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Transient global amnesia (TGA) is a generally benign syndrome lasting less than 1 hour characterized by sudden loss of anterograde and retrograde memory and repetitious queries in the absence of changes of the personality and of detectable neurological signs. This paper reports the case of a 41-year-old woman who developed two episodes of TGA in June 1994. During a sustained more than 6-month treatment with an estrogen/progestogen association containing 20 mg of ethinylestradiol and 150 mg of desogestogen the woman experienced a sudden onset of spatial disorientation and of retrograde and anterograde amnesia. During the attack she experienced neither speech disturbance nor features suggestive of epilepsy. After approximately 40 minutes the womans memory gradually returned to normal and she had no memory of her amnesic episode. A second shorter episode of memory loss occurred 15 minutes later. The womans clinical summary was negative for major risk factors for cerebrovascular disease and she had no history of seizures migraine or head injury. Her physical examination was normal as were her EEG ECG two-dimensional echocardiography Doppler ultrasound of the carotid and vertebral arteries and brain nuclear magnetic resonance. Liver and kidney functions were normal as were antiphospholipid antibodies and protein C levels. However functional antithrombin III was 77% protein S was 60% and n-APC-ratio was 0.78. These hemostatic test values returned to within normal levels 2 months after discontinuation of the drug. 15 months following the end of administration of the drug the patient had yet to experience another episode of TGA.

The uricosuric effect of tiaprofenic acid was evaluated in a group of normouricaemic inpatients with various rheumatic disorders. Six patients aged 26 to 60 years were maintained on a standardised low-purine diet and, after a washout period of 72 hours, tiaprofenic acid was administered in 3 oral doses of 300 mg 12-hourly. A normal renal function, as assessed by serum creatinine and creatinine clearance determinations, was considered mandatory for entry into the study. The following parameters were evaluated before and after treatment: haematological values, blood urea nitrogen (BUN), serum and urinary creatine concentrations, serum uric acid concentration and the fractional excretion rate of uric acid. Our preliminary results showed a substantial increase of urinary uric acid excretion in the samples collected after treatment, especially in the first 4 hours.

Increasing doses of prostaglandin E2 (PGE2) (5, 10, 20, 40, 60 ng/kg/min) were infused in 7 normal volunteers before and after angiotensin II synthesis inhibition by captopril (100 mg by mouth). PGE2 infusion alone did not alter blood pressure, while it increased the urinary excretion of both epinephrine and norepinephrine, enhanced p aminohyppuric clearance (CPAH), inulin clearance (CIn), sodium and water excretion and decreased urinary osmolality. No changes of CIn CPAH and catecholamines were observed after captopril alone, whilst there was a significant increase in urine output and sodium excretion and a decrease in urinary osmolality. In the presence of captopril, the infusion of PGE2 caused a significant fall in blood pressure and CIn, enhanced epinephrine excretion and sodium excretion, while it did not significantly reduce CPAH· Our findings suggest that an intact renin-angiotensin system is necessary to maintain GFR during PGE2 infusion.

The potencies of prostaglandins (PG) I2, PGD2 and PGE1 as inhibitors of human platelet aggregation induced by threshold concentrations of four aggregating agents were determined in platelet-rich plasma from normal individuals who had not ingested aspirin. The order of activity against ADP, adrenaline and collagen was always PGI2 greater than PGD2 greater than PGE1. However, PGD2 and PGE1 were almost equipotent with PGI2 when tested against arachidonic acid (AA). The threshold inhibitory effects of PGD2, PGE1 and PGI2 could be over come by increasing the concentrations of the aggregating agents AA, collagen or ADP. Adrenaline was found to be different from the other aggregating agents. It could overcome inhibition of platelet aggregation by PGD2 but could not overcome inhibition by PGI2 or PGE1. These facts support the hypothesis that platelet receptors for PGI2 and PGE1 are similar to each other and different from the receptor(s) for PGD2. PRP obtained from normal subjects after the ingestion of aspirin exhibited only one wave of aggregation in response to ADP, adrenaline or collagen, PGI2, PGD2 and PGE1 were all powerful inhibitors of this single wave of aggregation. The inhibitory activity of all three prostaglandins at threshold concentrations was overcome by increasing the concentration of ADP or collagen but not by increasing the concentration of adrenaline.

After i.p. injection of PyMLV, NIH/OLAC mice showed thrombi in tail veins, ears, muscles and nesenterium together with thrombi and hematomas of subcutaneous tissues. This was followed by infarctions of lungs, brain and heart, that caused death of the animals. Laboratory evaluations of the infected mice showed normochromic anemia, mild thrombocytosis and marked defects in the aggregation and in the secretion of ATP from platelets exposed to AJP, collagen, thrombin or A23137. About 10% of cells present in the bone marrow was formed by blasts; 20% by multinucleated cells identified as megakariocytes (M) by peroxidase and acethyl-cholinesterase staining, and the vast majority of the other cells by entities belonging to all stages of maturation of the myeloid lineage. Hybridization experiments showed that the blasts present in the bone marrow were the only cells in which viral replication takes place. Maturation of M in the bone marrow was completely normal, and as for M from non-infected mice, proliferation and maturation in vitro was dependent on the presence of interleukin 3. Finally, studies in other strains of mice showed that the Fv-2 locus is involved in the pathogenicity of PyMLV in NIH/OLAC mice. Ne conclude that, in addition to its obvious pathophysiological significance, the myeloproliferative disease that occurs in mice after i.p. injection of PyMLV, can serve as an important probe for understanding basic events leading to bleeding and thrombosis.

Blood pressure variability was studied in eight patients with renovascular hypertension (group 1) and eight patients with hypertension and partial deficiency of adrenal zona fasciculata 11-hydroxylase (group 2). Systolic, diastolic and mean blood pressure were found to be higher in group 1 than in group 2 when measured at home (P less than 0.001), while no difference was measured when blood pressure was taken in the outpatient clinic. Heart rate was higher in group 1 than in group 2 both at home and in the clinic (P less than 0.005 and P less than 0.05, respectively); a significant increase was observed only in the hospital clinic for group 2 (P less than 0.001). No difference in systolic function and left ventricular mass was found between the two groups. Finally, group 2 exhibited a significant increase (P less than 0.0001) in systolic, diastolic and mean blood pressure in the clinic, compared with that found at home, while group 1 showed a poor increase (P less than 0.05) in systolic blood pressure only. It is suggested that the enzymatic impairment of adrenal glands in group 2 may play a role in the disparity of blood pressure levels determined at home and in the outpatient clinic. Furthermore, our data suggest that differences in blood pressure measurement at home and in the hospital clinic should be taken into account when screening patients with partial deficiency of 11-hydroxylase.

Sensitivity to induction of platelet aggregation by arachidonic acid (AA) and changes in plasma and platelet polyunsaturated fatty acid distribution were studied in seven women before and after six months of oral contraceptive (OC) treatment with a combination of d-norgestrel (0.25 mg) and ethinylestradiol (0.05 mg). Special interest was focused on AA because certain metabolites of fatty acid induce platelets to aggregate and are considered to play a crucial role in thromboembolic processes. In plasma, AA concentrations increased slightly, but significantly, in both the free fatty acid (FFA) and phospholipid fractions; in platelets AA increased in the phospholipid and neutral lipid fractions. The threshold aggregating concentration (TAC) of AA was significantly reduced in platelets of women after six months of OC treatment (0.65 +/- 0.08 versus 0.30 +/- 0.04 mM). This suggests that changes in platelet fatty acid composition may be associated with in vitro changes in platelet sensitivity to AA. Such changes may contribute to the thrombotic tendency associated with OC treatment.Sensitivity to induction of platelet aggregation by (AA) arachidonic acid and changes in plasma and platelet polyunsaturated fatty acid distribution were studied in 7 women before and after 6 months of (OC) oral contraceptive treatment with a combination of d-norgestrel (0.25 mg) and ethinyl estradiol (0.05 mg). Special interest was focused on AA because certain metabolites of this fatty acid induce platelets to aggregate and are considered to play a crucial role in thromboembolic processes. In plasma, AA concentrations increased slightly, but significantly, in both the free fatty acid and phospholipid fractions; in platelets AA increased in the phospholipid and neutral lipid fractions. The threshold aggregating concentration of AA was signifcantly reduced in platelets of women after 6 months of OC treatment (0.65 + or - 0.08 versus 0.30 + or -0.04 mM). This suggests that changes in platelet fatty acid composition may be associated with in vitro changes in platelet sensitivity to AA. Such changes may contribute to the thrombotic tendency associated with OC treatment.

Prolonged thrombin time (partially corrected by calcium chloride) and normal reptilase time were found in the plasma of two siblings with arterial thrombosis. Their purified fibrinogen showed similar abnormalities as well as impaired fibrino-peptide release in response to thrombin, delayed polymerization of pre-formed fibrin monomers and normal sialic content. Binding of radiolabelled thrombin by patient's fibrin was 30% of normal. Supernatants from patients' fibrin clots contained abnormal amounts of thrombin (not adsorbed by fibrin) and caused abnormal enhancement of platelet aggregation and ATP secretion from platelets exposed to sub-threshold concentrations of ADP or epinephrine. Hirudin suppressed the enhancing effect of the supernatant and substitution of γ-thrombin for α-thrombin led to normalization of platelet response. Studies on fibrinolysis showed that the abnormal fibrinogen from these patients as well as its naturally occurring derivative fibrin are highly resistant to lysis by plasmin. Thus our data support the concept that, in addition to the enhanced activation of platelets by residual free thrombin, thrombosis in these patients is the result of an impaired sensitivity of fibrinogen the lytic effect of plasmin.

To evaluate whether the activity of Iloprost, a chemically stable prostacyclin analog, on platelet aggregation could be potentiated by aspirin (ASA), in vitro and ex vivo studies in human volunteers were performed. In vitro studies were carried out in human platelet rich plasma (PRP) incubated with different concentrations of ASA (25-150 μM). For ex vivo studies Iloprost (0.5 ng.Kg−1.min−1 for 30 min) was given intravenously to healthy volunteers. After 20 hour wash out a single 50 mg ASA dose was given to the same subjects. Two hours after ASA intake, a second infusion of Iloprost was carried out. Blood was collected at appropriate time intervals thereafter. Platelet aggregation and thromboxane B2 (TXB2) formation were determined in collagen stimulated PRP. ASA, in vitro , dose dependently reduced the concentrations oF Iloprost required to achieve 50% inhibition of platelet aggregation (IC50) in PRP stimulated by 1 g/ml collagen. Also, the IC50S for Iloprost were significantly reduced (p

Following exposure to calcium ionophore A23187, washed peripheral blood mononuclear cells (MNC) aggregated and formed thromboxane, like platelets. However, while aspirin strongly inhibited platelet aggregation and thromboxane formation, it had a little effect on the aggregation of MNC. In about 50% of the samples studied, aggregation of MNC was associated with the secretion of ATP. However studies in which exogenous ATP or ADP were used, suggested that the aggregation of MNC is independent of the secretion of nucleotides. MNC from 2 thrombasthenic patients failed to aggregate and bound 9–10 fold less radiolabelled fibrinogen than those from normals when challenged with A23187. However, fibrinogen, which plays a major role in the aggregation of platelets, did not appear to be involved in the aggregation of MNC. A differing behavior of these two types of cells was also found when the effect of plasma was studied on the aggregation response to A23187. Indeed, citrated plasma greatly enhanced the aggregation of platelets while it suppressed the response of MNC. This inhibitory effect of plasma was not detected when heparinized plasma was substituted for citrated plasma. We conclude that the aggregation of MNC in response to A23187 does not involve basic events known to play a major role in the aggregation of platelets. The response to A23187 may be an important probe for understanding basic mechanisms and pathophysiological significance of the aggregation of MNC.

Publisher Summary Participation of lipids in platelet function has been suspected. However, the elucidation of the metabolism of arachidonic acid (AA) in platelets and vessel walls has provided a rationale for this belief. Membrane phospholipids represent the main source of AA, the precursor of prostaglandins and thromboxane, which are believed to play a major role in platelet aggregation and adhesion. Other fatty acids, such as dihomo-7-linolenic acid (DHLA) and eicosapentenoic acid (EPA), give rise to prostaglandins and thromboxanes with potential antithrombotic properties. Compared to AA, DHLA and EPA make up a small and insignificant portion of the prostaglandin precursor fatty acids present in a western diet as well as in membrane phospholipids. However, because the fatty acid composition of the diet influences the fatty acid composition of the tissues, and DHLA and EPA can replace AA in membrane phospholipids, a dietary approach to the prevention of thrombosis has been suggested. This chapter reviews the evidence of a modulatory role of some AA metabolites in thromboembolic diseases, and the potential clinical relevance of a dietary approach to the prevention of thrombosis.

Following stimulation with arachidonic acid, collagen, U-46619 (a stable analogue of prostaglandin endoperoxide/thromboxane-A2), thrombin, or adenosine diphosphate (ADP), unstirred human platelet suspensions bound labeled factor VIII in a reaction that reached equilibrium within 10 min. Apyrase inhibited binding induced by arachidonic acid, collagen, U-46619, and thrombin by less than 40%, but inhibited ADP-induced binding by 95%. Binding to aspirin-treated platelets was normal in response to U-46619, reduced by 60%-70% in response to ADP, collagen, and thrombin, and absent in response to arachidonic acid. Binding in response to U-46619 was not altered by the combination of apyrase and aspirin. Binding of factor VIII was decreased by 90% when 10 mM EDTA was added before each agonist, but it was inhibited less than 30% when EDTA was added following platelet stimulation. We conclude that arachidonic acid, collagen, and thrombin can expose binding sites for factor VIII independently of released ADP; that Ca++ is required for activation but probably not for binding of factor VIII to platelets; and that platelet thromboxane synthesis plays a major role in the binding of factor VIII to platelets induced by thrombin, ADP, or collagen.

To elucidate the bleeding tendency that follows the administration of ticlopidine, we investigated the skin bleeding time and some ex vivo functions of platelets obtained from eight healthy volunteers before and 1 wk after daily administration of 500 mg of ticlopidine. We found the following: ticlopidine significantly (P less than 0.001) prolonged the skin bleeding time and impaired the binding of radiolabeled fibrinogen and von Willebrand Factor, the clot retraction and the aggregation of platelets in response to ADP, epinephrine, thrombin, ionophore A23187, collagen, or arachidonic acid. In contrast, the administration of this drug did not affect intraplatelet levels of cAMP, agglutination and binding of von Willebrand Factor in response to ristocetin, shape change in response to ADP, collagen, thrombin, or arachidonic acid, or binding of prostaglandin E1 to resting platelets. Secretion of ATP in response to ADP or epinephrine was completely inhibited, whereas secretion as well as thromboxane synthesis in response to high concentrations of collagen, arachidonic acid, calcium ionophore A23187, or thrombin was unaffected. Studies with monoclonal antibodies showed that the glycoprotein IIb-IIIa complex (the putative receptor for fibrinogen and von Willebrand Factor on the surface of platelets exposed to naturally occurring aggregating agents) was quantitatively unaffected by ticlopidine. This observation was further confirmed by densitometric scannings of Periodic Acid-Schiff-stained gels of platelet suspensions. The onset, as well as the cessation of the inhibitory effect of ticlopidine on platelets was very slow, and reached a maximum after a 3-5-d administration. In addition, ticlopidine appeared to be a much more potent inhibitor when administered to subjects than when added in vitro to platelets. Finally, abnormalities comparable to those found in volunteers taking ticlopidine were observed when platelets from untreated subjects were incubated in the plasma of ticlopidine-treated subjects. We conclude that ticlopidine induces a thrombasthenic state in normal platelets without affecting the glycoprotein IIb-IIIa complex quantitatively. Furthermore, our data suggest that one or more active metabolites rather than the native drug mediate the abnormalities of platelet function observed in ticlopidine-treated subjects.

To verify whether angiotensin II (ANG II) stimulates ADH release in humans and to evaluate whether endogenous prostaglandins influence the resulting renal effect of ADH, nonpressor and low pressor doses of ANG II were infused in nine normal volunteers under normal conditions (control study) and after prostaglandin synthesis inhibition with aspirin (ASA study). During ANG II infusion plasma ADH increased in both conditions. Plasma PGE2, urinary PGE2, and urinary 6-keto-PGF1 alpha increased only in the control study, whereas they were undetectable in the plasma and significantly reduced in the urine in the ASA study. ANG II caused a significant fall of glomerular filtration rate, renal plasma flow (with an increase in filtration fraction), fractional sodium excretion, and urine output in both studies. Despite the reduced urine output, urine osmolality decreased significantly in the control study, whereas it increased after aspirin administration. These results suggest that intravenous ANG II stimulates ADH release in humans but that the renal effects of the resulting increase in plasma ADH are different depending on the presence or absence of endogenous prostaglandins.

Platelet concentrates stored at 22 degrees C have a marked decrease in their aggregation response to adenosine diphosphate (ADP) or epinephrine but a normal response to these agents when used as a pair. Since platelet stimulation involves exposure of receptors for fibrinogen, we studied fibrinogen binding to platelets from fresh and stored concentrates. Following stimulation with 10 microM ADP or 20 microM epinephrine, platelet suspensions from fresh concentrates bound 125I-fibrinogen in a reaction that reached completion within 30 min. Significantly less binding occurred in suspensions from platelet concentrates that had been stored for 5 days at 22 degrees C. When stimulated by ADP and epinephrine as a pair (2 microM each), binding of fibrinogen to platelets was complete within 10–15 min and was not significantly decreased in suspensions from stored concentrates. We also investigated the effect of storage on the glycoprotein IIb-IIa complex, thought to be a specific receptor for fibrinogen on the platelet surface. Binding of a monoclonal antibody specific for this complex (B59.2) to platelet suspensions was unaffected by 5 days of storage. Furthermore, B59.2 inhibited aggregation, secretion, and fibrinogen binding of fresh and stored platelets stimulated with the pair of agents just as it did with single agents. We conclude that storage for 5 days at 22 degrees C impairs the exposure of fibrinogen receptors on platelets in response to ADP or epinephrine when used as single agents, without affecting the glycoprotein IIb-IIIa complex quantitatively. The function of the receptor is normal in response to the pair of agents.

SummaryU-46619 is a stable epoxymethano analogue of cyclic endoperoxide PGH2. We studied platelet aggregation, 14C-5HT release, LDH extrusion and prostaglandin and thromboxane production induced by this compound in platelet-rich plasma samples from 15 healthy volunteers. Each subject was tested both before and 90 min after aspirin (500 mg) ingestion. The threshold aggregating concentration (TAC) of U-46619 ranged between 0.18 and 0.90 µM. Aggregation was maximal between 40 and 60 min after venipuncture and was concentration-dependent. At concentrations below the TAC, U-46619 induced primary reversible aggregation with minimal 14C-5HT release. At TAC or higher concentrations aggregation and release proceded as parallel events. Neither prostaglandin or thromboxane production nor LDH loss could be detected in any of the situations tested. Aspirin ingestion did not modify the pattern of platelet responses. In unstirred, not aggregated platelet samples 14C-5HT release by U-46619 occurred to a similar extent as in stirred, aggregated platelet samples. Addition to citrated PRP of 0.3 mM Na2 EDTA blocked both aggregation and release induced by U-46619. This compound, however, aggregated washed platelets resuspended in Ca++-free-tyrode-albumin containing fibrinogen. The mechanism by which U-46619 activates platelets differs from that of all other common aggregating agents.

Following stimulation with adenosine diphosphate (ADP), collagen, or arachidonic acid, unstirred human platelet suspensions bind 125I- fibrinogen in a reaction that reaches completion within 30 min. Scatchard analysis of these binding data reveals two sets of binding sites with all 3 agents: a high affinity site (Kd 0.029–0.045 microM) binding 1000–1600 fibrinogen molecules per platelet, and a lower affinity site (Kd 1.2–2.0 microM) binding 46,000–76,000 fibrinogen molecules per platelet. At a concentration of apyrase that inhibited ADP-induced fibrinogen binding by greater than 85%, fibrinogen binding induced by collagen and arachidonic acid was only partially affected. This suggests that fibrinogen binding induced by collagen or arachidonic acid does not require released ADP. We isolated a monoclonal antibody, B59.2, which precipitated the glycoprotein IIb- IIIa complex from solubilized platelet membranes. Binding of labeled antibody to platelets before or after exposure to ADP, collagen, or arachidonic acid showed a single class of approximately 22,000 binding sites with Kd 0.019 microM. Binding of B59.2 was complete within 1 min and was not inhibited by EDTA. Preincubation of platelet suspensions with a 2.1 microM concentration of B59.2 caused inhibition of secretion and aggregation, but not of thromboxane-B2 synthesis, in response to 1 microgram/ml collagen, 40 microM arachidonic acid, or 4 microM ADP, concentrations of aggregating agents that produced complete aggregation and secretion in the absence of B59.2. At this concentration of B59.2, fibrinogen binding to stimulated platelets was inhibited by approximately 45%-55%. These data demonstrate that collagen and arachidonic acid can expose fibrinogen binding sites independently of released ADP; and that the glycoprotein IIb-IIIa complex is involved in secretion, aggregation, and fibrinogen binding, but not in thromboxane synthesis occurring in response to collagen, arachidonic acid, or ADP.

Oral contraceptive (OC) treatment is associated with an increased thrcmbotic risk, the mechanism of which remains still unclear. We have investigated the platelet response to exogenous AA and the plasma and platelet lipid composition in eight healthy women (aged 18-29 y) before and after six month OC treatment with a canbination of d-norgestrel (0.25 mg) and ethynilestradiol (0.05 mg). The threshold aggregating concentration of AA was significantly reduced in platelets of women after 6 months of OC treatment (0.30±0.04 mM versus 0.65 ± 0.08 mM, p < 0.001) whereas it was unchanged in a group of 10 control women, matched for age with the OC treated group and tested twice, with a 6-month interval (0.48 ± 0.04 versus 0.50 ± 0.04). In the same group of patients, seme clotting and fibrinolytic parameters, namely the Normotest, the biological and antigenic activity of Antithranbin III, the anti factor Xa biological activity, the diluted whole blood clot lysis time, were completely unmodified by OC treatment.In platelets AA increased both in phospholipid and neutral lipid fractions. In plasma the pool of polyunsaturated fatty acids increased at the expense of the saturated pool. In particular, AA increased slightly but significantly in free-fatty acids and phospholipid fractions.This data indicates that during OC treatment changes in in vitro platelet aggregatory responses may be associated with modifications of platelet and plasma fatty acid composition. Such changes may contribute to the thrcmbotic tendency associated with OC treatment.

Captopril (C) causes ARF in hypertensive patients with renal artery stenosis (RAS) with a single functioning kidney (SK). Retrospective studies in two patients showed that episodes of C-induced ARF were preceded by a rise in urinary Na+ excretion and a rapid decrease in body weight. These observations prompted us to investigate whether extracellular fluid volume depletion secondary to C-induced natriuresis can be responsible for ARF. Prospective studies were performed in four patients with RAS-SK treated with C. These studies have shown that: ARF is associated with negative Na+ balance and is corrected by salt replacement, even without interrupting C; ARF is preceded by a rise in urinary prostaglandin (PG) E2 and 6-keto-F1 alpha; ARF is prevented by either saline infusion or aspirin administration; ARF does not occur when the dose of C is not sufficient to raise PGs and urinary N + excretion. We conclude therefore that C-induced ARF in patients with RAS-SK can be secondary to salt depletion dependent on a raised secretion of PGs.

Using captopril (C), an angiotensin (ANG) I converting-enzyme inhibitor, to increase endogenous prostaglandins (PGs) and to decrease endogenous ANG II synthesis, we studied the relationship between endogenous ANG II, PG, and antidiuretic hormone (ADH) release in seven normal volunteers before (control study) and after inhibition of PG synthesis by a single dose of aspirin (ASA study). In the control study, following the administration of 100 mg of C, there was a significant increase of plasma PGE2, plasma-renin activity (PRA), and urinary PGE2 and 6-keto-PGF1 alpha and a decrease of plasma ADH. Glomerular filtration rate (GFR) and renal plasma flow (RPF) were unaffected by C; urine output, fractional sodium excretion (FENa), and osmolal clearance (Cosmol) increased; and urinary osmolality (Uosmol) decreased significantly after C. In the ASA study PG were undetectable in plasma and significantly reduced in urine 1 h after aspirin and did not increase when C was added. Plasma ADH decreased and PRA increased, as in the control study, after C, whereas GFR, RPF, urine output, FENa, Cosmol, and Uosmol were unchanged. These results suggest that the effect of C on ADH release may be mediated, to a large extent, by a fall in endogenous circulating ANG II, since ADH decreased in the presence of both high or undetectable levels of PGE2. The results also suggest that the increase in PGE2 induced by C may precipitate the diuretic and natriuretic effects of acute C administration.

The purpose of this study was to evaluate both the natriuretic and the renal hernodynamic effects of Enalapril (E), a new ACE-inhibitor, in hypertensive patients.

The physiopathological role of platelet aggregation in some thromboembolic and atherosclerotic complications is strongly suggested on the basis of many indirect findings. The qualitative methodological approach to this problem generally used until recently is rapidly giving way to a quantitative, biochemical approach. Platelet aggregation, however, even if expressed in terms of nanomoles of a product obtained in a sophisticated reaction system, will continue to deceive investigators and clinicians who fail to view it in the adequate (although still uncertain) context of rheological and vascular interactions.

Isoprostanes represent a newly characterized family of vasoactive eicosanoids that are formed by a non-cyclooxygenase, free radical-catalyzed mechanism involving peroxidation of arachidonic acid (Morrow et al, 1990). One of these compounds is 8-epi-PGF2α which has attracted considerable attention because of its biological activity. 8-epi-PGF2α is a potent vasoconstrictor in the rat and its action is mediated, at least in part, through the vascular PGH2/TXA2 receptor (Takahashi et al., 1992). Moreover, 8-epi-PGF2α induces platelet shape change but not aggregation (Morrow et al., 1992).

In an attempt to elucidate the antithrombotic potential of defibrotide (D) we have evaluated several functions of monocytes from 7 healthy subjects before and after in vitro incubation of the cells with increasing concentrations of this drug. At concentrations as high as 40 μg/ml, D hardly affected the expression of both the procoagulant activity of monocytes and the formation of superoxide anion in response to 1 mg/ml zymosan (STZ). In contrast, at concentrations that may be achieved in vivo following the administration of the drug (5–20 μg/ml), D impaired in a dose-dependent manner (p < 0.05) the generation of O-2 in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP, 1 μM) or calcium ionophore A23187 (10 μM). Regardless of the agonist employed, at concentrations between 1 and 5 mM, extracellular Ca2+ had little effect on the impairment of superoxide anion generation by D. In contrast, the inhibitory effect was time-dependent, the maximum impairment ( > 30%) being observed when the cells were preincubated with the drug for 20 h. These data support the concept that the antithrombotic potential of D involves the ability of the drug to affect the generation of free radicals by leukocytes and suggest that future in vivo studies for the evaluation of the activity of D should take into account the role of monocytes in hemostasis and thrombosis.

U-46619 is a stable analogue of cyclic prostaglandin endoperoxides which induces human platelet aggregation independently from nucleotide secretion. We studied platelet aggregation, 14C-5 HT release and malondialdehyde (MDA) production induced by this compound in stirred or unstirred platelet-rich plasma (PRP) samples from 11 healthy volunteers. Each subject was tested both before and 90 min after aspirin ingestion (500 mg). The threshold aggregating concentration (TAC) of U-466l9 ranged between 240 and 900 nM. Aggregation was maximal between 30 and 60 min after venepuncture and was concentration-dependent (60-7, 200nM). At concentrations below the TAC, U-466l9 induced primary reversible aggregation without detectable l4C-5 HT release. At TAC or higher concentrations aggregation and release proceeded as parallel events. Neither MOA production nor intracellular LDH loss could be detected in any of the tested situations. Aspirin ingestion did not modify the above pattern of platelet responses. In unstirred samples l4C - 5 HT release occurred to the same extent as in stirred platelet suspensions. Addition to citrated PRP of Na2 - EDTA did not affect either aggregation or release. It is suggested that aggregation and secretion may be independent, parallel responses of platelet activation by U-466l9 and do not require either extracellular calcium or activation of endogenous arachidonic acid metabolism. (Supported by the Italian CNR and NIH).

SummaryWe have evaluated plasma fibrinogen levels in 171 subjects attending a metabolic ward. As in the general population, a significant difference in plasma fibrinogen concentrations (p The relationships between certain fibrinogen genotypes and familial risk have then been evaluated. Analysis of a locus (1.3 kb, HAE III digestion) of the promoter region of the BB fibrinogen gene, identified a polymorphic cutting site. The allele with the alternative restriction site (HI) was associated with mean fibrinogen levels which were 0.1-0.3 g/1 lower than those associated with the other allele (H2). This difference was not statistically significant. No obvious association was found between the familial risk and the presence of the H2 allele. We conclude that in a group of subjects from a metabolic ward, a positive family history for ischemic complications of atherosclerosis is consistently associated with high plasma fibrinogen levels. Interaction with hypertension significantly strengthens the association.

Mice inoculated with an artificially constructed retrovirus carrying the middle T gene of polyomavirus develop acute myeloproliferative disease with severe thrombotic and hemorrhagic disorder and impaired platelet function. The megakaryocytic lineage appears to be a target for polyoma-murine leukemia virus infection and middle T gene expression. This newly described disease represents a unique model system for studying disorders of the megakaryocytic lineage.

Risk of Myocardial Infarction in Carriers of Mutations in the Hemochromatosis-associated Gene

There have been reports of a 24–48-hr delay in the recovery of platelet cyclooxygenase activity and platelet function after the ingestion of aspirin. However, these studies employed a single aggregating agent to stimulate enzymatic or functional activity. We investigated the effects of some pairs of aggregating agents on 14 platelet-rich plasmas (PRP) from normal subjects 2 and 4 hr after ingestion of 650 mg aspirin and daily up to 72 hr. We studied platelet aggregation and secretion with a lumiaggregometer and thromboxane-B2 formation by radioimmunoassay. Aggregation and secretion occurred as early as 4 hr after aspirin ingestion in response to combinations of arachidonic acid with epinephrine, collagen, or adenosine diphosphate (ADP). Thromboxane formation was detected as early as 4 hr after ingestion of aspirin in response to 1 mM arachidonic acid in combination with 1 microgram/ml collagen. Up to 72 hr, there was a linear return of thromboxane formation stimulated by this combination, reflecting the entry of new platelets into the circulation. In vitro experiments with mixtures of aspirin-free and aspirin-treated platelets showed that the combination of collagen and arachidonic acid (AA) could produce full aggregation and secretion when only 2.5% of aspirin-free platelets were present. Use of the combination of collagen plus AA demonstrates the early entry into the circulation of platelets originating from megakaryocytes whose cyclooxygenase has not been completely acetylated.

We evaluated 106 subjects with and 109 subjects without a history of ischemic stroke. All were attending a metabolic ward. The two groups were compared for major risk factors for ischemic events. A positive family history for ischemic complications of atherosclerosis was more common in subjects with a history of stroke than in those without; moreover, plasma levels of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (TPA) were higher in patients with documented previous events. A strong positive significant correlation was found between TPA and PAI-1 levels, and an interaction between age and TPA was observed when the sample was stratified according to ages being above or below 70 years. When the patient population was analyzed according to the number of ischemic events, it was found that 62 of the 106 subjects with a history of stroke had experienced more than one ischemic event. Under these conditions, the levels of TPA and PAI-1 still correlated with the occurrence of previous ischemic episodes. As in the whole patient sample, TPA was the strongest discriminator. We conclude that in subjects attending a metabolic ward, TPA and PAI-1 levels consistently help identify subjects with a history of cerebral ischemic episodes and that TPA is the strongest discriminator.

18 rats were treated with L-ASA before heart transplantation and daily thereafter until death or rejection. 22 animals acted as controls. A significantly higher post-operative mortality rate, without any significant modification of the transplant survival time, was found in L-ASA-treated group.