Factor VII polymorphisms associated with plasma factor VII coagulant activity levels in healthy Tunisians.

Factor VII gene polymorphisms may contribute to elevations in factor VII coagulant (FVIIc) levels that have been associated with cardiovascular risk. We therefore studied the association of two polymorphisms--R353Q polymorphism at codon 353 involving the catalytic region and the 10 base pair (bp) insertion polymorphism involving the promoter region--with FVllc levels in 176 healthy Tunisians. The variant Q allele had a frequency of 0.213 (SD 0.021) whereas the frequency of the 10 bp insert allele was 0.250 (SD 0.023). Subjects with R/R genotype had significantly higher FVllc levels than Q353 heterozygote and homozygote subjects (96.36 versus 59.52). FVIIc levels with the 10 bp insertion polymorphism were not significantly different. The Q353 allele of the factor VII gene polymorphism is associated with decreased factor VII and could be protective against cardiovascular disease.

Mechanism of thrombin inhibition by antithrombin and heparin cofactor II in the presence of heparin.

The kinetics of thrombin inhibition by antithrombin (AT) and heparin cofactor II (HC II) were analysed as a function of the heparin concentration, from 10(-9) to 10(-4) M. The initial concentrations of inhibitor (l) and thrombin (E) were set at equimolar levels (CI = CE = 10(-8) M). The experimental data indicate that the reaction of thrombin inhibition was second-order both in the absence and in the presence of heparin, and that the apparent rate constant increased at heparin concentrations ranging from 10(-9) to 10(-6) M and decreased at higher concentrations. The data fit with the kinetic model established by Jordan et al. [J. Biol. Chem. 1979, 254, 2902-2913] for the catalysis of the thrombin-AT reaction by a low-molecular-weight heparin fraction. In this model, heparin (H) binds quickly to the inhibitor (I) and forms a heparin-inhibitor complex (HI), which is more reactive than the free inhibitor towards thrombin, leading to the formation of an inactive inhibitor-thrombin complex (I*E) and the release of free heparin, in a second step which is rate limiting. KH,I, the dissociation constant of HI, and k, the second-order rate constant of free thrombin inhibition by HI, were found to be 3.7 x 10(-7) M and 1.3 x 10(9) M-1 min-1, respectively, for AT, compared to a KH,I of 2.0 x 10(-6) M and k of 6.4 x 10(9) M-1 min-1 for HC II. These data indicate that heparin-HC II complex reactivity is greater than that of the heparin-AT complex towards thrombin, whereas heparin affinity is stronger for AT. At heparin concentrations higher than 10(-6) M, the decrease in the reaction rate was in keeping with the formation of a heparin-thrombin complex (HE), whose inactivation by the heparin-inhibitor complex (HI) is slower than that of the free protease.

Mechanism of thrombin inhibition by heparin cofactor II in the presence of dermatan sulphates, native or oversulphated, and a heparin-like dextran derivative.

The kinetics of thrombin inhibition by heparin cofactor II (HC II) in the presence of dermatan sulphates, native (DS), or oversulphated (DSS 1 and DSS 2) and a biospecific dextran derivative substituted with carboxymethyl, carboxymethyl-benzylamide and carboxymethyl benzylamide-sulphonate functional groups (CMDBS), has been studied as a function of the sulphated polysaccharide concentration. The initial HC II and thrombin concentrations were set at equimolar levels. Analysis of the experimental data obtained for DS, DSS1 and DSS2 was performed using a previously described model which allows computation of the dissociation constant (KPS,HC) of the polysaccharide-HC II complex and the rate constant of thrombin inhibition by the polysaccharide-HC II complex (k). A KPS.HC of 9.6 x'10(-7) M and a k of 4.5 x 10(9) M-1 min-1 were found for DS, whereas KPS,HC 2.1 x 10(-6) M, k 1.1 x 10(10) M-1 min-1 and KPS,HC 4.3 x 10(-7) M, k 1.4 x 10(10) M-1 min-1 were found for DSS1 and DSS2, respectively. Knowing that DSS1 has a sulphur content per disaccharide of 7.8%, compared with 11.5% for DSS2, these results indicate that the polysaccharide affinity for HC II is increased only in the case of DSS 2, whereas the oversulphation increases the reactivities towards thrombin of both complexes DSS1-HC II and DSS2-HC II. A better conformation of these complexes may favour a faster interaction with the protease. Unlike heparin, DS at concentrations higher than 10(-5) M does not modify the reaction rate of thrombin inhibition, a fact which can be explained by the absence of complex formation between DS and thrombin. The experimental data obtained for CMDBS fit a kinetic model in which the biospecific dextran derivative rapidly forms a complex with thrombin which is more reactive towards HC II than the free protease. The reaction rate remained unchanged for CMDBS concentrations equal to or higher than 10(-5) M, whereas CMDBS was found to interfere strongly with the fibrinogen-thrombin interaction. These data suggest that CMDBS has a strong affinity for the protease and no affinity for HC II. The computed dissociation constant of the CMDBS-thrombin complex (KPS,E) was 2.4 x 10(-7) M and the rate constant of the reaction of this complex with HC II (k) was 1.7 x 10(8) M-1 min-1. These findings indicate that CMDBS exerts its catalytic effect through a unique mechanism of action and may constitute a new class of anticoagulant drugs.

Influence of the oversulfation method and the degree of sulfation on the anticoagulant properties of dermatan sulfate derivatives.

Six oversulfated dermatan sulfate (DS) derivatives, differing in their tissue origin (porcine skin, bovine and porcine intestinal mucosa), and the oversulfation method of preparation, have been tested for their anticoagulant properties. In the first method, the SO3-trimethylamine complex is added to a DS sodium salt dissolved in formamide while it is added to a DS-benzethonium salt dissolved in dimethyl formamide in the second method. The rate of sulfation of these compounds ranged from 7.8 to 11.5 percent of sulfur on a weight basis, whereas it is 6% and 12% for natural DS and for heparin respectively. The anticoagulant potency was assessed by determining the catalytic effect of each glycosaminoglycan on the inhibition of thrombin added to (i) plasma (ii) purified heparin cofactor II(HC II) or (iii) purified antithrombin III(AT III). The catalytic effect on Factor Xa inhibition in the presence of AT III has also been investigated. The increased sulfation is found to enhance the antithrombin activity of the native dermatan sulfate whatever the method used, while the Factor Xa inhibition by AT III could be catalysed only by the most sulfated derivative obtained by the second method. The two derivatives which were less oversulfated, by the first oversulfation method, exhibit equal or even higher catalytic effects on thrombin inhibition when compared to the four other derivatives. The use of the first oversulfation method provides slightly oversulfated derivatives which exhibit strong anticoagulant properties and may constitute effective antithrombotic drugs with no bleeding tendency, a side effect perhaps related to a high rate of sulfation.

Antiphospholipid syndrome and retinal vein occlusion in adults.

Antiphospholipid antibodies may play and important role in the pathogenesis of retinal vascular occlusions; investigated the prevalence among 33 patients with retinal vein and artery occlusions and 80 controls. Prevalence was 33% and 5% respectively. Ophthalmic examination and fluorescein angiography showed that occlusions were due to ischaemic events. The 11 patients were diagnosed with antiphospholipid syndrome: 9 patients were treated successfully with laser photocoagulation and anticoagulant and anti-aggregant therapy. Two patients with antiphospholipid antibodies associated with resistance to activated protein C had unfavourable outcomes. Our results suggest a correlation between antiphospholipid syndrome and retinal vein occlusions; we recommend a systematic search for antiphospholipid antibodies in occlusions of unexplained origin and laser photocoagulation treatment and long-term oral anticoagulant and anti-aggregant therapy.

Factor VII polymorphisms associated with plasma factor VII coagulant activity levels in healthy Tunisians

Factor VII gene polymorphisms may contribute to elevations in factor VII coagulant [FVIIc] levels that have been associated with cardiovascular risk. We therefore studied the association of two polymorphisms--R353Q polymorphism at codon 353 involving the catalytic region and the 10 base pair [bp] insertion polymorphism involving the promoter region--with FVllc levels in 176 healthy Tunisians. The variant Q allele had a frequency of 0.213 [SD 0.021] whereas the frequency of the 10 bp insert allele was 0.250 [SD 0.023]. Subjects with R/R genotype had significantly higher FVllc levels than Q353 heterozygote and homozygote subjects [96.36 versus 59.52]. FVIIc levels with the 10 bp insertion polymorphism were not significantly different. The Q353 allele of the factor VII gene polymorphism is associated with decreased factor VII and could be protective against cardiovascular disease

Antiphospholipid syndrome and retinal vein occlusion in adults

Antiphospholipid antibodies may play and important role in the pathogenesis of retinal vascular occlusions; investigated the prevalence among 33 patients with retinal vein and artery occlusions and 80 controls. Prevalence was 33% and 5% respectively. Ophthalmic examination and fluorescein angiography showed that occlusions were due to ischaemic events. The 11 patients were diagnosed with antiphospholipid syndrome: 9 patients were treated successfully with laser photocoagulation and anticoagulant and anti-aggregant therapy. Two patients with antiphospholipid antibodies associated with resistance to activated protein C had unfavourable outcomes. Our results suggest a correlation between antiphospholipid syndrome and retinal vein occlusions; we recommend a systematic search for antiphospholipid antibodies in occlusions of unexplained origin and laser photocoagulation treatment and long-term oral anticoagulant and anti-aggregant therapy