Introduction: Neonates born with congenital cardiac defects often undergo complex corrective surgery requiring cardiopulmonary bypass (CPB) within a few days following birth. CPB is known to contribute significantly to surgery associated bleeding through induction of massive hemodilution, thrombocytopenia and platelet dysfunction; these bleeding risks are exacerbated in neonates due to preexisting platelet dysfunction and an immature coagulation system. CPB greatly hinders platelet function; therefore, materials that recapitulate platelet hemostatic functions could be utilized as an alternative to transfusion of adult blood products for augmentation of clotting in patients post-CPB. We have recently developed platelet-like-particles (PLPs) created from highly deformable microgels conjugated to molecularly evolved wound-targeting motifs which enable specific and high affinity binding to the provisional matrix protein fibrin[1]. These PLPs are capable of recapitulating a number of functions of natural platelets, including augmentation of clotting of adult plasma in vitro, decreased bleeding times in vivo in rodent models of traumatic injury, specific homing to injury sites and clot contraction. Here, we characterize the ability of PLPs to augment clotting in CPB-patient plasma samples in vitro and in vivo in a rat model of impaired platelet activity.
Materials and Methods: Ultra-low crosslinked (ULC) poly(N-isopropylacrylamide) (pNIPAM) microgels containing 95% pNIPAM and 5% acrylic acid (AAc) were produced through precipitation polymerization. PLPs were produced by conjugating fibrin-specific single domain variable fragment antibodies to ULC microgels through EDC/NHS coupling. Clotting was assessed in vitro in patient platelet poor plasma (PPP) samples collected from neonatal cardiac surgery patients at baseline, post-CPB (+/- PLPs) and post transfusion through an endothelialized microfluidics device that mimics the microenvironment of microcirculation[2]. Fibrin microarchitecture was analyzed using confocal microscopy. Clotting was assessed in vivo in a rodent platelet deficiency model, which mimics the gross dysfunction of platelets in pediatric cardiac patients. To induce platelet hyporeactivity, male Sprague Dawley rats received a daily oral gavage of aspirin (5 mg/kg) for seven days. On the seventh day, bleeding times following induction of a femoral vein injury were assessed in animals receiving intravenous administration of PLPs or saline.
Results and Discussion: In vitro clotting experiments in an endothelialized microfluidic device demonstrate minimal clotting in post-CPB PPP alone, while the addition of PLPs induces significant fibrin formation (Figure 1A). To determine the effect of PLPs on fibrin network microstructure, confocal microscopy was performed; incorporation of PLPs into post-CPB PPP results in a denser fibrin network compared to post-CBP PPP alone or samples collected post-transfusion (Figure 1B). In an in vivo model of platelet hyporeactivity, PLPs significantly decreased bleeding following injury compared to control animals (Figure 1C).
Conclusions: PLPs enhance clot formation in neonatal patient samples in vitro and decrease bleeding times in vivo in a rodent model of platelet dysfunction. These results highlight the utility of PLPs for treatment of postsurgical bleeding in coagulopathic neonatal patients.
1R21EB019068, NIH/NIBIB
References:
[1] Brown, A.C. et al., Ultrasoft microgels displaying emergent platelet-like behaviours. Nature Materials 13, 1108–1114 (2014) doi:10.1038/nmat4066
[2] Myers, D. R. et al. Endothelialized microfluidics for studying microvascular interactions in hematologic diseases. J. Vis. Exp. 64, e3958 (2012).