Thrombotic tendency in diabetes mellitus. Revisiting and revising a study initiated 30 years ago.

Studies initiated 30 years ago emphasized that dilute blood clot lysis time was longer in obese diabetic patients than in normal weight diabetics. It was also later reported that when compared to obese women with gluteal and femoral adiposity, the age matched men with abdominal obesity displayed a more delayed clot lysis, higher triglyceride levels and higher cholinesterase activity, as well as more increased concentration of plasminogen activator inhibitor-1 (PAL-1). According to authors' investigations and data in the literature, impaired fibrinolysis in overweight hypertriglyceridemic subjects are mainly due to increased plasma levels of coagulation factor XIII and PAI-1. It could also be demonstrated that plasma clotting factors VII and VIII activities as well as plasma fibrinogen and von Willebrand factor levels were higher in patients with type 2 diabetes and abdominal obesity than in diabetics without obesity. Such findings are supporting data in the literature, insisting on the pathogenic relevance of intraabdominal obesity and of the subsequently enhanced release of fatty acids and of proinflammatory cytokines in the portal flow. Surprisingly anticoagulant plasma proteins C and S levels were found to be increased in overweight and hyperlipidemic patients considered to be at risk for thrombotic complications. Recent data in the literature had however demonstrated that circulating protein C zymogen acquires anticoagulant activity only after its binding to specific receptors on endothelial cell membrane, while proinflammatory cytokines may disrupt this activating interaction with vascular endothelia.

Controversy on high density lipoprotein raising therapy.

Decreased high density lipoproteins (HDL) plasma levels are a recognized independent risk factor for atherosclerotic cardiovascular disease. Attempts were therefore initiated to pharmacologically raise plasma HDL cholesterol, and the most impressive increase was obtained by inhibiting cholesteryl ester transfer protein (CETP) by means of the synthetic compound torcetrapib. Clinical trials were however disappointing, as torcetrapib increased mortality and did not reduce the progression of atherosclerosis. According to some view, it was claimed that CETP inhibition is unfavourable and that development of this class of compounds should be abandoned. Controversy nevertheless stimulated research on HDL structure, heterogeneity and functions which are not limited to reverse cholesterol transport and exert antioxidant and antiinflammatory actions. It could also be demonstrated that the deleterious effects of torcetrapib are compound specific, including its tight binding to CETP on HDL particles, thereby blocking both neutral lipids and phospholipid transfer from HDL to other lipoproteins, and would also exert an off-target effect by increasing plasma sodium and decreasing potasium concentrations (an aldosterone-like effect). As the structure of CETP was elucidated, it became possible to design CETP inhibitors that lack such off-target toxicity and may successfully slow the progression of atherosclerosis. Noteworthy, mice and rats naturally lacking CETP are resistant to diet induced atherosclerosis, while rabbits with high CETP levels are very susceptible. Families with deficient activity and exceptional longevity had also been reported.

Lipolytic enzymes, metablic syndrome and atherosclerosis (revisiting and revising a 40 years old study).

More than 40 years ago, our laboratory reported that post-heparin lipolytic activity was decreased in patients with severe atherosclerotic disease, while values recorded in obese and hyperlipidemic subjects without clinically detectable atherosclerotic lesions did not significantly differ from normal weight normolipidemic controls. Because in 1967 data on pathophysiology of lipolytic enzymes were rather scarce, and mainly because our information facilities were limited in those years, we had difficulties in interpreting these results, and the study was to some extent awkwardly approached, as the investigated subjects were not considered according to their gender, body fat patterning and type of hyperlipoproteinemia, and the lipolytic activities of lipoprotein lipase and hepatic lipase had not been selectively assessed. Reviewing recent data in the literature it was noted that pre-heparin lipoprotein lipase mass assessed by ELISA was indeed significantly lower in insulin resistant coronary patients than in patients with no lesions, and correlated negatively with the severity of atherosclerotic lesions. Noteworthy hypoadiponectinemia, a hallmark of insulin resistance, was associated with decreased lipoprotein lipase and increased hepatic lipase activities. Clustering of increased plasma VLDL-triglyceride, cholesteryl-ester transfer protein and hepatic lipase would remodel HDL and LDL particles, generating an atherogenic lipoprotein profile. In opposition to atherogenic dyslipidemia related to an enhanced hepatic secretion of VLDL, cases with important hypertriglyceridemia subsequent to deficient lipolytic clearance are at a rather low risk for coronary artery disease. It may therefore be suggested that the decreased lipoprotein lipase noted in atherosclerotic patients is not a major pathogenic link, being rather related to the inflammatory component of the disease, its expression being reduced by proinflammatory cytokines.

Reverse cholesterol transport and atherosclerosis. A mini review.

Reverse cholesterol transport (RCT) is a complex process ensuring the efflux of cholesterol from peripheral cells and its transport back in the liver for its metabolism and biliary excretion. Cholesterol efflux results by the interaction of a cellular free cholesterol and phospholipid transporter, the ABC-AI, with lipid poor apoAI, endowed HDL particles. The free cholesterol taken up by HDL is then esterified by lecithin:cholesterol acyltransferase (LCAT) and the hydrophobic cholesteryl esters are retained into the core of HDL, so that new cholesterol molecules can be translocated on the HDL surface. The generated cholesteryl esters are partially transferred to triglyceride rich apoB containing lipoprotein through a nonenzymatic process mediated by cholesteryl ester transfer protein (CETP) in exchange for triglyceride. The hepatic uptake of the cholesterol released from peripheral cells may thus proceed via an HDL-receptor, the SR-BI and through the LDL receptor route. Hepatic lipase (HL) facilitates the selective uptake of cholesteryl esters by the hepatocytes by exerting a lipolytic effect and a ligand-binding effect, bridging the lipoprotein particles to the heparan sulfate proteoglycans on cells surface and allowing the transcytosis of cholesteryl esters. Studies on genetically modified animals and on humans with severe genetic deficiencies demonstrated that abnormalities of the various components of RCT would accelerate atherogenesis. Clinical studies revealed that the development of coronary artery disease (CAD) may by delayed by increased HL activity in patients with familial hypercholesterolemia (heterozygotes), while in hypertriglyceridemic patients an increased plasma CETP and HL levels would favor the generation of less lipidated HDL and of small dense atherogenic LDL particles.

Pathogenic role of abnormal fatty acids and adipokines in the portal flow. Relevance for metabolic syndrome, hepatic steatosis and steatohepatitis.

Evidence has been provided that increased levels of non esterified fatty acids (NEFA) in the portal flow would produce insulin resistance and would also stimulate the hepatic protein synthesis, thereby explaining the increased plasma levels not only of apolipoprotein B, but also of other liver-derived enzymes and proteins occurring in overweight and hypertriglyceridemic patients. The high plasma concentration of triglyceride-rich lipoprotein would facilitate the transfer of cholesteryl esters from HDL and LDL to VLDL in exchange for triglycerides, a process mediated by liver-derived cholesteryl ester transfer protein (CETP). The triglyceride thereby acquired in HDL and LDL would then be hydrolyzed by hepatic lipase. The resulting association of increased triglycerides, low HDL cholesterol and small dense LDL is considered to be an atherogenic profile. The prothrombotic state, another feature of the metabolic syndrome, may also be explained by an enhanced hepatic synthesis of clotting factors and of the inhibitors of fibrinolysis. It was recently shown that adipocyte synthesized adiponectin reduces the release of fatty acids from the adipose tissue and would also enhance their uptake and oxidation in the muscle, thereby limiting their uptake in the liver. Decreased adiponectin production in obesity would therefore promote the development of insulin resistance, of atherogenic dyslipidemia and of the prothrombotic state. Because adiponectin also exerts an antiinflammatory activity by antagonizing TNFalpha, hypoadiponectinemia may be involved in atherogenesis and in the progression of hepatic steatosis to steatohepatitis.

The discovery by Gh. Benga of the first water channel protein in 1985 in Cluj-Napoca, Romania, A few years before P. Agre (2003 Nobel Prize in Chemistry).

The first water channel protein, now called aquaporin 1, was identified or "seen" in situ in the human red blood cell membrane by Benga's group in 1985. It was again "seen" when it was by chance purified by Agre'group in 1988 and was again identified when its main feature, the water transport property, was found by Agre's group in 1992. Consequently, the omission of Gh. Benga from the 2003 Nobel Prize in Chemistry (half of which was awarded to P. Agre "for the discovery of the water channels") is a new mistake in the award of Nobel Prizes. The growing recognition of the priority of Gh. Benga over P. Agre in the discovery of water channels is documented in this paper.

Von Willebrand factor and cardiovascular disease. Restricted relevance.

Von Willebrand's disease, a bleeding disorder, is caused by deficiencies of an endothelia-derived multimeric glycoprotein receiving the deserved name von Willebrand factor (vWf). Since this factor mediates the interaction between blood platelets and the vessels wall, it was presumed that an increase of its plasma level could be a marker of endothelial lesions and may be also involved in the pathogeny of cardiovascular disease including thrombotic complications. Actually, the unchecked release of ultralarge vWf multimers may cause widespread platelet aggregation into the microcirculation, thereby producing thrombotic thrombocytopenic purpura. More recently, it was demonstrated that such a dramatic condition is normally prevented by the intervention of a protease (ADAMTS-13) which cleaves the ultralarge and large vWf multimers into smaller multimers less active functionally although endowed with antigenic epitopes. The relevance of an immunologic assessment of vWf plasma levels as vWf:Ag should therefore be cautiously considered because this test, more accessible to clinical laboratories, does not provide information about the actual level of large multimers which are functionally active and possibly prothrombotic. It was also reported that an acute phase reaction occurring in various disease states is accompanied by increased plasma vWf levels as the result of endothelial response to adrenergic or proinflammatory stimuli. Clinical and laboratory investigations are highly suggestive that an increased plasma vWf level does not necessarily reflect its leakage from injured endothelia and the laboratory data should be considered in context with the clinical condition and the overall hemostatic balance.

Effect of abdominal obesity on prothrombotic tendency in type 2 diabetes. Behavior of clotting factors VII and VIII, fibrinogen and von Willebrand Factor.

Patients with type 2 diabetes and abdominal fat patterning displayed higher plasma activities of clotting factors VII and VIII as well as increased plasma levels of fibrinogen and von Willebrand factor antigen, when compared with not only healthy normal weight controls, but also with diabetic patients at normal body weight. Mechanisms associating abdominal obesity with the above mentioned prothrombotic changes are only partially elucidated and may differ according to the individual haemostatic variable. Actually, according to data in the literature and authors' observations increased plasma factor VII activity is mainly associated with increased plasma triglyceride level, while the hepatic synthesis of fibrinogen as well as the synthesis and release of endothelia-derived von Willebrand factor are stimulated by cytokines originating in the visceral adipose tissue. The relationship between the presently investigated hemostatic variables and features of the metabolic syndrome are less obvious than in the previously recorded association of metabolic risk factors with plasma levels of plasminogen activator inhibitor.

Coagulation factor XIII and atherothrombosis. A mini-review.

Coagulation factor XIII is a transglutaminase catalysing the crosslinking of fibrin chains as well as the formation of covalent links between several extracellular matrix proteins such as fibronectin, vitronectin and collagen. By mediating the incorporation of alpha2 antiplasmin into the fibrin network, this factor also interferes with fibrinolysis. Increased plasma factor XIII activity was reported by our laboratory 30 years ago in hypertriglyceridemic subjects who also displayed increased activity of serum cholinesterase, a marker of hepatic protein synthesis, and a delayed diluted, blood clot lysis time. Recent data in the literature emphasize a relationship between insulin resistance (metabolic syndrome) and increased plasma levels of factor XIII, confirming our results. It was also reported that a faster activation of this factor related to the Val 34 leu polymorphism provides protective effect against myocardial infarction and stroke, this effect being however negated in patients with insulin resistance and high plasma levels of plasminogen activator inhibitor-1. The pathogenic role of factor XIII in atherothrombosis seems to be bivalent. On the one side, an increased activity would favor the persistence of fibrin depositions and increase plaque burden, while on the other side it would reduce plaque vulnerability and the risk of downstream embolization.

Serum cholinesterase activity correlates with serum insulin, C-peptide and free fatty acids levels in patients with type 2 diabetes.

When compared with values recorded in 14 control subjects, the 15 overweight patients with type 2 diabetes displayed significantly increased activities of serum alanineaminotransferase (172% of mean values in controls), gamma-glutamyltransferase (253%) and cholinesterase (139%). A much wider dispersion of individual values for the two firstly mentioned enzymes was however noted so that their correlation with serum triglycerides levels were weaker (r = 0.373; p < 0.05 and r = 0.451; p < 0.05 respectively) than the same correlation obtained for serum cholinesterase (r = 0.760; p < 0.001). In two other studies including 28 controls and 30 diabetic patients serum cholinesterase was found to be significantly correlated with serum levels of insulin (r = 0.622; p < 0.001), C-peptide (r = 0.652; p < 0.001) and free fatty acid (r = 0.821; p < 0.001). Circumstantial evidence is provided that insulin resistance and an increased flux of free fatty acids from adipose tissue to the liver would stimulate the hepatic synthesis of serum cholinesterase.

Apolipoproteins A1 and B levels and serum cholinesterase activity in hyperlipidemic subjects.

Serum apolipoprotein B (apo B) levels were found to be significantly (p < 0.001) higher in the 27 patients with combined hyperlipidemia (144 m./dl +/- 27.6) than in the 17 normal weight normolipidemic control subjects (92 mg/dl +/- 20.6; X +/- SD). When compared to apolipoprotein A1 (apo A1) levels obtained in controls (168.5 mg/dl +/- 28.4), hyperlipidemic subjects displayed a moderate yet significant (p < 0.02) decrease of this apolipoprotein (140 mg/dl +/- 24.2). Serum apo B levels were significantly (p < 0.001) correlated with serum cholesterol concentrations and also, to a lesser degree (p < 0.01), with serum cholinesterase activity. A highly significant correlation (p < 0.001) between apo A1 and HDL cholesterol levels was also noted. The decrease ofHDL cholesterol occurring in hyperlipidemic men (-30%) was however more accentuated than the decrease of apo A1 (-18%) suggesting an enhanced transfer of cholesterol esters from HDL to VLDL and LDL. It is considered that the determination of apolipoproteins may be useful not only for the detection of risk factors for atherosclerosis, but also for a better insight concerning the mechanisms involved in the development of an atherogenic dyslipidemia.

Plasma fibronectin in overweight men and women: correlation with serum triglyceride levels and serum cholinesterase activity.

When compared with 67 age- and sex-matched normal weight control subjects, the 71 overweight patients displayed obviously higher levels of plasma fibronectin. For a similar body mass index (BMI) the 16 overweight men younger than 45 years had a significantly (P < 0.01) higher plasma fibronectin level (455 +/- 99.3 mg/l; mean +/- SD) than the 16 age-matched overweight women (351 +/- 105 mg/l) while there was no significant difference between the 22 overweight men (446 +/- 89.2 mg/l) and the 17 overweight women (475 +/- 111 mg/l) older than 45 years. Particularly high plasma fibronectin levels were noted in the five women with upper body (android) obesity. Plasma fibronectin was positively correlated with BMI, serum triglyceride concentration, plasma fibrinogen and serum cholinesterase activity. It is considered that metabolic disturbances related to upper body obesity may lead to an enhanced hepatic secretion of VLDL and of several plasma proteins including fibronectin.

[The role of the endothelia in hemostasis]. / Rolul endoteliilor în hemostaza.

Unless associated with endothelial lesions or dysfunction, the increased plasma levels of fibrinogen, clotting factors VII and X as well as of fibrin stabilizing factor XIII and of the inhibitors of fibrinolysis, displayed by hyperlipidemic patients, could explain neither the localization nor the mechanisms triggering thrombotic events. Actually, endothelia are provided with both prohemostatic factors (tissue factor, von Willebrand factor, platelet activating factor) and antithrombotic mechanisms (tissue factor pathway inhibitor, proteoglycans activating antithrombin III, thrombodulin activating the protein C system as well as prostacyclin inhibiting platelet aggregation). Endothelia also modulate fibrinolytic activity by producing both activators and inhibitors of plasminogen activation. Evidence was provided that proinflammatory cytokines, anion superoxide and oxidized LDL would cause an upregulation of prohemostatic mechanisms, while down regulating the antithrombotic ones. Attempts made to detect endothelial dysfunction by methods available to the clinical laboratory and author's own observations concerning the behaviour of endothelia-derived plasma von Willebrand factor in various pathological conditions are briefly presented.

Serum lactate dehydrogenase and alkaline phosphatase activities and serum cholesterol level in bone marrow blood.

Serum cholesterol level as well as serum lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) were measured in 65 samples of bone marrow blood and in matched peripheral blood taken from patients with various hematological diseases. As expected, serum LDH activities were higher and serum total cholesterol levels were lower in the bone marrow blood than in the blood taken from the cubital vein. More interestingly, an important increase of heat-labile ALP, but not of serum GGT, was found in the bone marrow blood obtained from patients characterized by a proliferating bone marrow. Actually, both LDH and ALP activities were obviously higher in the bone marrow blood of patients with megaloblastic anemia, myelodysplastic syndrome and chronic myeloid leukemia than in samples taken from patients with chronic lymphocytic leukemia, a disease characterized by a slower proliferation rate. While the expected increased LDH activity is the result of an accelerated turnover of bone marrow cells implying the release of this enzyme from the dividing and/or decaying cells, the much higher activity of the heat-labile alkaline phosphatase found in the bone marrow blood would reflect an enhanced local remodeling of bone structures, probably related to an expanded proliferating bone marrow. The lower serum cholesterol level in the bone marrow blood could be subsequent to an enhanced uptake of low density lipoproteins by specific receptors on the bone marrow cells.

Acute phase reaction and the hemostatic balance.

Data in the literature as well as authors own observations concerning a possible relationship between the acute phase reaction and changes of the hemostatic variables, which may favour or trigger a thrombotic event, are reviewed. Acute phase reaction is usually accompanied by increased plasma levels of fibrinogen, von Willebrand factor and clotting factor VIII, while endothelial cells in culture added proinflammatory cytokines were found to express tissue factor activity, to release von Willebrand factor and to increase the production of plasminogen activator inhibitor. Evidence is also provided that inflammation would lead to an increase of plasma antithrombin III while the protein C system is down regulated. It is also considered that the above-mentioned changes of hemostatic variables would favour the local deposition of fibrin and platelets while attempting to prevent an intravascular dissemination of fibrin formation.

Increased plasma factor VIII:c activity in patients with unstable angina pectoris.

Plasma factor VIII:c activity was found to be significantly (p < 0.01) higher in the 17 patients with unstable angina pectoris (201% +/- 121; x +/- SD) than in the 10 healthy control subjects (97% +/- 16). Plasma fibrinogen level was also significantly (p < 0.003) higher in patients (455 mg/dl +/- 188) than in controls (260 mg/dl +/- 35) but there was no significant correlation between these two variables within the group of patients with unstable angina. No difference could be noted between plasma antithrombin III activities in patients and in controls. It is considered that the increased factor VIII:c activity in patients with unstable angina pectoris could be subsequent to the acute phase reaction induced by cytokines and/or by an enhanced adrenergic stimulation, although the possible presence of genetically-conditioned hyperactive factor VIII:c molecules can not be excluded. Since the outcome of a ruptured plaque may also depend on the systemic thrombotic propensity at the time of rupture, the presently reported findings could be pathogenically relevant.

Hemostatic balance during the acute inflammatory reaction. (II). With special reference to plasma protein C.

While plasma fibrinogen level and clotting factor VIII activity obviously increased during the acute inflammatory reaction caused by intramuscular injections of turpentine in 10 rabbits, the activity of plasma protein C, with anticoagulant and antithrombotic effects, was found to display a slight yet significant (p < 0.02) decrease. When compared to protein C activity in humans, this vitamin K-dependent serine protease was found to be lower in rabbits (52, 7% +/- 3,63 of standard human plasma) and this activity decreased to 45,8% +/- 2,80 (mean +/- SEM), 48 hours after the injection of turpentine. This slight decrease should however be interpreted in the context of other disturbances of the hemostatic balance caused by inflammation and may contribute to the intravascular deposition of fibrin.