Coagulation protein function VI: augmentation of anticoagulant function by acetaldehyde-treated heparin.

Acetaldehyde (AcH) at preincubation concentrations of 447, 89.4, and 17.9 mM potentiates the effects of heparin on the clotting time of plasma. While control plasma clotted in the range of 12.6+/-0.1 to 13.8+/-0.1 sec, and heparin-treated plasma clotted in a range from 131.5+/-2.5 to 168.2+/-1.2 sec, heparin that was preincubated at room temperature for 30 min with 89.4 or 447 mM AcH did not clot plasma in 300 sec. Heparin exposed to 17.9 mM AcH clotted plasma in 193+/-1.1 sec. Ethanol at a 404 mM concentration also prolonged the clotting time of heparin-treated plasma >300 sec, while 202 mM ethanol prolonged the clotting time of heparin-treated plasma from 149.0+/-2.0 sec to 219.5+/-1.7 sec. It is suggested that AcH alters the tertiary structure of heparin by adduct formation, possibly by formation of cyclic acetals with iduronic and glucuronic acids, thereby more readily affecting binding of the glycosaminoglycan to antithrombin III and/or thrombin, prolonging clotting time. Ethanol, which does not react covalently with heparin, might affect its conformation as a consequence of an organic solvent effect. Protamine sulfate prolonged the clotting time of plasma from 13.6+/-0.1 sec to 17.9+/-0.2 sec. Protamine sulfate-treated heparin clotted plasma in 21.0+/-0.4 sec relative to heparin-treated plasma (160.4+/-1.7 sec). In subsequent experiments, AcH-treated protamine sulfate extended the clotting time of protamine sulfate from 17.9+/-0 sec to 33.7+/-0.6 sec. Prior addition of protamine sulfate to AcH-heparin mixtures or heparin to protamine sulfate-AcH mixtures before addition to plasma resulted in clotting times of 22.0+/-0.4 sec and 24.1+/-0.5 sec, respectively, relative to control clotting times of 162.3+/-2.6 sec for plasma-heparin mixtures. These results confirm both the reduction in coagulation time of heparin-treated plasma by protamine sulfate and the prolongation of clotting time of plasma by protamine sulfate. Furthermore, and importantly, they indicate that acetaldehyde-treated protamine sulfate is a more effective anticoagulant than protamine sulfate. It is suggested that reversible adduct formation between acetaldehyde, heparin, and protamine sulfate may occur as a means explaining the essentially identical coagulation time of these mixtures when added to plasma regardless of the order of premixing. Ethanol (404 mM) did not influence protamine sulfate effects. Lastly, the potentiation of the anticoagulant function of heparin by acetaldehyde suggests that a structural modification of the glycosaminoglycan may occur in alcoholics.

Coagulation protein function VII: diametric effects of acetaldehyde on factor VII and factor IX function.

The first metabolite of ethanol, acetaldehyde, has the ability to form adducts with proteins and alter their function. It has been shown that acetaldehyde reacts with various proteins of the blood coagulation pathway and, subsequently, produces a prolongation of the clotting time. This study evaluated the function of clotting proteins from the extrinsic coagulation pathway (factor VII) and the intrinsic coagulation pathway (factor IX) when preincubated with acetaldehyde as compared to a control and compared to preincubation with ethanol. Prior to use in a clotting assay, incubation times with acetaldehyde, ethanol, and the control were the same for both factors VII and IX. An automatic fibrometer measured the clotting times. Factor VII preincubated with acetaldehyde prolonged the clotting time. However, factor IX preincubated with acetaldehyde actually decreased the clotting time. Of interest, both factors VII and IX preincubated with acetaldehyde produced statistically significant results when compared to the control and ethanol. This experiment indicates that acetaldehyde, in forming an adduct with proteins of the blood coagulation pathway, may induce a conformational change of factors VII and IX so as to either increase or decrease the clotting time. Therefore, it is possible that some of the deranged coagulation in alcohol abusers may be a final net result of the interaction of acetaldehyde and proteins of the coagulation pathway.

Coagulation protein function V: diminution of antithrombin III function by acetaldehyde.

The anticoagulant activity of antithrombin III (ATIII), as observed in a plasma-free system consisting of thrombin and fibrinogen, is readily reduced by acetaldehyde (AcH) at concentrations of 447, 89.4, and 17.9 mM. Whereas control thrombin-fibrinogen mixtures clotted in 17.7+/-0.75 sec, ATIII prolonged clotting time to 55.0+/-1.75 sec on preincubation with thrombin for 30 min at room temperature. On subsequent preincubation of ATIII with the AcH for 30 min at room temperature and passage of the mixture through Sephadex G-25 minicolumns to remove excess AcH, the eluates were tested for anticoagulant activity. Clotting times of 20.9+/-1.0, 32.3+/-1.0, and 45.3+/-1.6 sec were obtained with 447, 89.4, and 17.9 mM AcH-ATIII mixtures, respectively. These data suggest that functional groups on ATIII, such as guanidiniums, aminos, and others are susceptible to adduct formation with AcH, thereby altering the shape and charge of the anticoagulant. As a consequence of this type of reaction, an altered molecule of reduced biological activity may be produced. These experimental results may explain, in part, the reduction in ATIII levels reported by others in patients with alcoholic liver disease.

A perspective on acetaldehyde concentrations and toxicity in man and animals.

Acetaldehyde (AcH) at a concentration of 593 mM lowers the natural fluorescence of commercial human serum by 12%. It also lowers the fluorescence of a beta-naphthylamine standard curve (recovery) in serum by 17%. These results contrast with earlier reports showing that 447 mM AcH had no effect upon fluorescence of serum or a beta-naphthylamine standard curve in serum. Because 447 mM AcH and 593 mM AcH represent 2.5% and 3.3% AcH, it is apparent that there is a narrow window between which AcH may affect fluorescence by adduct formation with blood components and exogenous fluorophores. Nonetheless, serum has the capacity to bind > 2.5% (> 447 mM) AcH without alteration in fluorescence, suggesting that serum has a great carrying capacity for AcH, undoubtedly in the form of adducts to nucleophiles. These results are discussed in the light of toxicity of AcH and ethanol, the probable significance of the approximately 30 microM free AcH that is reported in chronic alcoholics and the planning of in vitro and in vivo studies with AcH.

Coagulation protein function. IV. Effect of acetaldehyde upon factor X and factor Xa, the proteins at the gateway to the common coagulation pathway.

Acetaldehyde (AcH) (447 mM) exerts an inhibition on Factor Xa, as followed by a clotting assay, but does not inhibit the hydrolysis of the synthetic fluorogenic substrate, N-tBOC-Ile-Glu-Gly-Arg-7-amido-4-methylcoumarin. These data suggest that AcH, although not reacting at the catalytic site of Factor Xa nor at the binding site for the synthetic substrate, does interact with the functional groups on the enzyme that bind to its natural substrate, prothrombin. As a consequence of such interaction, the charge and conformation of Factor Xa is altered, thereby limiting effective activation of prothrombin. Additionally, alkylation of factor Xa may also affect its capacity to associate with Factor Va for the activation of prothrombin. AcH also reacts with Factor X, prolonging clotting times when the zymogen is activated with Russell's viper venom (RVV). It also reduces the rate of hydrolysis of the fluorogenic substrate after activation of the alkylated zymogen by RVV. These data lead to the considerations that AcH-modified Factor X is no longer as effectively activated by RVV due to an alteration of its charge/conformation. Additional possibilities include a likely alkylation of the Factor Xa moiety of Factor X by AcH such that the activation product has an altered charge/conformation compared to native Factor Xa, including possible alkylation of its binding site(s) for prothrombin. The reduced rate of hydrolysis of the synthetic fluorogenic substrate for Factor Xa by the alkylated, activated Factor X lends further support to the generation of a modified Factor Xa by RVV, which may have a lower binding or catalytic rate for the fluorogenic substrate. These results support the suggestion that chronic consumption of alcohol may prolong the reported coagulation times as a result of reaction of alcohol's primary metabolite, AcH, with clotting factors, thereby reducing their physiological potential.

Coagulation protein function. III. Effect of acetaldehyde upon the activation of prothrombin.

When prothrombin is mixed with acetaldehyde (AcH), the primary metabolite of ethanol, at room temperature for 30 min, dialyzed to remove excess AcH, and then activated with Echis carinatus venom (ECV), a prolonged clotting time is observed. Similarly treated prothrombin, however, readily hydrolyzes the synthetic substrate, benzoyl-DL-arginine-beta-naphthylamide (BANA). These results suggest that AcH does not react with the catalytic site of thrombin, which is protected in the prothrombin molecule. However, it does react with susceptible sites on the prothrombin surface which remain alkylated during extensive dialysis to remove excess AcH and subsequent activation of the molecule by ECV. These alkylated sites on the newly formed thrombin molecule may inhibit or prevent the effective/efficient binding of fibrinogen at its binding sites, causing prolonged clotting times. The binding sites for accommodating fibrinogen on the thrombin molecule are apparently quite different from those that bind BANA. These data further suggest that prolonged clotting times that are reported in chronic alcoholics may be due, in part, to ready interaction of acetaldehyde with circulating prothrombin. Fibrinogen, which has been exposed to AcH and subsequently dialyzed to remove excess AcH, also responds with a prolonged clotting time in the presence of added thrombin, signifying that sites on fibrinogen that are essential for its function/conformation are susceptible to AcH.

Acetaldehyde-modified lysozyme function: its potential implication in the promotion of infection in alcoholics.

Incubation of lysozyme with acetaldehyde (0.44 M) at room temperature for 2 h produces a 62% inhibition of enzymic activity. Because the active site cleft contains tryptophyls, asparagine, glutamine, and an arginine residue, and because acetaldehyde reacts with indoles, amides, and guanidines, it is suggested that these sites are likely ones for alkylation. The epsilon-amino groups of lysines on the surface of the molecule are also susceptible to covalent modification. Total acetylation of lysozyme has been reported to inactivate the enzyme. These results suggest the possibility that inactivation of a fraction of the lysozyme activity by acetaldehyde may decrease the effectiveness of the enzyme in chronic alcoholics, thereby leading to an increased potential for susceptibility to bacterial infection.

Coagulation protein function. II. Influence of thiols upon acetaldehyde effects.

It has been reported that prolonged prothrombin time may be a result of the interaction of acetaldehyde (AcH) with clotting proteins to form alkylated inactive products. The current investigation focuses on the influence of L-cysteine (CysH), DL-homocysteine (HC), D-penicillamine, N-acetyl-L-cysteine (NAC), L-serine and L-alanine at 0.01 M concentrations, lactalbumin hydrolysate (2 mg/ml), and 1.0 mM dithiothreitol (DTT) on clotting time as well as their interaction with AcH. The sulfhydryl amino acids, as well as DTT prolonged clotting upon preincubation with plasma. Cysteine and NAC, upon addition to plasma prior to the addition of AcH, exhibited a prolongation of clotting time compared to that of AcH alone. On sequential addition of serine, alanine, or lactalbumin hydrolysate to plasma followed by the addition of acetaldehyde, a prolongation of clotting time comparable to that of AcH alone was exhibited. When HC and penicillamine were added to plasma prior to the addition of AcH, a prolonged clotting time was observed, which was significantly less than that of AcH alone. Premixing of serine, alanine, and lactalbumin hydrolysate with AcH for 20 min prior to addition to the plasma reduced the effectiveness of AcH in prolonging clotting time as compared to successive additions of the amino acid and AcH. Since CysH and penicillamine have been reported to form cyclic adducts with AcH, it is suggested that a similar possibility exists for penicillamine and for HC. The reversible cyclic adduct formation reported for CysH may explain why cysteine did not lower the prolonged clotting time induced by AcH.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaldehyde alters coagulation protein function.

Acetaldehyde is the first metabolite of ethanol and has the potential to react with proteins and alter their function. This study evaluated the function of clotting proteins that had been preincubated with acetaldehyde as compared to those incubated with buffer or ethanol as controls. Thrombin, fibrinogen, thromboplastin, or whole plasma were preincubated with 1.8-447 mM acetaldehyde, 1.7-429 mM ethanol, or buffer for varying time periods prior to use in a clotting assay. Clot formation was measured with an automatic fibrometer. Acetaldehyde prolonged the clotting time but ethanol did not. These experiments indicate that circulating acetaldehyde would have the potential to react with proteins of the clotting system and alter their function. Therefore, it is possible that not all of the abnormalities in coagulation in alcohol abusers result from inadequate hepatic synthesis. Perhaps some of the deranged coagulation may be the result of the interaction of acetaldehyde with coagulation proteins.

Effect of isolated portal hypertension on Kupffer cell function.

The increased incidence of infection in cirrhotics may in part be attributable to dysfunction of the reticuloendothelial system (RES) in removing pathogens from the circulation. The portosystemic shunting (PSS) that results from portal hypertension in cirrhotics may compromise RES function by allowing enteric pathogens to be shunted away from the Kupffer cells. A well-characterized model of portal hypertension induced by partial portal vein ligation (PVL), in which there is no hepatic parenchymal cell damage, was used. Kupffer cell function is unaltered and the effect of PSS alone on overall RES function can be evaluated. In addition to the usual immunologically inert [99mTc]sulfur colloid, an actual pathogen was also evaluated. PVL and sham-ligated rats were given either [99mTc]sulfur colloid or E. coli via the ileocolic vein. The right femurs, lungs, livers and spleens of the animals receiving 99mTc were excised and the radioactivity counted. The lungs, livers, and spleens of the animals receiving E. coli were liquefied and the bacteria were quantified. For both groups the ratios of 99mTc or E. coli in the lung, spleen, and femur to liver were calculated. PVL rats had significantly more 99mTc in the lung, spleen, and femur than the sham rats. There were also significantly more E. coli in the lungs for PVL rats but no significant difference in the spleen counts. These results imply that even in the absence of Kupffer cell dysfunction, PSS alters reticuloendothelial system function by causing a greater distribution of pathogens to the periphery. This altered distribution may contribute to an increased susceptibility to infection in cirrhotics.

Effect of ethanol on Kupffer cell function.

Kupffer cells are resident macrophages in the liver and are important in both local and systemic immune responses. We evaluated the ability of Kupffer cells in vitro to respond to immune stimulation after both acute exposure to ethanol and after long-term ethanol consumption of ethanol. Triplets of female Wistar rats were fed a liquid diet containing 0, 12, or 36% ethanol isocalorically for 112 days. When killed, the Kupffer cells were isolated by collagenase perfusion and adhered to plastic 24-well plates. They were then stimulated with 10 micrograms/ml lipopolysaccharide for 4.5 hr. Synthesis of procoagulant activity (PCA) and tumor necrosis factor (TNF), expressions of macrophage response to immune stimuli, were measured by a one-step clotting assay and L929 cytotoxicity assay, respectively. Within each of the 10 triplets, PCA and TNF levels were normalized and expressed as a percentage of the zero ethanol isocaloric control rat. The high ethanol group had significantly lower baseline and stimulated PCA and TNF levels than the low ethanol group. For evaluation of the effect of acute exposure to ethanol, Kupffer cells were stimulated with lipopolysaccharide and varying concentrations (0-400 mg/dl) of ethanol. Cells were incubated for 4.5 hr and assayed for PCA and TNF activity. There was dose-dependent inhibition of PCA and TNF, with increasing concentrations of ethanol. These results indicate that whereas exposure to high levels of ethanol depresses Kupffer cell function, lower levels may be immunostimulatory.

Procoagulant activity and tumor necrosis factor in rat hepatic allograft rejection.

We used a model of rat hepatic allograft rejection to evaluate levels of procoagulant activity and tumor necrosis factor during acute cellular rejection. ACI livers were transplanted into Lewis rats, and Lewis-to-Lewis isografts and unoperated animals served as controls. Animals were killed on days 1, 2, 3, 4, 5, 6, 7 and 9. Splenic mononuclear cells were obtained by Ficoll-Hypaque gradients. Collagenase perfusion, metrizamide gradients and centrifugal elutriation were used to isolate Kupffer cells. Procoagulant activity assay of the splenic and Kupffer cells was done using a one-step clotting assay. Tumor necrosis factor was assayed using an L929 cytotoxicity assay. Histological evidence of acute rejection began on the 4th postoperative day, and rats died by the 9th or 10th postoperative day. Splenic procoagulant activity was significantly elevated in rejecting rats on day 4 and remained elevated until death. In contrast, Kupffer-cell procoagulant activity was elevated over controls by day 3 and remained significantly elevated until death. The tumor necrosis factor levels were elevated from day 1 and remained so until death. The data indicate that procoagulant activity is synthesized both by peripheral monocytes and locally by Kupffer cells and that procoagulant activity and tumor necrosis factor levels rise during hepatic allograft rejection. Because procoagulant activity and tumor necrosis factor mediate immune functions that are postulated to be important in acute rejection (immune cell adherence, vascular thrombosis and delayed-type hypersensitivity), these elevations may contribute to the pathogenesis of acute rejection.

Orthotopic liver transplantation for alcoholic liver disease.

Alcohol abuse is the most common cause of end-stage liver disease in the United States, but many transplant centers are unwilling to accept alcoholic patients because of their supposed potential for recidivism, poor compliance with the required immunosuppression regimen and resulting failure of the allograft. There is also concern that alcohol-induced injury in other organs will preclude a good result. From July 1, 1982, to April 30, 1988, 73 patients received orthotopic liver transplants at the University of Pittsburgh for end-stage alcoholic liver disease. Fifty-two (71%) of these were alive at 25 +/- 9 mo (mean +/- S.D.) after transplantation, when a phone survey of these patients, their wives/husbands, and their physicians was performed to evaluate their subsequent use of alcohol, current medical condition and employment. Data obtained were compared with those for nonalcoholic patients selected as transplant controls. The recidivism rate has been 11.5%, with most patients drinking only socially. Fifty-four percent of the survivors are employed, 21% classify themselves as homemakers and only 11 (21%) are unable to work. Twenty-one patients died after transplantation; the most frequent cause of death was sepsis (43%), and intraoperative death was the next most common cause (28.6%). These data demonstrate that alcoholic patients can be transplanted successfully and achieve good health not significantly different from that of individuals transplanted for other causes. Thus orthotopic liver transplantation is a therapeutic option that should be considered for individuals with end-stage alcoholic liver disease who desire such therapy.

A case presentation of hemolytic anemia in ulcerative colitis and review of the literature.

Autoimmune hemolytic anemia associated with ulcerative colitis is a rare occurrence, and its management poses a therapeutic dilemma. Herein we present a case of ulcerative colitis involving an autoimmune hemolytic anemia and discuss the proposed mechanisms for the anemia. A review of past reported cases and their outcomes is included. In the past, a patient with autoimmune anemia unresponsive to corticosteroids was subjected first to splenectomy and then to colectomy if splenectomy failed to resolve the anemia. In view of the high failure rate for splenectomy, we recommend that ulcerative colitis patients with anemia unresponsive to corticosteroids proceed directly to total colectomy.