The importance of the interaction of platelets with “foreign” materials has been recognized (e.g., see Barr1) since their discovery.2–4 Platelets, as the blood cells that mediate coagulation in response to breaches to the vasculature, have been shown to activate when they contact a wide variety of foreign materials, including metals,5,6 glass,7–9 and plastics10,11 (e.g., see Packham12 for a review). An understanding of the interaction of platelets with foreign surfaces has played a fundamental role in the development of biomedical devices, such as stents13 and cardiopulmonary extracorporeal machinery,14 that minimize platelet activation. Alternatively, the optimization of activating interactions between platelets and foreign materials holds promise for the rational design of hemostatic materials for hemorrhage control.
Existing evidence indicates that the interaction of platelets with foreign materials involves two steps. First, plasma proteins,15 including adhesion proteins such as fibrinogen,16–18 fibronectin,19 and von Willebrand factor,20 change conformation when adsorbed onto surfaces. These interactions probably involve multiple points of attachment of the macromolecules to the foreign materials that are random in nature, but the result is an alteration in solution-phase protein conformation. Second, the altered conformations of the adsorbed proteins expose structural domains that activate platelet surface receptors. For example, fibrinogen might assume a fibrin-like conformation and thus initiate outside-in signaling through the αIIbβ3 complex. Similarly, adsorbed von Willebrand factor might assume a “sheared” conformation and bind to GPIb-IX complexes. A substantial body of evidence indeed indicates that the integrin αIIbβ3 8,9,21 and GPIb-IX22 are important for the interaction of platelets with adsorbed plasma proteins. Although the processes through which αIIbβ3, GPIb-IX, and other platelet surface receptors are activated by adsorbed ligands are largely undefined, it is reasonable to hypothesize that high local levels of the adsorbed plasma proteins interact with glycoprotein receptors on the platelet membrane to organize cytoskeletal signaling machinery for active outside-in signaling complexes. The interaction of platelets with foreign materials might also activate the Hageman contact activation factor (factor XII) for activation of the intrinsic coagulation cascade. Factor XII, which binds and proteolytically activates on contact with many anionic surfaces, is associated with the platelet surface for activation of the intrinsic coagulation pathway in the microenvironment of the cell.23 Although the series of proteolytic steps involving factor XII that occurs on the platelet surface is poorly understood, the protective effect of aprotinin on platelets during cardiopulmonary bypass has been hypothesized to originate in part from an inhibition of kallikrein-like proteolytic events as platelets contact foreign surfaces in the extracorporeal device.24
Intense efforts have focused on the development of hemostatic agents that are applied to wound sites for the control of hemorrhage. Formulations of the carbohydrate polymers have proven particularly useful for providing hemostasis. The biomedical uses of N-acetyl glucosamine and glucosamine-containing polymers (chitins and chitosans) from natural sources, usually crustacean outer shells and fungal mycelial mats, have been in development for decades.25 Earlier applications in coagulation therapy26 and wound healing27 were envisioned. However, chemical variability and contamination issues have hindered clinical progress. These issues were overcome with the development of methods of producing highly homogeneous and pure poly-N-acetyl glucosamine (p-GlcNAc) from diatom cultures.28 This marine polymer, with average molecular masses of 2.8 × 106 Da, is assembled by the diatom in vivo to form ordered and regular (crystalline) fiber structures that are stabilized by intra- and interchain hydrogen bonding.28 Formulations of poly-N-acetyl glucosamine fibers have been found to reduce bleeding in several animal models, including porcine splenic injury,29 porcine liver injury,30 and a canine model of variceal hemorrhage.31 The marine polymer has also proven useful in human clinical practice for the cessation of bleeding.32 The mechanism through which poly-N-acetyl glucosamine fiber material promote hemostasis has not yet been defined. Poly-N-acetyl glucosamine has been shown to promote red blood cell aggregation33 and to mediate endothelial-dependent vasoconstriction.34 However, an understanding of the mechanism for poly-N-acetyl glucosamine–mediated hemostasis, particularly with respect to the role of platelet interaction with the marine polymer, is unknown.35
The experiments reported here are designed to elucidate critical elements of the mechanism thorough which platelets interact with poly-N-acetyl glucosamine fibers to provide hemostasis. Experiments were performed in two stages. First, the extent to which platelet contact with poly-N-acetyl glucosamine produces an activation response was ascertained. Second, the effect of platelet contact with poly-N-acetyl glucosamine on fibrin network formation was investigated.
MATERIALS AND METHODS
Poly-N-acetyl glucosamine, chitosan, and chemically modified forms of poly-N-acetyl glucosamine were obtained from Marine Polymer Technologies, Inc. (Danvers, MA). Surface protein biotinylation kits, Alexa-Annexin V assay kits, and calcium orange were purchased from Molecular Probes (Eugene, OR). Mouse anti-β3 integrin (whole molecule), mouse anti-CD41a, mouse anti-CD41b, mouse anti-CD49e, and rat anti-CD49f were obtained from BD Biosciences (San Diego, CA). Mouse anti-β3 clone 61C101 and mouse anti-GP1b (CD42b) clone SZ2 were obtained from Biomeda (Foster City, CA) and Immunotech (Marseilles, France). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted.
Fresh human platelet-rich plasma was isolated from sodium citrated blood by differential centrifugation. For some experiments, the platelets were separated from plasma proteins with gel filtration or differential centrifugation, as reported by Fischer et al.36
Examination of Platelet Morphology by Scanning Electron Microscopy
The samples were fixed in an ice-cold mixture of 4% paraformaldehyde with 0.5% glutaraldehyde in phosphate-buffered saline (PBS). Scanning electron micrographs were taken with a Cambridge S200 scanning electron microscope at 20 kV.
Fluorescent Imaging of Platelet Mixtures with Poly-N-Acetyl Glucosamine for Activation Markers
Intracellular calcium indicators and Annexin-based phosphatidylserine probes were examined with multiphoton imaging and semiquantitative fluorescence measurements with a BioRad MRC 1024ES multiphoton imaging system (BioRad, Hercules, CA) coupled with a mode-locked titanium:sapphire laser (Spectra-Physics, Mountain View, CA) tuned to 810 nm, in transmission and epifluorescence mode as previously described.37 A Zeiss Axiovert S100 inverted microscope equipped with a high-quality water immersion 63×/1.2 NA, C-apochroma objective was used to image the samples. The 512 × 512-pixel images were collected in direct detection configuration at a pixel resolution of 0.484 μm with a Kalman 5 collection filter.37 Fluorescent antibody probes for P selectin and activated integrin αIIbβ3 complex were detected with a Nikon Microphot FXA upright fluorescence microscope.
Calcium mobilization in the platelets was measured by using calcium-sensitive calcium orange dye.37 The platelets were incubated with calcium orange in 1 mL PBS (10 μmol/L final concentration) for 30 minutes at 21°C. After the platelets had been gently washed twice with PBS to remove the excess dye, they were resuspended in Hank’s balanced salt solution (HBSS) and further incubated with poly-N-acetyl glucosamine fibers (0.35 mg/mL) or 10% bovine serum albumin (BSA) in HBSS for 10 minutes at 21°C. After incubation, the samples were mounted on slides coated with BSA (1% in PBS). Calcium mobilization as envisioned by an increase in intracellular calcium orange fluorescence was measured in real time by multiphoton microscopy.37
Citrate anticoagulated whole blood, diluted to 1:20 v/v in 1 mL HBSS, was incubated with the poly-N-acetyl glucosamine fiber slurry (0.35 mg/mL) or BSA (30% in HBSS) as control for 15 minutes at 21°C. After incubation, the samples were washed twice with PBS, and the cell pellets were resuspended in 100 μL of HBSS and incubated 1:1 with Alexa-Annexin solution (1 part Component A: Alexa Fluor 488 Annexin V to 10 parts HBSS) for 30 minutes at 21°C. The samples were then washed twice with PBS and resuspended in 100 μL of HBSS. The samples were mounted on BSA (1%)-coated slides and imaged by multiphoton microscopy.37
Surface P Selectin and Activated Integrin αIIbβ3 Complex
Platelets were isolated from fresh blood obtained from normal volunteers by differential centrifugation and gel filtration and were then mixed with poly-N-acetyl glucosamine fiber slurry in Tyrode’s buffer with divalent ions for a final polymer concentration of 1.1 mg/mL. After a 5-minute incubation at room temperature, Mab anti-P selectin–phycoerythrin conjugate (Mab AC1.2) and anti-integrin αIIbβ3 complex–fluorescein isothiocyanate conjugate (Mab PAC-1, activation-dependent) were diluted 1:200 into the platelet mixtures. The samples were allowed to incubate for an additional 5 minutes and quenched with ice-cold 2% paraformaldehyde for fluorescent microscopy.
Spectrophotometric Measurements of Poly-N-Acetyl Glucosamine/Platelet Fibrin-Network Formation Kinetics
To determine the optimal wavelength for measurement of the absorbance of platelet solutions, a human platelet-rich plasma sample was diluted with HBSS so that the final concentration of platelets was adjusted to a consistent value of 1.9 × 106 /mL The platelet solution was scanned in a spectrophotometer (BioRad SmartSpec 3000), and the optimal wavelength was determined to be 419 nm. The optical densities of 1-mL solutions containing platelet-rich plasma and varying concentrations of poly-N-acetyl glucosamine fibers (0.1–0.5 mg/mL, final concentration) were measured over a 10-minute period at 21°C, with measurements taken at 1-minute intervals. The spectrophotometer was blanked with the concentration of poly-N-acetyl glucosamine used in the corresponding test solution before each measurement. These experiments were repeated using structurally and chemically modified samples of poly-N-acetyl glucosamine fibers and chitosan, described below.
Treatment of Platelets with Antibody Probes for Inhibition
Platelet-rich plasma was diluted with PBS and divided into six samples of 100-μL aliquots each. One aliquot was left untreated as a control or treated with a sham antibody (1:100 dilution), and the remaining aliquots were treated with one of the following antibodies: anti-β3 integrin, CD41a, CD41b, CD49e, and CD49f, at 1:100 final dilution. All six samples were incubated at 21°C for 90 minutes, with occasional gentle shaking. After the incubation, antibody-treated samples were then mixed independently with poly-N-acetyl glucosamine fiber slurry and reconstituted to 1 mL with HBSS, so that the final concentration of poly-N-acetyl glucosamine in the solution was 0.35 mg/mL. Changes in optical density were measured at a wavelength of 419 nm with the spectrophotometer and recorded over a 10-minute period. In addition, six slides were coated with poly-N-acetyl glucosamine slurry (0.35 mg/mL) and allowed to air dry. A 10-μL drop of individual antibody-treated platelet solution was deposited in the center of the slide and covered with a coverslip. The slide was imaged immediately by using multiphoton microscopy in transmission mode. The recruitment of platelets on the poly-N-acetyl glucosamine fibers in the center of the slide was monitored over a period of 10 minutes.
Preparation of Poly-N-Acetyl Glucosamine Derivatives
To better characterize the hemostatic properties of poly-N-acetyl glucosamine, and also to obtain a better formulation of the product, samples of poly-N-acetyl glucosamine (acetylated linear polymer of poly-N-acetyl glucosamine residues organized in β-1,4 linkage, with an altered tertiary structure) were chemically modified to change the three-dimensional structure, expose the positive and/or negative charges, and also to alter the solubility characteristics of the polymer, as described before.28 Modified poly-N-acetyl glucosamine samples were coded so that the investigators were blinded to the nature of their chemical modification. After the completion of all sets of experiments and data analysis, the codes were opened. The samples tested were native poly-N-acetyl glucosamine fibers (beta-tertiary structure; control, no modification), poly-N-acetyl glucosamine particles (alpha-tertiary structure; more positive charges are exposed), 70% deacetylated poly-N-acetyl glucosamine (more positive charges are exposed, but the fiber structure is lost, and hence poly-N-acetyl glucosamine becomes soluble), 100% deacetylated poly-N-acetyl glucosamine (the positive charges are fully exposed; the fiber structure is completely lost, and thus poly-N-acetyl glucosamine becomes soluble), chitosan (a polysaccharide polymer similar to deacetylated poly-N-acetyl glucosamine), carboxymethylated poly-N-acetyl glucosamine (more negative charges are exposed), and sulfated poly-N-acetyl glucosamine (negative charges exposed).
Analysis of Fibrin Clot-Formation Times
Whole blood was drawn into citrate (10 mm citrate, pH 6.8) and then platelet-rich plasma was prepared by differential centrifugation. Platelet counts were measured, and then the level of platelets was adjusted to 150,000 platelets/μL by diluting with platelet-poor plasma. Clot formation assays were initiated by adding CaCl2 (1:100 dilution of aqueous stock) and poly-N-acetyl glucosamine (1:10 dilution of saline slurry) for respective concentrations of 10 mmol/L and 1.1 mg/mL. The samples were placed on a rocker at 20 rpm at room temperature, and the time required for the formation of a macroscopic gel was measured.
Studies were performed to define how platelets interact with poly-N-acetyl glucosamine in vitro. The extent to which platelet contact with poly-N-acetyl glucosamine results in an activation response and the effect of platelet/poly-N-acetyl glucosamine contact on fibrin network formation were investigated.
How Does the Interaction of Platelets with Poly-N-Acetyl Glucosamine Affect the Platelet Activation State?
The effect of platelet contact with p-GlcNAc fibers on their morphology, surface membrane activation, and intracellular calcium was investigated. The effect of native p-GlcNAc on platelet morphology was ascertained by mixing platelets with the marine polymer in the absence or presence of plasma proteins. Platelet/p-GlcNAc fiber mixtures were allowed to incubate for 3 minutes (before macroscopic fibrin network formation occurs in plasma samples) and then quenched and processed for scanning electron microscopy. Figure 1A shows that in the absence of excess plasma proteins, platelet contact with the marine polymer fibers resulted in shape changes and pseudopodia extensions that are characteristic of a full irreversible activation response. In the presence of plasma proteins (Fig. 1B), the platelets are seen to aggregate and form clusters on the poly-N-acetyl glucosamine fibers.
Further evidence that poly-N-acetyl glucosamine fiber contact leads to platelet activation is provided by the fluorescent microscopic observations that the intracellular calcium concentration was elevated, phosphatidylserine was exposed on the outer surface of the platelet membrane, and the adhesive properties of the surface membrane were increased by P selectin exposure and αIIbβ3 complex expression. There was a significant increase in intracellular calcium orange fluorescence in platelets in the presence of poly-N-acetyl glucosamine, demonstrating intracellular signaling for an increase in intracellular calcium concentration (Fig. 2A). In contrast, the change in intracellular calcium was negligible in control (BSA-treated) platelets (Fig. 2A). Similarly, platelets incubated with poly-N-acetyl glucosamine demonstrated a large increase in binding of Alexa-Annexin to the plasma membranes, indicating phosphatidylserine exposure (Fig. 2B). In contrast, platelets incubated with BSA in the absence of poly-N-acetyl glucosamine exhibited minimal Alexa-Annexin binding to the plasma membranes (Fig. 2B). The interaction of platelets with the marine polymer also resulted in surface exposure of P selectin from alpha-granule pools, as well as expression of integrin αIIbβ3 complexes (Fig. 2C). These results clearly demonstrate that the interaction of platelets with poly-N-acetyl glucosamine leads to platelet activation.
How Does Poly-N-Acetyl Glucosamine Affect Fibrin Polymerization?
The kinetics of fibrin polymerization was followed by measuring changes in turbidity. A spectrophotometric assay that measured the increase in optical density (OD419) of p-GlcNAc/platelet suspensions at 419 nm as a result of fibrin polymer growth was used. As shown in Figure 3, a time-dependent increase in OD419 of the p-GlcNAc fiber/platelet mixture was observed. The increase was saturable with a half-life for poly-N-acetyl glucosamine/platelet gel formation of 240 ± 5 seconds (mean ± SEM; n = 10). At the concentration of platelets used, the formation of a gel was also poly-N-acetyl glucosamine fiber concentration-dependent. The dose-response curve of poly-N-acetyl glucosamine/platelet gel formation (not shown) gave an extinction coefficient at 50% aggregation of 0.33 ± 0.02 mg of poly-N-acetyl glucosamine per milliliter (mean ± SEM; n = 7). However, the strongest gel (maximum OD419) was formed at a poly-N-acetyl glucosamine concentration of 0.5 mg/mL after 600 to 900 seconds of incubation. Platelet interaction with p-GlcNAc fibers leads to gel formation only in the presence of calcium.
The interaction between poly-N-acetyl glucosamine fibers, various chemically modified poly-N-acetyl glucosamine derivatives, chitosan, and platelets is shown in Figure 4. Platelets in combination with alpha-modified poly-N-acetyl glucosamine (0.5 mg/mL) formed a strong gel similar to that formed with unmodified, control beta–poly-N-acetyl glucosamine (0.5 mg/mL). Formation of a gel using platelets and alpha-modified p-GlcNAc was also time- and concentration-dependent, and required the presence of calcium in the reaction mixture. The half-life of this interaction was 239 ± 7 seconds (mean ± SEM; n = 3) for both alpha-modified and native beta–p-GlcNAc. In contrast, under similar assay conditions, all other chemically modified p-GlcNAc derivatives and chitosan did not demonstrate any substantial interaction for gel formation.
The role of cell-surface integrins in interactions between platelets and poly-N-acetyl glucosamine fibers was also investigated. Platelets (platelet-rich plasma) were separately treated with various anti-integrin antibodies (Fig. 5). The antibody-treated platelets were then incubated with poly-N-acetyl glucosamine (0.5 mg/mL) in calcium-containing buffer, and the change in OD419 was measured in real time over a period of 900 seconds. Antibodies derived against various components of β3 integrins (whole molecule, calcium-dependent binding domain CD41a, and CD41b or GPIb) inhibited poly-N-acetyl glucosamine/platelet interactions and thus significantly decreased gel formation. In contrast, anti-β1 integrin antibodies gave mixed results. As shown in Figure 4B, anti-CD49e (or VLA5) antibody did not inhibit the poly-N-acetyl glucosamine/platelet interaction, and the gel formation after treatment with these antibodies was similar to that observed in the control, untreated platelet sample. However, a partial but significant inhibition of gel formation was achieved by pretreating platelets with anti-CD49f (anti-VLA6) antibody. These results clearly indicate the differential involvement of cell-surface integrins in p-GlcNAc fiber/platelet interactions.
The results demonstrate that contact of platelets with poly-N-acetyl glucosamine fibers results in a full irreversible activation of the platelet. Contact of platelets with the marine polymer resulted in shape changes, including pseudopodia extension, that characterize an irreversible activation response. Furthermore, an intracellular calcium signaling response was measured in response to contact, as was calcium-dependent scramblase activity for surface presentation of phosphatidylserine.38 Interaction of the p-GlcNAc fibers with platelets also resulted in exposure of P selectin on the platelet surface membrane from alpha-granule sources39 and the activation of αIIbβ3 complexes for fibrinogen binding. Thus, on contact with poly-N-acetyl glucosamine fibers, platelets are activated for the subsequent catalysis of prothrombin to thrombin conversion as well as primed for αIIbβ3 complex stimulation of fibrin matrix formation. These results indicate that the interaction of p-GlcNAc fibers with platelets is similar to the interaction of this platelet with other materials, such as glass,8,9 polylysine,40,41 plastics,11 and metals.6
Adsorption experiments demonstrated that p-GlcNAc fibers tightly bound a wide variety of plasma proteins (data not shown). Furthermore, the adhesion of platelets to the marine polymer matrix (concurrent with plasma protein adsorption) is mediated by the interaction of multiple surface proteins on the platelet with the marine polymer matrix. This result suggests that the interaction of plasma proteins and platelet surface proteins with poly-N-acetyl glucosamine is in part driven by tight but nonspecific chemical and physical adsorption processes. However, the demonstration that αIIbβ3 and GPIb are among the surface proteins that tightly adsorb to the matrix suggests that the ligands for these receptors (fibrinogen and von Willebrand factor) are adsorbed to the fiber polymer matrix.
A functional consequence of platelet contact with poly-N-acetyl glucosamine in the presence of plasma proteins and calcium is the acceleration of the kinetics of fibrin matrix formation. This was demonstrated by optically monitoring the kinetics of fibrin polymerization as well as by measuring the times required for macroscopic clot formation when the marine polymer was mixed with platelet-rich plasma. The reported results indicate that two mechanisms are involved in this acceleration of fibrin polymerization that involve integrins and factor XII. First, the involvement of integrins is evidenced by the finding that eptifibatide slowed fibrin polymerization and delayed the time required for macroscopic gel formation (Fischer et al., data not shown). Integrin inhibition also resulted in poly-N-acetyl glucosamine/fibrin/platelet macrogels with relatively thick (approximately 400 nm as compared with native thickness of approximately 100 nm) fibrin strands, indicating that the thrombin concentration was reduced during the chain-elongation phase of the gel-formation process.42 On the basis of scanning electron microscopic analysis, in the presence of eptifibatide, the platelets were not intermeshed with the poly-N-acetyl glucosamine/fibrin copolymer matrix, as is the case in the absence of integrin inhibition. A similar observation has been made when platelets interact with glass in the presence of RGD peptide inhibitors of integrin function; the cells adsorb, but with a greatly reduced number of contact points and a morphology that is less “spread.”11 These observations are consistent with an interaction mechanism between platelets and poly-N-acetyl glucosamine that involves clustering of integrins on the platelet surface as a result of the formation of multiple contacts with the marine polymer strands. The integrin clustering activates the platelets through outside-in signaling43 to yield a cell surface for the catalysis of conversion of prothrombin to thrombin. Integrin inhibition would be expected to inhibit the contact of platelets with the p-GlcNAc fibers, resulting in reduced levels of thrombin and thicker fibrin strands.
To summarize, the reported results are consistent with a three-step interaction mechanism between platelets and poly-N-acetyl glucosamine fibers to produce hemostasis. First, platelets physically bind directly to poly-N-acetyl glucosamine and/or poly-N-acetyl glucosamine binds to immobilized plasma proteins like fibrinogen. Second, the integrin-mediated platelet activation then initiates the intrinsic (contact) coagulation factor pathway by factor XII activation to generate thrombin and fibrin polymerization to form a stabilized clot. The process of poly-N-acetyl glucosamine/platelet-driven fibrin clot formation is augmented by a tendency of platelets to agglutinate on the poly-N-acetyl glucosamine matrix and generate vasoconstrictive substances like thromboxane and serotonin.35 Third, the resulting poly-N-acetyl glucosamine/fibrin/platelet structures undergo platelet-mediated clot retraction in concert with local vasoconstriction34 for accelerated wound closure. The kinetics of in vitro clot formation correspond well with the time required for p-GlcNAc fiber-mediated in vitro aggregation of red blood cells33 and rapid in vivo hemostasis achieved using the poly-N-acetyl glucosamine–derived SyvekPatch bandages in patients undergoing cardiac catheterization.32 The p-GlcNAc fiber–mediated effect is a multifactorial process that is driven by the interaction of the polymer fibers with platelets, plasma clotting factors, and red blood cells to achieve rapid hemostasis at the wound site.
We thank John Vournakis and Marina Demcheva for their generous gifts of poly-N-acetyl glucosamine materials, chemically modified poly-N-acetyl glucosamine, and chitosan, and for their useful suggestions and discussions; and Nancy Healey for editorial assistance.
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