Acquired von Willebrand syndrome in congenital heart disease surgery: results from an observational case-series.

Essentials Bleeding complications during congenital heart disease surgery in neonatal age are very common. We report the perioperative incidence of acquired von Willebrand syndrome (aVWS) in 12 infants. aVWS was detected in 8 out of 12 neonates and infants intraoperatively after cardiopulmonary bypass. Ten patients received von Willebrand factor concentrate intraoperatively and tolerated it well. SUMMARY: Background Cardiac surgery of the newborn and infant with complex congenital heart disease (CHD) is associated with a high rate of intraoperative bleeding complications. CHD-related anatomic features such as valve stenoses or patent arterial ducts can lead to enhanced shear stress in the blood stream and thus cause acquired von Willebrand syndrome (aVWS). Objective To evaluate the intraoperative incidence and impact of aVWS after cardiopulmonary bypass (CPB) in neonates and infants with complex CHD. Patients/Methods We conducted a survey of patients aged < 12 months undergoing complex cardiac surgery in our tertiary referral center. Twelve patients, whose blood samples were analyzed for aVWS before CPB and immediately after discontinuation of CPB on a routine basis, were eligible for the analysis. von Willebrand factor antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo), collagen binding activity (VWF:CB), VWF:multimers and factor VIII activity (FVIII:C) were determined. Results aVWS was diagnosed by VWF multimer analysis in 10 out of 12 patients (83%) prior to surgery and intraoperatively at the end of CPB in 8 out of 12 patients (66%). Ten patients received VWF/FVIII concentrate intraoperatively as individual treatment attempts during uncontrolled bleeding. They tolerated it well without intraoperative thrombotic events. One patient suffered a transient postoperative cerebral sinuous vein thrombosis. Conclusions aVWS is of underestimated incidence in complex CHD surgery. These data may offer a new approach to reduce the risk of severe bleedings and to achieve hemostasis during high-risk pediatric cardiac surgery by tailoring the substitution with von Willebrand factor concentrate.

Platelet prostacyclin binding in coronary artery disease.

Reduced responsiveness of platelets to prostacyclin, reported in vitro in patients with coronary artery disease, has been thought to be a factor predisposing toward coronary thrombosis and vasospasm as a result of enhanced in vivo release of cyclic endoperoxides and thromboxane A2 by the platelets. In this study, specific binding of prostacyclin to intact platelets was determined in patients with coronary artery disease by direct binding studies using 9-3H-prostacyclin sodium salt. In addition, the inhibitory effect of prostacyclin on primary aggregation induced by adenosine diphosphate and cyclic adenosine monophosphate (cyclic AMP) accumulation stimulated by prostacyclin was examined. Twenty patients with angiographically documented coronary artery disease and stable angina, 8 patients with acute myocardial infarction, 14 healthy volunteers and 10 patients with normal angiograms were studied. In patients with stable angina, binding capacity and affinity of platelet prostacyclin binding sites and prostacyclin-induced cyclic AMP accumulation were not different from those of control subjects. In patients with acute myocardial infarction, however, binding capacity of platelet prostacyclin receptors was significantly reduced (0.69 +/- 0.45 versus 1.35 +/- 0.37 pmol/10(9) platelets, p = 0.001) and the postreceptor response, represented by platelet responsiveness to prostacyclin and prostacyclin-induced cyclic AMP synthesis, was impaired. Because all patients with myocardial infarction were receiving intravenous heparin and nitroglycerin, which might interfere with platelet prostacyclin binding, competition experiments were performed in vitro. Neither heparin (3 to 250 IU/ml) nor nitroglycerin (0.8 to 22 microM) displaced specifically bound 9-3H-prostacyclin. L-Epinephrine in concentrations up to 10 microM also exhibited no competition with specific platelet prostacyclin binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular cytosine arabinoside accumulation and cytosine arabinoside triphosphate formation in leukemic blast cells is inhibited by etoposide and teniposide.

Cytosine arabinoside (ara-C) is one of the most active compounds in the treatment of acute leukemias. In the majority of current protocols ara-C is combined with other cytotoxic agents in an attempt to increase antileukemic activity. The present study investigated the impact of etoposide, teniposide, amsacrine, mitoxantrone, anthracyclines, and asparaginase on the cellular accumulation of ara-C and its intracellular metabolism in order to provide a better rationale for combination therapy. Intracellular accumulation and phosphorylation of ara-C were determined in peripheral blast cells from twenty patients with acute leukemias after exposure to 1 and 10 mumol/l ara-C alone and after preincubation with 1 and 10 micrograms/ml etoposide, 10 and 100 micrograms/ml teniposide, 10 mumol/l amsacrine, 500 ng/ml mitoxantrone (or daunorubicin or doxorubicin) or 10 mumol/l asparaginase. Ara-C accumulation at 10 mumol/l was decreased by 1 microgram/ml etoposide (67 +/- 18% of control), 10 micrograms/ml etoposide (30 +/- 22%), 10 micrograms/ml teniposide (12 +/- 23%), 100 micrograms/ml teniposide (10 +/- 18%), and amsacrine (51 +/- 21%). Intracellular ara-CTP formation was determined at an extracellular concentration of 10 mumol/l and preincubation with these drugs. The intracellular formation of ara-CTP was decreased by 1 microgram/ml etoposide (77 +/- 15% of control), 10 micrograms/ml etoposide (32 +/- 22%), 10 micrograms/ml teniposide (10 +/- 9%), 100 micrograms/ml teniposide (0 +/- 0%), but not by amsacrine. These data indicate that prior exposure to etoposide and teniposide influence ara-C metabolism and possibly cytotoxicity, and thus should not immediately precede ara-C administration in clinical trials.

Fish oil preparations rich in docosahexaenoic acid modify platelet responsiveness to prostaglandin-endoperoxide/thromboxane A2 receptor agonists.

The effects of daily dietary supplementation for 6 weeks with either 4.5 g eicosapentaenoic acid (EPA) and 3.35 g docosahexaenoic acid (DHA) (group I, EPA/DHA = 1.33) or 3.5 g EPA and 6.4 g DHA (group II, EPA/DHA = 0.54) on platelet responsiveness to the stable prostaglandin (PG)-endoperoxide analogue 9,11-dideoxy,9 alpha-11 alpha-methanoepoxy-PGF2 alpha (U 46619) were studied in healthy volunteers. Dose-response curves (DRC) of U 46619-induced platelet aggregation were analysed by computerized non-linear curve fitting. In group I, the concentration of U 46619 required for half-maximum platelet aggregation (EC50) remained unchanged, whereas the Hill coefficient decreased from 6.2 to 3.3 (P < 0.02). In group II, characterized by a high intake of DHA, a considerable increase of EC50 from 0.3 to 1.4 microM was found (P < 0.02). These results suggest different effects of EPA and DHA on the platelet thromboxane/endoperoxide-amplifying system. The considerable shift of the DRC in group II suggests a direct effect of DHA on the presentation of the endoperoxide receptor and/or post-receptoral events.

Platelet endoperoxide/thromboxane A2 (PGH2/TXA2) receptors in patients with myeloproliferative disorders.

In patients with myeloproliferative disorders (MPD) an altered sensitivity of platelets to antiaggregatory prostaglandins and to the endoperoxide analogue U 46619 has been found. In this study we examined U 46619-induced platelet aggregation and binding of the endoperoxide/thromboxane A2 (TXA2) receptor antagonist SQ 29548 in 11 patients with MPD and 11 healthy controls. Although platelet responsiveness to U 46619 was significantly enhanced (p less than 0.05) in MPD, binding affinity and binding capacity of the corresponding endoperoxide/TXA2 receptor were not altered (Bmax 0.67 +/- 0.20 vs. 0.58 +/- 0.14 pmol/10(9) platelets, Kd 0.41 +/- 0.11 vs. 0.55 +/- 0.09 nM). These data exclude the possibility that changes in the presentation of endoperoxide/TXA2 receptors are responsible for the enhanced platelet sensitivity to endoperoxides found in MPD.

Aspirin non-responder status in patients with recurrent cerebral ischemic attacks.

BACKGROUND: Antiplatelet agents such as acetylsalicylic acid (aspirin) reduce the relative risk for cerebrovascular events in patients with cardiovascular or cerebrovascular disorders by approximately 23 %. Recent observations raise the possibility that aspirin resistance may contribute to the failure of aspirin treatment in a significant proportion of patients (aspirin non-responders). To evaluate the clinical relevance of aspirin non-responder status, we analysed platelet functions in symptomatic and asymptomatic patients treated with aspirin for secondary prevention of cardiovascular and cerebrovascular events. METHODS: A total of 53 patients on 100 mg aspirin daily for secondary prevention (mean treatment duration > 60 months) were included. Patients were categorized as asymptomatic if they were free of cerebrovascular incidents for at least 24 months (n = 18). Symptomatic patients had suffered ischemic strokes or transient ischemic attacks within the previous 3 days (n = 35). Platelet function was assessed using the PFA-100 system that allows for quantitative assessment of platelet function, reporting platelet aggregatability as the time required to close a small aperture in a biologically active membrane. RESULTS: Collagen/epinephrine closure times were significantly shorter in symptomatic patients than in asymptomatic patients (p < 0.01). Individual closing times were normal in 12 of 35 symptomatic patients (34 % non-responders) whereas all asymptomatic patients had prolonged closure times. CONCLUSIONS: Aspirin non-responder status may contribute to failure of aspirin therapy in the secondary prevention of cerebrovascular incidents in as much as 30-40 % of patients. Quantitative assessment of platelet functions may provide a means to predict aspirin treatment failure in individual patients and to re-direct therapeutic strategies.

Desensitization of platelets to iloprost. Loss of specific binding sites and heterologous desensitization of adenylate cyclase.

The binding characteristics of [3H]prostacyclin and [3H]iloprost ([3H]5-[(E)-(1S,5S,6R,7R)-7-hydroxy-6-[(E)-(3S,4RS) -3-hydroxy-4-methyl-1-octen-6-inyl]-bicyclo[3.3.0]octan-3-yl idene] -pentanoic acid) and platelet adenylate cyclase activities were investigated in platelet-rich plasma preincubated with iloprost. The exposure of platelets to 0.1 microM iloprost (12 h, 20 degrees C) caused a significant loss of iloprost binding sites (P less than 0.01) without causing changes in binding affinity. This loss of specific [3H]iloprost binding was time- and dose-dependent. The reduction of iloprost receptor density was accompanied by an impaired responsiveness of platelet adenylate cyclase to iloprost, prostaglandin D2 and forskolin. In contrast, basal adenylate cyclase activity was not affected by iloprost pretreatment. The diminished response of the enzyme to GTP and NaF pointed to an involvement of the stimulatory guanyl nucleotide-binding protein (Gs) in iloprost-induced heterologous desensitization. Consequently, [32P]NAD+ and cholera toxin were used for the direct labelling of Gs. Platelet membranes desensitized to iloprost incorporated less label into the 45 kD subunit of Gs. These data suggest that the site of action of iloprost for heterologous desensitization of human platelet adenylate cyclase is located on Gs.

Platelets from patients with liver cirrhosis exhibit a defect in the von Willebrand factor-binding domain.

In this study, we examined the platelet von Willebrand factor (vWF)-binding domain in patients with liver cirrhosis. Direct binding studies were performed with the monoclonal antibody (mab) AN51 with specificity for the glycoprotein (GP) lb alpha. Specific binding of AN51 to intact washed platelets was monitored by an enzyme-linked immunoassay (ELISA). Half-maximum binding to intact washed platelets occurred at concentrations as low as 94 +/- 24 ng/ml; N = 12). Binding saturation analysis of with AN51 revealed a more than fifty percent reduction of AN51-binding sites (p < 0.001) in cirrhosis (N = 13) as compared to sex- and age-matched healthy controls (N = 12). These data demonstrate an impairment of the platelet surface vWF-binding domain in patients with liver cirrhosis. The resulting defect in primary haemostasis, i.e. the vWF-mediated attachment of platelets to the exposed subendothelium, is expected to contribute to the increased risk of haemorrhagic complications in these patients.

The binding of 6-oxo-prostaglandin E1 to platelet prostanoid receptors.

6-oxo-PGE1 has been shown to cause a dose dependent stimulation of platelet adenylate cyclase (Km 1.6 X 10(-7)M). Since this compound also displaced specifically bound 9-3H-PGI2 from intact washed platelets (Ki 1.6 X 10(-6)M), the coupling to platelet PGI2 receptors has been suggested to account for the observed platelet cyclase stimulation. However, the Ki/Km-ratio of 6-oxo-PGE1 appears to be high (10.3) as compared to PGI2, ZK 36 374, and PGE1 (1.9, 2.4 and 3.1, resp.). Thus, one might expect that 6-oxo-PGE1 interacts with a heterogeneous population of platelet receptors mediating the same biological response, eg cyclase activation. In this study, we present evidence supporting this concept and report that 6-oxo-PGE1 also displaces specifically bound 3H-PGD2 from its platelet receptor (Ki 1.6 X 10(-5)M).

Platelet and vessel associated prostacyclin and thromboxane A2/prostaglandin endoperoxide receptors.

Synthetic stable analogues of thromboxane A2 (TXA2), cyclic endoperoxides (PGH2) and prostacyclin (PGI2) opened up new opportunities for investigating the mechanisms of action of these compounds. They proved to be useful pharmacological probes for characterizing PGI2 and TXA2/PGH2 receptors. Over the past few years, new synthetic antagonists with high specificity allowed the modulation of biological responses to endogenous eicosanoids. These compounds will, therefore, considerably promote our understanding of the biological function and significance of arachidonate metabolites. The present review summarizes current concepts that have arisen concerning platelet and vascular PGI2 and TXA2/PGH2 receptors, their transmembrane signal transduction, as well as their possible implications in the pathophysiology of cardiovascular disease.

The anti-fungal agent itraconazole exerts immunosuppressive effects on alloreactivity but not on natural immunity in vitro.

The anti-fungal azole drug itraconazole was compared with fluconazole regarding immunosuppressive effects in a model of human alloreactivity in vitro (the mixed lymphocyte culture, MLC) and in assays of non-adaptive immunity (natural killing, NK, and lymphokine activated killing, LAK). Itraconazole, but not fluconazole, strongly inhibited lymphocyte proliferation and the generation of allospecific cytolytic activity, but had no effect on the development of major histocompatibility complex (MHC)-unrestricted ("natural killer-like") cytotoxicity or of alloindifferent suppressive activity in MLC. Neither drug blocked LAK cell induction, nor the effector phase of either NK or LAK activity. These results suggest that itraconazole might represent a new class of immunosuppressive agent which specifically blocks alloreactivity without affecting natural immunity.

Effects of heparin on prostacyclin binding and platelet adenylate cyclase activity stimulated by iloprost and forskolin.

Heparin is known to impair the antiaggregatory effectiveness of prostacyclin (PGI2) for adenosine diphosphate-induced aggregation in citrated platelet rich plasma. Thus porcine mucosal heparin (PMH) from different commercial sources was investigated for its ability to compete with specific platelet prostacyclin binding. In concentrations up to 250 IU/ml PMH itself did not interfere with the binding of 3 X 10(-9) M 9-3H-PGI2-sodium salt to intact washed platelets. The displacement of specifically bound PGI2 observed by PMH (Ki 60 IU/ml) containing 4-chloro-m-cresol (4-CC) was caused by the preservative (Ki 4-CC 3.0 X 10(-4) M). Although 60 IU/ml PMH (free of 4-CC) have been shown to inhibit basal 3 X 10(-5) M forskolin-, and 10(-8)M Iloprost-stimulated adenylate cyclase in platelet membranes (-63%, -62%, -83% respectively), no effect of PMH on 3H-cyclic-AMP formation has been observed when intact platelets were studied by 3H-adenine prelabeling technique. There is also no evidence that the preincubation of PGI2 (5 X 10(-7) M) with 1000 IU/ml PMH might neutralize the effectiveness of this eicosanoid. In contrast, PGI2 preincubated with PMH (10 min, 37 degrees C) caused a more pronounced increase of platelet 3H-cyclic AMP (+65%) compared with PGI2 incubated in 5 X 10(-2) M Tris-HCl, pH 7.4. Thus our data provide no evidence that PMH interferes with i) specific platelet PGI2 binding, ii) PGI2-stimulated cyclic AMP synthesis or iii) neutralizes PGI2 by formation of a biologically less active PGI2-PMH complex.

Metabolism of 14-C-arachidonic acid in platelets and antiaggregatory potency of prostacyclin in women taking oral contraceptives.

PIP: Arachidonic acid (AA) concentration in platelet lipids is known to be altered by the intake of oral contraceptives (OCs). Conflicting results have been reported on the influence of OCs on eicosanoid metabolism in platelets. The metabolism of exogenous 14-C-AA in platelets and synthesis of TXB2 from endogenous AA sources under OC medication was investigated. The antiaggregatory potency of prostacyclin also was determined. Citrated PRP from healthy women without and with intake of OCs was investigated (low dose estrogen 54%, medium dose 33%, high dose 13%, combined with various preparations). Age, smoking habits, and hematocrit values were comparable in both groups. Metabolism of exogenous 14-C-AA was determined in gel-filtered platelets. Metabolities were separated by thin layer chromatography. Synthesized TXB2 from endogenous AA sources was measured by radioimmunoassay after collagen-induced aggregation. Therewas no influence of OC intake on metabolism of exogenous AA in platelets. No increase in 12-HETE levels under combined estrogen/progestogen medication was observed, which may be due to the antiestrogenic effect of progestogens. OC intake causes a slight decrease of TXB2 synthesis from endogenous sources without changing platelet aggregability measured in citrated PRP. In contrast to earlier findings, these data do not indicate reduced antiagregatory potency of prostacyclin in women taking OCs. The results suggest that the observed increase in platelet reactivity ex vivo cannot be explained by changes in AA metabolism. Changes of plasmatic coagulation and consecutive activation of platelets may be an alternative explanation.^ieng