Endocan: A biomarker for endothelial dysfunction and inflammation, linking maternal obesity and pediatric obesity in a cohort of preterm neonates.

OBJECTIVES: Numerous animal and epidemiologic studies have demonstrated a positive association between maternal obesity in pregnancy and obesity in offspring. The biologic mechanisms of this association remain under investigation. One proposed mechanism includes fetoplacental endothelial dysfunction secondary to inflammation. Endocan is a relatively new biomarker for endothelial dysfunction and inflammation. Our objectives were to examine (1) the association between maternal obesity and neonatal serum endocan at birth, and (2) the association between neonatal serum endocan at birth and pediatric obesity at 24-36 months of age. STUDY DESIGN: This was a secondary analysis of a prospective cohort of neonates born < 33 weeks gestation. Serum endocan was collected within 48 hours of birth. Serum endocan levels were compared in neonates born to obese mothers vs. those born to non-obese mothers. BMI data were retrospectively collected from cohort neonates between 24 and 36 months of age. RESULTS: The analysis included 120 mother/neonate dyads. Neonates born to obese mothers had higher median serum endocan at birth compared to neonates born to non-obese mothers (299 ng/L [205-586] vs. 251 ng/L [164-339], p = 0.045). In a linear regression modeled on neonatal serum endocan level, maternal obesity had a statistically significant positive association (p = 0.021). Higher mean serum endocan level at birth was associated with pediatric obesity between 24 and 36 months (obese vs. non-obese offspring; 574 ng/L (222) vs. 321 ng/L (166), p = 0.005). CONCLUSIONS: In our cohort of preterm neonates, elevated serum endocan at birth was associated with both maternal obesity and downstream pediatric obesity. More research is needed to understand intergenerational transmission of obesity. A large focus has been on epigenetic modification. Endothelial dysfunction and inflammation may play important roles in these pathways. Effective biomarkers, including endocan, may also serve as intermediate outcomes in future pregnancy research.

Exposure to the Amino Acids Histidine, Lysine, and Threonine Reduces mTOR Activity and Affects Neurodevelopment in a Human Cerebral Organoid Model.

Evidence of the impact of nutrition on human brain development is compelling. Previous in vitro and in vivo results show that three specific amino acids, histidine, lysine, and threonine, synergistically inhibit mTOR activity and behavior. Therefore, the prenatal availability of these amino acids could be important for human neurodevelopment. However, methods to study the underlying mechanisms in a human model of neurodevelopment are limited. Here, we pioneer the use of human cerebral organoids to investigate the impact of amino acid supplementation on neurodevelopment. In this study, cerebral organoids were exposed to 10 mM and 50 mM of the amino acids threonine, histidine, and lysine. The impact was determined by measuring mTOR activity using Western blots, general cerebral organoid size, and gene expression by RNA sequencing. Exposure to threonine, histidine, and lysine led to decreased mTOR activity and markedly reduced organoid size, supporting findings in rodent studies. RNA sequencing identified comprehensive changes in gene expression, with enrichment in genes related to specific biological processes (among which are mTOR signaling and immune function) and to specific cell types, including proliferative precursor cells, microglia, and astrocytes. Altogether, cerebral organoids are responsive to nutritional exposure by increasing specific amino acid concentrations and reflect findings from previous rodent studies. Threonine, histidine, and lysine exposure impacts the early development of human cerebral organoids, illustrated by the inhibition of mTOR activity, reduced size, and altered gene expression.

Leptin and ghrelin concentrations and weight loss in Parkinson's disease.

OBJECTIVES: To investigate the role of leptin, ghrelin, GH and IGF-1 in energy balance disturbances in Parkinson's disease (PD). MATERIALS AND METHODS: Thirty-nine patients were included: 11 PD patients with unintentional weight loss, 16 PD patients without weight loss and 12 controls. UPDRS, MMSE, MADRS, appetite scale, BMI, adipose tissue content, plasma leptin and active ghrelin concentrations and serum GH, IGF-1, TSH, T3 and T4, concentrations were evaluated. RESULTS: A lower plasma leptin concentration and a higher serum IGF-1 concentration were found in PD patients with weight loss. BMI and the content of adipose tissue were positively correlated with leptin concentration in all PD patients. Paradoxically, the lower BMI was, the lower plasma active ghrelin concentration was in PD patients with the weight loss. CONCLUSION: These findings confirm that changes of plasma leptin concentration occur in PD patients with loss of weight.

The association between AB blood group and neonatal disease.

BACKGROUND: Numerous studies have examined the association between ABO blood groups and adult disease states, but very few have studied the neonatal population. The objective of this study was to determine the relationship between AB blood group and the occurrence of common neonatal disorders such as neutropenia at birth, sepsis, respiratory distress syndrome (RDS), intraventricular hemorrhage (IVH), retinopathy of prematurity (ROP), and patent ductus arteriosus (PDA) compared to all other blood groups. METHODS: We performed a retrospective review on 3,981 infants born at 22 0/7 to 42 6/7 weeks' gestational age and compared the relative risk of neonatal diseases in infants with AB blood group to that of infants with all other blood groups (A, B, and O). RESULTS: When compared to all other blood groups, AB infants demonstrated an increased risk for developing negative clinical outcomes. AB blood group was significantly associated with a 14-89% increased risk of neutropenia at birth, sepsis, RDS, and ROP. Risks for IVH and PDA were not significant. CONCLUSION: We hypothesize that the phenotypic expression of A and B antigens, rather than the antigens themselves, in the AB group may reveal an enhanced susceptibility to injury at the endothelial level resulting in an increased risk for disease development.

1,161 patients with adrenal incidentalomas: indications for surgery.

BACKGROUND AND AIMS: The aim of this study is to analyze the clinical data and criteria for surgery in a group of over 1,100 patients with adrenal incidentalomas (AI) observed at the Department of Endocrinology. PATIENTS AND METHODS: The material consisted of 1,161 patients (842 women and 319 men, 10-87 years old) with AI ranging in size from 1.0 to 23.0 cm. The methods included clinical examination, imaging studies, hormonal determinations in the blood and in the urine as well as histological and immunocytochemical investigations in 390 patients treated by surgery. RESULTS: Basing on these studies, we diagnosed 112 patients with primary malignant adrenal tumors (100 with carcinoma), 45 with metastatic infiltrations, and 1,004 with probable benign AI. Imaging phenotypes (especially high density on computed tomography, CT) were characteristic of malignant and chromaffin tumors. Subclinical adrenal hyperactivity was found in 8% of the patients with pre-Cushing's syndrome as the most frequent form (6.5%). Chromaffin tumors were detected in 3%. CONCLUSIONS: (1) Indications for surgery include malignant tumors (both primary and metastatic), tumors with subclinical hyperfunction, and chromaffin tumors. High density on CT, >20 HU, appeared to be an important indication for surgery. (2) A slight prevalence of oncological indications over endocrinological indications (14 vs. 11%) was found.

Molecular profiling of heparinase-I resistant glycosaminoglycans in contaminated heparins. Comparative studies with uncontaminated heparin and porcine oversulfated chondroitin sulfate.

AIM: Heparin is a widely used anticoagulant which is usually obtained from porcine mucosal tissue. The structure of heparin is comparable to other naturally occurring glycosaminoglycans such as chondroitin sulfate and dermatan sulfate. The commercially available heparin preparations may contain small amounts of dermatan sulfate as a carry-over impurity. More recently (November 2007 to April 2008), an increased incidence of adverse events and deaths associated with the use of heparin alerted regulatory agencies to investigate the composition of heparin. As a result, oversulfated chondroitin sulfate was found to be the main determinant of the observed adverse reactions. This glycosaminoglycan is not usually found in the mammalian tissues. METHODS: This investigation reports on the comparison of contaminant free and contaminated heparins and their digestion by heparinase-I. It also describes the molecular profile of the contaminant isolated from the recalled heparin preparations in comparison to oversulfated chondroitin sulfate. The anticoagulant and anti-Xa activities are also reported. RESULTS: The contaminant is found to be comparable to the synthesized OSCS as both were resistant to heparinase-I digestion. The contaminant and OSCS exhibited weaker anticoagulant activities than heparin and did not have any anti-Xa effects. CONCLUSION: This data strongly suggests that such glycosaminoglycans as chondroitin sulfate can be structurally modified to exhibit anticoagulant activities and their molecular weight can be adjusted to mimic heparin.

Structural features and anticoagulant activities of a novel natural low molecular weight heparin from the shrimp Penaeus brasiliensis.

A natural low molecular weight heparin (8.5 kDa), with an anticoagulant activity of 95 IU/mg by the USP assay, was isolated from the shrimp Penaeus brasiliensis. The crustacean heparin was susceptible to both heparinase and heparitinase II from Flavobacterium heparinum forming tri- and di-sulfated disaccharides as the mammalian heparins. (13)C and (1)H NMR spectroscopy revealed that the shrimp heparin was enriched in both glucuronic and non-sulfated iduronic acid residues. The in vitro anticlotting activities in different steps of the coagulation cascade have shown that its anticoagulant action is mainly exerted through the inhibition of factor Xa and heparin cofactor II-mediated inhibition of thrombin. The shrimp heparin has also a potent in vivo antithrombotic activity comparable to the mammalian low molecular weight heparins.

Activation of platelets by heparin-induced thrombocytopenia antibodies in the serotonin release assay is not dependent on the presence of heparin.

The serotonin release assay (SRA) tests for antibodies responsible for heparin-induced thrombocytopenia (HIT). By definition, SRA-positive antibodies cause platelet serotonin release in vitro, in the presence of low concentrations of heparin, but not with excess heparin. Many SRA-positive sera activate platelets in the presence of saline without drug, either as a result of residual heparin in the specimen, or because of intrinsic features of the HIT antibodies. The present experiments show that neither exhaustive heparinase treatment, nor chromatographic removal of heparin abrogates the spontaneous platelet activation caused by these HIT antibodies. This is the first study to systematically demonstrate that in vitro activity of HIT antibodies can be independent of heparin. In addition, T-gel chromatography demonstrated differences among fractions of enzyme-linked-immunosorbent assay (ELISA)-positive HIT antibodies within individual specimens. Certain ELISA-positive fractions had SRA activity while others did not, and the SRA activity was not proportional to HIT antibody ELISA titer. These data suggest that antibodies formed as a result of heparin treatment are heterogeneous, and that some can contribute to the pathogenesis of HIT even when heparin is no longer present.

Comparative tissue factor pathway inhibitor release potential of heparins.

Tissue factor pathway inhibitor (TFPI) is released following the administration of unfractionated heparin, low-molecular-weight heparins, defibrotide and PI-88. In this study, the comparative effects of heparin, a low-molecular-weight heparin-gammaparin and a heparin-derived oligosaccharide mixture-subeparin (C3) were studied on functional and immunologic tissue factor pathway inhibitor activity levels in a non-human primate (Macaca mulatta) model. The dose-dependent effect was studied following intravenous and subcutaneous administration. Following the administration of 1 mg/kg of heparin, gammaparin, and C3, the functional levels of TFPI at 5 minutes were 2.40, 2.56, and 1.08 U/mL and the corresponding TFPI immunologic levels were 4.3-, 4.0-, and 2.1-fold, increased, respectively, over the baseline value. From these results, it can be concluded that heparin and gammaparin produced similar levels of TFPI release. Hence, gammaparin and heparin have similar TFPI release potential despite their differences in molecular weight. The influence of molecular weight, charge density, and interactions with heparin cofactor II on TFPI release are also discussed.

The role of endogenous opiates in the mechanism of inhibited luteinizing hormone (LH) secretion in women with anorexia nervosa: the effect of naloxone on LH, follicle-stimulating hormone, prolactin, and beta-endorphin secretion.

The aim of this study was to evaluate the role of endogenous opiates in the mechanism of decreased LH secretion in women with anorexia nervosa. For this purpose the effect of opiate receptor blockade with naloxone on LH, FSH, PRL, and beta-endorphins secretion was studied in 24 women with anorexia nervosa and 7 normal women. Serum LH, FSH, PRL, beta-endorphin-like substance, ACTH, and cortisol concentrations were measured before and after opiate receptor blockade after a single iv dose of 0.2 mg/kg naloxone or saline. Mean serum LH and FSH concentrations increased significantly after naloxone in the normal women. Eleven patients had a significant increase in serum LH concentrations in response to naloxone and 13 did not respond to naloxone with an increase in LH concentration. In the first group the basal LH values were higher than those in the second group. In the majority of patients in the first group amenorrhea preceded the wt loss, whereas in most patients in the second group amenorrhea appeared during the phase of wt loss. Naloxone did not alter pulsatile LH secretion in 6 women. No effect of naloxone on serum FSH and PRL concentrations was found. A significant increase in beta-endorphin-like substance levels after naloxone administration occurred in patients with anorexia nervosa. However, serum ACTH and cortisol concentrations were not altered in response to naloxone. In conclusion, the increase in LH release after opiate receptor blockade by naloxone suggests that endogenous opiates may play a role in the mechanism of inhibited LH secretion at least, in the majority of those women with anorexia nervosa in whom amenorrhea preceded wt loss. The results also point to a different mechanism of ACTH and beta-endorphin secretion in patients with anorexia nervosa.

An abnormality of the growth hormone/insulin-like growth factor-I axis in women with polycystic ovary syndrome due to coexistent obesity.

In order to evaluate the GH/insulin-like growth factor-I (IGF-I) axis in the polycystic ovary syndrome (PCO), 21 women aged 18-38 yr were studied. The GH responses to the GH-releasing hormone (GHRH), and plasma concentrations of IGF-I were measured in seven obese women with PCO, seven obese healthy controls without PCO, and in seven nonobese subjects. Total GH secretion, as expressed by the integrated GH response to GHRH, in PCO obese women (617.4 +/- 150 micrograms/L.min) and in obese women without PCO (327.1 +/- 161.4 micrograms/L.min) were lower than that in nonobese healthy controls (3181.4 +/- 644.3 micrograms/L.min, P less than 0.001 and P less than 0.001, respectively). Plasma concentrations of IGF-I in obese PCO women (199.5 +/- 39.1 micrograms/L), and in obese women without PCO (192.4 +/- 36.8 micrograms/L) were similar to the IGF-I levels in nonobese controls (224.3 +/- 33.2 micrograms/L). In obese women with and without PCO, a negative correlation was found between the body mass index and the peak GH responses to GHRH (r = -0.639, P less than 0.02) and between age and IGF-I levels (r = -0.520, P less than 0.05). These findings suggest that an abnormality of the GH/IGF-I axis in PCO women may be due to coexistent obesity.

Laboratory tests for heparin-induced thrombocytopenia: a multicenter study.

A multicenter clinical trial of the thrombin inhibitor argatroban (Novastan; Texas Biotechnology, Houston, TX; Smith-Kline Beecham Pharmaceuticals, Philadelphia, PA) was recently conducted in patients with heparin-induced thrombocytopenia (HIT) and HIT that had progressed to thrombosis (HITTS). In patients defined by the inclusion/exclusion criteria, the utility of three diagnostic HIT assays was investigated: the platelet aggregation assay, the serotonin release assay (SRA), and the enzyme-linked immunosorbent assay (ELISA) for the antibody to the heparin-platelet factor 4 (H-PF4) complex. Confirmation was made in 26%, 55%, and 64% of the patients, respectively (n = 199 patients; 512 to 606 samples; P < .001 platelet aggregation assay v SRA v ELISA). Patients who progressed to HITTS (n = 98) were more often confirmed than were HIT patients without associated thrombosis (n = 101) (P < .05). Confirmation by platelet aggregation assay and SRA results generally was associated with a higher antibody titer. However, a minimum critical titer could not be identified, because all patterns of positive and negative results by the platelet aggregation assay, SRA, and ELISA were observed, and clinically ill patients had a wide range of antibody titers. Over a 30-day period, the percentage of positive responses did not change. Although multiple testing over several days enhanced the chance of confirmation, this difference was not significant. Combined results of the three assays enhanced the positive response to 83% of the total population (P < .005). These data demonstrate that there is no direct correlation between the positive response of these assays, and that clinically positive HIT patients can be missed by all three assays. With these limitations, the combination of platelet aggregation assay, SRA, and ELISA testing with multiple samples offers the best chance of confirming a positive HIT patient. Caution is advised, however, in interpreting all assay results, as no assay is optimal.

Effect of SR121566A, a potent GP IIb-IIIa antagonist, on the HIT serum/heparin-induced platelet mediated activation of human endothelial cells.

Heparin-induced thrombocytopenia (HIT) is a common adverse effect of heparin therapy that carries a risk of serious thrombotic events. This condition is caused by platelet aggregation, which is mediated by anti-heparin/platelet factor 4 antibodies. Sera from patients with HIT in the presence of platelets, induced the expression of E-selectin, VCAM, ICAM-1 and tissue factor and the release of IL1beta, IL6, TNFalpha and PAI-1 by human umbilical vein endothelial cells (HUVECs) in vitro and initiated platelet adhesion to activated HUVECs. These effects which occurred in a time-dependent manner were significant in the first 1-2 h of incubation and reached a maximum after 6 to 9 h. The GP IIb-1IIa receptor antagonist SR121566A which has been shown to block platelet aggregation induced by a wide variety of agonists including HIT serum/heparin, reduced in a dose-dependent manner the HIT serum/heparin-induced, platelet mediated expression and release of the above mentioned proteins. The IC50 for inhibition of HIT serum/ heparin-induced platelet dependent HUVEC activation by SR121566A was approximately 10-20 nM. ADP, but not serotonin release, also appeared to be involved as apyrase and ATPgammaS blocked platelet-dependent, HIT serum/heparin-induced cell surface protein expression and cytokine release by HUVECs. Increased platelet adherence to HIT serum/heparin-activated HUVECs was inhibited by SR121566A and, to a lesser extent, by apyrase and ATPgammaS, showing that platelet activation and release was at the origin of the HIT serum/heparin-induced expression of these proteins by HUVECs. Thus, sera from patients with HIT induced the expression of adhesive and coagulation proteins and the release of cytokines by HUVECs through the activation of platelets which occurred in a GP IIb-IIIa-dependent manner, a process that could be selectively blocked by SR121566A.

Low-molecular-weight heparins: pharmacologic profile and product differentiation.

The interchangeability of low-molecular-weight heparins (LMWHs) has been the subject of discussion since these products were first introduced for the prophylaxis of deep vein thrombosis. Experimental evidence now exists to show that LMWHs differ from each other in a number of characteristics. Products have been differentiated on the basis of molecular weight and biologic properties, but only limited information derived from the clinical setting is available. Potency has been described on the basis of anti-Factor Xa activity, but at equivalent anti-Xa activities, the anti-Factor IIa activity of different products shows marked variations. At the relatively small doses used for the management of postsurgical deep vein thrombosis, the effect of these interproduct differences may be relatively minor, but as LMWHs are developed for therapeutic use at much higher doses, such differences may become clinically important. Variations in safety and efficacy reported in clinical trials of LMWHs may reflect the known differences in their molecular composition and pharmacologic properties.

Early diagnosis of Nelson's syndrome.

Nelson's syndrome is a specific form of Cushing's disease treated by bilateral adrenalectomy, presenting with a deep hyperpigmentation caused by a pituitary adenoma (corticotropinoma). These ACTH-secreting tumors are frequently aggressive, so early diagnosis is of prime importance. We have studied 33 patients with Nelson's syndrome, 28 women and 5 men, aged 14-56 yr at the time of adrenalectomy and 16-58 yr at the time of Nelson's syndrome diagnosis (observed for 5-32 yr). Methods of examination included simultaneous adrenocorticotropic hormone (ACTH) and cortisol measurements during routine hydrocortisone replacement therapy, computed tomography (CT), pituitary magnetic resonance imaging (MRI), and visual field examination. The results obtained in a group of six patients diagnosed in the last 3 yr were compared with those obtained in a group of 27 patients examined before 1992. High plasma ACTH levels accompanied by normal serum cortisol concentration were characteristic for a late stage of the disease. Absolute temporal scotomas were an early finding. MRI, especially with the gadolinium enhancement, was superior to CT in demonstrating pituitary microadenomas in Nelson's syndrome. Thus, MRI diagnosis allowed for an early neurosurgical treatment of the patients with Nelson's tumors.

Statin drugs and dietary isoprenoids as antithrombotic agents.

Statin drugs and various isoprenoids from plant origins inhibit mevalonic acids, cholesterol, and other isoprenoid products. Among these, reduction of farnesyl and geranylgeranyl prenylated proteins impedes signal transduction at the cellular level. The authors envision that limiting such prenylated proteins downregulates thrombin-stimulated events, including decreasing the expression and availability of protease-activated receptor-1 mitigating thrombin stimulation of cells, tissue factor preventing additional thrombin generation, and plasminogen activator inhibitor-1 allowing thrombosis. Additional processes may enhance nitric oxide production and induce other processes. Downregulation of thrombin-stimulated events should promote hypothrombotic or quiescent conditions that reduce cardiovascular disease, thus contributing to longevity.

Molecular weight profiling of low molecular weight heparins utilizing a heparinase degraded oligosaccharide mixture as a calibrator.

Heparinase degraded heparin fragments (HDHF) are enriched with a UV chromophore and can be utilized as calibrators to determine the molecular weight profile of low molecular weight heparins (LMWH's). In a standard protocol (1), the second to the last peak is assumed to be a hexasaccharide with a molecular weight of 1.8 kDa. All other peak molecular weights in the elution profile are assigned based upon this assumption. In this study, synthetic analogues of heparin with a defined molecular weight have been used to investigate the validity of this assumption. These compounds included a sulfated bis-lactobionic acid amide (2.4 kDa), a pentasaccharide (1.7 kDa), and lactose polysulfate (1.2 kDa). With reference to these internal standards, the second to the last peak of the HDHF mixture was found to elute after the pentasaccharide and close to the lactose polysulfate suggesting that it is a tetrasaccharide. For additional validation, calibration curves were constructed using individual calibrators as the reference method as well as the HDHF method. Several low molecular weight heparins were profiled for their molecular weight using these methods. In comparison to the reference method, the molecular weights obtained with the HDHF method were 25-40% higher. When the results were recalculated assuming the molecular weight of the second to the last peak as 1.2 kDa, the HDHF results compared well with the reference method. Additionally, it is seen that the HDHF method does not identify molecular components greater than 8.0 kDa.

Inhibitory effects of TFPI variants on thrombin and factor Xa generation in fibrinogen-deficient human plasma.

Using a fast kinetic centrifugal analyzer, the inhibitory effects of glycosylated and unglycosylated full-length and truncated forms of TFPI on protease generation were studied in fibrinogen-deficient human plasma after extrinsic (EA) or intrinsic (IA) activation of coagulation. When the assay system was supplemented with increasing amounts of the TFPI variants the generation of both thrombin and factor Xa was inhibited in a concentration-dependent manner. Clear differences in the effectiveness of the TFPI variants were found. After EA, the unglycosylated full-length TFPI was most effective followed by the glycosylated full-length form. The C-terminal truncated TFPI showed the lowest inhibitory activity in this system. However, its efficiency increased several fold when coagulation was activated via the intrinsic pathway. Comparing the IC50 values after IA, the truncated TFPI was more effective than the unglycosylated full-length form and nearly as effective as the glycosylated full-length TFPI. After both EA and IA the thrombin generation inhibition by TFPI variants was more pronounced than the inhibition of factor Xa generation. The results show that chemical modifications of the TFPI structure can result in changes of TFPI's inhibitory properties to activated clotting factors leading to differences in protease generation inhibition.

Protamine sulfate neutralization of the anticoagulant activity of Aprosulate, a synthetic sulfated lactobionic acid amide.

Aprosulate or lactobionic acid is a highly sulfated analogue of heparin which is currently undergoing clinical trials in Europe as a potential antithrombotic drug. Aprosulate exerts a strong anticoagulant effect in plasma as a result of its interaction with heparin cofactor II. In this study, the ability of protamine sulfate to neutralize the anticoagulant activity of Aprosulate was investigated. In vitro, ex vivo, and in vivo coagulation studies were performed using various clotting assays such as the APTT, Heptest, and thrombin time as a measure of the anticoagulant activity of Aprosulate. In the first study, protamine sulfate when administered in vitro to plasma samples containing various concentrations of Aprosulate was found to effectively neutralize the anticoagulant activity of the Aprosulate in both normal human and normal monkey plasma systems. However, the relative index of neutralization of Aprosulate was assay dependent. Protamine sulfate was also found to antagonize the anticoagulant effects of Aprosulate in an ex vivo study. The ex vivo supplementation of protamine sulfate to plasma samples collected at various time intervals following the subcutaneous administration of Aprosulate to a group of primates completely neutralized the anticoagulant activity of the Aprosulate. In a third in vivo study, protamine sulfate when injected intravenously into the bloodstream of a group of primate was found to completely neutralize the anticoagulant effects of a previously administered dosage of Aprosulate. The results of these three studies clearly suggest that protamine sulfate can be used to effectively neutralize the anticoagulant activity of Aprosulate.

Anticoagulant and antithrombotic actions of a semisynthetic beta-1,3-glucan sulfate.

Sulfation of the natural polysaccharide curdlan results in anticoagulantly active beta-1,3-glucan sulfates whose activity depends on various structural parameters. In this study the anticoagulant and antithrombotic effects of one of these beta-1,3-glucan sulfates (GS) were compared with those of a porcine mucosal heparin. GS produced a concentration dependent anticoagulant effect in all the global coagulation assays with the exception of the anti-Xa assay. The best activity was found in the APTT and the thrombin time assays indicating that protease generation and the direct inhibition of thrombin may be sites of actions of this agent. Whereas the anticoagulant activity of GS was approximately 5 fold lower compared to heparin, a 32 fold higher concentration (ED50 = 550 micrograms/kg) was needed for an antithrombotic effect similar to heparin (ED50 = 17.2 micrograms/kg) in a rabbit model of stasis thrombosis. In contrast to this, when a rat model of clamping induced jugular vein occlusion was used to produce vascular obstruction, GS produced similar antithrombotic actions to heparin. At a 250 micrograms/kg dosage, both agents doubled the number of clampings required for complete vascular obstruction. Since the mechanical injury to the blood vessel is the primary determinant of the thrombogenic response, GS may inhibit some of the pathophysiologic mechanisms responsible for the occlusion of the blood vessel. The current study also points to the fact that the global anticoagulant effects may not reflect the antithrombotic potential of newer sulfated carbohydrate derived drugs.