is sufficient stable during transit to the target while the external trigger can effectively release the agent. Methods: The tPA and low molecular-weight heparin (LMWH) was conjugated via thiolation chemistry, followed by ion-affinity chromatography. Separately, human serum albumin (SA) and salmine protamine conjugate was prepared via site-specific chemistry. The N-terminus of protamine was thiolated using either succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or the same type of heterobifunctional crosslinker with PEG 34kDa spacer. Following the complexation of the two conjugates, amidolytic activities were spectrophotometrically measured at 405 nm via an indirect chromogenic assay using S-2251, a plasmin-specific substrate in the presence of varying amounts of unfractionated heparin. Results: The tPA activity was fully released from the construct using SA-protamine (no spacer) at the heparin concentration of 0.4 U/mL whereas it took 0.6 U/mL to fully release tPA from the construct using SA-PEG-protamine. Conclusions: The presence of the PEG spacer in the construct resulted in the firmer binding between the LMWH and the protamine moieties from each conjugate, requiring higher concentration of heparin for the full release of tPA activity, compared to the construct without the spacer. For each construct, the heparin concentration required to trigger tPA release was still within the therapeutic range of heparin (0.2-0.7 U/mL). Grant: PFRDG (FY22)