Platelet Attachment Research Articles

Enhanced biocompatibility of CD47-functionalized vascular stents

The effectiveness of endovascular stents is hindered by in-stent restenosis (ISR), a secondary re-obstruction of treated arteries due to unresolved inflammation and activation of smooth muscle cells in the arterial wall. We previously demonstrated that immobilized CD47, a ubiquitously expressed transmembrane protein with an established role in immune evasion, can confer biocompatibility when appended to polymeric surfaces. In present studies, we test the hypothesis that CD47 immobilized onto metallic surfaces of stents can effectively inhibit the inflammatory response thus mitigating ISR. Recombinant CD47 (recCD47) or a peptide sequence corresponding to the Ig domain of CD47 (pepCD47), were attached to the surfaces of both 316L-grade stainless steel foils and stents using bisphosphonate coordination chemistry and thiol-based conjugation reactions to assess the anti-inflammatory properties of CD47-functionalized surfaces. Initial in vitro and ex vivo analysis demonstrated that both recCD47 and pepCD47 significantly reduced inflammatory cell attachment to steel surfaces without impeding on endothelial cell retention and expansion. Using a rat carotid stent model, we showed that pepCD47-functionalized stents prevented fibrin and platelet thrombus deposition, inhibited inflammatory cell attachment, and reduced restenosis by 30%. It is concluded that CD47-modified stent surfaces mitigate platelet and inflammatory cell attachment, thereby disrupting ISR pathophysiology.

Surface-modified poly(vinyl alcohol) vascular grafts improve endothelialization while maintaining hemocompatibility

Event Abstract Back to Event Surface-modified poly(vinyl alcohol) vascular grafts improve endothelialization while maintaining hemocompatibility Deirdre Anderson1, Marie F. Cutiongco2, Evelyn K. Yim2, 3, 4 and Monica T. Hinds1, 5, 6 1 Oregon Health & Science University, Biomedical Engineering, United States 2 National University of Singapore, Biomedical Engineering, Singapore 3 National University of Singapore, Department of Surgery, Singapore 4 National University of Singapore, Mechnobiology Institue, Singapore 5 Oregon Health & Science University, Knight Cardiovascular Institute, United States 6 Oregon Health & Science University, Department of Neuroscience, Oregon National Primate Research Center, United States Introduction: Synthetic vascular grafts are needed in ~20% of coronary artery bypass cases, where autologous grafts are not available. However, current biomaterials fail at small diameters due to the compliance mismatch between the native artery and the stiff material and the lack of an endothelial layer. These issues lead to tissue ingrowth and long-term stenosis. Attempts to tissue engineer a vascular graft have yet to achieve success, and cell-based therapies are often plagued by the long and costly process of cell isolation and expansion before use. This work aims to create an off-the-shelf, compliant vascular graft capable of stimulating endothelialization and reducing intimal hyperplasia in vivo. To complete these goals we used chemical and topographical surface-modified poly(vinyl) alcohol (PVA) biomaterials to eliminate the compliance mismatch and encourage endothelial cell (EC) growth. Materials and Methods: PVA biomaterial grafts: An aqueous solution of sodium trimetaphosphate crosslinker and PVA was cast onto 4mm diameter cylindrical molds, which were either smooth or patterned. Samples were cast with protein solution in the lumen or plasma treated with N2 gas (Fig 1). Cell attachment and quantification: Human umbilical vein endothelial cells were seeded on PVA for 1 or 6days. After fixation, they were stained for F-actin, VE-cadherin, and nuclei. Cell attachment was quantified at 24hrs. Hemocompatibility testing: A well-established, non-human primate arteriovenous shunt model was used to quantify hemocompatibility. A chronic shunt was surgically implanted between the femoral artery and vein. Autologous platelets were labeled with 111In and infused with 125I-fibrinogen prior to study. Platelets were quantified every 5min for 60min using a gamma camera. After the 111In decayed, fibrin was quantified. A clinical standard ePTFE graft was used as a negative control and collagen-coated ePTFE was used as a positive control. Results and Discussion: Cell counts were significantly increased on plasma-treated samples for all surface topographies with confluent monolayers seen by day 6 (Fig 2). All grafts remained patent for the duration of the shunt study. While no significant differences were seen in platelet attachment, PVA samples, even with plasma treatment, exhibited minimal platelet or fibrin accumulation (Fig 3). This reflects our previous results, which indicated the promise of PVA as a blood-contacting material[1]. Conclusions: The PVA grafts have excellent hemocompatibility properties showing minimal reactions in a stringent ex vivo shunt model lacking any anti-coagulation. The addition of plasma treatment encouraged endothelialization in vitro. Future studies will examine additional topographies with the plasma treatment to encourage a healthy EC phenotype, migration, proliferation, and retention under flow. We will also implant these grafts to determine their in vivo potential for endothelialization and preventing tissue ingrowth. Jennifer Greisel; Dr. Michael Wallisch

The ABCC4 membrane transporter modulates platelet aggregation

AbstractControlling the activation of platelets is a key strategy to mitigate cardiovascular disease. Previous studies have suggested that the ATP-binding cassette (ABC) transporter, ABCC4, functions in platelet-dense granules. Using plasma membrane biotinylation and super-resolution microscopy, we demonstrate that ABCC4 is primarily expressed on the plasma membrane of both mouse and human platelets. Platelets lacking ABCC4 have unchanged dense-granule function, number, and volume, but harbor a selective impairment in collagen-induced aggregation. Accordingly, Abcc4 knockout (KO) platelet attachment to a collagen substratum was also faulty and associated with elevated intracellular cyclic AMP (cAMP) and reduced plasma membrane localization of the major collagen receptor, GPVI. In the ferric-chloride vasculature injury model, Abcc4 KO mice exhibited markedly impaired thrombus formation. The attenuation of platelet aggregation by the phosphodiesterase inhibitor EHNA (a non-ABCC4 substrate), when combined with Abcc4 deficiency, illustrated a crucial functional interaction between phosphodiesterases and ABCC4. This was extended in vivo where EHNA dramatically prolonged the bleeding time, but only in Abcc4 KO mice. Further, we demonstrated in human platelets that ABCC4 inhibition, when coupled with phosphodiesterase inhibition, strongly impaired platelet aggregation. These findings have important clinical implications because they directly highlight an important relationship between ABCC4 transporter function and phosphodiesterases in accounting for the cAMP-directed activity of antithrombotic agents.

Von Willebrand factor propeptide: biology and clinical utility

Abstractvon Willebrand factor (VWF) is a large multimeric glycoprotein that mediates the attachment of platelets to damaged endothelium and also serves as the carrier protein for coagulation factor VIII (FVIII), protecting it from proteolytic degradation. Quantitative or qualitative defects in VWF result in von Willebrand disease (VWD), a common inherited bleeding disorder. VWF is synthesized with a very large propeptide (VWFpp) that is critical for intracellular processing of VWF. VWFpp actively participates in the process of VWF multimerization and is essential for trafficking of VWF to the regulated storage pathway. Mutations identified within VWFpp in VWD patients are associated with altered VWF structure and function. The assay of plasma VWFpp has clinical utility in assessing acute and chronic vascular perturbation associated with diseases such as thrombotic thrombocytopenic purpura, sepsis, and diabetes among others. VWFpp assay also has clear utility in the diagnosis of VWD subtypes, particularly in discriminating true type 3 subjects from type 1C (reduced plasma survival of VWF), which is clinically important and has implications for therapeutic treatment.

Enhancement of bioactivity of titanium carbonitride nanocomposite thin films on steels with biosynthesized hydroxyapatite.

Thin films of titanium carbonitride (TiCN) were fabricated by DC magnetron sputtering on medical grade steel. The biocompatibility of the coating was further enhanced by growing hydroxyapatite crystals over the TiCN-coated substrates using biologically activated ammonia from synthetic urine. The coatings were characterized using X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy (SEM)-energy dispersive spectroscopy, and Raman spectroscopy. The electrochemical behavior of the coatings was determined in simulated body fluid. In addition, hemocompatibility was assessed by monitoring the attachment of platelets on the coating using SEM. The wettability of the coatings was measured in order to correlate with biocompatibility results. Formation of a coating with granular morphology and the preferred orientation was confirmed by SEM and X-ray diffraction results. The hydroxyapatite coating led to a decrease in thrombogenicity, resulting in controlled blood clot formation, hence demonstrating the hemocompatibility of the coating.

Hemocompatible biomaterials of zwitterionic sulfobetaine hydrogels regulated with pH-responsive DMAEMA random sequences

ABSTRACTPolyzwitterionic biomaterial with pH-responsive and hemocompatible dual functions is formulated by the copolymerization of 2-(N, N-dimethyl amino) ethyl methacrylate (DMAEMA) sequences and sulfobetaine methacrylate (SBMA) moieties. Hydrogels are salt responsive, due to the interplay between hydrogen bonds formation, ions solvation, and antipolyelectrolyte effect, while DMAEMA sequences provide pH-responsiveness. Their low-fouling nature is demonstrated using plasma proteins and bacteria. Improved hemocompatibility with SBMA content is unveiled by the resistance to platelet and erythrocyte attachment, as well as from delayed plasma clotting time. Overall, this study suggests that dual-functional zwitterionic hydrogels are promising pH-responsive and hemocompatible polymeric biomaterials.

Facile fabrication of bactericidal and antifouling switchable chitosan wound dressing through a ‘click’-type interfacial reaction

A facile approach to functionalize chitosan (CS) non-woven surface with the bactericidal and antifouling switchable moieties is presented. Azlactone-cationic carboxybetaine ester copolymer was firstly prepared, then chemically attached onto CS non-woven surface through the fast and efficient ‘click’-type interfacial reaction between CS primary amines and azlactone moieties. The CS non-woven surface functionalized with cationic carboxybetaine esters is able to kill bacteria effectively. Upon the hydrolysis of carboxybetaine esters into zwitterionic groups, the resulting zwitterionic surface can further prevent the attachment of proteins, platelets, erythrocytes and bacteria. This CS non-woven that switches from bactericidal performance during storage to antifouling property before its service has great potential in wound dressing applications.

Cilostazol, Not Aspirin, Prevents Stenosis of Bioresorbable Vascular Grafts in a Venous Model.

Despite successful translation of bioresorbable vascular grafts for the repair of congenital heart disease, stenosis remains the primary cause of graft failure. In this study, we investigated the efficacy of long-term treatment with the antiplatelet drugs, aspirin and cilostazol, in preventing stenosis and evaluated the effect of these drugs on the acute phase of inflammation and tissue remodeling. C57BL/6 mice were fed a drug-mixed diet of aspirin, cilostazol, or normal chow during the course of follow-up. Bioresorbable vascular grafts, composed of poly(glycolic acid) mesh sealed with poly(l-lactide-co-ε-caprolactone), were implanted as inferior vena cava interposition conduits and followed up for 2 weeks (n=10 per group) or 24 weeks (n=15 per group). Both aspirin and cilostazol suppressed platelet activation and attachment onto the grafts. On explant at 24 weeks, well-organized neotissue had developed, and cilostazol treatment resulted in 100% graft patency followed by the aspirin (67%) and no-treatment (60%) groups (P<0.05). Wall thickness and smooth muscle cell proliferation in the neotissue of the cilostazol group were decreased when compared with that of the no-treatment group at 24 weeks. In addition, cilostazol was shown to have an anti-inflammatory effect on neotissue at 2 weeks by regulating the recruitment and activation of monocytes. Cilostazol prevents stenosis of bioresorbable vascular graft in a mouse inferior vena cava implantation model up to 24 weeks and is accompanied by reduction of smooth muscle cell proliferation and acute inflammation.

Development of a Polyester Coating Combining Antithrombogenic and Cell Adhesive Properties: Influence of Sequence and Surface Density of Adhesion Peptides.

Biofunctionalization strategies have been developed to improve small-diameter vascular grafts. However, a fully successful coating featuring antithrombogenic properties while allowing for endothelialization has not been achieved yet. In this report, we explored the combination of low-fouling polyethylene glycol (PEG) and adhesion peptides, namely, RGD, YIGSR, and REDV, grafted on top of polyvinylamine (PVAm)-coated polyester. The peptides were grafted over a wide range of density (ca. 20-2000 pmol/cm(2)) on top of a dense PEG underlayer. The coating performances were assessed through HUVEC adhesion, platelet attachment, and protein adsorption, which were all drastically diminished on PEG-coated samples. RGD exhibited the expected high adhesive properties, toward both HUVEC and platelets. REDV had no effect neither on platelet attachment, as expected, nor on HUVEC adhesion, in contrast with previous reports. YIGSR was the most promising sequence even though its combination with other agents should be explored to further decrease thrombogenicity for vascular graft applications.

Abstract 55: Chronic Loss of Serotonin Transporter Function Alters Platelet Adhesion and Spreading

Clinical observations indicate that patients taking selective serotonin reuptake inhibitors(SSRIs) have an increased risk of bleeding. However it remains unclear how SSRIs, which inhibit the serotonin transporter(SERT), modulate homeostasis and platelet activation. There are two primary mechanisms by which serotonin(5HT) affects platelet function: activation of the 5HT2A receptor and uptake by SERT. It has also been demonstrated that SERT regulates aIIbB3(GPIIbIIIa) function, the common signaling pathway in platelet activation and crucial for platelet aggregation. We sought to elucidate how the serotonergic system modulates aIIbB3 function by examining platelet spreading and attachment to immobilized fibrinogen. To model altered serotonergic tone, we used mice with a genetic deletion of SERT(SERT KO). As observed with SSRI use, the blood 5HT concentration in SERT KO mice is miniscule. We found that SERT KO mice display increased tail bleed time and thrombin time. Interestingly, SERT KO platelets showed reduced spreading but increased attachment on immobilized fibrinogen. 5HT(100nM) was able to rescue the reduced spreading on immobilized fibrinogen. To determine the mechanism, we examined platelet spreading with kentanserin(5HT2A antagonist) or citalopram(SSRI). We found that acute administration of citalopram did not alter spreading at physiologically relevant levels. Blockade of 5HT2A activation however, caused WT platelets to behave similarly to SERT KO with reduced spreading. Src is a tyrosine kinase that plays a major role in platelet spreading. Using in-cell western, P-Src(416) was decreased in SERT KO mice following spreading on immobilized. After addition of 5HT, P-Src(416) in SERT KO platelets was normalized to WT P-Src levels. Furthermore, treatment with ketanserin caused WT P-Src(416) to decreased to SERT KO P-Src(416) levels. These finding suggest that the peripheral serotonergic system is “recalibrated” in the absence of 5HT and leads to altered function of aIIbB3 via 5HT2A. From this data, we have elucidated a novel regulatory mechanism of 5HT for platelet fibrinogen binding that could regulate the recruitment of platelets by fibrinogen binding to a growing thrombus.

Biocompatible and antifouling coating of cell membrane phosphorylcholine and mussel catechol modified multi-arm PEGs.

The design and easy fabrication of biocompatible and antifouling coatings on different materials are extremely important for biotechnological and biomedical devices. Here we report a substrate-independent biomimetic modification strategy for fabricating a biocompatible and antifouling ultra-thin coating. Cell membrane antifouling phosphorylcholine (PC) and/or mussel adhesive catechol (c) groups are grafted at the amino-ends of an 8-armed poly(ethylene glycol). The PC groups are introduced by grafting a random copolymer bearing both PC and active ester groups. The modified 8-arm PEGs (PEG-2c-23PC, PEG-6c-23PC and PEG-8c) anchor themselves onto various substrates from aqueous solution and form cell outer membrane mimetic surfaces. Static contact angle, atomic force microscope (AFM) and X-ray photoelectron spectra (XPS) measurements confirm the successful fabrication of coatings on polydopamine (PDA) precoated surfaces. Real-time interaction results between proteins/bacteria and the coatings measured by surface plasmon resonance (SPR) technique suggest excellent anti-protein adsorption and short-term anti-bacteria adhesion performance. The long-term bacteria adhesion, platelet and L929 cell attachment results strongly support the SPR conclusions. Furthermore, the cell membrane mimetic and mussel adhesive protein mimetic PEG-2c-23PC shows hardly any toxicity to L929 fibroblasts, and the coating surface demonstrates the best anti-biofouling performance. This PDA-assisted immobilization of PC and/or catechol modified multi-arm PEGs provides a convenient and universal way to produce a biocompatible and fouling-resistant surface with tailor-made functions, which hopefully can be expanded to a wider range of applications based on both structure and surface superiorities.

Thrombin-Dependent Incorporation of Von Willebrand Factor into a Fibrin Network

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components and coagulation factors. It has been indicated that VWF crosslinks to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). The aim of our study was to investigate the mechanism of VWF incorporation into a fibrin network and thereby characterize the role of VWF in arterial thrombus growth.We monitored the interactions of von Willebrand factor with fibrin(ogen) using different techniques and enzymes in purified test system at high shear stress. Applying surface techniques, ellipsometry and surface plasmon resonance, we real-time observed that VWF does not bind to either a fibrinogen monolayer or to a polymerized fibrin layer formed on the solid surface. However, we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin. Using either Arvin (cleaves FpA but not FpB) or protease III from snake venom Crotalus atrox (cleaves 42 amino acids, where FpB is located) we were able to show that VWF interacts with fibrin monomers. Furthermore, using transmittance measurements we observed that in the presence of VWF the density of the fibrin clot increases suggesting that VWF incorporates into fibrin network in solution. These findings indicate that incorporation of VWF into a fibrin network occurs via the E domain of fibrinogen where FpA and/or FpB were situated. Using a domain deletion mutant deltaC1C2-VWF we demonstrated the involvement of the C1C2 domain of VWF in the binding to fibrin monomers. Our results did not show any interaction of deltaC1C2-VWF with fibrin monolayer in the presence of thrombin. Application of the inhibitor K9-DON against factor XIIIa in the presence of calcium and thrombin slightly decreased the amount of VWF adsorbed on fibrin monolayer but not completely abolished it, illustrating that cross linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Additionally, under high shear conditions, we were able to show that platelets adhere to fibrin only if VWF had been incorporated. Addition of a GPIb blocking antibody almost completely abolished the adhesion of platelets to the fibrin monomers surface with bound VWF. These data provided evidence that the binding of platelets to a fibrin monomer layer under high shear rate is completely dependent on the VWF-GPIb interaction. All experiments were performed in the presence of abciximab suggesting that the GPIIb/IIIa receptor is not involved in this binding.In conclusion, our experiments show that the C1C2 domain of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate. Additionally it may help to explain the observed phenotype in types I and III of von Willebrand Disease and the Bernard-Soulier Syndrome. Low amount of (functional) VWF or deficiency in GPIb-IX-V, here lead to disruption in thrombus formation and wound healing. DisclosuresNo relevant conflicts of interest to declare.

Thrombin-dependent Incorporation of von Willebrand Factor into a Fibrin Network

Attachment of platelets from the circulation onto a growing thrombus is a process involving multiple platelet receptors, endothelial matrix components, and coagulation factors. It has been indicated previously that during a transglutaminase reaction activated factor XIII (FXIIIa) covalently cross-links von Willebrand factor (VWF) to polymerizing fibrin. Bound VWF further recruits and activates platelets via interactions with the platelet receptor complex glycoprotein Ib (GPIb). In the present study we found proof for binding of VWF to a fibrin monomer layer during the process of fibrinogen-to-fibrin conversion in the presence of thrombin, arvin, or a snake venom from Crotalus atrox. Using a domain deletion mutant we demonstrated the involvement of the C domains of VWF in this binding. Substantial binding of VWF to fibrin monomers persisted in the presence of the FXIIIa inhibitor K9-DON, illustrating that cross-linking via factor XIII is not essential for this phenomenon and suggesting the identification of a second mechanism through which VWF multimers incorporate into a fibrin network. Under high shear conditions, platelets were shown to adhere to fibrin only if VWF had been incorporated. In conclusion, our experiments show that the C domains of VWF and the E domain of fibrin monomers are involved in the incorporation of VWF during the polymerization of fibrin and that this incorporation fosters binding and activation of platelets. Fibrin thus is not an inert end product but partakes in further thrombus growth. Our findings help to elucidate the mechanism of thrombus growth and platelet adhesion under conditions of arterial shear rate.

Fabrication of micro‐patterned titanium dioxide nanotubes thin film and its biocompatibility

In this study, in order to obtain a novel biomaterials surface which possesses the functions of an anticoagulant, inhibiting smooth muscle proliferation and pro-endothelialisation simultaneously, a micro-patterned titanium dioxide (TiO2)-nanotubes (P-TiO2-nanotubes) thin film was prepared on titanium (Ti) surface by photolithography combining with anodising method. The authors found that the P-TiO2-nanotubes could reduce platelet attachment and conformational change of fibrinogen (FGN) as well as promoting endothelial cells (ECs) growth compared with the TiO2-nanotubes. Notably, P-TiO2-nanotubes could significantly reduce smooth muscle cells (SMCs) proliferation compared with TiO2-nanotubes and TiO2, and the ECs and SMCs cultured on the P-TiO2-nanotubes thin film were elongated, which was suggested to be beneficial for maintaining the cell function. Therefore it is suggested that the P-TiO2-nanotubes thin film can contribute more to biocompatible functions of regulating and coordinating the behaviour of platelets, ECs and SMCs.

The use of the ex vivo Chandler Loop Apparatus to assess the biocompatibility of modified polymeric blood conduits.

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the subsequent attachment and activation of platelets, monocyte/macrophage adhesion, and inflammatory cell signaling events, leading to post-procedural complications. The Chandler Loop Apparatus is an experimental system that allows researchers to study the molecular and cellular interactions that occur when large volumes of blood are perfused over polymeric conduits. To that end, this apparatus has been used as an ex vivo model allowing the assessment of the anti-inflammatory properties of various polymer surface modifications. Our laboratory has shown that blood conduits, covalently modified via photoactivation chemistry with recombinant CD47, can confer biocompatibility to polymeric surfaces. Appending CD47 to polymeric surfaces could be an effective means to promote the efficacy of polymeric blood conduits. Herein is the methodology detailing the photoactivation chemistry used to append recombinant CD47 to clinically relevant polymeric blood conduits and the use of the Chandler Loop as an ex vivo experimental model to examine blood interactions with the CD47 modified and control conduits.

Platelet Membrane Glycoproteins: A Historical Review

The search for the components of the platelet surface that mediate platelet adhesion and platelet aggregation began for earnest in the late 1960s when electron microscopy demonstrated the presence of a carbohydrate-rich, negatively charged outer coat that was called the "glycocalyx." Progressively, electrophoretic procedures were developed that identified the major membrane glycoproteins (GP) that constitute this layer. Studies on inherited disorders of platelets then permitted the designation of the major effectors of platelet function. This began with the discovery in Paris that platelets of patients with Glanzmann thrombasthenia, an inherited disorder of platelet aggregation, lacked two major GP. Subsequent studies established the role for the GPIIb-IIIa complex (now known as integrin αIIbβ3) in binding fibrinogen and other adhesive proteins on activated platelets and the formation of the protein bridges that join platelets together in the platelet aggregate. This was quickly followed by the observation that platelets of patients with the Bernard-Soulier syndrome, with macrothrombocytopenia and a distinct disorder of platelet adhesion, lacked the carbohydrate-rich, negatively charged, GPIb. It was shown that GPIb, through its interaction with von Willebrand factor, mediated platelet attachment to injured sites in the vessel wall. What follows is a personal reflection on the studies that were performed in the early pioneering days.

Chondroitin sulfate coatings display low platelet but high endothelial cell adhesive properties favorable for vascular implants.

This study highlights the advantages of chondroitin sulfate (CS) as a sublayer combining selective low-fouling properties, low-platelet adhesion and pro-adhesive properties on endothelial cells, making CS promising for vascular graft applications. These properties were evaluated by comparing CS with well-known low-fouling coatings such as poly(ethylene glycol) (PEG) and carboxymethylated dextran (CMD), which were covalently grafted on primary amine-rich plasma polymerized (LP) films. Protein adsorption studies by quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence measurements showed that CS is as effective as PEG in reducing fibrinogen adsorption (~90% reduction). CS also largely reduced adsorption of bovine serum albumin (BSA) as well as fetal bovine serum (FBS) but to a lower extent than PEG and CMD surfaces (72% vs 85% for BSA and 66% vs 89% for FBS). Whole blood perfusion assays indicated that, while LP surfaces were highly reactive with platelets, PEG, CMD, and CS grafted surfaces drastically decreased platelet adhesion and activation to levels significantly lower than polyethylene terephthalate (PET) surfaces. Finally, while human umbilical vein endothelial cell (HUVEC) adhesion and growth were found to be very limited on PEG and CMD, they were significantly increased on CS compared to that on bare PET and reached similar values as those for tissue culture polystyrene positive controls. Interestingly, HUVEC retention during perfusion with blood was found to be excellent on CS but poor on PET. Overall, our results suggest that the CS surface has the advantage of promoting HUVEC growth and resistance to flow-induced shear stress while preventing fibrinogen and platelet attachment. Such a nonthrombogenic but endothelial-cell adhesive surface is thus promising to limit vascular graft occlusion.

The Effect of Platelet Proteins Released in Response to Titanium Implant Surfaces on Macrophage Pro-Inflammatory Cytokine Gene Expression.

Platelets are one of the earliest cell types to interact with surgically inserted titanium implants. This in vitro study investigated the effect of titanium surface-induced platelet releasate on macrophage cytokine gene expression. To mimic the in vivo temporal sequence of platelet arrival and protein production at the implant surface and the subsequent effect of these proteins on mediators of the immune response, the levels of platelet attachment and activation in response to culture on smooth polished, sandblasted and acid-etched (SLA), and hydrophilic-modified SLA (modSLA) titanium surfaces were first determined by microscopy and protein assay. The subsequent effect of the platelet-released proteins on human THP-1 macrophage cytokine gene expression was determined by polymerase chain reaction array after 1 and 3 days of macrophage culture on the titanium surfaces in platelet-releasate conditioned media. Platelet attachment was surface dependent with decreased attachment observed on the hydrophilic (modSLA) surface. The platelet releasate, when considered independently of the surface effect, elicited an overall pro-inflammatory response in macrophage cytokine gene expression, that is, the expression of typical pro-inflammatory cytokine genes such as TNF, IL1a, IL1b, and CCL1 was significantly up-regulated whereas the expression of anti-inflammatory cytokine genes such as IL10, CxCL12, and CxCL13 was significantly down-regulated. However, following platelet exposure to different surface modifications, the platelet releasate significantly attenuated the macrophage pro-inflammatory response to microrough (SLA) titanium and hastened an anti-inflammatory response to hydrophilic (modSLA) titanium. Theses results demonstrate that titanium surface topography and chemistry are able to influence the proteomic profile released by platelets, which can subsequently influence macrophage pro-inflammatory cytokine expression. This immunomodulation may be an important mechanism via which titanium surface modification influences osseointegration.

Heparin surfaces: Impact of immobilization chemistry on hemocompatibility and protein adsorption

Immobilization of heparin to surfaces has been used for decades to reduce the thrombogenicity of blood contacting devices. This study evaluates how the mode of covalent heparin bonding affects the hemocompatibility and uptake of antithrombin on surfaces in whole blood. End-point attached (EPA) heparin, using the proprietary Carmeda Bioactive Surface (CBAS Surface), was compared with other methods of covalent heparin bonding that typically yield multiple covalent linkages (using reductive amination of periodate oxidized native heparin or EDC coupling of native heparin). All heparin surfaces were immobilized on flexible polyvinyl chloride tubing and exposed to fresh non-anticoagulated blood in an in vitro recirculating Chandler loop blood model. After exposure, biomarkers for coagulation and platelet activation were analyzed in the solution phase, and adsorbed plasma proteins were eluted from the heparin surfaces and measured for surface concentration of antithrombin and total adsorbed protein. Only the EPA-heparin surface conferred thromboresistance, as observed by the absence of clotting. Attachment and activation of platelets as well as activation of the clotting cascade was significantly lower on the EPA-heparin surface when compared with the other heparin surfaces. In addition, antithrombin constituted ∼40% of the total adsorbed plasma protein concentration on the EPA-heparin surfaces.

Pro32Pro33 Mutations in the Integrinβ3PSI Domain Result inαIIbβ3Priming and Enhanced Adhesion: Reversal of the Hypercoagulability Phenotype by the Src Inhibitor SKI-606

The plasma-membrane integrin αIIbβ3 (CD41/CD61, GPIIbIIIa) is a major functional receptor in platelets during clotting. A common isoform of integrin β3, Leu33Pro is associated with enhanced platelet function and increased risk for coronary thrombosis and stroke, although these findings remain controversial. To better understand the molecular mechanisms by which this sequence variation modifies platelet function, we produced transgenic knockin mice expressing a Pro32Pro33 integrin β3. Consistent with reports utilizing human platelets, we found significantly reduced bleeding and clotting times, as well as increased in vivo thrombosis, in Pro32Pro33 homozygous mice. These alterations paralleled increases in platelet attachment and spreading onto fibrinogen resulting from enhanced integrin αIIbβ3 function. Activation with protease-activated receptor 4- activating peptide, the main thrombin signaling receptor in mice, showed no significant difference in activation of Pro32Pro33 mice as compared with controls, suggesting that inside-out signaling remains intact. However, under unstimulated conditions, the Pro32Pro33 mutation led to elevated Src phosphorylation, facilitated by increased talin interactions with the β3 cytoplasmic domain, indicating that the αIIbβ3 intracellular domains are primed for activation while the ligand-binding domain remains unchanged. Acute dosing of animals with a Src inhibitor was sufficient to rescue the clotting phenotype in knockin mice to wild-type levels. Together, our data establish that the Pro32Pro33 structural alteration modifies the function of integrin αIIbβ3, priming the integrin for outside-in signaling, ultimately leading to hypercoagulability. Furthermore, our data may support a novel approach to antiplatelet therapy by Src inhibition where hemostasis is maintained while reducing risk for cardiovascular disease.