Matthew T. Rondina

Signal-dependent splicing of tissue factor pre-mRNA modulates the thrombogenecity of human platelets

Tissue factor (TF) is an essential cofactor for the activation of blood coagulation in vivo. We now report that quiescent human platelets express TF pre-mRNA and, in response to activation, splice this intronic-rich message into mature mRNA. Splicing of TF pre-mRNA is associated with increased TF protein expression, procoagulant activity, and accelerated formation of clots. Pre-mRNA splicing is controlled by Cdc2-like kinase (Clk)1, and interruption of Clk1 signaling prevents TF from accumulating in activated platelets. Elevated intravascular TF has been reported in a variety of prothrombotic diseases, but there is debate as to whether anucleate platelets—the key cellular effector of thrombosis—express TF. Our studies demonstrate that human platelets use Clk1-dependent splicing pathways to generate TF protein in response to cellular activation. We propose that platelet-derived TF contributes to the propagation and stabilization of a thrombus.

Platelets as Cellular Effectors of Inflammation in Vascular Diseases

Platelets are chief effector cells in hemostasis. In addition, they are multifaceted inflammatory cells with functions that span the continuum from innate immune responses to adaptive immunity. Activated platelets have key thromboinflammatory activities in a variety of vascular disorders and vasculopathies. Recently identified inflammatory and immune activities provide insights into the biology of these versatile blood cells that are directly relevant to human vascular diseases.

The effects of drilling force on cortical temperatures and their duration: an in vitro study.

Bone loss due to thermonecrosis may weaken the purchase of surgically placed screws and pins, causing them to loosen post-operatively. The goal of this study was to determine how differences in applied drilling forces affect the temperature of cortical tissue near the drilling site. Results from thermocouples placed into fresh cortical bone indicate that increasing the applied drilling force resulted in a significant decrease (P=0.001) of maximum cortical temperatures. Furthermore, increasing the drilling force resulted in a significant decrease (P=0.001) in the average duration of temperature elevations above 50 degrees C. The results of the current study demonstrate that by the application of a larger force to the drill, both maximum cortical temperatures and their duration above 50 degrees C may be effectively reduced, decreasing the potential for thermal necrosis in the neighboring cortical bone.

Treatment of submassive pulmonary embolism with tenecteplase or placebo: cardiopulmonary outcomes at 3 months: multicenter double‐blind, placebo‐controlled randomized trial

Acute pulmonary embolism (PE) can worsen quality of life due to persistent dyspnea or exercise intolerance.

Mammalian target of rapamycin regulates neutrophil extracellular trap formation via induction of hypoxia-inducible factor 1 α.

Neutrophils are highly specialized innate immune effector cells that evolved for antimicrobial host defense. In response to inflammatory stimuli and pathogens, they form neutrophil extracellular traps (NETs), which capture and kill extracellular microbes. Deficient NET formation predisposes humans to severe infection, but, paradoxically, dysregulated NET formation contributes to inflammatory vascular injury and tissue damage. The molecular pathways and signaling mechanisms that control NET formation remain largely uncharacterized. Using primary human neutrophils and genetically manipulated myeloid leukocytes differentiated to surrogate neutrophils, we found that mammalian target of rapamycin (mTOR) regulates NET formation by posttranscriptional control of expression of hypoxia-inducible factor 1 α (HIF-1α), a critical modulator of antimicrobial defenses. Next-generation RNA sequencing, assays of mRNA and protein expression, and analysis of NET deployment by live cell imaging and quantitative histone release showed that mTOR controls NET formation and translation of HIF-1α mRNA in response to lipopolysaccharide. Pharmacologic and genetic knockdown of HIF-1α expression and activity inhibited NET deployment, and inhibition of mTOR and HIF-1α inhibited NET-mediated extracellular bacterial killing. Our studies define a pathway to NET formation involving 2 master regulators of immune cell function and identify potential points of molecular intervention in strategies to modify NETs in disease.

Weight-based dosing of enoxaparin for VTE prophylaxis in morbidly obese, medically-Ill patients

INTRODUCTION In clinical trials, fixed-dose enoxaparin (40 mg once daily) reduces the risk of venous thromboembolism (VTE) in medically-ill patients. However, morbidly obese patients were under-represented in these trials and using fixed-dose enoxaparin in obese patients may be inadequate. We completed a pharmacokinetic study in morbidly obese, medically-ill patients to determine if weight-based dosing of enoxaparin for VTE prophylaxis was feasible, without excessive levels of anticoagulation, as determined by peak anti-Xa levels. MATERIALS AND METHODS Twenty eight morbidly obese (BMI>or=35 kg/m(2)) patients were enrolled and completed the study protocol. Enoxaparin 0.5 mg/kg was administered once daily subcutaneously and peak anti-Xa levels were measured approximately 4-6 hours after the enoxaparin dose. RESULTS AND CONCLUSIONS Overall, 46% of patients were female, the average age (+/-SD) was 54 (+/-11) years, and the average weight and BMI were 135.6 kg (+/-25.3) and 48.1 kg/m(2) (+/-11.1), respectively. The average daily dose of enoxaparin was 67 mg (+/-12). The average peak anti-Xa level was 0.25 (SD+/-0.11, range 0.08 to 0.59) units/mL. Peak anti-Xa levels did not significantly correlate with weight or BMI. There were no bleeding events, symptomatic VTE, or significant thrombocytopenia. In morbidly obese, medically-ill patients, use of weight-based enoxaparin dosed at 0.5 mg/kg once daily is feasible and results in peak anti-Xa levels within or near recommended range for thromboprophylaxis, without any evidence of excessive anti-Xa activity. These data suggest that this weight-based regimen may be more effective than standard fixed-dose enoxaparin. Clinical outcome studies are warranted to determine the clinical safety and efficacy of this regimen.

The septic milieu triggers expression of spliced tissue factor mRNA in human platelets

Summary.  Background: Activated platelets have previously‐unrecognized mechanisms of post‐transcriptional gene expression that may influence hemostasis and inflammation. A novel pathway involves splicing of pre‐mRNAs in resting platelets to mature, translatable mRNAs in response to cellular activation. Objectives: We asked if bacterial products and host agonists present in the septic milieu induce tissue factor pre‐mRNA splicing in platelets from healthy subjects. In parallel, we asked if spliced tissue factor (TF) mRNA is present in platelets from septic patients in a proof‐of‐principle analysis. Patients/methods: TF pre‐mRNA and mRNA expression patterns were characterized in platelets from septic patients and in platelets isolated from healthy subjects activated with bacteria, toxins and inflammatory agonists. Procoagulant activity was also measured. Results and conclusions: Live bacteria, staphylococcal α‐toxin and lipopolysaccharide (LPS) induced TF pre‐mRNA splicing in platelets isolated from healthy subjects. Toxin‐stimulated platelets accelerated plasma clotting, a response that was blocked by a previously‐characterized splicing inhibitor and by an anti‐tissue factor antibody. Platelets from septic patients expressed spliced TF mRNA, whereas it was absent from unselected and age‐matched control subjects. Tissue factor‐dependent procoagulant activity was elevated in platelets from a subset of septic patients. Thus, bacterial and host factors induce splicing of TF pre‐mRNA, expression of TF mRNA and tissue factor‐dependent clotting activity in human platelets. TF mRNA is present in platelets from some septic patients, indicating that it may be a marker of altered platelet phenotype and function in sepsis and that splicing pathways are induced in this syndrome.

Prevention of venous thromboembolism in obesity

Venous thromboembolism (VTE) is a significant cause of morbidity and mortality in hospitalized patients. Where appropriate, evidence-based methods of prophylaxis are implemented and the burden of VTE can be reduced substantially. Obesity, including morbid obesity, is associated with a high risk of VTE and, unfortunately, fixed doses of US FDA-approved anticoagulant regimens, including unfractionated heparins, low-molecular-weight heparins and factor Xa inhibitors, may not provide optimal VTE prophylaxis in these patients. Although the data are still limited, a rapidly growing body of literature and cumulative evidence suggests that anticoagulant dose adjustments in morbidly obese patients may optimize pharmacodynamic activity and reduce VTE risk. With the prevalence of morbid obesity continuing to rise, more high-quality clinical data are needed to better understand the pathobiology of VTE in obesity and provide effective, yet safe, prevention strategies.

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

Prospective comparison of three enoxaparin dosing regimens to achieve target anti-factor Xa levels in hospitalized, medically ill patients with extreme obesity.

Enoxaparin is commonly used to prevent venous thromboembolism(VTE) [1,2] but has not been well-studied in patients with extreme obesity,a population at high risk for VTE. We prospectively compared three enoxaparin dosing regimens for the achievement of goal peak anti-Factor Xa levels in medically ill patients (n 5 31) with extreme obesity (body mass index (BMI) ‡ 40 kg/m2). Patients were assigned to receive fixed-dose (FD) enoxaparin 40 mg daily (QDay, n 5 11), weight based,lower-dose (LD) enoxaparin 0.4 mg/kg QDay (n 5 9), or weight based,higher-dose (HD) enoxaparin 0.5 mg/kg QDay (n 5 11). The average BMI and weight of the entire cohort was 62.1 kg/m2 (range40.5–82.4) and 176 kg (range 115–256 kg) and did not differ between groups. Peak anti-Factor Xa levels were significantly higher in the HD group compared to either LD or FD groups. Patients in the HD group achieved target anti-Factor Xa levels more frequently than the LD and FD groups (P < 0.05). Peak anti-Factor Xa levels did not correlate with age, weight, BMI, or creatinine clearance, demonstrating the predictability of weight-based enoxaparin dosing. There were no adverse events (e.g., bleeding, thrombosis, thrombocytopenia). To our knowledge this is the first prospective comparative study demonstrating that in extremely obese, medically ill patients enoxaparin 0.5 mg/kg QDay is superior to FD and LD enoxaparin for the achievement of target anti-Factor Xa levels.