Longitudinal Assessment of Cytokine Expression and Plasminogen Activation in Hantavirus Cardiopulmonary Syndrome Reveals Immune Regulatory Dysfunction in End-Stage Disease.

Pathogenic New World orthohantaviruses cause hantavirus cardiopulmonary syndrome (HCPS), a severe immunopathogenic disease in humans manifested by pulmonary edema and respiratory distress, with case fatality rates approaching 40%. High levels of inflammatory mediators are present in the lungs and systemic circulation of HCPS patients. Previous studies have provided insights into the pathophysiology of HCPS. However, the longitudinal correlations of innate and adaptive immune responses and disease outcomes remain unresolved. This study analyzed serial immune responses in 13 HCPS cases due to Sin Nombre orthohantavirus (SNV), with 11 severe cases requiring extracorporeal membrane oxygenation (ECMO) treatment and two mild cases. We measured viral load, levels of various cytokines, urokinase plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1). We found significantly elevated levels of proinflammatory cytokines and PAI-1 in five end-stage cases. There was no difference between the expression of active uPA in survivors' and decedents' cases. However, total uPA in decedents' cases was significantly higher compared to survivors'. In some end-stage cases, uPA was refractory to PAI-1 inhibition as measured by zymography, where uPA and PAI-1 were strongly correlated to lymphocyte counts and IFN-γ. We also found bacterial co-infection influencing the etiology and outcome of immune response in two cases. Unsupervised Principal Component Analysis and hierarchical cluster analyses resolved separate waves of correlated immune mediators expressed in one case patient due to a sequential co-infection of bacteria and SNV. Overall, a robust proinflammatory immune response, characterized by an imbalance in T helper 17 (Th17) and regulatory T-cells (Treg) subsets, was correlated with dysregulated inflammation and mortality. Our sample size is small; however, the core differences correlated to survivors and end-stage HCPS are instructive.

Low level of plasminogen increases risk for mortality in COVID-19 patients.

The pathophysiology of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and especially of its complications is still not fully understood. In fact, a very high number of patients with COVID-19 die because of thromboembolic causes. A role of plasminogen, as precursor of fibrinolysis, has been hypothesized. In this study, we aimed to investigate the association between plasminogen levels and COVID-19-related outcomes in a population of 55 infected Caucasian patients (mean age: 69.8 ± 14.3, 41.8% female). Low levels of plasminogen were significantly associated with inflammatory markers (CRP, PCT, and IL-6), markers of coagulation (D-dimer, INR, and APTT), and markers of organ dysfunctions (high fasting blood glucose and decrease in the glomerular filtration rate). A multidimensional analysis model, including the correlation of the expression of coagulation with inflammatory parameters, indicated that plasminogen tended to cluster together with IL-6, hence suggesting a common pathway of activation during disease's complication. Moreover, low levels of plasminogen strongly correlated with mortality in COVID-19 patients even after multiple adjustments for presence of confounding. These data suggest that plasminogen may play a pivotal role in controlling the complex mechanisms beyond the COVID-19 complications, and may be useful both as biomarker for prognosis and for therapeutic target against this extremely aggressive infection.

Assessing Plasmin Generation in Health and Disease.

Fibrinolysis is an important process in hemostasis responsible for dissolving the clot during wound healing. Plasmin is a central enzyme in this process via its capacity to cleave fibrin. The kinetics of plasmin generation (PG) and inhibition during fibrinolysis have been poorly understood until the recent development of assays to quantify these metrics. The assessment of plasmin kinetics allows for the identification of fibrinolytic dysfunction and better understanding of the relationships between abnormal fibrin dissolution and disease pathogenesis. Additionally, direct measurement of the inhibition of PG by antifibrinolytic medications, such as tranexamic acid, can be a useful tool to assess the risks and effectiveness of antifibrinolytic therapy in hemorrhagic diseases. This review provides an overview of available PG assays to directly measure the kinetics of plasmin formation and inhibition in human and mouse plasmas and focuses on their applications in defining the role of plasmin in diseases, including angioedema, hemophilia, rare bleeding disorders, COVID-19, or diet-induced obesity. Moreover, this review introduces the PG assay as a promising clinical and research method to monitor antifibrinolytic medications and screen for genetic or acquired fibrinolytic disorders.

Alterations of serum levels of plasminogen, TNF-α, and IDO in granulomatosis with polyangiitis patients.

BACKGROUND: Granulomatosis with polyangiitis (GPA) is a representative of vasculitides associated with anti-neutrophil cytoplasmic autoantibodies. "Classical" antibodies directed against proteinase 3 are involved in the pathogenesis and are part of the GPA diagnosis at the same time. Along with them, however, antibodies against Lysosomal-Associated Membrane Protein-2 (LAMP-2) and antibodies directed against plasminogen have been described in GPA.Objectives and methodology: We performed a cross-sectional study enrolling 34 patients diagnosed with GPA. Our study was aimed at looking for correlations between serum levels of LAMP-2 and plasminogen and the clinical manifestations of the GPA. Furthermore, we examined serum levels of tumor necrosis factor-alpha (TNF-α) and its associated indoleamine-pyrrole 2,3-dioxygenase (IDO), as well as we looked for a correlation between these cytokines and the clinical manifestations of GPA. RESULTS: The results showed that in GPA, serum plasminogen levels were negatively associated with renal involvement (receiver operating characteristic (ROC) area under the curve (AUC) of 0.78) (95% CI 0.53-0.91), p = 0.035, and the extent of proteinuria, Spearman's Rho = -0.4, p = 0.015. Increased levels of TNF-α and IDO correlated with disease activity, Spearman's Rho =0.62, p = 0.001 and Spearman's Rho = 0.4, p = 0.022, respectively, whereas only TNF-α was increased in severe forms of GPA with lung involvement (ROC AUC of 0.8) (95% CI 0.66-0.94), p = 0.005. CONCLUSIONS: In this study, we demonstrate the alteration of soluble factors, which play an important role in the pathogenesis of GPA and their relationship with the clinical manifestations of the disease. Our main results confirm the associations of increased secretory TNF-α and some clinical manifestations, and we describe for the first time decreased serum plasminogen levels and their association with renal involvement.

Reductions in plasmin inhibitor and fibrinogen predict the improved fibrin clot lysis 6 months after obesity surgery.

Prothrombotic and metabolic variables are decreased after obesity surgery, and fibrin clot lysis is increased. It is unknown how fibrinolytic variables are affected, and whether fibrinolytic and metabolic changes predict the enhanced clot lysis. Study aims were to determine fibrinolytic biomarkers before and 6 months after Roux-en-Y gastric bypass (RYGB) and to identify predictors of the RYGB-induced increase in clot lysis. Women (n = 42) and men (n = 18) with obesity underwent RYGB, and factor XIII (FXIII), thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen and plasmin inhibitor (PI) were measured before and 6 months after surgery. Regression analyses identified determinants of the RYGB-induced increase in clot lysis among changes in fibrinogen and in fibrinolytic and metabolic variables. Results showed that after RYGB, FXIII, TAFI, plasminogen and PI were reduced (P < .0005). Reductions in PI (ß = -0.59) and fibrinogen (ß = -0.35), together with age (ß = -0.22) and male sex (ß = 0.22), predicted the enhanced clot lysis with the model explaining 56% (P < .0005). Predictors of the reduction in PI were reductions in cholesterol (ß = 0.37) and glucose (ß = 0.29), together with male sex (ß = -0.28), whereas reductions in fibrinogen were predicted by lowering of interleukin-6 (IL-6) (ß = 0.32). In conclusion, fibrinolytic variables were reduced 6 months after RYGB. Targeting PI and fibrinogen, by reducing metabolic variables such as glucose, cholesterol and IL-6, has a profibrinolytic effect in obesity.

[Biomarkers of angiogenesis and endothelial dysfunction in children and adolescents with chronic viral hepatitis.]

At some works, it has been shown there are signs of damage and endothelium dysfunction in patients with chronic viral hepatitis (CVH) and liver cirrhosis of viral etiology the severity of these conditions depends on the severity of the pathological process. Evaluation of the role of angiogenic factors and endothelial dysfunction in persistent of CVH in children and adolescents. 35 patients were examined: of which 11 with chronic hepatitis B (CHB) and 24 with chronic hepatitis C (CHC). The reference group consisted of 120 practically healthy persons of the corresponding age and sex. VEGF-A, angiotensin (ANG), soluble receptors of VEGF-A (sVEGF-R1 и sVEGF-R2) and trombomodulin (TM) have been investigated in serum by enzyme immunoassay using special kits (BCM Diagnostics, USA). Other endothelial dysfunction markers as von Willebrand factor (vWf) was determined in blood plasma by immunoturbidimetry (Siemens, Germany), plasminogen (PLG) was investigated due to extended coagulation. In children with CVH, regardless of etiology, the concentration of VEGF-A was significantly lower, and sVEGF-R2, sVEGF-R1 and TM was higher than in children without liver disease (p <0.001, p <0.05, p <0.01, p <0.001, respectively). The concentration of TM and the level of PLG activity in patients with CHC were slightly higher than in CHB. Decreased level of VEGF-A and increased expression of its soluble receptors indicate enhanced inhibition of angiogenesis in CVH, which may indicate the pathogenetic role of this phenomenon in the development of liver damage in CHC.

Integrated Lipidomics and Proteomics Point to Early Blood-Based Changes in Childhood Preceding Later Development of Psychotic Experiences: Evidence From the Avon Longitudinal Study of Parents and Children.

BACKGROUND: The identification of early biomarkers of psychotic experiences (PEs) is of interest because early diagnosis and treatment of those at risk of future disorder is associated with improved outcomes. The current study investigated early lipidomic and coagulation pathway protein signatures of later PEs in subjects from the Avon Longitudinal Study of Parents and Children cohort. METHODS: Plasma of 115 children (12 years of age) who were first identified as experiencing PEs at 18 years of age (48 cases and 67 controls) were assessed through integrated and targeted lipidomics and semitargeted proteomics approaches. We assessed the lipids, lysophosphatidylcholines (n = 11) and phosphatidylcholines (n = 61), and the protein members of the coagulation pathway (n = 22) and integrated these data with complement pathway protein data already available on these subjects. RESULTS: Twelve phosphatidylcholines, four lysophosphatidylcholines, and the coagulation protein plasminogen were altered between the control and PEs groups after correction for multiple comparisons. Lipidomic and proteomic datasets were integrated into a multivariate network displaying a strong relationship between most lipids that were significantly associated with PEs and plasminogen. Finally, an unsupervised clustering approach identified four different clusters, with one of the clusters presenting the highest case-control ratio (p < .01) and associated with a higher concentration of smaller low-density lipoprotein cholesterol particles. CONCLUSIONS: Our findings indicate that the lipidome and proteome of subjects who report PEs at 18 years of age are already altered at 12 years of age, indicating that metabolic dysregulation may contribute to an early vulnerability to PEs and suggesting crosstalk between these lysophosphatidylcholines, phosphatidylcholines, and coagulation and complement proteins.

Serum plasminogen as a potential biomarker for the effects of low-dose benzene exposure.

Exposure to low-dose benzene may lead to hematotoxicity and cause health problems. Though peripheral blood cell count is widely used in benzene exposure assessment and health risk assessment, the reports regarding the effects of low-dose benzene exposure on blood cell count remain inconsistent. To uncover more sensitive biomarkers for low-dose benzene exposure, our previous study screened out three potential serum proteins-plasminogen (PLG), platelet basic protein (PBP) and apolipoprotein B100 (ApoB100)-as biomarkers from chronic benzene poisoning patients by using proteomic analysis. In the present study, we verify the three serum proteins as biomarkers for the effects of low-dose benzene exposure in a large low-dose benzene exposure population. The study showed that serum PLG increased in benzene exposed workers and was positively correlated with benzene exposure levels. However, no significant changes in serum PBP or ApoB100 were found in the benzene exposed workers. To explore whether the candidate serum proteins are associated with hematotoxicity, the study population was regrouped into two groups, based on their WBC counts. Our results showed that the workers with high serum PLG levels suffered higher risk of WBC abnormalities than did workers with low serum PLG levels. Taken together, these findings indicate that the increase in serum PLG might be associated with low-dose benzene exposure and benzene-induced hematotoxicity. Thus, we suggest serum PLG could be used as a potential biomarker for the effects of low-dose benzene exposure.

Value of anti-plasminogen binding peptide, anti-carbonic anhydrase II, immunoglobulin G4, and other serological markers for the differentiation of autoimmune pancreatitis and pancreatic cancer.

The diagnosis of autoimmune pancreatitis (AIP) and its differential diagnosis from pancreatic cancer (PC) can be challenging. In this retrospective study, we aimed to evaluate the value of anti-plasminogen binding peptide (a-PBP), immunoglobulin G4 (IgG4), and anti-carbonic anhydrase-II (a-CA-II), together with other serological markers whose value is not fully elucidated.The serum levels of a-PBP, IgG4, IgG, anti-nuclear antibodies (ANA), anti-lactoferrin (a-LF), a-CA-II, and rheumatoid factor (RF) were evaluated in patients with AIP (n = 29), PC (n = 17), pancreatic neuroendocrine neoplasm (P-NEN, n = 12), and alcoholic chronic pancreatitis (ACP, n = 41). ANCA were measured in the AIP patients.There was no statistically significant difference in mean a-PBP values in AIP compared with PC. A ROC curve showed that, when using a cut-off of 38.3 U, low values of a-PBP had a sensitivity and specificity of 45% and 71% for differentiating AIP from PC. The sensitivity and specificity of IgG4 (cut-off 1.4 g/L) for differentiating AIP from PC was 45% and 88%, but rose to 52% and 88% when using a cut-off of 1.09 g/L. When using this cut-off, the sensitivity and specificity for differentiating type 1 AIP from PC was 68% and 88%. None of the other markers were significantly changed in AIP versus PC. For differentiation of type 1 and type 2 AIP, the only significant differences were IgG4 in type 1 AIP (P < .01), with a sensitivity of 68% and a specificity of 80%, and c-ANCA elevations found in some type 2 AIP patients (P < .05).The only serological marker for which we found a statistically significant difference in mean values between AIP and PC was IgG4. However, the value of IgG4 for the distinction of AIP from PC was limited, probably in part due to the relatively high number of type 2 AIP patients in our study. In accord with recent publications, our data do not support a role of increased serum a-PBP for the diagnosis of AIP.

Tissue factor levels and the fibrinolytic system in thin and thick intraluminal thrombus and underlying walls of abdominal aortic aneurysms.

BACKGROUND: The hemostatic system cooperates with proteolytic degradation in processes allowing abdominal aortic aneurysm (AAA) formation. In previous studies, it has been suggested that aneurysm rupture depends on intraluminal thrombus (ILT) thickness, which varies across each individual aneurysm. We hypothesized that hemostatic components differentially accumulate in AAA tissue in relation to ILT thickness. Thick (A1) and thin (B1) segments of ILTs and aneurysm wall sections A (adjacent to A1) and B (adjacent to B1) from one aneurysm sac were taken from 35 patients undergoing elective repair. METHODS: Factor levels were measured using enzyme-linked immunosorbent assay of protein extract. RESULTS: Tissue factor (TF) activities were significantly higher in thinner segments of AAA (B1 vs A1, P = .003; B vs A, P < .001; B vs A1, P < .001; B vs B1, P = .001). Significantly higher tissue plasminogen activator was found in thick thrombus-covered wall segments (A) than in B, A1, and B1 (P = .015, P < .001, and P < .001, respectively). Plasminogen concentrations were highest in ILT. Concentrations of α2-antiplasmin in thin ILT adjacent walls (B) were higher compared with wall (A) adjacent to thick ILT (P = .021) and thick ILT (A1; P < .001). Significant correlations between levels of different factors were mostly found in thick ILT (A1). However, no correlations were found at B sites, except for a correlation between plasmin and TF activities (r = 0.55; P = .004). CONCLUSIONS: These results suggest that higher TF activities are present in thinner AAA regions. These parameters and local fibrinolysis may be part of the processes leading to destruction of the aneurysm wall.

Plasminogenemia asociada a conjuntivitis leñosa / Plasminogen Deficiency and Ligneous Conjunctivitis

El artículo presenta el caso clínico de un joven de 18 años de edad sin antecedente familiares de enfermedad crónica o hereditaria, nacido a término, sin complicaciones, quien desde los dos días de vida presentó una lesión tipo masa en la conjuntiva tarsal. Inicialmente, recibió tratamiento farmacológico tópico y seguimiento por oftalmología, por conjuntivitis bilateral persistente, masas tarsales recidivantes y cataratas en ambos ojos, por lo que requirió siete intervenciones con pobre respuesta al manejo farmacológico y quirúrgico. A los seis meses de vida se le diagnosticó hidrocefalia, que requirió manejo con derivación del ventrículo peritoneal. Dada la persistencia de la sintomatología y la refractariedad al tratamiento, se ampliaron los estudios y en una junta médica se sugirió el diagnóstico de conjuntivitis leñosa asociada a alteración del plasminógeno. Este diagnóstico fue confirmado por laboratorio clínico, que mostró sus bajas concentraciones de plasminógeno en muestras tomadas con intervalos de dos meses en tres ocasiones: 16,9%, 11,1%, 18,6% (valores de referencia: 70-150%). Se le indicó heparina de bajo peso molecular antes de procedimientos quirúrgicos mayores y triamcinolona tópica según síntomas oculares.

We present a case of an 18-year-old patient without a family history of ocular disease, born full term without complications, within his first 2 days a mass in the tarsal conjunctiva appeared. Initially he received topical treatment and follow-up by the ophthalmology department with a diagnosis of persistent bilateral conjunctivitis, relapsing tarsal masses and cataracts in both eyes requiring a total of 7 surgical interventions with a poor response. At the age of 6 months he was diagnosed with hydrocephalus and required a ventricular-peritoneal shunt. Giren the persistence of the symptoms, further studies were made and a medical board made the diagnosis of ligneous conjunctivitis associated to low levels of plasminogen. The diagnosis was confirmed by decreased levels of plasminogen in serum measured three times with 2 months intervals: 16.9%, 11.1%, 18.6% (reference valúes 70'150%). Low molecular weight heparin was ordered before surgical procedures, and topical triamcinolone applied according to ocular symptoms.

A sustained decrease in plasma fibrinolytic potential following partial liver resection or pancreas resection.

BACKGROUND: Patients undergoing partial hepatectomy have a substantial risk for postoperative venous thrombosis even in the presence of optimal thromboprophylaxis. Recently we demonstrated a hypercoagulable state following a partial hepatectomy which was related to decreased plasma levels of natural anticoagulants and elevated levels of FVIII. The fibrinolytic status following partial hepatectomy has not been studied, but may display unique features as a result of temporarily decreased synthesis of fibrinolytic proteins. METHODS: We included 17 patients undergoing a partial hepatectomy and determined plasma fibrinolytic potential and measured plasma levels of individual fibrinolytic proteins in serial samples taken perioperatively. Results were compared to ten patients undergoing pancreas resection and twenty-four healthy volunteers. RESULTS AND CONCLUSION: Following both partial hepatectomy and pancreas resection plasma fibrinolytic potential decreased at the end of surgery, normalized on post-operative day 1, and decreased again on post-operative day 3 after which the hypofibrinolytic state gradually resolved. The hypofibrinolytic state on day 1 associated with increased plasma levels of PAI-1 in both groups. Plasma levels of plasminogen, α2-antiplasmin and TAFI all decreased following partial hepatectomy and pancreas resection and levels recovered over time. The kinetics of recovery were different for the different proteins and were slower in the liver resection group, resulting in a unique ratio of pro-to-anti-fibrinolytic proteins at each time point. This may explain the hypofibrinolytic status from day 3 onwards. A sustained plasma hypofibrinolytic state in combination with the hypercoagulable state we previously identified may contribute to the increased risk of thrombotic complications after partial liver resection.

Pathogenic potential of Tannerella forsythia enolase.

Although enolases are cytosolic enzymes involved in the glycolytic pathway, they can also be secreted or expressed on the surface of a variety of eukaryotic cells and bacteria. Surface-exposed enolases of eukaryotes and bacteria can function as plasminogen receptors. Furthermore, antibodies raised against bacterial enolases can react with host enolases, suggesting molecular mimicry between bacterial and host enzymes. In this study, we analyzed an enolase of the major periodontopathogen Tannerella forsythia, which is either secreted or present on the cell surface, via matrix-assisted laser desorption ionization time-of-flight mass spectrometry and immunofluorescence, respectively. The T. forsythia enolase retained the enzymatic activity converting 2-phosphoglycerate to phosphoenolpyruvate and showed plasminogen binding and activating ability, which resulted in the degradation of fibronectin secreted from human gingival fibroblasts. In addition, it induced proinflammatory cytokine production, including interleukin-1ß (IL-1ß), IL-6, IL-8, and tumour necrosis factor-α (TNF-a) in the human THP-1 monocytic cell line. Taken together, our results demonstrate that T. forsythia enolase plays a role in pathogenesis in the host by plasminogen activation and proinflammatory cytokine induction, which has the potential to exaggerate inflammation in periodontitis.

Role of Endogenous Factors in Response of Erythrocyte Membrane in Patients with Cardiovascular Diseases under Conditions of Ischemic Exposure.

We studied specific features of erythrocyte membrane response to short-term occlusion of the brachial artery in patients with cardiovascular pathology. Under ischemic conditions, processes of sorption were primarily intensified in patients with effort angina and processes of hemoglobin binding with erythrocyte membrane predominated in patients with essential hypertension. These changes in the cell membrane were related to modulation of aggregation properties of erythrocytes (in patients with angina) and plasminogen activity (in patients with essential hypertension). They can also be associated with changes in glucose levels (effort angina) and uric acid (essential hypertension) whose effects can be significantly modified by other endogenous factors.

A controlled, cross-sectional exploratory study on markers for the plasminogen system and inflammation in crevicular fluid samples from healthy, mucositis and peri-implantitis sites.

PURPOSE: To investigate expression of gene markers for the plasminogen system, inflammation, and bone resorption/remodelling in peri-implant crevicular fluid samples from healthy subjects, subjects with mucositis and subjects with peri-implantitis. A possible inhibitory effect of suppuration on the analysis of gene expression in samples from subjects with peri-implantitis was also analysed. MATERIALS AND METHODS: Peri-implant crevicular fluid (PICF) was sampled from 25 healthy subjects (H), 25 subjects with mucositis (M) and 25 subjects with peri-implantitis (P) using paper points and suction tips. The samples were analysed by quantitative polymerase chain reaction (qPCR). The following biomarkers associated with the plasminogen system, inflammation and bone resorption/ remodelling were investigated: interleukin-1 beta (IL-1ß), interleukin 8 (IL-8), tissue plasminogen activator (tPA), plasminogen activator inhibitor 2 (PAI-2), tartrate-resistant acid phosphatase (TRAP) and cathepsin K (CatK). RESULTS: IL-1ß and IL-8 were significantly upregulated in the P group, and tPA and PAI-2 were significantly upregulated in the M group. These four genetic markers were oppositely regulated in samples from the subjects in the mucositis compared with the peri-implantitis group. TRAP and CatK showed no differences between the groups. The presence of suppuration did not have a detectable effect on gene analysis in samples from subjects with peri-implantitis. CONCLUSIONS: Markers for the plasminogen system and inflammation could be used to distinguish between mucositis and peri-implantitis. The results suggested that the plasminogen system was sufficiently upregulated allowing for resolution of inflammation and healing at the inflamed implant site in subjects with mucositis, whereas such upregulation was insufficient resulting in impaired healing and prolonged inflammation in subjects with peri-implantitis. The combination of tissue inflammation and low levels of tPA was a strong predictor of marginal bone loss in this study. It may be an interesting candidate for the unambiguous diagnosis of mucositis and peri-implantitis independent of radiographs and could possibly constitute a powerful future tool for rapid assessment of the periimplant tissue condition and the effect of subject treatment.

Effects of storage and thawing conditions on coagulation testing.

INTRODUCTION: Current recommendations for coagulation testing storage and thawing are based on historical studies that were performed using unbuffered 3.8% sodium citrate. We sought to measure the effects of freezing and thawing conditions 3.2% buffered sodium citrate plasma samples that have been stored in vials with either snap or sealed screw tops, frozen in -70 °C freezer or dry ice and thawed either capped or uncapped. METHODS: Shed blood samples were pooled and then aliquoted into four snap top and four screw tops vials. Half the vials were stored in a -70 °C freezer, and half on dry ice for at least 16 h. Afterwards, half the frozen samples were thawed in 37 °C waterbath capped, and other half were thawed capped. After thawing cycles, samples were tested for PT, activated partial thromboplastin time (APTT), fibrinogen, D-dimer, factor assays, von Willebrand factor activity, plasminogen, antithrombin, protein C and lupus anticoagulant. RESULTS: Prothrombin time, APTT, factor X, and lupus anticoagulant testing were affected by all vials, freezing and thawing conditions, whereas fibrinogen, D-dimer, von Willebrand activity or protein C were not affected by any vial, freezing or storage condition. CONCLUSIONS: Storage vials, freezing and thawing condition affect coagulation testing, although these differences may not be clinically significant.

Cautious enthusiasm about GWAS findings.

In this issue of Blood, Ma and colleagues report the first agnostic investigation by a genome-wide association study (GWAS) in search of genetic determinants of a variation in plasma plasminogen levels.

Genetic variants in PLG, LPA, and SIGLEC 14 as well as smoking contribute to plasma plasminogen levels.

Plasminogen is the precursor of the serine protease plasmin, a central enzyme of the fibrinolytic system. Plasma levels of plasminogen vary by almost 2-fold among healthy individuals, yet little is known about its heritability or genetic determinants in the general population. In order to identify genetic factors affecting the natural variation of plasminogen levels, we performed a genome-wide association study and linkage analysis in a sample of 3456 young healthy individuals who participated in the Genes and Blood Clotting Study (GABC) or the Trinity Student Study (TSS). Heritability of plasminogen levels was 48.1% to 60.0%. Tobacco smoking and female sex were associated with higher levels of plasminogen. In the meta-analysis, 11 single-nucleotide polymorphisms (SNPs) in 2 regions reached genome-wide significance (P < 5.0E-8). Of these, 9 SNPs were near the PLG or LPA genes on Chr6q26, whereas 2 were on Chr19q13 and 5' upstream of SIGLEC14. These 11 SNPs represented 4 independent signals and collectively explained 6.8% of plasminogen level variation in the study populations. The strongest association was observed for a nonsynonymous SNP in the PLG gene (R523W). Individuals bearing an additional copy of this allele had an average decrease of 13.4% in plasma plasminogen level.