Fibrinogen-Coated Nanospheres Prevent Thrombocytopenia-Related Bleeding

Abstract

s / Biol Blood Marrow Transplant 21 (2015) S108eS113 S112 Benny J. Chen . 1 Division of Hematologic Malignancies and Cellular Therapy, Duke University Medical Center, Durham, NC; 2 Fibroplate Inc., Las Vegas, NV; 3 Division of Hematology, Duke University Medical Center, Durham, NC; 4 Department of Pathology, Duke University Medical Center, Durham, NC 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 coatedwith fibrinogen.We hypothesize that FCN bind to platelets through fibrinogen-GPIIb/IIIa interactions, contributing to hemostasis in the setting of TCP. We used twomurinemodels 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. Figure 2. FCN were labeled with Alexa Fluor 488 (green); platelets with anti-CD31 collagen (col) (2D, 2E, 2F), a platelet agonist. In the absence of collagen, FCN forms dis and green particles on overlay (2C). In the presence of collagen, FCN clumps on green (2F). Mixing control spheres (CS, albumin spheres without fibrinogen, also labeled wi suggesting that the fibrinogen coating is necessary for nanosphere-platelet interactio 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. PE antibody (red), and both mixed in the absence (2A, 2B, 2C) or presence of crete particles on the green channel (2A), platelets on red (2B), and discrete red (2D) correspond to platelet clumps on red (2E) and yellow clumps on overlay th Alexa Fluor 488) with platelets failed to form yellow clumps on overlay (21),