Development of Coagulation Factor XII Antibodies for Inhibiting Vascular Device-Related Thrombosis

Abstract

Introduction: Vascular devices such as stents, hemodialyzers, and membrane oxygenators can activate blood coagulation and often require the use of systemic anticoagulants to selectively prevent intravascular thrombotic/embolic events or extracorporeal device failure. Coagulation factor (F)XII of the contact activation system has been shown to play an important role in initiating vascular device surface-initiated thrombus formation. As FXII is dispensable for hemostasis, targeting the contact activation system holds promise as a significantly safer strategy than traditional antithrombotics for preventing vascular device-associated thrombosis.

Objective: Generate and characterize anti-FXII monoclonal antibodies that inhibit FXII activation or activity.

Methods: Monoclonal antibodies against FXII were generated in FXII-deficient mice and evaluated for their binding and anticoagulant properties in purified and plasma systems, in whole blood flow-based assays, and in an in vivo non-human primate model of vascular device-initiated thrombus formation.

Results: A FXII antibody screen identified over 400 candidates, which were evaluated in binding studies and clotting assays. One non-inhibitor and six inhibitor antibodies were selected for characterization in functional assays. The most potent inhibitory antibody, 1B2, was found to prolong clotting times, inhibit fibrin generation on collagen under shear, and inhibit platelet deposition and fibrin formation in an extracorporeal membrane oxygenator deployed in a non-human primate.

Conclusion: Selective contact activation inhibitors hold potential as useful tools for research applications as well as safe and effective inhibitors of vascular device-related thrombosis.

Keywords: Contact activation; Hemostasis; Platelet.

Figure 1

Human FXII. (a) Primary and secondary structure of FXII. (b) Schematic diagrams comparing the domain structures of FXII, α- FXIIa, and β- FXIIa. (c) Chimera and deletion constructs. Dark gray shading indicates the HGFA domains introduced into FXII to create FXII /HGFA chimeras.

Figure 2

(a) Western blots of FXII unreduced and reduced human recombinant FXII (R), human plasma FXII (P), and mouse recombinant FXII (M)-fractionated by sodium dodecyl sulfate-PAGE. (b) Western blots of human α- and β- FXII. (c) Western blots of FXII and FXII/HGFA chimeras.

Figure 3

Western Blots of FXII from mammalian plasmas. Western blots of non-reducing 7.5% polyacrylamide gels of FXII for a variety of animal species (a) and commonly used preclinical research animal models (b).

Figure 4

Serially diluted normal pooled human plasma with FXII-depleted plasma alone was used as reference data (a). Platelet-poor plasma from (b) human, (c) baboon, (d) cynomolgus, (e) rhesus, (f) mouse, (g) rat, or (h) rabbit was serially diluted into FXII-deficient human plasma. These dilutions were incubated with 20 µg/mL of FXII antibody candidates. Clotting times were measured following addition of an aPTT reagent. Changes in aPTT were plotted as a function of FXII levels on a logarithmic scale.

Figure 5

Thrombus formation and growth was measured using non-activated thromboelastometry (NATEM). Human plasma from healthy volunteers was inhibited with 40 µg/mL antibody. (a) FXII inhibition confirmed by aPTT; (b) thromboelastometry curve explaining the parameters reported below; (c) clotting time; (d) clot formation time, (e) time to 20 mm clot firmness; (f) time to maximum clot firmness; (g) maximum clot firmness; (h) time to maximum velocity. Data represents mean ± SEM and were analyzed using one-way ANOVA (GraphPad Software, San Diego, CA) with Dunnett’s posthoc analysis versus control. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

Anti-FXII antibodies inhibit FXIIa activity. Increasing concentrations of anti-FXII antibodies were added to a solution of purified FXII. FXII was activated by either long PolyP (a), short PolyP (b), or dextran sulfate (c). Amidolytic activity was quantified as hydrolysis of the chromogenic substrate, Spectrozyme FXIIa. Activated FXII (FXIIa; 20 nM) was incubated with increasing concentrations of anti-FXII antibodies for 5 min before addition of Spectrozyme FXIIa. The velocity of hydrolysis was measured for 20 minutes and V_max was calculated for each reaction (d). Each data point was measured in duplicate. Data represent mean ± SEM from n = 2.

Figure 7

Effect of anti-FXII antibodies on fibrin and thrombus formation under shear. Re-calcified, citrated whole blood pretreated with anti-FXII antibody (100 µg/mL, 10 min) was perfused through collagen-coated chambers at 300 s⁻¹. Representative images from n = 3.

Figure 8

Time course of coagulation parameters after 1B2 or 1D7 administration in a non-human primate. Baboons were administered i.v. doses of 1 mg/kg 1B2 or 1D7 every 40 min over a 4-h period. Blood samples were collected for up to three weeks post administration. (a) aPTT, (b) ACT, (c) PT, and (d) TAT levels were measured. Complete blood count (CBC) was measured: (e) platelet counts, (f) hematocrit, (g) white blood cell count, and (h) red blood cell count. The upper and lower limits of reference values are indicated by dashed lines. aPTT, activated partial thromboplastin time; ACT activated clotting time, PT prothrombin time, TAT thrombin-antithrombin complex.

Figure 9

Effect of 1B2 on platelet deposition and fibrin formation in ECMO. (a) Real-time platelet deposition was monitored in an ECMO device inserted in the extended loop of a chronic AV shunt. Study arms included vehicle control (n = 2) or 1B2 (5 mg/kg initial day + 2 mg/kg on the following days 30 min before perfusion, n = 4). (b) Average platelet deposition rate was calculated from 30 to 60 min. (c) Measurement of terminal fibrin content in the oxygenator and (d) correlation of fibrin and platelet content. (e) Two Surgicutt® bleeding time measurements were performed during each experiment and each data point represents the average of the two measurements. ECMO extracorporeal membrane oxygenator, AV arteriovenous, FXII factor XII.