Biocompatibility of Polysulfone Hemodialysis Membranes and Its Mechanisms: Involvement of Fibrinogen and Its Integrin Receptors in Activation of Platelets and Neutrophils.

Activation of blood cells during hemodialysis is considered to be a significant determinant of biocompatibility of the hemodialysis membrane because it may affect patient health adversely through microvascular inflammation and oxidative stress. This study found very different cell activation among various polysulfone (PSf) hemodialysis membranes. For example, CX-U, a conventional PSf membrane, induced marked adhesion of platelets to its surface and increased surface expression of activated CD11b and production of reactive oxygen species (ROS) by neutrophils; while NV-U, a hydrophilic polymer-immobilized PSf membrane, caused little platelet adhesion and slight CD11b expression and ROS production by neutrophils. Analysis of the molecular mechanisms of the above phenomena on CX-U and NV-U indicated that anti-integrin GPIIb/IIIa antibody blocked platelet adhesion, and that the combination of anti-CD11b (integrin α subunit of Mac-1) and anti-integrin αvß3 antibodies blocked ROS production by neutrophils. Plasma-derived fibrinogen, a major ligand of GPIIb/IIIa, Mac-1, and αvß3 on membranes, was thus analyzed and found to be more adsorbed to CX-U than to NV-U. Moreover, comparison between five PSf membranes showed that the number of adherent platelets and neutrophil ROS production increased with increasing fibrinogen adsorption. These results suggested that fibrinogen, adsorbed on membranes, induced GPIIb/IIIa-mediated platelet activation and Mac-1/αvß3-mediated neutrophil activation, depending on the amount of adsorption. In conclusion, the use of biocompatible membranes like NV-U, which show lower adsorption of fibrinogen, is expected to reduce hemodialysis-induced inflammation and oxidative stress by minimizing cell activation.

Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation.

As platelets aggregate and activate at the site of vascular injury to stem bleeding, they are subjected to a myriad of biochemical and biophysical signals and cues. As clot formation ensues, platelets interact with polymerizing fibrin scaffolds, exposing platelets to a large range of mechanical microenvironments. Here, we show for the first time (to our knowledge) that platelets, which are anucleate cellular fragments, sense microenvironmental mechanical properties, such as substrate stiffness, and transduce those cues into differential biological signals. Specifically, as platelets mechanosense the stiffness of the underlying fibrin/fibrinogen substrate, increasing substrate stiffness leads to increased platelet adhesion and spreading. Importantly, adhesion on stiffer substrates also leads to higher levels of platelet activation, as measured by integrin αIIbß3 activation, α-granule secretion, and procoagulant activity. Mechanistically, we determined that Rac1 and actomyosin activity mediate substrate stiffness-dependent platelet adhesion, spreading, and activation to different degrees. This capability of platelets to mechanosense microenvironmental cues in a growing thrombus or hemostatic plug and then mechanotransduce those cues into differential levels of platelet adhesion, spreading, and activation provides biophysical insight into the underlying mechanisms of platelet aggregation and platelet activation heterogeneity during thrombus formation.

Interleukin 1 receptor 1 and interleukin 1ß regulate megakaryocyte maturation, platelet activation, and transcript profile during inflammation in mice and humans.

OBJECTIVE: Interleukin 1 Receptor 1 (IL1R1) and its ligand, IL1ß, are upregulated in cardiovascular disease, obesity, and infection. Previously, we reported a higher level of IL1R1 transcripts in platelets from obese individuals of the Framingham Heart Study (FHS), but its functional effect in platelets has never been described. Additionally, IL1ß levels are increased in atherosclerotic plaques and in bacterial infections. The aim of this work is to determine whether IL1ß, through IL1R1, can activate platelets and megakaryocytes to promote atherothrombosis. APPROACH AND RESULTS: We found that IL1ß-related genes from platelets, as measured in 1819 FHS participants, were associated with increased body mass index, and a direct relationship was shown in wild-type mice fed a high-fat diet. Mechanistically, IL1ß activated nuclear factor-κB and mitogen-activated protein kinase signaling pathways in megakaryocytes. IL1ß, through IL1R1, increased ploidy of megakaryocytes to 64+ N by 2-fold over control. IL1ß increased agonist-induced platelet aggregation by 1.2-fold with thrombin and 4.2-fold with collagen. IL1ß increased adhesion to both collagen and fibrinogen, and heterotypic aggregation by 1.9-fold over resting. High fat diet-enhanced platelet adhesion was absent in IL1R1(-/-) mice. Wild-type mice infected with Porphyromonas gingivalis had circulating heterotypic aggregates (1.5-fold more than control at 24 hours and 6.2-fold more at 6 weeks) that were absent in infected IL1R1(-/-) and IL1ß(-/-) mice. CONCLUSIONS: In summary, IL1R1- and IL1ß-related transcripts are elevated in the setting of obesity. IL1R1/IL1ß augment both megakaryocyte and platelet functions, thereby promoting a prothrombotic environment during infection and obesity; potentially contributing to the development of atherothrombotic disease.

Hydrogen peroxide promotes aging-related platelet hyperactivation and thrombosis.

BACKGROUND: The incidence of thrombotic events increases during aging, but the mechanisms are not well understood. To investigate the prothrombotic role of oxidative stress during aging, we tested the hypothesis that aged mice overexpressing the antioxidant enzyme glutathione peroxidase-1 (Gpx1) are protected from experimental thrombosis. METHODS AND RESULTS: Susceptibility to carotid artery thrombosis was first examined in wild-type C57BL/6J mice. After photochemical injury of the carotid artery, the time to stable occlusion was significantly shorter in 12- and 18-month-old mice compared with 4-month-old mice (P<0.01). Unlike wild-type mice, transgenic mice overexpressing Gpx1 (Gpx1 Tg) did not exhibit shortened times to occlusion of the carotid artery at 12 or 18 months of age. Wild-type mice also exhibited increased susceptibility to venous thrombosis after inferior vena cava ligation at 12 or 18 months of age (P<0.05 versus 4 months of age). Gpx1 Tg mice were protected from this aging-related enhanced susceptibility to venous thrombosis. Age-dependent platelet hyperactivation, evidenced by increased hydrogen peroxide, fibrinogen binding, and activation of fibrinogen receptor αIIbß3, was observed in thrombin-activated platelets from wild-type but not Gpx1 Tg mice (P<0.05). Enhanced platelet activation responses in aged mice were also prevented by polyethylene glycol-catalase or apocynin, an inhibitor of NADPH oxidase. Aged mice displayed increased intraplatelet expression of p47(phox) and superoxide dismutase-1, suggesting a mechanistic pathway for increased hydrogen peroxide generation. CONCLUSIONS: Our findings demonstrate that hydrogen peroxide is a key mediator of platelet hyperactivity and enhanced thrombotic susceptibility in aged mice.

Platelet-rich preparations to improve healing. Part II: platelet activation and enrichment, leukocyte inclusion, and other selection criteria.

Multiple platelet-rich preparations have been reported to improve wound and bone healing, such as platelet-rich plasma (PRP) and platelet rich fibrin (PRF). The different methods employed during their preparation are important, as they influence the quality of the product applied to a wound or surgical site. Besides the general protocol for preparing the platelet-rich product (discussed in Part 1 of this review), multiple choices need to be considered during its preparation. For example, activation of the platelets is required for the release and enmeshment of growth factors, but the method of activation may influence the resulting matrix, growth factor availability, and healing. Additionally, some methods enrich leukocytes as well as platelets, but others are designed to be leukocyte-poor. Leukocytes have many important roles in healing and their inclusion in PRP results in increased platelet concentrations. Platelet and growth factor enrichment reported for the different types of platelet-rich preparations are also compared. Generally, TGF-ß1 and PDGF levels were higher in preparations that contain leukocytes compared to leukocyte-poor PRP. However, platelet concentration may be the most reliable criterion for comparing different preparations. These and other criteria are described to help guide dental and medical professionals, in large and small practices, in selecting the best procedures for their patients. The healing benefits of platelet-rich preparations along with the low risk and availability of simple preparation procedures should encourage more clinicians to incorporate platelet-rich products in their practice to accelerate healing, reduce adverse events, and improve patient outcomes.

Designing an experimental method for assessing biocompatibility of circuit coatings using biomarkers for platelet activation during cardiopulmonary bypass.

INTRODUCTION: Cardiopulmonary bypass is an essential component of cardiothoracic surgeries. However, significant complications such as systemic inflammatory response syndrome (SIRS) resulting from cardiopulmonary bypass (CPB) are a common occurrence due to contact between circulating blood and foreign surfaces that leads to platelet activation. It is suggested that different available CPB circuit coatings can potentially reduce platelet activation. However, there have been no published evidence-based reports confirming these claims. In addition, there is no well-established protocol for studying platelet activation biomarkers during CPB in vitro in a laboratory setting. METHODS: CPB was simulated in the laboratory using bovine blood in two different types of coated CPB circuits: Trillium® Biosurface by Medtronic, and XcoatingTM Surface by Terumo. Fresh bovine blood samples were collected and circulated through the CPB circuit following the standard protocol used in the operation rooms. Blood samples were then collected at 5 min, 30 min, and 55 min during the circulation. Blood plasmas were separated and subjected to enzyme-linked immunosorbent assay to measure most established platelet activation markers P-selectin, Platelet Factor 4 (PF4), Glycoprotein IIb/IIIa (GPIIb/IIIa), and ß-thromboglobulin (ß-TG) at different time points. RESULTS: The biomarker values at 30 min and 55 min were compared to the base values at 5 min for each type of CPB circuit. The results of the means from all measured biomarkers showed data measurements that indicated no significant variability within each coating. All collected data points fell within ±2 SD of the means, which was considered acceptable variations across technical replicates.  Conclusion: In this study, we were able to establish an in vitro protocol in the laboratory setting that is precise and reliable with minimum intra-variability. This established protocol will allow for future studies in which different coated CPB circuits can be compared for their effectiveness in blocking platelet activation during the CPB.

A novel in vitro model for preclinical testing of the hemocompatibility of intravascular stents according to ISO 10993-4.

Subacute stent thrombosis, caused by undesired interactions between blood and the stent surface, is a major concern in the first few weeks following coronary artery stent implantation. The aim of this study was to establish a novel in vitro model for hemocompatibility testing of coronary artery stents according to ISO 10993-4. The model consists of a modified Chandler-Loop design with closed heparin-coated PVC Loops and a thermostated water bath. The tests were performed with anticoagulated human whole blood. After incubation in the loop, blood was analyzed for coagulation and inflammatory activation markers (TAT, ß-TG, sP-selectin, SC5b-9 and PMN-elastase). Three different stent types with varying thrombogenicity were tested; statistically significant differences were found between the three stent types in measures of coagulation and platelet activation. The new Chandler-Loop model can be used as an alternative to animal and current in vitro models, especially for the determination of early events after stent implantation.

The Effects of the Combined Argatroban/Nitric Oxide-Releasing Polymer on Platelet Microparticle-Induced Thrombogenicity in Coated Extracorporeal Circuits.

Clotting, anticoagulation, platelet consumption, and poor platelet function are major factors in clinical extracorporeal circulation (ECC). We have shown that nitric oxide-releasing (NOReL) coatings prevent thrombosis in a rabbit model of ECC without systemic anticoagulation. Nitric oxide-releasing prevents platelet adhesion and activation, resulting in preserved platelet count and function. Previous work has shown that activated platelets form platelet-derived microparticles (PMPs). These experiments were designed to determine if PMPs can identify platelet function during ECC. The objective of this study is to investigate the effects of NOReL on platelet activation and PMP formation during ECC. Uncoated ECCs, including with and without systemic heparin, and NOReL-coated ECCs, including DBHD/N2O2 and argatroban (AG)/DBHD/N2O2-coated ECCs without systemic heparin, were tested in a 4-hour rabbit thrombogenicity model. Before and after ECC exposure, platelets were stimulated with collagen, and PMPs were measured using flow cytometry. The uncoated ECCs clotted within the first hour, while the NOReL-coated ECCs circulated for 4 hours. During pre-ECC blood exposure, platelets stimulated with collagen produced PMPs. With post-ECC exposure, platelets from uncoated circuits generated less PMPs than baseline (mean ± SDs: 23246 ± 3611 baseline vs. 1300 ± 523 uncoated post circuit, p = 0.018) when stimulated with collagen. However, platelets from the AG/DBHD/N2O2-coated ECCs generated a greater number of PMPs as baseline values (23246 ± 3611 baseline vs. 37040 ± 3263 AG/DBHD/N2O2 post 4 hours circuit, p = 0.023). Blood exposure during ECC results in platelet activation and clotting in uncoated ECCs. The remaining circulating platelets have lost function, as demonstrated by the low PMP formation in response to collagen. AG/DBHD/N2O2-coated ECCs prevented significant platelet activation and clotting, while DBHD/N2O2 trended towards prevention of platelet activation. In addition, function of the circulating platelets was preserved, as demonstrated by PMP formation in response to collagen. These results indicate that PMPs may be an important measure of platelet activation during ECC. Platelet-derived microparticles may provide a simplified way to measure platelet function during clinical ECC.

The platelet actin cytoskeleton associates with SNAREs and participates in alpha-granule secretion.

Following platelet activation, platelets undergo a dramatic shape change mediated by the actin cytoskeleton and accompanied by secretion of granule contents. While the actin cytoskeleton is thought to influence platelet granule secretion, the mechanism for this putative regulation is not known. We found that disruption of the actin cytoskeleton by latrunculin A inhibited alpha-granule secretion induced by several different platelet agonists without significantly affecting activation-induced platelet aggregation. In a cell-free secretory system, platelet cytosol was required for alpha-granule secretion. Inhibition of actin polymerization prevented alpha-granule secretion in this system, and purified platelet actin could substitute for platelet cytosol to support alpha-granule secretion. To determine whether SNAREs physically associate with the actin cytoskeleton, we isolated the Triton X-100 insoluble actin cytoskeleton from platelets. VAMP-8 and syntaxin-2 associated only with actin cytoskeletons of activated platelets. Syntaxin-4 and SNAP-23 associated with cytoskeletons isolated from either resting or activated platelets. When syntaxin-4 and SNAP-23 were tested for actin binding in a purified protein system, only syntaxin-4 associated directly with polymerized platelet actin. These data show that the platelet cytoskeleton interacts with select SNAREs and that actin polymerization facilitates alpha-granule release.

Comparison of the Thrombogenicity of a Bare and Antithrombogenic Coated Flow Diverter in an In Vitro Flow Model.

BACKGROUND: Dual antiplatelet therapy is a pre-requisite for flow diverter (FD) implantation. The purpose of this study was to assess the thrombogenicity of the p48 FD, coated with the newly developed phenox Hydrophilic Polymer Coating (p48_HPC, phenox GmbH, Germany) in comparison with uncoated p48 FDs in an in vitro flow model (Chandler Loop). METHODS: p48 and p48_HPC FDs were implanted into silicon tubes filled with whole human blood and incubated at 37 °C under pulsating flow. After 120 min, platelet count was determined in the blood. Platelet activation markers (PAR1) and formation of microparticles were analyzed in a flow cytometer. Fluorescence microscopy of CD42a positive cells and scanning electron microscopy was used to detect adherent platelets on the wire surface. RESULTS: Platelets in contact with the uncoated p48 FDs are significantly more activated than those incubated with p48_HPC (73 ± 9% vs. 65 ± 6%, p < 0.05) and release more microparticles (1.8 ± 0.5 vs. 1.4 ± 0.4, p < 0.05). The platelet count after 120-min circulation in the Chandler Loop was significantly lower for the uncoated p48 compared to the p48_HPC indicating significantly greater adherence of the platelets to the p48 (71 ± 8% vs. 87 ± 5%, p < 0.05). SEM and fluorescent antibody imaging revealed minimal platelet adherence to the surface of the p48_HPC compared to the uncoated p48. CONCLUSION: The pHPC coating significantly reduces thrombogenicity of the p48 FD. This may help to reduce the risk of thromboembolic complications when using these devices. A reduction in antiplatelet therapy may be possible.

Comparison of Blood Compatibility in Various Membrane Materials for Continuous Renal Replacement Therapy.

Because hemofilters used for continuous renal replacement therapy contact with blood over a prolonged period during treatments, platelet activation may occur stronger. The purpose of this study is to clarify the blood compatibility in three hemofilters mostly used in Japan. We compared the blood compatibility of the two polysulfone (AEF: Asahi Kasei Medical Co., Tokyo, Japan and SHG: Toray Medical Co., Ltd., Tokyo, Japan) and one polymethylmethacrylate membranes (CH: Toray Medical Co., Ltd.). First, test blood was collected from healthy volunteers. Subsequently, the blood was circulated by a roller pump at the rate of 100 mL/min. We measured the platelet counts and platelet factor 4 (PF4). The platelet counts at 48 h in polymethylmethacrylate membrane were significantly less than that in polysulfone membranes. Levels of the PF4 after the circulation were 978.5 ± 200.0 ng/dL with AEF, 863.0 ± 233.9 ng/dL with SHG and 1780.0 ± 465.1 ng/dL with CH, respectively. Hemofilters with polysulfone membranes showed less platelet activation. It was inferred that the amount of PVP, the smoothness of the membrane surface, and the inner diameter of the hollow fiber affect the blood compatibility in the hemofilter.

Aptamers recognizing fibronectin confer improved bioactivity to biomaterials and promote new bone formation in a periodontal defect in rats.

The use of alloplastic materials in periodontal regenerative therapies is limited by their incapacity to establish a dynamic dialog with the surrounding milieu. The aim of the present study was to control biomaterial surface bioactivity by introducing aptamers to induce the selective adsorption of fibronectin from blood, thus promoting platelets activation in vitro and bone regeneration in vivo. A hyaluronic acid/polyethyleneglycole-based hydrogel was enriched with aptamers selected for recognizing and binding fibronectin. In vitro, the capacity of constructs to support osteoblast adhesion, as well as platelets aggregation and activation was assessed by chemiluminescence within 24 h. Matrices were then evaluated in a rat periodontal defect to assess their regenerative potential by microcomputed tomography (µCT) and their osteogenic capacity by Luminex assay 5, 15 and 30 d postoperatively. Aptamers were found to confer matrices the capacity of sustaining firm cell adhesion (p = 0.0377) and to promote platelets activation (p = 0.0442). In vivo, aptamers promoted new bone formation 30 d post-operatively (p < 0.001) by enhancing osteoblastic lineage commitment maturation. Aptamers are a viable surface modification, which confers alloplastic materials the potential capacity to orchestrate blood clot formation, thus controlling bone healing.

Preventive effect of alpha-tocopherol and glycyrrhizin against platelet-neutrophil complex formation induced by hemodialysis membranes.

BACKGROUND: The intradialytic activation of leukocytes is a major cause of hemodialysis (HD)-associated complications. Contact between blood and HD membranes frequently induces the formation of microaggregates composed of activated platelets and leukocytes, causing leukocyte activation that includes the generation of reactive oxygen species (ROS). This complex formation is mediated primarily by the interaction between P-selectin on activated platelets and its counter-ligands on leukocytes. OBJECTIVE: We examined the preventive effects of alpha-tocopherol and glycyrrhizin in vitro against platelet- neutrophil microaggregate formation and neutrophil ROS production induced by HD membranes. METHODS AND RESULTS: Microaggregate formation induced by the incubation of heparinized whole blood with polysulfone (PS) HD membranes was effectively inhibited by alpha-tocopherol and glycyrrhizin. alpha-Tocopherol, but not glycyrrhizin, was found to inhibit PS membrane-induced P-selectin expression on the platelet surface; however, glycyrrhizin did inhibit both the formation of neutrophil-platelet microaggregates induced by adenosine diphosphate (ADP) and the adhesion of HL60 leukemic cells to P-selectin-expressing Chinese hamster ovary (CHO) cells, suggesting that glycyrrhizin acts as a competitive inhibitor of P-selectin-mediated cell adhesion. Finally, these compounds almost completely abrogated PS membrane-induced and platelet-dependent ROS production by neutrophils. CONCLUSIONS: These results suggest that alpha-tocopherol and glycyrrhizin may function as preventive agents of HD-associated leukocyte activation though the modulation of platelet-leukocyte interaction.

Shear-induced platelet adherence and activation in an in-vitro dynamic multiwell-plate system.

Circulating blood cells are prone to varying flow conditions when contacting cardiovascular devices. For a profound understanding of the complex interplay between the blood components/cells and cardiovascular implant surfaces, testing under varying shear conditions is required. Here, we study the influence of arterial and venous shear conditions on the in vitro evaluation of the thrombogenicity of polymer-based implant materials.Medical grade poly(dimethyl siloxane) (PDMS), polyethylene terephthalate (PET) and polytetrafluoroethylene (PTFE) films were included as reference materials. The polymers were exposed to whole blood from healthy humans. Blood was agitated orbitally at low (venous shear stress: 2.8 dyne · cm-2) and high (arterial shear stress: 22.2 dyne · cm-2) agitation speeds in a well-plate based test system. Numbers of non-adherent platelets, platelet activation (P-Selectin positive platelets), platelet function (PFA100 closure times) and platelet adhesion (laser scanning microscopy (LSM)) were determined.Microscopic data and counting of the circulating cells revealed increasing numbers of material-surface adherent platelets with increasing agitation speed. Also, activation of the platelets was substantially increased when tested under the high shear conditions (P-Selectin levels, PFA-100 closure times). At low agitation speed, the platelet densities did not differ between the three materials. Tested at the high agitation speed, lowest platelet densities were observed on PDMS, intermediate levels on PET and highest on PTFE. While activation of the circulating platelets was affected by the implant surfaces in a similar manner, PFA closure times did not reflect this trend.Differences in the thrombogenicity of the studied polymers were more pronounced when tested at high agitation speed due to the induced shear stresses. Testing under varying shear stresses, thus, led to a different evaluation of the implant thrombogenicity, which emphasizes the need for testing under various flow conditions. Our data further confirmed earlier findings where the same reference implants were tested under static (and not dynamic) conditions and with fresh human platelet rich plasma instead of whole blood. This supports that the application of common reference materials may improve inter-study comparisons, even under varying test conditions.

Surface modification of metallic materials designed for a new generation of artificial heart valves.

PURPOSE:: The main goal of this work was to develop haemocompatibile thin film materials dedicated to novel flexible mechanical heart valves intended for pulsatile ventricle assist devices. METHODS:: The studies performed have led to the selection of a material for the surface modification of the metallic scaffold. Haemocompatible, biofunctional, ultra-elastic, thin carbon-based coatings were proposed. The surface was designed to eliminate thrombogenic and microbial construction by a reduction in turbulence and sufficient washing of the biofunctional-adapted surfaces, thus allowing for extended use for temporary heart support. The article presents the influence of the mechanical properties of coatings and their influence on the haemocompatibility. In this study, we investigated a simplified model of the whole blood shear stress based on a cone and plate rotational viscometer. Several indices of platelet activation were analysed, including platelet and granulocyte-platelet aggregates, platelet activation markers and platelet-derived microparticles. RESULTS:: The shear stress induced a platelet aggregate count in the range from 2% to 30% of the CD61 positive cells. For polyurethane (PU), the average value of platelet aggregates was on the level of 7%. The analyses have demonstrated that the cytometric methods of the direct determination of platelet-derived microparticles in plasma are reproducible and reliable. Considering the generation of microparticles on the tested coatings under hydrodynamic conditions, the best properties were observed for the coating a-C:H,N. CONCLUSION:: The results indicate that a-C:H-based coatings with the thickness of 110 nm do not induce an immune response and do not influence the origin of platelet microparticle formation; thus, these type of coatings are the most promising for the parts which are planned to withstand blood contact under the high value of shear stress.

The effect of gas plasma modification on platelet and contact phase activation processes.

Medical-grade polytetrafluoroethylene (PTFE), polydimethylsiloxane (PDMS), polyetherurethane (PEU) and ultrahigh molecular weight polyethylene (UHMWPE) were plasma treated with O2, Ar, N2 and NH3. Their surface properties were characterised using X-ray photoelectron spectroscopy (XPS), static secondary ion mass spectroscopy (SSIMS), atomic force microscopy (AFM) and dynamic contact angle (DCA) analysis. Platelet adhesion, aggregation, activation and release of microparticles were determined after contact with whole blood in a cone and plate viscometer. Activation of the coagulation system was quantified in a static environment using a partial thromboplastin time (PTT) assay. The chemical compositions of the untreated surfaces were found to be very similar to those of the bulk material except for PEU, whose surface was comprised almost entirely of soft ether segments. For all materials, the different plasma treatments resulted in moderate etching with the incorporation of functional groups and removal of side groups: defluorination, dehydrogenation, cleavage of methyl side groups and soft segments for PTFE, UHMWPE, PDMS and PEU, respectively. Consequently, plasma treatment resulted in increased wettability in all cases. Blood contact with the virgin materials resulted in activation of platelets and the clotting cascade. Plasma treatment resulted in a significant reduction in platelet adhesion for all materials and all treatments. In the case of PTFE and PEU, the activation status of these cells was also reduced. Plasma treatment of all materials reduced fluid-phase CD62P expression. Platelet aggregate size correlated well with degree of aggregate formation, but many treatments increased the degree of aggregation, as was the case for microparticle shedding. There was no correlation between CD62P expression, aggregate formation and platelet microparticle (PMP) shedding. It is concluded that despite incorporation of the same chemical groups, the pattern of response to blood in vitro is not the same across different polymers.

The role of whole blood in thrombin generation in contact with various titanium surfaces.

Understanding of the thrombotic response (activation of the intrinsic coagulation system followed by platelet activation) from blood components upon contact with a titanium dental implant is important and not fully understood. The aims of this study were to evaluate: (1) the thrombogenic response of whole blood, platelet-rich plasma (PRP) and platelet-poor plasma (PPP) in contact with a highly thrombogenic surface as titanium, (2) the thrombogenic response of clinically used surfaces as hydroxyapatite (HA), machined titanium (mTi), TiO2 grit-blasted titanium (TiOB) and fluoride ion-modified grit-blasted titanium (TiOB-F). An in vitro slide chamber model, furnished with heparin, was used in which whole blood, PRP or PPP came in contact with slides of the test surfaces. After incubation (60 min rotation at 22 rpm in a 37 degrees C water bath), blood/plasma was mixed with EDTA or citrate, further centrifuged at +4 degrees C (2200 g at 10 min). Finally, plasma was collected pending analysis. Whole blood in contact with Ti alloy resulted in the binding of platelets to the material surface and in the generation of thrombin-antithrombin (TAT) complexes. With whole blood TAT levels increased 1000-fold compared with PRP and PPP, in which both almost no increase of TAT could be detected. In addition, the platelet activation showed a similar pattern with a 15-fold higher release of beta-TG in whole blood. In the in vitro chamber model with the clinically relevant materials, the fluoride-modified surface (TiOB-F) showed pronounced TAT generation compared with TiOB, mTi and HA. Similar results were achieved for platelet consumption and activation markers of the intrinsic coagulation system. Taken together these results implicate first that whole blood is necessary for sufficient thrombin generation and platelet activation during placement of implants. Second, a fluoride ion modification seems to augment the thrombogenic properties of titanium.

Redistribution of activated pp60c-src to integrin-dependent cytoskeletal complexes in thrombin-stimulated platelets.

Thrombin stimulation of platelets induces a transient increase in the specific activity of pp60c-src followed by a redistribution of pp60c-src to the Triton X-100-insoluble, cytoskeleton-rich fraction. Concomitant with the observed increase in pp60c-src activity was a rapid dephosphorylation of tyrosine 527 in 10 to 15% of pp60c-src molecules. In addition, we found that pp60c-src from the Triton-insoluble fraction was phosphorylated on tyrosine 416, the autophosphorylation site which is phosphorylated in activated oncogenic variants of pp60src. Furthermore, in platelets from patients with Glanzmann's thrombasthenia (which are deficient in the integrin receptor GPIIb-IIIa), pp60c-src was not translocated to the Triton-insoluble fraction, and there was a sustained increase in pp60c-src activity following thrombin treatment. These results suggest that pp60c-src is rapidly activated in thrombin-stimulated platelets, potentially by a protein tyrosine phosphatase, before it translocates to a cytoskeletal fraction, where many of its potential substrates are found. The evidence that the cytoskeletal association of pp60c-src is dependent upon engagement of the integrin receptor GPIIb-IIIa suggests that integrin-cytoskeletal complexes may serve to compartmentalize and anchor activated enzymes involved in signal transduction.

The influence of intrinsic coagulation pathway on blood platelets activation by oxidized cellulose.

Oxidized cellulose is an effective hemostat that works naturally to aid in blood coagulation. The mechanism of its action is not very well understood. Little effect on blood coagulation, but a pronounce decrease in platelet count has been reported upon the addition of the oxidized cellulose to the whole blood. As a marker of platelet activation and aggregation we used serotonin release reaction and turbidity changes in time. We found that oxidized cellulose did not activate washed platelets reconstituted in plasma-free medium or plasma-free medium with fibrinogen; no reduction of platelet count was observed. Serotonin release in platelet-rich plasma incubated with oxidized cellulose started in the range from 5 to 10 min. Serotonin release from platelets reconstituted in plasma deficient in either coagulation factor V, VIII, IX, or XII was delayed. Blood platelets activation by oxidized cellulose requires calcium ions present in dispersion of oxidized cellulose. Factor XIII deficiency had no influence on blood platelets activation by oxidized cellulose. Our results clearly indicate the significance of intrinsic coagulation pathway activation on blood platelets activation by oxidized cellulose and so indirectly on the hemostyptic effect of oxidized cellulose.