Sexual dimorphism in platelet-like particle mediated clotting response

Introduction: : Uncontrolled hemorrhage remains a primary cause of preventable death in trauma patients under the age of 40, with trauma-induced coagulopathy (TIC) affecting up to one-third of cases. TIC presents with multifaceted disruptions in coagulation, including platelet depletion and dysfunction, disrupted fibrin formation, and hyperfibrinolysis. Notably, emerging research in TIC indicates a sexual dimorphism in coagulation, with females often exhibiting a hypercoagulable profile with enhanced clot formation, reduced bleeding, and improved survival compared to males. While the underlying mechanisms are not fully understood, estrogen is believed to play a key role. Elevated circulating estrogen levels have been observed in hormonally active females post-injury, and animal models lacking endogenous estrogen (e.g., following ovariectomy) demonstrate impaired coagulation responses.
Advancements in synthetic intravenous hemostats for rapid hemorrhage control, such as platelet-like particles (PLPs), offer promising strategies for restoring clot function in TIC. PLPs mimic platelet-fibrin interactions using ultra-low crosslinked poly(N-isopropylacrylamide) microgel cores functionalized with fibrin-binding nanobodies. Upon binding, PLPs induce clot contraction via Brown-driven collapse, improving the mechanobiological stability of fibrin clots in TIC settings and demonstrating hemostatic efficacy in preclinical liver laceration animal models. Despite their therapeutic potential, the impact of sex as a biological factor on their performance is unknown. This study evaluates sex-specific and hormone-driven differences in PLP-mediated clotting dynamics, with the goal of advancing tailored strategies for trauma resuscitation.

Materials and

Methods: : PLPs were synthesized via precipitation polymerization of 90% N-isopropylacrylamide (NIPAM) and 10% acrylic acid (AAc), forming ultra-low crosslinked microgels. Fibrin fragment E polyclonal antibodies were conjugated to microgels to enable fibrin binding. Particle size was characterized by atomic force microscopy (AFM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS).
Human male or female platelet-poor plasma (PPP) was acquired from pooled donors (ZenBio). Porcine platelet-rich plasma (PRP) and PPP samples were also collected longitudinally from a cohort of six female pigs across multiple estrous cycles. PRP was isolated via centrifugation at 150 xg for 15 minutes and normalized to 2×10⁷ platelets/mL. PPP was obtained by centrifugation at 1000 xg for 15 minutes and stored at -80 °C.
Clots were polymerized with 0.1 U/mL thrombin, 2.5mM CaCl2 , and 10µg/mL Alexa-Fluor 488 labeled fibrinogen overnight. A Zeiss Laser Scanning Microscope (LSM 880) was used to acquire 5µm z-stacks images of fibrin architecture. Image J was used to calculate fibrin density by binarizing images and calculating fibrin-to-background pixel ratios. Endogenous fibrinolysis assays were performed to measure clot polymerization and degradation kinetics. 100µL clots were polymerized in triplicate in a 96-well plate with 0.1U/mL thrombin, 5mM CaCl2, and 10 µg/mL tPA for 3 hours at 405 nm. Statistical analyses were conducted in GraphPad Prism using two-way ANOVA with Tukey’s post hoc test. All data sets are reported as mean ± SD.

Results, Conclusions, and Discussions:: Human female PPP samples exhibited hypercoagulability, with greater fibrin fiber density, increased maximum turbidity, and reduced time to half-max in clot formation compared to male PPP samples. Notably, female samples showed reduced sensitivity to PLP treatment, with significant enhancements observed only at the highest PLP concentrations tested. In contrast, male samples demonstrated a greater response to PLPs, with increased fibrin density and clot formation parameters approaching levels observed in untreated female samples. Polymerization rates were comparable between sexes, though rates of fibrinolysis were slightly heightened in male samples. Endogenous hormone fluctuations across the estrous cycle revealed that porcine samples with elevated levels of estrogen exhibited enhanced clot structure, elevated max absorbance, faster clot formation, and decreased degradation rates in both PRP and PPP conditions. PLPs were most effective in enhancing coagulation parameters in samples with decreased estrogen levels but had limited impact when estrogen levels - and endogenous coagulability - were high, suggesting hormone-driven modulation of PLP efficacy. Interestingly, PLP effects were more pronounced in PPP than in PRP, with PLP-treated PPP displaying enhanced coagulation parameters compared to untreated PRP, indicating potential interaction or competition with healthy native platelets. Together, these results demonstrate that PLP-mediated clot reinforcement is modulated by sex and hormone levels, with heightened efficacy in hypocoagulable, platelet-diminished settings and reduced effects in hypercoagulable settings. This highlights the importance of considering sex-specific strategies in the design of synthetic hemostatic therapies, particularly in trauma settings where platelet count of function is compromised. Ongoing studies will further investigate the interplay between PLPs, endogenous platelets, and fibrinolytic activity on clot mechanics.

Acknowledgements and/or References (Optional)::