The blood coagulation system plays the critical role of maintaining hemostasis in the body by generating thrombus (i.e., a blood clot) to stop the loss of blood when a blood vessel is broken. However, the natural behavior of blood is also to initiate a thrombotic response when it comes in contact with a synthetic material surface. This response is thus problematic when synthetic materials are used in medical devices to treat cardiovascular and vascular problems, which can lead to the failure of the device, blockage of blood flow, or the generation of emboli leading to stroke or organ failure. This chapter addresses these topics by first providing an overview of the blood coagulation system and its connection with the complement and inflammatory systems of the body. An overview of the interactions of blood when it comes in contact with synthetic material surfaces is then provided. This topic is then followed by an overview of the approaches that have been taken in attempts to improve the blood compatibility of medical devices that are used in blood-contacting applications.

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.

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.

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.

Thrombin generation is required for hemostasis at sites of vascular injury. This process is typically initiated when extravascular and subendothelial proteins, including tissue factor (TF) and collagen, are exposed to flowing blood, triggering platelet activation and formation of the TF/factor VIIa (FVIIa) complex. TF/FVIIa, in turn, activates FIX and FX that catalyze the production of thrombin. Thrombin clots fibrinogen and activates platelets and FXIII, contributing to the formation of a stable hemostatic plug. In contrast to hemostatic mechanisms that cause the arrest of bleeding, thrombosis that can restrict or block normal blood flow occurs within the lumen of blood vessels in response to vascular injury, altered blood flow patterns (eg, stenoses), and predisposing functional abnormalities of platelets and coagulation pathways. The coagulation protein FXI plays a limited role compared with FVIIa in hemostasis. Humans lacking FXI have a relatively mild disorder characterized by excessive trauma induced hemorrhage in tissues with high-fibrinolytic activity. However, several recent studies in rodents and nonhuman primates suggest that FXI contributes significantly to thrombosis. Furthermore, humans with increased levels of FXI are at an increased risk for venous thrombosis, myocardial infarction, and stroke, whereas patients with severe FXI deficiency have a reduced incidence of ischemic stroke and deep-vein thrombosis. The observation that FXI plays a significant role in thrombosis, with only a modest contribution to hemostasis, makes it an attractive target for pharmacological anticoagulant therapy.

Protein S (ProS) is an essential plasma protein that enhances the anticoagulant activity of activated protein C (APC). In vitro , purified native human Zn2+-containing ProS also exerts direct anticoagulant activity by inhibiting prothrombinase and extrinsic FXase activities independently of APC. We investigated antithrombotic effects of ProS infused without APC in a baboon shunt model of thrombogenesis that employs a device consisting of arterial and venous shear flow segments. In in vitro experiments, the Zn2+-containing human ProS used for the studies displayed >10-fold higher prothrombinase inhibitory activity and anticoagulant activity in tissue factor-stimulated plasma, and four-fold higher inhibition of the intrinsic pathway than the Zn2+-deficient ProS used. In the thrombosis model, ProS (33 μg/minute for 1 hour) or saline was infused locally; platelet and fibrin deposition in the shunt were measured over 2 hours. During experiments performed at 50 ml/minute blood flow, Zn2+-containing ProS inhibited platelet deposition 73-96% in arterial-type flow segments and 90-99% in venous-type flow segments; Zn2+-deficient ProS inhibited platelet deposition 52% in arterial-type flow segments and 65-73% in venous-type flow segments. At 100 ml/min blood flow rate, Zn2+-containing ProS inhibited platelet deposition by 39% and 73% in the respective segments; Zn2+-deficient ProS inhibited platelet deposition by 5% and 0% in the respective segments. Zn2+-containing ProS suppressed fibrin deposition by 67-90%. Systemic APC-independent ProS activity was significantly increased and thrombin-antithrombin complex levels were significantly decreased after infusion of ProS. Thus, infused human Zn2+-containing ProS is antithrombotic in primates, and may have therapeutic potential even in protein C-deficient human patients.

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.

Activation of hemostatic pathways by blood‐contacting materials remains a major hurdle in the development of clinically durable artificial organs and implantable devices. Here, it is postulated 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 coagulation 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, 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 is presented. 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.

Abstract Abstract 3361 We have developed a semi-synthetic antithrombotic heparin complex, APL001, to mimic mast cell-derived natural heparin proteoglycans (HepPG). HepPG attenuate platelet-collagen interactions under blood flow by inhibiting VWF- and GPIIb/IIIa -mediated platelet aggregation. In addition, rat-derived HepPG arrest platelet thrombus growth on collagen surfaces or at vascular injury sites, both in vitro and in vivo (Lassila et al.ATVB 1997, Kauhanen et al. ATVB 2000, Olsson et al. Thromb Haemost 2002). Our objective was to study the inhibitory capacity of APL001 for preventing human platelet aggregation in vitro and acute thrombosis in a baboon model in vivo. The effects of unfractionated heparin (UFH) and APL001 were compared in relevant coagulation assays (APTT, PT, thrombin time, anti-FXa activity, fibrinogen, FVIII:C and VWF activity (VWF:RCo) and antigen). Additionally, agonist-induced (collagen, ristocetin and ADP) platelet aggregation in citrate or hirudin-anticoagulated whole blood (Multiplate®) (n=10 healthy subjects), and platelet function analysis (PFA100®) in citrated platelet rich plasma (PRP) were assessed. In a well-established baboon thrombosis model a collagen-coated PTFE graft (length 2 cm, lumen 4 mm) was placed in an arterio-venous shunt. Prior to blood contact the thrombogenic surface was treated for 10 min with UFH or APL001 (both at 4 mg/mL). Thrombus formation was initiated by exposing the surface to blood flow (100 mL/min, shear rate 265−1), and the deposition of 111-In-labeled platelets and of fibrin was quantified continuously over 1h. Fibrin thrombus accumulation was assessed from the incorporation of circulating 125-I-fibrinogen. In the heparin-relevant coagulation tests APL001 was comparable or 20–30% more potent than UFH while FVIII, fibrinogen and VWF variables remained unaltered. In contrast to UFH, APL001 (300 μg/mL) consistently inhibited collagen- and ristocetin-induced platelet aggregation, whereas UFH had only a modest effect in comparison with PBS control (Table). ADP-induced aggregation was unaffected. Comparable results were observed in the PRP aggregation assay. PFA100 testing also demonstrated inhibitory effects. In the in vivo thrombosis model (n=4) APL001 reduced platelet deposition on collagen (vs. the results with UFH) by 34% (p=0.01), while platelet accumulation in distal propagated thrombus was reduced by 61% (p=0.16). APL001-treated surfaces accumulated 45% less fibrin than the UFH-treated surfaces (p=0.008). In conclusion, when compared with UFH APL001 inhibited both collagen- and ristocetin-induced platelet aggregation in human blood, while anticoagulant properties were comparable. In the absence of systemic antithrombotic drugs, exposure of APL001 to a highly thrombogenic collagen surface arrested thrombus formation in an in vivo baboon model. This finding suggests that locally administered APL001 alone, due to its dual antiplatelet and anticoagulant effects, may limit the growth and size of thrombus and thereby prevent subsequent thrombo-occlusion.TableAnticoagulantInhibition-% of platelet aggregation ± SDConc. 300 μg/mLnColl (3.2 μg/mL)Ristocetin (0.77 mg/mL)ADP (6.4 μM)CitrateAPL0011033 ± 1543 ± 166 ± 24UFH1011 ± 1323 ± 153 ± 7p value0.0030.0100.700HirudinAPL0011032 ± 1043 ± 178 ± 10UFH108 ± 1116 ± 166 ± 9p value0.0000.0020.600 Disclosures: Lassila: Aplagon: Chief Scientific Advisor.

Abstract Abstract 2250 It has recently been demonstrated that the intrinsic pathway is an important contributor to pathologic intravascular thrombosis, suggesting that targeting this pathway may yield effective antithrombotic agents. Furthermore, FXI deficiency is not associated with spontaneous bleeding in humans, and deficiency of a contact factor does not cause abnormal bleeding. We have previously shown that treatment with FXI Antisense Oligonucleotides (ASOs) produces potent, dose-dependent antithrombotic activity in various venous and arterial murine thrombosis models. In the current study we characterize the antithrombotic effects of FXI ASOs in a non-human primate model. Subcutaneous administration of FXI ASOs in cynomologus monkeys resulted in a dose-and time-dependant decrease in FXI mRNA levels in liver (up to 90%), decreased FXI protein and activity levels measured in plasma, and prolonged activated partial thromboplastin times (aPTT). Importantly, FXI depletion in monkeys was not associated with an increased risk of bleeding. Recent studies demonstrated that complete inhibition (>99%) of FXI by a monoclonal antibody reduced thrombin generation and prevented vascular occlusion in a collagen-coated graft inserted into a baboon arteriovenous shunt (Tucker et al., Blood 2009). In the present study, we set out to determine the relationship between FXI levels and antithrombotic activity, and in particular, the minimal level of FXI reduction required to produce an antithrombotic effect in a baboon thrombosis model. Using the FXI monoclonal antibody, we demonstrate that ∼50% inhibition of FXI levels produces antithrombotic activity in baboons. Next we applied baboon-specific FXI ASOs and demonstrated reductions in FXI protein levels and activity, with corresponding increases in aPTT levels. Baboons were then treated with FXI ASOs in a manner that would produce ∼50% reduction of FXI protein levels and measured anti-thrombotic activity. Similar to the antibody approach, ASO-mediated reduction of FXI plasma levels ≥ 50% resulted in a potent antithrombotic effect. Furthermore, reductions in FXI levels do not increase bleeding times in baboons. These results further support the development of FXI ASOs for antithrombotic therapy with the potential for a superior safety profile compared to currently available anticoagulants. Disclosures: Crosby: Isis Pharmaceuticals: Employment. Zhao:Isis Pharmaceuticals: Employment. Gao:Isis Pharmaceuticals: Employment. Revenko:Isis Pharmaceuticals: Employment. MacLeod:Isis Pharmaceuticals: Employment. Gruber:Aronora, LLC: Consultancy, Equity Ownership. Monia:Isis Pharmaceuticals: Employment.

Abstract 1173 This icon denotes a clinically relevant abstract

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.

Optical coherence tomography (OCT) has shown promise at non-destructively characterizing engineered tissues such as collagen gels. However, as the collagen gels develop, the OCT images lose contrast of structures as the gels develop, making visual assessment difficult. Our group proposed quantitatively characterizing these gels by fitting the optical properties from the OCT signals. In this paper, we imaged collagen gels seeded with smooth muscle cells (SMCs) over a 5-day period and used the data to measure their optical properties. Our results showed that over time, the reflectivity of the samples increased 10-fold, corresponding to a decrease in anisotropy factor g, without much change in the scattering coefficient μs. Overall, the optical properties appeared to be dominated by scattering from the collagen matrix, not the cells. However, SMCs remodeled the collagen matrix, and this collagen remodeling by the cells is what causes the observed changes in optical properties. Moreover, the data showed that the optical properties were sensitive to the activity of matrix metalloproteinases (MMPs), enzymes that break down local collagen fibrils into smaller fragments. Blocking MMPs in the SMC gels greatly impeded both the remodeling process and change in optical properties at day 5. Treating day 1 acellular gels with MMP-8 for 3 hr managed to partially reproduce the remodeling observed in SMC gels at day 5. Altogether, we conclude that matrix remodeling in general, and MMPs specifically, greatly affect the local optical properties of the sample, and OCT is a unique tool that can assess MMP activity in collagen gels both non-destructively and label free.

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.

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. (C) 2010 Elsevier Ltd. All rights reserved.

Thrombogenesis depends on biochemical reactions affected by blood flow dynamics. While mathematical models of mural thrombogenesis provide a means of understanding how blood flow affects thrombus growth, comparisons to experimental data are needed to validate the models and enable prediction of thrombus growth under diverse conditions. In this paper, we present mathematical models of mural thrombogenesis under flow and validation of the models with experimental data collected from a thrombogenic vascular graft segment. The grafts were placed in exteriorized high-flow arteriovenous (AV) shunts in baboons. Radiolabeled platelet deposition onto the thrombogenic segment, a marker of thrombus size, and plasma thrombin-antithrombin (TAT) concentration downstream of the graft, a marker of local thrombin generation, were monitored over time. The mathematical model of mural thrombogenesis consisted of transport-reaction equations in which platelets and thrombin were explicitly considered. We found that the transport-reaction model captured the order of magnitude of TAT sampled levels, while calculated rates of platelet deposition agreed well with radioimaging results. Analysis of experimental and modeling data indicates that, at least during part of thrombus growth progression, thrombin generation is in excess and platelet adhesion rates would be sustained even at lower local thrombin concentrations.