Fibrinogen-Coated Nanospheres Prevent Thrombocytopenia-Related Bleeding

Introduction: Thrombocytopenia (TCP) may cause severe and life-threatening bleeding. While TCP-related bleeding may be prevented by platelet transfusions, transfusions are associated with significant costs and complications. There is an urgent need for a synthetic alternative.

Methods and Results: We evaluated the ability of fibrinogen-coated nanospheres (FCN) to prevent TCP-related bleeding. FCN are made of human albumin polymerized into a 400 nm sphere and coated with fibrinogen. We hypothesize that FCN bind to platelets through fibrinogen-GPIIb/IIIa interactions, contributing to hemostasis in the setting of TCP.

We used two murine models to test the hemostatic effects of FCN: in the first, BALB/c mice received 7.25 Gy total body irradiation (TBI) on day 0. This dose was selected after titration to induce fatal hemorrhage in 2/3 of animals. In the second model, to more selectively look at the effects of TCP, we used a lower dose of radiation (7.0 Gy TBI), but this was combined with an anti-platelet antibody (anti-CD41 5 mcg ip) on Days 0, 5, 10 to induce severe TCP. FCN 8 mg/kg iv or saline (control) were injected Days 1, 5, and 10 in both models to correspond with the period of TCP nadir.

FCN significantly improved survival compared to saline control in both models (Fig 1A, 1B; both p<0.001). All deaths were due to gastrointestinal or intracranial bleeding, suggesting FCN improved survival by improving hemostasis. In particular, addition of anti-platelet antibody to 7.0 Gy TBI significantly increased mortality (Fig 1B, blue) compared to just 7.0 Gy TBI (Fig 1B, green), suggesting that deaths were primarily due to severe TCP. As FCN did not improve platelet numbers compared to control (Fig 1C, 1D), we inferred that FCN improved hemostasis by enhancing function. Additionally, in a saphenous vein bleeding model of antibody-induced TCP, FCN shortened bleeding times in a TCP-dependent manner (Fig 1F). There were no clinical signs of thrombosis or laboratory findings of disseminated intravascular coagulation after FCN. Also of support of safety, fluorescence microscopy suggests that FCN bind to platelets only upon platelet activation with collagen (Fig 2), limiting activity to areas of endothelial damage.

Interestingly, no differences in platelet aggregation or clot strength were detectable on light aggregometry (PAP-8E), impedance aggregometry (Multiplate), or thromboelastography (TEG, ROTEM). Nor were differences seen in fibrin formation (Fig 3A). However, FCN significantly inhibited clot lysis in a dose-dependent manner compared to fibrinogen or control spheres (albumin, no fibrinogen) (Fig 3B).

Conclusion: FCN may prevent TCP-related bleeding by interacting with platelets and inhibiting clot lysis. FCN may reduce the need for platelet transfusions.