Decreased Platelet Adhesion Research Articles

Estrogen‐mediated nitric oxide (NO) production in cultured human vascular endothelial cells

Estrogen (E2) is reported to induce the rapid activation of endothelial nitric oxide synthase (eNOS) and NO release, resulting in vasodilation, decreased platelet adhesion and angiogenesis in the vessel wall. In this study, we have used a cell line (EA.hy926) derived from human umbilical vein endothelial cells (HUVECs) to investigate the cellular mechanism underlying E2-induced NO synthesis. Experimentally, NO production and cytosolic free [Ca2+] were monitored using the NO-sensitive dye DAF-FM and Fluo-3, respectively, and agonist-induced changes in membrane potential were recorded using patch clamp techniques. E2 dose-dependently induced rapid increases in NO synthesis, and only modest changes in cytosolic [Ca2+]. The PI3 kinase inhibitor LY294002, the E2 receptor antagonist ICI 182,780, and the eNOS inhibitor L-NAME all blocked E2-mediated NO synthesis. Interestingly, removal of extracellular Ca2+, buffering of cytosolic Ca2+ by BAPTA or inhibition of SK and IK Ca-activated K+ channels by apamin and charybdotoxin did not affect NO production in response to E2, but attenuated ATP-mediated NO synthesis. Unlike ATP or histamine, E2 did not induce hyperpolarization of endothelial cell membrane potential. These observations indicate that in human vascular endothelial cells, E2 induces rapid NO production via a cellular pathway distinct from that utilized by Ca-mobilizing stimuli. (Funded by CIHR)

ADAMTS-13 regulates platelet adhesion under flow

ADAMTS-13 cleaves large and ultra-large von Willebrand factor multimers normally secreted by endothelial cells. Severe deficiency of this enzyme leads to thrombotic thrombocytopenic purpura (TTP). We applied the Impact-R system [Cone and plate(let) Analyzer, CPA] to determine optimal conditions for ADAMTS-13 function, to assess it's activity in TTP patients and to distinguish inherited TTP (inTTP) from acquired TTP (acTTP). The role of ADAMTS-13 in platelet adhesion under different conditions was investigated applying recombinant forms of VWF and ADAMTS-13. rVWF was first treated by rADAMTS-13 either in solution or when immobilized on the surface of the well, under static or flow conditions, in the presence or absence of BaCl2. The resulting cleaved VWF fragments were then immobilized and served to assess type 3 von Willebrand disease whole blood platelet adhesion under flow. Maximal effect of the rADAMTS-13 (decrease of platelet adhesion in the absence compared to the presence of BaCl2), was observed when the rVWF was pre-immobilized and the cleavage step took place under flow (85%). Mixing plasma ofTTP patients with normal blood (1:3) yielded a 1.6- to 2-fold increase of platelet adhesion under flow compared to mixing normal plasma with normal blood, at shear rate range of 1,800 - 2,500 s(-1) . Maximal increase of platelet adhesion was observed under 2,050 s(-1) . Under these conditions, the extent of adhesion was similarly higher in patients with inTTP and acTTP versus control [surface coverage (SC) 14.5 +/- 2.8% and 14.6 +/- 2.5% vs.7.4 +/- 1.7%, respectively]. ADAMTS-13 activity measured by collagen-binding test was similarly low (4.2 +/- 3.8% and 3.5 +/- 2.4% vs. 72.2 +/- 8.0%, respectively). An inverse correlation between SC and ADAMTS-13 activity was observed in a patient with inTTP assayed eight times during plasma infusion treatment. Addition of BaCl2 to the mixture of TTP plasma and normal whole blood yielded a decrease in platelet adhesion in inTTP (by 51%) but not in acTTP. The lack of reduction of platelet adhesion in acTTP could presumably be due to the presence of ADAMTS-13 inhibitor in these patients. These results suggest that VWF immobilization and high shear flow yielded optimal conditions for ADAMTS-13 activity, and that introduction of BaCl2 in the Impact-R (CPA) test may be useful for differentiation between inherited and acquired TTP.

The C-Terminal α-Helix of von Willebrand Factor Domain A2 Interacts with ADAMTS13 C-Terminal Domains To Regulate Substrate Cleavage.

ADAMTS13 is a member of the A Disintergrin And Metalloprotease with ThromboSpondin type I repeat family, and it cleaves the Tyr1605-Met1606 bond in the von Willebrand factor (VWF) central A2 domain, thereby decreasing platelet adhesion mediated by VWF. Recently a minimal substrate for ADAMTS13 was characterized that consists of GST linked to Asp1596-Arg1668 with a C-terminal 6×His tag (VWF73). Further removal of 9 amino acids that comprise a predicted C-terminal α-helix (VWF64) appeared to eliminate cleavage by plasma ADAMTS13, suggesting a critical role for this helix in substrate recognition. We obtained similar results, but VWF64 was cleaved significantly at long reaction times. For example, plasma ADAMTS13 (0.3 nM, one-tenth of normal plasma) cleaved 50% of 65 nM VWF73 in 2 hours and 50% of 62 nM VWF64 in 24 hours. Similar results were obtained in either 50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl2, 0.1 μM ZnCl2, or 5 mM Tris-HCl, pH 8.0, 10 mM BaCl2. By amino acid sequencing, ADAMTS13 was shown to cleave the Tyr1605-Met1606 bond of VWF64. Truncation of ADAMTS13 after certain structural domains had different effects on substrate cleavage. Using 3 nM enzyme for 30 min, VWF73 was cleaved ~2-fold faster by ADAMTS13 truncated after the spacer domain (MDTCS) than by ADAMTS13 truncated after the cysteine-rich domain (MDTC). Conversely, VWF64 was cleaved ~3-fold faster by MDTC than by MDTCS. Also, in 30 min MDTCS cleaved 70% of VWF73 and <10% of VWF64, whereas MDTC cleaved 40% of VWF73 and 30% of VWF64. ADAMTS13 truncated after the first TSP1 repeat (MDT) or the disintegrin domain (MD) had markedly reduced activity, but with prolonged incubation (24 h) at increased concentration (30 nM) both enzymes cleaved most of VWF64 and VWF73 at the expected site. No aberrant cleavage products were detected by Western blotting. The metalloproteinase domain alone (M) was inactive. The selective effects of deleting the cysteine-rich or spacer domain suggest that the C-terminal α-helix of the VWF A2 domain is not essential but facilitates substrate recognition by interacting with specific C-terminal domains of ADAMTS13, particularly the cysteine-rich and spacer domains.

Development of Novel Submicron Textured Polyether(Urethane Urea) for Decreasing Platelet Adhesion

Ventricular assist devices have proven to be a useful clinical option for providing circulatory support as a bridge to transplantation and a mode of destination therapy. Thromboembolism is prevented by designing devices that use blood interfaces that either encourage biological material deposition and strong adhesion, or discourage deposition via surface chemistry, surface finish, and fluid flow fields. Minimum continuous or periodic wall shear forces and maximum time at reduced shear are important, and sometimes difficult-to-satisfy, design constraints. We present an approach to reducing platelet adhesion via surface topography, reducing surface area for platelet-material interaction. Large areas of polyether(urethane urea) were textured with two different sizes of ordered pillar arrays via two-stage replication molding without affecting surface chemistry. Pillars had subplatelet dimensions designed to reduce the surface area a platelet may contact. Platelet adhesion was assessed in a physiologically relevant shear stress range from 0-10 dyn/cm2 using a rotating disk and compared to smooth control. Adhesion was highest from 0-5 dyn/cm2. Surface texturing reduced platelet adhesion without increasing platelet activation in bulk suspension. This study demonstrates that material surface texture is an additional variable that may be used to reduce platelet adhesion under low shear stresses potentially reducing thromboembolism.

Pharmacologic cerebral capillary blood flow improvement after deep hypothermic circulatory arrest: An intravital fluorescence microscopy study in pigs

Despite meticulous investigation of bypass techniques for deep hypothermic circulatory arrest, unfavorable long-term neurologic deficits have been well documented. Our aim was to improve brain perfusion by reducing platelet plugging with a glycoprotein IIb/IIIa inhibitor (eptifibatide) in an experimental model of deep hypothermic circulatory arrest-reperfusion in pigs. Two groups of 12 piglets each (eptifibatide group [eptifibatide + unfractionated heparin] vs UFH group [only unfractionated heparin]) underwent 10 minutes of normothermic bypass, 40 minutes of cooling during cardiopulmonary bypass (hematocrit, 30%; cardiopulmonary bypass flow, 100 mL x kg(-1) x min(-1)), 60 minutes of circulatory arrest at 15 degrees C, and a 40-minute rewarming period. Intravital fluorescence microscopy of pial vessels at set intervals was performed. During the cooling period, there was a tendency toward reduced functional capillary density values without statistical significance in both groups. During reperfusion, the eptifibatide group demonstrated a significantly decreased platelet adhesion and aggregation (at 30 minutes of reperfusion: functional capillary density, 104% +/- 3% vs 77% +/- 4% relative to baseline, P = .02; red blood cell velocity, 0.65 vs 0.30 mm/s, P < .004). A more rapid recovery of tissue oxygenation (P < .001) was documented. Furthermore, a significant microvascular permeability reduction was achieved compared with that seen in the UFH group (P < .02). The use of eptifibatide resulted in fewer ultrastructural changes in hippocampal tissue, which is demonstrated by histologic examination. Platelet plugging reduction with the glycoprotein IIb/IIIa inhibitor eptifibatide improves cerebral capillary blood flow and reduces cerebral ischemia in the setting of deep hypothermic circulatory arrest. Furthermore, significant endothelial cell injury and perivascular edema reduction can be achieved.

Is Platelet Adhesion Assay Able To Quantify Drug-Induced Platelet Dysfunction?

The platelet adhesion assay (PADA) is an innovative method for the detection of both normal, pathologically increased or decreased platelet adhesiveness. Adhesion is the first important phase of platelet activation, followed by shape change and aggregation. Adhesion is triggered by glycoproteins (GP) on the platelet surface, mainly by GPIIb/IIIa, and to a lesser extent also by GPIb/V/IX. Since fibrinogen serves as adhesive protein for GPIIb/IIIa receptors, and since the PADA uses polymer particles that become coated with fibrinogen, the PADA is able to monitor GPIIb/IIIa receptor antagonists and to detect overdosing, potentially leading to bleeding complications. Ex vivo, citrated whole blood from healthy volunteers and patients was spiked with increasing GPIIb/IIIa inhibitor concentrations and PADA was measured. Comparing these results with GPIIb/IIIa receptor occupancy, determined by FACS, a basic consistency of the data was shown. Via intracellular signaling, the adenosine diphosphate (ADP) receptor mechanism is closely involved in the activation of GPIIb/IIIa receptors so that also ADP receptor antagonists of the thienopyridine type, especially clopidogrel, can be quantitatively determined by the PADA. In patients under clopidogrel therapy, the therapeutic effect was monitored and also individual dose adjustments were realized. Furthermore, patients having partial or full clopidogrel resistance were identified. Overdoses can be detected as well.

Measurement of adhesion of human platelets in plasma to protein surfaces in microplates

Introduction Platelet adhesion is an initial, crucial and complex event for inhibiting blood loss upon vascular injury. Activation and adhesion of platelets also play a fundamental role in the development of thrombosis. A combination of exposed extracellular matrix proteins in the vascular wall and release of activating compounds from the participating cells activate the platelets. New potent anti-platelet agents are in progress but there is a shortage of methods that measure the concerted action of adhesive surfaces and soluble compounds upon platelet adhesion in vitro. The aim of this work was to develop a method to measure adhesion of platelets in plasma with standard laboratory equipment. Methods Platelet-rich plasma from healthy humans was used in studies to optimise the conditions of the present assay. Different proteins were coated in microplate wells and various soluble platelet activators and inhibitors were added to establish the ability of the current method to detect increased as well as decreased platelet adhesion. The amount of platelet adhesion was measured by the reaction between p-nitrophenyl phosphate and the intracellular enzyme acid phosphatase. Results Adhesion of platelets in plasma to microplate wells coated with albumin, collagen, fibrinogen and activated plasma showed significant surface dependency. The known soluble platelet activators adenosine diphosphate, adrenaline and ristocetin enhanced the levels of adhesion. Available anti-platelet agents such as prostacyclin, forskolin, acetylsalicylic acid and RGD containing peptides caused dose-dependent inhibition of platelet adhesion. Discussion This report describes a further development of a previously described method and offers the advantage to use platelets in plasma to measure platelet adhesion to protein surfaces. The assay is simple and flexible and is suitable in basic research for screening and characterisation of platelet adhesion responsiveness.

Inhibition of blood platelet adhesion and secretion by different phenolics from Yucca schidigera Roezl. bark

Objective Yucca schidigera is a plant that grows in Mexico, and it has a very high level of saponins and phenolic compounds with antioxidant action. The products of Y. schidigera are used as food additives and have a generally recognized as safe label. This study investigated the antiplatelet mechanisms of four phenolic compounds. Methods We investigated antiplatelet mechanisms of the phenolic compounds trans-3,4′,5-trihydroxystilbene ( trans-resveratrol), trans-3,3′,5,5′-tetrahydroxy-4′-methoxystilbene, and yuccaols A and C that had been isolated from the bark of Y. schidigera by studying their effects on the first step of platelet activation, i.e., platelet adhesion to collagen and fibrinogen. The effects of these compounds on the release of adenine nucleotides, proteins, and β- N-acetyl-glycosaminidase (a marker of lysosomal secretion) from blood platelets activated by thrombin were also studied. Results These different phenolic compounds (1 to 25 μg/mL) and their extracts decreased platelet adhesion and secretion. Conclusions Resveratrol and yucca extract were more reactive in decreasing these processes than were other tested phenolic compounds.

Improvement of Function and Morphology of Tumor Necrosis Factor-.ALPHA. Treated Endothelial Cells With 17-.BETA. Estradiol A Preliminary Study for a Feasible Simple Model for Atherosclerosis

Dysfunction of endothelial cells (EC) to produce endothelial nitric oxide synthase (eNOS) by tumor necrosis factor-alpha (TNF-alpha) causes critical features of vascular inflammation associated with several disease states (eg, atherosclerosis including increased platelet aggregation and adhesion on EC, elevated adhesion molecules and enhanced inflammatory cells binding to EC). 17-beta estradiol (E2) can stimulate eNOS production and improve the critical features of atherosclerosis. Using TNF-alpha and E2, we attempted to develop an in vitro vascular model for studying atherosclerosis. Human umbilical vein endothelial cells (HUVEC) grown in transwells were cocultured with smooth muscle cells in a 24-well plate to mimic the major components of the vascular wall. The model was incubated with TNF-alpha (10 ng/ml) for 12 h, prior exposed to E2 (100 pg/ml) for 6-12 h, then investigated by transmission and scanning electron microscopes. The result indicated recovered morphology with good tight junction, and decreased platelet adhesion was noted in defective HUVEC after E2 treatment. 17-beta estradiol was represented as an antiatherosclerogenic agent to demonstrate feasibility of the model. Although our finding focused only on the endothelium, this would be the basis for our future studies to develop ex vivo continuous perfusion of human vessel segments for a further atherosclerosis study.

Improved hemocompatibility of poly(ethylene terephthalate) modified with various thiol-containing groups

Thiol groups were attached to polyethylene terephthalate (PET) to promote the transfer of a known platelet inhibitor, nitric oxide (NO), from nitrosated thiols naturally found in the body to PET, followed by the release of NO from PET to prevent platelet adhesion. In order to immobilize the most thiols on the modified polymer, the processing parameters used to attach the following three thiol containing groups were assessed: l-cysteine, 2-iminothiolane, and a cysteine polypeptide. When comparing the immobilized concentrations of thiol groups from each of the optimized processes the amount of immobilized thiol groups increased in order with the following groups: cysteine polypeptide <2-iminothiolane < l-cysteine. The effect of each optimized polymer on platelet adhesion was studied by in vitro experiments utilizing a parallel plate perfusion chamber. Platelets in the following solutions were tested: Tyrode's buffer, 7 μ m nitrosated bovine serum albumin in Tyrode's buffer, 50% plasma in Tyrode's buffer, and 50% whole blood in Tyrode's buffer. All of the polymers demonstrated a significant decrease in platelet adhesion compared to controls when exposed to the BSANO, plasma and whole blood solutions. The most significant decrease was for the l-cysteine modified polymer in the plasma solution with a 65% decrease.

Platelet adhesion to radiofrequency glow-discharge-deposited fluorocarbon polymers preadsorbed with selectively depleted plasmas show the primary role of fibrinogen

Fluorocarbon radio-frequency glow-discharge (RFGD) treatment has previously been shown to cause decreased platelet adhesion despite the presence of adsorbed fibrinogen on the surfaces. In this study platelet adhesion to fluorocarbon RFGD-treated surfaces preadsorbed with human plasma was further examined. A series of plasma deposited fluorocarbon thin films were made by varying the C3F6/CH4 ratio in the monomer feed. The surfaces were preadsorbed with plasma, serum, or plasma selectively depleted of fibronectin, vitronectin, or Von Willebrand factor, and platelet adhesion was measured. We also measured fibrinogen adsorption to the surfaces from plasma, monoclonal antibody binding to adsorbed fibrinogen and SDS elutability of the adsorbed fibrinogen. The antibodies used bind to the three putative platelet binding sites on fibrinogen, namely, M1 antibody binds to the dodecapeptide at the C-terminus of the gamma chain, γ (402–411), R1 antibody binds to a sequence in the Aα chain (87–100) which includes RGDF at Aα (95–98) and R2 antibody binds a sequence in the Aα chain (566–580) which includes RGDS at Aα (572–575). Fibrinogen was found to play a decisive role in mediating platelet adhesion to the fluorocarbon surfaces contacting plasma. Few platelets adhered to the fluorocarbon surfaces preadsorbed with serum, while preadsorption with plasma selectively-depleted of either fibronectin, vitronectin, or von Willebrand factor did not decrease platelet adhesion significantly. Replenishment of exogenous fibrinogen to serum restored platelet adhesion, while replenishment of the other proteins had no effect. Platelet adhesion to the fluorocarbon surfaces was lower than to PET or the methane glow-dischargetreated PET. However, there was no apparent correlation between platelet adhesion and the amount of fibrinogen adsorption or monoclonal antibody binding to surface-bound fibrinogen.

Bioactive coatings of endovascular stents based on polyelectrolyte multilayers.

Layer-by-layer self-assembly of two polysaccharides, hyaluronan (HA) and chitosan (CH), was employed to engineer bioactive coatings for endovascular stents. A polyethyleneimine (PEI) primer layer was adsorbed on the metallic surface to initiate the sequential adsorption of the weak polyelectrolytes. The multilayer growth was monitored using a radiolabeled HA and shown to be linear as a function of the number of layers. The chemical structure, interfacial properties, and morphology of the self-assembled multilayer were investigated by time-of-flight secondary ions mass spectrometry (ToF-SIMS), contact angle measurements, and atomic force microscopy (AFM), respectively. Multilayer-coated NiTi disks presented enhanced antifouling properties, compared to unmodified NiTi disks, as demonstrated by a decrease of platelet adhesion in an in vitro assay (38% reduction; p = 0.036). An ex vivo assay on a porcine model indicated that the coating did not prevent fouling by neutrophils. To assess whether the multilayers may be exploited as in situ drug delivery systems, the nitric-oxide-donor sodium nitroprusside (SNP) was incorporated within the multilayer. SNP-doped multilayers were shown to further reduce platelet adhesion, compared to standard multilayers (40% reduction). When NiTi wires coated with a multilayer containing a fluorescently labeled HA were placed in intimate contact with the vascular wall, the polysaccharide translocated on the porcine aortic samples, as shown by confocal microscopy observation of a treated artery. The enhanced thromboresistance of the self-assembled multilayer together with the antiinflammatory and wound healing properties of hyaluronan and chitosan are expected to reduce the neointimal hyperplasia associated with stent implantation.

Protein adsorption and platelet adhesion of polysulfone membrane immobilized with chitosan and heparin conjugate

AbstractPolysulfone (PSF) membranes were treated with ozone to introduce peroxides, and then grafted with either acrylic acid or chitosan, followed by the immobilization of heparin. The effect of spacer arm on blood compatibility was investigated using three chitosans of different molecular weight [1170 (water soluble), 160 000, and 400 000] and similar degrees of deacetylation (75%). The hydrophilicity was evaluated by measuring the contact angle of water. Blood compatibility was evaluated using the activated partial thromboplastin time (APTT) as well as the adhesion of platelets. The protein affinity was determined by the absorption of human serum albumin (HSA) and human plasma fibrinogen (HPF). The results show that by the coupling of chitosan, the amount of heparin immobilized can be increased by four times. Water contact angle (from 78 ° to 41 °) decreased with the increase of the amount of heparin immobilized, showing increased wettability. The heparinized PSF membrane showed longer APTT and decreasing platelet adhesion, compared to that of unmodified PSF membrane. The adsorption of HSA and HPF were reduced to 17 and 6%, respectively. This suggests that longer spacer binding to heparin can increase the opportunity of anti‐coagulation on contacting blood. These results demonstrated that the hydrophilicity and blood compatibility of PSF membrane could be improved by chitosan and heparin conjugate. Copyright © 2003 John Wiley &amp; Sons, Ltd.

Platelet adhesion to polystyrene-based surfaces preadsorbed with plasmas selectively depleted in fibrinogen, fibronectin, vitronectin, or von Willebrand's factor.

Four plasma proteins have been shown to be able to mediate platelet adhesion to synthetic materials when they are adsorbed as purified proteins: fibrinogen (Fg), fibronectin (Fn), vitronectin (Vn), and von Willebrand factor (vWF). Among them, Fg is thought to play a leading role in mediating platelet adhesion to plasma-preadsorbed biomaterials, but this has been established for only three types of materials so far in our laboratory. Furthermore, the role of Fn, Vn, and vWF in mediating platelet adhesion to plasma-preadsorbed surfaces is still unclear. The aim of the current study was to assess the importance of Fg, Fn, Vn, and vWF in mediating platelet adhesion to a series of polystyrene-based surfaces. The strategy applied in the present investigation was to compare platelet adhesion to surfaces preadsorbed with normal plasma, plasma selectively depleted in Fn or Vn or both Fn and Vn, plasma from donors who were genetically deficient in vWF, and serum. Few platelets adhered to the surfaces preadsorbed with serum, whereas depletion of Fn, Vn, or vWF from plasma did not decrease platelet adhesion significantly. Replenishment of exogenous Fg to serum before protein adsorption restored platelet adhesion to the surfaces, suggesting that Fg was the major plasma protein that mediated platelet adhesion. Also, we found that a surface density of adsorbed Fg far below the amount that usually adsorbs to synthetic surfaces was sufficient to support full-scale platelet adhesion.

Platelet adhesion to photodynamic therapy-treated extracellular matrix proteins.

Photodynamic therapy (PDT) produces reactive species that alter vascular wall biology and vessel wall proteins. In this study, we examined platelet adhesion to PDT-treated (photosensitizer = Photofrin; fluence 100 J/cm2; lambda = 630 nm) extracellular matrix (ECM), fibrinogen, von Willebrand factor (vWF) and collagen Types I and III, under flow conditions in a recirculating perfusion chamber. Platelet adhesion was quantified by image analysis. The effect of PDT on vWF was assessed by measuring the binding of domain-specific antibodies to treated vWF. PDT significantly decreased platelet adhesion to the ECM, fibrinogen and vWF. However, PDT of collagen resulted in significantly increased platelet adhesion, with large aggregate formation. PDT affected mostly the A1 (glycoprotein [GP]-Ib-binding site), A2 and A3 (collagen-binding site) domains of vWF but not the D'-D3 (factor VIII-binding site) and B-C1 (GP-IIb/IIIa-binding site) domains. In conclusion, PDT can alter the ECM, resulting in decreased platelet adhesion. However, vessels with high collagen content, such as veins and small arteries, may become increasingly prone to thrombosis. The results of this study may thus play a role in understanding the thrombogenic properties and mechanisms of vascular PDT.

Saratin, an inhibitor of von Willebrand factor–dependent platelet adhesion, decreases platelet aggregation and intimal hyperplasia in a rat carotid endarterectomy model

Purpose: Post–carotid endarterectomy, thrombosis, and intimal hyperplasia may be decreased by the inhibition of platelet adhesion and activation. In this study, a novel agent, saratin, was used to inhibit platelet-to-collagen adhesion in a rat carotid endarterectomy model. Saratin is a recombinant protein isolated from the saliva of the medicinal leech Hirudo medicinalis , which is thought to act by binding to collagen, and inhibits von Willebrand factor–collagen interaction under conditions of increased shear and therefore, the adherence and activation of platelets at the vessel wall. Saratin has the advantage of being a nonsystemic, site-specific topical application. Methods: A rat carotid endarterectomy model was used in which an open technique with arteriotomy and intimectomy was used. Saratin was applied to the endarterectomized surface of the carotid artery before arterial closure. End point measurements included platelet adhesion, thrombosis rate, intimal hyperplasia development, bleeding times, and platelet counts. Electron micrographs of carotid arteries were used for quantitative analysis of platelet aggregation and platelet counts. Intimal hyperplasia and thrombosis were assessed with computer-assisted morphometric analysis of elastin-stained carotid artery sections with direct measurement of the intimal hyperplasia area. Results: The topical application of saratin significantly decreased platelet adhesion compared with controls at 3 hours after carotid endarterectomy (64 ± 17 vs 155 ± 33 platelets per grid, P =.05), and 24 hours after carotid endarterectomy (35 ± 11 vs 149 ± 37 platelets per grid, P =.0110), respectively. A percent luminal stenosis, as a measure of intimal hyperplasia, was significantly decreased with saratin application compared with controls (10.9% ± 1.8% vs 29.8% ± 6.8%, P =.0042). This decrease in intimal hyperplasia formation correlated with the inhibition of platelet adhesion. Thirty-three percent of control arteries were found to be thrombosed 2 weeks after carotid endarterectomy compared with a 0% thrombosis rate in the saratin-treated group (P =.0156). No increased bleeding was encountered along the arterial suture line in the saratin group. Bleeding times and systemic platelet counts were not found to change significantly in the saratin-treated rats compared with control rats at 3 and 24 hours after endarterectomy. Conclusion: Saratin significantly decreased platelet adhesion, intimal hyperplasia, luminal stenosis, and thrombosis after carotid endarterectomy in rats. Saratin did not increase suture line bleeding or bleeding times, and did not decrease platelet counts. Saratin may serve as a topical agent to be used for the site-specific inhibition of thrombosis and intimal hyperplasia after vascular manipulation. (J Vasc Surg 2001;34:724-9.)

Immobilization of poly(ethylene glycol) or its sulfonate onto polymer surfaces by ozone oxidation

A novel surface modification method has been developed to improve biocompatibility of polymeric biomaterials. This approach involves ozonation and then followed by graft polymerization with acrylates containing PEG, sulfonated PEG or by coupling of PEG derivatives. All the reactions were confirmed by ATR FT-IR and ESCA. The degree of ozonation measured by the iodide method was dependent on the ozone permeability of the polymers used. Surface hydrophilicity was investigated by measuring the contact angles. Ozonation itself yielded a slight increase in hydrophilicity and a decrease in platelet adhesion, but PEG immobilization showed a significant effect on surface hydrophilicity and platelet adhesion to confirm well-known PEG’s passivity which minimize the adhesion of blood components on polymer surfaces. Both graft polymerization and coupling were effective for PU. In contrast, only grafting gave enough yields for PMMA and silicone. Platelet adhesion results demonstrated that all PEG modified surfaces adsorbed lower platelet adhesion than untreated or ozonated ones. Polymers coupled with sulfonated PEG exhibited the lowest platelet adhesion when compared with control and PEG coupled ones by virtue of the synergistic effect of non-adhesive PEG and negatively charged SO 3 groups. This PEG or sulfonated PEG immobilization technology using ozonation is relatively simple for introducing uniform surface modification and therefore very useful for practical application of blood contacting medical devices.

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.

Mutations on fibrinogen (gamma 316-322) are associated with reduction in platelet adhesion under flow conditions.

In this paper we report on studies of platelet adhesion to several fibrinogen gamma chain variants under physiological flow conditions. Reduced platelet adhesion was found to patient dysfibrinogen Vlissingen and its recombinant form (deletion of gamma 319-320). Furthermore, substitutions of the amino acids 318, 320, or both in the recombinant fibrinogen gamma chain showed a strong decrease in platelet adhesion under flow conditions in our perfusion system. Antibodies raised against peptides covering these sequences inhibited platelet adhesion completely, which suggested that the gamma 316-322 sequence could be involved in platelet adhesion in flowing blood.

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