Inhibition of thiol isomerase activity diminishes endothelial activation of plasminogen, but not of protein C.

INTRODUCTION: Cell surface thiol isomerase enzymes, principally protein disulphide isomerase (PDI), have emerged as important regulators of platelet function and tissue factor activation via their action on allosteric disulphide bonds. Allosteric disulphides are present in other haemostasis-related proteins, and we have therefore investigated whether thiol isomerase inhibition has any influence on two endothelial activities relevant to haemostatic regulation, namely activation of protein C and activation of plasminogen, with subsequent fibrinolysis. MATERIALS AND METHODS: The study was performed using the human microvascular endothelial cell line HMEC-1. Thiol isomerase gene expression was measured by RT-PCR and activation of protein C and plasminogen by cell-based assays using chromogenic substrates S2366 and S2251, respectively. Cell mediated fibrinolysis was measured by monitoring absorbance at 405 nm following fibrin clot formation on the surface of HMEC-1 monolayers. RESULTS AND CONCLUSIONS: A variety of thiol isomerase enzymes, including PDI, were expressed by HMEC-1 cells and thiol reductase activity detectable on the cell surface was inhibited by both RL90 anti-PDI antibody and by the PDI inhibitor quercetin-3-rutinoside (rutin). In cell-based assays, activation of plasminogen, but not of protein C, was inhibited by RL90 antibody and, to a lesser extent, by rutin. Fibrin clot lysis occurring on a HMEC-1 monolayer was also significantly slowed by RL90 antibody and by rutin, but RL90-mediated inhibition was abolished in the presence of exogenous tissue plasminogen activator (tPA). We conclude that thiol isomerases, including PDI, are involved in fibrinolytic regulation at the endothelial surface, although not via a direct action on tPA. These findings broaden understanding of haemostatic regulation by PDI, and may aid in development of novel anti-thrombotic therapeutic strategies targeted via the fibrinolysis system.

S-nitrosothiols as selective antithrombotic agents - possible mechanisms.

S-nitrosothiols have a number of potential clinical applications, among which their use as antithrombotic agents has been emphasized. This is largely because of their well-documented platelet inhibitory effects, which show a degree of platelet selectivity, although the mechanism of this remains undefined. Recent progress in understanding how nitric oxide (NO)-related signalling is delivered into cells from stable S-nitrosothiol compounds has revealed a variety of pathways, in particular denitrosation by enzymes located at the cell surface, and transport of intact S-nitrosocysteine via the amino acid transporter system-L (L-AT). Differences in the role of these pathways in platelets and vascular cells may in part explain the reported platelet-selective action. In addition, emerging evidence that S-nitrosothiols regulate key targets on the exofacial surfaces of cells involved in the thrombotic process (for example, protein disulphide isomerase, integrins and tissue factor) suggests novel antithrombotic actions, which may not even require transmembrane delivery of NO.

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery.

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 microM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) (100 microM) or inhibiting PDI with bacitracin (5mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 microM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 microM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.

Rapid S-nitrosothiol metabolism by platelets and megakaryocytes.

RSNOs (S-nitrosothiols) regulate platelet and megakaryocyte function, and may act in vivo as a nitric oxide reservoir. There is a discrepancy between the spontaneous rate of NO release from different RSNO compounds and their pharmacological effects, implying that target cells may mediate biological activity either by metabolism of RSNOs or by displaying cell surface receptors. In the present study, we sought evidence for rapid cell-mediated metabolism of RSNOs. Exposure of platelets to GSNO (S-nitrosoglutathione) for as little as 5 s inhibited thrombin-induced platelet aggregation by >95%; however, AlbSNO (S-nitrosoalbumin) was much less effective over these short time periods. Incubation of 1 microM GSNO or AlbSNO with platelets and megakaryocytes resulted in a 25-34% loss of RSNO recoverable from the supernatant (P <0.02) within 30 s. This rapid cell-mediated RSNO decay did not progress further over 5 min, and could not be accounted for by release of free NO. The gamma-glutamyl transpeptidase inhibitor acivicin (100 microM) partially decreased GSNO decay, whereas the membrane-impermeable thiol-blocking agent 5,5'-dithiobis-(2-nitrobenzoic acid) (100 microM) completely blocked cell-mediated GSNO decay and partially blocked AlbSNO decay. Our results highlight differences between high- and low-molecular-mass RSNOs with regard to their rapid metabolism/uptake and subsequent cellular responses, and indicate a critical role for extracellular thiols in RSNO metabolism by platelets and megakaryocytes.

Mitochondrial superoxide production during oxalate-mediated oxidative stress in renal epithelial cells.

Crystals of calcium oxalate monohydrate (COM) in the renal tubule form the basis of most kidney stones. Tubular dysfunction resulting from COM-cell interactions occurs by mechanism(s) that are incompletely understood. We examined the production of reactive oxygen intermediates (ROI) by proximal (LLC-PK1) and distal (MDCK) tubular epithelial cells after treatment with COM (25-250 microg/ml) to determine whether ROI, specifically superoxide (O(2)(*-)), production was activated, and whether it was sufficient to induce oxidative stress. Employing inhibitors of cytosolic and mitochondrial systems, the source of ROI production was investigated. In addition, intracellular glutathione (total and oxidized), energy status (ATP), and NADH were measured. COM treatment for 1-24 h increased O(2)(*-) production 3-6-fold as measured by both lucigenin chemiluminescence in permeabilized cells and dihydrorhodamine fluorescence in intact cells. Using selective inhibitors we found no evidence of cytosolic production. The use of mitochondrial probes, substrates, and inhibitors indicated that increased O(2)(*-) production originated from mitochondria. Treatment with COM decreased glutathione (total and redox state), indicating a sustained oxidative insult. An increase in NADH in COM-treated cells suggested this cofactor could be responsible for elevating O(2)(*-) generation. In conclusion, COM increased mitochondrial O(2)(*-) production by epithelial cells, with a subsequent depletion of antioxidant status. These changes may contribute to the reported cellular transformations during the development of renal calculi.

Uremic concentrations of guanidino compounds inhibit neutrophil superoxide production.

BACKGROUND: In uremia, diminished reactive oxygen intermediate (ROI) production is an important consequence of impaired neutrophil function. We have studied the effect of guanidino compounds, known uremic toxins, on neutrophil ROI production in vitro. METHODS: Neutrophils from healthy volunteers were exposed for three hours to individual or mixed guanidino compounds (GCmix) at concentrations encountered in uremic plasma. After removal of guanidino compounds, neutrophils were activated by adhesion, N-formyl-methionyl-leucyl-phenyalanine (fMLP), phorbol 12-myristate 13-acetate (PMA), or opsonized zymosan, and superoxide production was measured by lucigenin chemiluminescence (CL). The direct effect of guanidino compounds on superoxide production in activated neutrophils was also measured. The energy status (ATP and creatine phosphate), antioxidant status (total glutathione), and glycolytic flux (lactate production) were measured. RESULTS: The GCmix pretreatment decreased the superoxide production in activated neutrophils (fMLP or zymosan) by 50% (P < 0.01) and the ATP concentration by 60% (P < 0.05), and it inhibited glycolytic flux (lactate production) by 45% (P < 0.01), but did not alter glutathione concentration. Simultaneous exposure to GCmix and activation did not inhibit nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in cell lysates, but inhibited superoxide formation in zymosan-activated intact neutrophils, and this inhibition was reversed following removal of the guanidino compounds. CONCLUSION: Guanidino-succinate, -propionate, and -butyrate were individually as potent as the GCmix. Inhibition of neutrophil superoxide generation by guanidino compounds results from a depressed energy status. Uremic concentrations of guanidino compounds significantly inhibit neutrophil metabolism, and this has serious implications for their function in host defense.

S-nitrosothiols are stored by platelets and released during platelet-neutrophil interactions.

Interaction between platelets and neutrophils is important in vascular injury. We have investigated the storage and release of nitric oxide (NO) by platelets interacting with neutrophils. Shear-activated platelets were added to neutrophils in suspension and both superoxide and peroxynitrite formations monitored by lucigenin- and luminol-enhanced chemiluminescence. In addition, intraplatelet S-nitrosothiols were measured by dichlorofluorescein fluorescence following displacement of NO by mercuric chloride. Addition of activated platelets to neutrophils caused free radical production and platelet-neutrophil rosette formation. Pretreatment of platelets with 20 microM S-nitrosoglutathione changed the balance between luminol and lucigenin chemiluminescence in favor of luminol, whereas S-nitrosoglutathione in platelet-free plasma did not produce these changes. This pattern was also observed both following inhibition of neutrophil NO synthase and in a neutrophil-free superoxide-generating system. Inhibition of platelet NO synthase decreased luminol and increased lucigenin chemiluminescence. These effects were reversed by L-arginine. Platelet activation increased intraplatelet S-nitrosothiols from 1.93+/-0.19 (mean +/- SD) to 4.9+/-1.10 x 10(-18) mol/platelet (P < 0.01); this increase halved following NO synthase inhibition, but was enhanced by approximately 220% following incubation with S-nitrosoglutathione. These results show that during shear stress platelets store S-nitrosothiols, which can be derived either endogenously from NO synthesis or exogenously by sequestration of S-nitrosoglutathione. Release of stored NO during platelet-neutrophil interaction alters the interaction of NO with superoxide and could modulate vascular inflammation.

Evidence for a cyclic GMP-independent mechanism in the anti-platelet action of S-nitrosoglutathione.

1. We have measured the ability of a range of NO donor compounds to stimulate cyclic GMP accumulation and inhibit collagen-induced aggregation of human washed platelets. In addition, the rate of spontaneous release of NO from each donor has been measured spectrophotometrically by the oxidation of oxyhaemoglobin to methaemoglobin. The NO donors used were five s-nitrosothiol compounds: S-nitrosoglutathione (GSNO), S-nitrosocysteine (cysNO), S-nitroso-N-acetyl-DL-penicillamine (SNAP), S-nitroso-N-acetyl-cysteine (SNAC), S-nitrosohomocysteine (homocysNO), and two non-nitrosothiol compounds: diethylamine NONOate (DEANO) and sodium nitroprusside (SNP). 2. Using 10 microM of each donor compound, mean+/-s.e.mean rate of NO release ranged from 0.04+/-0.001 nmol min(-1) (for SNP) to 3.15+/-0.29 nmol min(-1) (for cysNO); cyclic GMP accumulation ranged from 0.43+/-0.05 pmol per 10(8) platelets (for SNP) to 2.67+/-0.31 pmol per 10(8) platelets (for cysNO), and inhibition of platelet aggregation ranged from 40+/-6.4% (for SNP) to 90+/-3.8% (for SNAC). 3. There was a significant positive correlation between the rate of NO release and the ability of the different NO donors to stimulate intra-platelet cyclic GMP accumulation (r = 0.83; P = 0.02). However, no significant correlation was observed between the rate of NO release and the inhibition of platelet aggregation by the different NO donors (r= -0.17), nor was there a significant correlation between cyclic GMP accumulation and inhibition of aggregation by the different NO donor compounds (r = 0.34). 4. Comparison of the dose-response curves obtained with GSNO, DEANO and 8-bromo cyclic GMP showed DEANO to be the most potent stimulator of intraplatelet cyclic GMP accumulation (P < 0.001 vs both GSNO and 8-bromo cyclic GMP), but GSNO to be the most potent inhibitor of platelet aggregation (P < 0.01 vs DEANO, and P < 0.001 vs 8-bromo cyclic GMP). 5. The rate of NO release from GSNO, and its ability both to stimulate intra-platelet cyclic GMP accumulation and to inhibit platelet aggregation, were all significantly diminished by the copper (I) (Cu+) chelating agent bathocuproine disulphonic acid (BCS). In contrast, BCS had no effect on either the rate of NO release, or the anti-platelet action of the non-nitrosothiol compound DEANO. 6. Cyclic GMP accumulation in response to GSNO (10(-9) 10(-5) M) was undetectable following treatment of platelets with ODQ (100 microM), a selective inhibitor of soluble guanylate cyclase. Despite this abolition of guanylate cyclase stimulation, GSNO retained some ability to inhibit aggregation, indicating the presence of a cyclic GMP-independent component in its anti-platelet action. However, this component was abolished following treatment of platelets with a combination of both ODQ and BCS, suggesting that Cu+ ions were required for the cyclic GMP-independent pathway to operate. 7. The cyclic GMP-independent action of GSNO, observed in ODQ-treated platelets, could not be explained by an increase in intra-platelet cyclic AMP. 8. The impermeable thiol modifying agent p-chloromercuriphenylsulphonic acid (CMPS) produced a concentration-dependent inhibition of aggregation of ODQ-treated platelets, accompanied by a progressive loss of detectable platelet surface thiol groups. Additional treatment with GSNO failed to increase the degree of aggregation inhibition, suggesting that a common pathway of thiol modification might be utilized by both GSNO and CMPS to elicit cyclic GMP-independent inhibition of platelet aggregation. 9. We conclude that NO donor compounds mediate inhibition of platelet aggregation by both cyclic GMP-dependent and -independent pathways. Cyclic GMP generation is related to the rate of spontaneous release of NO from the donor compound, but transfer of the NO signal to the cyclic GMP-independent pathway may depend upon a cellular system which involves both copper (I) (Cu+) ions and surface membrane thiol groups. The potent anti-platelet action of GSNO

Interaction of macrophage-migration-inhibitory factor with haematin.

Macrophage-migration-inhibitory factor (MIF) is retained by S-hexylglutathione-agarose but is not specifically eluted in high yield. Human liver MIF was purified in high yield using retention by phenyl-agarose at low ionic strength and cation-exchange FPLC as described for bovine lens MIF [Rosengren, Bucala, Aman, Jacobsson, Odh, Metz and Rorsman (1996) Mol. Med. 2, 143-149]. The l-dopachrome methyl ester tautomerase activity of human liver MIF was not inhibited by a variety of glutathione S-conjugates, eicosanoids or glucocorticoids but was very sensitive to inhibition by haematin (IC50 100-200 nM). The inhibition was non-competitive and showed positive co-operativity (h=5.8). Similar sensitivity to haematin was obtained with purified recombinant human MIF. The sensitivity of MIF to haematin is approx. 1000-fold greater than for any previously described ligands, and is within its physiological range. Therefore the interaction is likely to be important in modulating the function of MIF in the initiation of immune responses.

How cytotoxic is nitric oxide?

Nitric oxide (NO) shows an unusual divergence of action, being utilised both as a physiological signalling molecule, and as a toxic mediator. NO-mediated cellular injury may arise by a variety of mechanisms, including disruption of mitochondrial respiration, enzyme inhibition, lipid peroxidation and genetic mutation. Toxicity is largely mediated via intermediates such as N2O3 and peroxynitrite, arising from the reaction of NO with either molecular oxygen or reactive oxygen species. In general, such reactions become significant only when high concentrations of NO are generated by the induction of nitric oxide synthase.

Cell-mediated biotransformation of S-nitrosoglutathione.

Spontaneous release of nitric oxide (NO) from S-nitrosothiols cannot explain their bioactivity, suggesting a role for cellular metabolism or receptors. Using immortalised cells and human platelets, we have identified a cell-mediated mechanism for the biotransformation of the physiological S-nitrosothiol compound S-nitrosoglutathione (GSNO) into nitrite. We suggest the name "GSNO lyase" for this activity. GSNO lyase activity varied between cell types, being highest in a fibroblast cell line and lowest in platelets. In NRK 49F fibroblasts, GSNO lyase mediated a saturable, GSNO concentration-dependent accumulation of nitrite in conditioned medium, which was inhibited both by transition metal chelators, and by subjecting cells to oxidative stress using a combination of the thiol oxidant diamide and Zn2+, a glutathione reductase inhibitor. Activity was resistant, however, to both acivicin, an inhibitor of gamma-glutamyl transpeptidase (EC 2.3.2.2), and to ethacrynic acid, an inhibitor of Pi class glutathione-S-transferases (EC 2.5.1.18), thus neither of these enzymes could account for NO release. Although GSNO lyase does not explain the platelet-selective pharmacological properties of GSNO, cellular biotransformation suggests therapeutic avenues for targeted delivery of NO to other tissues.

Role of a copper (I)-dependent enzyme in the anti-platelet action of S-nitrosoglutathione.

1. S-nitrosoglutathione (GSNO) is a potent and selective anti-platelet agent, despite the fact that its spontaneous rate of release of nitric oxide (NO) is very slow. Our aim was to investigate the mechanism of the anti-aggregatory action of GSNO. 2. The biological action of GSNO could be mediated by NO released from S-nitrosocystylglycine, following enzymatic cleavage of GSNO by gamma-glutamyl transpeptidase. The anti-aggregatory potency of GSNO was not, however, altered by treatment of target platelets with the gamma-glutamyl transpeptidase inhibitor acivicin (1 mM). gamma-Glutamyl transpeptidase is not, therefore, involved in mediating the action of GSNO. 3. The rate of breakdown of S-nitrosoalbumin was increased from 0.19 +/- 0.086 nmol min-1 to 1.52 +/- 0.24 nmol min-1 (mean +/- s.e.mean) in the presence of cysteine (P < 0.05, n = 4). Inhibition of platelet aggregation by S-nitrosoalbumin was also significantly increased by cysteine (P < 0.05, n = 4), suggesting that the biological activity of S-nitrosoalbumin is mediated by exchange of NO from the protein carrier to form the unstable compound cysNO. Breakdown of GSNO showed a non-significant acceleration in the presence of cysteine, from 0.56 +/- 0.22 to 1.77 +/- 0.27 nmol min-1 (mean +/- s.e.mean) (P = 0.064, n = 4), and its ability to inhibit platelet aggregation was not enhanced by cysteine. This indicates that the anti-platelet action of GSNO is not dependent upon transnitrosation to form cysNO. 4. Platelets pretreated with the copper (I)-specific chelator bathocuproine disulphonic acid (BCS), then resuspended in BCS-free buffer, showed resistance to the inhibitory effect of GSNO. These findings suggest that BCS impedes the action of GSNO by binding to structures on the platelet, rather than by chelating free copper in solution. 5. Release of NO from GSNO was catalysed enzymatically by ultrasonicated platelet suspensions. This enzyme had an apparent K(m) for GSNO of 12.4 +/- 2.64 microM and a Vmax of 0.21 +/- 0.03 nmol min-1 per 10(8) platelets (mean +/- s.e.mean, n = 5). It was inhibited by BCS, but not by the iron chelator bathophenathroline disulphonic acid, nor by acivicin. 6. We conclude that the stable S-nitrosothiol compound GSNO may exert its anti-platelet action via enzymatic, rather than spontaneous release of NO. This is mediated by a copper-dependent mechanism. The potency and platelet-selectivity of GSNO may result from targeted NO release at the platelet surface.

Copper chelation-induced reduction of the biological activity of S-nitrosothiols.

1. The effect of copper on the activity of the S-nitrosothiol compounds S-nitrosocysteine (cysNO) and S-nitrosoglutathione (GSNO) was investigated, using the specific copper chelator bathocuproine sulphonate (BCS), and human washed platelets as target cells. 2. Chelation of trace copper with BCS (10 microM) in washed platelet suspensions reduced the inhibition of thrombin-induced platelet aggregation by GSNO; however, BCS had no significant effect on the anti-aggregatory action of cysNO. BCS inhibited cyclic GMP generation in response to both cysNO and GSNO. 3. The effect of BCS was rapid (within 30 s), and could be abolished by increasing the platelet concentration to 500 x 10(9) l-1. 4. In BCS-treated platelet suspensions, the addition of Cu2+ ions (0.37-2.37 microM) led to a restoration of both guanylate cyclase activation and platelet aggregation inhibition by GSNO. 5. The anti-aggregatory activity of GSNO was reduced in a concentration-dependent manner by the copper (I)-specific chelators BCS and neocuproine, and to a smaller extent by desferal. No effect was observed with the copper (II) specific chelator, cuprizone, the iron-specific chelator, bathophenanthroline sulphonate, or the broader-specificity copper chelator, D-penicillamine. 6. In both BCS-treated and -untreated platelet suspensions, cys NO was more potent than GSNO as a stimulator of guanylate cyclase. In BCS-treated platelet suspensions there was no significant difference between the anti-aggregatory potency of cysNO and GSNO; however, in untreated suspensions, GSNO was significantly more potent than cysNO. Thus, when copper was available, GSNO produced a greater inhibition of aggregation than cysNO, despite being a less potent activator of guanylate cyclase. 7. The breakdown of cysNO and GSNO was measured spectrophotometrically by decrease in absorbance at 334 nm. In Tyrode buffer, cysNO (10 microM) broke down at a rate of 3.3 microM min-1. BCS (10 microM)reduced this to 0.5 microM min-1. GSNO, however, was stable, showing no fall in absorbance over a period of 7 min even in the absence of BCS.8. We conclude that copper is required for the activity of both cysNO and GSNO, although its influence on anti-aggregatory activity is only evident with GSNO. The stimulatory effect of copper is unlikely to be explained solely by catalysis of S-nitrosothiol breakdown. The enhancement by copper of the anti-aggregatory activity of GSNO, relative to cysNO, suggests that copper may be required for biological activity of GSNO which is independent of guanylate cyclase stimulation.

Loss of biological activity of arginine vasopressin during its degradation by vasopressinase from pregnancy serum.

BACKGROUND AND OBJECTIVE: Degradation of AVP by placental vasopressinase may precipitate gestational diabetes insipidus, which in some cases is accompanied by pre-eclampsia. Abnormally elevated vasopressinase has also been reported in pre-eclampsia without diabetes insipidus. This association between excessive vasopressinase production and pre-eclampsia might be explained if the products of AVP degradation by vasopressinase retained pressor activity even after anti-diuretic activity had been destroyed. Recent evidence indicates that such products may raise blood pressure in rats. The objective of this study was, therefore, to purify vasopressinase and investigate its action on both the V1 and V2 receptor-stimulating activity of AVP. DESIGN: Vasopressinase was purified from pooled pregnancy serum by ammonium sulphate precipitation, followed by sequential ion exchange, lentil lectin affinity and gel filtration chromatography. Purified enzyme was then used to degrade AVP and the loss of both immunoreactivity and biological activity monitored. Loss of V1 receptor-stimulating activity and V2 receptor-stimulating activity was compared by two-way ANOVA. PATIENTS: Blood was obtained from healthy women between week 34 and the end of pregnancy. Pooled serum from 20-30 patients was used as starting material for the purification of vasopressinase. MEASUREMENTS: AVP immunoreactivity was measured by radioimmunoassay, V1 receptor-stimulating activity by a platelet aggregation bioassay, and V2 receptor-stimulating activity by adenylate cyclase stimulation in LLC-PK1 target cells. RESULTS: Purified vasopressinase was a dimeric protein of molecular weight 330 kDa, which cleaved the synthetic substrate S-benzyl-L-cysteine-4-nitroanilide with a Km of 0.33 mM. Incubation of AVP (0.1 mM) with vasopressinase (0.66 milligrams) at 37 degrees C led to a parallel loss of both AVP immunoreactivity and biological activity. The rates of loss of V1 and V2 receptor mediated activities were not significantly different. CONCLUSIONS: We report the first direct comparison between the loss of V1 and V2 receptor mediated activities of vasopressinase degraded AVP. There was no significant retention of V1, relative to V2, receptor mediated activity. AVP degradation products are unlikely to be pathogenic in hypertensive pregnancy.

Serum Vasopressinase and Platelet Responses to Arginine Vasopressin in Normal Pregnancy, Pregnancy-induced Hypertension and Pre-eclampsia.

Pregnancy is marked by the placental production of vasopressinase, an enzyme which accelerates the metabolic clearance of arginine vasopressin (AVP), and serum vasopressinase is reported to be abnormally elevated in hypertensive pregnancy. Since platelet AVP binding capacity is influenced by the plasma AVP concentration, we sought to determine whether alterations in vasopressinase and AVP concentrations affect platelet responsiveness to AVP in normotensive and hypertensive pregnancy. Four groups of 10 women were studied: non-pregnant subjects, normotensive pregnancy, hypertensive pregnancy, and pre-eclampsia (PE). AVP-induced platelet aggregation was measured turbidometrically, serum vasopressinase by chromogenic assay and plasma AVP by radioimmunoassay. Platelet responses to AVP were similar in all groups, as were plasma concentrations of AVP. Vasopressinase was raised in all pregnant patients compared with non-pregnant subjects, and levels were significantly higher in pregnancy-induced hypertension than in either the normotensive or PE groups (p<0.05). No significant correlation was observed between platelet responsiveness to AVP and circulating concentrations of either AVP or vasopressinase. Thus circulating vasopressinase is increased in pregnancy and abnormally so in hypertensive pregnancy. This does not, however, appear to influence ex vivo platelet responsiveness to AVP.

Platelets from patients on haemodialysis show impaired responses to nitric oxide.

1. Platelets from patients on haemodialysis showed a loss of sensitivity to nitric oxide, reflected by a reduction in the ability of nitric oxide both to inhibit thrombin-induced aggregation and to increase intraplatelet cyclic GMP levels. Responses of platelets from patients on continuous ambulatory peritoneal dialysis were slightly, but not significantly, impaired. Platelets from both groups of uraemic patients showed normal sensitivity to the cyclic AMP-dependent inhibitor prostacyclin. 2. Reduced levels of cyclic GMP in response to nitric oxide in platelets from patients on haemodialysis were due to a defect in its generation, rather than to accelerated breakdown. 3. Basal levels of intra-platelet cyclic GMP were significantly increased in both patients on haemodialysis and patients on continuous ambulatory peritoneal dialysis. 4. The activity of nitric oxide was more stable in plasma than in buffer; its survival in plasma from patients on haemodialysis was similar to that in plasma from healthy control subjects.

Haemostatic activation and proteinuria as factors in the progression of chronic renal failure.

Haemostatic activation was measured in patients with either non-diabetic chronic renal failure (CRF) or diabetic nephropathy. We have investigated the relationship between these haemostatic markers and the rate of progression of renal failure. When compared with age- and sex-matched healthy controls, both patient groups showed significantly elevated plasma concentrations of D dimer, von Willebrand factor antigen (vWFAg), and C-reactive protein (CRP) (all P less than 0.001), as well as an increase in spontaneous platelet aggregation (P less than 0.01). Plasma concentration of platelet factor 4 was slightly but not significantly increased. Serum thromboxane was subnormal (P less than 0.01). Multiple regression analysis showed that in non-diabetic CRF proteinuria and serum TxB2 were independently related to the rate of progression of renal failure; in diabetic nephropathy proteinuria and vWFAg were independently related to the rate of progression. In both groups the relationship was stronger with proteinuria (standardised regression coefficients 0.56 and 0.45 respectively) than with serum TxB2 (0.29) or with vWFAg (0.37). We have found haemostatic activation in both non-diabetic and diabetic progressive renal failure. Proteinuria, and also in this study serum TxB2 and vWFAg, appear to be determining factors in the progression of renal failure, and their measurement may have prognostic value.

Platelet function in uraemia.

Haemostasis is abnormal in uraemic patients with end-stage chronic renal failure. There is a risk of bleeding due to a failure of primary haemostasis,(1) yet at the same time arteriovenous shunt thrombosis is common(2) and the incidence of occlusive arterial disease is increased.(3) Both bleeding and thrombotic tendencies thus co-exist. There is a great deal of information about the former, less about the latter.