thromboplastin and Hyperglycemia

Atherothrombotic vascular disease is a complex disorder in which inflammation and coagulation play a pivotal role. Rupture of high-risk, vulnerable plaques with the subsequent tissue factor (TF) exposure is responsible for coronary thrombosis, the main cause of unstable angina, acute myocardial infarction, and sudden cardiac death. Tissue factor (TF), the key initiator of coagulation is an important modulator of inflammation. TF is widely expressed in atherosclerotic plaques and found in macrophages, smooth muscle cells, extracellular matrix and acellular lipid-rich core. TF expression can be induced by various stimulants such as C-reactive protein, oxLDL, hyperglycemia and adipocytokines. The blood-born TF encrypted on the circulating microparticles derived from vascular cells is a marker of vascular injury and a source of procoagulant activity. Another form of TF, called alternatively spliced has been recently identified in human and murine. It is soluble, circulates in plasma and initiates coagulation and thrombus propagation. Evidence indicates that elevated levels of blood-borne or circulating TF has been associated with metabolic syndrome, type 2 diabetes and cardiovascular risk factors and is a candidate biomarker for future cardiovascular events. Therapeutic strategies have been developed to specifically interfere with TF activity in the treatment of cardiovascular disease.

Topics: Coronary Artery Disease; Coronary Thrombosis; Diabetes Mellitus; Endothelium, Vascular; Humans; Hyperglycemia; Inflammation; Obesity; Thromboplastin

Tissue factor (TF) is the primary initiator of blood coagulation. Circulating TF procoagulant activity (TF-PCA) is associated with blood cells and microparticles and is elevated in patients with type 2 diabetes mellitus. Combined hyperinsulinemia and hyperglycemia and to a lesser degree selective hyperinsulinemia for 24 hours in healthy volunteers increased circulating TF-PCA, monocyte TF surface expression and mRNA, plasma thrombin generation, and coagulation factors VII and VIII activities, suggesting that the coagulation system had been activated. In addition, platelet CD40L and platelet-monocyte aggregates increased, indicating platelet activation. Somatostatin abolished these changes. We conclude that hyperinsulinemia, but particularly the combination of hyperinsulinemia and hyperglycemia, creates a prothrombotic state and may, in addition, be proinflammatory and proatherogenic by virtue of the actions of CD40L and TF.

Topics: Blood Coagulation; Diabetes Mellitus, Type 2; Humans; Hyperglycemia; Hyperinsulinism; Somatostatin; Thromboplastin

Individuals with chronically elevated glucose and/or insulin levels, i.e., most patients with type 2 diabetes, have accelerated atherosclerosis and are prone to acute vascular events. We have tested the hypothesis that hyperglycemia and/or hyperinsulinemia singly or combined may increase tissue factor, the primary initiator of blood coagulation. We have determined changes in circulating tissue factor procoagulant activity (PCA) and other procoagulation proteins in healthy volunteers exposed to 24 h of selective hyperinsulinemia, selective hyperglycemia, or combined hyperinsulinemia and hyperglycemia. Combined elevations of plasma insulin and glucose levels for 24 h produced a ninefold increase in tissue factor PCA, which was associated with an increase in monocyte tissue factor protein (flow cytometry) and mRNA (RT-PCR), increases in plasma thrombin-antithrombin complexes, prothrombin fragment 1.2, factor VIII coagulant activity, and platelet CD40 ligand as well as decreases in factor VIIa, factor VII coagulant activities, and factor VII antigen. Effects of selective hyperinsulinemia and selective hyperglycemia were less striking but appeared to be additive. We conclude that hyperinsulinemia and hyperglycemia but particularly the combination of both create a prothrombotic state and in addition may be proinflammatory and proatherogenic because of the proinflammatory actions of CD40 ligand and tissue factor.

Topics: Adult; Antigens; Blood Platelets; CD40 Ligand; Factor VII; Factor VIIa; Factor VIII; Female; Gene Expression Regulation; Glucose Clamp Technique; Humans; Hyperglycemia; Hyperinsulinism; Male; Monocytes; Prothrombin; Thromboplastin

Early growth response-1 (Egr-1) protein upregulation is reported in diabetes and vascular disorders. This study aims at deciphering its role in hyperglycemia induced changes of retinal endothelium. Human retinal endothelial cells (hRECs) were exposed to hyperglycemia (25mM) and normoglycemia (5.5mM). Gene silencing was done using siRNA against Egr-1. Transcript and protein level analysis of Egr-1 and gene targets were done using qPCR and immunoblotting respectively in hRECs, diabetic and nondiabetic human retina and immunofluorescence for localization in retinal sections. Hyperglycemia induced Egr-1 and vascular endothelial growth factor-A (VEGF-A) but not pigment epithelium derived factor (PEDF) in hRECs. Expression of Egr-1 repressor NGFI-A binding protein-2 (NAB-2) was unaltered. Egr-1 downstream gene targets, tissue factor (TF) and intercellular adhesion molecule-1 (ICAM-1) expression were increased in hRECs which was reduced by Egr-1 silencing in hyperglycemia. Diabetic retina, showed an increase in Egr-1, VEGF-A and gene target TF, ICAM-1 but not NAB-2 and PEDF similar to the changes seen in hyperglycemic hRECs. Hyperglycemic induction of Egr-1 and absence of NAB-2 repression in retinal endothelium, up-regulates downstream genes involved in pro-thrombotic and pro-inflammatory pathways linking Egr-1 in diabetes mediated vascular aberration of retina.

Topics: Cells, Cultured; Diabetic Retinopathy; Early Growth Response Protein 1; Endothelial Cells; Eye Proteins; Glucose; Humans; Hyperglycemia; Intercellular Adhesion Molecule-1; Nerve Growth Factors; Repressor Proteins; Retinal Vessels; Serpins; Signal Transduction; Thromboplastin; Up-Regulation; Vascular Endothelial Growth Factor A

Inflammation and hyperglycaemia are associated with a prothrombotic state. Cell-derived microparticles (MPs) are the conveyors of active procoagulant tissue factor (TF) and circulate at high concentration in diabetic patients. Liraglutide, a glucagon-like peptide (GLP)-1 analogue, is known to promote insulin secretion and β-cell preservation. In this in vitro study, we examined the link between insulin impairment, procoagulant activity and plasma membrane remodelling, under inflammatory conditions. Rin-m5f β-cell function, TF activity mediated by MPs and their modulation by 1 μM liraglutide were examined in a cell cross-talk model. Methyl-β-cyclodextrine (MCD), a cholesterol depletor, was used to evaluate the involvement of raft on TF activity, MP shedding and insulin secretion as well as Soluble N-éthylmaleimide-sensitive-factor Attachment protein Receptor (SNARE)-dependent exocytosis. Cytokines induced a two-fold increase in TF activity at MP surface that was counteracted by liraglutide. Microparticles prompted TF activity on the target cells and a two-fold decrease in insulin secretion via protein kinase A (PKA) and p38 signalling, that was also abolished by liraglutide. Large lipid raft clusters were formed in response to cytokines and liraglutide or MCD-treated cells showed similar patterns. Cells pre-treated by saturating concentration of the GLP-1r antagonist exendin (9-39), showed a partial abolishment of the liraglutide-driven insulin secretion and liraglutide-decreased TF activity. Measurement of caspase 3 cleavage and MP shedding confirmed the contribution of GLP-1r-dependent and -independent pathways. Our results confirm an integrative β-cell response to GLP-1 that targets receptor-mediated signalling and membrane remodelling pointing at the coupling of insulin secretion and inflammation-driven procoagulant events.

Topics: Animals; Caspase 3; Cell Membrane; Cell-Derived Microparticles; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Exocytosis; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Hyperglycemia; Inflammation; Insulin; Insulin-Secreting Cells; Liraglutide; MAP Kinase Signaling System; Peptide Fragments; Rats; SNARE Proteins; Thromboplastin

Diabetes mellitus (DM) patients have an increased incidence of cardiovascular events. Blood tissue factor-procoagulant activity (TF-PCA), the initiating mechanism for blood coagulation, is elevated in DM. We have shown that hyperglycaemia (HG), hyperinsulinaemia (HI) and combined HG+HI (induced using 24-hour infusion clamps) increases TF-PCA in healthy and type 2 DM (T2DM) subjects, but not in type 1 DM (T1DM) subjects. The mechanisms for this are unknown. DM patients have elevated plasma lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 ligand. We postulated that TLR4 plays a role in modulating TF levels. We studied the effect of HG+HI on TLR4 and TF-PCA in vivo during 24-hour HG+HI infusion clamps in healthy subjects, and T1DM and T2DM subjects, and in vitro in blood. In vivo, in healthy subjects, 24-hour HG + HI infusion increased TLR4 six-fold, which correlated with TF-PCA (r= 0.91, p<0.0001). T2DM patients showed smaller increases in both. In T1DM subjects, TLR4 declined (50%, p<0.05) and correlated with TF-PCA (r=0.55; p<0.05). In vitro, HG (200 mg/dl added glucose) and HI (1-100 nM added insulin) increased TF-PCA in healthy subjects (~2-fold, 2-4 hours). Insulin inhibited by ~30% LPS-induced increase in TF-PCA and high glucose reversed it. TLR4 levels paralleled TF-PCA (r=0.71, p<0.0001); HG and HI increased TLR4 and insulin inhibited LPS-induced TLR4 increase. This is first evidence that even in healthy subjects, HG of short duration increases TLR4 and TF-PCA, key players in inflammation and thrombosis. TLR4-TF interplay is strikingly different in non-diabetic, T1DM and T2DM subjects.

Topics: Adult; Biomarkers; Blood Coagulation; Blood Coagulation Tests; Blood Glucose; Case-Control Studies; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Female; Glucose Clamp Technique; Humans; Hyperglycemia; Hyperinsulinism; Inflammation; Insulin; Lipopolysaccharides; Male; Middle Aged; Thromboplastin; Thrombosis; Time Factors; Toll-Like Receptor 4

To determine tissue factor procoagulant activity (TF-PCA) in patients with type 1 diabetes and to examine effects of hyperglycemia and hyperglycemia plus hyperinsulinemia on TF-PCA.. We have determined circulating TF-PCA and other coagulation factors under basal (hyperglycemic) conditions, after acute correction of hyperglycemia, in response to 24 h of selective hyperglycemia, and in response to 24 h of hyperglycemia plus hyperinsulinemia in nine type 1 diabetic patients and in seven nondiabetic control subjects.. As shown previously in patients with type 2 diabetes, basal TF-PCA and plasma coagulation factor VIIa (FVIIa) were higher in patients with type 1 diabetes than in nondiabetic control subjects. However, in contrast with type 2 diabetes, normalizing glucose did not decrease the elevated TF-PCA levels, and raising glucose or glucose plus insulin levels did not increase TF-PCA.. Patients with type 1 diabetes have elevated circulating TF-PCA and FVIIa levels and are in a procoagulant state that may predispose them to acute cardiovascular events. The mechanisms regulating TF-PCA in patients with type 1 and type 2 diabetes are different and should be further explored.

Topics: Adult; Analysis of Variance; Blood Coagulation; Cardiovascular Diseases; Diabetes Mellitus, Type 1; Diabetic Angiopathies; Factor VIIa; Female; Glucose Clamp Technique; Humans; Hyperglycemia; Hyperinsulinism; Male; Middle Aged; Thromboplastin; Young Adult

Alterations in blood coagulation may explain the poorer neurological outcome with diabetes mellitus and hyperglycemia after acute ischemic stroke. We studied the relationships between diabetes mellitus, hyperglycemia, whole blood tissue factor procoagulant activity (TF-PCA) and plasma factorVIIa (FVIIa) in ten patients with type 2 diabetes mellitus and 11 non-diabetic patients at baseline and 6, 12, 24, and 48 hours (h) after presentation for acute stroke. In addition, we examined plasma prothrombin fragment 1+2 (F1.2) and thrombin-antithrombin complexes (TAT) as markers of thrombin generation. Stroke severity, assessed by National Institute of Health Stroke Scale (NIHSS), was similar at baseline (p=0.26) but worse in diabetic (8.20+/-4.3) than nondiabetic patients (2.67+/-2.1, p=0.023) at 48 h. At presentation, diabetic patients had higher FVIIa (p=0.004) and lower TF-PCA (p=0.027) than non-diabetic patients but both were higher than in normal control subjects. FVIIa levels remained higher in diabetic patients at 6, 12 and 24 h after stroke. In diabetic patients, FVIIa (r=0.40, p=0.02) and TF-PCA (r=0.50, p=0.02) correlated with blood glucose; and, FVIIa correlated with plasma F1.2 (r=0.34, p=0.002) and TAT levels (r=0.62, p<0.0001). In non-diabetic patients, TF-PCA, but not FVIIa, correlated with F1.2 (r=0.402, p=0.010) and TAT (r=0.39, p=0.011). Combining both groups, NIHSS scores were positively related to FVIIa levels (r=0.50, p=0.021) and inversely related to TF-PCA levels (r=-0.498, p=0.02). Acute ischemic stroke patients with diabetes and hyperglycemia have a more intense procoagulant state compared with nondiabetic patients. This is related to glucose levels and provides a potential mechanism for the observed worse prognosis in such patients after acute stroke.

Topics: Aged; Aged, 80 and over; Biomarkers; Case-Control Studies; Diabetes Complications; Diabetes Mellitus, Type 2; Factor VIIa; Female; Humans; Hyperglycemia; Male; Middle Aged; Prognosis; Severity of Illness Index; Stroke; Thrombophilia; Thromboplastin

Elevated plasma homocysteine (Hcy) is a risk factor for coronary disease. The objective of this study was to investigate whether Hcy either alone or in high glucose conditions induces endothelin-1 (ET-1) synthesis via the production of reactive oxygen species (ROS).. Bovine aortic endothelial cells were grown in high (25 mmol/l) and low (5 mmol/l) glucose medium.. In high glucose, Hcy caused a time-dependent increase in ET-1 release, which was greatest with 50 micromol/l Hcy at 24 h (p < 0.01). This effect was not seen in low glucose conditions. In high glucose and 50 micromol/l Hcy, ET-1 mRNA levels were maximal after 1 h (p < 0.05). Tissue factor mRNA levels were raised at 4 h (p < 0.05) and functional activity was raised at 6 h (p < 0.01). Intracellular ROS production was increased by 50 micromol/l Hcy after 24 h (p < 0.05) but only in high glucose. To investigate the role of mitochondrial metabolism in ROS production, cells were incubated with thenoyltrifluoroacetone (inhibitor of complex II) or carbonyl cyanide m-chlorophenylhydrazone (uncoupler of oxidative phosphorylation). Both compounds abolished the Hcy-induced increase in ROS production and ET-1 release. There was an alteration in intracellular glutathione (GSH) levels with Hcy treatment with more oxidised GSH present.. The combined metabolic burden of Hcy and high glucose stimulates ET-1 synthesis in bovine aortic endothelial cells via a mechanism dependent on the production of mitochondrial ROS, but may not be generalisable to all types of endothelial cells.

Topics: Animals; Aorta; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cattle; Cells, Cultured; Electron Transport Complex II; Endothelin-1; Endothelium, Vascular; Glucose; Glutathione; Homocysteine; Hyperglycemia; Oxidative Stress; Reactive Oxygen Species; Thenoyltrifluoroacetone; Thromboplastin; Uncoupling Agents

Patients with diabetes have an increased prevalence of premature atherosclerotic vascular disease, and alterations in plasma coagulation proteins have been incriminated as a possible cause. The roles of hyperglycemia and hyperinsulinemia in the pathogenesis of these changes are unknown. To examine the effects of prolonged hyperglycemia and of selective hyperinsulinemia on the tissue factor pathway of blood coagulation, nine healthy young men were infused with glucose to maintain levels at 11.1 mmol/l (approximately 200 mg/dl) for 18-72 h (hyperglycemia-hyperinsulinemia group). Five normal men were infused with regular insulin to maintain levels comparable to that in the previous group (900 pmol/l, approximately 150 microU/ml) and with glucose to maintain levels at 5.6 mmol/l (approximately 100 mg/dl) (euglycemia-hyperinsulinemia group). Measured were plasma activated factor VII activity (FVIIa), FVII coagulant (FVIIC) activity, FVIII coagulant (FVIIIC) activity, tissue factor pathway inhibitor (TFPI) antigen, and thrombin markers; and serum glucose, insulin, and electrolytes. Plasma FVIIa, FVIIC, FVIIIC, and TFPI rose during hyperglycemic-hyperinsulinemia but not during euglycemic-hyperinsulinemia. Markers of thrombin generation rose transiently and inconsistently during hyperglycemia-hyperinsulinemia. We concluded that in normal subjects, hyperglycemia-hyperinsulinemia induced activation of the tissue factor pathway, reflected by increases in plasma FVIIa, FVIIC, and TFPI. This activation was independent of hyperinsulinemia, hypertriglyceridemia, and hyperosmolality. The elevations in plasma coagulation factors during hyperglycemia-hyperinsulinemia, characteristic of type 2 diabetes, may constitute a potential for enhanced thrombin generation and thrombosis when triggered by exposure of tissue factor, such as during arterial plaque rupture.

Topics: Adult; Blood Coagulation; Factor VII; Factor VIIa; Factor VIII; Glucose; Glucose Clamp Technique; Humans; Hyperglycemia; Hyperinsulinism; Insulin; Lipoproteins; Male; Middle Aged; Thrombin; Thromboplastin

Phospholipid asymmetry in biological membranes is maintained by an aminophospholipid-specific Mg(2+)-ATPase that transports PS and PE from the outer to the inner monolayer. Recent evidence indicates that a loss of phospholipid asymmetry occurs in erythrocytes from diabetic individuals, resulting in the appearance of PS in the membrane outer leaflet. We show that hyperglycemic treatment of normal erythrocytes duplicates this effect. Erythrocytes incubated for 18-24 h in the presence of glucose were assayed for PS transport and transmembrane phospholipid asymmetry. Phospholipid asymmetry in erythrocytes treated with high concentrations of glucose (> 5 mM) showed a time-dependent (t1/2 approximately 12 h) and concentration-dependent (half-maximal concentration approximately 7.5 mM) increase in the accessibility of PS and PE, and a decrease in the accessibility of SM and PC, to exogenous phospholipases. After an 18 h incubation with 20 mM glucose, 40% of the endogenous PS and PE was found in the outer monolayer concomitant with a decrease in the outer monolayer content of SM (from 80% to 50%) and PC (from 75% to 65%). These values are consistent with an almost complete transbilayer scrambling of erythrocyte phospholipids. The loss of PS asymmetry was verified using an assay based on the activation of the prothrombinase complex. The observed loss of asymmetry is not due to inhibition of PS transport or glucose-induced Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine Triphosphate; Adult; Biological Transport; Calcium; Erythrocyte Membrane; Erythrocytes; Glucose; Glutathione; Glycated Hemoglobin; Humans; Hyperglycemia; In Vitro Techniques; Kinetics; Membrane Lipids; Phospholipids; Thromboplastin; Time Factors