Number Of Adherent Platelets Research Articles

Platelet adhesiveness and lipoprotein lipase activity in patients with benign and malignant disease of the prostate.

Abstract Platelet adhesiveness and lipoprotein lipase (clearing factor lipase) activity have been measured in 22 patients with benign prostatic obstruction and in 20 patients with prostatic carcinoma. The total platelet counts and the number of adherent platelets were significantly higher and the lipoprotein lipase responses to heparin were significantly lower in the patients with carcinoma.

Plasma Fibronectin Enhances Adhesion and Aggregation of Platelets by Both Its Incorporation into Adherent Platelets and Its Crosslinking to Fibrin Networks.

Plasma fibronectin has recently been demonstrated to stabilize growth of platelet-rich thrombi in injured mesenteric arterioles in vivo. We have determined the impact of two forms of insolubilized plasma fibronectin on adhesion and aggregation of platelets - fibronectin-crosslinked fibrin by activated factor XIII (fibronectin-fibrin) and fibronectin assembled by adherent platelets. In static adhesion assays, 2-fold more platelets adhered to fibronectin-fibrin than to fibrin. Antibody blocking studies demonstrated that a different mix of αIIbβ3, αvβ3, and α5β1 integrins mediate platelet adhesion to both substrates. At a shear rate of 300 or 1,250s-1 in a parallel plate flow chamber, more platelets adhered and aggregated on surfaces coated with fibronectin-fibrin than surfaces coated with fibrin. The number of adherent platelets and the volume of platelet thrombi increased by 3- to 4-fold when soluble fibronectin was co-perfused with platelets, which resulted in assembly of fibronectin by adherent platelets. Increased adhesion and aggregation of platelets were dependent on the amounts of fibronectin crosslinked to fibrin prior to perfusion or included in the perfusate. The effects of perfused fibronectin were blocked by two inhibitors of fibronectin assembly, the N-terminal 70-kD fragment of fibronectin or the functional upstream domain of protein F1 of Streptococcus pyogenes, indicating that assembly of soluble fibronectin by adherent platelets is mediated by the binding of the N-terminal region of fibronectin to the platelet surface. Thus, plasma fibronectin has the potential to enhance adhesion and aggregation of platelets in vivo as a result of being crosslinked to fibrin networks and its subsequent assembly by adherent platelets.

New Method for Evaluating Material Blood Compatibility Using Microchannel Array

Microchannel array chips modified by metal deposition or polymer coating were contacted with blood. Titanium (Ti), chromium (Cr) and gold (Au) films were deposited onto the microchannel array chips. Albumin (Alb) and MPC polymer (MPC) were coated onto other chips. Non-treated Si chips were used as a control. Whole blood was collected with 1000 units/ml heparin solution from young healthy volunteers. The passing time of a 100 µl portion of human whole blood through these channels was measured under a pressure difference of 20 cm H2O. Simultaneously, the flow behavior of blood cells in channel was observed by an optical microscope and recorded by a video recorder. Platelet adhesion was observed on Si, Ti, Cr, and especially on Au. The blood pass-through time (BPT) increased in order of Ti, Si, Cr and Au. On the Alb- and MPC-coated chips, platelets were seldom observed and the BPTs were short in comparison with the metal chips. From these consequences, platelet adhesion depended on the materials. The BPT correlated well to the number of adherent platelets on the materials. Therefore, the blood coagulation reaction originated in platelet activation could be detected using microchannel array. We concluded that this method could be applied to evaluate initial blood compatibility of materials within several minutes in vitro.

Platelet function in acute experimental pancreatitis induced by ischaemia–reperfusion

Ischaemia-reperfusion (IR)-associated microcirculatory changes play a major role in acute post-transplantation pancreatitis. The pathophysiological role of platelets in these events is unknown. The aim of this study was to examine platelet adhesion and function during early reperfusion after pancreatic ischaemia. Rats were subjected to warm pancreatic ischaemia by cross-clamping of the pancreatic vessels for 1 h. After 1 h of reperfusion, platelet-endothelium interaction was evaluated after platelet separation and staining by fluorescence microscopy. Amylase levels and pancreatic histology were evaluated 24 h after reperfusion. Animals treated according to an identical protocol, but without ischaemia, served as controls. Mild pancreatitis had developed by 24 h after IR; serum amylase levels were significantly higher than those in control animals. The numbers of adherent platelets in capillaries and venules were significantly increased, and platelet velocity in capillaries was significantly decreased, in the IR group compared with controls. There was significantly more oedema and inflammation in pancreatic tissue after IR. Warm ischaemia for 1 h followed by reperfusion for 24 h caused mild pancreatitis in this experimental model. The pancreatic microcirculation was characterized by pronounced platelet-endothelium interaction in capillaries and venules. These results suggest that platelet activation may play an important role in acute post-transplantation pancreatitis.

Biomechanical Properties of Bonds Mediated by the von Willebrand Factor A1 Domain during Platelet Adhesion under Flow Conditions.

The initial attachment of platelets to vascular lesions exposed to a high wall shear rate (γw) depends on the interaction between the membrane glycoprotein (GP) Ibα, a component of the GP Ib-IX-V receptor complex, and the A1 domain of surface-immobilized von Willebrand factor (VWFA1). We performed perfusion experiments under different flow conditions to measure transient platelet contacts onto immobilized recombinant VWFA1 and determine the probability of bond formation (capturing) and resistance to tensile stress (bond lifetime) of VWFA1-GP Ibα interactions. To define how molecular conformations influence the biomechanical properties of the bonds, we compared fragments exhibiting the native dimeric assembly of A1 domain with monomeric fragments obtained by selective purification of recombinant protein expressed in stable D. melanogaster cell lines. The minimum coating concentration of dimeric VWFA1 at which platelet adhesion events were statistically significantly different from nonspecific interactions on uncoated glass was 1 μg/ml. In the range of γw between 30 and 30,000 s−1, there was no threshold value for the initiation of adhesion, as seen for selectins. The number of adhering platelets first increased and then decreased with monotonically increasing γw, indicating the effect of transport phenomena as well as hydrodynamic forces on the VWFA1-GP Ibα interaction. Maximum event number was at 5,000 s−1 for dimeric and 1,500 s−1 for monomeric VWFA1. The platelet count had no statistically relevant influence on the efficiency of capturing and duration of adhesive contacts. As γw increased, a higher coating concentration of the VWF A1 domain was required to initiate platelet adhesion. The coating concentration determined the number of individual adhesion events that could occur over a defined period of time but did not affect the residence time, which is a measure of the strength of the bond between the receptor and the ligand. Hydrodynamic forces generated by blood flow shortened the duration of VWF-GP Ibα interactions. The percentage of platelets that had a residence time of less than 0.1 s increased almost linearly with increasing γw. Dimeric A1 domain was more efficient than the monomeric counterpart in promoting platelet adhesion as it displayed activity at lower coating concentrations. At permissible γw, a 10-fold higher monomer than dimer coating concentration (2 vs. 20 μg/ml) was required to obtain a similar capturing efficiency. Moreover, the upper limit of γw compatible with the initiation of adhesion was significantly higher for dimeric as compared to monomeric A1 domain. Doubling the dimer coating concentration resulted in a 5-fold increase in the γw limit for adhesion, but the same increase in the monomer coating concentration did not enhance the probability of bond formation at higher γw. In spite of the substantial difference in capturing efficiency, monomeric and dimeric VWFA1 supported platelet adhesion events of similar duration at any given γw, indicating that a different molecular conformation did not affect the lifetime of the interaction with GP Ibα. These results indicate that the dimeric assembly of A1 domains in VWF multimers may be crucial to support the initiation of platelet adhesion at high shear rates, but the duration of each adhesion event is limited by intrinsic properties of the individual VWFA1-GP Ibα bond.

In vitro and in vivo hemocompatibility evaluation of graphite coated polyester vascular grafts

Attempts have been made in this study to prepare a homogeneous and stable coating of graphite on polyester vascular grafts (GPVG) using an electrophoresis method to evaluate thromboresistant and blood compatibility of GPVG in comparison to non-coated PVG and InterGard (collagen sealed PVG) as control. Lactate dehydrogenase (LDH) activity measurement was carried out on all PVG types to evaluate platelet adhesion. To examine tissue reaction GPVG and non-coated sheets of knitted polyester fabric were implanted simultaneously in the dorsal flank of rats subcutaneously. The GPVG, non-coated and control were implanted in descending aorta as end-to-end or end-to-side implantation substitution in 25 sheep for 4–60 weeks. Results showed that the graphite coating on polyester vascular grafts reduced the number of adherent platelets and prevent platelet activation and spreading on the surface in comparison with non-coated and control. Pathological investigation showed inflammatory reactions were totally resolved after 12 weeks and there was no difference in the tissue reaction between graphite coated, non-coated and control patches. All grafts remained patent and there was no significant difference in patency rate between these three types of PVG. We found that GPVG has no need for pre-clotting and it showed lower platelet aggregation, thinner capsule formation and lower calcification after 15 months. However, suturing of GPVG was more difficult in comparison with the other types.

5-Aminoisoquinolinone, a novel inhibitor of poly(adenosine disphosphate-ribose) polymerase, reduces microvascular liver injury but not mortality rate after hepatic ischemia-reperfusion.

The aim of this study was to investigate the impact of the novel, potent, water-soluble inhibitor of poly(adenosine diphosphate-ribose) polymerase (PARP) 5-aminoisoquinolinone (5-AIQ) on hepatic microcirculation, hepatocellular injury, and survival in a murine model of hepatic ischemia-reperfusion. Randomized animal study. Research laboratory. C57BL6 mice were subjected to warm either partial (90 mins) or total (75 mins) ischemia of the liver. Either PARP inhibitor 5-AIQ (3 mg/kg) or vehicle was administered to mice intravenously immediately before the start of reperfusion. Sham-operated animals served as controls. As shown by intravital fluorescence microscopy after 30-60 mins of reperfusion, ischemia-reperfusion significantly enhanced platelet- and leukocyte-endothelial cell interactions in hepatic microvessels and impaired sinusoidal perfusion. Hepatocellular injury was characterized by an increase in the number of necrotic and apoptotic cells, dramatic elevation of aspartate aminotransferase/alanine aminotransferase serum activity, and lipid peroxidation in liver tissue. 5-AIQ treatment attenuated ischemia-reperfusion-induced increases in the numbers of adherent platelets and leukocytes as well as of necrotic and apoptotic cells and ameliorated perfusion failure. Furthermore, PARP inhibition prevented the increase in aspartate aminotransferase activity after ischemia-reperfusion but did not affect postischemic alanine aminotransferase release. However, no protective impact of 5-AIQ on postischemic oxidative stress was observed. Although PARP inhibition did not alter the survival percentage after ischemia-reperfusion (22% in both groups), this approach prolonged survival from 1 to 24 hrs (ischemia-reperfusion + vehicle) up to 48-72 hrs in the treated group. PARP inhibition with 5-AIQ during hepatic ischemia-reperfusion attenuates microvascular injury and reduces the extent of necrotic/apoptotic cell damage but does not protect from oxidative injury and does not improve postoperative survival rate.

Serum protein adsorption and platelet adhesion on aspartic-acid-immobilized polysulfone membranes

Polysulfone (PSf) membranes that covalently conjugated with aspartic acid (ASP-PSf) were prepared and analyzed for hemocompatability. Compared to PSf or other types of surface-modified PSf membranes, the ASP-PSf membranes had a reduced ability to adsorb protein from either a plasma solution or a mixed solution of albumin, globulin and fibrinogen. This appears to be due to the creation of a hydrophilic surface by the aspartic acid zwitterion immobilized on the ASP-PSf membranes. Furthermore, the analyses of membrane protein adsorption showed that a mixed protein solution recapitulates the cooperative adsorption of proteins that occurs in plasma. We also found that the number of adhering platelets was the lowest on the ASP-PSf membranes and, in general, that platelet adhesion decreased in parallel with fibrinogen adsorption. In summary, aspartic acid immobilized on the ASP-PSf membranes, which have zwitterions with a net zero charge, effectively contributes to the hydrophilic and hemocompatible sites on the surface of the hydrophobic PSf membranes.

Development of a YIGSR-peptide-modified polyurethaneurea to enhance endothelialization

Polyurethanes have been investigated for use as vascular grafts due to their excellent mechanical properties and relatively good biocompatibility. However, poor retention of endothelial cells and thrombogenicity in vivo remain problematic for vascular graft applications. The peptide YIGSR has been shown to increase endothelial cell adhesion but not attachment of platelets, suggesting its possible utility for vascular graft applications. In this study, a bioactive polyurethaneurea has been synthesized by incorporating GGGYIGSRGGGK peptide sequences into the polymer backbone. Successful incorporation of the peptides was confirmed by NMR, contact angle measurement and ESCA. Uniform distribution of peptides on the surface was observed using a fluorescent probe capable of reacting with tyrosine residues on the peptides. Hard segment domains were visualized using tapping mode AFM. Endothelial cell adhesion, spreading, proliferation, migration and extra-cellular matrix production were improved on bioactive polyurethaneurea compared to control polyurethaneurea. Competitive inhibition of endothelial cell attachment and spreading by soluble YIGSR peptides indicated that cell adhesion and spreading were specifically mediated by YIGSR-sensitive cell adhesion receptor, not just by changed surface properties. There was no significant difference in the number of adherent platelets. Therefore, this bioactive polyurethanurea may improve vascular graft endothelialization without increasing thrombogenicity.

Exposure of fibrinogen-adherent platelets to plasma proteins: a new method for studying protein interactions with platelets.

To understand the effects of mediators in coagulating blood at biomaterial surfaces, it is important to use methods that resemble the normal sequence of events in wound healing around implants. The initial adhesion of platelets from whole blood onto material surfaces is mediated by the fibrinogen receptor glycoprotein IIb/IIIa, as shown in a previous study (Broberg et al., J Lab Clin Med 2002; 139:163-172). In this study, isolated platelets were adhered to fibrinogen and exposed to IgG, von Willebrand factor, or thrombin. The response was detected as the number of adherent platelets, the spreading of platelets, the exposure of CD62P (P-selectin), and the release of platelet factor 4 (PF4), ADP, and ATP. These results were compared to the response of platelets adhering to surfaces coated with the same proteins. Fibrinogen-adherent platelets exposed to thrombin generated the significantly highest exposure of CD62P and release of PF4, ADP, and ATP. When platelets were adhered to different protein coatings, von Willebrand factor generated the most CD62P exposure, IgG generated the most PF4 release, and thrombin generated the highest concentration of ADP. These results indicate that protein interactions with platelets may generate different results, depending on the mode of protein exposure.

Adhesive Properties of Platelets from Different Animal Species

The use of large animals (e.g., pig and sheep) in human medicine, and the need to develop new therapeutic strategies for domestic animal diseases related to platelet disorders, require better characterization of the physiology of animal platelets. In this study, the ability of platelets from buffaloes, horses, pigs and sheep to adhere to immobilized autologous fibrinogen was compared with that of human platelets. Blood samples were collected from the jugular vein of six healthy subjects of each species and platelet-rich plasma (PRP) was obtained by centrifugation. Platelets, isolated by further centrifugation of PRP, were washed by gel-filtration on Sepharose-2B, counted and added to the wells of 96-well plates pre-coated with autologous fibrinogen. After different times of incubation, non-adherent platelets were removed, and the number of adherent platelets was assessed by measuring endogenous acid phosphatase activity. Horse platelets showed the strongest ability to adhere to autologous immobilized fibrinogen, being 1·7-, 3·1- and 2·3-fold more active than human, buffalo and porcine platelets, respectively. Sheep platelets were unable to adhere to autologous immobilized fibrinogen. Platelet activation by adenosine 5-diphosphate (ADP) increased both human and animal platelet adhesive response. ADP-stimulated sheep platelets were able to adhere to autologous immobilized fibrinogen, albeit to a lesser extent than platelets from the other animal species. The observed interspecies variability in adhesive properties of platelets may reflect structural differences, or differences in the availability of the fibrinogen receptor (glycoprotein IIb/IIIa) on the platelet surface.

Cell adhesion on poly(propylene fumarate-co-ethylene glycol) hydrogels.

We synthesized poly(propylene fumarate-co-ethylene glycol) block copolymers [P(PF-co-EG)] that were crosslinked to form hydrogels and investigated the effect of copolymer composition on cell adhesion to the hydrogels. These copolymers were water soluble when the molar ratio of ethylene glycol repeating unit to propylene fumarate repeating unit was higher than 4.4. The water content of swollen hydrogels increased from 29 to 63% and the water contact angle decreased from 38 to 21 degrees as the molar ratio increased from 0.6 to 4.4. No significant change in either property was observed for ratios higher than 4.4. In a cell adhesion assay under serum-free conditions, the number of adherent platelets and smooth muscle cells decreased from 21 to 2% and from 78 to 20% of the initial seeding density, respectively, as the molar ratio increased from 0.6 to 7.8. Adherent smooth muscle cells did not spread on the hydrogels of the compositions tested. Adherent platelets did not show any filopodia. These results suggest that the hydrophilicity of P(PF-co-EG) hydrogels is one of the factors affecting cell adhesion, and that copolymer modification may be required for enhancing cell adhesion for an application involving the copolymers as in situ crosslinkable cell carriers.

ANTITHROBOCENIC CHARACTERISTICS OF TITANIA-GEL

BACKGROUND: Titanium alloy (Ti) has been used for long-term blood pumps as a good blood compatible material. However, thrombus formation induced by activation of coagulation cascade or platelet sometimes contributes to fatal pump failure. In this study, we formed titania-gel (Ti-Gel) on the Ti surface and evaluated its feasibility as a new blood compatible material in a static condition. METHODS: The Ti plate was chemically treated to form the gel on the surface. Whole blood was used for blood coagulation test. The ratio of blood coagulation was calculated from free hemoglobin in red blood cells, which were not trapped in the thrombus. Platelet rich plasma was used for platelet adhesion test and platelets were activated with adenosine diphosphate. The status and number of adhering platelets on the surface of plates were evaluated by scanning electron microscope (SEMI and enzymatic reaction. RESULTS: Blood coagulation ratios on the Ti and Ti-gel were 80.5 % and 52.1 % at 15 minutes, 99.9 % and 97.1 % at 30 minutes. Deformation to spinate shapes of platelets for adhering on the surface of plates and its enhancement of platelet aggregation were observed by SEM studies. Compared with the Ti, Ti-gel reduced the adhering platelets by 17.3%. CONCLUSION: Ti-Gel has an appropriate antithrombogenic property because of its anti-platelet and anti-blood coagulation effect.

The effect of fluid shear and co-adsorbed proteins on the stability of immobilized fibrinogen and subsequent platelet interactions

The conformation adopted by the plasma protein fibrinogen upon its adsorption onto synthetic surfaces has been implicated to play an important role in determining the blood compatibility of biomaterials. It has recently been shown that adsorbed fibrinogen undergoes biologically significant conformational changes with increasing residence time on the surface of selected biomaterials. The purpose of this study was to examine the effects of co-adsorbed proteins and shear forces on such timedependent functional changes in fibrinogen adsorbed onto polyethylene (PE), polytetrafluoroethylene (PTFE), and silicone rubber (SR). Fibrinogen was adsorbed onto these materials for 1 min and then allowed to 'reside' on these surfaces for up to 2 h prior to assessing its biological activity. Changes in fibrinogen reactivity were determined by measuring the adhesion of 51Cr-labeled platelets and the ability of blood plasma to displace previously adsorbed fibrinogen. The magnitude of platelet adhesion to substrates adsorbed with pure fibrinogen increased in the presence of shear, compared with static conditions; at the lowest shear rate of 200 s-1, samples exhibited a 20-fold increase in adhered platelet levels. In contrast, at a higher shear rate of 1000 s-1, the three polymers supported minimal levels of platelet attachment. Surfaces pre-adsorbed with 10% plasma did not promote a significant increase in the number of adherent platelets with increasing shear when compared with the pure fibrinogencoated substrates. The presence of shear also significantly altered the materials' ability to retain fibrinogen. Under static conditions, the amount of fibrinogen retained following incubation in blood plasma increased on all materials with increasing fibrinogen residence time. However, the materials varied distinctly in their ability to retain adsorbed fibrinogen with increasing fibrinogen residence time, shear rate, and nature of the co-adsorbed proteins. Thus, the results from this study indicate that fluid shear, residence time of the adsorbed protein, nature of the co-adsorbed proteins, and surface chemistry of the material all play important roles in influencing platelet–surface interactions and that they act in a complex manner to influence the biocompatibility of a material.

Platelet adhesion onto a polystyrene surface under static conditions: role of specific platelet receptors and effect of divalent cations

An experimental model was used to elucidate the basic mechanisms involved in the interaction of platelets with an artificial surface. The role of divalent cations and the involvement of specific platelet membrane receptors were evaluated. Isolated platelets were allowed to interact with a polystyrene surface for 20 min in the presence of divalent cations (Ca 2+ , Mg 2+ or Zn 2+ ), a chelating agent (ethylenediaminetetraacetic, EDTA), and specific antibodies to the main platelet receptors, glycoproteins (GP) Ib and IIb-IIIa. The degree of platelet interaction was evaluated using light and electron microscopy. Morphometric analysis was performed to follow up the progression of platelet shape changes after surface activation. Neither Ca 2+ nor Mg 2+ influenced the number of adherent platelets or the degree of spreading on the polymer. Only Zn 2+ induced a statistically significant increase in the rate of platelet adhesion ( P <0.01) with higher proportion of fully spread platelets ( P <0.01). Chelation of internal pools of divalent cations did not modify the rates of platelet adhesion but prevented platelet spreading. Presence of monoclonal antibodies to GPIb and GP IIb-IIIa did not result in significant differences in the studied parameters. These results suggest that platelet adhesion onto artificial surfaces, in the absence of flow and plasma proteins, is more dependent on cellular motility, where Zn 2+ could play an important role, and less dependent on major receptorial mechanisms.

Blood Compatibility of Metal Oxide Layers on Stainless-steel

ABSTRACTWe examined blood compatibility of titanium oxide layer on stainless-steel (SUS316L). The oxide layers with varied thickness were yielded on SUS316L plates by dip-coating of sol-gel solution starting from tetraethyltitanate. The blood compatibility was evaluated in term of platelet adhesion using platelet rich plasma. With increase in the thickness of the oxide layer, the number of adherent platelets decreased rapidly, reached minimum around 150nm. This indicated that the thickness of titanium oxide layer affected platelet adhesion.

Adherence properties of Staphylococcus aureus under static and flow conditions: roles of agr and sar loci, platelets, and plasma ligands.

Global regulatory genes in Staphylococcus aureus, including agr and sar, are known to regulate the expression of multiple virulence factors, including cell wall adhesins. In the present study, the adherence of S. aureus RN6390 (wild type), RN6911 (agr), ALC136 (sar), and ALC135 (agr sar) to immobilized fibrinogen, fibronectin, von Willebrand factor (vWF), extracellular matrix (ECM), and human endothelial cells (EC) EAhy.926 was studied. Bacteria grown to postexponential phase were subjected to light oscillation (static condition) or to shear stress at 200 s(-1) (flow condition) on tissue culture polystyrene plates coated with either protein ligands, ECM, or EC. Adherence of nonlabeled bacteria to immobilized ligands was measured by an image analysis system, while adherence of [(3)H]thymidine-labeled S. aureus to ECM and EC was measured by a beta-scintillation counter. The results showed increased adherence of agr and agr sar mutants to immobilized fibrinogen and higher potential of these mutants to induce platelet aggregation in suspension, decreased adherence of sar and agr sar mutants to immobilized fibronectin and vWF as well as to ECM and EC, increased adherence of both S. aureus wild type and sar mutant to EC treated with platelet-rich plasma (PRP) compared to platelet-poor plasma (PPP) and to EC treated with PPP compared to the control, and increased adherence of S. aureus wild type to EC coated with PRP in which platelets were activated with phorbol 12-myristate 13-acetate compared to intact PRP. This finding paralleled the increased adherence to EC of activated compared to intact platelets. It is suggested that platelet-mediated S. aureus adherence to EC depends on platelet activation and the number of adherent platelets and available receptors on the platelet membrane. In conclusion, the agr locus downregulates S. aureus adherence to fibrinogen, while the sar locus upregulates S. aureus adherence to fibronectin, vWF, ECM, and EC. The effect of both agr and sar on S. aureus adherence properties develops primarily under flow conditions, which suggests different adhesion mechanisms in static and flow conditions.

Platelet adhesion to heparin coated oxygenator fibers under in vitro static conditions: impact of temperature.

Heparin coating of cardiopulmonary bypass (CPB) circuitry may attenuate the platelet consumption associated with CPB. We investigated the effect of temperature on the interaction between platelet and heparin coated surfaces under in vitro static conditions. Heparin coated and non coated oxygenator fibers were incubated with heparinized whole blood at 37 degrees C and 22 degrees C. The incubation time was set at 30, 60, 180, and 300 minutes. The number of platelets adhering to each fiber was assessed with enzyme immunoassay using monoclonal antibody against platelet receptor protein CD 61(GPIIbIIIa). As an index of platelet activation, plasma soluble(s) P-selectin levels were measured by enzyme-linked immunosorbent assay. Under normothermia, the number of adherent platelets on the non coated surface increased significantly after 300 min of incubation. Platelet adhesion was reduced significantly by heparin coating of the surface and was kept constant after 300 min. Under hypothermia, heparin coating was also associated with significant reduction of platelet adhesion. The levels of sP-selectin did not correlate with the extent of platelet adhesion. Our results suggest that heparin coating is effective in decreasing platelet adhesion to the synthetic surface tested regardless of the temperature under static conditions. Inhibition of platelet activation on the heparin coated surface may be masked by standard dose heparinization.

Evaluation of the permeability and blood‐compatibility properties of membranes formed by physical interpenetration of chitosan with PEO/PPO/PEO triblock copolymers

AbstractIn order to develop blood compatible membranes with controlled porosity, we have fabricated and examined the properties of physical interpenetrating network (PIN) of chitosan and poly(ethylene oxide)/poly(propylene oxide)/poly(ethylene oxide) (PEO/PPO/PEO) triblock copolymers (Pluronics®). Degree of equilibrium swelling, scanning electron microscopy, and electron spectroscopy for chemical analysis (ESCA) were used to characterize the bulk and surface properties. Vitamin B12 and human serum albumin were used as permeability markers. Platelet adhesion and activation were used to determine the blood‐interaction properties of the PIN membranes. Unlike chitosan membranes that were nonporous, the chitosan‐Pluronic PIN membranes were highly porous with the pore size, depending on the type of incorporated Pluronic polyol. ESCA results showed a significant increase in the CO signal of C1s spectra on the PIN membranes that correlates with the presence of PEO chains on the surface. The permeability coefficients of vitamin B12 and albumin were higher in the chitosan‐Pluronic PIN membranes than in the control. The number of adherent platelets and the extent of activation were significantly reduced on the chitosan‐Pluronic PIN membranes. The decrease in platelet adhesion and activation correlated positively with the PEO chain length of the incorporated Pluronic polyols. The results of this study show that chitosan‐Pluronic PIN membranes offer a blood‐compatible alternative with a higher‐molecular‐weight cutoff for use in hemodialysis and related applications. © 2001 John Wiley &amp; Sons, Inc. J Appl Polym Sci 80: 1274–1284, 2001

Surface characterization and platelet adhesion studies on polyethylene surface with hirudin immobilization.

Hirudin, a protein composed of 65 or 66 amino acid, is a newly risen anticoagulant agent and has been considered as the most potent thrombin inhibitor. Hirudin can block the active site of thrombin by means of its carboxylic acid reaction with the active regime of thrombin, and becomes a tightly bound complex, and thus controls the formation of thrombus. Hirudin was covalently immobilized onto the water-soluble carbodiimide preactivated and chromic acid oxidized PE surface. Surface chemistry analysis indicated that a certain amount of carboxylic acid groups was generated on the polyethylene surface after oxidation with chromic acid solution. The amount of carboxylic acid functional group increased with the oxidation time. In addition, polyethylene surface was etched by chromic acid solution, and a rougher surface was created. The morphology of oxidized polyethylene surface was similar to each other among the samples with oxidation time from 1 to 8 min. ESCA results indicated the number of hirudin molecules immobilized was constant at the reaction time studied. In vitro platelet adhesion assay indicated the number of adhered platelets on the oxidized polyethylene surface increased significantly after oxidation. In contrast, surface with hirudin immobilization showed a reduction in adhered platelet density than the chromic acid oxidized counterpart due to the decrease of platelet-activating capability by the hirudin-thrombin complex and the differences in the adsorbed protein composition.