Mark W. Swaim

Methionine sulfoxide and the oxidative regulation of plasma proteinase inhibitors.

The sensitivity of methionine residues to oxidation is a mechanism by which many proteins, including plasma proteinase inhibitors, may be oxidatively inactivated. Much evidence suggests that methionine oxidation and concurrent losses of protein activity not only occur widely in living systems but are physiologic, homeostatic processes. Neutrophils, macrophages and other leukocytes secrete large quantities of powerful oxidants at sites of inflammation and may readily bring about methionine oxidative inactivation of proteins. In particular, oxidation of proteinase inhibitors may favorably alter the proteinase‐antiproteinase balance to facilitate tissue remodeling and protection from invading organisms. Leukocyte‐mediated inhibitor oxidation also appears to regulate local immunosuppressive activity. Pathophysiologic processes such as emphysema and rheumatoidal disease involve derangements of these homeostatic mechanisms.

Ascites fluid as a possible origin for hyperfibrinolysis in advanced liver disease

OBJECTIVES:Advanced liver disease is associated with both exaggerated fibrinolysis and with ascites. This study was undertaken to determine whether fibrinolytic activity exists in the ascites fluid of patients with liver disease and to see whether such activity is associated with evidence of plasma fibrinolysis.METHODS:Both the ascites fluid and plasma from 15 patients with cirrhotic ascites (group A) were evaluated for markers of fibrinolysis: fragment D-dimer, plasminogen, fibrinogen, and fibrin split products. In addition, the euglobulin lysis time, a test highly specific for fibrinolysis, was evaluated in the ascites fluid samples. As a control group, the plasma from 15 cirrhotic patients without ascites (group B) was evaluated for markers of fibrinolysis.RESULTS:In group A, elevated fragment D-dimer and fibrin split products were uniformly found in ascites fluid in concentrations that would be considered pathologically elevated if in plasma. Ascites fluid was also depleted, compared with plasma, of both plasminogen and fibrinogen. These results, along with the short euglobulin lysis time in 83% of the patients, suggest that increased fibrinolytic activity is present in ascites fluid. In 93% of these patients, plasma D-dimer was elevated. The mean plasma plasminogen was also low in these patients. In group B, only 33% of patients had elevated plasma D-dimer.CONCLUSIONS:Ascites fluid has fibrinolytic activity. Because ascites fluid reenters the systemic circulation via the thoracic duct, via a natural peritoneovenous shunt, ascites fluid warrants serious consideration as a pathological fluid that contributes to the systemic fibrinolytic state found in the majority of our patients with ascites.

Selectivity and stereospecificity of the reactions of dichlorodiammineplatinum (II) with three purified plasma proteins

The reactions of cis- and trans-dichlorodiammineplatinum(II) (cis- and trans-DDP) with albumin and two plasma proteinase inhibitors were compared. Reaction with alpha 2-macroglobulin (alpha 2M) resulted in subunit crosslinking and loss of proteinase binding activity. The reaction also modified a receptor recognition site present on each alpha 2M subunit. While more trans-DDP was incorporated into alpha 2M than cis-DDP, cis-DDP was more effective at blocking receptor recognition, alpha 1-proteinase inhibitor was also inactivated by reaction with either cis- or trans-DDP. These reactions resulted in binding of platinum to methionine-358 at the reactive center of this inhibitor. Trans-DDP, however, was less selective and also bound to the single cysteine residue (Cys-232) of alpha 1PI. Reaction of albumin with cis-DDP resulted in incorporation of about 1 mol platinum per mol protein, and this platinum modified the single cysteine (Cys-34) in the molecule. Albumin incorporated twice as much trans-DDP, but the binding did not involve cysteine-34. In general, reactions of cis-DDP with proteins appear to be more selective than those observed for modification with the trans isomer.

Characterization of platelet aggregation induced by human colon adenocarcinoma cells and its inhibition by snake venom peptides, trigramin and rhodostomin

SUMMARY. SW‐480 cells, derived from a primary human colon adenocarcinoma, caused dose‐dependent platelet aggregation in heparinized human platelet‐rich plasma. SW‐480 tumour cell‐induced platelet aggregation (TCIPA) was completely inhibited by hirudin (5 U/ml) but unaffected by apyrase (10 U/ml). This TCIPA was also unaffected by cysteine proteinase inhibition with E‐64 (10 μM) but was limited by cell pretreatment with phospholipase A2. SW‐480 cell suspension caused marked dose‐dependent decreases in plasma recalcification times using normal, factor VIII‐deficient and factor IX‐deficient human plasma. This effect was potentiated with cell lysates but inhibited in intact cells pretreated with sphingosine. SW‐480 cell suspension did not affect the recalcification time of factor VII‐deficient plasma. Moreover, monoclonal antibody against human tissue factor completely abolished SW‐480 TCIPA. Taken together, these data suggest that SW‐480 TCIPA arises from SW‐480 tissue factor activity expression. Trigramin and rhodostomin, RGD‐containing snake venom peptides, which antagonize the binding of fibrinogen to platelet membrane glycogen IIb/IIIa, prevented SW‐480 TCIPA. Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against platelet membrane glycoprotien IIb/IIIa and Ib prevent SW‐480 TCIPA, which was unaffected by control peptide GRGES. On a molar basis, trigramin (IC50 0.09 μM) and rhodostomin (IC50 0.03 μM) were about 6000 and 18000 times, respectively, more potent than GRGDS (IC50 0.56mM).

Case Report: Reversible Nefazodone-Induced Liver Failure

In a comprehensive review of data involving more than 3500 patients treated with nefazodone worldwide, no fatalities or cases of hepatitis or fulminant or subfulminant liver failure were reported (1). Nefazodone is a newer antidepressant that differs from selectively acting serotonergic agents used to treat depression. It inhibits synaptosomal uptake of serotonin and norepinephrine (2, 3). Because it has less anticholinergic, a1-adrenolytic, and antihistaminic activities than other agents, nefazodone is highly attractive for the treatment of depression due to its high safety profile. However, a recent report described three cases of fulminant liver failure attributed to the use of nefazodone (4). We report a case of severe hepatocellular jaundice that was successfully managed by withdrawal of nefazodone.

Characterization of platelet aggregation induced by human breast carcinoma and its inhibition by snake venom peptides, trigramin and rhodostomin

SummaryMCF-7 cells, a metastatic human breast carcinoma line, caused dose-dependent platelet aggregation in heparinized human platelet-rich plasma (PRP). MCF-7 tumor cell-induced platelet aggregation (TCIPA) was almost blocked by apyrase (0.5 U/ml) and completely inhibited by hirudin (5 U/ml). This TCIPA was unaffected by cysteine proteinase inhibition with E-64 (10 µM), but was limited by cell pretreatment with phospholipase A2. MCF-7 cell suspension caused marked, dose-dependent decrease in plasma recalcification times using normal, Factor VIII-deficient, and Factor IX-deficient human plasma. This effect was potentiated in cell lysates but was inhibited in intact cells preincubated with sphingosine. MCF-7 cell suspension did not affect the recalcification time of Factor VII-deficient plasma. Taken together, these data suggest that MCF-7 TCIPA arises from MCF-7 tisssue factor activity expression. Trigramim and rhodostomin, RGD-containing snake venom peptides which antagonized the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa, prevented MCF-7 TCIPA. Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against human tissue factor, platelet membrane glycoprotein IIb/IIIa and Ib prevented MCF-7 TCIPA, which was unaffected by control peptide GRGES. On a molar basis, trigramin (IC50, 0.1 µM) and rhodostomin (IC50, 0.03 µM), were about 5,000 and 18,000 times, respectively, more potent than GRGDS (IC50, 0.54 mM).

Characterisation of platelet aggregation induced by PC-3 human prostate adenocarcinoma cells and inhibited by venom peptides, trigramin and rhodostomin

PC-3 cells, a metastatic human prostate adenocarcinoma line, caused dose-dependent platelet aggregation in heparinised human platelet-rich plasma (PRP). PC-3 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin (5 U/ml) and limited by increasing concentrations of apyrase. This TCIPA was unaffected by cysteine proteinase inhibition with E-64 (10 microM), but was limited by cell pretreatment with phospholipase A2. PC-3 cell suspension caused marked, dose-dependent decreases in plasma recalcification times using normal, Factor VIII-deficient and Factor IX-deficient, but not Factor VII-deficient, human plasma. This effect was potentiated in cell lysates, but was inhibited in intact cells preincubated with sphingosine. Overall, these data suggest that PC-3 TCIPA arises from PC-3 tissue factor activity expression. Trigramin and rhodostomin, RGD-containing snake venom peptides which antagonise the binding of fibrinogen to platelet membrane glycoprotein IIb-IIIa, prevented PC-3 TCIPA. Similarly, synthetic peptide GRGDS as well as monoclonal antibodies against platelet membrane glycoproteins IIb-IIIa and Ib prevented PC-3 TCIPA, which was unaffected by control peptide GRGDS. On a molar basis, trigramin (IC50, 0.11 microM) and rhodostomin (IC50, 0.03 microM) were approximately 5000 and 18000 times, respectively, more potent than GRGDS (IC50, 0.56 mM).