Abstract
Abstract 1171
Thrombolytic therapies are limited in their use to acute life- or limb-threatening thrombosis due to the high risk of significant bleeding. To overcome this limitation, we propose a novel strategy incorporating two targeting features: 1) an N-terminal single-chain antibody variable region (scFv) chain that binds with high affinity to human platelet alphaIIb, and 2) a C-terminal thrombin-specific activatable low molecular weight urokinase (uPA-T) that has its plasmin-activation site replaced by a thrombin cleavage site. We anticipate that such pro-drugs would preferentially bind and become activated at sites of active clot propagation. Two distinct anti-alphaIIb scFvs were studied: one (312.8) derived from a monoclonal antibody that bound human activated or quiescent alphaIIb whether complexed to human or mouse beta3, and one (LIBS) that bound preferentially to activated human or mouse alphaIIb bound to either human or mouse beta3. We anticipated that the 312.8/uPA-T chimera would have a more prolonged half-life, but that the LIBS version would be more effective at the nascent clot where activated platelets predominate. All proteins studied (uPA-T, 312.8/uPA-T and LIBS/uPA-T) were expressed and isolated from Drosophila S2 insect cells. Flow cytometric studies confirmed the expected binding of the chimera proteins to resting and activated human platelets, wildtype (WT) mouse platelets and mouse platelets that expressed only human alphaIIb/mouse beta3 (haIIb/mb3) on their surface. Neither chimeric protein interfered with platelet aggregation stimulated by adenosine diphosphate (10 μM). Further studies using diverse serine proteases confirmed that induction of fibrinolytic activities of both chimeras was thrombin-specific, and both were activated by thrombin to the same extent as isolated uPA-T. In haIIb/mb3 mice, both flow cytometric studies and tracking of 125I-labeled chimeric proteins showed 312.8/uPA-T had a longer half-life than uPA-T or LIBS/uPA-T. Ex vivo studies of thromboprophylactic efficacy of the chimeric proteins relative to uPA-T were performed using a microfluidic system with the surfaces coated with species-specific von Willebrand Factor. Platelets were isolated from whole human or haIIb/mb3 murine blood, exposed to a fibrinolytic agent or control, gel filtered to remove unbound drug, and added back to reconstitute whole blood. 312.8/uPA-T decreased platelet aggregation and virtually eliminated fibrin formation at drug concentrations <0.5 μg/ml, while both uPA-T and LIBS/uPA-T were ineffective even at the highest concentration tested (50 μg/ml). This microfluidic system was then modified to simulate the prothrombotic state seen in HIT by adding the well-characterized HIT-like monoclonal antibody KKO plus recombinant platelet factor 4 (PF4), markedly enhancing fibrin accumulation in the model. Again, low doses of 312.8/uPA-T enhanced fibrinolysis, while neither uPA-T nor LIBS/uPA-T were effective at a 100-fold higher concentration, consistent with both uPA-T and LIBS/uPA-T not binding well to resting platelets. In vivo studies of cremaster laser arteriolar injury were performed in haIIb/mb3 mice that were also transgenic for human PF4 and FcgammaRIIa before and after inducing a HIT-like state by injecting KKO. In this prothrombotic setting, pre-infusions of either chimeric proteins prevented fibrin accumulation and decreased platelet accumulation for up to 1 hour (latest time point studied). This demonstrates that LIBS/uPA-T is effective in thrombolysis despite its shorter half-life than 312.8/uPA-T. In summary, these studies support a novel approach towards thromboprophylaxis by combining a pro-drug that requires activation by thrombin with platelet delivery to sites of incipient thrombosis. The preferred epitope for platelet targeting needs further study, as targeting to all platelets or specifically to activated platelets each appears to have advantages depending on the setting. These chimeric pro-fibrinolytic agents may be useful in systemic prothrombotic states such as HIT, but whether targeting and safety are preserved in vivo in such a setting needs further examination.
Disclosures:
Cines: Amgen: Consultancy; Genzyme: Consultancy; Sanofi: Consultancy; Eisai: Consultancy.
Antithrombotic Effect of Platelet Glycoprotein Ib–Blocking Monoclonal Antibody Fab Fragments in Nonhuman Primates