Enhancing hemostasis in glanzmann thrombasthenia via protein S inhibition Free

Introduction: Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder caused by a deficiency of the platelet integrin αIIbβ3, essential for platelet aggregation and clot formation. Patients commonly experience mucocutaneous bleeding, while severe cases present with gastrointestinal (GI) hemorrhage, chronic anemia, and life-threatening bleeds. Acute bleeds are primarily managed with platelet transfusions, and recombinant factor VIIa is used for refractory cases or perioperative support. HMB-001, a novel bispecific antibody, is under investigation as the first prophylactic therapy for GT.

Protein S (PS) is a natural anticoagulant that functions as a cofactor for activated protein C, and tissue factor pathway inhibitor, and independently regulates the prothrombinase and tenase complexes, thereby reducing thrombin generation. We previously showed that hepatic PS silencing via small interfering RNA (PS-siRNA) enhances thrombin generation and rebalances hemostasis in hemophilia models. This strategy may also offer therapeutic benefit in GT.

Aim: To evaluate the efficacy of PS-siRNA in enhancing thrombin generation and providing prophylactic protection against bleeding in a murine model of Glanzmann thrombasthenia.

Methods:αIIbβ3⁻/⁻ mice, a genetic GT model, received a single subcutaneous injection of PS-siRNA (10 mg/kg) or phosphate-buffered saline (PBS) as control. After seven days, blood was collected for platelet-rich plasma (PRP) isolation and thrombin generation assay using a calibrated automated thrombogram. In parallel, spleen weights were measured as an index of splenomegaly, serving as an indirect marker of bleeding severity. In GT, splenomegaly reflects extramedullary erythropoiesis driven by chronic GI bleeding and hemostatic dysfunction.

In parallel, PRP from a GT patient was spiked with an anti–PS antibody to reduce free PS levels to 0% or 50%, or vehicle. Thrombin generation was measured in the presence of 1 pM tissue factor and 2 nM thrombomodulin. Clot retraction was assessed in healthy donor PRP treated with anti-PS antibody, anti-αIIbβ3 antibody (abciximab), both antibodies, or vehicle. Clots were monitored for 90 min at 37°C, and extruded serum volume was measured.

Results: A single PS-siRNA injection in αIIbβ3⁻/⁻ mice prevented overt bleeding at necropsy, unlike PBS-treated controls, which showed visible mucocutaneous and GI bleeding. This correlated with a significant increase in endogenous thrombin potential (ETP): 774 ± 125 nM in PS-siRNA–treated mice vs. 438 ± 81 nM in PBS controls (n = 2–3), indicating that ^~50% PS silencing enhances thrombin generation in GT. Spleen weights were lower in PS-siRNA–treated αIIbβ3⁻/⁻ mice (148 ± 28 mg) compared to PBS-treated mice (168 ± 43 mg), suggesting partial reversal of splenomegaly, likely due to reduced extramedullary erythropoiesis from decreased bleeding. In GT patient PRP, anti-PS antibody–mediated reduction of free PS to 0% and 50% increased ETP by 46% and 44%, respectively, vs. untreated PRP (1503 ± 44 nM and 1489 ± 153 nM vs. 1032 ± 38 nM). In clot retraction assays using healthy donor PRP, combined inhibition with abciximab and anti-PS antibody enhanced clot retraction vs. abciximab alone, as shown by greater serum extrusion (abciximab: 277 ± 31 µL; anti-PS + abciximab: 300 ± 30 µL; untreated: 297 ± 91 µL), suggesting PS inhibition can partially restore clot contraction under αIIbβ3 blockade.

Conclusion and ongoing investigations: These findings demonstrate that PS inhibition enhances hemostasis and reduces splenomegaly in αIIbβ3⁻/⁻ mice. In GT patient PRP, complete or partial PS inhibition increased thrombin generation by ^~45%. Dual blockade of αIIbβ3 and PS also improved clot retraction, suggesting PS targeting may partially compensate for defective platelet function.

Ongoing studies are investigating the impact of PS inhibition on clot formation, stability, and fibrinolysis resistance. Saphenous vein bleeding studies in PS-siRNA–treated αIIbβ3⁻/⁻ mice are underway. To extend these findings, PS-siRNA will be administered to pregnant αIIbβ3⁻/⁻ and αIIbβ3⁺/⁻ females to assess its effects on intrauterine bleeding and pregnancy outcomes. Postnatal survival of treated αIIbβ3⁻/⁻ pups will also be monitored, given ^~8% mortality from spontaneous bleeding reported between 3 and 7 weeks of age (Kairbaan D. et al., JCI, 1999).