Introduction: Abdominal aortic aneurysm (AAA) is a progressive infrarenal aortic dilation accounting for >167,200 deaths annually. In ~80% of AAA patients, a platelet laden intraluminal thrombus (ILT) forms in the aneurysm. While thrombosis may stabilize the aorta initially, progressive ILT accumulation is positively associated with aortic rupture, but a causal relationship has not yet been established. Platelet activation is linked to faster AAA growth and anti-platelet therapy shows pre-clinical benefit, yet no pharmacologic therapies are approved for AAA patients. Here, we characterize the temporal relationship between aneurysm growth and ILT formation in a murine model featuring ILT and test whether aspirin (ASA) intervention limits ILT expansion. Methods and Results: Male C57BL/6J mice (10-14 weeks) received 0.2% BAPN in drinking water for 1 week before laparotomy, when 10mg/mL elastase (El+B, n=13) or heat inactivated elastase (Hl+B, n=5) was applied to the infrarenal aorta. BAPN water was maintained throughout the study. Bi-weekly 3D color Doppler imaging (VisualSonics Vevo F2) showed severe aortic dilation with disturbed and stagnant blood flow at the elastase-treated segment. ILTs formed distal to perturbed blood flow in 3/13, 9/13 and 12/13 mice at 2-, 4-, and 8-weeks post laparotomy respectively. Aneurysms with ILT were larger and grew faster. After 8 weeks, mice were euthanized and aneurysms were OCT-embedded, serially sectioned, and stained for CD41-platelets, CD68-macrophages, MPO-neutrophils, and αSMA-VSMCs. Confocal imaging confirmed ILT formation with marked platelet, macrophage, and neutrophil accumulation. Next, at 4 weeks post-laparotomy, El+B mice with similar aortic diameters were randomized to low dose ASA (30mg/L, El+B+ASA) or vehicle (El+B+Pl) in 0.2% BAPN water and followed for 16 weeks post intervention with bi-weekly ultrasounds. Aneurysm burden did not differ between El+B+ASA and El+B+Pl groups. Surprisingly, El+B+ASA mice experienced aortic rupture (5/7) at a significantly higher rate compared to El+B+Pl mice (0/6). Mice that underwent rupture exhibited an acute increase in aortic growth rate prior to death. Conclusions: In this ILT murine AAA model, progressive thrombus formation parallels accelerated aneurysm growth and precedes rupture, but delayed low-dose ASA fails to reduce aneurysm burden while markedly increasing rupture, suggesting ASA may destabilize the aneurysmal aorta in this setting.

BACKGROUND:Cardiovascular disease is a significant burden globally and, despite current therapeutics, remains the leading cause of death. PAR (protease-activated receptor) 4 is a receptor highly expressed by hematopoietic cells, strongly activated by thrombin, and plays a vital role in platelet activation and aggregation. However, the role of PAR4 in atherothrombotic disease remains understudied. METHODS:Mice on a low-density lipoprotein receptor-deficient (Ldlr-/-) background were bred with Par4-deficient (Par4-/-) mice to create Ldlr-/-/Par4+/+ and Ldlr-/-/Par4-/- cousin lines. Mice were fed high-fat (42%) and high-cholesterol (0.2%) Western diet for 12 weeks for all studies. Bone marrow transplant studies were conducted by irradiating Ldlr-/-/Par4+/+ and Ldlr-/-/Par4-/- mice with 550 rads (2×, 4 hours apart) and then repopulated with Par4+/+ or Par4-/- bone marrow. To determine whether the effects of thrombin were mediated solely by PAR4, the thrombin inhibitor dabigatran was added to the Western diet. RESULTS:We observed higher abundance of PAR4 in arteries with atherosclerosis in mouse and human lesions versus healthy controls. Using a global deletion of PAR4, we observed an attenuation in atherosclerosis versus Par4+/+ mice. Bone marrow transplant studies demonstrated these effects were due to hematopoietic cells. When observing whether PAR4 activation via thrombin contributes to atherosclerotic development, Ldlr-/-/Par4-/- mice given dabigatran did not further decrease their atherosclerotic burden. Differences between apoE-deficient (apoE-/-) and Ldlr-/- platelets were assessed for changes in reactivity. PAR4 appeared to be acting independent of PAR1, as there were no changes with the addition of dabigatran to Par4-/- mice. apoE-/- platelets were hyperreactive compared with Ldlr-/- platelets. CONCLUSIONS:We conclude that hematopoietic-derived PAR4 plays a vital role in the development and progression of atherosclerosis. More specifically, we observe thrombin-activated PAR4 contributes to the progression. Specifically, targeting of PAR4 may be a potential therapeutic target for cardiovascular disease.

Objective: Abdominal aortic aneurysms (AAA) frequently feature the formation of a nonocclusive intraluminal thrombus (ILT) along the site of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA associated ILT is still poorly understood. Thus, we sought to investigate how platelet activation via the glycoprotein VI receptor pathway impacts the pathogenesis of AAA. Methods and Results: RNA sequencing was performed on age-matched control human aortic tissue, AAA aortic tissue, and AAA ILT. Platelet transcripts comprised 25% of significantly enriched genes in the comparison of AAA thrombus to AAA tissue and control aortic wall, with GPVI increased by 9.1 Log2-FC (P = 3.21E−08). Circulating GPVI was significantly elevated in platelets isolated from AAA patients versus age-matched controls. Soluble GPVI (sGPVI—indicator of platelet activation), was significantly increased in the plasma of patients with fast-growing AAAs compared to slow-growing AAAs and healthy control subjects. To determine if GPVI blockade was protective from AAA progression, mice underwent either the angiotensin II (AngII) infusion or the topical elastase models of aneurysm formation. Following model initiation, aortic diameter was monitored by weekly ultrasounds. After two weeks, mice which developed an aneurysm (defined as an aortic diameter of >1.2mm) were randomized into control (IgG—50μg) or treatment (JAQ1—50μg, monoclonal GPVI antibody) groups. As expected, platelets isolated from JAQ1 treated mice did not activate in response to the GPVI specific agonist convulxin. Furthermore, JAQ1 treatment attenuated aneurysm progression, increased type I collagen deposition, attenuated macrophage accumulation, and increased survival in both murine AAA models versus IgG control treated mice. Conclusions: Platelets play a critical role in AAA pathophysiology and specific blockade of the GPVI-mediated activation pathway attenuates progression of established aneurysms in two independent murine models. Thus, our work demonstrates that specific targeting of GPVI to blunt platelet activation in AAA could be a potential therapeutic for a pathology with no current pharmacological treatments. R01HL147171-04 and R01HL158801-02. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Abdominal aortic aneurysm (AAA) is a disease associated with the pathophysiologic degradation of the tunica media resulting in aortic dilatation, systemic inflammation, and dysregulated hemostasis. Beyond role its role in initiating primary hemostasis, platelets are a source of ROS, inflammatory cytokines and growth factors necessary for angiogenesis and vascular remodeling. Although platelets contribute to the progression of established aneurysms, their role in the initiation of AAA remains undefined. Methods: Low density lipoprotein receptor deficient (Ldlr-/-) mice were examined for platelet accumulation in the angiotensin II (AngII) model of AAA utilizing in vivo labeling techniques. Two platelet antagonists (clopidogrel and aspirin), a thrombin inhibitor (dabigatran), or genetic deficiencies (protease-activated receptor 4, P2Y12, Lnk) were administered to AngII-infused mice to determine the role of platelets in the initiation of AAA. The effect of platelet depletion was examined in multiple mouse strains of AngII-induced AAA and two additional aneurysm models. PheWAS and meta-analysis was analyzed in humans for platelet gene SNPs associated with AAA. Results: We show that platelets are recruited rapidly to the aorta after the initiation of AngII infusion. Genetic deficiency of platelet receptors had no effect on abdominal aortic diameter but augmented rupture-induced death in littermate versus placebo controls during AngII-induced AAA. Moreover, Ldlr-/- mice receiving anti-platelet inhibitors or a thrombin inhibitor also had augmented rupture-induced death. Platelet depletion preceding aneurysm formation resulted in pervasive rupture-induced death in several mouse strains and with three different mouse models of AAA. Conclusions: Inhibition of platelet function is detrimental in an early expanding aortic lumen, resulting in catastrophic rupture and hemodynamic failure in murine AAA models. ### Competing Interest Statement SRS reports personal fees as a consultant for Eko Health and Prolaio Health and grant support from Janssen Research & Development AD is a consultant for Regeneron

ABSTRACT:A common feature in patients with abdominal aortic aneurysms (AAAs) is the formation of a nonocclusive intraluminal thrombus (ILT) in regions of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA-associated ILT is still poorly understood. Thus, we sought to investigate how platelet activation affects the pathogenesis of AAA. Using RNA sequencing, we identified that the platelet-associated transcripts are significantly enriched in the ILT compared with the adjacent aneurysm wall and healthy control aortas. We found that the platelet-specific receptor glycoprotein VI (GPVI) is among the top enriched genes in AAA ILT and is increased on the platelet surface of patients with AAAs. Examination of a specific indicator of platelet activity, soluble GPVI (sGPVI), in 2 independent cohorts of patients with AAAs is highly predictive of an AAA diagnosis and associates more strongly with aneurysm growth rate than D-dimer in humans. Finally, intervention with the anti-GPVI antibody (JAQ1) in mice with established aneurysms blunted the progression of AAA in 2 independent mouse models. In conclusion, we show that the levels of sGPVI in humans can predict a diagnosis of AAA and AAA growth rate, which may be critical in the identification of high-risk patients. We also identify GPVI as a novel platelet-specific AAA therapeutic target, with minimal risk of adverse bleeding complications, for which none currently exists.

Background: Platelets are rapidly recruited to the forming intraluminal thrombus in abdominal aortic aneurysm (AAA), stabilizing the aortic wall and preventing rupture. Adhesion and activation of platelets releases a mix of inflammatory mediators such as transforming growth factor β (TGFβ), which mediates cellular proliferation, inflammatory signaling, and fibrosis. Importantly, while TGFβ neutralization results in increased rupture and disease progression in mouse models of AAA, the source of protective TGFβ signaling remains controversial. We hypothesized that platelet-derived TGFβ1 attenuates aortic growth in the elastase AAA model. Methods and Results: Ldlr -/- mice with platelet-specific deletion of Tgfβ1 were produced by breeding hemizygous male mice expressing Cre under the control of the Pf4 promoter to female Tgfβ1 floxed mice (Jackson Labs). Mice were subjected to laparotomy and topical elastase application to the abdominal aorta (10 mg/mL porcine pancreatic elastase for 5 minutes) and sacrificed at day 28 to determine AAA progression via aortic diameter. Male TGFβ1-Pf4 Cre+ mice (n=8) had significantly increased abdominal aortic diameters compared to Male TGFβ1-Pf4 Cre- mice (n=7) ( Cre- : 1.70 ± 0.12 mm; Cre+ : 2.66 ± 0.25 mm; P < 0.004). Additionally, 25% of male TGFβ1-Pf4 Cre+ mice died of AAA rupture compared to 0% in the TGFβ1-Pf4 Cre- cohort. To determine if these actions were occurring via TGFβ1 activation of the TGFβ receptor 2 (βr 2 ), Ldlr -/- /βr2 Flox /PF4 Cre+ and Cre- mice were created, similarly to the previously described Tgfβ1 mice. Male βr2-Pf4 Cre+ (n=7) had increased aortic diameter compared to βr2-Pf4 Cre- (n=6) ( Cre- : 1.70± 0.20 mm; Cre+ : 2.45 ± 0.38 mm; P < 0.138). Additionally, female βr2-Pf4 Cre+ (n=9) also had significantly larger aortic diameters compared to βr2-Pf4 Cre- (n = 5) ( Cre- : 1.27± 0.04 mm; Cre+ : 1.90 ± 0.23 mm; P < 0.01). Conclusions: Our results further support TGFβ signaling playing a protective role in AAA. More specifically, platelet-derived TGFβ1 ligand and signaling via platelet TGFβr2 mitigates aortic diameter expansion. Further research into the pertinent TGFβ isoforms and the mechanism of TGFβr2 activation will ideally yield insights into therapeutic targets for an, as yet, intractable disease.

Objective: Abdominal aortic aneurysms (AAA) frequently feature the formation of a nonocclusive intraluminal thrombus (ILT) along the site of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA associated ILT is still poorly understand. Thus, we sought to investigate how platelet activation via the glycoprotein VI (GPVI) platelet receptor impacts the pathogenesis of AAA. Methods and Results: RNA sequencing of age-matched control human aortic tissue, AAA aortic tissue, and AAA ILT demonstrates that platelet transcripts comprised 25% of significantly enriched genes AAA thrombus compared to AAA tissue and control aortic wall, with GPVI increased by 9.1 Log2-FC (P = 3.21E -08 ). Examination of soluble GPVI (sGPVI), a specific marker of platelet activity, in 2 independent AAA patient cohorts is predictive of AAA case status, associates strongly with aneurysm growth rate, and better predicts the presence of a fast-growing AAA when compared to D-dimer. To determine if GPVI blockade ameliorates AAA progression, mice were subjected to either the angiotensin II (AngII) infusion or the topical elastase models of aneurysm formation. Following model initiation, aortic diameter was monitored by weekly ultrasounds. After two weeks, mice which developed an aneurysm (defined as an aortic diameter of >1.2mm) were randomized into control (IgG-50μg) or treatment (JAQ1-50μg, monoclonal GPVI antibody) groups. Treatment with JAQ1 blunted GPVI mediated platelet activation, attenuated aneurysm progression, and increased survival in both murine AAA models versus control. Conclusions: The highly specific platelet activation biomarker soluble glycoprotein VI strongly predicts both AAA case status and growth rate with potential use in identifying high risk patients. Specific blockade of GPVI-mediated platelet activation attenuates progression of established aneurysms in two independent murine models. Thus, our work demonstrates that specific targeting of GPVI to blunt platelet activation in AAA could be a potential therapeutic for a pathology with no current pharmacological treatments.

A common feature in patients with abdominal aortic aneurysms (AAA) is the formation of a nonocclusive intraluminal thrombus (ILT) in regions of aortic dilation. Platelets are known to maintain hemostasis and propagate thrombosis through several redundant activation mechanisms, yet the role of platelet activation in the pathogenesis of AAA associated ILT is still poorly understood. Thus, we sought to investigate how platelet activation impacts the pathogenesis of AAA. Using RNA-sequencing, we identify that the platelet-associated transcripts are significantly enriched in the ILT compared to the adjacent aneurysm wall and healthy control aortas. We found that the platelet specific receptor glycoprotein VI (GPVI) is among the top enriched genes in AAA ILT and is increased on the platelet surface of AAA patients. Examination of a specific indicator of platelet activity, soluble GPVI (sGPVI), in two independent AAA patient cohorts is highly predictive of a AAA diagnosis and associates more strongly with aneurysm growth rate when compared to D-dimer in humans. Finally, intervention with the anti-GPVI antibody (J) in mice with established aneurysms blunted the progression of AAA in two independent mouse models. In conclusion, we show that levels of sGPVI in humans can predict a diagnosis of AAA and AAA growth rate, which may be critical in the identification of high-risk patients. We also identify GPVI as a novel platelet-specific AAA therapeutic target, with minimal risk of adverse bleeding complications, where none currently exist.KEY POINTS:Soluble glycoprotein VI, which is a platelet-derived blood biomarker, predicts a diagnosis of AAA, with high sensitivity and specificity in distinguishing patients with fast from slow-growing AAA.Blockade of glycoprotein VI in mice with established aneurysms reduces AAA progression and mortality, indicating therapeutic potential.

Background: We have previously linked the microbiome-derived metabolite trimethylamine N-oxide (TMAO) to abdominal aortic aneurysm (AAA) in both associative human and mechanistic mouse studies. TMAO is reported to bind protein kinase R-like endoplasmic reticulum kinase (PERK), resulting in selective activation of the unfolded protein response (UPR). Concordantly, our previous RNA sequencing results indicate that TMAO augments PERK-mediated UPR pathways in the aneurysm wall. Thus, the current study will determine the functional role of TMAO-mediated PERK activation in aortic VSMCs to AAA pathogenesis. Methods & Results: Ldlr -/- mice with VSMC-specific deletion of Perk were produced by breeding hemizygous male mice expressing Cre under the control of the Transgelin promoter ( B6.Cg-TG(Tagln-cre)1Her/J , Jackson Labs) to female Perk floxed mice ( Eif2ak3 tm1.2Drc /J , Jackson Labs). Specific deletion of PERK in VSMCs was validated by western blot. Male mice were subjected to angiotensin II (AngII) infusion via osmotic mini pump (1,000 ng/kg/min; 28 days), while female mice underwent laparotomy and application of topical elastase (10 mg/mL porcine pancreatic elastase 5 minutes) to induce AAA. To augment circulating TMAO levels, mice were fed a high choline diet (0.2% total cholesterol + 1.2% choline supplementation) for one week prior to and throughout the study. Male mice with VSMC specific PERK knockout demonstrated significantly reduced AngII-induced aortic dilation (Cre+: 0.90 ± 0.048 mm; Cre-: 1.57 ± 0.156 mm; P = 0.001) and rupture induced death relative to control. Similarly, female mice with VSMC specific PERK knockout displayed significantly reduced aortic diameter (Cre+: 1.17 ± 0.069 mm; Cre-: 2.65 ± 0.432 mm; P = 0.002) and rupture induced mortality as compared to control. Additionally, male and female mice with VSMC specific PERK knockout had significantly greater Type I collagen and reduced macrophage accumulation compared to control. Conclusions: These results indicate that VSMC specific knockout of PERK blunts AAA formation in two independent murine models. Further studies are needed to elucidate the mechanism of PERK mediated ER stress in AAA, which may reveal novel therapeutic targets for AAA treatment.

Background: Surgeon choice of the appropriate staple height has been cited as a factor in the mechanical integrity of a staple line. However, tissue measured at the industry standard 8 g/mm(2) is usually thicker than the formed staple height of the staples that hold it together. This means that the pressure that the staples apply must be greater than 8g/mm(2). Additionally, formed staple heights in tissue may be different than formed staple heights of the same cartridge type when fired without tissue. This means that there is likely a compressive limit to the individual staples deployed by the stapling system. The purpose of this study is to establish the degree to which staple heights of endocutter staples auto-adjust to tissue and the compressive limit to tissue that this infers. Materials and methods: Excised gastric remnants from laparoscopic sleeve gastrectomy were measured for tissue thickness at different external pressures. An optimized experimental staple line was then created in parallel to the clinical staple line. The doubly-stapled gastric sliver then underwent computed tomography with solid modeling software to measure staple heights. Results: Staple heights fired in gastric tissue were significantly different than industry labelled and control staple heights. Clinical staple heights were significantly shorter than measured tissue thickness at 8 g/mm(2). Staple height more closely approximated tissue thickness under 15 g/mm(2) of pressure, rather than the 8 g/mm(2) loading pressure used by industry for tissue thickness range labelling. Conclusions: Staples deployed in human gastric tissue are taller than commercial labelling. The closed staple height corresponds to tissue thickness under 15g/mm(2) of pressure, not the labelled staple height. These results demonstrate that staple heights from modern endocutter staplers adjust to tissue, approximating a maximum compressive force just above 15g/mm(2). (C) 2021 Elsevier Inc. All rights reserved.

Background: There is interest in methods of measuring noninvasive intracranial pressure (ICP), including pupillometry, ultrasonographic transcranial Doppler (TCD), and optic nerve sheath diameter (ONSD), for diagnosing traumatic brain injury (TBI) in limited resource environments. Whether these technologies have diagnostic agreement is unknown. We hypothesized that ONSD, pupillometry, and TCD could both distinguish severe TBI and correlate with ICP.Methods: A prospective study of 135 patients was conducted at a level 1 trauma center. Four test groups were established: nontrauma patients with ICP monitoring, trauma patients without TBI, trauma patients with mild TBI, and trauma patients with severe TBI with ICP monitoring. All patients underwent daily measurements of ONSD, pupillometry, and TCD with both CX50 Sonosite and the Spencer ST3 Yi Pencil probe.Results: ONSD differed significantly in patients with severe TBI compared with patients with mild and no TBI, but did not correlate with ICP. Pupillometric constriction velocity, dilation velocity, and percent change in pupil diameter were significantly different in patients with severe TBI, but also did not correlate with ICP. TCD did not differ among TBI severities, but middle cerebral artery peak systolic velocity, middle cerebral artery flow velocity, and carotid flow velocity correlated with ICP.Conclusions: This is a novel study of four noninvasive tests to screen for severity of TBI and measure ICP. Our analysis indicates that no single device can do both. However, ONSD and pupillometry may be used as a supplementary screening tool for severe TBI, whereas TCD could be used to estimate and follow ICP in patients with severe TBI.(c) 2020 Elsevier Inc. All rights reserved.

BACKGROUND:Optimal stapler selection during laparoscopic sleeve gastrectomy requires careful balance between tissue compression, hemostasis, and mechanical integrity. Junctions along a staple line can further increase the risks of technical or mechanical staple line failures. The aim of this study was to compare two commonly utilized laparoscopic linear gastrointestinal staplers (Ethicon, Medtronic) with a novel linear stapler (Titan) designed to perform a sleeve gastrectomy with a single stapler firing.METHODS:Excised gastric remnants from laparoscopic sleeve gastrectomy were utilized and tissue thickness was measured from fundus to antrum. An optimized experimental staple line was then created. The greater curve remnant was insufflated to determine the staple line burst pressure and location. The doubly stapled (clinical and experimental) gastric specimen underwent staple analysis for junctional location, malformation, and height.RESULTS:The Titan stapler withstood a significantly higher burst pressure than both Ethicon and Medtronic linear cutting staplers. While the Medtronic and Ethicon staplers had a similar percentage of staples in junctions, the Titan stapler has no junctions. In considering the formation of all staples outside of junctions, the Medtronic and Titan staplers had no difference in percentage of malformed staples, while the Ethicon stapler had a significantly higher percentage. Additionally, there were no differences in mismatch between staple height and tissue thickness between experimental groups.CONCLUSIONS:The Titan stapler conveys the mechanical benefits of higher burst pressure with the advantage of single load functionality. This single staple load eliminates staple line junctions without sacrificing the integrity of staple formation.