Biomaterials and Advanced Technologies for Hemostatic Management of Bleeding

Abstract

Bleeding complications arising from trauma, surgery, and as congenital, disease-associated, or drug-induced blood disorders can cause significant morbidities and mortalities in civilian and military populations. Therefore, stoppage of bleeding (hemostasis) is of paramount clinical significance in prophylactic, surgical, and emergency scenarios. For externally accessible injuries, a variety of natural and synthetic biomaterials have undergone robust research, leading to hemostatic technologies including glues, bandages, tamponades, tourniquets, dressings, and procoagulant powders. In contrast, treatment of internal noncompressible hemorrhage still heavily depends on transfusion of whole blood or blood's hemostatic components (platelets, fibrinogen, and coagulation factors). Transfusion of platelets poses significant challenges of limited availability, high cost, contamination risks, short shelf-life, low portability, performance variability, and immunological side effects, while use of fibrinogen or coagulation factors provides only partial mechanisms for hemostasis. With such considerations, significant interdisciplinary research endeavors have been focused on developing materials and technologies that can be manufactured conveniently, sterilized to minimize contamination and enhance shelf-life, and administered intravenously to mimic, leverage, and amplify physiological hemostatic mechanisms. Here, a comprehensive review regarding the various topical, intracavitary, and intravenous hemostatic technologies in terms of materials, mechanisms, and state-of-art is provided, and challenges and opportunities to help advancement of the field are discussed.

Keywords: biomaterials; hemostasis; platelets; transfusion; wound dressings.

Figure 1

Schematic of the complex mechanism of blood vessel hemostasis. Vessel injury can lead to endothelial activation and denudation resulting in secretion and deposition of von Willebrand Factor (vWF) and exposure of collagen at the injury site, as well as, exposure of tissue factor (TF) bearing cells at the site; vWF and collagen exposure allows platelet adhesion and activation, while TF exposure allows extrinsic pathway of coagulation to propagate and produce moderate amounts of thrombin (FIIa) that activates other coagulation factors in the intrinsic pathway; activated platelets aggregate via fibrinogen (Fg) mediated interaction with platelet surface integrin GPIIb-IIIa to form a platelet plug (primary hemostasis) that staunches bleeding; the surface of aggregated active platelets exposes negatively charged phospholipids that allow co-localization and further activation of coagulation factors to form the prothrombinase (FVa + FXa + FII) complex in presence of calcium (Ca⁺⁺), leading to amplified generation of thrombin (FIIa) that breaks down fibrinogen (Fg) to fibrin; fibrin self-assembles and undergoes further crosslinking by action of FXIIIa to form a dense biopolymeric mesh that forms the hemostatic clot and arrests flow of blood components (secondary hemostasis).

Figure 2

Representative chemical structures of cotton (cellulose) biopolymers and its derivatives that have undergone extensive research in the development of hemostatic technologies like gauze and wound dressings.

Figure 3

Schematic of concomitant roles of thrombin and fibrin(ogen) in propagating the formation of hemostatic clots via activation of platelets and formation of fibrin mesh.

Figure 4

Multiscale schematic representation of fibrillar collagen structure where triple-helical microfibrils formed of Gly-X-Y amino acid repeat units assemble in staggered orientation to form collagen fibrils, which in turn further assemble to form high molecular weight collagen fibers and bundles; denaturation of collagen disrupts this assembled structures to form gelatin, which can be partly reassembled into helical components to form lower molecular weight gels.

Figure 5

Chemical structures of some polysaccharide polymers, namely alginate, chitosan and dextrin that have been extensively used in development of hemostatic bandages and dressings.

Figure 6

Selected results from hemostasis-relevant studies carried out with PolyP-coated silica particle systems (6A and 6B) and thrombin-loaded CaCO₃-based particles mixed in TXA-NH3⁺ that can self-propel themselves into wound depth (6C, 6D and 6E). 6A demonstrates that PolyP loaded in silica nanoparticles (PolyP-SNP) accelerates thrombin generation compared to SNP alone while 6B demonstrates that PolyP delivered via SNP particles (PO₃ in SNP) reduces clotting time of blood (i.e. speeds up coagulation) compared to PolyP in solution; 6C shows representative fluorescence and scanning electron micrograph images of CaCO₃ particles along with a schematic of experimental set-up to administer thrombin-loaded ‘self-propelled’ particles in TXA-NH₃⁺ medium to a liver puncture site; 6D shows presence of green fluorescent particles deep within the liver injury site in mice; 6E demonstrates that liver injury site-localized delivery of thrombin-loaded ‘self-propelled’ CaCO3 particles in TXANH3+ results in significant reduction of blood loss, compared to non-propelled thrombin delivery or control treatment. Figure components adapted and reproduced with permission.^[355,356] Copyright 2015, John Wiley & Sons Inc. and 2015, AAAS Science Advances CC-BY NC.

Figure 7

Schematic of fibrinogen conversion to fibrin and assembly of cross-linked fibrin biopolymeric mesh catalyzed by thrombin (FIIa) and FXIIIa, and hemostatic materials and technologies inspired by these mechanisms.

Figure 8

Selected results from hemostatic studies carried out with platelet-like microgel particles (PLPs, 8A–8D) and with polySTAT injectable polymer systems (8E–8G). 8A shows magnified images of fibrin-binding soft ultralow crosslinked microgel particles (PLPs), natural platelets (PRP), non-binding control microgel particles (S11–ULCs), and fibrin-binding rigid polystyrene particles (H6-PS) within fibrin matrices 1 hr post fibrin polymerization and 8B shows the calculated spread area of PLPs, PRP platelets, S11-ULCs and H6-PS particles within the fibrin matrices, demonstrating that soft PLPs have spreading behavior comparable to natural platelets (PRP) and greater than control particles; 8C and 8D show bleeding time and blood loss analysis data in rat femoral vein injury model where administration of PLPs resulted in reduced bleeding time and blood loss comparable to administration of FVIIa and significantly lower than treatment with vehicle or control particles; 8E shows the homogenous incorporation of the fibrin-binding polySTAT polymer (green fluorescence) within native fibrin matrix (red fluorescence); 8F shows that polySTAT incorporation within fibrin results in crosslinking based strengthening of fibrin and thus reduction of clot lysis over time (i.e. increased clot stability) compared to treatment with control saline and control polymer (polySCRAM); 8G demonstrates that intravenous administration of polySTAT results in significant reduction of blood loss rate in a rat femoral artery hemorrhage model. Figure components adapted and reproduced with permission.^[365,368] Copyright 2014, Macmillan Publishers Ltd and 2015, AAAS.

Figure 9

Schematic of platelet’s injury site-selective adhesion mechanisms (platelet GPIbα of the GPIb-IX-V complex binding to vWF, and GPIa-IIa as well as GPVI binding to collagen) and aggregation mechanism (fibrinogen-mediated bridging of active platelet surface integrin GPIIb-IIIa), and various hemostatic technologies inspired by these mechanisms.

Figure 10

Selected results from hemostatic studies with infusible platelet membrane (IPM) technology, where (10 A) addition of IPM in thrombocytopenic blood perfused ex vivo in rabbit aorta segments dose-dependently increased fibrin deposition at low and high shear rates and (10B) in vivo administration of IPM in thrombocytopenic rabbits resulted in significant reduction of bleeding time. Figure components adapted and reproduced with permission.^[401,402] Copyright 2000, John Wiley & Sons Inc. and 2012, IJPSR.

Figure 11

Selected results from studies involving technologies that leverage platelet’s bleeding site-selective adhesion mechanisms. 11A shows platelet aggregometry studies to demonstrate that addition of rGPIbα-decorated albumin microsphere particles significantly enhances the aggregation level of platelets in presence of vWF and ristocetin, compared to without ristocetin, thereby confirming that the aggregation enhancement is due to the binding of rGPIbα to ristocetin-induced unfolded vWF; 11B shows that polystyrene microparticles surface-decorated with vWF’s A1 domain or platelet’s GPIbα fragment can undergo shear-responsive enhanced adhesion on GPIbα–coated or vWF-coated surfaces respectively, under flow, mimicking platelet-relevant adhesion mechanisms; 11C shows an experimental schematic of flowing rGPIbα-decorated red fluorescent liposomes over vWF-coated surfaces, with the corresponding fluorescence microscopy results demonstrating enhanced adhesion and accumulation of these liposomes over time at higher shear, mimicking the shear-responsive interaction of platelet GPIbα to vWF. Figure components adapted and reproduced with permission^[413,414]. Copyright 2012, John Wiley & Sons Inc. and 2000, American Chemical Society.

Figure 12

Selected results from studies carried out with various fibrinogen-coated particle designs. 12A shows the effect of administering fibrinogen-coated albumin microcapsules (Synthocyte™) in reducing ear punch bleeding time in thrombocytopenic rabbits, where induction of thrombocytopenia (TCP) significantly increased bleeding time from normal levels and administration of Synthocyte™ particles (SC) could significantly reduce bleeding time in these animals, compared to administration of control particles (CP) or saline; 12B shows scanning electron micrograph of the Synthocyte™ particles incorporated with platelets and fibrin in hemostatic clots; 12C shows the ability of fibrinogen-coated albumin particles to bind to platelet-immobilized surfaces in absence versus presence of Ca⁺⁺ ions, compared to binding of unmodified albumin particles and 12D shows representative fluorescence microscopy images of these binding studies, confirming high binding of fibrinogen-coated particles and establishing that these particles can interact with active platelets under flow environment, possibly via interaction with platelet surface integrin GPIIb-IIIa, thereby mimicking and amplifying the active platelet aggregation component of hemostasis. Figure components adapted and reproduced with permission.^[419,420] Copyright 1999, Macmillan Publishers Ltd and 2001, American Chemical Society.

Figure 13

Selected results from studies carried out with various particle platforms surface-decorated with fibrinogen-relevant RGD or H12 peptides to mimic platelet aggregation mechanism. 13A shows platelet aggregometry studies with RGD-decorated RBCs (Thromboerythrocytes), where unmodified control erythrocytes were unable to enhance aggregation of ADP-activated platelets and Thromboerythrocytes were unable to enhance aggregation of inactive platelets or integrin GPIIb-IIIa-blocked platelets, but they could significantly enhance the aggregation of ADP-activated platelets, thereby mimicking fibrinogen and GPIIb-IIIa mediated platelet aggregation mechanism; 13B shows representative fluorescent images as well as quantitative surface-coverage data of H12 peptide-decorated, or RGD peptide-decorated or undecorated fluorescently-labeled latex beads suspended in reconstituted blood and flowed over platelet-immobilized surfaces, indicating the enhanced GPIIb-IIIa-binding capability of H12-decorated and RGD-decorated beads to platelets; 13C shows the capability of H12-decorated PEGylated albumin particles (H12-PEG-Alb) to significantly reduce ear punch bleeding time upon intravenous administration in thrombocytopenic rabbits, compared to administration of control undecorated PEG-albumin (PEG-Alb) particles; 13D shows the capability of H12-decorated PEGylated liposomal vesicles (H12-PEG-vesicles) to significantly reduce tail bleeding time in thrombocytopenic rats, compared to administration of control undecorated PEG-vesicles; 13E shows quantitative bleeding time reduction data and representative fluorescence image of hemostasized vessel, where administration of RGD-decorated polymeric nanoparticles (GRGDS-PLGA-PLL particles) significantly reduced bleeding time in a rat femoral artery injury model, compared to undecorated particles or saline. Figure components adapted and reproduced with permission.^{[424,425,426,431,432]} Copyright 1992, American Society for Clinical Investigation; 2003, Elsevier; 2008, John Wiley & Sons Inc.; 2005, American Chemical Society and 2009, AAAS.

Figure 14

Selected results from hemostatic studies carried out with platelet-inspired ‘integrative’ particle technologies that combine platelet-relevant adhesion and aggregation mechanisms on a single particle platform. 14A shows platelet aggregometry studies where undecorated latex beads, or H12 peptide-decorated latex beads, or latex beads co-decorated with H12 peptides and rGPIbα fragments were added to ADP-activated platelets and the results showed that the co-decorated beads had no additional aggregation enhancement effects, possibly due to the masking of small H12 peptides by the large rGPIbα fragments; This masking effect could be resolved by utilizing heteromultivalent decoration of small peptides, as shown in the example in 14B where intravenous administration of liposomes heteromultivalently decorated with vWF-binding, collagen-binding and fibrinogen-mimetic peptides (functionally integrated particles) resulted in significant reduction in tail bleeding time in normal mice, compared to administration of liposomes bearing only pro-adhesive peptides (‘adhesion only’ particles) or liposomes bearing only pro-aggregatory peptides (‘aggregation only’ particles); such heteromultivalent decoration could be adapted onto other particle platforms, as shown in the example in 14C where discoid albumin particles (red fluorescent) surface-decorated heteromultivalently with all three peptides showed higher co-localization with active platelets (green fluorescent) to form visible aggregates in collagen-coated microfluidic channels, compared to particles bearing proaggregatory peptides only (‘FMP only’) or pro-adhesive peptides only (‘VBP + CBP’ only); 14D shows representative results from tail bleeding studies in thrombocytopenic mice where intravenously administered synthetic platelet (SynthoPlate™) particles (liposomes surface-decorated heteromutivalently with VBP, CBP and FMP) could dose-dependently reduce tail bleeding time, compared to administration of saline or control particles. Figure components adapted and reproduced with permission.^{[438,440,448,451]} Copyright 2006, Springer; 2013, Elsevier; 2014, American Chemical Society and 2017, International Society on Thrombosis and Haemostasis.