Regulation of the phosphoinositide cycle by Na+/H+ exchange and intracellular pH in human platelets.

We have found that thrombin-induced activation of protein kinase C (PKC) in platelets, measured by phosphorylation of the 47 kDa protein, is synergistically enhanced by the amiloride analogue ethylisopropylamiloride (EIA), a specific inhibitor of Na+/H+ exchange. This EIA effect was further synergistically enhanced by lowering intracellular pH (pHi) with either nigericin or sodium propionate, and reversed by raising pHi with monensin or ammonium chloride. The synergistic enhancement of thrombin-activated PKC by EIA plus nigericin was not observed when PKC was directly activated by phorbol esters. EIA and EIA plus nigericin caused a 3- to 6-fold increase in thrombin-induced diacylglycerol (DAG), but not phosphatidic acid (PA), production. EIA and nigericin also caused a marked increase in thrombin-induced breakdown and inhibition of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2). In summary, we have presented evidence that inhibition of Na+/H+ exchange causes primarily a H(+)-mediated interruption of the phosphoinositide cycle in activated platelets, including the accumulation of DAG associated with the enhancement of PKC activation, the inhibition of conversion of DAG to PA, and increased PIP2 breakdown. These data suggest a model in which Na+/H+ and pHi play an important regulatory role in permitting the phosphoinositide cycle to proceed in thrombin-activated platelets.

Platelet count, anti-heparin/platelet factor 4 antibodies and tissue factor pathway inhibitor plasma antigen level in chronic dialysis.

We studied 50 chronic dialysis patients with end-stage renal disease. Mean platelet count was within normal limits. An inverse linear correlation was observed between pre-dialysis platelet count and serum creatinine (r=0.304, p=0.038). Dialysis caused a decrease in platelet count (216+/-80x10(9)/L, pre; 198+/-68, post; p=0.0001), and the higher the pre-dialysis platelet count, the greater the decrease (r=0.623, p=0.0001). Post-dialysis triglyceride decreased (1.67+/-1.27 mmol/L, pre; 1.23+/-0.96, post; p=0.0001). Tissue factor pathway inhibitor (TFPI) antigen plasma level was higher in uremic patients than in controls (114+/-42 ng/ml vs. 64+/-12, p=0.0001). TFPI increased 2.3 times following dialysis and such an increase was directly correlated with post-dialysis plasma heparin concentration (r=0.571, p=0.0002) and inversely correlated with post-dialysis triglyceride variation (r=0.407, p=0.005). Six of fifty patients (12%) had anti-heparin/platelet factor 4 antibodies (Hab), 3 IgG, and 3 IgM. Female sex and the use of cuprophane membranes were more frequent among Hab-positive patients (p=0.0001), while a lower percentage of them were on anti-aggregating drugs as compared to Hab-negative patients (p=0.002). Only one Hab-positive patient was slightly thrombocytopenic and none showed bleeding or thrombotic manifestations. Serum albumin and y globulin decreased following dialysis in Hab-positive patients, while the opposite was seen in those Hab-negative (-2.47+/-1.72 g/L, vs. 0.21+/-1.77, p=0.001 and -0.48+/-0.60 g/L vs. 0.64+/-0.97, p=0.007, respectively). In vivo factors other than Hab are involved in the development of heparin-induced thrombocytopenia. Besides a blunted immunological response, increased levels of TFPI, the use of anti-aggregating drugs, and the observed behavior of serum proteins might play a role in this regard.

Effect of age on oral contraceptive-induced venous thrombosis.

This study was undertaken to investigate the effect of age on oral contraceptive-induced venous thrombosis. All women seen in the University of Padua Department of Medical and Surgical Science who had had two courses of oral contraceptive therapy at different ages were included. A total of 28 subjects met these criteria. Fifteen patients had a congenital or acquired prothrombotic condition, whereas 13 women were normal subjects. The mean age at which thrombosis occurred was 33.3 and 36.3 years for women with or without a prothrombotic condition, respectively. The ages during which the women remained asymptomatic were 23.1 and 23.3 years for women with or without a predisposing defect, respectively. Thrombosis occurred, during the second course of oral contraceptive therapy, after the mean duration of 6.5 cycles or 18.4 cycles in women with or without prothrombotic defects, respectively. During the asymptomatic course, approximately the same number of women took old progestins or third-generation compounds. On the contrary, during the second period, 21 of 28 women took progestins with third-generation compounds. Age seems to plays an important role in oral contraceptive-induced venous thrombosis. In normal women, thrombosis occurred after a greater number of oral contraceptive cycles as compared with the women with prothrombotic defects. Because the majority of women took preparations that contained third-generation progestins during the second course of therapy, concomitant contributing effects of these compounds cannot be excluded.

Effects of glycosaminoglycans and protamine chloridrate on platelet aggregation induced by collagen and thrombin.

The effects of heparin (HE), dermatan sulfate (DS), heparan sulfate (HS) and protamine chloridrate (PC) on platelet aggregation were studied. Both PC and the three glycosaminoglycans (GAGs) did not influence collagen-induced platelet aggregation. In contrast, all the tested GAGs blocked thrombin-induced platelet aggregation. HE and HS were equivalent and very effective, while DS was also but to a lesser extent. This could be because HE and HS act via both antithrombin III and heparin-cofactor II, whereas DS exerts its action on the latter only. PC, too, inhibited, in a dose-dependent fashion, thrombin-induced platelet aggregation, probably by competing with the thrombin affinity binding sites on the platelet surface. When the GAGs were tested together with PC, HE was shown to be the most effective: on a weight-for-weight basis, an identical amount of PC was unable to counteract the inhibitory effect of HE, while it partially reversed those of DS and HS. A full reversal of the inhibitory effect of DS and HS was never observed, in spite of adding increasing amounts of PC. It seems likely that plasma components may preserve the bindings of such GAGs with their cofactors.

Interaction between histidine-rich glycoprotein and platelet factor 4 with dermatan sulfate and low-molecular-weight dermatan sulfate.

There is a controversy about whether or not histidine-rich glycoprotein (HRG), the most abundant plasma protein with glycosaminoglycans-neutralizing capacity, is able to prevent the inhibition of human thrombin by heparin cofactor II (HC II) in the presence of dermatan sulfate (DS). The authors studied the interaction of DS and low molecular weight DS, in a purified system with HRG, platelet factor 4 (PF 4), and with HC II. Their results show that HRG, like PF 4, has an affinity, not only for heparin, but also for DS. However, this affinity seems very weak. In fact, HRG is 10 times less effective than PF 4 in neutralizing the 50% antithrombin activity of HC II in the presence of DS.

[Symptomatic relief of carcinoid syndrome by ketanserin. A case]. / Soulagement symptomatique du syndrome carcinoïde par la kétansérine. Une observation.

The carcinoid syndrome is a rare clinical entity mainly characterized by flushing and diarrhoea. It is due to different biological mediators produced by tumours that arise from enterochromaffin cells. Such tumours are typically located in the ileum, have a long course and become symptomatic only in the presence of overt liver metastases. Among the involved mediators, the role of serotonin (5-hydroxytryptamine, 5-HT) has been ascertained in the pathogenesis of diarrhoea, while it remains controversial in that of flushing. Ketanserin is a 5HT-2 antagonist with no mixed receptor agonist-antagonist activity. We report the case of a severely distressing carcinoid syndrome fully dominated by ketanserin. The patient was a 75-year-old man, who came to our attention because of marked weight loss, impossibility to feed and almost continuous diarrhoea due to liver colonization of a mid ileum carcinoid tumour, previously resected at the age of 65. Sustained facial and trunk flushing also presented several times daily. Ketanserin, 20 mg twice a day orally, was administered and then increased up to 40 mg daily with no side effects and progressive complete control of both diarrhoea and flushing. It is suggested that ketanserin, due to its availability and tolerability, should first be considered for palliative relief of carcinoid syndrome. The literature on this subject is extensively reviewed.

[Use and misuse of hospital admission in a department of medicine in Padua]. / Uso appropriato e inappropriato del ricovero in un reparto di medicina a Padova.

We evaluated retrospectively 357 consecutive inpatients (M = 157; F = 200) 14=93 wars old (mean 60.9 +/- 1.0, SF: M = 59.5 +/- 1.4: F = 61.9 +/- 1.4) treated by a same physician at the 2nd Division of Medicine, Padua University Hospital, from January 1993 to July 1995. Such Division, which is representative of the Internal Medicine of an important Center has a high medical standard, as evaluated on the basis of academical and scientific titles of its staff, but no specific geriatric facilities. Patients had been assigned to the Division and, within it, to a given physician at random. 38.1% of patients were older than 70 (M = 30.0; F = 44.5, p < 0.01). Apart from the previous group, 29.1% of admissions (M = 28.0; F = 30.0) were inappropriate. 4.5% of admissions were terminally-ill cancer patients, already diagnosed and treated at other divisions and not further susceptible of specific therapy. Only 28.3% of patients (M = 38.2; F = 20.5, p < 0.0005) did not fall within any of the previous categories, but 33% of the days of their hospital stay were inappropriate (M = 30; F = 37). The mean hospital stay was 10.2 +/- 0.5 SE days (M = 10.4 +/- 0.6; F = 10.1 +/- 0.6). Considering patients altogether, the mean hospital stay was longer for patients older than 70 than for the others (12 +/- 0.9 vs. 9.3 +/- 0.5 days, p < 0.01) and a significant correlation was observed between age and stay length (r = 0.249, p < 0.0001). The educational level of inpatients was as follows: no education and elementary 65.8%; junior-high 20.7%; high 8.9%; university 4.4% with no difference between sexes, nor as compared to the general population, adjusted for age. It is concluded that a highly qualified division, to which it would appear appropriate applying only for cases not manageable as outpatients or by less qualified institutions, is suboptimally utilized with waste of money and resources. This is due to a deficiency in geriatric and residential facilities as well as in community and domiciliary care, to a noxious culture subordinating internal medicine to specialty branches and to the inadequate use of resources by both physicians and patients, in particular, among them, by females.

Main clinical manifestations of a bleeding diathesis: an often disregarded aspect of medical and surgical history taking.

A suitable clinical evaluation of a bleeding diathesis is often forgone. The young doctor is often unprepared to describe in an accurate way the different types of bleeding. An adequate classification and adequate clinical information about a bleeding diathesis are instead of paramount importance. Bleeding may be cutaneous, mucous, articular, muscular, parenchymal, intracavitary, orificial. Each of these sites and forms may have diagnostic implications. An accurate description of the several forms of cutaneous bleeding (petechiae, purpuric spots, ecchymosis, haematomas, etc.) is needed for referrals and for controls. The correct evaluation of cutaneous bleeding manifestations of children (battered child syndrome) is absolutely important for clinical and medico-legal purposes. The same is true for the battering syndrome seen in women abused by their spouses. The grading of haemarthrosis in haemophilia patients is important for the follow-up. A proper description of haematuria is essential in suggesting the probable site of bleeding (kidney or bladder or urethra). A proper evaluation of bleeding may give also useful information on the general health status of the patients (presence of anaemia, poor nutrition, renal insufficiency, etc.). The combination of bleeding and thrombosis in the same patient is also a clinical challenge. The relationship between haemorrhage and thrombosis may be sequential or concomitant. Sequential thrombosis may occur in a patient confined in bed for a brain haemorrhage. Concomitant thrombosis and bleeding occur in DIC and in patients with thrombosis being treated with anticoagulants. Finally, it should be kept in mind that a proper evaluation of the bleeding diathesis of a given patient may help the caring doctor in ordering appropriate laboratory tests (e.g. a platelet count for petechiae, a PTT for a patient with haemarthrosis, etc.).

Normal thrombopoietin and its receptor (c-mpl) genes in children with essential thrombocythemia.

BACKGROUND: Following the observation of thrombopoietin (TPO) gene abnormalities as the cause of familiar cases of thrombocythemia similar derangements of TPO and/or its receptor (c-mpl) might be surmised to be at the root of increased platelet count also in non-familiar (sporadic) cases. Although this was not demonstrated in adults, little data exist about childhood. PROCEDURES: We studied the molecular biology of TPO and c-mpl in seven children with non-familiar essential thrombocythemia (ET) and one child with secondary thrombocytosis (ST). Plasma TPO content was measured using a commercially available kit. Genomic DNA was extracted from whole blood by standard methods and TPO and c-mpl genes were amplified by polymerase chain reaction (PCR) and sequenced. RESULTS: Plasma TPO levels were normal in all our patients. No alteration was detected in either coding region, including the flanking intronic sequences of TPO and c-mpl genes. As compared to the published normal sequence of the TPO gene, one allelic base change in a non-coding region of intron 1 was found in all children with ET and ST, but this was reported as a common finding in normal subjects as well. CONCLUSIONS: High platelet count in our series of sporadic ET of childhood is not due to an abnormality either of TPO or c-mpl gene.

Lack of increase or reduction in circulating platelet factor 4 and heparin-releasable platelet factor 4 in symptomatic patients with asthma.

We measured circulating platelet factor 4 (PF4) and heparin-releasable platelet factor 4 (HR-PF4) in 13 symptomatic patients suffering from bronchial asthma and in 10 matched normal controls. Neither a difference between patients and controls, nor a correlation between platelet count and HR-PF4 was found (PF4: controls 2.1 +/- 2.9; patients 3.6 +/- 4.4 ng/ml (n.s.); HR-PF4: controls 127 +/- 49, patients 101 +/- 34 ng/ml (n.s.). Therefore, the pathogenetic role of PF4 in the mechanism of bronchial asthma, elsewhere hypothesized, cannot definitely be established.

PF 4 assay by an ELISA method: a good correlation with the usual RIA method.

PF 4 is a specific platelet protein. This protein is released from alpha granules during the platelet activation and later it adheres to endothelium. Intravenous heparin injection displaces PF 4 from vessels wall. Thus, PF 4 levels are an index of in act or past platelet activation. We have compared two methods of PF 4 dosage on 39 blood samples taken from healthy volunteers and patients. The samples has been shared out tree groups according to the procedure of collecting; so the values of PF 4 are widely enough distributed. There was no difference between the mean values of each group obtained with two methods. Equally the mean value of all samples processed with radioimmunoassay was similar to the mean value obtained with immunoenzymatic method. The correlation index between the values of PF 4 obtained with radioimmunoassay and immunoenzymatic method was 0.97. Therefore the new immunoenzymatic method for the dosage of PF 4 is as sensitive and precise as the radioimmunoassay.

PF 4 versus beta TG as evidence for platelet activation in myeloproliferative disorders.

19 patients with MPD have been studied. As described in normals, an age-related increase in beta thromboglobulin (beta TG) release is observed. Such release, however, is greater in patients with myeloproliferative disorders (MPD). MPD seem therefore to cause platelet activation, allowing an earlier and more evident manifestation of physiologic ageing phenomena. PF 4 levels are near zero both in controls and patients, regardless of platelet number. This suggests that increased levels of PF 4 represent only a laboratory artifact, caused by platelet activation in vitro. Mean ability in producing thromboxane B2 (TxB2) is increased, but is perfectly normal in patients with normal platelet count and decreased in 3 thrombocythaemic patients, who seem to present an increased thrombotic risk. TxB2 is reduced almost to zero by the administration of aspirin plus dipyridamole; contrarily, all other parameters were unaffected, either by such drugs or by AD 6, a new coumarin derivative with antiplatelet properties.

A new family with classical factor X deficiency as demonstrated by electroimmunoassay.

A new family with classical factor X deficiency is described. The proposita is a 6 year old girl who presented with occasional epistaxis and a hematoma after an intramuscular injection. The main laboratory features consisted in a prolongation of partial thromboplastin, prothrombin and Stypven clotting times corrected by the addition of normal serum. Factor X activity varied between 3 and 6%. Factor X amydolytic activity was 15% of normal. Electroimmunoassay failed to show the presence of factor X antigen. No inhibitor was found in the proposita plasma. Parents and other family members showed intermediate levels of factor X activity and antigen and were considered to be heterozygotes. No consanguineity was found in the family.