An acquired antithrombin autoantibody directed toward the catalytic center of the enzyme.

Antibody inhibitors against human thrombin are rare and have remained poorly characterized. We report the case of a 40-yr-old patient who developed a potent thrombin inhibitor revealed by mild bleeding symptoms and marked prolongation of most laboratory clotting times. After two years of evolution, he died from cerebral hemorrhage. The inhibitor, a polyclonal IgG, was associated with hematological and immunological criteria of autoimmune disorder. Antithrombin IgG was isolated from the patient's plasma by protein A- and thrombin-affinity chromatography. Fab fragments inhibited amidolytic activity of alpha thrombin, and thrombin-thrombomodulin catalyzed protein C activation with a Ki of approximately 10(-8) M in a noncompetitive manner. Alpha to gamma conversion of thrombin resulted in a moderate loss of affinity for the inhibitor. Upon complex formation of thrombin with staphylocoagulase or alpha 2-macroglobulin (alpha 2M), inhibition was decreased by two orders of magnitude and acquired an apparent competitive character. In Western blot experiments, the antibody reacted with active alpha-thrombin, did not react with chloromethylketone-inhibited thrombin and reacted with a lower affinity with iPr2P-thrombin. The inhibitor did not block thrombin binding to benzamidine-, heparin-, or fibrin-Sepharose, but displaced proflavin from its complex with thrombin. Taken together, these results indicate that the patient's autoantibody recognized a conformational structure which includes, at least in part, the apolar binding site adjacent to the catalytic site of thrombin.

Studies on the structural requirements of heparin for the catalysis of thrombin inhibition by heparin cofactor II.

The structural requirements of heparin for the catalysis of thrombin inhibition by heparin cofactor II (HC II) were investigated. A series of well characterized heparin derivatives were prepared and their activities were measured using human thrombin in the presence of an excess of purified human HC II and, for comparison, antithrombin III (AT III). The 50% inhibitory concentrations of each derivative were calculated and compared with those of unmodified heparin. Heparin activity was strongly dependent on molecular weight (Mr) in a manner grossly comparable for the two inhibitors. High-Mr fractions were the most active. Below 10 kDa, the activity dropped rapidly. A minimum size of 26 residues appeared to be required for HC II activation (against 16-18 for AT III). Below 5 kDa, a residual activity two orders of magnitude lower than that of high-Mr species remained with HC II (but not with AT III). Heparin was selectively desulfated or oversulfated in the O- and/or N-position. When an N-acetyl group was substituted for the original N-sulfate in the glucosamine and the derivative was oversulfated in the O-position, a strong activity with HC activities with both inhibitors decreased when the overall sulfate content (i.e., the charge density) was reduced, and vice-versa. Carboxyl-reduced heparin was also inactive but activity could be restored after O-sulfation. Our results thus suggest that, unlike the case of AT III, no functional group in heparin is critical for optimal thrombin inhibition by HC II. Sulfate and carboxylate are important in as much as they contribute to the global charge of the molecule.

Early potent antithrombotic effect with combined aspirin and a loading dose of clopidogrel on experimental arterial thrombogenesis in humans.

BACKGROUND: We conducted a double-blind, randomized, crossover study to assess the antithrombotic effects of the combination of aspirin (acetylsalicylic acid, ASA) and clopidogrel, with or without a loading dose, versus ASA alone in a model of arterial thrombosis in humans. METHODS AND RESULTS: Eighteen male volunteers received the following 3 regimens for 10 days separated by a 1-month period: (1) 325 mg ASA daily, (2) 325 mg ASA+75 mg clopidogrel daily, (3) 325 mg ASA daily+300-mg clopidogrel loading dose on day 1 and +75 mg clopidogrel per day on days 2 to 10. The antithrombotic effect was measured 1.5, 6, and 24 hours after drug intake on day 1 and 6 hours after drug intake on day 10. Arterial thrombus formation was induced ex vivo by exposing a collagen-coated coverslip in a parallel-plate perfusion chamber to native blood for 3 minutes at an arterial wall shear rate. Without a loading dose, clopidogrel+ASA developed an antithrombotic effect within 6 hours after the first intake. It was superior to that produced by ASA, but it was moderate (P

The specific thromboxane receptor antagonist S18886: pharmacokinetic and pharmacodynamic studies.

OBJECTIVES AND PATIENTS: We conducted a multicenter double-blind pharmacokinetic/pharmacodynamic (PK/PD) study of the new oral thromboxane receptor antagonist S18886 in 30 patients with peripheral artery disease, who were randomized to receive five different oral dosages of S18886 (1, 2.5, 5, 10 or 30 mg) for 12 weeks (83 days). Primary objective was to determine the effect of S18886 on platelet aggregation ex vivo. RESULTS: Pharmacokinetics of S18886 was linear, with peak plasma levels being reached between 30 min and 2 h and a terminal half-life of 5.8-10 h. No significant accumulation of S18886 in plasma was observed after repeated dosing. The relationship between the S18886 concentration and platelet inhibition was examined in terms of U46619-induced platelet aggregation. Over the range of doses studied, there was a predictable relation between the plasma drug concentration and the degree of platelet inhibition at each dose. Maximal inhibition of U46619-induced platelet aggregation was achieved within 1 h with all oral doses of S18886, and this effect was maintained for at least 12 h. The PK/PD relationship was direct, and U46619-induced platelet aggregation was strongly inhibited by S18886 plasma concentrations above 10 ng mL(-1). This concentration was thus the minimal effective antiplatelet level in this population, and was maintained only by the dosages of 10 and 30 mg. The safety profile of S18886 was excellent, whatever the unit dose, with no attributable adverse events. CONCLUSION: The results of this study, which included modeling and simulation, help identify the minimal effective plasma concentration of S18886 required for potent antiplatelet efficacy in patients with stable peripheral arterial disease.

Enhancement of enzymatic fibrinolysis with 2-MHz ultrasound and microbubbles.

BACKGROUND: Microbubbles used for echo-contrast agents accelerate enzymatic fibrinolysis of clots exposed to low-frequency ultrasound (US). It is not known whether microbubbles are also effective in enhancing high-frequency US-driven enzymatic fibrinolysis. METHODS AND RESULTS: Calibrated whole blood clots were exposed to US, or US and galactose-based microbubbles (Levovist), with or without recombinant tissue plasminogen activator (rt-PA) in an in-vitro flow system. We used low-intensity, 2-MHz, pulsed wave US. Relative weight reduction of clot +/- SD was 30.7 +/- 9.5% after exposure to microbubbles, rt-PA and US, 13.1 +/- 2.6% after exposure to rt-PA and US, 10.9 +/- 3.6% after exposure to microbubbles and US, and 6.1 +/- 1.9% after exposure to US alone. anova demonstrated a significant effect of rt-PA (P =0.001), microbubbles (P = 0.012), and interaction of both (P = 0.022). CONCLUSIONS: The application of galactose-based microbubbles (Levovist) strongly accelerates lysis of clots exposed to 2 MHz, low-intensity US in vitro both with and without rt-PA. The findings suggest a synergy between microbubbles and rt-PA. These methods routinely used for transcranial diagnostic applications have the potential to improve the efficacy of intravenous rt-PA in acute ischemic stroke.

Low molecular weight heparin therapy: is monitoring needed?

Recent meta-analyses indicate that low molecular weight heparins (LMWH) are more effective than unfractionated heparin (UH) in preventing and treating deep vein thrombosis. This article presents the arguments for and against the need for laboratory monitoring. At the present time, the only tests currently available for monitoring LMWH therapy are those which measure the anti Xa activity in the plasma. Due to lower binding to plasma proteins and to cell surfaces, the plasma anti Xa activity generated by a given dose of LMWH is more predictable than for UH. Some clinical trials suggest that LMWH delivered at the recommended dose expose the patient to less bleeding risk than UH. Several meta-analyses indicate comparable risk while any overdose unacceptably increases the haemorrhagic risk. The lowest dose of LMWH still effective in treating established DVT is presently unknown; some reports indicate that inadequate doses of LMWH are associated with a lack of efficacy for prevention. An overview of the published clinical trials indicates that the LMWH dose has never been monitored for prevention of DVT. In the treatment of established DVT, several trials have been performed without any monitoring, while in others the dose was adapted to target a given anti Xa activity. These considerations suggest that in prevention of DVT, monitoring the dose is not required.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet anti-aggregating activity and tolerance of clopidogrel in atherosclerotic patients.

The anti-aggregating activity of five rising doses of clopidogrel has been compared to that of ticlopidine in atherosclerotic patients. The aim of this study was to determine the dose of clopidogrel which should be tested in a large scale clinical trail of secondary prevention of ischemic events in patients suffering from vascular manifestations of atherosclerosis [CAPRIE (Clopidogrel vs Aspirin in Patients at Risk of Ischemic Events) trial]. A multicenter study involving 9 haematological laboratories and 29 clinical centers was set up. One hundred and fifty ambulatory patients were randomized into one of the seven following groups: clopidogrel at doses of 10, 25, 50, 75 or 100 mg OD, ticlopidine 250 mg BID or placebo. ADP and collagen-induced platelet aggregation tests were performed before starting treatment and after 7 and 28 days. Bleeding time was performed on days 0 and 28. Patients were seen on days 0, 7 and 28 to check the clinical and biological tolerability of the treatment. Clopidogrel exerted a dose-related inhibition of ADP-induced platelet aggregation and bleeding time prolongation. In the presence of ADP (5 microM) this inhibition ranged between 29% and 44% in comparison to pretreatment values. The bleeding times were prolonged by 1.5 to 1.7 times. These effects were non significantly different from those produced by ticlopidine. The clinical tolerability was good or fair in 97.5% of the patients. No haematological adverse events were recorded. These results allowed the selection of 75 mg once a day to evaluate and compare the antithrombotic activity of clopidogrel to that of aspirin in the CAPRIE trial.

A simple method to measure dermatan sulfate at sub-microgram concentrations in plasma.

A simple method for biological assay of dermatan sulfate (DS) in plasma is described. DS accelerates thrombin inhibition by heparin cofactor II (HC II). The principle of the assay is to measure the residual amidolytic thrombin activity after a short period of incubation with HC II in defibrinated plasma at low ionic strength. For this method we take advantage of two observations. Firstly, at fixed concentrations of DS and of HC II, the rate of thrombin inhibition increases when the ionic strength of the medium decreases. Secondly, defibrination by bentonite absorption also removes antithrombin III, HC II and for a large part alpha-2 macroglobulin from the plasma, so that no other thrombin inhibitor competes with HC II added as a reagent in a second step. In the conditions described, there is a linear relationship between DS concentrations in plasma from 0 to 2 micrograms/ml and the log of residual thrombin activity. The limit of sensitivity is 0.1 micrograms/ml. The assay displays an acceptable reproducibility in intra-assay, inter-assay and inter-individual experiments. It can be used to measure DS in human, rabbit and rat plasmas. The assay is also sensitive to other HC II activators such as heparin and pentosan polysulfate. DS is effective in experimental thrombosis without any detectable anticoagulant effect ex vivo. Pharmacological concentrations of DS in plasma fall into the range of sensitivity of this assay, which would be helpful in experimental or clinical studies of DS and related glycosaminoglycans.

Turnover study of heparin cofactor II in healthy man.

Heparin cofactor II (HC II) is a heparin-dependent inhibitor of thrombin, distinct from antithrombin III (AT III). This study was designed to evaluate its metabolism in healthy subjects. Purified HC II was labelled with 125I by the lactoperoxidase-glucose oxidase technique. The biological activity of the HC II was unchanged after labelling as was its migratory pattern by crossed immunoelectrophoresis in the presence of heparin or dermatan sulfate. Three healthy volunteers were injected with 10 microCi and the plasma radioactivity was measured daily. The data were approximated by a sum of two exponential terms and the metabolism of HC II was described by a two compartment mamillary system. The mean values of fractional catabolic rate, intravascular fraction and half-life of the elimination phase were respectively: 0.44 d-1, 0.60 and 2.53 d. These parameters are of the same order of magnitude as those reported in the literature for AT III. The plasma HC II concentration in the 3 subjects ranged from 61 to 82 micrograms/ml as estimated using our purified preparation. Accordingly, the absolute catabolic rate ranged from 1.17 to 1.36 mg X kg-1 X d-1.

Effect of pentosan polysulphate administration in man on the formation of covalent complexes between heparin cofactor II and thrombin generated in plasma by contact activation.

An assay for the quantification of pentosan polysulphate (PPS) in plasma is described. As PPS has been shown to potentiate thrombin inhibition by the second heparin cofactor (HC II), the principle of this assay was to measure the formation of covalent complexes between HC II and the thrombin generated in plasma after contact activation and recalcification. The complexes were quantified by using purified 125I-HC II added to the plasma as a tracer and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The assay was sensitive to low PPS concentrations (limit of detection: 0.1 microgram/ml) and therefore suitable for the measurement of PPS in plasma after its administration to man. The clearance of PPS was studied in 3 subjects receiving respectively 10, 50 and 100 mg intravenously (IV) and in 3 subjects receiving 35 mg subcutaneously (SC). PPS was still detectable 8 h after 50 and 100 mg IV and 6 h after 35 mg SC. The activated partial thromboplastin time (APTT) was, in comparison, relatively insensitive but for concentrations above 1 microgram/ml the values derived from the APTT and from the SDS-PAGE method fitted. The results were also in general agreement with those reported by McGregor et al. (5) who used a sensitive competitive binding assay. This indicates that low concentrations of PPS previously measured chemically are also pharmacologically active in plasma.

Dermatan sulfate is a more potent inhibitor of clot-bound thrombin than unfractionated and low molecular weight heparins.

Clot-bound thrombin proteolyses fibrinogen and amplifies the coagulation cascade at its close vicinity, thereby ensuring the growth of fibrin-rich thrombus. The present study compares the ability of various glycosaminoglycans (GAGs) to inhibit these 2 properties. Unfractionated heparin (UH), 3 low molecular weight heparins (LMWHs) with increasing antifactor Xa/antifactor IIa ratio, the synthetic pentasaccharide (PS), devoid of antifactor IIa activity, and dermatan sulfate (DS), a catalyst of thrombin inhibition by heparin cofactor II, were selected on the basis of their different properties. Proteolysis of fibrinogen by clot-bound thrombin was evaluated by measuring fibrinopeptide A (FPA) generation after an incubation of standardized washed clots in plasma for 120 min in absence or in presence of increasing concentrations of heparins or of DS. The results were compared to those obtained when free alpha-thrombin (0.4 nM) was added to plasma in the same experimental conditions. On the basis of equivalent antithrombin units, UH and LMWHs gave identical results. To inhibit by 70% fibrinogen proteolysis induced by clot-bound thrombin (IC 70), 5- to 9-fold higher concentrations of UH or of LMWHs were required in comparison with those required to inhibit free thrombin. For DS, only a 1.3 times higher concentration was required. PS (final concentration 1 anti Xa U.ml-1) was devoid of any inhibitory effect. The amplification of the coagulation cascade induced by clot-bound thrombin was evaluated by measuring the shortening of whole blood clotting time (WBCT) resulting from the incubation of washed clots in native blood. In absence of GAG, clot-bound thrombin reduced WBCT from 18 +/- 2 min to 9 +/- 1 min.(ABSTRACT TRUNCATED AT 250 WORDS)

The activity of unfractionated heparin and low molecular weight heparins in rabbit plasma--the need for using absolute anti-factor Xa and antithrombin activities.

Rabbit being a common animal model to evaluate the antithrombotic effect of heparins, our purpose was to apply the heparin Standard Independent Unit (SIU) approach to rabbit plasma. To take into account the specificities of the enzymes we have measured the decay constants of factor Xa and of thrombin from autologous and heterologous origins, in presence and in absence of heparin. Different heparins or heparin fractions with a mean molecular weight from 1.7 to 10.5 kDa were used. We found that: a) the decay constants varied strongly between species and between enzymes; b) the decay constants of thrombin were always higher than those of factor Xa; c) the specific anti-factor Xa activity of heparins increased with the molecular weight and was 1.33 times higher when determined with bovine factor Xa than with rabbit factor Xa; d) the specific antithrombin activity of heparins also increased with the molecular weight but was similar when determined with rabbit and human thrombin; e) the specific anti-factor Xa activity was always lower than the specific antithrombin activity; f) the calibration of the heparins and heparin fractions against the 4th International Standard of Heparin expressed in International Units (IU) lead to a systematic overestimation of the anti-factor Xa activity and to an under-estimation of the antithrombin activity. These observations indicate that it may be very important to use the SIU approach and to know the accurate activities to better understand the mechanism of the antithrombotic activity of heparins in experimental models.

Up-regulation of tissue factor expression by platelet-derived growth factor in human vascular smooth muscle cells in culture--role of mitogen-activated protein kinase and effects of intracellular cyclic AMP.

Human vascular smooth muscle cells (SMCs) isolated from normal adult arteries were investigated for the expression of Tissue Factor (TF) procoagulant activity at their membrane surface and TF antigen in lysed cells. In growing conditions, SMCs expressed high levels of TF activity and antigen. When cells were made quiescent by 72 h of subculture in serum-poor medium, these levels fell to about one third of the initial values. Platelet-derived growth factor BB (PDGF) up-regulated in a dose-dependent manner TF expression with a significant increase (1.8 fold) within five hours. PD 98059, a specific inhibitor of mitogen-activated protein kinase (MAPK) pathway involved in cell response to PDGF, also prevented TF up-regulation. Short-term treatment by Pentoxifylline and dibutyryl cAMP also completely prevented induced TF up-regulation, but remained without effect on baseline levels of quiescent, unstimulated cells. At their effective concentrations, pentoxifylline and dibutyryl cAMP also inhibited the activation of MAPK induced by PDGF. The rapid induction of TF upon growth factor stimulation may be important in circumstances where SMCs proliferate, such as vascular events or vessel development.

D-Dimers, thrombin antithrombin III complexes and prothrombin fragments 1+2: diagnostic value in clinically suspected deep vein thrombosis.

This study was performed to determine the accuracy of D-Dimer fibrin derivatives, thrombin-antithrombin III (TAT) complexes and prothrombin fragments 1 + 2 (F 1 + 2) determinations for the diagnosis of deep vein thrombosis (DVT). One hundred and sixteen consecutive patients referred to the angiology unit of our hospital for a clinically suspected DVT were investigated. They were submitted to mercury strain gauge plethysmography and to ultrasonic duplex scanning examination; in cases of inconclusive results or of proximal DVT (n = 35), an ascending phlebography was performed. After these investigations were completed, the diagnosis of DVT was confirmed in 34 and excluded in 82. One half of the patients were already under anticoagulant therapy at the time of investigation. The 3 biological markers were assayed using commercially available ELISA techniques and the D-Dimer was also assayed with a fast latex method. The normal distribution of these markers was established in 40 healthy blood donors. The most accurate assay for the diagnosis of DVT was the D-Dimer ELISA which had both a high sensitivity (94%) and a high negative predictive value (95%). The D-Dimer latex, TAT complexes and F 1 + 2 were far less sensitive and provided negative predictive values which ranged between 78 and 85%. In spite of positive and significant correlations between the levels of the 3 markers, their association did not improve their overall accuracy for detecting DVT. Therefore, with the exception of the D-Dimer ELISA, these markers were of little value for the diagnosis of DVT in this specific population.

Comparative turn-over of heparin cofactor II and antithrombin III in baboons. Influence of heparin and pentosan polysulfate administration.

Heparin and pentosan polysulfate (PPS) interact in plasma with antithrombin III (AT III) and Heparin cofactor II (HC II) respectively. To assess the influence of heparin or PPS treatment on the metabolism of their respective cofactors, we performed a double tracer study in baboons receiving heparin or PPS. Purified AT III and HC II from human plasma were labelled with 131I and 125I respectively by the lactoperoxidase-glucose oxidase technique. The tracers had unchanged biological activities, were homogeneous in SDS-PAGE, migrated as native proteins by crossed immunoelectrophoresis in the presence of heparin or PPS and virtually coeluted with endogenous baboon proteins from heparin-agarose. Nine animals were randomly allocated to receive, during the metabolic study, heparin (500 IU/kg/d, n = 3), PPS (5 mg/kg,d, n = 3) or a placebo (n = 3) given in 2 daily subcutaneous injections. Heparin levels and anticoagulant effects were similar in extent and duration to those usually achieved in man. The plasma concentrations of AT III and HC II did not vary under treatment. The half-life of the elimination phase in the placebo group ranged from 1.95 to 2.33 d for AT III and from 1.96 to 2.21 d for HC II. There was no significant difference in the half-lives of the 2 inhibitors between the placebo group and the animals receiving heparin or PPS. This suggest that clinical conditions associated with heparin treatment may be important for the effect of heparin on AT III metabolism previously reported in patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of tissue factor synthesis by disruption of ERK kinases and PKC signaling pathways in human vascular SMCs.

Tissue Factor (TF), the receptor for plasma VII/VIIa and the initiator of blood coagulation, is inducible in vascular smooth muscle cells (SMCs) by growth factors and bacterial lysopolysaccharides (LPS) and is expressed in vivo after vascular injury. As TF expression is a determinant of the thrombogenicity of vascular lesions, we investigated the signal pathways involved in this process. Human vascular SMCs were obtained from normal arteries and made quiescent by serum deprivation. Baseline TF antigen and activity were up-regulated by various agonists: fetal calf serum (FCS), LPS, and platelet derived growth factor (PDGF) being the most effective but with different kinetics. TF expression induced by LPS was transient with a maximum 6 h after stimulation and returned to baseline levels after 24 h whereas TF expression induced by serum or PDGF was sustained for at least 24 h. Rapid and transient activation of Extracellular signal-Regulated Kinase (ERK) was observed after stimulation by PDGF and FCS, but not by LPS. The role of ERK, Ras and protein kinase C activities were investigated using specific inhibitors, PD 98059, manumycin A and calphostin C respectively. For TF induction by LPS, PKC activity was required and the ERK/Ras pathway was not involved. In contrast, the effect of PDGF was strictly ERK and Ras dependent, but partially prevented by PKC inhibitors. TF induction by FCS was ERK dependent but partially Ras and PKC dependent. In conclusion, TF expression appears to be a non-specific response of SMCs to numerous stimuli through multiple signal pathways which differ according to the inducing agent.

Effects of heparin, dermatan sulfate and of their association on the inhibition of venous thrombosis growth in the rabbit.

This study compares the ability of unfractionated heparin, of dermatan sulfate, and of their simultaneous administration delivered as continuous intravenous infusion or as a single bolus injection to inhibit the growth of a standardized venous thrombosis in the rabbit. When delivered as continuous intravenous infusion for 4 h, heparin and dermatan sulfate inhibited thrombus growth in a dose dependent manner. The maximum antithrombotic effect of heparin was achieved at the dose of 0.15 mg kg-1 h-1 (25 U kg-1 h-1) which generated a mean plasma concentration of 1.8 micrograms ml-1 (0.31 U ml-1) and a 1.8 fold prolongation of the activated partial thromboplastin time (APTT) in comparison to the pretreatment value. A comparable antithrombotic effect was obtained with dermatan sulfate at the dose of 2 mg kg-1 h-1. This dose generated a mean plasma concentration of 30 micrograms ml-1 and a 1.3 fold APTT prolongation. Increasing these doses up to 10 fold did not improve the antithrombotic effect which did not overpass 60-70% of the controls. When the compounds were delivered simultaneously, the maximum antithrombotic effect (64%) was obtained with the following association: 0.06 mg kg-1 h-1 (10 U kg-1 h-1) for heparin and 1 mg kg-1 h-1 for dermatan sulfate. Increasing these doses up to 4 to 5 fold did not improve the antithrombotic effect. Heparin, dermatan sulfate and the association of both were also delivered as single bolus injections and the resultant antithrombotic effect was determined 4 h after saline infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombotic and bleeding effects of a new synthetic direct thrombin inhibitor and of standard heparin in the rabbit.

This study reports on the anticoagulant, antithrombotic and bleeding effects of a new synthetic direct thrombin inhibitor (SDTI) in comparison with standard heparin (SH). The anticoagulant effect was determined with the thrombin clotting time (TCT) and the activated partial thromboplastin time (APTT). SDTI was more potent than SH in prolonging the TCT, but as potent as SH in prolonging the APTT. The antithrombotic effect was determined using a modified Wessler model in the rabbit, either 30 min after a continuous IV infusion of increasing doses or at various times after a single SC injection (20 mg/kg). After continuous IV infusion of 187 micrograms/kg/h of SDTI and of 60 micrograms/kg/h of SH, significant thrombus prevention effects were obtained (59 and 57% respectively). Increasing the dose of SDTI up to 3000 micrograms/kg/h did not significantly improve the antithrombotic effect. After SC injection, a significant antithrombotic effect was observed for 12 h with SDTI but for more than 24 h with SH. The bleeding effect was studied using the rabbit ear model 15 min after a continuous infusion of 7.5 and 15 mg/kg/h: the amounts of blood loss were dose-dependent and comparable for SDTI and SH. These studies also indicated that SDTI possesses a considerable shorter half-life in comparison with SH. Accordingly, the ex vivo concentrations generated after continuous IV infusion or SC injection of the same dose were higher for SH than for the SDTI.

Unfractionated heparin and CY 216: pharmacokinetics and bioavailabilities of the antifactor Xa and IIa effects after intravenous and subcutaneous injection in the rabbit.

This report compares the pharmacokinetics and the bioavailabilities of the antifactor Xa and of the antifactor IIa activities generated by intravenous (IV) and subcutaneous (SC) injections of increasing doses of unfractionated heparin (UH) and of a low molecular weight heparin (CY 216). Rabbits were injected with 500, 1,000, 2,500, and 5,000 antifactor Xa u/kg of both heparins and their biological activities were followed at various time intervals. After IV injection the clearance of the antifactor Xa activities was independent of the dose and the clearance of UH was significantly higher than that of CY 216; after SC injection the bioavailability estimated from the antifactor Xa effect was consistently over 100% for CY 216 while that of UH increased from 27% at the lowest dose to 93% at the highest dose. The pharmacokinetic parameters estimated by the antifactor IIa activity of UH were superimposable to those calculated with the antifactor Xa activity. For CY 216 no direct comparison between the two activities was made since the dose injected expressed in antifactor IIa units was 3.4 times lower. UH and CY 216 were therefore injected intravenously to other animals at equivalent and increasing doses expressed in antifactor IIa units (50-5,000 u/kg). The pharmacokinetic parameters calculated from the curves of the antifactor IIa activities were basically identical except at the two lower doses (50 and 100 u/kg) for which UH was cleared faster than CY 216.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal platelet serotonin uptake and binding sites in myeloproliferative disorders.

A simultaneous investigation of the kinetics of serotonin (5 HT) uptake and of binding sites was carried out in the platelets of normal subjects and of 10 patients affected with various types of myeloproliferative disorders (MD). The 5 HT uptake was analysed according to the Lineweaver-Burk and the Eadie-Hofstee methods. With the two methods, the patient's platelets exhibited a dramatic reduction of the Vi max and of the Km; in some patients the Eadie-Hofstee analysis revealed that a passive diffusion phenomenon is superimposed on the active 5 HT uptake at least for the higher concentration used. The binding data were analysed with the Scatchard method. Two classes of binding sites (high affinity - low capacity, low affinity - high capacity) were found in normal subjects and patients. Pharmacological studies with imipramine, a specific inhibitor of 5 HT uptake, suggested that both the sites are involved in 5 HT uptake. The number of both binding sites was significantly decreased in patient's platelets while the affinity constants of these binding sites were not significantly reduced in comparison with those of the control subjects. No correlations were found between Vi max, Km and the number of binding sites. These results suggest that a reduction in the number of platelet membrane acceptors for 5 HT commonly occurs in myeloproliferative disorders but does not provide a full explanation of the uptake defect.