A rare missense mutation in CHRNA4 associates with smoking behavior and its consequences.

Using Icelandic whole-genome sequence data and an imputation approach we searched for rare sequence variants in CHRNA4 and tested them for association with nicotine dependence. We show that carriers of a rare missense variant (allele frequency=0.24%) within CHRNA4, encoding an R336C substitution, have greater risk of nicotine addiction than non-carriers as assessed by the Fagerstrom Test for Nicotine Dependence (P=1.2 × 10(-4)). The variant also confers risk of several serious smoking-related diseases previously shown to be associated with the D398N substitution in CHRNA5. We observed odds ratios (ORs) of 1.7-2.3 for lung cancer (LC; P=4.0 × 10(-4)), chronic obstructive pulmonary disease (COPD; P=9.3 × 10(-4)), peripheral artery disease (PAD; P=0.090) and abdominal aortic aneurysms (AAAs; P=0.12), and the variant associates strongly with the early-onset forms of LC (OR=4.49, P=2.2 × 10(-4)), COPD (OR=3.22, P=2.9 × 10(-4)), PAD (OR=3.47, P=9.2 × 10(-3)) and AAA (OR=6.44, P=6.3 × 10(-3)). Joint analysis of the four smoking-related diseases reveals significant association (P=6.8 × 10(-5)), particularly for early-onset cases (P=2.1 × 10(-7)). Our results are in agreement with functional studies showing that the human α4ß2 isoform of the channel containing R336C has less sensitivity for its agonists than the wild-type form following nicotine incubation.

Experimental haemorrhagic effect of two-domain non-glycosylated tissue factor pathway inhibitor compared to low molecular weight heparin.

The glycosylated multivalent three-domain Kunitz inhibitor TFPI is a natural inhibitor of tissue factor-FVIIa complex in the presence of FXa. TFPI has an experimental antithrombotic capacity indistinguishable from LMWH in a prophylactic dose, regardless of glycosylation and of the third domain. An inherited equilibrium between antithrombosis and haemorrhage exists. The aim of the study was to evaluate whether a two-domain non-glycosylated TFPI (117QTFPI1-161) has a bleeding potential in a rat gastric mucosa model. Groups; placebo, LMWH (tinzaparin) 60 and 250 anti-Xa IU/kg and 117 QTFPI1-161 1.0 and 10.0 mg/kg, given i.v. (bolus injection), randomised double dummy design. All actively treated groups significantly prolonged both the bleeding volume (493-984 microliters) and the bleeding time (10-20 min) compared to placebo (41 microliters, 2 min). It was not possible to distinguish a difference between the lower dose of LMWH and 117QTFPI1-161 in either parameter (p = 0.23-0.71). The two doses of 117QTFPI1-161 caused elevation of plasma-TFPI, 18 and 150 times baseline value. Both LMWH doses (0.6-3.2 anti-Xa IU/ml) and both 117QTFPI1-161 doses (0.2-2.7 anti-Xa IU/ml), caused significant effect in the anti-Xa assay, however 117QTFPI1-161 significantly less. Only the largest dose of 117QTFPI1-161 caused significant prolongation in the APTT assay (34 s). Both doses of LMWH caused significant prolongation (60-300 s). LMWH was the only substance to prolong the dilute-PT assay. Non-glycosylated two-domain 1.0 mg/kg TFPI, yielding supraphysiological plasma concentration, has an experimental haemorrhagic potential indistinguishable from LMWH in a prophylactic dose. The effect mediated by this type of TFPI could primarily be due to an inhibition of FXa.

The haemorrhagic effect of low molecular weight heparins, dermatan sulphate and hirudin.

In a randomized, blind study the primary effect on haemostasis after intravenous administration of dermatan sulphate (DS), recombinant hirudin (r-hirudin) and four commercial low molecular weight heparins (LMWHs) (nadroparine, enoxaparin, dalteparin and tinzaparin) was investigated in rats and compared with saline (control). The tail bleeding time, the bleeding from the gastric mucosa [the mucosal bleeding time (min) and the mucosal bleeding (microliter)] as well as changes in activated partial thromboplastin time, antifactor IIa and Xa activities were investigated. DS and r-hirudin were investigated in a dose potentially suitable in thomboprophylaxis and the LMWHs in doses recommended by the manufacturers for thromboprophylaxis, adjusted to body weight. All substances significantly prolonged the mucosal bleeding time. Dalteparin, tinzaparin, DS and r-hirudin increased the mucosal bleeding when compared with controls whereas nadroparine and enoxaparin did not. The effect of r-hirudin was also significantly more pronounced compared with other treatments. Moreover, r-hirudin prolonged the tail bleeding time significantly whereas the other substances did not. The antifactor Xa activity in plasma correlated well with the given dose of the LMWHs (rs = 0.7). However, the monitored bleeding parameters in the LMWH groups did not correlate with the plasma activities of antifactor IIa or Xa. The results indicate that the tested LMWHs are not equipotent in their effect on haemostasis in this model and that antifactor IIa or Xa activities do not directly correlate with their effect on haemostasis although increased haemorrhage was observed in the LMWHs with lower antifactor Xa/antifactor IIa ratios.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of experimental venous thrombosis in rabbits with different low molecular weight heparins, dermatan sulphate and hirudin.

In a randomized, blind study the antithrombotic efficacy of dermatan sulphate (DS), recombinant hirudin (r-hirudin) and four commercially available low molecular weight heparins (LMWHs) was investigated after intravenous administration in a jugular vein thrombus model in rabbits utilizing a combination of endothelial damage and flow reduction. The parameters observed were the frequency of visible thrombosis, the frequency of occlusive thrombosis and thrombus weights. DS and r-hirudin were investigated in one fixed dose and the LMWHs in prophylactic doses, adjusted to body weight, recommended in high-risk situations by the manufacturers. All substances effectively reduced the three observed parameters. DS and r-hirudin were equally or even more effective than the LMWHs. Although significant differences were not found between the individual LMWH treatments a visible trend cannot be overlooked in all three observed parameters. This cannot solely be explained by the different antifactor Xa activity doses given but increased antithrombotic activity was observed with declining antifactor Xa/antifactor IIa ratio. Our findings indicate that the different commercial LMWHs may differ in their antithrombotic activity when given in recommended doses adjusted to body weight in this experimental model. Dose range studies are required to clarify the thromboprophylactic efficacy of DS and r-hirudin in relation to the tested LMWHs.

Effect of dextran and enoxaparin on early ePTFE graft thrombogenicity in sheep.

OBJECTIVES: To evaluate the effect of low molecular weight heparin (LMWH), dextran 70 and their combination on platelet adhesion and fibrinogen uptake in ePTFE grafts in an experimental sheep model. DESIGN: Prospective open study. SETTING: Animal Laboratory of a University Hospital. MATERIALS: Early thrombogenicity of ePTFE grafts was studied after interposition in the two common carotid arteries of 40 adult sheep. The animals received one of four different treatment regimens in a double blind randomised way: enoxaparin and polygeline, saline and dextran 70, enoxaparin and dextran 70 or saline and polygeline (control). The substances were administered i.v. with a total dose of 73 antifactor-Xa U/kg for enoxaparin and 1.0 g/kg for dextran 70. Polygeline and saline were used as placebo substances in equivalent volumes. On one side (random allocation) the carotid blood flow was restricted to 25 ml/min, on the other side it was left unrestricted. CHIEF OUTCOME MEASURES: The following variables were studied: 1) fibrinogen uptake; 2) platelet uptake; 3) early graft patency; 4) blood flow in patient grafts; 5) visible presence of graft thrombus; 6) thrombus weight. MAIN RESULTS: The results verified the importance of adequate blood flow as only 30% of grafts with restricted blood flow in the control group were patent compared with 80% of those with unrestricted blood flow (p = 0.038). Dextran 70, enoxaparin and the combination of the two increased early graft patency (p < 0.05) and reduced thrombus weights (p < 0.05) in grafts with restricted blood flow. The relative number of grafts with thrombus free surface was increased in the unrestricted blood flow situation. CONCLUSIONS: Dextran 70 and enoxaparin appeared to be equally effective in decreasing fibrinogen and platelet uptake in the grafts. Their combination was not significantly more effective although there was a favourable trend.

Study of the interaction of dextran and enoxaparin on haemostasis in humans.

The effect on haemostatic variables by dextran 70, enoxaparin and their combinations, given in doses of 500 ml i.v. and 40 mg s.c. respectively, was studied in a randomised cross-over fashion in twelve healthy male volunteers. Antifactor-IIa activity, antifactor-Xa activity, APTT, factor VIII, vWF, bleeding time and blood counts were analysed over a 24-h period. Dextran alone did not affect antifactor-IIa activity and antifactor-Xa activity. No difference in antifactor-IIa and antifactor-Xa activity was found for Amax, tmax, AUC0-8 h and AUC0-24 h in the groups treated with enoxaparin or the combination of enoxaparin and dextran. Only minor changes in APTT were observed without statistical significance between the treatment groups. Factor VIII did not change significantly in the three treatment groups. However, vWF was significantly reduced in the dextran and the dextran/enoxaparin group (p = 0.046 and 0.01 respectively) but no difference was found between the two groups. Bleeding time was not significantly increased four hours after administration of the test substances and no difference was found between the individual treatment groups. Our findings indicate that dextran can be combined with enoxaparin, when used in thromboprophylactic doses, without increased risk for bleeding.

Prevention of experimental venous thrombosis in rabbits with low molecular weight heparin, dextran and their combinations, administered before or during induction of venous endothelial trauma.

An in vivo experimental venous thrombosis model based on endothelial damage and flow reduction was used to investigate the effect of low molecular weight heparin (LMWH) alone and in combination with dextran and the effect of surgical and endothelial trauma on thrombus formation, formation of occlusive thrombi and thrombus weights. Five groups with 15 rabbits in each were studied. Two groups received dalteparin (50 anti-Xa IU/kg i.v.) before surgical trauma or after, during the endothelial trauma and two groups received dalteparin (50 anti-Xa IU/kg i.v.) with dextran 70 (1 g/kg i.v.) before surgical trauma or after, during the endothelial trauma. Compared to a control group (saline) all treatment regimes reduced significantly the frequency of thrombosis and occlusive thrombi as well as thrombus weights. No significant difference was observed between the identical treatment groups when the substances were introduced before respective after surgical trauma. It is concluded, from the present study that thromboprophylaxis with LMWH in this particular in vivo model, given before or after surgical trauma is equally effective. Dextran has a certain augmenting thromboprophylactic effect when added to LMWH in this model.

Effect of low molecular weight heparin, dextran and their combinations on experimental venous thrombosis in rabbits.

An experimental model based on the combination of endothelial damage and flow reduction was used to induce jugular vein thrombosis in rabbits. The effect on thrombosis of a low molecular weight heparin (LMWH [Fragmin]), dextran 70, placebo and their combination was studied in a double-blind fashion with actual doses used in clinical thromboprophylaxis. Saline and polygeline were used as placebo in the control group. Four groups with 120 isolated vein segments in 60 animals were studied for presence of thrombus formation, occlusive thrombi and thrombus weights. Dextran reduced the thrombus weights (p = 0.048) and the formation of occlusive thrombi (p = 0.01), but not the formation of thrombi when compared with the placebo control group. Similarly, LMWH reduced the thrombus weights (p = 0.046), the formation of thrombi (p = 0.007) and occlusive thrombi (p = 0.0001). Compared with the LMWH group the group treated with the combination of LMWH and dextran was found to reduce the frequency of occlusive thrombi (p = 0.03) and numerically, but not significantly, further reduce the overall frequency of thrombosis (p = 0.18) and thrombus weights (p = 0.11). The results are consistent with an augmentation of the antithrombotic effect of LMWH by dextran 70. The need for further evaluation of the combined efficacy of LMWH and dextran is apparent from this study.