Stratification of Bleeding Risk Using Thromboelastography in Children on Extracorporeal Membrane Oxygenation Support.

OBJECTIVES: Patients undergoing extracorporeal membrane oxygenation are at high risk for bleeding and thrombotic complications. Current laboratory methods for assessing the coagulation system may be imprecise and complicate clinical decision-making. We hypothesize that thromboelastography may be more strongly associated with bleeding events than traditional methods and can aid extracorporeal membrane oxygenation coagulation management. DESIGN: In a retrospective study, 40 patients with congenital heart disease requiring extracorporeal membrane oxygenation support yielded a total of 159 patient days of data for thromboelastography analysis. SETTING: Pediatric cardiac ICU at a single institution. SUBJECTS: Pediatric patients (≤ 18 yr) with congenital heart disease requiring extracorporeal membrane oxygenation support. INTERVENTIONS: None. METHODS: Thromboelastography was performed on whole blood samples collected 6-12 hours following extracorporeal membrane oxygenation initiation and daily for the duration of extracorporeal membrane oxygenation. Bleeding during each 24-hour period was defined as need for re-exploration or need for blood transfusion. Associations between thromboelastography variables and bleeding over each 24-hour period (bleeding vs nonbleeding days) were assessed using mixed effects logistic regression and classification and regression tree analysis. MEASUREMENTS AND MAIN RESULTS: Bleeding occurred in 25 patients (63%), contributing 87 bleeding days (55% extracorporeal membrane oxygenation days) for analysis. The probability of bleeding within the 24-hour period was not associated with activated partial thromboplastin time (p = 0.6) or anti-Xa levels (p = 0.3) on that day. The strongest correlate of bleeding was a maximum amplitude less than 55.4 mm on thromboelastography (odds ratio, 3.28; 95% CI, 1.63-6.60; p < 0.001). Bleeding occurred on 73% versus 35% of extracorporeal membrane oxygenation days for maximum amplitude less than 55.4 mm versus greater than or equal to 55.4 mm, respectively. Bleeding occurred on all days when a combination of maximum amplitude less than 55.4 mm and a reaction time greater than 12.9 minutes was present. The lowest risk of bleeding (28% of patient days) was associated with maximum amplitude greater than or equal to 55.4 mm and plasma fibrinogen greater than 345 mg/dL. CONCLUSIONS: Thromboelastography-derived variables maximum amplitude and reaction time, along with plasma fibrinogen levels, can help predict bleeding events in children on extracorporeal membrane oxygenation support. Research based on larger patient samples is needed to confirm the specific thresholds identified for bleeding risk stratification for extracorporeal membrane oxygenation anticoagulation management.

Thromboelastography Is Associated With Surrogates for Bleeding After Pediatric Cardiac Operations.

BACKGROUND: Perioperative bleeding is a common complication in pediatric patients undergoing cardiac operation. Although thromboelastography (TEG) has been used in patients undergoing adult cardiac operation, limited data are available in pediatric patients. We hypothesize that TEG variables may be associated with surrogate end points for postoperative bleeding in pediatric patients undergoing complex cardiac operation. METHODS: In a retrospective study, TEG was obtained after protamine administration and on admission to the intensive care unit (ICU) in pediatric patients (≤18 years) undergoing cardiac operation that required cardiopulmonary bypass. A composite end point of extended blood product transfusion or surgical re-exploration for bleeding was used as a surrogate for perioperative bleeding. TEG variables were compared between patients who did or did not reach the composite end point. RESULTS: The study included 511 pediatric patients undergoing complex cardiac operation. The composite end point was reached in 52% of patients with maximum amplitude (MA) less than 45 mm compared with 31% with MA of 45 mm or more (p < 0.001). With the use of multivariable regression analysis, MA less than 45 mm was independently associated with the composite end point (p < 0.001). Patients with MA less than 45 mm who received platelet transfusion in the operating room (OR) were less likely to reach the composite end point within the subsequent 24 hours (8%) compared with patients who did not receive intraoperative platelet transfusion (24%) (p = 0.02). CONCLUSIONS: Intraoperative TEG MA less than 45 mm is associated with a surrogate end point for intraoperative bleeding in pediatric patients undergoing complex cardiac operation. In patients with MA less than 45 mm, prophylactic platelet transfusion in the OR may be associated with reduction in bleeding end points in the ICU.

Platelet testing to guide aspirin dose adjustment in pediatric patients after cardiac surgery.

OBJECTIVES: Thrombosis is associated with increased morbidity and mortality in pediatric patients undergoing cardiac surgery. Although aspirin commonly is used for thromboprophylaxis, the utility of laboratory-based tests that assess aspirin efficacy have not been evaluated. We sought to determine the relationship between platelet aggregation testing and aspirin dose adjustment on thrombosis rates in this population. METHODS: Pediatric patients undergoing cardiac surgery who received aspirin and underwent platelet testing were studied retrospectively. Patients were excluded if they were treated with multiple agents or experienced thrombosis before the initiation of aspirin. Thrombosis events within 30 days after initiation of aspirin were recorded. Associations between aspirin responsiveness and thrombosis rate and aspirin dose adjustment and thrombosis rate were assessed with the use of multivariable logistic regression analysis. RESULTS: Suboptimal platelet response to aspirin was detected in 64 of 430 patients (15%) and thrombosis was detected in 11 patients (2.6%). Lack of aspirin responsiveness on initial testing was a significant risk factor for thrombosis (P < .001) independent of age, weight, diagnosis, and initial aspirin dose. Dose escalation based on aspirin testing was performed in 40 of 64 patients, and significantly lower rate of thrombosis was observed in patients who underwent dose escalation compared with those without dose escalation (0/40 vs 9/24, P < .001). By multivariable analysis, the only significant independent risk factor for thrombosis was failure to increase aspirin dose after initial unresponsiveness (P < .001). CONCLUSIONS: Current practice of weight-based aspirin dosing may lead to subtherapeutic platelet inhibition in some children. Aspirin unresponsiveness is associated with increased risk of thrombosis after specific pediatric cardiac surgical procedures. Aspirin dose increase in unresponsive patients is associated with reduced risk of thrombosis.

Integration of proteomic and transcriptomic profiles identifies a novel PDGF-MYC network in human smooth muscle cells.

BACKGROUND: Platelet-derived growth factor-BB (PDGF-BB) has been implicated in the proliferation, migration and synthetic activities of smooth muscle cells that characterize physiologic and pathologic tissue remodeling in hollow organs. However, neither the molecular basis of PDGFR-regulated signaling webs, nor the extent to which specific components within these networks could be exploited for therapeutic benefit has been fully elucidated. RESULTS: Expression profiling and quantitative proteomics analysis of PDGF-treated primary human bladder smooth muscle cells identified 1,695 genes and 241 proteins as differentially expressed versus non-treated cells. Analysis of gene expression data revealed MYC, JUN, EGR1, MYB, RUNX1, as the transcription factors most significantly networked with up-regulated genes. Forty targets were significantly altered at both the mRNA and protein levels. Proliferation, migration and angiogenesis were the biological processes most significantly associated with this signature, and MYC was the most highly networked master regulator. Alterations in master regulators and gene targets were validated in PDGF-stimulated smooth muscle cells in vitro and in a model of bladder injury in vivo. Pharmacologic inhibition of MYC and JUN confirmed their role in SMC proliferation and migration. Network analysis identified the diaphanous-related formin 3 as a novel PDGF target regulated by MYC and JUN, which was necessary for PDGF-stimulated lamellipodium formation. CONCLUSIONS: These findings provide the first systems-level analysis of the PDGF-regulated transcriptome and proteome in normal smooth muscle cells. The analyses revealed an extensive cohort of PDGF-dependent biological processes and connected key transcriptional effectors to their regulation, significantly expanding current knowledge of PDGF-stimulated signaling cascades. These observations also implicate MYC as a novel target for pharmacological intervention in fibroproliferative expansion of smooth muscle, and potentially in cancers in which PDGFR-dependent signaling or MYC activation promote tumor progression.

JunB mediates basal- and TGFß1-induced smooth muscle cell contractility.

Smooth muscle contraction is a dynamic process driven by acto-myosin interactions that are controlled by multiple regulatory proteins. Our studies have shown that members of the AP-1 transcription factor family control discrete behaviors of smooth muscle cells (SMC) such as growth, migration and fibrosis. However, the role of AP-1 in regulation of smooth muscle contractility is incompletely understood. In this study we show that the AP-1 family member JunB regulates contractility in visceral SMC by altering actin polymerization and myosin light chain phosphorylation. JunB levels are robustly upregulated downstream of transforming growth factor beta-1 (TGFß1), a known inducer of SMC contractility. RNAi-mediated silencing of JunB in primary human bladder SMC (pBSMC) inhibited cell contractility under both basal and TGFß1-stimulated conditions, as determined using gel contraction and traction force microscopy assays. JunB knockdown did not alter expression of the contractile proteins α-SMA, calponin or SM22α. However, JunB silencing decreased levels of Rho kinase (ROCK) and myosin light chain (MLC20). Moreover, JunB silencing attenuated phosphorylation of the MLC20 regulatory phosphatase subunit MYPT1 and the actin severing protein cofilin. Consistent with these changes, cells in which JunB was knocked down showed a reduction in the F:G actin ratio in response to TGFß1. Together these findings demonstrate a novel function for JunB in regulating visceral smooth muscle cell contractility through effects on both myosin and the actin cytoskeleton.

FosB regulates stretch-induced expression of extracellular matrix proteins in smooth muscle.

Fibroproliferative remodeling in smooth muscle-rich hollow organs is associated with aberrant extracellular matrix (ECM) production. Although mechanical stimuli regulate ECM protein expression, the transcriptional mediators of this process remain poorly defined. Previously, we implicated AP-1 as a mediator of smooth muscle cell (SMC) mechanotransduction; however, its role in stretch-induced ECM regulation has not been explored. Herein, we identify a novel role for the AP-1 subunit FosB in stretch-induced ECM expression in SMCs. The DNA-binding activity of AP-1 increased after stretch stimulation of SMCs in vitro. In contrast to c-Jun and c-fos, which are also activated by the SMC mitogen platelet-derived growth factor, FosB was only activated by stretch. FosB silencing attenuated the expression of the profibrotic factors tenascin C (TNC) and connective tissue growth factor (CTGF), whereas forced expression of Jun~FosB stimulated TNC and CTGF promoter activity. Chromatin immunoprecipitation revealed enrichment of AP-1 at the TNC and CTGF promoters. Bladder distension in vivo enhanced nuclear localization of c-jun and FosB. Finally, the distension-induced expression of TNC and CTGF in the detrusor smooth muscle of bladders from wild-type mice was significantly attenuated in FosB-null mice. Together, these findings identify FosB as a mechanosensitive regulator of ECM production in smooth muscle.

An Akt- and Fra-1-dependent pathway mediates platelet-derived growth factor-induced expression of thrombomodulin, a novel regulator of smooth muscle cell migration.

Overdistension of hollow organs evokes pathological changes characterized by smooth muscle remodeling. Mechanical stimuli induce smooth muscle cell (SMC) growth through acute activation of signaling cascades and by increased expression of soluble mitogens. Physical forces have also been implicated in ligand-independent activation of receptor tyrosine kinases, including the platelet-derived growth factor (PDGF) receptor, although the extent to which this occurs in intact tissue is unknown. Previously, we implicated Akt and activator protein-1 (AP-1) as mediators of growth and gene expression in SMC exposed to cyclic stretch or PDGF. Here we show that bladder wall distension leads to PDGFR activation and identify thrombomodulin (TM) as an Akt and AP-1 target in SMC. We demonstrate that TM, also induced by bladder stretch injury, is regulated at the transcriptional level by the AP-1 components c-jun and Fra1. Mutation of an AP-1 motif at -2010/-2004 abolished both AP-1 binding and PDGF responsiveness of the TM promoter. Fra1 silencing diminished PDGF-induced TM expression and SMC cell cycle transit. In contrast, TM knockdown did not affect cell growth but attenuated PDGF-stimulated SMC migration. Taken together, these results reveal new facets of TM regulation in SMC and provide the first demonstration of a role for endogenous TM in PDGF-induced cell migration. Moreover, TM induction on bladder injury suggests that it may be a biomarker for pathological smooth muscle remodeling.