Immobilization of actively thromboresistant assemblies on sterile blood-contacting surfaces.

Rapid one-step modification of thrombomodulin with alkylamine derivatives such as azide, biotin, and PEG is achieved using an evolved sortase (eSrtA) mutant. The feasibility of a point-of-care scheme is demonstrated herein to site-specifically immobilize azido-thrombomodulin on sterilized commercial ePTFE vascular grafts, which exhibit superior thromboresistance compared with commercial heparin-coated grafts in a primate model of acute graft thrombosis.

Thrombotic responses of endothelial outgrowth cells to protein-coated surfaces.

There is significant clinical need for viable small-diameter vascular grafts. While there are many graft biomaterials in development, few have been clinically successful. Evaluation of grafts with a clinically relevant model is needed to drive development. This work examined extracellular matrix coatings on the thrombotic phenotype of endothelial outgrowth cells (EOCs). EOCs were tested on flat plates and tubular grafts. Flat plate studies examined collagen I, collagen IV, fibronectin and α-elastin coatings. EOCs attached or proliferated more readily on collagen I and fibronectin surfaces as determined by total DNA. The production of activated protein C (APC) by EOCs was also dependent on the surface coating, with collagen I and fibronectin displaying a higher activity than both collagen IV and α-elastin on flat plate studies. Based on these results, only collagen I and fibronectin coatings were tested on expanded polytetrafluoroethylene (ePTFE) in the ex vivo model. Tubular samples showed significantly greater tissue factor pathway inhibitor gene expression on collagen I than on fibronectin. Platelet adhesion was not significantly different, but EOCs on collagen I produced significantly lower APC than on fibronectin, suggesting that differences exist between the flat plate and tubular cultures. Overall, while the hemostatic phenotype of EOCs displayed some differences, cell responses were largely independent of the matrix coating. EOCs adhered strongly to both fibronectin- and collagen-I-coated ePTFE grafts under ex vivo (100 ml/min) flow conditions suggesting the usefulness of this clinically relevant cell source, testing modality, and shunt model for future work examining biomaterials and cell conditioning before implantation.

Antithrombotic effect of antisense factor XI oligonucleotide treatment in primates.

OBJECTIVE: During coagulation, factor IX (FIX) is activated by 2 distinct mechanisms mediated by the active proteases of either FVIIa or FXIa. Both coagulation factors may contribute to thrombosis; FXI, however, plays only a limited role in the arrest of bleeding. Therefore, therapeutic targeting of FXI may produce an antithrombotic effect with relatively low hemostatic risk. APPROACH AND RESULTS: We have reported that reducing FXI levels with FXI antisense oligonucleotides produces antithrombotic activity in mice, and that administration of FXI antisense oligonucleotides to primates decreases circulating FXI levels and activity in a dose-dependent and time-dependent manner. Here, we evaluated the relationship between FXI plasma levels and thrombogenicity in an established baboon model of thrombosis and hemostasis. In previous studies with this model, antibody-induced inhibition of FXI produced potent antithrombotic effects. In the present article, antisense oligonucleotides-mediated reduction of FXI plasma levels by ≥ 50% resulted in a demonstrable and sustained antithrombotic effect without an increased risk of bleeding. CONCLUSIONS: These results indicate that reducing FXI levels using antisense oligonucleotides is a promising alternative to direct FXI inhibition, and that targeting FXI may be potentially safer than conventional antithrombotic therapies that can markedly impair primary hemostasis.

Capture of circulatory endothelial progenitor cells and accelerated re-endothelialization of a bio-engineered stent in human ex vivo shunt and rabbit denudation model.

AIMS: The Genous™ Bio-engineered R™ stent (GS) aims to promote vascular healing by capture of circulatory endothelial progenitor cells (EPCs) to the surface of the stent struts, resulting in accelerated re-endothelialization. Here, we assessed the function of the GS in comparison to bare-metal stent (BMS), when exposed to the human and animal circulation. METHODS AND RESULTS: First, 15 patients undergoing coronary angiography received an extracorporeal femoral arteriovenous (AV) shunt containing BMS and GS. Macroscopical mural thrombi were observed in BMS, whereas GS remained visibly clean. Confocal and scanning electron microscopic (SEM) analysis of GS showed an increase in strut coverage. Quantitative polymerase chain reaction (qPCR) analysis of captured cells on the GS demonstrated increased expression of endothelial markers KDR/VEGFR2 and E-selectin, and a decrease in pro-thrombogenic markers tissue factor pathway inhibitor and plasminogen activator inhibitor-1 compared with BMS. Secondly, a similar primate AV shunt model was used to validate these findings and occlusion of BMS was observed, while GS remained patent, as demonstrated by live imaging of indium-labelled platelets. Thirdly, in an in vitro cell-capture assay, GS struts showed increased coverage by EPCs, whereas monocyte coverage remained similar to BMS. Finally, the assessment of re-endothelialization was studied in a rabbit denudation model. Twenty animals received BMS and GS in the aorta and iliac arteries for 7 days. Scanning electron microscopic analysis showed a trend towards increased strut coverage, confirmed by qPCR analysis revealing increased levels of endothelial markers (Tie2, CD34, PCD31, and P-selectin) in GS. CONCLUSION: In this proof-of-concept study, we have demonstrated that the bio-engineered EPC-capture stent, Genous™ R™ stent, is effective in EPC capture, resulting in accelerated re-endothelialization and reduced thrombogenicity.

Fluid shear stress alters the hemostatic properties of endothelial outgrowth cells.

Surface endothelialization is an attractive means to improve the performance of small diameter vascular grafts. While endothelial outgrowth cells (EOCs) are considered a promising source of autologous endothelium, the ability of EOCs to modulate coagulation-related blood activities is not well understood. The goal of this study was to assess the role of arterial flow conditions on the thrombogenic phenotype of EOCs. EOCs derived from baboon peripheral blood, as well as mature arterial endothelial cells from baboons, were seeded onto adsorbed collagen, then exposed to physiologic levels of fluid shear stress. For important hemostatic pathways, cellular responses to shear stress were characterized at the gene and protein level and confirmed with a functional assay for activated protein C (APC) activity. For EOCs, fluid shear stress upregulated gene and protein expression of anticoagulant and platelet inhibitory factors, including thrombomodulin, tissue factor pathway inhibitor, and nitric oxide synthase 3 (eNOS). Fluid shear stress significantly altered the functional activity of EOCs by increasing APC levels. This study demonstrates that fluid shear stress is an important determinant of EOC hemostatic properties. Accordingly, manipulation of EOC phenotype by mechanical forces may be important for the development of thrombo-resistant surfaces on engineered vascular implants.

Numerical investigation of the effects of channel geometry on platelet activation and blood damage.

Thromboembolic complications in Bileaflet mechanical heart valves (BMHVs) are believed to be due to the combination of high shear stresses and large recirculation regions. Relating blood damage to design geometry is therefore essential to ultimately optimize the design of BMHVs. The aim of this research is to quantitatively study the effect of 3D channel geometry on shear-induced platelet activation and aggregation, and to choose an appropriate blood damage index (BDI) model for future numerical simulations. The simulations in this study use a recently developed lattice-Boltzmann with external boundary force (LBM-EBF) method [Wu, J., and C. K. Aidun. Int. J. Numer. Method Fluids 62(7):765-783, 2010; Wu, J., and C. K. Aidun. Int. J. Multiphase flow 36:202-209, 2010]. The channel geometries and flow conditions are re-constructed from recent experiments by Fallon [The Development of a Novel in vitro Flow System to Evaluate Platelet Activation and Procoagulant Potential Induced by Bileaflet Mechanical Heart Valve Leakage Jets in School of Chemical and Biomolecular Engineering. Atlanta: Georgia Institute of Technology] and Fallon et al. [Ann. Biomed. Eng. 36(1):1]. The fluid flow is computed on a fixed regular 'lattice' using the LBM, and each platelet is mapped onto a Lagrangian frame moving continuously throughout the fluid domain. The two-way fluid-solid interactions are determined by the EBF method by enforcing a no-slip condition on the platelet surface. The motion and orientation of the platelet are obtained from Newtonian dynamics equations. The numerical results show that sharp corners or sudden shape transitions will increase blood damage. Fallon's experimental results were used as a basis for choosing the appropriate BDI model for use in future computational simulations of flow through BMHVs.

A biologically active surface enzyme assembly that attenuates thrombus formation.

Activation of hemostatic pathways by blood-contacting materials remains a major hurdle in the development of clinically durable artificial organs and implantable devices. We postulate that surface-induced thrombosis may be attenuated by the reconstitution onto blood contacting surfaces of bioactive enzymes that regulate the production of thrombin, a central mediator of both clotting and platelet activation cascades. Thrombomodulin (TM), a transmembrane protein expressed by endothelial cells, is an established negative regulator of thrombin generation in the circulatory system. Traditional techniques to covalently immobilize enzymes on solid supports may modify residues contained within or near the catalytic site, thus reducing the bioactivity of surface enzyme assemblies. In this report, we present a molecular engineering and bioorthogonal chemistry approach to site-specifically immobilize a biologically active recombinant human TM fragment onto the luminal surface of small diameter prosthetic vascular grafts. Bioactivity and biostability of TM modified grafts is confirmed in vitro and the capacity of modified grafts to reduce platelet activation is demonstrated using a non-human primate model. These studies indicate that molecularly engineered interfaces that display TM actively limit surface-induced thrombus formation.

Non-destructive label-free monitoring of collagen gel remodeling using optical coherence tomography.

Matrix remodeling plays a fundamental role in physiological and pathological processes, as well as in tissue engineering applications. In this paper, optical coherence tomography (OCT), a non-destructive optical imaging technology, was used to image collagen gel remodeling by smooth muscle cells (SMCs). The optical scattering properties of collagen-SMC gels were characterized quantitatively by fitting OCT data to a theoretical model. Matrix remodeling over 5 days produced a 10-fold increase in the reflectivity of the collagen gels, corresponding to a decrease in scattering anisotropy from 0.91 to 0.46. The increase in reflectivity was corroborated in confocal mosaic images. Blocking matrix degradation in collagen-SMC gels with doxycycline, a non-specific matrix metalloproteinases (MMPs) inhibitor, impeded the decrease in scattering anisotropy and resulted in few macroscopic signs of remodeling. Causing matrix degradation in acellular gels with a 3 h treatment of MMP-8 (collagenase 2) partially mimicked the decrease in anisotropy measured in collagen-SMC gels after 5 days. These results suggest that the decrease in scattering anisotropy in the collagen-SMC gels was due to MMP activity that degrades collagen fibrils into smaller fragments.

A role for factor XIIa-mediated factor XI activation in thrombus formation in vivo.

Mice lacking factor XII (fXII) or factor XI (fXI) are resistant to experimentally-induced thrombosis, suggesting fXIIa activation of fXI contributes to thrombus formation in vivo. It is not clear whether this reaction has relevance for thrombosis in pri mates. In 2 carotid artery injury models (FeCl(3) and Rose Bengal/laser), fXII-deficient mice are more resistant to thrombosis than fXI- or factor IX (fIX)-deficient mice, raising the possibility that fXII and fXI function in distinct pathways. Antibody 14E11 binds fXI from a variety of mammals and interferes with fXI activation by fXIIa in vitro. In mice, 14E11 prevented arterial occlusion induced by FeCl(3) to a similar degree to total fXI deficiency. 14E11 also had a modest beneficial effect in a tissue factor-induced pulmonary embolism model, indicating fXI and fXII contribute to thrombus formation even when factor VIIa/tissue factor initiates thrombosis. In baboons, 14E11 reduced platelet-rich thrombus growth in collagen-coated grafts inserted into an arteriovenous shunt. These data support the hypothesis that fXIIa-mediated fXI activation contributes to thrombus formation in rodents and primates. Since fXII deficiency does not impair hemostasis, targeted inhibition of fXI activation by fXIIa may be a useful antithrombotic strategy associated with a low risk of bleeding complications.

Safety and antithrombotic efficacy of moderate platelet count reduction by thrombopoietin inhibition in primates.

Most heart attacks and strokes are caused by blood clots (thrombi) that block the vasculature. Because disease-causing arterial thrombosis depends on blood platelets, platelet inhibitors such as aspirin and clopidogrel effectively decrease the risk of thrombosis; however, they also impair platelet-dependent hemostasis that staunches bleeding from wounds and can therefore produce excessive bleeding. Experimental studies show that a reduction in the number of platelets also inhibits thrombosis, but these treatments also interfere with platelet function. Because normal hemostasis requires that the platelet concentration remain within a physiological range in the circulation, we evaluated whether lowering the number of circulating platelets--but only to a value still within the normal range--by inhibiting platelet formation in the bone marrow inhibits acute thrombogenesis in baboons. We reduced the platelet count with an inhibitor against the megakaryocyte-promoting hormone thrombopoietin and then showed that experimental occlusive thrombogenesis on collagen-coated vascular grafts was reduced, without impairment of primary hemostasis. These results suggest that suppressing platelet production without interfering with the hemostatic function of platelets may offer a safe alternative to current therapies for prevention of stroke and heart attack triggered by blood clotting.

Quantitative characterization of developing collagen gels using optical coherence tomography.

Nondestructive optical imaging methods such as optical coherence tomography (OCT) have been proposed for characterizing engineered tissues such as collagen gels. In our study, OCT was used to image collagen gels with different seeding densities of smooth muscle cells (SMCs), including acellular gels, over a five-day period during which the gels contracted and became turbid with increased optical scattering. The gels were characterized quantitatively by their optical properties, specified by analysis of OCT data using a theoretical model. At 6 h, seeded cell density and scattering coefficient (mu(s)) were correlated, with mu(s) equal to 10.8 cm(-1)(10(6) cells mL). Seeded cell density and the scattering anisotropy (g) were uncorrelated. Over five days, the reflectivity in SMC gels gradually doubled with little change in optical attenuation, which indicated a decrease in g that increased backscatter, but only a small drop in mu(s). At five days, a subpopulation of sites on the gel showed substantially higher reflectivity (approximately a tenfold increase from the first 24 h). In summary, the increased turbidity of SMC gels that develops over time is due to a change in the structure of collagen, which affects g, and not simply due to a change in number density of collagen fibers due to contraction.

Cytoskeletal structure regulates endothelial cell immunogenicity independent of fluid shear stress.

The cardiovascular disease atherosclerosis is directly linked to the functions of endothelial cells (ECs), which are affected by fluid shear stress (FSS). High, unidirectional FSS causes EC elongation with aligned cytoskeletal components and nonimmunogenic EC functions that protect against atherosclerosis. In contrast, low, oscillatory FSS is associated with cobblestone-shaped ECs with randomly oriented cytoskeletons and proinflammatory EC functions that promote atherosclerosis. Whether EC shape plays a role in EC immunogenic functions, independent of FSS, has not been previously determined. The goal of this study was to determine the effect of EC elongation and cytoskeletal alignment on the expression of inflammatory genes and functions. With the use of micropatterned lanes, EC elongation and cytoskeletal alignment were achieved in the absence of FSS. EC gene expression of key inflammation markers determined that the elongation and cytoskeletal alignment of micropattern-elongated ECs (MPECs) alone significantly downregulated VCAM-1 while having no effect on E-selectin and ICAM-1. The positive control of FSS-elongated ECs promoted E-selectin and VCAM-1 downregulation and upregulation of ICAM-1. Functionally, monocytic U937 cells formed weaker interactions on the surface of MPECs compared with cobblestone ECs. Interestingly, MPEC expression of the known FSS-dependent transcription factor krüppel-like factor 2 (KLF2), which promotes a nonimmunogenic EC phenotype, was significantly upregulated in MPECs compared with cobblestone ECs. Cytoskeletal regulation of KLF2 expression was shown to be dependent on microtubules. Therefore, the cellular elongation and cytoskeletal alignment of MPECs regulated immunogenic gene expression and functions and may act synergistically with FSS to create an EC surface with reduced inflammatory capability.

Distinct extracellular matrix microenvironments of progenitor and carotid endothelial cells.

Endothelial cells (ECs) produce and maintain the local extracellular matrix (ECM), a critical function that contributes to EC and blood vessel health. This function is also crucial to vascular tissue engineering, where endothelialization of vascular constructs require a cell source that readily produces and maintains ECM. In this study, baboon endothelial progenitor cell (EPC) deposition of ECM (laminin, collagen IV, and fibronectin) was characterized and compared to mature carotid ECs, evaluated in both elongated and cobblestone morphologies typically found in vivo. Microfluidic micropatterning was used to create 15-microm wide adhesive lanes with 45-microm spacing to reproduce the elongated EC morphology without the influence of external forces. Both EPCs and ECs elongated on micropatterned lanes had aligned actin cytoskeleton and readily deposited ECM. EPCs deposited and remodeled the ECM to a greater extent than ECs. Since a readily produced ECM can improve graft patency, EPCs are an advantageous cell source for endothelializing vascular constructs. Furthermore, EC deposition of ECM was dependent on cell morphology, where elongated ECs deposited more collagen IV and less fibronectin compared to matched cobblestone controls. Thus micropatterned surfaces controlled EC shape and ECM deposition, which ultimately has implications for the design of tissue-engineered vascular constructs.

Prevention of vascular graft occlusion and thrombus-associated thrombin generation by inhibition of factor XI.

The protease thrombin is required for normal hemostasis and pathologic thrombogenesis. Since the mechanism of coagulation factor XI (FXI)-dependent thrombus growth remains unclear, we investigated the contribution of FXI to thrombus formation in a primate thrombosis model. Pretreatment of baboons with a novel anti-human FXI monoclonal antibody (aXIMab; 2 mg/kg) inhibited plasma FXI by at least 99% for 10 days, and suppressed thrombin-antithrombin (TAT) complex and beta-thromboglobulin (betaTG) formation measured immediately downstream from thrombi forming within collagen-coated vascular grafts. FXI inhibition with aXIMab limited platelet and fibrin deposition in 4-mm diameter grafts without an apparent increase in D-dimer release from thrombi, and prevented the occlusion of 2-mm diameter grafts without affecting template bleeding times. In comparison, pretreatment with aspirin (32 mg/kg) prolonged bleeding times but failed to prevent graft occlusion, supporting the concept that FXI blockade may offer therapeutic advantages over other antithrombotic agents in terms of bleeding complications. In whole blood, aXIMab prevented fibrin formation in a collagen-coated flow chamber, independent of factor XII and factor VII. These data suggest that endogenous FXI contributes to arterial thrombus propagation through a striking amplification of thrombin generation at the thrombus luminal surface.

Capture of flowing endothelial cells using surface-immobilized anti-kinase insert domain receptor antibody.

In humans, self-endothelialization of synthetic grafts is severely limited, but a recent interesting idea is to attract endothelial progenitor cells (EPCs) from peripheral blood onto grafts via antibodies directed at proposed EPC markers. Results with anti-CD34 antibodies have shown some promise, but it is unclear whether CD34 is the best marker for cells with re-endothelializing potential. Much evidence points to kinase insert domain receptor (KDR) as an important indicator of endothelial potential if not a definitive marker. Because KDR is not an adhesion molecule (like CD34), we first demonstrated the ability to use adsorbed and protein G-oriented antibody to this receptor to capture flowing cells onto a solid surface. Using endothelial cells and smooth muscle cells, we show in a model system under low shear rates the ability to selectively capture cells by this receptor. Furthermore, our results indicate that concomitant flow of cells lacking the receptor does not affect the efficiency of capture of KDR(+) cells but that orienting the antibody significantly increases the efficiency of capture.

Survival advantage of coagulation factor XI-deficient mice during peritoneal sepsis.

Anticoagulation is a rational approach to the treatment of sepsis-associated consumptive coagulopathy, but its application is limited because of the risk of excessive bleeding. Factor XI (FXI) contributes substantially to pathological blood coagulation (thrombosis), whereas it contributes only modestly to normal hemostasis. We found that FXI-deficient mice have reduced coagulopathy and increased survival relative to FXI-expressing wild-type mice during cecal ligation and puncture-induced acute peritonitis/sepsis. This finding suggests that FXI contributes to coagulopathy and/or inflammation during sepsis and that pharmacologic inhibition of FXI activity may alter the course and outcome of some infections.

Endothelial cell cytoskeletal alignment independent of fluid shear stress on micropatterned surfaces.

Endothelial cells (ECs) in athero-protective regions are elongated with actin and microtubule fibers aligned parallel to the direction of blood flow. Fluid shear stress (FSS) affects EC shape and functions, but little is known about shape-dependent EC properties that are independent of FSS. To evaluate these properties, ECs were elongated on micropatterned (MP) 25mum wide collagen-coated lanes (MPECs) and characterized by cell shape index, actin and microtubule alignment, and polarization of the microtubule-organizing center (MTOC). ECs on non-patterned surfaces were also exposed to FSS. MPEC elongation was microtubule-dependent (and actin-independent); shape indices and cytoskeletal alignment were comparable to FSS-elongated ECs. Cytoskeletal alignment was lost when MPECs were exposed to perpendicular FSS, but not parallel FSS. MTOC polarization was FSS-dependent. Thus, by isolating EC elongation and cytoskeletal alignment from FSS, micropatterning creates a platform for studying EC shape-related cellular functions that are independent of FSS.

Procoagulant properties of flow fields in stenotic and expansive orifices.

In the United States, over 125,000 mechanical heart valves (MHVs) are implanted each year. Flow through the MHV hinge can cause thromboemboli formation. The purpose of this study was to examine various orifice geometries representing the MHV hinge region and how these geometries may contribute to platelet activation and thrombin generation. We also characterized these flow fields with digital particle image velocimetry (DPIV). Citrated human blood at room temperature was forced through the orifices (400 and 800 microm ID) with a centrifugal bypass pump, continuously infusing calcium chloride to partially reverse the citrate anticoagulant. Blood samples were tested for the presence of thrombin-antithrombin complex (TAT) and platelet factor 4 (PF4). Velocity and shear stress were measured with DPIV using a blood analog fluid seeded with fluorescent microbeads. The results indicate that small changes in geometry, although they do not affect the bulk flow, change the coagulation propensity as blood flows through the orifices. A more abrupt geometry allows more stagnation to occur resulting in more thrombin generation. PF4 measurements indicated similar levels of platelet activation for all orifices. DPIV showed differences in the jets with respect to entrainment of stagnant fluid. These results help to pinpoint the important parameters that lead to flow stasis and subsequent thrombus formation.

Potential of baboon endothelial progenitor cells for tissue engineered vascular grafts.

Thrombosis and intimal hyperplasia limit the usefulness of small caliber vascular grafts. While some improvements have been reported for grafts seeded with mature endothelial cells (EC), the harvesting of ECs from autologous sources, for example, veins or adipose tissue, remains problematic. More recently, endothelial progenitor cells (EPCs) have been considered a promising source of ECs because EPCs can be readily isolated from whole blood then rapidly expanded in vitro. Additionally, EPCs are increasingly recognized to play important roles in hemostasis, angiogenesis, and arterial injury repair. However, the characterization of EPCs in relevant animal models remains poorly defined. Accordingly, we have characterized the isolation, growth, and functional characteristics of Baboon EPCs (BaEPCs) to evaluate their potential for an autologous cell source for tissue engineered vascular grafts. BaEPCs were successfully cultured from the peripheral blood with an average population doubling time of 1.17 +/- 0.43 days. While the BaEPCs were positive for typical EC markers of vWF, CD31, VE-cadherin, VEGF-R2, Thrombomodulin, and E-selectin, there was reduced eNOS expression. The BaEPCs cell body and actin filaments align in the direction of flow typical of mature ECs. Thus while the lack of eNOS expression is worthy of investigation, EPCs are an attractive cell source for tissue engineered vascular grafts and the baboon model has great potential for continuing evaluations of these cells.