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Low-density lipoprotein (LDL) contributes to atherogenesis and cardiovascular disease through interactions with peripheral blood cells, especially platelets. However, mechanisms by which LDL affects platelet activation and atherothrombosis, and how to best therapeutically target and safely prevent such responses remain unclear. Here, we investigate how oxidized low-density lipoprotein (oxLDL) enhances glycoprotein VI (GPVI)-mediated platelet hemostatic and procoagulant responses, and how traditional and emerging antiplatelet therapies affect oxLDL-enhanced platelet procoagulant activity ex vivo. Human platelets were treated with oxLDL and the GPVI-specific agonist, crosslinked collagen-related peptide, and assayed for hemostatic and procoagulant responses in the presence of inhibitors of purinergic receptors (P2YR), cyclooxygenase (COX), and tyrosine kinases. Ex vivo, oxLDL enhanced GPVI-mediated platelet dense granule secretion, α-granule secretion, integrin activation, thromboxane generation and aggregation, as well as procoagulant phosphatidylserine exposure and fibrin generation. Studies of washed human platelets, as well as platelets from mouse and nonhuman primate models of hyperlipidemia, further determined that P2YR antagonists (eg, ticagrelor) and Bruton tyrosine kinase inhibitors (eg, ibrutinib) reduced oxLDL-mediated platelet responses and procoagulant activity, whereas COX inhibitors (eg, aspirin) had no significant effect. Together, our results demonstrate that oxLDL enhances GPVI-mediated platelet procoagulant activity in a manner that may be more effectively reduced by P2YR antagonists and tyrosine kinase inhibitors compared with COX inhibitors.

Deep vein thrombosis (DVT) and post-thrombotic syndrome (PTS) remain highly prevalent despite modern medical therapy. Contact activation is a promising target for safe antithrombotic anticoagulation. The anti-factor XI (FXI) monoclonal antibody 14E11 reduces circulating levels of FXI without compromising hemostasis. The human recombinant analog, AB023, is in clinical development. The role of FXI in mediation of inflammation during DVT resolution is unknown.Investigate the effects of pharmacological targeting of FXI with 14E11 in an experimental model of venous thrombosis.Adult wild-type CD1 mice were treated with subcutaneous anti-FXI antibody (14E11, 5 mg/kg) versus saline prior to undergoing surgical constriction of the inferior vena cava (IVC). Mice were evaluated at various time points to assess thrombus weight and volume, as well as histology analysis, ferumoxytol enhanced magnetic resonance imaging (Fe-MRI), and whole blood flow cytometry.14E11-treated mice had reduced thrombus weights and volumes after IVC constriction on day 7 compared to saline-treated mice. 14E11 treatment reduced circulating monocytes by flow cytometry and macrophage content within thrombi as evaluated by histologic staining and Fe-MRI. Collagen deposition was increased at day 3 while CD31 and smooth muscle cell actin expression was increased at day 7 in the thrombi of 14E11-treated mice compared to saline-treated mice.Pharmacologic targeting of FXI enhances the early stages of experimental venous thrombus resolution in wild-type CD1 mice, and may be of interest for future clinical evaluation of the antibody in DVT and PTS.

Cannabis usage has steadily increased as acceptance is growing for both medical and recreational reasons. Medical cannabis is administered for treatment of chronic pain based on the premise that the endocannabinoid system signals desensitize pain sensor neurons and produce anti-inflammatory effects. The major psychoactive ingredient of cannabis is Δ9-tetrahydrocannabinol (THC) that signals mainly through cannabinoid receptor-1 (CBr), which is also present on nonneuron cells including blood platelets of the circulatory system. In vitro, CBr-mediated signaling has been shown to acutely inhibit platelet activation downstream of the platelet collagen receptor glycoprotein (GP)VI. The systemic effects of chronic THC administration on platelet activity and function remain unclear. This study investigates the effects of chronic THC administration on platelet function using a nonhuman primate (NHP) model. Our results show that female and male NHPs consuming a daily THC edible had reduced platelet adhesion, aggregation, and granule secretion in response to select platelet agonists. Furthermore, a change in bioactive lipids (oxylipins) was observed in the female cohort after THC administration. These results indicate that chronic THC edible administration desensitized platelet activity and function in response to GPVI- and G-protein coupled receptor-based activation by interfering with primary and secondary feedback signaling pathways. These observations may have important clinical implications for patients who use medical marijuana and for providers caring for these patients.

Hemorrhage remains a major complication of anticoagulants, with bleeding leading to serious and even life-threatening outcomes in rare settings. Currently available anticoagulants target either multiple coagulation factors or specifically coagulation factor (F) Xa or thrombin; however, inhibiting these pathways universally impairs hemostasis. Bleeding complications are especially salient in the medically complex population who benefit from medical devices. Extracorporeal devices—such as extracorporeal membrane oxygenation, hemodialysis, and cardiac bypass—require anticoagulation for optimal use. Nonetheless, bleeding complications are common, and with certain devices, highly morbid. Likewise, pharmacologic prophylaxis to prevent thrombosis is not commonly used with many medical devices like central venous catheters due to high rates of bleeding. The contact pathway members FXI, FXII, and prekallikrein serve as a nexus, connecting biomaterial surface-mediated thrombin generation and inflammation, and may represent safe, druggable targets to improve medical device hemocompatibility and thrombogenicity. Recent in vivo and clinical data suggest that selectively targeting the contact pathway of coagulation through the inhibition of FXI and FXII can reduce the incidence of medical device-associated thrombotic events, and potentially systemic inflammation, without impairing hemostasis. In the following review, we will outline the current in vivo and clinical data encompassing the mechanism of action of drugs targeting the contact pathway. This new class of inhibitors has the potential to herald a new era of effective and low-risk anticoagulation for the management of patients requiring the use of medical devices.

Thrombogenesis remains the primary failure of synthetic vascular grafts. Endothelial coverage is crucial to provide an antithrombogenic surface. However, most synthetic materials do not support cell adhesion, and transanastomotic endothelial migration is limited. Here, a surface modification strategy using fucoidan and topography was developed to enable fast

In the initial stages of atherosclerosis, vascular adhesion molecule-1 (VCAM-1) is a surface protein that mediates leukocyte adhesion to the endothelium’s luminal surface. VCAM-1 expression is upregulated on endothelial cells (ECs) under pro-inflammatory conditions and is known to be modulated by fluid shear stress (FSS). High, pulsatile FSS induces endothelial elongation and cytoskeletal alignment and downregulates pro-inflammatory induced VCAM-1 expression, which is associated with an athero-protective EC phenotype. In contrast, athero-prone ECs under low, oscillatory FSS fail to elongate and maintain a cobblestone morphology with random cytoskeletal alignment, while VCAM-1 expression is upregulated. Whether EC shape and cytoskeletal alignment play a role in the regulation of VCAM-1 protein expression independent of FSS has not been previously determined. The goal of this study was to determine the effect of EC morphology, specifically cell elongation and alignment, and cytoskeletal alignment on VCAM-1 protein expression using topographical micropatterning of an endothelial monolayer and single cell image analysis techniques. Elongated ECs with an aligned cytoskeleton significantly downregulated VCAM-1 protein expression in the absence of FSS compared to planar controls. In addition, linear correlations between morphological metrics and protein expression showed that actin alignment had a significantly stronger effect on VCAM-1 expression than cell elongation. Functionally, monocytic U937 cells statically adhered less on micropatterns compared to planar substrates, in a VCAM-1 dependent manner. Therefore, endothelial cellular elongation and alignment as well as cytoskeletal alignment regulate VCAM-1 protein expression and immunogenic functions to produce a less inflammatory phenotype in the absence of hemodynamic effects.

Spleen tyrosine kinase (Syk) and Bruton’s tyrosine kinase (BTK) play critical roles in platelet physiology, facilitating intracellular immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling downstream of platelet glycoprotein VI (GPVI) and GPIIb/IIIa receptors. Small molecule tyrosine kinase inhibitors (TKIs) targeting Syk and BTK have been developed as antineoplastic and anti-inflammatory therapeutics and have also gained interest as antiplatelet agents. Here, we investigate the effects of 12 different Syk and BTK inhibitors on GPVI-mediated platelet signaling and function. These inhibitors include four Syk inhibitors, Bay 61-3606, R406 (fostamatinib), entospletinib, TAK-659; four irreversible BTK inhibitors, ibrutinib, acalabrutinib, ONO-4059 (tirabrutinib), AVL-292 (spebrutinib); and four reversible BTK inhibitors, CG-806, BMS-935177, BMS-986195, and fenebrutinib. In vitro, TKIs targeting Syk or BTK reduced platelet adhesion to collagen, dense granule secretion, and alpha granule secretion in response to the GPVI agonist cross-linked collagen-related peptide (CRP-XL). Similarly, these TKIs reduced the percentage of activated integrin αIIbβ3 on the platelet surface in response to CRP-XL, as determined by PAC-1 binding. Although all TKIs tested inhibited phospholipase C γ2 (PLCγ2) phosphorylation following GPVI-mediated activation, other downstream signaling events proximal to phosphoinositide 3-kinase (PI3K) and PKC were differentially affected. In addition, reversible BTK inhibitors had less pronounced effects on GPIIb/IIIa-mediated platelet spreading on fibrinogen and differentially altered the organization of PI3K around microtubules during platelets spreading on fibrinogen. Select TKIs also inhibited platelet aggregate formation on collagen under physiological flow conditions. Together, our results suggest that TKIs targeting Syk or BTK inhibit central platelet functional responses but may differentially affect protein activities and organization in critical systems downstream of Syk and BTK in platelets.

Background: Platelet role in primary clot formation in response to vessel wall injury is well established; however, the role of systemic platelet levels in deep vein thrombosis (DVT) and its sequelae is unclear. Their interaction with the venous endothelium under low shear stress, regulation of thrombosis, and mediation of both innate and adaptive immune responses through selectin and toll-like receptor expression, respectively, warrants further investigation into their role in DVT formation and resolution. In this study, we present a role for systemic platelet levels in regulating thrombus resolution. Methods: Wildtype, male or female CD1 mice were treated with a thrombopoietin antisense oligonucleotide (TPO-ASO) at 10 mg/kg, eltrombopag at 75 mg/ kg, saline (control), or a scrambled TPO-ASO (control) at 8 mg/ kg prior to surgical creation of a venous thrombus by ligation of the infra-renal inferior vena cava (IVC). Peak scaled thrombus weights (i.e., linear density measured using a ratio of the clot and IVC weight scaled to the length of the IVC) were measured to characterize the early resolution of the thrombus through involution and recanalization. Mice were euthanized on postoperative days 3 and 7 to measure the formation and resolution of the thrombus, respectively. Results: Platelet levels were elevated and decreased by over 30% by the day of surgical treatment after TPO-ASO and eltrombopag treatments, respectively. Scaled thrombus weights were significantly different (p = 0.045, Kruskal Wallis) between groups on day 7 after IVC constriction. Post-hoc analysis (Dunn Test with Benjamini-Hochberg correction) revealed that the TPO-ASO group (mean = 25.4 +/- 0.9 mg/cm) had significantly decreased (p = 0.018) scaled thrombus weights compared to the mice treated with eltrombopag (mean = 35.3 +/- 6.5 mg/cm) on day 7. Thrombus formation on day 3 was not statistically different between treatment groups. Conclusion: Achieving a minimum threshold of 30% reduction of systemic platelet levels by TPO-ASO enhanced thrombus resolution, but not thrombus formation, in our stasis model of rodent DVT resolution. These data suggest a potential role for systemic platelet levels in regulating thrombus resolution.

Background & Objective: Oxidized low density lipoprotein (oxLDL) contributes to atherogenesis and cardiovascular disease through interactions with peripheral blood cells, especially platelets. However, the mechanisms by which oxLDL affects platelet activation, and how to best therapeutically target and safely prevent such responses remains unclear. Here, we investigate how oxLDL upregulates glycoprotein VI (GPVI) mediated platelet hemostatic and procoagulant responses, and how traditional and emerging antiplatelet therapies affect oxLDL-enhanced platelet activity ex vivo . Methods: Human platelets were treated with oxLDL and GPVI specific agonist, crosslinked collage-related peptide (CRP-XL) and assayed for hemostatic and procoagulant responses in the presence of inhibitors of purinergic receptors (P2Y; ticagrelor, AR-C 66096, MRS2179), cyclooxygenase (COX; aspirin, indomethacin), Bruton’s tyrosine kinase (BTK; ibrutinib), and spleen tyrosine kinase (Syk; fostamatinib/R406). Results: Ex vivo , oxLDL enhanced GPVI-mediated platelet dense granule secretion, alpha granule secretion, integrin activation, thromboxane generation, and platelet aggregation, as well as phosphatidylserine exposure and fibrin generation. Our study further demonstrated that CD36 signaling from oxLDL activation overlaps with the GPVI signaling pathway, in such that the phosphorylation of proteins along the Syk-BTK-PI3K/Akt axis was increased. P2Y antagonists (e.g., ticagrelor), BTK inhibitors (e.g., ibrutinib), and Syk inhibitors (e.g. fostamatinib) reduced oxLDL-mediated platelet responses and procoagulant activity, whereas COX inhibitors (e.g., aspirin) had no significant effect. Conclusion: Together, our results demonstrate that oxLDL enhances ex vivo platelet responses and procoagulant activity downstream of GPVI signaling in a manner that may be reduced by P2Y antagonists and tyrosine kinase inhibitors, but not significantly affected by COX inhibitors.

This article describes the properties and performance of a rotary total artificial heart (TAH) that produces inherently pulsatile flow. The hydraulic performance of the TAH was characterized using a mock circulatory loop to simulate four physiologically relevant conditions: baseline flow, increased flow, systemic hypertension, and pulmonary hypertension. The pump has a variable shuttle rate (beats per minute), percentage dwell time, and angular velocity on either side (revolutions per minute), which allows for full control of the flow rate and pulsatility over a range of healthy and pathologic pressures and flow rates. The end-to-end length and displacement volume of the TAH are 9.8 cm and 130 mL, respectively, allowing it to fit in smaller chest cavities including those of smaller adults and juvenile humans.

INTRODUCTION: Vascular devices such as stents, hemodialyzers, and membrane oxygenators can activate blood coagulation and often require the use of systemic anticoagulants to selectively prevent intravascular thrombotic/embolic events or extracorporeal device failure. Coagulation factor (F)XII of the contact activation system has been shown to play an important role in initiating vascular device surface-initiated thrombus formation. As FXII is dispensable for hemostasis, targeting the contact activation system holds promise as a significantly safer strategy than traditional antithrombotics for preventing vascular device-associated thrombosis. OBJECTIVE: Generate and characterize anti-FXII monoclonal antibodies that inhibit FXII activation or activity. METHODS: Monoclonal antibodies against FXII were generated in FXII-deficient mice and evaluated for their binding and anticoagulant properties in purified and plasma systems, in whole blood flow-based assays, and in an in vivo non-human primate model of vascular device-initiated thrombus formation. RESULTS: A FXII antibody screen identified over 400 candidates, which were evaluated in binding studies and clotting assays. One non-inhibitor and six inhibitor antibodies were selected for characterization in functional assays. The most potent inhibitory antibody, 1B2, was found to prolong clotting times, inhibit fibrin generation on collagen under shear, and inhibit platelet deposition and fibrin formation in an extracorporeal membrane oxygenator deployed in a non-human primate. CONCLUSION: Selective contact activation inhibitors hold potential as useful tools for research applications as well as safe and effective inhibitors of vascular device-related thrombosis.

PURPOSE-: Crosslinked poly(vinyl alcohol) (PVA) is a biomaterial that can be used for multiple cardiovascular applications. The success of implanted biomaterials is contingent on the properties of the material. A crucial consideration for blood-contacting devices is their potential to incite thrombus formation, which is dependent on the material surface properties. The goal of this study was to quantify the effect of different crosslinking methods of PVA hydrogels on in vitro thrombogenicity. METHODS-: PVA was manufactured using three different crosslinking methods: 30% sodium trimetaphosphate (STMP), three 24hr freeze-thaw cycles (FT), and 2% glutaraldehyde-crosslinked (GA) to produce STMP-PVA, FT-PVA and GA-PVA, respectively. Expanded polytetrafluoroethylene (ePTFE) was used as a clinical control. As markers of thrombus formation, the degree of coagulation factor (F) XII activation, fibrin formation, and platelet adhesion were measured. RESULTS-: The GA-PVA material increased FXII activation in the presence of cofactors compared to vehicle and increase platelet adhesion compared to other PVA surfaces. The STMP-PVA and FT-PVA materials had equivalent degrees of FXII activation, fibrin formation and platelet adhesion. CONCLUSION-: This work supports crosslinker dependent thrombogenicity of PVA hydrogels and advances our understanding of how the manufacturing of a PVA hydrogel affects its hemocompatibility.

Although thrombin is a key enzyme in the coagulation cascade and is required for both normal hemostasis and pathologic thrombogenesis, it also participates in its own negative feedback via activation of protein C, which downregulates thrombin generation by enzymatically inactivating factors Va and VIIIa. Our group and others have previously shown that thrombin’s procoagulant and anticoagulant activities can be effectively disassociated to varying extents through site-directed mutagenesis. The thrombin mutant W215A/E217A (WE thrombin) has been one of the best characterized constructs with selective activity toward protein C. Although animal studies have demonstrated that WE thrombin acts as an anticoagulant through activated protein C (APC) generation, the observed limited systemic anticoagulation does not fully explain the antithrombotic potency of this or other thrombin mutants. AB002 (E-WE thrombin) is an investigational protein C activator thrombin analog in phase 2 clinical development (clinicaltrials.gov NCT03963895). Here, we demonstrate that this molecule is a potent enzyme that is able to rapidly interrupt arterial-type thrombus propagation at exceedingly low doses (

Background The contact factor XII (FXII) activates upon contact with a variety of charged surfaces. Activated FXII (FXIIa) activates factor XI, which activates factor IX, resulting in thrombin generation, platelet activation, and fibrin formation. In both in vitro and in vivo rabbit models, components of medical devices, including extracorporeal oxygenators, are known to incite fibrin formation in a FXII‐dependent manner. Since FXII has no known role in hemostasis and its inhibition is therefore likely a safe antithrombotic approach, we investigated whether FXII inhibition also reduces accumulation of platelets in extracorporeal oxygenators. Objectives We aimed to determine the effect of FXII inhibition on platelet deposition in perfused extracorporeal membrane oxygenators in nonhuman primates. Methods A potent FXII neutralizing monoclonal antibody, 5C12, was administered intravenously to block contact activation in baboons. Extracorporeal membrane oxygenators were temporarily deployed into chronic arteriovenous access shunts. Radiolabeled platelet deposition in oxygenators was quantified in real time using gamma camera imaging. Biochemical assays were performed to characterize the method of action of 5C12. Results The anti‐FXII monoclonal antibody 5C12 recognized both the alpha and beta forms of human and baboon FXII by binding to the protease‐containing domain, and inhibited FXIIa activity. Administration of 5C12 to baboons reduced platelet deposition and fibrin formation in the extracorporeal membrane oxygenators, in both the presence and absence of systemic low‐dose unfractionated heparin. The antiplatelet dose of 5C12 did not cause measurable increases in template bleeding times in baboons. Conclusions FXII represents a possible therapeutic and safe target for reducing platelet deposition and fibrin formation during medical interventions including extracorporeal membrane oxygenation.

INTRODUCTION: Inflammatory activation of the vascular endothelium leads to overexpression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), contributing to the pro-thrombotic state underpinning atherogenesis. While the role of TEC family kinases (TFKs) in mediating inflammatory cell and platelet activation is well defined, the role of TFKs in vascular endothelial activation remains unclear. We investigated the role of TFKs in endothelial cell activation in vitro and in a nonhuman primate model of diet-induced atherosclerosis in vivo. METHODS AND RESULTS: In vitro, we found that ibrutinib blocked activation of the TFK member, BMX, by vascular endothelial growth factors (VEGF)-A in human aortic endothelial cells (HAECs). Blockade of BMX activation with ibrutinib or pharmacologically distinct BMX inhibitors eliminated the ability of VEGF-A to stimulate VCAM-1 expression in HAECs. We validated that treatment with ibrutinib inhibited TFK-mediated platelet activation and aggregation in both human and primate samples as measured using flow cytometry and light transmission aggregometry. We utilized contrast-enhanced ultrasound molecular imaging to measure platelet GPIbα and endothelial VCAM-1 expression in atherosclerosis-prone carotid arteries of obese nonhuman primates. We observed that the TFK inhibitor, ibrutinib, inhibited platelet deposition and endothelial cell activation in vivo. CONCLUSION: Herein we found that VEGF-A signals through BMX to induce VCAM-1 expression in endothelial cells, and that VCAM-1 expression is sensitive to ibrutinib in vitro and in atherosclerosis-prone carotid arteries in vivo. These findings suggest that TFKs may contribute to the pathogenesis of atherosclerosis and could represent a novel therapeutic target.

In the native vasculature, flowing blood produces a frictional force on vessel walls that affects endothelial cell function and phenotype. In the arterial system, the vasculature's local geometry directly influences variations in flow profiles and shear stress magnitudes. Straight arterial sections with pulsatile shear stress have been shown to promote an athero-protective endothelial phenotype. Conversely, areas with more complex geometry, such as arterial bifurcations and branch points with disturbed flow patterns and lower, oscillatory shear stress, typically lead to endothelial dysfunction and the pathogenesis of cardiovascular diseases. Many studies have investigated the regulation of endothelial responses to various shear stress environments. Importantly, the accurate in vitro simulation of in vivo hemodynamics is critical to the deeper understanding of mechanotransduction through the proper design and use of flow chamber devices. In this review, we describe several flow chamber apparatuses and their fluid mechanics design parameters, including parallel-plate flow chambers, cone-and-plate devices, and microfluidic devices. In addition, chamber-specific design criteria and relevant equations are defined in detail for the accurate simulation of shear stress environments to study endothelial cell responses.

Arterial bypass grafts are a standard preclinical model for evaluating physiology and pathophysiology at graft-material interfaces. Implantations of vascular grafts are commonly done as end-to-end grafts in small animal models. Here we detail bilateral end-to-side aortoiliac graft implantation, which requires open surgery and the creation of vascular anastomoses between the graft material and the infrarenal aorta and iliac artery in a nonhuman primate model. In this model, the aortoiliac graft configuration is created using two 4 mm inner diameter vascular grafts (e.g., ePTFE). After exposure and control of the infrarenal aorta and bilateral common iliac arteries and heparinization, the proximal aortic-graft anastomosis is sewn on the lateral wall of the aorta, and subsequently the distal graft-common iliac anastomosis is sewn on the anterior wall of the common iliac artery with one tube graft. Another tube graft is sewn on the contralateral side in the same manner.

Arterial bypass grafts are a standard preclinical model for evaluating physiology and pathophysiology at graft-material interfaces. Implantations of vascular grafts are commonly done as end-to-end grafts in small animal models. Here we detail bilateral end-to-side aortoiliac graft implantation, which requires open surgery and the creation of vascular anastomoses between the graft material and the infrarenal aorta and iliac artery in a nonhuman primate model. In this model, the aortoiliac graft configuration is created using two 4 mm inner diameter vascular grafts (e.g., ePTFE). After exposure and control of the infrarenal aorta and bilateral common iliac arteries and heparinization, the proximal aortic-graft anastomosis is sewn on the lateral wall of the aorta, and subsequently the distal graft-common iliac anastomosis is sewn on the anterior wall of the common iliac artery with one tube graft. Another tube graft is sewn on the contralateral side in the same manner.

Cardiovascular diseases remain the leading cause of death worldwide. Patency rates of clinically-utilized small diameter synthetic vascular grafts such as Dacron® and expanded polytetrafluoroethylene (ePTFE) to treat cardiovascular disease are inadequate due to lack of endothelialization. Sodium trimetaphosphate (STMP) crosslinked PVA could be potentially employed as blood-compatible small diameter vascular graft for the treatment of cardiovascular disease. However, PVA severely lacks cell adhesion properties, and the efforts to endothelialize STMP-PVA have been insufficient to produce a functioning endothelium. To this end, we developed a one-pot method to conjugate cell-adhesive protein via hydroxyl-to-amine coupling using carbonyldiimidazole by targeting residual hydroxyl groups on crosslinked STMP-PVA hydrogel. Primary human umbilical vascular endothelial cells (HUVECs) demonstrated significantly improved cells adhesion, viability and spreading on modified PVA. Cells formed a confluent endothelial monolayer, and expressed vinculin focal adhesions, cell-cell junction protein zonula occludens 1 (ZO1), and vascular endothelial cadherin (VE-Cadherin). Extensive characterization of the blood-compatibility was performed on modified PVA hydrogel by examining platelet activation, platelet microparticle formation, platelet CD61 and CD62P expression, and thrombin generation, which showed that the modified PVA was blood-compatible. Additionally, grafts were tested under whole, flowing blood without any anticoagulants in a non-human primate, arteriovenous shunt model. No differences were seen in platelet or fibrin accumulation between the modified-PVA, unmodified PVA or clinical, ePTFE controls. This study presents a significant step in the modification of PVA for the development of next generation in situ endothelialized synthetic vascular grafts.

Objective— Activation of coagulation FXI (factor XI) by FXIIa (activated factor XII) is a prothrombotic process. The endothelium is known to play an antithrombotic role by limiting thrombin generation and platelet activation. It is unknown whether the antithrombotic role of the endothelium includes sequestration of FXIa (activated factor XI) activity. This study aims to determine the role of endothelial cells (ECs) in the regulation of the intrinsic pathway of coagulation. Approach and Results— Using a chromogenic assay, we observed that human umbilical veins ECs selectively blocked FXIa yet supported kallikrein and FXIIa activity. Western blotting and mass spectrometry analyses revealed that FXIa formed a complex with endothelial PAI-1 (plasminogen activator inhibitor-1). Blocking endothelial PAI-1 increased the cleavage of a chromogenic substrate by FXIa and the capacity of FXIa to promote fibrin formation in plasma. Western blot and immunofluorescence analyses showed that FXIa–PAI-1 complexes were either released into the media or trafficked to the early and late endosomes and lysosomes of ECs. When baboons were challenged with Staphylococcus aureus to induce a prothrombotic phenotype, an increase in circulating FXIa–PAI-1 complex levels was detected by ELISA within 2 to 8 hours postchallenge. Conclusions— PAI-1 forms a complex with FXIa on ECs, blocking its activity and inducing the clearance and degradation of FXIa. Circulating FXIa–PAI-1 complexes were detected in a baboon model of S. aureus sepsis. Although ECs support kallikrein and FXIIa activity, inhibition of FXIa by ECs may promote the clearance of intravascular FXIa. Visual Overview— An online visual overview is available for this article.

Objective— Factor XI (FXI) contributes to thrombotic disease while playing a limited role in normal hemostasis. We generated a unique, humanized anti-FXI antibody, AB023, which blocks factor XIIa-mediated FXI activation without inhibiting FXI activation by thrombin or the procoagulant function of FXIa. We sought to confirm the antithrombotic activity of AB023 in a baboon thrombosis model and to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics in healthy adult subjects. Approach and Results— In a primate model of acute vascular graft thrombosis, AB023 reduced platelet and fibrin accumulation within the grafts by >75%. To evaluate the safety of AB023, we performed a first-in-human study in healthy adult volunteers without any serious adverse events. Overall, 10 of 21 (48%) subjects experienced 20 treatment-emergent adverse events, with 7 of 16 (44%) subjects following active treatment and 3 of 5 (60%) subjects following placebo. AB023 did not increase bleeding or prothrombin times. Anticoagulation was verified by a saturable ≈2-fold prolongation of the partial thromboplastin time for over 1 month after the highest dose. Conclusions— AB023, which inhibits contact activation-initiated blood coagulation in vitro and experimental thrombus formation in primates, produced a dose-dependent duration of limited anticoagulation without drug-related adverse effects in a phase 1 trial. When put in context with earlier observations suggesting that FXI contributes to venous thromboembolism and cardiovascular disease, although contributing minimally to hemostasis, our data further justify clinical evaluation of AB023 in conditions where contact-initiated FXI activation is suspected to have a pathogenic role. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT03097341.

End-stage renal disease (ESRD) patients on chronic hemodialysis have repeated blood exposure to artificial surfaces that can trigger clot formation within the hemodialysis circuit. Dialyzer clotting can lead to anemia despite erythropoietin and iron supplementation. Unfractionated heparin prevents clotting during hemodialysis, but it is not tolerated by all patients. Although heparin-free dialysis is performed, intradialytic blood entrapment can be problematic. To address this issue, we performed a randomized, double-blind, phase 2 study comparing AB023, a unique antibody that binds factor XI (FXI) and blocks its activation by activated FXII, but not by thrombin, to placebo in 24 patients with ESRD undergoing heparin-free hemodialysis. Patients were randomized to receive a single predialysis dose of AB023 (0.25 or 0.5 mg/kg) or placebo in a 2:1 ratio, and safety and preliminary efficacy were compared with placebo and observations made prior to dosing within each treatment arm. AB023 administration was not associated with impaired hemostasis or other drug-related adverse events. Occlusive events requiring hemodialysis circuit exchange were less frequent and levels of thrombin-antithrombin complexes and C-reactive protein were lower after AB023 administration compared with data collected prior to dosing. AB023 also reduced potassium and iron entrapment in the dialyzers, consistent with less blood accumulation within the dialyzers. We conclude that despite the small sample size, inhibition of contact activation–induced coagulation with AB023 was well tolerated and reduced clotting within the dialyzer. This trial was registered at www.clinicaltrials.gov as #NCT03612856.

BACKGROUND: Human coagulation factor (F) XI deficiency, a defect of the contact activation system, protects against venous thrombosis, stroke, and heart attack, whereas FXII, plasma prekallikrein, or kininogen deficiencies are asymptomatic. FXI deficiency, inhibition of FXI production, activated FXI (FXIa) inhibitors, and antibodies to FXI that interfere with FXI/FXII interactions reduce experimental thrombosis and inflammation. FXI inhibitors are antithrombotic in patients, and FXI and FXII deficiencies are atheroprotective in apolipoprotein E–deficient mice. OBJECTIVES: Investigate the effects of pharmacological targeting of FXI in experimental models of atherogenesis and established atherosclerosis. METHODS AND RESULTS: Low-density lipoprotein receptor-knockout (Ldlr(−/−)) mice were administered high-fat diet (HFD) for 8 weeks; concomitantly, FXI was targeted with anti-FXI antibody (14E11) or FXI antisense oligonucleotide (ASO). 14E11 and FXI-ASO reduced atherosclerotic lesion area in proximal aortas when compared with controls, and 14E11 also reduced aortic sinus lesions. In an established disease model, in which therapy was given after atherosclerosis had developed, Ldlr(−/−) mice were fed HFD for 8 weeks and then administered 14E11 or FXI-ASO weekly until 16 weeks on HFD. In this established disease model, 14E11 and FXI-ASO reduced atherosclerotic lesion area in proximal aortas, but not in aortic sinus. In cultures of human endothelium, FXIa exposure disrupted VE-Cadherin expression and increased endothelial lipoprotein permeability. Strikingly, we found that 14E11 prevented the disruption of VE-Cadherin expression in aortic sinus lesions observed in the atherogenesis mouse model. CONCLUSION: Pharmacological targeting of FXI reduced atherogenesis in Ldlr(−/−) mice. Interference with the contact activation system may safely reduce development or progression of atherosclerosis.

This study investigated the effects of terminal sterilization of polyvinyl alcohol (PVA) biomaterials using clinically translatable techniques, specifically ethylene oxide (EtO) and gamma (γ) irradiation. While a few studies have reported the possibility of sterilizing PVA with γ-radiation, the use of EtO sterilization of PVA requires additional study. PVA solutions were chemically crosslinked with trisodium trimetaphosphate and sodium hydroxide. The three experimental groups included untreated control, EtO, and γ-irradiation, which were tested for the degree of swelling and water content, and mechanical properties such as radial compliance, longitudinal tensile, minimum bend radius, burst pressure, and suture retention strength. In addition, samples were characterized with scanning electron microscopy, differential scanning calorimetry, X-ray photoelectron spectroscopy, and water contact angle measurements. Cell attachment was assessed using the endothelial cell line EA.hy926, and the sterilized PVA cytotoxicity was studied with a live/dead stain. Platelet and fibrin accumulation was measured using an ex vivo shunt baboon model. Finally, the immune responses of PVA implants were analyzed after a 21-day subcutaneous implantation in rats and a 30-day implantation in baboon. EtO sterilization reduced the PVA graft wall thickness, its degree of swelling, and water content compared with both γ-irradiated and untreated PVA. Moreover, EtO sterilization significantly reduced the radial compliance and increased Young's modulus. EtO did not change PVA hydrophilicity, while γ-irradiation increased the water contact angle of the PVA. Consequently, endothelial cell attachment on the EtO-sterilized PVA showed similar results to the untreated PVA, while cell attachment significantly improved on the γ-irradiated PVA. When exposing the PVA grafts to circulating whole blood, fibrin accumulation of EtO-sterilized PVA was found to be significantly lower than γ-irradiated PVA. The immune responses of γ-irradiated PVA, EtO-treated PVA, and untreated PVA were compared. Implanted EtO-treated PVA showed the least MAC387 reaction. The terminal sterilization methods in this study changed PVA hydrogel properties; nevertheless, based on the characterizations performed, both sterilization methods were suitable for sterilizing PVA. We concluded that EtO can be used as an alternative method to sterilize PVA hydrogel material. IMPACT STATEMENT: Polyvinyl alcohol (PVA) hydrogels have been used for a variety of tissue replacements, including neural, cardiac, meniscal, cartilage, muscle, pancreatic, and ocular applications. In addition, PVA can be made into a tubular shape and used as a small-diameter vascular graft. Ethylene oxide (EtO) is one of the Food and Drug Administration-approved methods for sterilization, but its effect on PVA has not been studied extensively. The outcome of this study provides the effects of EtO and γ-irradiation of PVA grafts on both the material properties and the in vivo responses, particularly for vascular applications. Knowledge of these effects may ultimately improve the success rate of PVA vascular grafts.

BACKGROUND In vitro studies with ultrasound (US) and microbubbles (MB) have reported that sono-thrombolysis can be achieved at high peak rarefactional acoustic pressure amplitudes (PRAPAs) using 0.25 and 1.05 MHz US frequencies. OBJECTIVE The aim of the current study was to determine if these parameters work on an ex vivo physiological model of thrombosis. METHODS A thrombogenic device was placed in an ex vivo chronic arteriovenous shunt in juvenile baboons. Platelet accumulation was measured by dynamic imaging of the device and the 10 cm thrombus tail with 111 In-labeled platelets. After 15 minutes of thrombus formation, treatment with either low-dose recombinant tissue plasminogen activator (rtPA) or low-dose rtPA + MB+US was performed for 20 minutes. Four US settings at 0.25% duty cycle were used: 0.25 MHz at PRAPAs of 1.20 and 2.20 MPa, and 1.05 MHz at 1.75 and 4.75 MPa. RESULTS Platelet accumulation was not inhibited by low-dose rtPA or MB with US alone. Platelet accumulation was significantly reduced with 0.25 MHz US at 2.20 PRAPA (P

Artificial small-diameter vascular grafts remain an unmet need in modern medicine, due to the thrombosis and neointimal hyperplasia that plague currently available synthetic devices. Tissue engineering techniques, including in vitro endothelialization, could offer a solution to this problem. A potential minimally invasive source of patient autologous endothelium is endothelial colony-forming cells (ECFCs), endothelial-like outgrowth products of circulating progenitors. While ECFCs respond to shear stress similar to mature endothelial cells (ECs), their response to luminal topographic micropatterning (TMP), a biomaterial modification with the potential to flow-independently, enhance the attachment, migration, gene expression, and function of mature ECs, remains unstudied. In this study, case-matched carotid endothelial cells (CaECs) and blood-derived ECFCs are statically cultured on polyurethane substrates with micropatterned pitches (pitch = peak to peak distance) ranging from 3–to 14 μm. On all pattern pitches tested, both CaECs and ECFCs showed significant and robust alignment to the angle of the micropatterns. Using a novel cell-by-cell image analysis technique, it was found that actin fibers similarly and significantly aligned to the angle of micropatterned features on all pitches tested. Microtubules analyzed through the same novel approach showed significant alignment on most pitches examined, with a greater variation in fiber angle overall. Interestingly, only CaECs showed significant cellular elongation, and notably to a lower degree than previously seen either in vivo due to flow or in vitro due to spatial growth restriction micropatterning, but consistent with earlier studies of TMP. Neither cell type displayed any significant micropattern-driven changes in the expression of KLF-2 or the downstream adhesion molecules it regulates. These results demonstrate that TMP flow-independently affects ECFC morphology, but that alignment alone is insufficient to drive protective changes in EC and ECFC function. IMPACT STATEMENT: Endothelialization aided by luminal topography may increase the biocompatibility of small-diameter artificial vascular grafts, a current unmet clinical need. We quantified the effects of luminal anisotropic topographic micropatterning on the morphology and immunogenic gene expression of blood-derived endothelial colony-forming cells (ECFCs), which have substantial tissue engineering potential. We found that topography drives whole cell and cytoskeletal alignment, demonstrating flow-independent control of ECFC morphology. However, topography does not elongate ECFCs, and neither ECFC nor mature endothelial cell immunogenic gene expression is affected by topography, raising questions about the relative importance of the morphological metrics used in micropatterning literature.

Coronavirus disease 2019 (COVID‐19) is predicted to overwhelm health care capacity in the United States and worldwide, and, as such, interventions that could prevent clinical decompensation and respiratory compromise in infected patients are desperately needed. Excessive cytokine release and activation of coagulation appear to be key drivers of COVID‐19 pneumonia and associated mortality. Contact activation has been linked to pathologic upregulation of both inflammatory mediators and coagulation, and accumulating preclinical and clinical data suggest it to be a rational therapeutic target in patients with COVID‐19. Pharmacologic inhibition of the interaction between coagulation factors XI and XII has been shown to prevent consumptive coagulopathy, pathologic systemic inflammatory response, and mortality in at least 2 types of experimental sepsis. Importantly, inhibition of contact activation also prevented death from Staphylococcus aureus–induced lethal systemic inflammatory response syndrome in nonhuman primates. The contact system is likely dispensable for hemostasis and may not be needed for host immunity, suggesting it to be a reasonably safe target that will not result in immunosuppression or bleeding. As a few drugs targeting contact activation are already in clinical development, immediate clinical trials for their use in patients with COVID‐19 are potentially feasible for the prevention or treatment of respiratory distress.

Background: Factor XI (FXI) inhibition demonstrated strong efficacy in preventing thrombus formation in preclinical and clinical models of arterial and venous thrombosis. However, the effect of FXI inhibition in halting the progression of a formed clot remains largely unknown. Aims: This study aims to test whether abelacimab, a dual-acting FXI and activated FXI (FXIa) monoclonal antibody, is effective in halting clot formation and downstream growth when administered before or during active clot formation in an established baboon femoral arterio-venous (AV) shunt model. Methods: Three baboons had a chronic femoral AV shunt put in place; platelet and fibrin deposition inside and distal to collagen- or collagen + tissue factor (TF)-coated vascular grafts were measured at baseline (control), in a therapeutic setting, where abelacimab (1 mg/kg, intravenously) was administered 30 minutes after thrombus initiation, and in a preventative setting within the first 48 h and 1 week (144 - 216 h) post-administration. Pharmacodynamic effect was measured by activated partial thromboplastin time (aPTT). Results: Consistent with its half-life of 20 to 30 days, single iv administration of abelacimab at a dose of 1 mg/kg resulted in long-lasting (> 4-week) aPTT prolongation (> 2-fold). Administration of abelacimab 30 minutes after initiation of thrombosis using grafts coated either with collagen or with collagen + TF quickly halted downstream propagation of platelet and fibrin deposition compared to control. Further, downstream propagation of platelet and fibrin deposition was markedly reduced when clotting was induced by collagen, or collagen + tissue factor after abelacimab administration. Conclusions: These data suggest that abelacimab, a dual-acting anti-FXI/FXIa monoclonal antibody with a single long-lasting iv injection has the potential to slow down the growth and reduce the size of thrombi when admistered before or after clot induction. Data indicate a potential for therapeutic benefit of targeting FXI both in therapeutic and preventive settings. Sponsored by: Anthos Therapeutics Inc., 55 Cambridge Parkway, Suite 103, Cambridge, MA 02142 Figure 1 Figure 1. Disclosures Wallisch: Aronora Inc,: Current Employment. Khder: Anthos Therapeutics: Consultancy; Novartis: Current equity holder in publicly-traded company, Other: Retiree. Bloomfield: Anthos Therapeutics: Current Employment. Gruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment.

Background: End stage renal disease (ESRD) patients on chronic hemodialysis (HD) repeatedly have their blood exposed to artificial surfaces within the hemodialysis circuit, which triggers contact system-initiated coagulation. Clot formation within the HD circuit results in blood loss, decreased HD efficiency, and device circuit failure. Heparin reduces circuit clotting but it is not tolerated by all patients. Mounting experimental data suggest that contact system inhibition is antithrombotic without significantly compromising hemostasis. AB023 is a unique recombinant anti-factor (F) XI antibody that interferes with the interactions of FXI and FXII without inhibiting FXI activation by thrombin or the activation of FIX by activated FXI. This study evaluated the safety, tolerability, pharmacokinetics, and preliminary efficacy of AB023 in patients with ESRD on chronic HD. Methods: In this phase 2, randomized, double-blind, placebo-controlled, single-administration study, AB023 (0.25 or 0.5 mg/kg) or placebo was injected into the HD circuit of 24 ESRD patients at the start of heparin-free HD (NCT03612856). All patients underwent three pre-dose HD procedures and four monitored post-dose HD sessions. In total, the study encompassed 168 individual HD sessions, 72 of which occurred pre-dose and 96 of which occurred post-dose. Safety parameters, including time to hemostasis at HD access site, prothrombin time, physical examinations, vital signs, electrocardiograms, clinical chemistry, immunogenicity and other adverse events (AEs), were recorded. Clotting within the hemodialyzer circuit was assessed using a visual clotting scale by investigators who were blinded to treatment. The frequency of circuit clot obstructions requiring dialyzer exchange was also recorded. Pharmacokinetic and pharmacodynamic parameters, including activated partial thromboplastin times, were also assessed. The study was approved by an ethics review board and all subjects provided written informed consent. Results: AB023 demonstrated a favorable safety profile in ESRD patients. No drug-related AEs were noted. Clinically relevant bleeding did not occur in any patient, and the time to hemostasis at the HD access site was unchanged after AB023 administration. As expected, the aPTT was prolonged after AB023 administration, reaching saturation at both dose levels (about 2-fold prolongation) immediately after dosing, and remaining prolonged for up to 9 days with the high dose. Compared with pretreatment, the frequency of occlusive events requiring circuit exchange decreased by 68% and 50% in subjects given the 0.25 and 0.5 mg/kg AB023 respectively, while the number of occlusive events was numerically unchanged in the placebo arm. Likewise, the number of saline flushes required to maintain circuit patency decreased by 44% and 85%, and the incidence of high-grade clotting in the hemodialyzer as assessed by visual scoring decreased by 15% and 43% on day 1, 8% and 35% by day 3, and 0% and 19% by day 5 in the 0.25 and 0.5 mg/kg cohorts, respectively. Conclusions: Anticoagulation with AB023 was well-tolerated and reduced clot formation within the HD circuit of ESRD patients. These findings suggest that inhibiting contact system activation could reduce medical device-initiated blood clot formation in patients. Disclosures Lorentz: Aronora, Inc.: Current Employment. Verbout:Aronora, Inc.: Current Employment. Shatzel:Aronora, Inc.: Consultancy. Wallisch:Aronora, Inc.: Current Employment. Markway:Aronora, Inc.: Current Employment. Tucker:Aronora, Inc.: Current Employment. Gruber:Aronora, Inc.: Current Employment, Current equity holder in private company.

INTRODUCTION: Tissue factor (TF) and factor (F) VII, components of the extrinsic pathway of blood coagulation, are essential for hemostatic plug formation in response to injury; less clear are their roles in propagating thrombosis, as observational data in humans with congenital FVII deficiency suggests persistent thrombotic and bleeding risk even at significantly decreased FVII levels. We aimed to define the contribution of FVII to thrombus formation and hemostasis using a non-human primate model. METHODS: We treated baboons with a FVII antisense oligonucleotide (ASO) and measured platelet and fibrin deposition inside and distal to collagen- or TF-coated vascular grafts. We assessed hemostasis by measuring bleeding time (BT) and prothrombin time (PT). Enoxaparin and vehicle treatments served as controls. RESULTS: FVII–ASO treatment reduced FVII levels by 95% and significantly increased both the PT and BT. Lowering FVII levels did not decrease platelet deposition in collagen- or TF-coated grafts, in thrombi distal to the grafts, or fibrin content of either collagen- and TF-coated grafts. Lowering FVII levels were associated with a modest 25% reduction in platelet deposition at 60 min in the distal thrombus tail of TF-coated grafts only. CONCLUSIONS: FVII inhibition by way of ASO is feasible yet significantly impairs hemostasis while only exhibiting antithrombotic effects when thrombosis is initiated by vessel wall surface-associated TF exposure.

BackgroundFlow diverters offer a promising treatment for cerebral aneurysms. However, they have associated thromboembolic risks, mandating chronic dual antiplatelet therapy (DAPT). Shield Technology is a phosphorylcholine surface modification of the Pipeline Embolization Device (PED) flow diverter, which has shown significant reductions in material thrombogenicity in vitro.ObjectiveTo compare the thrombogenicity of PED, PED with Shield Technology (PED+Shield), and the Flow-Redirection Endoluminal Device (FRED)—with and without single antiplatelet therapy and DAPT—under physiological flow.MethodsAn established non-human primate ex vivo arteriovenous shunt model of stent thrombosis was used. PED, PED+Shield, and FRED were tested without antiplatelet therapy, with acetylsalicylic acid (ASA) monotherapy, and with DAPT. Radiolabeled platelet deposition was quantified over 1 hour for each device and total fibrin deposition was also quantified.ResultsCumulative statistical analysis showed significantly lower platelet deposition on PED compared with FRED. The same statistical model showed significant decreases in platelet deposition when ASA, clopidogrel, or Shield Technology was used. Direct comparisons of device performances within antiplatelet conditions showed consistent significant decreases in platelet accumulation on PED+Shield relative to FRED. PED+Shield showed significant reductions in platelet deposition compared with unmodified PED without antiplatelet therapy and with DAPT. PED accumulated minimal fibrin with and without Shield Technology.ConclusionsIn this preclinical model, we have shown that the Shield Technology phosphorylcholine modification reduces the platelet-specific thrombogenicity of a flow diverter under physiologically relevant flow with and without DAPT. We have further identified increased fibrin-driven thrombogenicity associated with FRED relative to PED.

The performance of clinical synthetic small diameter vascular grafts remains disappointing due to the fast occlusion caused by thrombosis and intimal hyperplasia formation. Poly(vinyl alcohol) (PVA) hydrogels have tunable mechanical properties and a low thrombogenic surface, which suggests its potential value as a small diameter vascular graft material. However, PVA does not support cell adhesion and thus requires surface modification to encourage endothelialization. This study presents a modification of PVA with fucoidan. Fucoidan is a sulfated polysaccharide with anticoagulant and antithrombotic properties, which was shown to potentially increase endothelial cell adhesion and proliferation. By mixing fucoidan with PVA and co-crosslinked by sodium trimetaphosphate (STMP), the modification was achieved without sacrificing mechanical properties. Endothelial cell adhesion and monolayer function were significantly enhanced by the fucoidan modification. In vitro and ex-vivo studies showed low platelet adhesion and activation and decreased thrombin generation with fucoidan modified PVA. The modification proved to be compatible with gamma sterilization. In vivo evaluation of fucoidan modified PVA grafts in rabbits exhibited increased patency rate, endothelialization, and reduced intimal hyperplasia formation. The fucoidan modification presented here benefited the development of PVA vascular grafts and can be adapted to other blood contacting surfaces.

BackgroundEndothelialization of small diameter synthetic vascular grafts is a potential solution to the thrombosis and intimal hyperplasia that plague current devices. Endothelial colony forming cells, which are blood-derived and similar to mature endothelial cells, are a potential cell source. Anisotropic spatial growth restriction micropatterning has been previously shown to affect the morphology and function of mature endothelial cells in a manner similar to unidirectional fluid shear stress. To date, endothelial colony forming cells have not been successfully micropatterned. This study addresses the hypothesis that micropatterning of endothelial colony forming cells will induce morphological elongation, cytoskeletal alignment, and changes in immunogenic and thrombogenic-related gene expression.MethodsSpatially growth restrictive test surfaces with 25 μm-wide lanes alternating between collagen-I and a blocking polymer were created using microfluidics. Case-matched endothelial colony forming cells and control mature carotid endothelial cells were statically cultured on either micropatterned or non-patterned surfaces. Cell elongation was quantified using shape index. Using confocal microscopy, cytoskeletal alignment was visualized and density and apoptotic rate were determined. Gene expression was measured using quantitative PCR to measure KLF-2, eNOS, VCAM-1, and vWF.ResultsEndothelial colony forming cells were successfully micropatterned for up to 50 hours. Micropatterned cells displayed elongation and actin alignment. Micropatterning increased the packing densities of both cell types, but did not affect apoptotic rate, which was lower in endothelial colony forming cells. KLF-2 gene expression was increased in micropatterned relative to non-patterned endothelial colony forming cells after 50 hours. No significant differences were seen in the other genes tested.ConclusionsEndothelial colony forming cells can be durably micropatterned using spatial growth restriction. Micropatterning has a significant effect on the gross and subcellular morphologies of both cell types. Further study is required to fully understand the effect of micropatterning on endothelial colony forming cell gene expression.