Cell mimetic monolayer supported chitosan-haemocompatibility studies

Chitosan is a natural polymer, widely explored for biomedical and tissue engineering applications. However the thrombogenic nature limits their application in blood contacting devices and implants. Here, we have attempted to understand the haemocompatibility of chitosan by immobilizing a monolayer of cell mimetic lipid compositions. The phosphatidylcholine/cholesterol/galactocerebroside lipid composition (PC/Chol/GalC, 1:0.35:0.125) was deposited onto the chitosan films. Characterization of the modified surface was done by sessile drop contact angle measurement. The contact angle of the chitosan film reduced from 80.65 +/- 1.4 to 23.5 +/- 1.9 after the surface modification. Swelling nature of chitosan seemed to influence the orientation and packing of the lipid monolayer. In vitro calcification studies with metastable salt solution indicated increased calcification on the modified surface. This may be due to formation of nuclei for calcification on the expanding monolayer. The preliminary haemocompatibility studies with washed platelets, leukocytes and erythrocytes showed overall reduction in blood cell adhesion to the modified surfaces. Scanning electron microscopy was used for morphological characterization of platelet adhesion and activation on the surfaces. On the bare chitosan surface, fully spread platelets with extending pseudopodia indicated platelet activation. The smooth surface of the modified film did not activate platelets. These studies showed that, though the lipid monolayer on chitosan film is able to reduce the over all blood cell adhesion and platelet activation it is prone to calcification.     

The Procoagulant Properties of Hyaluronic Acid-Collagen (I)/Chitosan Complex Film

Biomaterial-induced human platelet activation remains one of the most crucial factors to determine the procoagulant properties of the biomaterial. In this experiment, a new type of biomacromolecule complex film (hyaluronic acid-collagen (I)/chitosan, HCC) was prepared using the electrostatic self-assembly method. Then the procoagulant properties of this complex film were characterized. Based on the nano-resolution of atomic force microscopy, the platelet-derived microparticles (PMPs) that present the activation of platelets were clearly visualized on the membrane surface of platelets for the first time, and the measurement indicated that the size of PMPs is around 50–110 nm. Furthermore, the results of AFM measurement were confirmed by flow cytometry analysis. The expression of CD62P (P-selectin) dramatically increased after the platelet-rich plasma interacted with the biomaterial solution. From the results, we could draw the conclusion that this biomacromolecule complex film has promising procoagulant properties, and has the potential to be practically used as procoagulant material.     

Activation of platelets by in vitro whole blood contact with materials: Increases in microparticle,procoagulant activity,and soluble P-selectin blood levels

Non-adherent platelets and plasma were analyzed for evidence of platelet activation after whole blood contact with materials under conditions of low shear for one hour at 37°C. The contact involved adding heparinized whole blood to small diameter tubes that were connected to two arms extending from a rocking platform. For all surfaces (polyethylene, polypropylene, Silastic?, PVA hydrogel) tested there was strong evidence of platelet activation in the bulk blood: platelet-derived microparticles, procoagulant platelet membranes and soluble P-selectin levels. Flow cytometric quantification of microparticles (MPs) was highly sensitive and entailed the direct determination of microparticle concentrations as opposed to the traditional quantification of microparticle percentages (relative to total number of MPs and platelets). Whole blood contact with polypropylene surfaces led to the greatest drops in bulk platelet counts and also to the lowest increases in microparticle concentrations. Flow cytometry was also used to assess procoagulant levels (annexin V binding) within a light scatter region known to contain platelets and some large microparticles. All surfaces were noted to generate a significant procoagulant population that was, based on forward light scatter, mostly very small platelets or large microparticles. In contrast, most of the P-selectin positive platelets were averaged sized. Lastly, all surfaces generated soluble P-selectin levels that were approximately double the level (25 ng ml^-1) noted in the resting whole blood samples. In addition to our previous reports, these findings support the observation that there is strong evidence of platelet activation in the bulk that we anticipate will ultimately lead to more relevant in vitro testing of the compatibility of platelets towards materials.     

Activation of platelets by in vitro whole blood contact with materials: increases in microparticle, procoagulant activity, and soluble P-selectin blood levels.

Non-adherent platelets and plasma were analyzed for evidence of platelet activation after whole blood contact with materials under conditions of low shear for one hour at 37 degrees C. The contact involved adding heparinized whole blood to small diameter tubes that were connected to two arms extending from a rocking platform. For all surfaces (polyethylene, polypropylene, Silastic, PVA hydrogel) tested there was strong evidence of platelet activation in the bulk blood: platelet-derived microparticles, procoagulant platelet membranes and soluble P-selectin levels. Flow cytometric quantification of microparticles (MPs) was highly sensitive and entailed the direct determination of microparticle concentrations as opposed to the traditional quantification of microparticle percentages (relative to total number of MPs and platelets). Whole blood contact with polypropylene surfaces led to the greatest drops in bulk platelet counts and also to the lowest increases in microparticle concentrations. Flow cytometry was also used to assess procoagulant levels (annexin V binding) within a light scatter region known to contain platelets and some large microparticles. All surfaces were noted to generate a significant procoagulant population that was, based on forward light scatter, mostly very small platelets or large microparticles. In contrast, most of the P-selectin positive platelets were averaged sized. Lastly. all surfaces generated soluble P-selectin levels that were approximately double the level (25 ng ml(-1)) noted in the resting whole blood samples. In addition to our previous reports, these findings support the observation that there is strong evidence of platelet activation in the bulk that we anticipate will ultimately lead to more relevant in vitro testing of the compatibility of platelets towards materials.     

Surface modification with PEO-containing triblock copolymer for improved biocompatibility: in vitro and ex vivo studies

Poly(ethylene oxide) (PEO) has been frequently used to modify biomaterial surfaces for improved biocompatibility. We have used PEO-polybutadiene-PEO triblock copolymer to graft PEO to biomaterials by gamma-irradiation for a total radiation dose of 1 Mrad. The molecular weight of PEO in the block copolymer was 5000. In vitro study showed that fibrinogen adsorption to Silastic, polyethylene, and glass was reduced by 70 to approximately 95% by PEO grafting. On the other hand, the reduction of fibrinogen adsorption was only 30% on expanded polytetrafluoroethylene (e-PTFE). In vitro platelet adhesion study showed that almost no platelets could adhere to PEO-coated Silastic, polyethylene, and glass, while numerous platelet aggregates were found on the ePTFE. The platelet adhesion in vitro corresponded to the fibrinogen adsorption. When the PEO-grafted surfaces were tested ex vivo using a series shunt in a canine model, the effect of the grafted PEO was not noticeable. Platelet deposition on ePTFE was reduced by PEO grafting from 8170 +/- 1030 to 5100 +/- 460 platelets 10(-3) microm2, but numerous thrombi were still present on the PEO-grafted surface. The numbers of platelets cumulated on Silastic, polyethylene, and glass were 100 +/- 80, 169 +/- 35, and 24 +/- 22 platelets 10(-3) microm2, respectively. This is about 35% reduction in platelet deposition by PEO grafting. While the numbers of deposited platelets were small, the decreases were not as large as those expected from the in vitro study. This may be due to a number of reasons which have to be clarified in future studies, but it appears that in vitro platelet adhesion and fibrinogen adsorption studies may not be a valuable predictor for the in vivo or ex vivo behavior of the PEO-grafted surfaces.     

Platelet adherence and detachment with adsorbed fibrinogen: a flow study with a series of hydroxyethyl methacrylate-ethyl methacrylate copolymers using video microscopy.

The adhesion and detachment of platelets were studied on glass coatings of a series of copolymers of hydroxyethyl methacrylate (HEMA) and ethyl methacrylate (EMA) with preadsorbed fibrinogen. Observations of the interactions of acridine-orange-labeled washed platelets with these surfaces from a flowing (500 s-1 wall shear rate) suspension in Tyrode's solution containing albumin and red cells were made with epifluorescent video microscopy (EVM). In some cases preadsorbed materials were incubated for 24 h, during which little or no loss of protein occurred. Protein surface concentration, by itself, was a poor indicator of expected cell adhesion and morphology. Surface chemistry was a second important factor which must be considered. A third observation is that for the 100% EMA copolymer, 24 h of incubation led to a large reduction in platelet adhesion when compared to the 100% EMA material without incubation. For the 0% and 100% EMA polymers, the percentage of contacting platelets which adhere and detach is greater for the 24-h incubation cases than for those not incubated. These results led to the conclusion that our most hydrophilic surface favors adhesion with detachment, transient cell contact, over long-term adhesion, as does incubation of adsorbed protein. A brief discussion is presented of a possible connection between this behavior and platelet consumption in vivo for hydrogels.     

生理性流动条件下血小板在玻璃表面的聚集行为

目的 研究生理性流动条件下血小板在玻璃表面的聚集行为。方法 采用软光刻工艺加工聚二甲基硅氧烷(polydimethylsiloxane, PDMS)-玻璃微通道芯片。将抗凝人外周全血以300、1 500 s^-1壁剪切率流过微通道芯片,倒置荧光显微镜拍摄荧光标记血小板在微通道玻璃表面形成的血小板聚集体荧光图像,通过图像分析得到血小板聚集体数量、平均尺寸、表面覆盖率和平均荧光强度。分别对玻璃表面进行亲水性处理、牛血清白蛋白(bovine serum albumin, BSA)封闭和胶原纤维蛋白修饰处理,调整血液样品红细胞比容(hematocrit, Hct),用抗血小板制剂处理血液样品,观察上述条件下血小板在玻璃表面的聚集行为;比较分析健康人群和糖尿病患者的血小板聚集情况。结果 流动条件下,血小板在玻璃表面发生聚集形成三维血小板聚集体;血小板聚集依赖于壁剪切率大小、玻璃表面亲疏水性和Hct;血小板聚集主要由血小板膜糖蛋白GPIIb/Ⅲa-纤维蛋白原和ADP-P2Y₁₂受体通路调控;血小板在玻璃表面的聚集能够反映糖尿病患者血小板的高活化状态。结论 ...     

Morphological characterization of surface-induced platelet activation

Morphological changes of platelets activated on glass and dimethyldichlorosilane-treated glass were investigated using video microscopy. The platelet morphological changes were quantified by measuring the area and circularity of spreading platelets. In addition, re-organization of cytoskeletal structures of spread platelets was examined. The effects of precoated albumin and fibrinogen on the platelet spreading kinetics were examined as a function of surface protein concentrations. Results showed that platelet shape changes were very sensitive to the surface concentration of precoated proteins. In general, platelets on fibrinogen-precoated surfaces spread fully to a circular shape and developed an extensive inner filamentous zone. In the presence of albumin on the surface, however, platelets could not spread fully and the development of the inner filamentous zone was very poor. For both albumin and fibrinogen, the maximum effects of precoated proteins on platelet shape changes were observed when the surface protein concentration reached the monolayer concentration.     

Assessing acute platelet adhesion on opaque metallic and polymeric biomaterials with fiber optic microscopy

The degree of platelet adhesion and subsequent thrombus formation is an important measure of biocompatibility for cardiovascular biomaterials. Traditional methods of quantifying platelet adhesion often are limited by the need for direct optical access, limited spatial resolution, or the lack of temporal resolution. We have developed a new imaging system that utilizes fiber optics and fluorescence microscopy for the quantification of platelet adhesion. This fiber optic remote microscope is capable of imaging individual fluorescently labeled platelets in whole blood on opaque surfaces. Using this method, platelet adhesion was quantified on a series of metallic [low-temperature isotropic carbon (LTIC); titanium alloy (Ti); diamond-like carbon (DLC); oxidized titanium alloy (TiO); and polycrystalline diamond (PCD)] and polymeric [woven Dacron (WD)] collagen-impregnated Dacron (HEM), expanded polytetrafluoroethylene (ePTFE), and denucleated ePTFE (dePTFE)] biomaterials designed for use in cardiovascular applications. These materials were perfused with heparinized whole human blood in an in vitro parallel plate flow chamber. Platelet adhesion after 5 min of perfusion ranged from 3.7 +/- 1.0 (dePTFE) to 16.8 +/- 1.5 (WD) platelets/1000 micrometer. The temporal information revealed by these studies provides a comparative measure of the acute thrombogenicity of these materials as well as some insight into their long-term hemocompatibilities. Also studied here were the effects of wall shear rate and axial position on platelet adhesion. A predicted increase in platelet adhesion with increased wall shear rate and a trend toward a decrease in platelet adhesion with increased axial distance was observed with the fiber optic microscope. Future applications for this imaging technique may include the long-term evaluation of thrombosis in blood-contacting devices in vitro and, in animal models, in vivo.     

Characterization of transient platelet contacts on a polyvinyl alcohol hydrogel by video microscopy.

Acridine orange labelled, washed human platelets were counted and tracked on polyvinyl alcohol (PVA), heparin-PVA and polyethylene (PE)-coated coverslips with a view to understand why transient contact on the PVA hydrogels lead to elevated platelet activation and consumption relative to polyethylene. Over the 4 min of initial contact that was studied, platelet adhesion was higher on PE than on PVA or heparin-PVA at both 40 and 200 s(-1), as expected, regardless of whether the surfaces were pre-treated with albumin or fibrinogen. Not all platelets appearing to make contact with the surface, actually attached. For example, less than 2% of the platelets contacting albumin pre-treated PVA (at 40 s(-1)) remained adherent at the end of the initial 60 s observation time, while the corresponding number for PE was greater than 9%. A greater fraction of the platelets remained adherent at the higher shear rate or with fibrinogen pre-treatment, but the difference between PVA and PE remained similar: for example, with fibrinogen pre-treatment at 200 s(-1), approximately 25% of the platelet contacts resulted in adhesion on PVA while 66% did so on PE. While net platelet adhesion was less for the hydrogels, than for PE, the total number of contacts (adherents + non-adherents) were more comparable and unexpectedly higher for albumin pre-treatment than for fibrinogen. Net platelet adhesion is but one component of the total platelet interaction with a material surface. Fluorescent video microscopy has been shown to be a useful, albeit not unequivocal, method for assessing the platelets that make contact with but do not adhere to a surface. reserved     

Platelet-derived growth factors stimulate proliferation and extracellular matrix synthesis of pancreatic stellate cells: implications in pathogenesis of pancreas fibrosis

At present, the cell-cell interactions and molecular mechanisms of pancreas fibrogenesis are largely unknown. The purpose of this study was to investigate paracrine stimulatory loops between platelets and pancreatic stellate cells (PSC). Human PSC were obtained by outgrowth from fibrotic human pancreas. Native platelet lysate (nPL) and transiently acidified platelet lysate (aPL) were added to cultured PSC (passage 4 to 7) in the absence of serum. The synthesis of collagen types I and III and c-fibronectin (cFN) was demonstrated on protein (immunofluorescence and quantitative immunoassay) and mRNA (Northern blot) level. Using sections of human pancreas with acute pancreatitis, platelet aggregates in capillaries were demonstrated by transmission electron microscopy. nPL, and to an even greater extent aPL, significantly increased the synthesis of collagen types I and III and of c-FN (120 microl/ml aPL increased collagen type I concentration in PSC supernatants by 1.99 +/- 0.17 times and c-FN of 2.49 +/- 0.28 times, mean +/- SD, n = 3). nPL and aPL also significantly stimulated cell proliferation (increased bromodeoxyuridine (BrdU) incorporation by 6.4 +/- 0.78 times and 10 +/- 0.29 times, respectively). By preincubating aPL with transforming growth factor beta (TGFbeta)- and platelet-derived growth factor (PDGF)-neutralizing antibodies and the TGFbeta-latency associated peptide, respectively, TGFbeta1 was identified as the main mediator stimulating matrix synthesis and PDGF as the responsible mitogen. Our data demonstrate that platelets contain fibrogenic mediators that stimulate proliferation (PDGF) and matrix synthesis (TGFbeta1) of cultured PSC. We suggest that platelets and PSC cooperate in the development of pancreas fibrosis.     

Morphological characteristics of bovine platelets activated with platelet activating factor or thrombin

The morphological alterations induced by the activation of bovine platelet rich plasma suspensions with the inflammatory mediator, platelet activating factor (PAF), and following the activation of washed bovine platelet suspensions with thrombin are described. The unstimulated bovine platelet exhibits a smooth oval or discoid shape and granules are randomly dispersed throughout the cytoplasm. The initial activation response to PAF is the development of irregularly shaped cells, the migration of granules to the periphery of the cell and the appearance of large pseudopodia devoid of membrane organelles. As activation continues and large platelet aggregates form, two zones of irregularly shaped, discrete platelets are evident within each large aggregate: an outer zone in which the cells are devoid of granules and an inner zone in which many of the platelets exhibit the typical ultrastructural features of a non-activated cell. In washed platelet preparations activated with thrombin, virtually all platelets undergo shape change and yet many cells retain their alpha granules. In addition, discrete irregularly shaped agranular platelets are also found. The distinctive morphological alterations observed in activated bovine platelets are likely associated with the absence of an open canalicular system, characteristic of many other types of mammalian platelets, and with the ability of the cytoplasmic microtubule coil to reorganise into a linear array following thrombin activation. It is postulated that the bovine platelet has evolved as a cell that can respond to various stimuli, for example inflammatory mediators, by releasing active metabolites from its granular stores without forming platelet-platelet bridges that can serve as a foci for thrombus formation. In this manner the bovine platelet can effectively function as an inflammatory cell without acting as a potent thrombogenic agent.     

Effects of leptospiral lipopolysaccharide on rabbit platelets

Leptospiral lipopolysaccharides (LPSs) extracted from Leptospira interrogans serovar copenhageni virulent strain Shibaura, serovar canicola virulent strain Moulton, and serovar hebdomadis strain Hebdomadis, were tested for their ability to induce platelet aggregation and/or lysis in rabbit platelet-rich plasma (PRP). All showed positive reactions with a release of adenosine triphosphate (ATP) and serotonin. The values, however, were different from each other. The ability of leptospiral LPS extracted from serovar copenhageni virulent strain Shibaura (I-LPS) to induce platelet aggregation was the highest of all. After treatment of I-LPS, the platelets developed a ruffled surface with appearance of pseudopodia as observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). I-LPS also showed cytotoxicity for the platelets. Degenerative or lytic changes were recognized in 44.5% of the platelets which were observed 60 min after I-LPS treatment.     

Adsorption and reactivity of human fibrinogen and immunoglobulin G on two types of hemodialysis membranes

Phase contrast microscopic examination of Cuprophane and polyacrylonitrile. After clinical ex vivo use by uremic patients revealed extensive cellular deposition of erythrocytes, platelets, and leukocytes on Cuprophane but not on polyacrylonitrile. In vitro studies with 125I-labeled human fibrinogen or immunoglobulin G (IgG) showed that the adsorption of fibrinogen or IgG was greater on polyacrylonitrile than on Cuprophane. Further studies on the reactivity of fibrinogen adsorbed on polyacrylonitrile surface indicated that the absorbed fibrinogen: (a) was not desorbed readily from the surface, (b) was not appreciably displaced by other plasma proteins such as albumin, IgG, and fibrinogen, (c) was not readily accessible for reaction with 125I-antifibrinogen-IgG, and (d) did not promote the adhesion of 51Cr-labeled platelets. IgG adsorbed on the surface produced essentially no effect on platelet adhesion to polyacrylonitrile and promoted only slightly on the adhesion of granulocytes to the same material. On the other hand, fibrinogen and IgG augmented greatly the platelet adhesion to Cuprophane and IgG enhanced the granulocyte adhesion moderately. These data indicate that fibrinogen and IgG, though at high concentration on polyacrylonitrile, may adsorb in a biologically inactive form. Our observations suggest that the thrombogenicity of an artificial surface may not be assessed entirely by the types and amount of the various protein species adsorbed but is likely determined by the reactivity of the specific protein species adsorbed on the material.     

Glow discharge plasma deposition of tetraethylene glycol dimethyl ether for fouling-resistant biomaterial surfaces.

The glow discharge plasma deposition (GDPD) of tetraethylene glycol dimethyl ether is introduced as a novel method for obtaining surfaces that are resistant to protein adsorption and cellular attachment. Analysis of films by x-ray photoelectron spectroscopy and several biological assays indicate the formation of a fouling-resistant, PEO-like surface on several substrata (e.g., glass, polytetrafluoroethylene, polyethylene). Adsorption of 125I-radiolabelled proteins (fibrinogen, albumin and IgG) from buffer and plasma was very low (typically less than 20 ng/cm2) when compared to the untreated substrata, which exhibited much higher levels of protein adsorption. Not all coated substrata adsorbed equal amounts of protein (e.g., coated glass samples typically adsorbed more protein than coated polyethylene or coated polytetrafluoroethylene samples), suggesting that the substratum used may affect the amount of protein adsorbed. Measurement of dynamic platelet adhesion, using epifluorescent video microscopy, and endothelial cell attachment further demonstrates the short-term nonadhesiveness of these surfaces.     

Temperature-dependent modulation of blood platelet movement and morphology on poly(N-isopropylacrylamide)-grafted surfaces

Poly(N-isopropylacrylamide) (PIPAAm) exhibits a reversible, temperature-dependent soluble/insoluble transition at its lower critical solution temperature (LCST) of 32 degrees C in aqueous media. The temperature-responsive PIPAAm was grafted onto tissue culture polystyrene (TCPS) dish surfaces by electron beam irradiation. Blood platelet behaviors on PIPAAm-grafted surface were examined by computerized image analysis and scanning electron microscopy. Platelet behaviors on this surface were dramatically dependent upon temperature, but those on poly(ethylene glycol)(PEG)-grafted or polystyrene remained unchanged. Below the 32 degrees C (LCST), platelets on PIPAAm-grafted surfaces retained a rounded shape and an oscillating vibratory microbrownian motion for extended times, similarly to those on PEG-grafted surfaces. Above the LCST, platelets readily adhered, spread and developed characteristic pseudopodia on PIPAAm-grafted surface similarly to those on TCPS. An ATP synthesis inhibitor failed to hinder prevention of platelet adhesion onto PIPAAm-grafted surface (below the LCST) suggesting that the preventive mechanism is ATP-independent similarly to that of PEG-grafted surfaces. These results correlate platelet surface activation state with the hydration and structure of polymer surfaces, and demonstrate the ability to modulate such reactions by a small temperature change in situ.     

The minimum surface fibrinogen concentration necessary for platelet activation on dimethyldichlorosilane-coated glass

Albumin and fibrinogen were competitively adsorbed onto dimethyldichlorosilane-coated glass (DDS-glass) and platelet activation was examined as a function of the surface fibrinogen concentration. The weight ratio of albumin to fibrinogen in the adsorption solution was varied from 10 to 700. Platelet activation was quantitated by the area and circularity of spread platelets. When the DDS-glass was coated with albumin alone, platelets were only contact adherent and could not spread at all. After competitive adsorption of fibrinogen and albumin, however, platelets were able to spread on the surface. Platelet activation increased linearly as the surface fibrinogen concentration increased up to 0.02 micrograms/cm2. Platelets were able to activate fully if the surface fibrinogen concentration was 0.02 micrograms/cm2 or higher, even though the surface was dominated by albumin. It appears that platelets can activate fully as long as only a small fraction (2-15%) of the surface is covered with tightly bound fibrinogen.     

Platelet interaction with damaged rabbit aorta.

We have quantified the accumulation of 51Cr-labeled washed rabbit platelets on the subendothelium of rabbit aortae following injury with a balloon catheter. The amount of radioactivity that became associated with the damaged wall within 10 minutes of the injury did not change appreciably during the following 24 hours, indicating that there was little turnover of platelets on the injured vessel wall. In addition, by injecting 51Cr-labeled platelets into rabbits at different times after injury, it was possible to estimate the reactivity of the exposed surface to newly injected platelets. Scanning electron microscopy showed that a monolayer of platelets initially formed on the injured surface; the number of platelets associated with the surface decreased over the 7-day observation period. The amount of 51Cr associated with the injured vessel wall also diminished during this period. The ability of the damaged surface to attract fresh platelets gradually decreased during the 7 days following injury. Platelet survival in rabbits was not significantly reduced following the removal of the aortic endothelium (balloon catheter injury 66.3 +/- 12.2 hours, sham operated 72.1 +/- 7.2 hours, untreated controls 76.2 +/- 3.8 hours). Thus, in rabbits, it cannot be assumed that platelet survival provides an estimate of endothelial injury in all circumstances.     

Secondary scanning electron microscopy of cells infected with murine oncornaviruses.

The surfaces of cells infected with murine oncornaviruses were examined by secondary scanning electron microscopy (SSEM). Virions were found budding at the plasma membrane of productively infected cells, attached to newly infected cells, and adsorbed to the glass substrate between virus-producing cells. Virions were not observed on control uninfected, mock-infected, or nonproducer cultures. The surface of virions was composed of symmetrically arrayed globular units 20–28 nm in diameter. The distribution of budding Kirsten murine sarcoma virus on individual cells appeared random over the cell surface.     

Effects of fibrinogen residence time and shear rate on the morphology and procoagulant activity of human platelets adherent to polymeric biomaterials

Fibrinogen readily adsorbs to the surface of biomaterials and, because of its demonstrated ability to support platelet adhesion and aggregation, plays a role in thrombotic events associated with the implantation of synthetic materials in the human body. Thus, understanding the factors influencing the interactions of fibrinogen with biomaterials, and how platelet responses are affected, is crucial for the development of synthetic materials exhibiting improved blood compatibility. In this study, the effects of fibrinogen residence time and shear rate on the procoagulant activity of adherent platelets, along with their morphologic status, as deduced from scanning electron microscopy, were investigated. To examine whether adherent platelets promoted the generation of thrombin, polymeric materials (polytetrafluoroethylene, polyethylene, and silicone rubber) preadsorbed with fibrinogen were exposed to platelet suspensions at different wall shear rates and then incubated with clotting factors for 5 minutes under static conditions. The amount of thrombin generated per platelet was calculated from the optical density of the color developed by adding substrate S-2238. Scanning electron microscopy images of the platelets revealed that the platelets exhibited different morphologies, depending on the shear rate and residence time of the adsorbed fibrinogen. Platelets ranged from their normal discoid shape observed primarily under static conditions, to that of fully spread platelets. Results from this study show that platelets, in the presence of shear forces, undergo activation on exposure to surfaces on which adsorbed fibrinogen has resided for short residence times rather than long residence times. Interestingly, studies examining the procoagulant responses of such adherent platelets demonstrated that the platelets attached to the fibrinogen coated materials did not promote significant thrombin generation. Such low prothrombinase activity of adherent platelets suggests that adsorbed fibrinogen, while capable of supporting platelet adhesion and spreading on biomaterials, does not necessarily enhance the procoagulant activity of adherent platelets.