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Question: How does the size of the heparin moiety in the anti-thrombin (AT)-heparin complex influence its anticoagulant properties?, Approach: Of 52 heparin fractions of precise Mr between 2800 and 37,000 we determined the dissociation constant (Kd) of the binding of the enzyme to the AT-heparin complex and the decay constant (kdec) of thrombin and factor Xa at 1 microM of that complex., Results: The Kd of thrombin or factor Xa is constant when expressed in terms of the concentration of sugar units, i.e. the enzymes bind the better the longer the heparin. Thrombin (Kd=1.86+/-0.13 microM) binds 11 times tighter than factor Xa (Kd=20.2 +/-1.5 microM). Factor Xa inactivation velocity is proportional to the concentration of pentasaccharide-bound AT if Mr<10,000 but decreases at higher Mr. Thrombin inactivation is constant per pentasaccharide with twelve adjacent monosaccharides (C-domain)., Conclusion: The data fit a model in which thrombin and factor Xa bind at a random site on the heparin chain and, via one-dimensional diffusion, reach the AT that is bound to its specific binding site on the heparin. Factor Xa, but not thrombin, can dissociate from heparin before reaching bound AT.

In rats, dietary fish oil causes a plasma triglyceride-lowering as well as hypocoagulant effect. The latter is apparent from reduced levels of vitamin K-dependent coagulation factors and a decreased thrombin-forming potential of the coagulating plasma. Here, we describe that intervention with low levels of n-3 polyunsaturated fatty acids (n-3 PUFAs, about 2.5% of digestible energy, en%) resulted in no more than a small reduction in coagulation factors, when supplied as part of a high-fat diet relatively rich in vitamin K. Plasma triglycerides also remained unchanged. On the other hand, when feeding rats with low- or high-fat diets restricted in vitamin K, intervention with 3 en% of n-3 PUFAs acids (fish oil) caused only a lowering in triglycerides in combination with high fat. The fish caused a reduction in coagulation potential and levels vitamin K-dependent coagulation factors (prothrombin and factor VII) that was most prominent with the low-fat diet. Fish oil, in combination with low fat but not with high fat, reduced the vitamin K levels in the liver of the animals. In addition, regardless of the fat content, the vitamin K-independent coagulation factor V was decreased in the fish oil groups. Taken together, these results indicate that, in the rat, the hypocoagulant effect of a low dose of n-3 PUFAs is most apparent at low intakes of both vitamin K and fat, is not linked to the triglyceride plasma level, but involves modulation of both vitamin K-dependent and -independent coagulation factors.

We proposed the endogenous thrombin potential (ETP) as an overall function test of the coagulation system. We recently introduced a routine test which requires defibrinated plasma. In order to develop an assay in which the ETP-value can be directly obtained by measuring the optical density, we investigated two methods to inhibit fibrinogen clottability and to inactivate alpha2-macroglobulin. The first method makes use of hydroxylamine to inactivate alpha2-macroglobulin and H-Gly-Pro-Arg-Pro-OH to inhibit fibrin polymerization. At pH 7.35, plasma incubated with 25 mM hydroxylamine and 1.5 mg/mL H-Gly-Pro-Arg-Pro-OH for 5 minutes at 37 degrees C resulted in a reduced endlevel of the amidolytic activity on small chromogenic substrates. The second method uses a metalloprotease purified from Crotalus basiliscus to remove alpha2-macroglobulin from plasma in combination with H-Gly-Pro-Arg-Pro-OH. Herein plasma is incubated with 3.5 LM protease during 15 minutes at 37 degrees C in the presence of 1 mg/mL polymerization inhibitor. The enzymatic method results in a zero endlevel of the amidolytic activity and this would imply that measurement of the ETP is reduced to an endpoint determination of the optical density. We show that the endpoint determination of the optical density correlates well with the calculated ETP in plasmas with different degrees of anticoagulation.

Pentosan polysulphate is an heparin analogue which acts via an antithrombin III (AT III) independent pathway. We compared the effect of this drug to that of heparin and AT III infusions in AT III deficient patients. Four patients with AT III congenital deficiency received on three different occasions: (i) an infusion of human AT III concentrate (20 U/kg or 40 U/kg), (ii) an intramuscular injection of pentosan polysulphate (2 mg/kg), (iii) a subcutaneous calcium heparin injection (100 U/kg). AT III infusion inhibits the excessive thrombin generation (46% of inhibition) observed in the plasma of AT III deficient patients during at least 12 hours, but does not modify the factor Xa formation. On the contrary, pentosan polysulphate has a marked effect on both thrombin (62% of inhibition) and factor Xa generation (57% of inhibition) still present 8 hours after injection. Heparin injection has the same effect, more prolonged, as pentosan polysulphate on thrombin generation but is not so effective on impairing factor Xa generation (27% of inhibition). The marked effect of pentosan polysulphate on thrombin and factor Xa generation in these patients is due to its AT III independent mechanism of action.