Ultrasoft platelet-like particles stop bleeding in rodent and porcine models of trauma.

Uncontrolled bleeding after trauma represents a substantial clinical problem. The current standard of care to treat bleeding after trauma is transfusion of blood products including platelets; however, donated platelets have a short shelf life, are in limited supply, and carry immunogenicity and contamination risks. Consequently, there is a critical need to develop hemostatic platelet alternatives. To this end, we developed synthetic platelet-like particles (PLPs), formulated by functionalizing highly deformable microgel particles composed of ultralow cross-linked poly (N-isopropylacrylamide) with fibrin-binding ligands. The fibrin-binding ligand was designed to target to wound sites, and the cross-linking of fibrin polymers was designed to enhance clot formation. The ultralow cross-linking of the microgels allows the particles to undergo large shape changes that mimic platelet shape change after activation; when coupled to fibrin-binding ligands, this shape change facilitates clot retraction, which in turn can enhance clot stability and contribute to healing. Given these features, we hypothesized that synthetic PLPs could enhance clotting in trauma models and promote healing after clotting. We first assessed PLP activity in vitro and found that PLPs selectively bound fibrin and enhanced clot formation. In murine and porcine models of traumatic injury, PLPs reduced bleeding and facilitated healing of injured tissue in both prophylactic and immediate treatment settings. We determined through biodistribution experiments that PLPs were renally cleared, possibly enabled by ultrasoft particle properties. The performance of synthetic PLPs in the preclinical studies shown here supports future translational investigation of these hemostatic therapeutics in a trauma setting.

Wound-triggered shape change microgels for the development of enhanced biomimetic function platelet-like particles.

Platelets play a pivotal role in hemostasis and wound healing and conditional shape change is an important component of platelet functionality. In normal circumstances, platelets travel through the circulatory system in an inactive rounded state, which enables platelets to easily move to vessel walls for attachment. When an injury occurs, platelets are prompted by molecules, such as thrombin, to shift into a stellate shape and increase exposure of fibrin-binding receptors. When active, platelets promote hemostasis and clot retraction, which enhances clot stability and promotes healing. However, in conditions where platelets are depleted or hyporeactive, these functions are diminished and lead to inhibited hemostasis and healing. To treat platelet depletion, our group developed platelet-like particles (PLPs) which consist of highly deformable microgels coupled to fibrin binding motif. However, first generation PLPs do not exhibit wound-triggered shape change like native platelets. Thus, the objective of these studies was to develop a PLP formulation that changes shape when prompted by thrombin. To create thrombin-sensitive PLPs (TS-PLPs), we incorporated a thrombin-cleavable peptide into the microgel body and then evaluated PLP properties before and after exposure to thrombin including morphology, size, and in vitro clot retraction. Once thrombin-prompted shape change ability was confirmed, the TS-PLPs were tested in vivo for hemostatic ability and subsequent wound healing outcomes in a murine liver trauma model. We found that TS-PLPs exhibit a wound-triggered shape change, induce significant clot retraction following exposure to thrombin and promote hemostasis and healing in vivo after trauma.

Doing more with less: low-titer group O whole blood resulted in less total transfusions and an independent association with survival in adults with severe traumatic hemorrhage.

BACKGROUND: Low-titer group O whole blood (LTOWB) or component therapy (CT) may be used to resuscitate hemorrhaging trauma patients. LTOWB may have clinical and logistical benefits and may improve survival. OBJECTIVES: We hypothesized LTOWB would improve 24-hour survival in hemorrhaging patients and would be safe and equally efficacious in non-group O compared with group O patients. METHODS: Adult trauma patients with massive transfusion protocol activations were enrolled in this observational study. The primary outcome was 24-hour mortality. Secondary outcomes included 72-hour total blood product use. A Cox regression determined the independent associations with 24-hour mortality. RESULTS: In total, 348 patients were included (CT, n = 180; LTOWB, n = 168). Demographics were similar between cohorts. Unadjusted 24-hour mortality was reduced in LTOWB vs CT: 8% vs 19% (P = .003), but 6-hour and 28-day mortality were similar. In an adjusted analysis with multivariable Cox regression, LTOWB was independently associated with reduced 24-hour mortality (hazard ratio, 0.21; 95% CI, 0.07-0.67; P = .004). LTOWB patients received significantly less 72-hour total blood products (80.9 [41.6-139.3] mL/kg vs 48.9 [25.9-106.9] mL/kg; P < .001). In stratified 24-hour survival analyses, LTOWB was associated with improved survival for patients in shock or with coagulopathy. LTOWB use in non-group O patients was not associated with increased mortality, organ injury, or adverse events. CONCLUSION: In this hypothesis-generating study, LTOWB use was independently associated with improved 24-hour survival, predominantly in patients with shock or coagulopathy. LTOWB also resulted in a 40% reduction in blood product use which equates to a median 2.4 L reduction in transfused products.

Cold-stored platelet hemostatic capacity is maintained for three weeks of storage and associated with taurine metabolism.

BACKGROUND: Platelet (PLT) product transfusion is a life-saving therapy for actively bleeding patients. There is an urgent need to maintain PLT function and extend shelf life to improve outcomes in these patients. Cold-stored PLT (CS-PLT) maintain hemostatic potential better than room temperature-stored PLT (RT-PLT). However, whether function in long-term CS-PLT is maintained under physiological flow regimes and/or determined by cold-induced metabolic changes is unknown. OBJECTIVES: This study aimed to (i) compare the function of RT-PLT and CS-PLT under physiological flow conditions, (ii) determine whether CS-PLT maintain function after 3 weeks of storage, and (iii) identify metabolic pathways associated with the CS-PLT lesion. METHODS: We performed phenotypic and functional assessments of RT- and CS-PLT (22 °C and 4 °C storage, respectively; N = 10 unique donors) at storage days 0, 5, and/or 21 via metabolomics, flow cytometry, aggregation, thrombin generation, viscoelastic testing, and a microfluidic assay to measure primary hemostatic function. RESULTS: Day 21 4 °C PLT formed an occlusive thrombus under arterial shear at a similar rate to day 5 22 °C PLT. Day 21 4 °C PLTs had enhanced thrombin generation capacity compared with day 0 PLT and maintained functionality comparable to day RT-PLT across all assays performed. Key metrics from microfluidic assessment, flow cytometry, thrombin generation, and aggregation were associated with 4 °C storage, and metabolites involved in taurine and purine metabolism significantly correlated with these metrics. Taurine supplementation of PLT during storage improved hemostatic function under flow. CONCLUSION: CS-PLT stored for 3 weeks maintain hemostatic activity, and storage-induced phenotype and function are associated with taurine and purine metabolism.

Hemostatic In Vitro Properties of Novel Plasma Supernatants Produced from Late-storage Low-titer Type O Whole Blood.

BACKGROUND: The use of low-titer group O whole blood is increasing. To reduce wastage, unused units can be converted to packed red blood cells. Supernatant is currently discarded post-conversion; however, it could be a valuable transfusable product. The aim of this study was to evaluate supernatant prepared from late-storage low-titer group O whole blood being converted to red blood cells, hypothesizing it will have higher hemostatic activity compared to fresh never-frozen liquid plasma. METHODS: Low-titer group O whole blood supernatant (n = 12) prepared on storage day 15 was tested on days 15, 21, and 26 and liquid plasma (n = 12) on 3, 15, 21, and 26. Same-day assays included cell counts, rotational thromboelastometry, and thrombin generation. Centrifuged plasma from units was banked for microparticle characterization, conventional coagulation, clot structure, hemoglobin, and additional thrombin generation assays. RESULTS: Low-titer group O whole blood supernatant contained more residual platelets and microparticles compared to liquid plasma. At day 15, low-titer group O whole blood supernatant elicited a faster intrinsic clotting time compared to liquid plasma (257 ± 41 vs. 299 ± 36 s, P = 0.044), and increased clot firmness (49 ± 9 vs. 28 ± 5 mm, P < 0.0001). Low-titer group O whole blood supernatant showed more significant thrombin generation compared to liquid plasma (day 15 endogenous thrombin potential 1,071 ± 315 vs. 285 ± 221 nM·min, P < 0.0001). Flow cytometry demonstrated low-titer group O whole blood supernatant contained significantly more phosphatidylserine and CD41+ microparticles. However, thrombin generation in isolated plasma suggested residual platelets in low-titer group O whole blood supernatant were a greater contributor than microparticles. Additionally, low-titer group O whole blood supernatant and liquid plasma showed no difference in clot structure, despite higher CD61+ microparticle presence. CONCLUSIONS: Plasma supernatant produced from late-storage low-titer group O whole blood shows comparable, if not enhanced, in vitro hemostatic efficacy to liquid plasma.

Dose-Dependent Von Willebrand Factor Inhibition by Aptamer BB-031 Correlates with Thrombolysis in a Microfluidic Model of Arterial Occlusion.

Von Willebrand Factor (VWF) plays a critical role in thrombus formation, stabilization, and propagation. Previous studies have demonstrated that targeted inhibition of VWF induces thrombolysis when administered in vivo in animal models of ischemic stroke. The study objective was to quantify dose-dependent inhibition of VWF-platelet function and its relationship with thrombolysis using BB-031, an aptamer that binds VWF and inhibits its function. VWF:Ac, VWF:RCo, T-TAS, and ristocetin-induced impedance aggregometry were used to assess BB-031-mediated inhibition of VWF. Reductions in original thrombus surface area and new deposition during administration of treatment were measured in a microfluidic model of arterial thrombolysis. Rotational thromboelastometry was used to assess changes in hemostasis. BB-031 induced maximal inhibition at the highest dose (3384 nM) in VWF:Ac, and demonstrated dose-dependent responses in all other assays. BB-031, but not vehicle, induced recanalization in the microfluidic model. Maximal lytic efficacy in the microfluidic model was seen at 1692 nM and not 3384 nM BB-031 when assessed by surface area. Minor changes in ROTEM parameters were seen at 3384 nM BB-031. Targeted VWF inhibition by BB-031 results in clinically measurable impairment of VWF function, and specifically VWF-GPIb function as measured by VWF:Ac. BB-031 also induced thrombolysis as measured in a microfluidic model of occlusion and reperfusion. Moderate correlation between inhibition and lysis was observed. Additional studies are required to further examine off-target effects of BB-031 at high doses, however, these are expected to be above the range of clinical targeted dosing.

Fibrin-specific poly(N-isopropylacrylamide) nanogels for targeted delivery of tissue-type plasminogen activator to treat thrombotic complications are well tolerated in vivo.

Targeted drug delivery for maintaining blood fluidity can reduce the risks associated with systemic anticoagulants that can lead to off-target bleeding. Recently, there has been much interest in targeted delivery of tissue-type plasminogen activator (tPA) for treating thrombotic complications. The work presented here characterizes a fibrin-specific nanogel (FSN) design for targeted delivery of tPA to treat thrombotic complications. Fibrin binding and clot degradation were characterized in vitro, and animal models of thrombosis were used to examine nanogel effects on coagulation parameters. In vitro assays showed tPA-FSNs attach to fibrin in a dose-dependent manner independent of tPA loading. In animal models of thrombosis, including an electrolytic injury to monitor clot properties in real time, and a lipopolysaccharide-induced disseminated intravascular coagulation (DIC) animal model, tPA-FSNs modulated fibrin/fibrinogen and platelet incorporation into clots and at optimized dosing could recover consumptive coagulopathy in DIC. Distribution of unloaded and tPA-loaded FSNs showed potential clearance of tPA-FSNs after 24 h, although unloaded FSNs may be retained at sites of fibrin deposits. Maximum tolerated dose studies showed tPA-FSNs have minimal toxicity up to 20 times the optimized therapeutic dose. Overall, these studies demonstrate the therapeutic efficacy of targeted fibrinolysis for systemic microthrombi and begin to evaluate key translational parameters for tPA-FSN therapeutics, including optimal tPA-FSN dosage in a DIC rodent model and safety of intravenous tPA-FSN therapeutics.

Synthetic platelet microgels containing fibrin knob B mimetic motifs enhance clotting responses.

Native platelets are crucial players in wound healing. Key to their role is the ability of their surface receptor GPIIb/IIIa to bind fibrin at injury sites, thereby promoting clotting. When platelet activity is impaired as a result of traumatic injury or certain diseases, uncontrolled bleeding can result. To aid clotting and tissue repair in cases of poor platelet activity, our lab has previously developed synthetic platelet-like particles capable of promoting clotting and improving wound healing responses. These are constructed by functionalizing highly deformable hydrogel microparticles (microgels) with fibrin-binding ligands including a fibrin-specific whole antibody or a single-domain variable fragment. To improve the translational potential of these clotting materials, we explored the use of fibrin-binding peptides as cost-effective, robust, high-specificity alternatives to antibodies. Herein, we present the development and characterization of soft microgels decorated with the peptide AHRPYAAK that mimics fibrin knob 'B' and targets fibrin hole 'b'. These "Fibrin-Affine Microgels with Clotting Yield" (FAMCY) were found to significantly increase clot density in vitro and decrease bleeding in a rodent trauma model in vivo. These results indicate that FAMCYs are capable of recapitulating the platelet-mimetic properties of previous designs while utilizing a less costly, more translational design.

Platelet-like particles reduce coagulopathy-related and neuroinflammatory pathologies post-experimental traumatic brain injury.

Coagulopathy may occur following traumatic brain injury (TBI), thereby negatively affecting patient outcomes. Here, we investigate the use of platelet-like particles (PLPs), poly(N-isopropylacrylamide-co-acrylic-acid) microgels conjugated with a fibrin-specific antibody, to improve hemostasis post-TBI. The objective of this study was to diminish coagulopathy in a mouse TBI model (controlled cortical impact) via PLP treatment, and subsequently decrease blood-brain barrier (BBB) permeability and neuroinflammation. Following an acute intravenous injection of PLPs post-TBI, we analyzed BBB permeability, ex vivo coagulation parameters, and neuroinflammation at 24 hr and 7 days post-TBI. Both PLP-treatment and control particle-treatment had significantly decreased BBB permeability and improved clot structure 24 hr post-injury. Additionally, no significant change in tissue sparing was observed between 24 hr and 7 days for PLP-treated cohorts compared to that observed in untreated cohorts. Only PLP-treatment resulted in significant reduction of astrocyte expression at 7 days and percent difference from 24 hr to 7 days. Finally, PLP-treatment significantly reduced the percent difference from 24 hr to 7 days in microglia/macrophage density compared to the untreated control. These results suggest that PLP-treatment addressed acute hypocoagulation and decreased BBB permeability followed by decreased neuroinflammation and fold-change tissue loss by 7 days post-injury. These promising results indicate that PLPs could be a potential therapeutic modality for TBI.

Multivalent Aptamer-Functionalized Single-Strand RNA Origami as Effective, Target-Specific Anticoagulants with Corresponding Reversal Agents.

Anticoagulants are commonly utilized during surgeries and to treat thrombotic diseases like stroke and deep vein thrombosis. However, conventional anticoagulants have serious side-effects, narrow therapeutic windows, and lack safe reversal agents (antidotes). Here, an alternative RNA origami displaying RNA aptamers as target-specific anticoagulant is described. Improved design and construction techniques for self-folding, single-molecule RNA origami as a platform for displaying pre-selected RNA aptamers with precise orientational and spatial control are reported. Nuclease resistance is added using 2'-fluoro-modified pyrimidines during in vitro transcription. When four aptamers are displayed on the RNA origami platform, the measured thrombin inhibition and anticoagulation activity is higher than observed for free aptamers, ssRNA-linked RNA aptamers, and RNA origami displaying fewer aptamers. Importantly, thrombin inhibition is immediately switched off by addition of specific reversal agents. Results for single-stranded DNA (ssDNA) and single-stranded peptide nucleic acid (PNA) antidotes show restoration of 63% and 95% coagulation activity, respectively. To demonstrate potential for practical, long-term storage for clinical use, RNA origami is freeze-dried, and stored at room temperature. Freshly produced and freeze-dried RNA show identical levels of activity in coagulation assays. Compared to current commercial intravenous anticoagulants, RNA origami-based molecules show promise as safer alternatives with rapid activity switching for future therapeutic applications.

Fibrin-modulating nanogels for treatment of disseminated intravascular coagulation.

Disseminated intravascular coagulation (DIC) is a pathological coagulopathy associated with infection that increases mortality. In DIC, excessive thrombin generation causes symptoms from formation of microthrombi to multiorgan failure; bleeding risks can also be a concern because of clotting factor consumption. Different clinical events lead to DIC, including sepsis, trauma, and shock. Treatments for thrombotic episodes or bleeding presentation in DIC oppose each other, thus creating therapeutic dilemmas in management. The objective of this study was to develop fibrin-specific core-shell nanogels (FSNs) loaded with tissue-type plasminogen activator (tPA) to treat the microcirculatory complications of DIC, which would facilitate targeted clot dissolution to manage microthrombi and the potential consumptive coagulopathy that causes bleeding. FSNs enhance formation of actively polymerizing clots by crosslinking fibrin fibers, but they can also target preexisting microthrombi and, when loaded with tPA, facilitate targeted delivery to lyse the microthrombi. We hypothesized that this dual action would simultaneously address bleeding and microthrombi with DIC to improve outcomes. In vivo, tPA-FSNs decreased the presentation of multiorgan microthrombi, recovered platelet counts, and improved bleeding outcomes in a DIC rodent model. When incorporated with human DIC patient plasma, tPA-FSNs restored clot structure and clot growth under flow. Together, these data demonstrate that a fibrinolytic agent loaded into fibrin-targeting nanogels could improve DIC outcomes.

Platelet-like particles improve fibrin network properties in a hemophilic model of provisional matrix structural defects.

Following injury, a fibrin-rich provisional matrix is formed to stem blood loss and provide a scaffold for infiltrating cells, which rebuild the damaged tissue. Defects in fibrin network formation contribute to impaired healing outcomes, as evidenced in hemophilia. Platelet-fibrin interactions greatly influence fibrin network structure via clot contraction, which increases fibrin density over time. Previously developed hemostatic platelet-like particles (PLPs) are capable of mimicking platelet functions including binding to fibrin fibers, augmenting clotting, and inducing clot retraction. In this study, we aimed to apply PLPs within a plasma-based in vitro hemophilia B model of deficient fibrin network structure to determine the ability of PLPs to improve fibrin structure and wound healing responses within hemophilia-like abnormal fibrin network formation. PLP impact on structurally deficient clot networks was assessed via confocal microscopy, a micropost deflection model, atomic force microscopy and an in vitro wound healing model of early cell migration within a provisional fibrin matrix. PLPs improved clot network density, force generation, and stiffness, and promoted fibroblast migration within an in vitro model of early wound healing under hemophilic conditions, indicating that PLPs could provide a biomimetic platform for improving wound healing events in disease conditions that cause deficient fibrin network formation.

Nanosilver composite pNIPAm microgels for the development of antimicrobial platelet-like particles.

Platelets crucially facilitate wound healing but can become depleted in traumatic injury or chronic wounds. Previously, our group developed injectable platelet-like particles (PLPs) comprised of highly deformable, ultralow crosslinked pNIPAm microgels (ULCs) coupled to fibrin binding antibodies to treat post-trauma bleeding. PLP fibrin-binding facilitates homing to sites of injury, promotes clot formation, and, due to high particle deformability, induces clot retraction. Clot retraction augments healing by increasing clot stability, enhancing clot stiffness, and promoting cell migration into the wound bed. Because post-traumatic healing is often complicated by infection, the objective of these studies was to develop antimicrobial nanosilver microgel composite PLPs to augment hemostasis, fight infection, and promote healing post-trauma. A key goal was to maintain particle deformability following silver incorporation to preserve PLP-mediated clot retraction. Clot retraction, antimicrobial activity, hemostasis after trauma, and healing after injury were evaluated via confocal microscopy, colony-forming unit assays, a murine liver trauma model, and a murine full-thickness injury model in the absence or presence of infection, respectively. We found that nanosilver incorporation does not affect base PLP performance while bestowing significant antimicrobial activity and enhancing infected wound healing outcomes. Therefore, Ag-PLPs have great promise for treating hemorrhage and improving healing following trauma.

Clot Structure and Implications for Bleeding and Thrombosis.

The formation of a fibrin clot matrix plays a critical role in promoting hemostasis and wound healing. Fibrin dynamics can become disadvantageous in the formation of aberrant thrombus development. Structural characteristics of clots, such as fiber diameter, clot density, stiffness, or permeability, can determine overall clot integrity and functional characteristics that have implications on coagulation and fibrinolysis. This review examines known factors that contribute to changes in clot structure and the presence of structural clot changes in various disease states. These insights provide valuable information in forming therapeutic strategies for disease states where alterations in clot structure are observed. Additionally, the implications of structural changes in clot networks on bleeding and thrombus development in terms of disease states and clinical outcomes are also examined in this review.

Targeted Treatment of Ischemic and Fibrotic Complications of Myocardial Infarction Using a Dual-Delivery Microgel Therapeutic.

Myocardial infarction (MI), commonly known as a heart attack, affects millions of people worldwide and results in significant death and disabilities. A major cause of MI is fibrin-rich thrombus formation that occludes the coronary arteries, blocking blood flow to the heart and causing fibrin deposition. In treating MI, re-establishing blood flow is critical. However, ischemia reperfusion (I/R) injury itself can also occur and contributes to cardiac fibrosis. Fibrin-specific poly( N-isopropylacrylamide) nanogels (FSNs) comprised of a core-shell colloidal hydrogel architecture are utilized in this study to design a dual-delivery system that simultaneously addresses the need to (1) re-establish blood flow and (2) inhibit cardiac fibrosis following I/R injury. These therapeutic needs are met by controlling the release of a fibrinolytic protein, tissue plasminogen activator (tPA), and a small molecule cell contractility inhibitor (Y-27632). In vitro, tPA and Y-27632-loaded FSNs rapidly degrade fibrin and decrease cardiac cell stress fiber formation and connective tissue growth factor expression, which are both upregulated in cardiac fibrosis. In vivo, FSNs localize to fibrin in injured heart tissue and, when loaded with tPA and Y-27632, showed significant improvement in left ventricular ejection fraction 2 and 4 weeks post-I/R as well as significantly decreased infarct size, α-smooth muscle actin expression, and connective tissue growth factor expression 4 weeks post-I/R. Together, these data demonstrate the feasibility of this targeted therapeutic strategy to improve cardiac function following MI.

Targeted repair of heart injury by stem cells fused with platelet nanovesicles.

Stem cell transplantation, as used clinically, suffers from low retention and engraftment of the transplanted cells. Inspired by the ability of platelets to recruit stem cells to sites of injury on blood vessels, we hypothesized that platelets might enhance the vascular delivery of cardiac stem cells (CSCs) to sites of myocardial infarction injury. Here, we show that CSCs with platelet nanovesicles fused onto their surface membranes express platelet surface markers that are associated with platelet adhesion to injury sites. We also find that the modified CSCs selectively bind collagen-coated surfaces and endothelium-denuded rat aortas, and that in rat and porcine models of acute myocardial infarction the modified CSCs increase retention in the heart and reduce infarct size. Platelet-nanovesicle-fused CSCs thus possess the natural targeting and repairing ability of their parental cell types. This stem cell manipulation approach is fast, straightforward and safe, does not require genetic alteration of the cells, and should be generalizable to multiple cell types.

Material Strategies for Modulating Epithelial to Mesenchymal Transitions.

Epithelial to mesenchymal transitions (EMT) involve the phenotypic change of epithelial cells into fibroblast-like cells. This process is accompanied by the loss of cell-cell contacts, increased extracellular matrix (ECM) production, stress fiber alignment, and an increase in cell mobility. While essential for development and wound repair, EMT has also been recognized as a contributing factor to fibrotic diseases and cancer. Both chemical and mechanical cues, such as tumor necrosis factor alpha, NF-κB, Wnt, Notch, interleukin-8, metalloproteinase-3, ECM proteins, and ECM stiffness can determine the degree and duration of EMT events. Additionally, transforming growth factor beta is a primary driver of EMT and, interestingly, can be activated through cell-mediated mechanoactivation. In this review, we highlight recent findings demonstrating the contribution of mechanical stimuli, such as tissue and material stiffness, in driving EMT. We then highlight material strategies for controlling EMT events. Finally, we discuss drivers of the similar process of endothelial to mesenchymal transition (EndoMT) and corresponding material strategies for controlling EndoMT.