α2M Levels Research Articles

High alpha-2-macroglobulin levels are a risk factor for cardiovascular disease events: A Moli-sani cohort study

Backgroundα2-macroglobulin (α2M) is a versatile endopeptidase inhibitor that plays a role in cell growth, inflammation and coagulation. α2M is an inhibitor of key coagulation enzyme thrombin. Hypercoagulability due to an excess of thrombin production can cause thrombotic events. Therefore, we investigated the association of α2M levels and cardiovascular events in a subset of the general Italian population. MethodsWe determined α2M levels in the baseline samples of a prospective cohort (n = 19,688; age: 55 ± 12 years; 47.8 % men) of the Moli-sani study and investigated the association with the cardiovascular events (n = 432, 2.2 %) in the median follow-up period of 4.3 years. Hazard ratios (HR) with 95 % confidence intervals (CI) were calculated by multivariable Cox regression and adjusted for a large panel of confounding factors. Resultsα2M levels above the 90th percentile were significantly associated with cardiovascular disease (CVD) events after full adjustment for age, sex, current smoking, BMI, oral contraceptive use, cardiovascular diseases, hypertension, hypercholesterolemia, diabetes and history of cancer (HR: 1.36; CI: 1.06–1.74). Moreover, high α2M was associated with coronary heart disease (CHD; HR: 1.47; CI: 1.12–1.91), but not stroke. Stratification for CVD at baseline showed that high α2M levels are associated with CHD events in subjects without CVD at baseline (HR: 1.40; CI: 1.00–1.95) and subjects with CVD at baseline (HR: 1.58; CI: 1.02–2.44). ConclusionWe show in a prospective cohort that high levels of α2M could be a risk factor for cardiovascular events, especially coronary heart disease events.

Effect of Short PSG Peptides on Inflammatory Markers in Allogeneic Bone Marrow Cell Transplantation in Wistar Rats.

Short linear peptide fragments of placental trophoblastic β1-glycoprotein (PSG) (YECE, YQCE, YVCS, and YACS) were studied in the context of their immunomodulatory effects at the level of inflammatory markers. The original host-versus-graft model was used in male Wistar rats without prior conditioning of recipient bone marrow. A composition of PSG peptide fragments was injected to animals after allogeneic transplantation of bone marrow cells in a dynamic experiment, inflammatory markers α1-acid glycoprotein (AGP, orosomucoid), α2-macroglobulin (α2M) were assayed by ELISA, and biochemical parameters (total protein, glucose, creatinine, and urea) were measured. The levels of α2M and AGP increased in response to allotransplantation, whereas administration of PSG peptides normalized serum α2M levels by the end of the experiment. The decrease in α2M level coincided with the independent effect of PSG peptide administration. The levels of total protein, glucose, creatinine, and urea in rat serum after allotransplantation were reduced throughout the experiment. Administration of PSG peptides contributed to normalization of serum total protein, creatinine, and urea levels by the end of the experiment. Administration of PSG peptides after allogeneic transplantation of bone marrow suspension contributed to normalization of the levels of α2M, total protein, creatinine, and urea, which can be interpreted as an anti-inflammatory effect of these peptides.

Proinflammatory phenotype in major depressive disorder with adulthood adversity: In line with social signal transduction theory of depression

BackgroundThe social signal transduction theory of depression proposes that life stress can be transformed into inflammatory signals, and ultimately lead to the development of major depressive disorder (MDD). The hypotheses of this study were: (1) The pro-inflammatory effect of life stress was only seen in patients with MDD, but not in healthy controls (HCs); (2) Inflammation can mediate the relationship between life stress and depressive symptoms. MethodsThis study included 170 MDD patients and 196 HCs, and 13 immune-inflammatory biomarkers closely related to MDD were measured, principal component analysis (PCA) was adopted to extract the inflammatory index. Life stress was assessed by Life Event Scale (LES), a total score of >32 points on the LES was considered as adulthood adversity (AA). Path analyses were used to explore the relationship among adulthood stress, inflammatory index, and severity of depression. ResultsAmong MDD patients, α2M, CXCL-1, IL-1β, and TLR-1 levels were higher in patients with AA than non-AA group (all FDR-adjusted P values <0.05), meanwhile, the levels of CCL-2 and IL-18 were lower. Path analyses suggested that pro- and anti-inflammatory index could mediate the association between AA and severity of depression in MDD patients. ConclusionThis study found that inflammatory signals can mediate the relationship between adulthood adversity and depression, however, the causal relationship need to be further confirmed. These findings shed light on further understanding the theory of social signal transduction in MDD and provide clues for stress management and controlling inflammation strategies in depression.Clinical trials: NCT02023567

Semi-automated thrombin dynamics applying the ST Genesia thrombin generation assay.

BackgroundThe haemostatic balance is an equilibrium of pro- and anticoagulant factors that work synergistically to prevent bleeding and thrombosis. As thrombin is the central enzyme in the coagulation pathway, it is desirable to measure thrombin generation (TG) in order to detect possible bleeding or thrombotic phenotypes, as well as to investigate the capacity of drugs affecting the formation of thrombin. By investigating the underlying processes of TG (i.e., prothrombin conversion and inactivation), additional information is collected about the dynamics of thrombin formation.ObjectivesTo obtain reference values for thrombin dynamics (TD) analysis in 112 healthy donors using an automated system for TG.MethodsTG was measured on the ST Genesia, fibrinogen on the Start, anti-thrombin (AT) on the STA R Max and α2Macroglobulin (α2M) with an in-house chromogenic assay.ResultsTG was measured using STG-BleedScreen, STG-ThromboScreen and STG-DrugScreen. The TG data was used as an input for TD analysis, in combination with plasma levels of AT, α2M and fibrinogen that were 113% (108–118%), 2.6 μM (2.2 μM−3.1 μM) and 2.9 g/L (2.6–3.2 g/L), respectively. The maximum rate of the prothrombinase complex (PCmax) and the total amount of prothrombin converted (PCtot) increased with increasing tissue factor (TF) concentration. PCtot increased from 902 to 988 nM, whereas PCmax increased from 172 to 508 nM/min. Thrombin (T)-AT and T-α2M complexes also increased with increasing TF concentration (i.e., from 860 to 955 nM and from 28 to 33 nm, respectively). PCtot, T-AT and T-α2M complex formation were strongly inhibited by addition of thrombomodulin (−44%, −43%, and −48%, respectively), whereas PCmax was affected less (−24%). PCtot, PCmax, T-AT, and T-α2M were higher in women using oral contraceptives (OC) compared to men/women without OC, and inhibition by thrombomodulin was also significantly less in women on OC (p < 0.05).ConclusionsTG measured on the ST Genesia can be used as an input for TD analysis. The data obtained can be used as reference values for future clinical studies as the balance between prothrombin conversion and thrombin inactivation has shown to be useful in several clinical settings.

Major depressive disorder comorbid with general anxiety disorder: Associations among neuroticism, adult stress, and the inflammatory index

Accumulating evidence shows that higher neuroticism and adult stress may be potential risk factors for major depressive disorder (MDD) comorbid with generalized anxiety disorder (GAD). Studies have shown that anxious and depressed patients have significantly more neurobiological abnormalities than non-anxious depressed patients. However, the biological mechanism of comorbidity remains unknown. A study of serum markers allows a better understanding of the mechanism. This was a multi-centre, cross-sectional study. A total of 169 MDD patients (42 MDD patients with comorbid GAD and 127 MDD patients without comorbid GAD) were studied to analyse the risk factors for MDD with comorbid GAD. Twenty-four peripheral serum markers were measured. Path analysis was applied to test the association among neuroticism, adult stress, inflammatory markers, and psychopathology. After Bonferroni correction, MDD patients with comorbid GAD had lower levels of CCL2 (P = 0.001) and higher levels of α2M (P < 0.001) and TLR-1 (P = 0.001) than MDD patients without comorbid GAD (adjusted P < 0.002). In the path analyses of the association among adult stress, the inflammatory index, and psychopathology, neuroticism had a direct effect (β = 0.238, P = 0.003) and an indirect effect (β = 0.068, P = 0.004) on MDD and GAD comorbidity through adult stress and the inflammatory index. Our results suggest that MDD with comorbid GAD is associated with higher levels of inflammatory markers, stress factors and personality traits, which may provide some cues for early identification or more tailored and comprehensive treatment for MDD with comorbid GAD.

Systemic biomarkers for the preclinical diagnosis of dementia

Abstract Background As a consequence of aging and the age-related cardiovascular risk, both of them closely associated to neurodegenerative diseases, dementia has become one of the major public health concerns for governments and the scientific and medical communities. Up to date, there isn't any front-line screening tool implemented at a population level. In this regard, we aim to assess the association of several systemic biomarkers related to neuroinflammation [lactate dehydrogenase (LDH), complement component 3, C-reactive protein, alpha-1-antitrypsin, alpha-2-macroglobulin (α2M) and pancreatic prohormone], lipid metabolism [apolipoprotein E, 24S-hydroxycholesterol], and neuronal integrity [tau protein], with dementia risk in an adult-elderly population at high cardiovascular risk. Methods We designed a case-cohort study nested in the PREDIMED cohort (110 incident cases of dementia, 251 non-cases). Participants were at high risk for cardiovascular disease (CVD), but had no clinical CVD neither dementia at the time of enrolment. Inclusion criteria were an age of 55-80 years old and the presence of diabetes type 2 or at least 3 cardiovascular risk factors (smoking, overweight or obesity, hypertension, dyslipidemia, and family history of premature CVD). We used Cox proportional hazard models to estimate hazard ratios (HR) of the association between these nine plasma biomarkers and dementia risk adjusted for several covariates. Results Dementia cases were older, presented higher levels of fasting glucose and were more likely to have type 2 diabetes. The prevalence of dyslipidemia and smoking in patients with dementia was lower. Dementia cases had higher levels of LDH and α2M. Higher LDH levels were associated with a higher risk of developing dementia after adjustment (HR = 1.19). Conclusions Higher LDH plasma levels are associated with a higher risk of dementia incidence in population at high cardiovascular risk. Key messages Higher levels of LDH are associated with higher risk of dementia incidence among adult-elderly individuals with a high cardiovascular risk profile. The early detection of this biomarker could enable an early intervention and to delay, or perhaps even prevent, the onset of clinical symptoms.

Survival, retention rate and immunity of the black shell colored stocks of pearl oyster Pinctada fucata martensii after grafting operation

We have developed a black shell colored selected line observed to have higher survival ability. In this study, to understand its immune capacity, total carotenoid content (TCC) of the black shell colored line (BG) and the control group (CG) were compared. Survival and retention rates, immunity and antioxidant capacity of BG were compared relative to CG at different times after grafting operation. The results showed that BG had significantly larger TCC than CG (P < 0.05). BG had significantly higher survival and retention rates than CG on days 7, 30 and 360 after grafting (P < 0.05). On days 360, BG had significantly larger pearl thickness than CG (P < 0.05). BG exhibited increased ACP, AKP, SOD, CAT, TAOC and LZ activity than the CG on 0 h, 12 h, 1 d, 3 d, 5 d, 7 d and 30 d after grafting. BG had higher expression levels of Fascin, SOD, CDK-7, CDAP-1, IRAK-1, α2m, GST-1, TRAF-3 and Caspase-2 than CG. The results suggested that BG had higher immune competence and pearl production performances, which is promising to improve pearl quality and production.

Ratio of Alpha 2-Macroglobulin Levels in Cerebrospinal Fluid and Serum: An Expression of Neuroinflammation in Acute Disseminated Encephalomyelitis

BackgroundAcute encephalitis and encephalopathy are life-threatening diseases in children. However, no laboratory examinations are performed for their early diagnosis and treatment. Alpha 2-macroglobulin (α2M) is a blood glycoprotein that increases during the early stages of inflammation. In the present study, we investigated the role of α2M levels in acute encephalitis and encephalopathy. MethodsWe analyzed the cerebrospinal fluid and serum samples from patients with acute disseminated encephalomyelitis, infection-related acute encephalopathy, febrile status epilepticus, and febrile seizure simplex type. Samples were collected from the pediatric department of hospitals throughout the Fukushima Prefecture between January 1, 1999, and May 31, 2012. Resultsα2M levels in the cerebrospinal fluid were 4.7 (3.8–8.4) μg/mL for acute disseminated encephalomyelitis, 2.1 (1.1–2.3) μg/mL for infection-related acute encephalopathy, 1.1 (0.9–6.4) μg/mL for febrile status epilepticus, and 1.0 (0.8–1.1) μg/mL for febrile seizure simplex type. α2M levels in patients with acute disseminated encephalomyelitis were significantly higher than those in patients with infection-related acute encephalopathy and febrile seizure simplex type (P = 0.019 and P = 0.002, respectively). The ratio of α2M level in the cerebrospinal fluid to that in the serum in patients with acute disseminated encephalomyelitis was significantly higher than the ratio in patients with febrile status epilepticus (P = 0.04). In patients with acute disseminated encephalomyelitis, α2M levels in the cerebrospinal fluid decreased with treatment. ConclusionsOur results suggest that α2M levels in the cerebrospinal fluid reflect the neuroinflammatory status of patients with acute disseminated encephalomyelitis.

Deciphering the binding of dutasteride with human alpha-2-macroglobulin: Molecular docking and calorimetric approach

Dutasteride is a pharmacologically important drug employed to treat prostate cancer. Alpha-2-macroglobulin (α2M) is the primary proteinase inhibitor and is abundant in vertebrate plasma. Previous studies have shown that α2M levels were down regulated in prostate cancer. Our results of functional assay shows 50% decrease in the antiproteolytic potential ofα2Mupon its interaction with dutasteride. Fluorescence quenching revealed that dutasteride binds with α2M via static mechanism, resulting in the formation of dutasteride-α2M complex. Synchronous fluorescence studies suggest alteration in the microenvironment around tryptophan residues. Changes in the UV–visible spectra hints at formation of complex between the drug and protein. Secondary structural perturbations in α2M are confirmed by circular dichroism studies. Molecular docking discloses the involvement of hydrogen bonding during the interaction process and suggests the site of interaction of dutasteride on α2M monomer as Asn173, Lys171, Asp1178, Lys1236, His1182, Lys1177, Ser1180 and Lys1240.Isothermal titration calorimetry affirms the binding process to be spontaneous and exothermic. The results of this study may potentially be important should it be shown that dutasteride interacts with α2M under physiological conditions.

Membrane-bound Glucose regulated protein 78 interacts with alpha-2-macroglobulin to promote actin reorganization in sperm during epididymal maturation.

Glucose regulated protein 78 (GRP78) is expressed on cell surface in exceptional conditions as seen in cancer cells and macrophages. We have reported its membrane localization in sperm. The functional significance of its surface localization in sperm is an enigma. Alpha-2-macroglobulin (α2M) reportedly binds surface GRP78, regulating macrophage motility. Additionally, seminal plasma α2M levels are low in cases of asthenozoospermia. We investigated the functional relevance of sperm surface GRP78 and α2M crosstalk using testicular sperm (Tsp; immature) and caudal sperm (Cdsp; mature) from adult Holtzman rats. α2M colocalized and interacted with GRP78 and was significantly higher in Cdsp. Cofilin pathway proteins were detected in Tsp and Cdsp, however the pathway was highly active in Cdsp. Tsp surface GRP78 tyrosine phosphorylation and [Ca2+]i levels increased significantly on exposure to activated α2M (α2M*). This binding activated Rac/Cdc42, and consequently PAK, leading to LIMK and cofilin phosphorylation and thus promoting actin reorganization. Cofilin translocation from the sperm tail to the head in the presence of α2M* possibly prevented F-actin depolymerization in the tail. Thus, profiles observed with Cdsp could be re-created upon exposure of Tsp with α2M*. We conclude that α2M secreted into seminiferous tubule fluid by Sertoli cells, may be activated by proteinases in the epididymis and may bind to sperm surface GRP78 during epididymal transit, thereby facilitating sperm motility via actin reorganization. As F-actin is required for maintaining structural integrity and hyperactivated motility in sperm, our finding has significant implications in light of our previous reports of reduced GRP78 phosphorylation and the actin-based motility pathway being significantly altered in asthenozoospermia.

Influence of electroacupuncture therapy of tonifying the kidney and regulating governor vessel on Aβ related degradation enzymes in the hippocampus of a rat model of Alzheimer's disease induced by Aβ1-42

ObjectiveTo explore influence of electroacupuncture (EA) therapy of tonifying the kidney and regulating governor vessel on amyloid beta (Aβ) related degradation enzymes in the hippocampus of a rat model of Alzheimer's disease (AD) induced by Aβ1-42. MethodsForty Wistar male rats were randomly divided into 4 groups: a normal group, a sham operation group, a model group and an EA group, 10 rats in each one. The rats in normal group were normally fed. The rats in sham operation group were bilaterally injected in the hippocampus with 5 µL of saline and they were normally fed after the injection. The rats in the model group and the EA group were bilaterally injected in the hippocampus with 5 µL of Aβ1-42 on each side. Rats in the EA group received EA of 5 Hz continuous wave at the “Băihuì (百会 GV20)” and bilateral “Shènshū (肾俞 BL23)” for a duration of 15 min per time every day and continuously for 15 days. After 15 days, the hippocampal expression levels of insulin degrading enzyme (IDE), lipoprotein (LPL), transthyretin (TTR), apolipoprotein E (APoE), α2 macroglobulin (α2M) and Aβ1-42 of the 4 groups were tested by Western blot. ResultsCompared with the sham operation group, the expression levels of IDE, LPL, TTR, APoE and α2M in the hippocampus were significantly lower (P < 0.05, P < 0.01) and the expression of Aβ1-42 was significantly higher(P < 0.01) in the model group. Compared with the model group, the expression levels of IDE, LPL, TTR, APoE and α2M in the hippocampus of these rats were significantly lower (P < 0.05, P < 0.01), the expression of Aβ1-42 was significantly higher(P < 0.01) in the EA group. ConclusionEA therapy of tonifying the kidney and regulating governor vessel can enhance the expression of IDE, LPL, TTR, APoE, and α2M in the hippocampus of AD rats injected by Aβ1-42, and may consequently promote the degradation of aβ1-42 to help improve the pathological manifestations of AD and therefore delay its progression.

Effect of epidural block on restoration of protease inhibitors in patients with local kidney cancer.

593 Background: The purpose of the study was to reveal the effect of the epidural block (EB) on the dynamics of the main universal inhibitors in the blood plasma and urine of patients with local kidney cancer. Methods: 58 patients aged 56.5±8.7 years underwent partial nephrectomy with warm ischemia for 15-20 minutes (EB – 35 patients, standard anesthesia (SA) - 23 patients). EB involved catheterization of the epidural space at Th10-L1 with the following infusion of a mixture of ropivacaine 2 mg/ml and epinephrine 2 μg/ml at 6-10 ml/hr using a syringe doser, intraoperatively and within the first three postoperative days. α2-Macroglobulin (α2M) and α1-proteinase inhibitor (α1PI) were studied by ELISA and spectrophotometry in the blood plasma and urine of patients before the surgery (b/s), 40 min after the start of the surgery (40' s/s) and on days 1 and 3 after the surgery (a/s). Results were compared to the levels in the blood plasma and urine of 29 healthy donors (N). Results: α2M and α1PI in the blood plasma and urine of all patients b/s were lower than N (p &lt; 0.05). α2M in the blood plasma of all patients 40' s/s was similar to N. Normal α2M levels were maintained in the blood plasma in EB on days 1-3 a/s, while in SA they increased by 2.8 times on day 1 and decreased by 3.4 times on day 3, compared to the previous measurement results. α1PI in EB began to increase since 40' s/s with its normalization by the day 3 a/s; α1PI in SA was decreased in the blood plasma and urine during the whole study period. The urine/blood plasma ratio for α2M (α2Mu/α2Mb) was increased b/s in all patients, with its normalization on days 1-3 in EB and its decrease by 9.5-3.1 times on days 1 and 3 in SA. The α1PIu/α1PIb ratio was increased b/s on average by 2.1 times in all patients, with its normalization 40' s/s in EB, and remained similar to N by the day 3. Normalization was not observed in SA; α1PIu/α1PIb was decreased since 40' s/s to the day 3 by 1.8-1.4 times (p &lt; 0.05), compared to N. The results showed that in SA, the proteolysis was not controlled in the kidney due to the inhibitor deficiency. Conclusions: The epidural block contributes to the restoration of the activity of α2M and α1PI universal endogenous inhibitors during and after surgery, in contrast to the standard anesthesia.

α2-Macroglobulin Is a Significant In Vivo Inhibitor of Activated Protein C and Low APC:α2M Levels Are Associated with Venous Thromboembolism.

Activated protein C (APC) is a major regulator of thrombin formation. Two major plasma inhibitors form complexes with APC, protein C inhibitor (PCI) and α1-antitrypsin (α1AT), and these complexes have been quantified by specific enzyme-linked immunosorbent assays (ELISAs). Also, complexes of APC with α2-macroglobulin (α2M) have been observed by immunoblotting. Here, we report an ELISA for APC:α2M complexes in plasma. Plasma samples were pre-treated with dithiothreitol and then with iodoacetamide. The detection range of the newly developed APC:α2M assay was 0.031 to 8.0 ng/mL of complexed APC. Following infusions of APC in humans and baboons, complexes of APC with α2M, PCI and α1AT were quantified. These complexes as well as circulating APC were also measured in 121 patients with a history of venous thromboembolism (VTE) and 119 matched controls. In all the in vivo experiments, α2M was a significant APC inhibitor. The VTE case-control study showed that VTE patients had significantly lower APC:α2M and APC levels than the controls (p < 0.001). Individuals in the lowest quartile of APC:α2M or the lowest quartile of APC had approximately four times more VTE risk than those in the highest quartile of APC:α2M or of APC. The risk increased for individuals with low levels of both parameters. The APC:α2M assay reported here may be useful to help monitor the in vivo fate of APC in plasma. In addition, our results show that a low APC:α2M level is associated with increased VTE risk.

Low paediatric thrombin generation is caused by an attenuation of prothrombin conversion.

Thrombin generation (TG) is decreased in children. TG is determined by two underlying processes: the conversion of prothrombin to thrombin and the inactivation of thrombin. Therefore, lower TG capacity in children can either be caused by a reduction of prothrombin conversion, an increase of thrombin inactivation, or both. In 36 children and 8 adults, TG and the factors that determine thrombin inactivation (antithrombin, α2Macroglobulin (α2M) and fibrinogen) were measured. Prothrombin conversion, thrombin inhibitor complex formation, and the overall thrombin decay capacity were determined. In silico modelling was performed to determine the contribution prothrombin conversion and thrombin inactivation to deviant paediatric TG. Both the amount of prothrombin converted and the maximal prothrombin conversion rate are significantly reduced in children as compared to adults. This is partly due to the prothrombin levels being lower and partly to a lower prothrombin conversion rate. The overall thrombin decay capacity is not significantly different in children, but α2Macroglobulin plays a more important role than it does in adults. In silico experiments demonstrate that reduced prothrombin conversion and to a lesser extent elevated α2M levels provide an explanation for low TG in children. Young age has a dual effect on prothrombin conversion. Lower plasma prothrombin levels result in decreased prothrombin conversion but the rate of prothrombin conversion is also decreased, i. e. the development of prothrombinase is lower than in adults.

Rebalanced Thrombin Generation in Liver Cirrhosis Calls for Tailored Transfusion Protocols

IntroductionMost coagulation factors are produced by the liver, and therefore in liver cirrhosis both pro- and anticoagulant factor levels are reduced. Cirrhosis patients show no clear systemic bleeding or thrombotic phenotype and thrombin generation (TG) is (almost) unaffected, therefore it has previously been proposed that coagulation is rebalanced in cirrhosis because both pro- and anticoagulant processes are affected to a similar extent. Nevertheless, clinical parameters such as the prothrombin time indicate a bleeding risk in cirrhosis patients, and based on these tests patients often receive blood product transfusion during surgery.In this study we investigated thrombin generation and its main underlying pro- and anticoagulant processes (prothrombin conversion and thrombin inactivation) to provide empirical evidence for the hypothesis of rebalanced thrombin generation in liver cirrhosis. In addition, we used in silico experimentation to study the consequences of rebalanced TG for current transfusion practices.Methods25 cirrhosis patients and 25 healthy subjects were enrolled in our study. The group of cirrhosis patients consisted of 22 Child-Pugh A and 3 Child-Pugh B patients. Prothrombin, antithrombin (AT) and α2Macroglobulin (α2M) levels were determined and TG was measured at 5 pM tissue factor by calibrated automated thrombinography. Prothrombin conversion and thrombin inactivation were quantified from each TG curve, and thrombin-antithrombin and thrombin-α2Macroglobulin complex formation was determined.The effect of transfusion of prothrombin complex concentrate (PCC) was simulated by an in silico increase of prothrombin conversion. The increase of prothrombin conversion was simulated by substituting the prothrombin conversion curve of a cirrhosis patient by the average healthy subject prothrombin conversion curve, to normalize prothrombin conversion in patients. Thrombin generation curves were calculated based on this normalized prothrombin conversion curve and the original thrombin inactivation parameters of each cirrhosis patient.ResultsProthrombin and antithrombin are significantly decreased in cirrhosis patients (74 % and 70 %, p<0.001), whereas the α2M level is significantly increased (p<0.001). Thrombin generation in healthy subjects and patients is similar (figure), although prothrombin conversion (65 %, p<0.001) and thrombin inactivation (73 % p<0.001) are markedly reduced in cirrhosis patients. Acquired AT deficiency results in a reduction of thrombin inactivation by AT (64 %, p<0.001), but the substitution of AT by α2M leads to increased thrombin inactivation by α2M (220 %, p<0.001).The effect of prothrombin complex concentrate transfusion on thrombin generation in cirrhosis patients was determined by computational modeling (figure). In silico normalization of prothrombin conversion, as would be achieved by PCC transfusion, causes TG to rise to levels that have been shown to be highly prothrombotic (ETP 2878 ± 1323 nM∙min, p<0.001).ConclusionsDespite large differences in prothrombin conversion and thrombin inactivation, TG in liver cirrhosis patients remains within the normal range (rebalanced), in contrast to the prothrombin time that predicts a bleeding risk in these patients. The transfusion of PCC based on a prolonged prothrombin time would result in a substantial elevation of thrombin generation. This indicates that standard transfusion protocols need to be tailored to the specific needs of cirrhosis patients. [Display omitted] DisclosuresKremers:Synapse bv: Employment. De Laat:Synapse bv: Employment. Wagenvoord:Synapse bv: Employment. Hemker:Synapse bv: Employment.

Thrombin Generating Capacity and Phenotypic Association in ABO Blood Groups.

Individuals with blood group O have a higher bleeding risk than non-O blood groups. This could be explained by the lower levels of FVIII and von Willebrand Factor (VWF) levels in O individuals. We investigated the relationship between blood groups, thrombin generation (TG), prothrombin activation and thrombin inactivation. Plasma levels of VWF, FVIII, antithrombin, fibrinogen, prothrombin and α2Macroglobulin (α2M) levels were determined. TG was measured in platelet rich (PRP) and platelet poor plasma (PPP) of 217 healthy donors and prothrombin conversion and thrombin inactivation were calculated. VWF and FVIII levels were lower (75% and 78%) and α2M levels were higher (125%) in the O group. TG is 10% lower in the O group in PPP and PRP. Less prothrombin was converted in the O group (86%) and the thrombin decay capacity was lower as well. In the O group, α2M plays a significantly larger role in the inhibition of thrombin (126%). In conclusion, TG is lower in the O group due to lower prothrombin conversion, and a larger contribution of α2M to thrombin inactivation. The former is unrelated to platelet function because it is similar in PRP and PPP, but can be explained by the lower levels of FVIII.

Alpha-2-Macroglobulin Levels in Gingival Crevicular Fluid Pre- and Post-scaling and Root Planing with Adjunctive Tetracycline Fibers in Chronic Periodontitis: A Randomized Controlled Trial.

This split-mouth clinical study aimed to investigate levels of alpha-2-macroglobulin (a2M) in gingival crevicular fluid (GCF) of chronic periodontitis patients pre- and post-scaling and root planing (SRP) with or without adjunctive use of tetracycline fibers. In 30 patients of chronic periodontitis, samples of GCF were collected from the gingival sulcus before SRP. Recording of clinical parameters was conducted. This was followed by local drug delivery (LDD) of tetracycline fibers in test sites. In control sites, no LDD was done. Second samples of GCF were taken 90 days after treatment. Samples of crevicular fluid were analyzed to determine the levels of a2m. A gain of clinical attachment (CAL) of 3.30 mm for SRP and LDD and for SRP alone was 1.62 mm (p < 0.001). The pocket probing depth was significantly decreased by 2.43 mm for SRP and LDD and for SRP alone was 1.61 mm (p < 0.001) after 90 days. Alpha-2-macroglobulin was significantly reduced in GCF by SRP and SRP and LDD after 90 days (p < 0.001). Clinical and biochemical variables showed a more favorable outcome when SRP was combined with LDD of tetracycline fibers in management of patients suffering from chronic periodontitis.

Identification of α2‐Macroglobulin as a Master Inhibitor of Cartilage‐Degrading Factors That Attenuates the Progression of Posttraumatic Osteoarthritis

ObjectiveTo determine if supplemental intraarticular α2‐macroglobulin (α2M) has a chondroprotective effect in a rat model of osteoarthritis (OA).MethodsUsing Western blotting, mass spectrometry, enzyme‐linked immunosorbent assay (ELISA), and immunohistochemistry, α2M was identified as a potential therapeutic agent through a comparison of α2M concentrations in serum, synovial fluid (SF), and cartilage from normal subjects and patients with OA. In cultured chondrocytes, the effects of α2M on interleukin‐1 (IL‐1)–induced cartilage catabolic enzymes were evaluated by Luminex assay and ELISA. In vivo effects on cartilage degeneration and matrix metalloproteinase 13 (MMP‐13) concentration were evaluated in male rats (n = 120) randomized to 1 of 4 treatments: 1) anterior cruciate ligament transection (ACLT) and saline injections, 2) ACLT and 1 IU/kg injections of α2M, 3) ACLT and 2 IU/kg injections of α2M, or 4) sham operation and saline injections. Rats were administered intraarticular injections for 6 weeks. The concentration of MMP‐13 in SF lavage fluid was measured using ELISA. OA‐related gene expression was quantified by real‐time quantitative polymerase chain reaction. The extent of OA progression was graded by histologic examination.ResultsIn both normal subjects and OA patients, α2M levels were lower in SF as compared to serum, and in OA patients, MMP‐13 levels were higher in SF than in serum. In vitro, α2M inhibited the induction of MMP‐13 by IL‐1 in a dose‐dependent manner in human chondrocytes. In the rat model of ACLT OA, supplemental intraarticular injection of α2M reduced the concentration of MMP‐13 in SF, had a favorable effect on OA‐related gene expression, and attenuated OA progression.ConclusionThe plasma protease inhibitor α2M is not present in sufficient concentrations to inactivate the high concentrations of catabolic factors found in OA SF. Our findings suggest that supplemental intraarticular α2M provides chondral protection in posttraumatic OA.

WE‐E‐BRE‐05: Ensemble of Graphical Models for Predicting Radiation Pneumontis Risk

Purpose:We propose a prior knowledge‐based approach to construct an interaction graph of biological and dosimetric radiation pneumontis (RP) covariates for the purpose of developing a RP risk classifier.Methods:We recruited 59 NSCLC patients who received curative radiotherapy with minimum 6 month follow‐up. 16 RP events was observed (CTCAE grade ≥2). Blood serum was collected from every patient before (pre‐RT) and during RT (mid‐RT). From each sample the concentration of the following five candidate biomarkers were taken as covariates: alpha‐2‐macroglobulin (α2M), angiotensin converting enzyme (ACE), transforming growth factor β (TGF‐β), interleukin‐6 (IL‐6), and osteopontin (OPN). Dose‐volumetric parameters were also included as covariates. The number of biological and dosimetric covariates was reduced by a variable selection scheme implemented by L1‐regularized logistic regression (LASSO). Posterior probability distribution of interaction graphs between the selected variables was estimated from the data under the literature‐based prior knowledge to weight more heavily the graphs that contain the expected associations. A graph ensemble was formed by averaging the most probable graphs weighted by their posterior, creating a Bayesian Network (BN)‐based RP risk classifier.Results:The LASSO selected the following 7 RP covariates: (1) pre‐RT concentration level of α2M, (2) α2M level mid‐ RT/pre‐RT, (3) pre‐RT IL6 level, (4) IL6 level mid‐RT/pre‐RT, (5) ACE mid‐RT/pre‐RT, (6) PTV volume, and (7) mean lung dose (MLD). The ensemble BN model achieved the maximum sensitivity/specificity of 81%/84% and outperformed univariate dosimetric predictors as shown by larger AUC values (0.78∼0.81) compared with MLD (0.61), V20 (0.65) and V30 (0.70). The ensembles obtained by incorporating the prior knowledge improved classification performance for the ensemble size 5∼50.Conclusion:We demonstrated a probabilistic ensemble method to detect robust associations between RP covariates and its potential to improve RP prediction accuracy. Our Bayesian approach to incorporate prior knowledge can enhance efficiency in searching of such associations from data.The authors acknowledge partial support by: 1) CREATE Medical Physics Research Training Network grant of the Natural Sciences and Engineering Research Council (Grant number: 432290) and 2) The Terry Fox Foundation Strategic Training Initiative for Excellence in Radiation Research for the 21st Century (EIRR21)

α2Macroglobulin Fails To Compensate For Decreased Thrombin Decay In Severe Antithrombin Deficiency In Liver Cirrhosis Patients

IntroductionIn liver cirrhosis patients antithrombin (AT) decreases proportionally to the severity of the disease and in a part of the cases approaches the dangerous levels of heterozygous AT deficiency (50%) associated with thrombosis risk. Moreover, α2Macroglobulin (α2M) has been reported to increase up to fourfold. We investigated in how far the decrease of AT was compensated by the simultaneous increase of α2M. MethodsThrombin decay was characterized in plasma samples from healthy controls (n=32) and liver cirrhosis patients (n=29) by measuring AT, α2M and fibrinogen levels as well as the overall thrombin inhibitory capacity of the plasma. Functional AT, α2M and fibrinogen levels were measured with in-house chromogenic assays and the von Clauss method respectively. The overall inhibitory capacity of a plasma sample (total thrombin decay rate) was obtained by triggering thrombin generation (TG) with 50 pM tissue factor and analysis of thrombin decay after prothrombin conversion was over (t = 3 min). From the same TG curves, the rate of thrombin decay by α2M was calculated, and thrombin decay by antithrombin was calculated as the difference between total and α2M-dependent thrombin decay.In addition, we used a validated computer model to predict thrombin decay in time based on measured plasma levels of AT, α2M and fibrinogen. This allows us to predict thrombin decay under conditions that do not exist in real patient populations, such as liver cirrhosis patients with decreased AT levels, but normal α2M levels. In this way we can quantify the potential protective effect of α2M increase during liver cirrhosis. ResultsMean AT and α2M concentrations in the healthy control group were 2.01 μM (± 0.36 μM) and 3.34 μM (± 0.89 µM), respectively. In liver cirrhosis patients, the average AT level was significantly decreased (1.62 µM; p = 0.002) and the average α2M level was significantly increased (5.05 µM; p<0.001). In accordance, thrombin decay by α2M is significantly increased in liver cirrhosis patients (180% of control; p<0.001), whereas AT-dependent thrombin decay is significantly decreased (88% of control; p=0.009). The overall thrombin decay was not significantly altered in cirrhosis patients (95% of control; p=0.345), showing that α2M increase indeed compensates for AT decrease. However, in the subset of patients with AT<75% of normal, the rate of thrombin decay is significantly lower than in the control group (76%, p<0.001). Computer simulation of thrombin decay in this subset showed a similarity with the experimental curves to within the limit of experimental error. Replacing the values for the a2M concentration in individual liver cirrhosis patient samples by the average normal value reveals that the elevation in α2M levels increases the thrombin decay rate significantly (10%, p=0.016), but not enough to completely restore physiological thrombin decay rates (76%, p<0.001). ConclusionWe show that thrombin decay in liver cirrhosis is substantially reduced by the decrease of plasma antithrombin levels, and that a simultaneous increase in α2M levels counteracts this effect. In liver cirrhosis patients with moderate AT loss (<25%) α2M increase fully compensates for AT deficiency, but in patients with severe AT deficiency (<75% of normal levels) this compensation is only partial. In conclusion, the increase of the plasma α2M concentration in liver cirrhosis can be considered to be a (partial) protective mechanism. Disclosures:No relevant conflicts of interest to declare.