A Novel Anti-Glycoprotein (GP) Iba-Targeting Chimeric Fibrinolytic Agent Demonstrates in Vivo Efficacy Against Human Platelet-Enriched Thrombi in Mice

We previously described (Fuentes, J Clin Invest 2016) a chimeric protein, GPIIb/uPA-T, that provided effective thromboprophylaxis composed of an N-terminal scFv specifically to human (h) GPIIb linked to a C-terminal variant of low molecular weight urokinase containing a thrombin cleavable activating site. uPA-T prevented FeCl3-induced carotid arterial thrombosis in immunocompromized NOD/SCID, IL2 receptor γ-chain deficient (NSG) mice that had been pre-infused with human platelets (hPlts) without causing rebleeding from pre-existing tail-clips clots. However, this construct and subsequent hGPIIb-targeting scFv uPA-T chimeric constructs caused significant thrombocytopenia in hPlt-infused NSG (hPlt/NSG) mice and in hGPIIb+ mice. Therefore, we developed a new chimeric uPA-T targeting hGPIbα based on 5G6, a monoclonal antibody that binds to the hGPIbα juxtamembrane ADAM17 cleavage site and is known to not cause thrombocytopenia. The chimera 5G6/uPA-T binds to the surface of hPlts and hGPIbα-expressing mouse platelets at levels ~30% of those seen with hGPIIb/uPA-T, consistent with the difference in expression of GPIIb vs. GPIbα. 5G6/uPA-T did not reduce platelet counts in hPlt/NSG or hGPIα+ mice at doses that caused significant thrombocytopenia after infusion of GPIIb/uPA-T (1 mg/kg each, bolus + 30-minute infusion). We then tested the efficacy of 5G6/uPA-T in hPlt/NSG mice that that also carried a single mouse-to-human arginine (R) to histidine (H) substitution at position 1326 in von Willebrand factor, VWFH, created using CRISPR/Cas9 technology in NSG mice. This substitution, described by T. Diacovo (Megalion, Circulation 2011), markedly impairs binding of mouse Plts that express murine GPIbα, but increases the affinity for hPlt expressing hGPIbα. We hypothesized that such mice would allow studies of hPlt-enriched thrombi in mice. To test this hypothesis, hPlts were infused into NSG mice to achieve a circulating level of 10-20% of the total platelet pool. Following FeCl3-induced carotid arterial injury, similar numbers of hPlts were incorporated into the thrombi in wildtype VWFR/R NSG and heterozygous VWFR/H littermate NSG mice, whereas 1.4-1.8-fold more hPlts were incorporated in VWFH/H NSG littermates over the 3-minute observation period (n = 12-17, p<0.04 between VWFH/H NSG mice vs. VWFR/R and VWFR/H mice.