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.

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.

Estrogen-based hormonal therapy and the risk of thrombosis in COVID-19 patients.

OBJECTIVE: Estrogen-containing contraceptives and hormone replacement therapy are used commonly, however, the risks of venous and arterial thrombosis imparted by such medications during COVID-19 infection or other similar viral infections remain undescribed. METHODS: To assess the risk of venous and arterial thrombosis in patients receiving oral estrogen-containing therapy (ECT) with COVID-19 as compared to those receiving non-estrogen-based hormonal therapy, we conducted a multicenter cohort study of 991 patients with confirmed COVID-19 infection, 466 receiving estrogen-containing hormonal therapy, and 525 receiving progestin-only or topical therapy. RESULTS: The use of estrogen-containing therapy was found to significantly increase the risk of venous thromboembolism (VTE) following COVID-19 diagnosis after controlling for age (HR 5.46 [95% CI 1.12-26.7, p = .036]). This risk was highest in patients over age 50, with 8.6% of patients receiving estrogen-containing therapy diagnosed with VTE compared to 0.9% of those receiving non-estrogen-based therapies (p = .026). The risk of arterial thrombosis was not significantly associated with oral estrogen use. CONCLUSIONS: These results suggest that estrogen-containing therapy is associated with a significantly increased risk of VTE in COVID-19 patients, especially in older individuals. These findings may guide provider counseling and management of patients with COVID-19 on estrogen-containing therapy.

Clinical outcomes of patients referred for asymptomatic neutropenia: A focus on racial disparities in hematology.

BACKGROUND: Asymptomatic neutropenia is a common hematology referral, though standardized reference ranges and published clinical outcomes are lacking. METHODS: In our retrospective analysis, we evaluated demographics, laboratory, and clinical outcomes of adult patients referred to an academic hematology practice for evaluation of neutropenia from 2010 to 2018. Primary and secondary outcomes included incidence of hematologic disorders and rates of Duffy-null positivity by race, respectively. In a separate analysis, we reviewed absolute neutrophil count (ANC) reference ranges from publicly available Association of American Medical Colleges Medical School Member laboratory directories to assess institutional variations. RESULTS: In total, 163 patients were included, with disproportionate number of Black patients referred compared to local demographics. Twenty-three percent of patients (n = 38) were found to have a clinically relevant hematologic outcome (mean ANC of 0.59 × 109 /L), and only six were identified with ANC ≥1.0 × 109 /L. Incidence of hematologic outcomes was lowest among Black patients (p = .05), and nearly all Blacks who underwent Duffy-null phenotype testing were positive (93%), compared to 50% of Whites (p = .04). In separate review of laboratory directories, we confirmed wide variation in ANC lower limit of normal (0.91-2.40 × 109 /L). CONCLUSION: Hematologic disorders were rare in patients with mild neutropenia and among Blacks, highlighting the need to standardize hematological ranges representative of non-White communities.

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.

Heparan sulfate proteoglycan glypican-1 and PECAM-1 cooperate in shear-induced endothelial nitric oxide production.

This study aimed to clarify the role of glypican-1 and PECAM-1 in shear-induced nitric oxide production in endothelial cells. Atomic force microscopy pulling was used to apply force to glypican-1 and PECAM-1 on the surface of human umbilical vein endothelial cells and nitric oxide was measured using a fluorescent reporter dye. Glypican-1 pulling for 30 min stimulated nitric oxide production while PECAM-1 pulling did not. However, PECAM-1 downstream activation was necessary for the glypican-1 force-induced response. Glypican-1 knockout mice exhibited impaired flow-induced phosphorylation of eNOS without changes to PECAM-1 expression. A cooperation mechanism for the mechanotransduction of fluid shear stress to nitric oxide production was elucidated in which glypican-1 senses flow and phosphorylates PECAM-1 leading to endothelial nitric oxide synthase phosphorylation and nitric oxide production.

The glycocalyx core protein Glypican 1 protects vessel wall endothelial cells from stiffness-mediated dysfunction and disease.

AIMS: Arterial stiffness is an underlying risk factor and a hallmark of cardiovascular diseases. The endothelial cell (EC) glycocalyx is a glycan rich surface layer that plays a key role in protecting against EC dysfunction and vascular disease. However, the mechanisms by which arterial stiffness promotes EC dysfunction and vascular disease are not fully understood, and whether the mechanism involves the protective endothelial glycocalyx is yet to be determined. We hypothesized that endothelial glycocalyx protects the endothelial cells lining the vascular wall from dysfunction and disease in response to arterial stiffness. METHODS AND RESULTS: Cells cultured on polyacrylamide (PA) gels of substrate stiffness 10 kPa (mimicking the subendothelial stiffness of aged, unhealthy arteries) showed a significant inhibition of glycocalyx expression compared to cells cultured on softer PA gels (2.5 kPa, mimicking the subendothelial stiffness of young, healthy arteries). Specifically, gene and protein analyses revealed that a glycocalyx core protein Glypican 1 was inhibited in cells cultured on stiff PA gels. These cells had enhanced endothelial cell dysfunction as determined by enhanced cell inflammation (enhanced inflammatory gene expression, monocyte adhesion, and inhibited nitric oxide expression), proliferation, and EndMT. Removal of Glypican 1 using gene-specific silencing with siRNA or gene overexpression using a plasmid revealed that Glypican 1 is required to protect against stiffness-mediated endothelial cell dysfunction. Consistent with this, using a model of age-mediated stiffness, older mice exhibited a reduced expression of Glypican 1 and enhanced endothelial cell dysfunction compared to young mice. Glypican 1 gene deletion in knockout mice (GPC1-/-) exacerbated endothelial dysfunction in young mice, which normally had high endothelial expression, but not in old mice that normally expressed low levels. Endothelial cell dysfunction was exacerbated in young, but not aged, Glypican 1 knockout mice (GPC1-/-). CONCLUSION: Arterial stiffness promotes EC dysfunction and vascular disease at least partly through the suppression of the glycocalyx protein Glypican 1. Glypican 1 contributes to the protection against endothelial cell dysfunction and vascular disease in endothelial cells.

Endothelial Glycocalyx-Mediated Nitric Oxide Production in Response to Selective AFM Pulling.

Nitric oxide (NO) is a regulatory molecule in the vascular system and its inhibition due to endothelial injury contributes to cardiovascular disease. The glycocalyx is a thin layer of glycolipids, glycoproteins, and proteoglycans on the surface of mammalian epithelial cells. Extracellular forces are transmitted through the glycocalyx to initiate intracellular signaling pathways. In endothelial cells (ECs), previous studies have shown the glycocalyx to be a significant mediator of NO production; degradation of the endothelial glycocalyx layer (EGL) drastically reduces EC production of NO in response to fluid shear stress. However, the specific EGL components involved in this process are not well established. Recent work using short-hairpin RNA approaches in vitro suggest that the proteoglycan glypican-1, not syndecan-1, is the dominant core protein mediating shear-induced NO production. We utilized atomic force microscopy (AFM) to apply force selectively to components of the EGL of confluent rat fat pad ECs (RFPECs), including proteoglycans and glycosaminoglycans, to observe how each component individually contributes to force-induced production of NO. 4,5-diaminofluorescein diacetate, a cell-permeable fluorescent molecule, was used to detect changes in intracellular NO production. Antibody-coated AFM probes exhibited strong surface binding to RFPEC monolayers, with 100-300 pN mean adhesion forces. AFM pulling on glypican-1 and heparan sulfate for 10 min caused significantly increased NO production, whereas pulling on syndecan-1, CD44, hyaluronic acid, and with control probes did not. We conclude that AFM pulling can be used to activate EGL-mediated NO production and that the heparan sulfate proteoglycan glypican-1 is a primary mechanosensor for shear-induced NO production.

Integrated decision-making in response to weapons of mass destruction incidents: development and initial evaluation of a course for healthcare professionals.

INTRODUCTION: Standardized, validated training programs for teaching administrative decision-making to healthcare professionals responding to weapons of mass destruction (WMD) incidents have not been available. Therefore, a multidisciplinary team designed, developed, and offered a four-day, functional exercise, competency-based course at a national training center. OBJECTIVE: This report provides a description of the development and initial evaluation of the course in changing participants' perceptions of their capabilities to respond to WMD events. METHODS: Course participants were healthcare professionals, including physicians, nurses, emergency medical services administrators, hospital administrators, and public health officials. Each course included three modified tabletop and/or real-time functional exercises. A total of 441 participants attended one of the eight course offerings between March and August 2003. An intervention group only, pre-post design was used to evaluate change in perceived capabilities related to administrative decision-making for WMD incidents. Paired evaluation data were available on 339 participants (81.9%). Self-ratings for each of 21 capability statements were compared before and after the course. A 19-item total scale score for each participant was calculated from the pre-course and post-course evaluations. Paired t-tests on pre- and post-course total scores were conducted separately for each course. RESULTS: There was consistent improvement in self-rated capabilities after course completion for all 21 capability statements. Paired t-tests of pre- and post-course total scale scores indicated a significant increase in mean ratings for each course (all p < 0.001). CONCLUSION: The tabletop/real-time-exercise format was effective in increasing healthcare administrators' self-rated capabilities related to WMD disaster management and response. Integrating the competencies into training interventions designed for a specific target audience and deploying them into an interactive learning environment allowed the competency-based training objectives to be accomplished.