Factor XI Inhibition for the Prevention of Catheter-Associated Thrombosis in Patients With Cancer Undergoing Central Line Placement: A Phase 2 Clinical Trial.

BACKGROUND: Despite the ubiquitous utilization of central venous catheters in clinical practice, their use commonly provokes thromboembolism. No prophylactic strategy has shown sufficient efficacy to justify routine use. Coagulation factors FXI (factor XI) and FXII (factor XII) represent novel targets for device-associated thrombosis, which may mitigate bleeding risk. Our objective was to evaluate the safety and efficacy of an anti-FXI mAb (monoclonal antibody), gruticibart (AB023), in a prospective, single-arm study of patients with cancer receiving central line placement. METHODS: We enrolled ambulatory cancer patients undergoing central line placement to receive a single dose of gruticibart (2 mg/kg) administered through the venous catheter within 24 hours of placement and a follow-up surveillance ultrasound at day 14 for evaluation of catheter thrombosis. A parallel, noninterventional study was used as a comparator. RESULTS: In total, 22 subjects (n=11 per study) were enrolled. The overall incidence of catheter-associated thrombosis was 12.5% in the interventional study and 40.0% in the control study. The anti-FXI mAb, gruticibart, significantly prolonged the activated partial thromboplastin time in all subjects on day 14 compared with baseline (P<0.001). Gruticibart was well tolerated and without infusion reactions, drug-related adverse events, or clinically relevant bleeding. Platelet flow cytometry demonstrated no difference in platelet activation following administration of gruticibart. T (thrombin)-AT (antithrombin) and activated FXI-AT complexes increased following central line placement in the control study, which was not demonstrated in our intervention study. CRP (C-reactive protein) did not significantly increase on day 14 in those who received gruticibart, but it did significantly increase in the noninterventional study. CONCLUSIONS: FXI inhibition with gruticibart was well tolerated without any significant adverse or bleeding-related events and resulted in a lower incidence of catheter-associated thrombosis on surveillance ultrasound compared with the published literature and our internal control study. These findings suggest that targeting FXI could represent a safe intervention to prevent catheter thrombosis. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04465760.

Venous thromboembolism: diagnostic advances and unaddressed challenges in management.

PURPOSE OF REVIEW: This review summarizes recent advances in developing targeted diagnostics for venous thromboembolism (VTE) and unaddressed knowledge gaps in patient management. Without addressing these critical data needs, the morbidity in VTE patients will persist. RECENT FINDINGS: Recent studies investigating plasma protein profiles in VTE patients have identified key diagnostic targets to address the currently unmet need for low-cost, confirmatory, point-of-care VTE diagnostics. These studies and a growing body of evidence from animal model studies have revealed the importance of inflammatory and vascular pathology in driving VTE, which are currently unaddressed targets for VTE therapy. To enhance the translation of preclinical animal studies, clinical quantification of thrombus burden and comparative component analyses between modeled VTE and clinical VTE are necessary. SUMMARY: Lead candidates from protein profiling of VTE patients' plasma offer a promising outlook in developing low cost, confirmatory, point-of-care testing for VTE. Additionally, addressing the critical knowledge gap of quantitatively measuring clinical thrombi will allow for an array of benefits in VTE management and informing the translatability of experimental therapeutics.

The effects of in utero exposure to delta-9-tetrahydrocannabinol (THC) on cardiac extracellular matrix expression and vascular transcriptome in rhesus macaques.

Delta-9-tetrahydrocannabinol (THC), the psychoactive component of cannabis, remains a schedule I substance, thus safety data regarding the effects on the cardiovascular and prenatal health are limited. Importantly, there is evidence showing prenatal cannabis exposure can negatively impact fetal organ development, including the cardiovascular system. THC can cross the placenta and bind to cannabinoid receptors expressed in the developing fetus, including on endothelial cells. To understand the impact of prenatal THC exposure on the fetal cardiovascular system, we used our rhesus macaque model of prenatal daily edible THC consumption. Prior to conception, animals were acclimated to THC (2.5mg/7kg/day, equivalent to a heavy medical cannabis dose) and maintained on this dose daily throughout pregnancy. Fetal tissue samples were collected at gestational day 155 (full term is 168 days). Our model showed that in utero THC exposure was associated with a decreased heart to body weight ratio in offspring, warranting further mechanistic investigation. Histological examination of the fetal cardiac and vascular tissues did not reveal any significant effect of THC exposure on the maturity of collagen within the fetal heart or the aorta. Total collagen III expression and elastin production and organization were unchanged. However, bulk RNA-sequencing of vascular cells in the umbilical vein, umbilical artery, and fetal aorta demonstrated that THC alters the fetal vascular transcriptome and is associated with upregulated expression of genes involved in carbohydrate metabolism and inflammation. The long-term consequences of these findings are unknown, but suggest that prenatal THC exposure may affect cardiovascular development in offspring.

Comparison of bleeding and thrombotic outcomes in veno-venous extracorporeal membrane oxygenation: Heparin versus bivalirudin.

OBJECTIVES: We aimed to evaluate thrombotic and hemorrhagic complications with heparin versus bivalirudin use in veno-venous extracorporeal membrane oxygenation (V-V ECMO). METHODS: We performed a retrospective cohort study of adult patients placed on V-V ECMO with intravenous anticoagulation with either heparin or bivalirudin. Time to thrombotic event and major bleed were analyzed in addition to related outcomes. RESULTS: We identified 95 patients placed on V-V ECMO: 61 receiving heparin, 34 bivalirudin. The bivalirudin group had a higher rate of severe COVID-19, higher BMI, and longer ECMO duration. Despite this, bivalirudin was associated with reduced risk of thrombotic event (HR 0.14, 95% CI 0.06-0.32, p < .001) and increased average lifespan of the circuit membrane lung (16 vs. 10 days, p = 0.004). While there was no difference in major bleeding, the bivalirudin group required fewer transfusions of packed red blood cells and platelets per 100 ECMO days (means of 13 vs. 39, p = 0.004; 5 vs. 19, p = .014, respectively). Lastly, the bivalirudin group had improved survival to ECMO decannulation in univariate analysis (median OS 53 vs. 26 days, p = .015). CONCLUSIONS: In this real-world analysis of bivalirudin versus heparin, bivalirudin is a viable option for V-V ECMO and associated with lower risk of thrombotic complications and fewer transfusion requirements.

Role of platelet count in a murine stasis model of deep vein thrombosis.

Platelets are core components of thrombi but their effect on thrombus burden during deep vein thrombosis (DVT) has not been fully characterized. We examined the role of thrombopoietin-altered platelet count on thrombus burden in a murine stasis model of DVT. To modulate platelet count compared to baseline, CD1 mice were pretreated with thrombopoietin antisense oligonucleotide (THPO-ASO, 56% decrease), thrombopoietin mimetic (TPO-mimetic, 36% increase), or saline (within 1%). Thrombi and vein walls were examined on postoperative days (POD) 3 and 7. Thrombus weights on POD 3 were not different between treatment groups (p = .84). The mean thrombus weights on POD 7 were significantly increased in the TPO-mimetic cohort compared to the THPO-ASO (p = .005) and the saline (p = .012) cohorts. Histological grading at POD 3 revealed a significantly increased smooth muscle cell presence in the thrombi and CD31 positive channeling in the vein wall of the TPO-mimetic cohort compared to the saline and THPO-ASO cohorts (p < .05). No differences were observed in histology on POD 7. Thrombopoietin-induced increased platelet count increased thrombus weight on POD 7 indicating platelet count may regulate thrombus burden during early resolution of venous thrombi in this murine stasis model of DVT.

Deep vein thrombosis (DVT) is a pathology in which blood clots form in the deep veins of our body. Usually occurring in the legs, these clots can be dangerous if they dislodge and travel to the heart and are pumped into the lungs. Often these clots do not travel and heal where they formed. However, as the body heals the clot it may also cause damage to the vein wall and predispose the patient to future clots, i.e., the biggest risk factor for a second clot is the first clot. DVT can also cause symptoms of pain, swelling, and redness in the long-term, leading to post-thrombotic syndrome where the initial symptoms of the clot persist for a long time. All blood clots have common components of red blood cells, white blood cells, platelets, and fibrin in varying concentrations. Humans maintain a platelet count between 150 and 400 thousand platelets per microliter of our blood. However, diseases like cancer or medications like chemotherapy can cause a change in our body's platelet count. The effect of a changing platelet count on the size (clot burden) of DVT clot and how platelet count could affect DVT as the clot heals is not fully understood. Studying this might help us develop better targets and treat patients with a wide range of platelet counts who experience DVT. In this study, we intentionally decreased, left unchanged, and increased platelet counts in mice and then created a DVT to study what the effect of low, normal, and high platelet counts, respectively, would be on the clot burden. We observed that mice with higher platelet counts had a higher clot burden during the early part of the healing process of the clot. Within this study, we can conclude that higher platelet counts may lead to higher clot burden in DVT which furthers our understanding of how platelet count affects clot burden during DVT.

Targeting the Contact Pathway of Coagulation for the Prevention and Management of Medical Device-Associated Thrombosis.

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.

The contact activation inhibitor AB023 in heparin-free hemodialysis: results of a randomized phase 2 clinical trial.

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.

Chronic edible dosing of Δ9-tetrahydrocannabinol (THC) in nonhuman primates reduces systemic platelet activity and function.

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.

The protein C activator AB002 rapidly interrupts thrombus development in baboons.

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 (<2 µg/kg, IV), yet without substantial systemic anticoagulation in baboons. We demonstrate that AB002 produces APC on platelet aggregates and competitively inhibits thrombin-activatable fibrinolysis inhibitor (carboxypeptidase B2) activation in vitro, which may contribute to the observed in vivo efficacy. We also describe its safety and activity in a phase 1 first-in-human clinical trial. Together, these results support further clinical evaluation of AB002 as a potentially safe and effective new approach for treating or preventing acute thrombotic and thromboembolic conditions. This trial was registered at www.clinicaltrials.gov as #NCT03453060.

In Vitro Flow Chamber Design for the Study of Endothelial Cell (Patho)Physiology.

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.

Assessment of the effects of Syk and BTK inhibitors on GPVI-mediated platelet signaling and function.

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 αIIbß3 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.

Single cell morphological metrics and cytoskeletal alignment regulate VCAM-1 protein expression.

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.

Characterization of a pulsatile rotary total artificial heart.

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.

Endothelial PAI-1 (Plasminogen Activator Inhibitor-1) Blocks the Intrinsic Pathway of Coagulation, Inducing the Clearance and Degradation of FXIa (Activated Factor XI).

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.

Contact Activation Inhibitor and Factor XI Antibody, AB023, Produces Safe, Dose-Dependent Anticoagulation in a Phase 1 First-In-Human Trial.

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.

Protease-activated receptor 4 activity promotes platelet granule release and platelet-leukocyte interactions.

Human platelets express two protease-activated receptors (PARs), PAR1 (F2R) and PAR4 (F2RL3), which are activated by a number of serine proteases that are generated during pathological events and cause platelet activation. Recent interest has focused on PAR4 as a therapeutic target, given PAR4 seems to promote experimental thrombosis and procoagulant microparticle formation, without a broadly apparent role in hemostasis. However, it is not yet known whether PAR4 activity plays a role in platelet-leukocyte interactions, which are thought to contribute to both thrombosis and acute or chronic thrombo-inflammatory processes. We sought to determine whether PAR4 activity contributes to granule secretion from activated platelets and platelet-leukocyte interactions. We performed in vitro and ex vivo studies of platelet granule release and platelet-leukocyte interactions in the presence of PAR4 agonists including PAR4 activating peptide, thrombin, cathepsin G, and plasmin in combination with small-molecule PAR4 antagonists. Activation of human platelets with thrombin, cathepsin G, or plasmin potentiated platelet dense granule secretion that was specifically impaired by PAR4 inhibitors. Platelet-leukocyte interactions and platelet P-selectin exposure the following stimulation with PAR4 agonists were also impaired by activated PAR4 inhibition in either a purified system or in whole blood. These results indicate PAR4-specific promotion of platelet granule release and platelet-leukocyte aggregate formation and suggest that pharmacological control of PAR4 activity could potentially attenuate platelet granule release or platelet-leukocyte interaction-mediated pathological processes.

Oral administration of Bruton's tyrosine kinase inhibitors impairs GPVI-mediated platelet function.

The Tec family kinase Bruton's tyrosine kinase (Btk) plays an important signaling role downstream of immunoreceptor tyrosine-based activation motifs in hematopoietic cells. Mutations in Btk are involved in impaired B-cell maturation in X-linked agammaglobulinemia, and Btk has been investigated for its role in platelet activation via activation of the effector protein phospholipase Cγ2 downstream of the platelet membrane glycoprotein VI (GPVI). Because of its role in hematopoietic cell signaling, Btk has become a target in the treatment of chronic lymphocytic leukemia and mantle cell lymphoma; the covalent Btk inhibitor ibrutinib was recently approved by the US Food and Drug Administration for treatment of these conditions. Antihemostatic events have been reported in some patients taking ibrutinib, although the mechanism of these events remains unknown. We sought to determine the effects of Btk inhibition on platelet function in a series of in vitro studies of platelet activation, spreading, and aggregation. Our results show that irreversible inhibition of Btk with two ibrutinib analogs in vitro decreased human platelet activation, phosphorylation of Btk, P-selectin exposure, spreading on fibrinogen, and aggregation under shear flow conditions. Short-term studies of ibrutinib analogs administered in vivo also showed abrogation of platelet aggregation in vitro, but without measurable effects on plasma clotting times or on bleeding in vivo. Taken together, our results suggest that inhibition of Btk significantly decreased GPVI-mediated platelet activation, spreading, and aggregation in vitro; however, prolonged bleeding was not observed in a model of bleeding.

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.

Temporal Changes in the Surface Chemistry and Topography of Reactive Ion Plasma-Treated Poly(vinyl alcohol) Alter Endothelialization Potential.

Synthetic small-diameter vascular grafts (<6 mm) are used in the treatment of cardiovascular diseases, including coronary artery disease, but fail much more readily than similar grafts made from autologous vascular tissue. A promising approach to improve the patency rates of synthetic vascular grafts is to promote the adhesion of endothelial cells to the luminal surface of the graft. In this study, we characterized the surface chemical and topographic changes imparted on poly(vinyl alcohol) (PVA), an emerging hydrogel vascular graft material, after exposure to various reactive ion plasma (RIP) surface treatments, how these changes dissipate after storage in a sealed environment at standard temperature and pressure, and the effect of these changes on the adhesion of endothelial colony-forming cells (ECFCs). We showed that RIP treatments including O2, N2, or Ar at two radiofrequency powers, 50 and 100 W, improved ECFC adhesion compared to untreated PVA and to different degrees for each RIP treatment, but that the topographic and chemical changes responsible for the increased cell affinity dissipate in samples treated and allowed to age for 230 days. We characterized the effect of aging on RIP-treated PVA using an assay to quantify ECFCs on RIP-treated PVA 48 h after seeding, atomic force microscopy to probe surface topography, scanning electron microscopy to visualize surface modifications, and X-ray photoelectron spectroscopy to investigate surface chemistry. Our results show that after treatment at higher RF powers, the surface exhibits increased roughness and greater levels of charged nitrogen species across all precursor gases and that these surface modifications are beneficial for the attachment of ECFCs. This study is important for our understanding of the stability of surface modifications used to promote the adhesion of vascular cells such as ECFCs.

Pharmacologic targeting of coagulation factors XII and XI by monoclonal antibodies reduces thrombosis in nitinol stents under flow.

BACKGROUND: Cardiovascular implantable devices, such as vascular stents, are critical for the treatment of cardiovascular diseases. However, their success is dependent on robust and often long-term antithrombotic therapies. Yet, the current standard-of-care therapies often pose significant bleeding risks to patients. Coagulation factor (F)XI and FXII have emerged as potentially safe and efficacious targets to safely reduce pathologic thrombin generation in medical devices. OBJECTIVES: To study the efficacy of monoclonal antibody-targeting FXII and FXI of the contact pathway in preventing vascular device-related thrombosis. METHODS: The effects of inhibition of FXII and FXI using function-blocking monoclonal antibodies were examined in a nonhuman primate model of nitinol stent-related thrombosis under arterial and venous flow conditions. RESULTS: We found that function-blocking antibodies of FXII and FXI reduced markers of stent-induced thrombosis in vitro and ex vivo. However, FXI inhibition resulted in more effective mitigation of thrombosis markers under varied flow conditions. CONCLUSION: This work provides further support for the translation of contact pathway of coagulation inhibitors for their adjunctive clinical use with cardiovascular devices.