Abstract
Abstract 3361
We have developed a semi-synthetic antithrombotic heparin complex, APL001, to mimic mast cell-derived natural heparin proteoglycans (HepPG). HepPG attenuate platelet-collagen interactions under blood flow by inhibiting VWF- and GPIIb/IIIa -mediated platelet aggregation. In addition, rat-derived HepPG arrest platelet thrombus growth on collagen surfaces or at vascular injury sites, both in vitro and in vivo (Lassila et al.ATVB 1997, Kauhanen et al. ATVB 2000, Olsson et al. Thromb Haemost 2002).
Our objective was to study the inhibitory capacity of APL001 for preventing human platelet aggregation in vitro and acute thrombosis in a baboon model in vivo. The effects of unfractionated heparin (UFH) and APL001 were compared in relevant coagulation assays (APTT, PT, thrombin time, anti-FXa activity, fibrinogen, FVIII:C and VWF activity (VWF:RCo) and antigen). Additionally, agonist-induced (collagen, ristocetin and ADP) platelet aggregation in citrate or hirudin-anticoagulated whole blood (Multiplate®) (n=10 healthy subjects), and platelet function analysis (PFA100®) in citrated platelet rich plasma (PRP) were assessed. In a well-established baboon thrombosis model a collagen-coated PTFE graft (length 2 cm, lumen 4 mm) was placed in an arterio-venous shunt. Prior to blood contact the thrombogenic surface was treated for 10 min with UFH or APL001 (both at 4 mg/mL). Thrombus formation was initiated by exposing the surface to blood flow (100 mL/min, shear rate 265−1), and the deposition of 111-In-labeled platelets and of fibrin was quantified continuously over 1h. Fibrin thrombus accumulation was assessed from the incorporation of circulating 125-I-fibrinogen.
In the heparin-relevant coagulation tests APL001 was comparable or 20–30% more potent than UFH while FVIII, fibrinogen and VWF variables remained unaltered. In contrast to UFH, APL001 (300 μg/mL) consistently inhibited collagen- and ristocetin-induced platelet aggregation, whereas UFH had only a modest effect in comparison with PBS control (Table). ADP-induced aggregation was unaffected. Comparable results were observed in the PRP aggregation assay. PFA100 testing also demonstrated inhibitory effects. In the in vivo thrombosis model (n=4) APL001 reduced platelet deposition on collagen (vs. the results with UFH) by 34% (p=0.01), while platelet accumulation in distal propagated thrombus was reduced by 61% (p=0.16). APL001-treated surfaces accumulated 45% less fibrin than the UFH-treated surfaces (p=0.008).
In conclusion, when compared with UFH APL001 inhibited both collagen- and ristocetin-induced platelet aggregation in human blood, while anticoagulant properties were comparable. In the absence of systemic antithrombotic drugs, exposure of APL001 to a highly thrombogenic collagen surface arrested thrombus formation in an in vivo baboon model. This finding suggests that locally administered APL001 alone, due to its dual antiplatelet and anticoagulant effects, may limit the growth and size of thrombus and thereby prevent subsequent thrombo-occlusion.TableAnticoagulantInhibition-% of platelet aggregation ± SDConc. 300 μg/mLnColl (3.2 μg/mL)Ristocetin (0.77 mg/mL)ADP (6.4 μM)CitrateAPL0011033 ± 1543 ± 166 ± 24UFH1011 ± 1323 ± 153 ± 7p value0.0030.0100.700HirudinAPL0011032 ± 1043 ± 178 ± 10UFH108 ± 1116 ± 166 ± 9p value0.0000.0020.600
Disclosures:
Lassila: Aplagon: Chief Scientific Advisor.
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