FVIIa as used pharmacologically is not TF dependent in hemophilia B mice.

Activated factor VII is approved for treating hemophilia patients with autoantibodies to their factor IX or FVIII; however, its mechanism of action remains controversial. Some studies suggest that FVIIa requires tissue factor (TF) for function and that the reason for the high dose requirement is that it must compete with endogenous FVII for tissue factor. Others suggest that FVIIa binds platelets where it activates FX directly; the high concentration required would result from FVIIa's weak affinity for phospholipids. We address this question by infusing a chimera of mouse FIX (Gla and EGF1) with FVIIa (EGF2 and catalytic domain) into hemophilia B mice. This mutant has no TF-dependent activity because it cannot functionally bind TF at physiologically relevant concentrations. In vivo, this mutant is as effective as mouse FVIIa in controlling bleeding in hemophilia B mice. Our results suggest that the hemostatic effect of pharmacologic doses of FVIIa is TF independent.

Interferon-α is not elevated in idiopathic thrombotic thrombocytopenic purpura patients.

Idiopathic thrombotic thrombocytopenic purpura (TTP) patients have ADAMTS13 deficiency, which is usually caused by ADAMTS13 autoantibodies. However, the triggering factors for the autoantibody production remain unclear. Interferon-α (IFN-α) is a cytokine involved with many autoimmune processes such as inducing the activation of peripheral dendritic cells and stimulating T cells and B cells. It also plays an important role in some autoimmune diseases. Elevated IFN-α levels have been observed in some TTP patients and previous case reports have shown the occurrence of TTP after IFN-α treatment. Thus, we hypothesized that high levels of IFN-α would correlate with presence of ADAMTS13 autoantibodies. However, we did not observe elevated IFN-α levels in 36 TTP patients (mean 5.29 pg/ml, standard deviation (SD) 26.56 pg/ml) compared to healthy controls (mean 0 pg/ml, SD 0 pg/ml), P = 0.59. IFN-α levels of most patients (94%) were undetectable. Only two patients had increased IFN-α levels and ADAMTS13 autoantibodies were detected in these two patients. Interestingly, both the patients had an underlying autoimmune disease. Although there have been cases of secondary TTP following IFN-α treatment, no evidence supports a role of IFN-α in the development of idiopathic TTP in our patient population.

Blood utilization in patients with burn injury and association with clinical outcomes (CME).

BACKGROUND: Uncontrolled bleeding is an important cause of increased transfusion in burn victims; however, description of blood utilization patterns in the burn population is lacking. STUDY DESIGN AND METHODS: We conducted a single-institution, retrospective cohort study to measure blood utilization in 89 consecutive burn patients with 15% to 65% total body surface area (TBSA) burn within 60 days of injury. We also evaluated the relationship of blood product utilization with clinical variables including anticoagulant usage and mortality. RESULTS: We determined that: 1) the predictors for increased red blood cells (RBCs) and plasma transfusions were high TBSA burn and the use of argatroban anticoagulation (for suspected heparin-induced thrombocytopenia [HIT]); 2) TBSA burn and patient age were independent predictors of mortality, but not RBC or plasma transfusion; and 3) the incidence of symptomatic venous thromboembolic events is not uncommon (11.2%), although HIT is rare (1.1%). CONCLUSION: Despite concerns about adverse correlation between increased number of transfusions and mortality in other clinical settings, we did not find this association in our study. However, we demonstrated that the type and intensity of anticoagulation carries substantial risk for increased RBC as well as plasma usage.

Overexpression of the cell cycle inhibitor p16INK4a promotes a prothrombotic phenotype following vascular injury in mice.

OBJECTIVE: Age-associated cellular senescence is thought to promote vascular dysfunction. p16(INK4a) is a cell cycle inhibitor that promotes senescence and is upregulated during normal aging. In this study, we examine the contribution of p16(INK4a) overexpression to venous thrombosis. METHODS AND RESULTS: Mice overexpressing p16(INK4a) were studied with 4 different vascular injury models: (1) ferric chloride (FeCl(3)) and (2) Rose Bengal to induce saphenous vein thrombus formation; (3) FeCl(3) and vascular ligation to examine thrombus resolution; and (4) lipopolysaccharide administration to initiate inflammation-induced vascular dysfunction. p16(INK4a) transgenic mice had accelerated occlusion times (13.1 ± 0.4 minutes) compared with normal controls (19.7 ± 1.1 minutes) in the FeCl(3) model and 12.7 ± 2.0 and 18.6 ± 1.9 minutes, respectively in the Rose Bengal model. Moreover, overexpression of p16(INK4a) delayed thrombus resolution compared with normal controls. In response to lipopolysaccharide treatment, the p16(INK4a) transgenic mice showed enhanced thrombin generation in plasma-based calibrated automated thrombography assays. Finally, bone marrow transplantation studies suggested increased p16(INK4a) expression in hematopoietic cells contributes to thrombosis, demonstrating a role for p16(INK4a) expression in venous thrombosis. CONCLUSIONS: Venous thrombosis is augmented by overexpression of the cellular senescence protein p16(INK4a).

Longitudinal SARS-CoV-2 Testing among the Unvaccinated Is Punctuated by Intermittent Positivity and Variable Rates of Increasing Cycle Threshold Values.

The coronavirus disease 2019 (COVID-19) pandemic is complicated by cases of vaccine breakthrough and reinfection and widespread transmission of variants of concern (VOCs). Consequently, the need to interpret longitudinal positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) tests is crucial in guiding clinical decisions regarding infection control precautions and treatment. Although diagnostic real-time reverse transcription (RT)-PCR tests yield CT values that are inversely correlated with RNA quantity, these tests are only approved for qualitative interpretation. In this study, we performed a retrospective review of 72,217 SARS-CoV-2 positive tests and identified 264 patients with longitudinal positivity prior to vaccination and VOC circulation. Patients with longitudinal positivity fell into two categories: short-term (207, 78%) or prolonged (57, 22%) positivity, defined as ≤28 (range, 1 to 28; median, 16) days and >28 (range, 29 to 152; median, 41) days, respectively. In general, CT values increased over time in both groups; however, 11 short-term-positive patients had greater amounts of RNA detected at their terminal test than at the first positive test, and 6 patients had RNA detected at CT values of <35 at least 40 days after initial infection. Oscillating positive and negative results occurred in both groups, although oscillation was seen three times more frequently in prolonged-positive patients. Patients with prolonged positivity had diverse clinical characteristics but were often critically ill and were discharged to high-level care or deceased (22%). Overall, this study demonstrates that caution must be emphasized when interpreting CT values as a proxy for infectivity, a predictor of severity, or a guide for patient care decisions in the absence of additional clinical context, particularly among the unvaccinated population. IMPORTANCE We describe the duration of positivity and the COVID-19 treatment and outcome characteristics of an unvaccinated population of patients with prolonged SARS-CoV-2 positivity. This investigation serves to highlight challenges in using CT values to guide clinical decisions among unvaccinated individuals.

COVID-19 symptoms at time of testing and association with positivity among outpatients tested for SARS-CoV-2.

INTRODUCTION: Symptoms associated with SARS-CoV-2 infection remain incompletely understood, especially among ambulatory, non-hospitalized individuals. With host factors, symptoms predictive of SARS-CoV-2 could be used to guide testing and intervention strategies. METHODS: Between March 16 and September 3, 2020, we examined the characteristics and symptoms reported by individuals presenting to a large outpatient testing program in the Southeastern US for nasopharyngeal SARS-CoV-2 RNA RT-PCR testing. Using self-reported symptoms, demographic characteristics, and exposure and travel histories, we identified the variables associated with testing positive using modified Poisson regression. RESULTS: Among 20,177 tested individuals, the proportion positive was 9.4% (95% CI, 9.0-9.8) and was higher for men, younger individuals, and racial/ethnic minorities (all P<0.05); the positivity proportion was higher for Hispanics (26.9%; 95% CI. 24.9-29.0) compared to Blacks (8.6%; 95% CI, 7.6-9.7) or Whites (5.8%; 95% CI, 5.4-6.3). Individuals reporting contact with a COVID-19 case had the highest positivity proportion (22.8%; 95% CI, 21.5-24.1). Among the subset of 8,522 symptomatic adults who presented for testing after May 1, when complete symptom assessments were performed, SARS-CoV-2 RNA PCR was detected in 1,116 (13.1%). Of the reported symptoms, loss of taste or smell was most strongly associated with SARS-CoV-2 RNA detection with an adjusted risk ratio of 3.88 (95% CI, 3.46-4.35). The presence of chills, fever, cough, aches, headache, fatigue and nasal congestion also significantly increased the risk of detecting SARS-CoV-2 RNA, while diarrhea or nausea/vomiting, although not uncommon, were significantly more common in those with a negative test result. Symptom combinations were frequent with 67.9% experiencing ≥4 symptoms, including 19.8% with ≥8 symptoms; report of greater than three symptoms increased the risk of SARS-CoV-2 RNA detection. CONCLUSIONS: In a large outpatient population in the Southeastern US, several symptoms, most notably loss of taste or smell, and greater symptom burden were associated with detection of SARS-CoV-2 RNA. Persons of color and those with who were a contact of a COVID-19 case were also more likely to test positive. These findings suggest that, given limited SARS-CoV-2 testing capacity, symptom presentation and host characteristics can be used to guide testing and intervention prioritization.

The heparin-binding exosite of factor IXa is a critical regulator of plasma thrombin generation and venous thrombosis.

The role of the factor IXa heparin-binding exosite in coagulation was assessed with mutations that enhance (R170A) or reduce (R233A) stability of the protease-factor VIIIa A2 domain interaction. After tissue factor (TF) addition to reconstituted factor IX-deficient plasma, factor IX R170A supported a 2-fold increase in velocity index (slope) and peak thrombin concentration, whereas factor IX R233A had a 4- to 10-fold reduction relative to factor IX wild-type. In the absence of TF, 5 to 100 pM of factor IXa increased thrombin generation to approach TF-stimulated thrombin generation at 100% factor IX. Factor IXa R170A demonstrated a 2- to 3-fold increase in peak thrombin concentration and 5-fold increase in velocity index, whereas the response for factor IXa R233A was blunted and delayed relative to wild-type protease. In hemophilia B mice, factor IX replacement reduced the average time to hemostasis after saphenous vein incision, and the time to occlusion after FeCl(3)-induced saphenous vein injury. At 5% factor IX, the times to occlusion for factor IX wild-type, R170A, and R233A were 15.7 minutes, 9.1 minutes (P

Coagulation activation and inflammation in sickle cell disease-associated pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PHT) is common in sickle cell disease (SCD). The purpose of this study was to determine whether markers of coagulation activation and inflammation are associated with PHT in SCD. DESIGN AND METHODS: This cross-sectional study was performed using a cohort of patients followed at an adult Sickle Cell Clinic. Pulmonary artery systolic pressure was determined by Doppler echocardiography, and the diagnosis of PHT was defined using age, sex and body mass index-adjusted reference ranges. Clinical laboratory examinations, including hematologic studies and biochemical tests, as well as various measures of coagulation activation, endothelial activation and inflammation, were conducted on SCD subjects and on healthy, race-matched control subjects without SCD. RESULTS: Patients with SCD (n=76) had higher plasma levels of markers of coagulation (thrombin-antithrombin complex, prothrombin fragment F1+2, D-dimer) and endothelial (soluble vascular endothelial cell adhesion molecule, sVCAM) activation compared with control subjects (n=6). SCD patients with PHT (n=26) had significantly higher levels of sVCAM compared with those patients without PHT (n=50). Although PHT patients showed increased plasma measures of coagulation activation, the differences were not statistically significant when compared to those of patients without PHT. HbSS patients with PHT also had a trend towards higher levels of other inflammatory cytokines (interleukins 6, 8 and 10) than HbSS patients without PHT. There was a modest negative correlation between hemoglobin and plasma measures of coagulation and endothelial activation, and modest positive correlations between markers of hemolysis and plasma measures of coagulation and endothelial activation. CONCLUSIONS: SCD patients with PHT have higher levels of markers of endothelial activation and other inflammatory markers than patients without PHT. A trend towards an increased level of markers of coagulation activation was observed in SCD patients with PHT compared with that in patients without PHT. Markers of hemolysis are associated with coagulation activation and endothelial dysfunction in SCD patients. Clinical trials of anticoagulants and anti-inflammatory agents are warranted in SCD patients with PHT.

Overview of murine thrombosis models.

There are a myriad of options on where and how to perform thrombosis studies in mice. Models have been developed for systemic thrombosis, larger and smaller vessels of both the arterial and venous systems as well as several different microvascular beds. However, there are important differences between the models and investigators need to be careful and thoughtful when they choose which model to use.

RNA aptamer to thrombin binds anion-binding exosite-2 and alters protease inhibition by heparin-binding serpins.

We studied the RNA aptamer Toggle-25/thrombin interaction during inhibition by antithrombin (AT), heparin cofactor II (HCII) and protein C inhibitor (PCI). Thrombin inhibition was reduced 3-fold by Toggle-25 for AT and HCII, but it was slightly enhanced for PCI. In the presence of glycosaminoglycans, AT and PCI had significantly reduced thrombin inhibition with Toggle-25, but it was only reduced 3-fold for HCII. This suggested that the primary effect of aptamer binding was through the heparin-binding site of thrombin, anion-binding exosite-2 (exosite-2). We localized the Toggle-25 binding site to Arg 98, Glu 169, Lys 174, Asp 175, Arg 245, and Lys 248 of exosite-2. We conclude that a RNA aptamer to thrombin exosite-2 might provide an effective clinical reagent to control heparin's anticoagulant action.

Effect of multielement intravascular ultrasound on the anticoagulant potency of enoxaparin.

Two patients, treated with enoxaparin and eptifibatide, developed significant guide catheter-associated thrombus while undergoing intravascular ultrasound (IVUS). Using an ex vivo assay, we found that activation of a multielement IVUS catheter resulted in a decrease in anti-Xa activity and a decrease in clot formation time. This effect occurred rapidly and repeatedly after activation of the IVUS catheter.

Major burn injury is not associated with acute traumatic coagulopathy.

BACKGROUND: The pathophysiology and time course of coagulopathy after major burns are inadequately understood. Our study objectives were to determine whether acute traumatic coagulopathy (ATC) is seen in burn patients at admission and to determine the changes in international normalized ratio (INR), activated partial thromboplastin time (aPTT), platelet count (PLT), and hemoglobin (Hgb) in the first 7 days after injury. METHODS: We conducted a retrospective study of patients with burn injury of at least 15% total body surface area who presented to the University of North Carolina. Data on patient demographics, injury characteristics, and laboratory data (INR, aPTT, PLT, and Hgb) at admission and within the first 7 days after injury were recorded. We defined ATC as INR of 1.3 or greater, aPTT of 1.5 or greater times the mean normal limit, and normal PLT at admission. RESULTS: We studied the hematologic profile of 102 patients with burn injury of 15% to 100% total body surface area but did not identify a single patient with ATC at admission. The screening hematologic profile at admission was not influenced by burn severity. In the first 7 days after injury, the INR and aPTT were relatively preserved, while the PLT quickly recovered to baseline after an early decline and the Hgb remained stable at around 10 g/dL; all these changes occurred during the time when the burn patients had received large amounts of fluid resuscitation. CONCLUSION: The screening hematologic profile of burn patients at admission is normal, and the standard screening assays do not suggest the existence of ATC at admission. While this is a relatively small study, it provides evidence to suggest that ATC is unique to trauma patients. LEVEL OF EVIDENCE: Prognostic study, level III.

Substitution of a heparin correlation value for activated partial thromboplastin time in heparin nomograms.

PURPOSE. Use of nomograms based on the "heparin correlation value" (HCV)-a value that corresponds to measured activated partial thromboplastin time (aPTT) and that removes the need to revise nomograms in response to a change in the aPTT reagent or coagulometer used-was evaluated as an alternative to traditional aPTT-based anticoagulation nomograms. SUMMARY. Data were collected on patients receiving heparin therapy for selected indications (thrombotic disorders, cardiac conditions, and acute coronary syndromes) during four-month periods before (n = 59) and after (n = 60) implementation of the HCV-based nomograms. The primary endpoints were the rate at which coagulation laboratory measurements were obtained at the appropriate time and the rate of appropriate dosage adjustment in response to reported laboratory values; secondary endpoints included the time to attainment of the first target anticoagulation value. After implementation of HCV-based nomograms, coagulation laboratory measurements were obtained at the appropriate time in (mean ± S.D.) 92.9% ± 12.8% of patients, compared with 80.1% ± 15.5% of patients who received aPTT-based monitoring (p < 0.0001). After implementation of HCV-based monitoring, the rate of correct heparin dosage adjustments was improved (mean ± S.D. 94.7% ± 7.8% versus 89.3% ± 14.0%, p = 0.01), and the time to attainment of the first target anticoagulation value was shorter (mean ± S.D. 16.4 ± 10.6 hours versus 21.5 ± 14.8 hours, p = 0.03). CONCLUSION. The HCV, which relates measured aPTT values to corresponding antifactor Xa concentrations, was substituted for aPTT in heparin nomograms and appeared to be a viable alternative to the aPTT.

Serpins (serine protease inhibitors).

Serpins are a class of proteins involved in the regulation of serine and other types of proteases. In humans, the majority of serpins regulate the functions of proteases involved in the body's response to injury. This includes roles in coagulation, fibrinolysis, inflammation, wound healing, and tissue repair. Serpins have been implicated in various animal and human pathologies by the loss of a functional serpin gene through deletion or mutation, which results in a defect in functional protein. Examples of sestorically called antithrombin III) are first described. Then, protocols to determine the second-order rate constant of AT inhibition of thrombin in the absence and presence of heparin are presented. Also provided is a partial list of other serpins and their purification methods.

Molecular mapping of the thrombin-heparin cofactor II complex.

We used 55 Ala-scanned recombinant thrombin molecules to define residues important for inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) in the absence and presence of glycosaminoglycans. We verified the importance of numerous basic residues in anion-binding exosite-1 (exosite-1) and found 4 additional residues, Gln24, Lys65, His66, and Tyr71 (using the thrombin numbering system), that were resistant to HCII inhibition with and without glycosaminoglycans. Inhibition rate constants for these exosite-1 (Q24A, K65A, H66A, Y71A) thrombin mutants (0.02-0.38 x 10(8) m(-1) min(-1) for HCII-heparin when compared with 2.36 x 10(8) m(-1) min(-1) with wild-type thrombin and 0.03-0.53 x 10(8) m(-1) min(-1) for HCII-dermatan sulfate when compared with 5.23 x 10(8) m(-1) min(-1) with wild-type thrombin) confirmed that the structural integrity of thrombin exosite-1 is critical for optimal HCII-thrombin interactions in the presence of glycosaminoglycans. However, our results are also consistent for HCII-glycosaminoglycan-thrombin ternary complex formation. Ten residues surrounding the active site of thrombin were implicated in HCII interactions. Four mutants (Asp51, Lys52, Lys145/Thr147/Trp148, Asp234) showed normal increased rates of inhibition by HCII-glycosaminoglycans, whereas four mutants (Trp50, Glu202, Glu229, Arg233) remained resistant to inhibition by HCII with glycosaminoglycans. Using 11 exosite-2 thrombin mutants with 20 different mutated residues, we saw no major perturbations of HCII-glycosaminoglycan inhibition reactions. Collectively, our results support a "double bridge" mechanism for HCII inhibition of thrombin in the presence of glycosaminoglycans, which relies in part on ternary complex formation but is primarily dominated by an allosteric process involving contact of the "hirudin-like" domain of HCII with thrombin exosite-1.