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Background: Low-molecular-weight heparins (LMWHs) are routinely given without the control of their effect on coagulation. The endogenous thrombin potential (ETP) is a sensitive detector of the heparin effect., Question: What is the interindividual variation in TG after a fixed dose of LMWH in normal volunteers, is it explained by variation in weight?, Methods: Subcutaneous (s.c.) injection, in 12 healthy volunteers, of 9000 aXa-units of unfractionated heparin (UFH) and of three heparins with narrow MW distribution around 10.5, 6.0 and 4.5 kD. Measurement of anti-thrombin (aIIa) and antifactor Xa (aXa)-activities and ETP at 11 time points over 24 h., Results: The coefficient of variation (CV) of the AUCs of aXa- and aIIa-activities is 50% for UFH and 22-37% for LMWHs. Because of the hyperbolic form of the dose-response curve, the CV of the inhibition of the ETP is lower: 32% for UFH and 13-21% for the LMWHs. Fixed dosage of LMWH caused under-dosage in 10-13% of the samples and over-dosage in 5-11%. High or low response is an individual property independent of the type of heparin injected and only partially explained by variation in body weight., Conclusion: Optimized individual dosage of LMWH is possible through recognition of high and low responders, which requires one measurement of the heparin concentration or, preferably, the heparin effect on the ETP, 2-5 h after a first injection.

Background: Heparins in clinical use differ considerably as to mode of preparation, molecular weight distribution and pharmacodynamic properties., Objectives: Find a common basis for their anticoagulant action., Methods: In 50 fractions of virtually single molecular weight (Mr), prepared from unfractionated heparin (UFH) and four low-molecular-weight heparins (LMWH), we determined: (i) the molar concentration of material (HAM) containing the antithrombin binding pentasaccharide (A-domain); (ii) the specific catalytic activity in thrombin and factor Xa inactivation; (iii) the capacity to inhibit thrombin generation (TG) and prolong the activated partial thromboplastin time (APTT). We also calculated the molar concentration of A-domain with 12 sugar units at its non-reducing end, i.e. the structure that carries antithrombin activity (C-domain)., Results: The antithrombin activity and the effects on TG and APTT are primarily determined by the concentration of C-domain and independent of the source material (UFH or LMWH) or Mr. High Mr fractions (>15 000) are less active, probably through interaction with non-antithrombin plasma proteins. Anti-factor Xa activity is proportional to the concentration of A-domain, it is Ca2+- and Mr-dependent and does not determine the effect on TG and APTT., Conclusion: For any type of heparin, the capacity to inhibit the coagulation process in plasma is primarily determined by the concentration of C-domain, i.e. the AT-binding pentasaccharide with 12 or more sugar units at its non-reducing end.

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.

Thrombin formation and blood platelet reactions are intimately linked in haemostasis and in thrombosis. In vivo, procoagulant phospholipids required for the coagulation mechanism are mainly provided by activated platelets, and thrombin is the most potent platelet activator. To study these interactions, an ancient tool of coagulation physiology, the thrombin generation test, was revived and the results obtained were reviewed. The amount of thrombin activity that develops, expressed as the endogenous thrombin potential (the area under the thrombin generation curve), is influenced by the clotting factors (except XII and XIII), the activated protein C system and natural inhibitors on the one hand and by platelet activity on the other. The platelet reactions that we found to be involved are induced by thrombin via glycoprotein (GP) IIb/IIIa activation and by fibrin via interaction with GPIb. von Willebrand factor is crucial in both reactions and therefore an obligatory factor for normal thrombin generation in the presence of platelets. All antithrombotics, be it anticoagulants (e.g. OAC, all heparins or hirudin) or antiplatelet drugs (aspirin, GPIIb/IIIa blockers) diminish thrombin generation.

Thrombin generation in platelet-rich plasma (PRP) involves complex interactions between platelets and coagulation proteins. We previously reported that the addition of fibrin to PRP enhances tissue-factor initiated thrombin generation by approximately 40%, and the current studies were designed to assess the mechanism(s) underlying thrombin generation in the absence and presence of fibrin. Blocking platelet GPIIb/IIIa + alphavbeta3 receptors with a monoclonal antibody (MoAb) inhibited basal thrombin generation, but did not affect the enhancement produced by fibrin. In contrast, blocking GPIb with any of three different MoAbs had no effect on basal thrombin generation, but essentially eliminated fibrin enhancement of thrombin generation. When thrombin generation was tested in PRP deficient in von Willebrand factor (vWF), both basal and fibrin-enhanced thrombin generation were markedly reduced, and the addition of factor VIII did not normalize thrombin generation. Botrocetin, which induces the binding of vWF to GPIb, enhanced thrombin generation. In all studies, the ability of PRP to support thrombin generation correlated with the production of platelet-derived microparticles and serum platelet-derived procoagulant activity. Thus, two separate mechanisms, both of which depend on vWF, appear to contribute to platelet-derived procoagulant activity: one is independent of fibrin and relies primarily on GPIIb/IIIa, but with a minor contribution from alphavbeta3; and the other is fibrin-dependent and relies on GPIb. These data may have implications for understanding the mechanisms of the abnormalities in serum prothrombin times reported in Bernard-Soulier syndrome, hemorrhage in von Willebrand disease (vWD), and the increased risk of thrombosis associated with elevated vWF levels.

In a final stage of activation, platelets become procoagulant because of the appearance of phosphatidylserine (PS) at the membrane outer surface. This PS exposure requires a rise in cytosolic [Ca(2+)](i), is accompanied by formation of membrane blebs, and stimulates the formation of thrombin from its precursor prothrombin. Here, we investigated whether thrombin, as a potent platelet agonist, can induce this procoagulant response in plasma-free platelets interacting with fibrin or fibrinogen through their integrin alpha(IIb)beta(3) receptors. First, in platelets that were stimulated to spread over fibrin or fibrinogen surfaces with adrenaline, addition of thrombin and CaCl(2) caused a potent Ca(2+) signal that in about 30% of the cells was accompanied by exposure of PS. At low doses, integrin alpha(IIb)beta(3) receptor antagonist (RGD peptide) inhibited platelet spreading as well as thrombin-evoked PS exposure. Second, in platelet-fibrinogen microaggregates that were preformed in the presence of adrenaline, thrombin/CaCl(2) induced PS exposure and bleb formation of about 35% of the cells. Third, a potent, thrombin-dependent stimulation of prothrombinase activity was measured in platelet suspensions that were incubated with a fibrin clot. These results indicate that, in the absence of coagulating plasma, thrombin is a moderate inducer of the procoagulant response of platelets, once integrin alpha(IIb)beta(3)-mediated interactions are stimulated (by adrenaline) and CaCl(2) is present.

We investigated the effects of dietary polyunsaturated fatty acids (PUFAs) on blood lipids and processes that determine hemostatic potential: platelet activation, coagulation, and fibrinolysis. For 8 to 10 weeks, Wistar rats were fed a high-fat diet containing various amounts (2% to 16%) of n-3 PUFAs derived from fish oil (FO) or a diet enriched in n-6 PUFAs from sunflower seed oil (SO). Only the FO diets caused a reduction in mean platelet volume, platelet arachidonate level, and formation of thromboxane B2 by activated platelets, but neither of the diets had a measurable effect on platelet activation. The FO-rich diets decreased the plasma concentrations of triglycerides and cholesterol, whereas the SO diet reduced triglycerides only. Parameters of fibrinolysis and standard coagulation times, ie, activated partial thromboplastin time and prothrombin time, were only marginally influenced by these diets. In contrast, dietary FO, but not SO, led to decreased levels of the vitamin K-dependent coagulation factors prothrombin and factor VII, while the level of antithrombin III was unchanged. The endogenous thrombin potential (ETP) was measured with an assay developed to detect the hypocoagulable state of plasma. After activation with tissue factor and phospholipids, the ETP was reduced by 23% or more in plasma from animals fed a diet with >4% FO. No significant effect of the SO diet on ETP was observed. Control experiments with plasma from warfarin-treated rats indicated that the ETP was more sensitive to changes in prothrombin concentration than in factor VII concentration. Taken together, these results indicate that in rats, prolonged administration of n-3 but not n-6 PUFAs can lead to a hypocoagulable state of plasma through a reduced capacity of vitamin K-dependent thrombin generation, with unchanged thrombin inactivation by antithrombin III.

We tested different types of clot for their ability to provoke procoagulant activity in platelets: normal clots from platelet poor plasma (des AABB- or fibrin II clots), similar clots in which the adsorbed thrombin has been inhibited by hirudin, and clots obtained by the action of two snake venom enzymes that release only fibrinopeptide A (des AA- or fibrin I clots). Analogous clots from fibrinogen solutions were also tested. In platelet rich plasma (PRP), where platelet coagulant phospholipids (PCP) are rate limiting for thrombin generation, the addition of any type of clot enhances the generation of thrombin thus it induces the appearance of PCP. Clots containing active adsorbed thrombin are the most potent ones in this respect. Lactate dehydrogenase (LDH) levels do not increase in the course of the thrombin generation so the platelets are not damaged in the process. Non-centrifugable PCP could be demonstrated to appear during the process, so the production of procoagulant phospholipid microparticles must be part of the mechanism. Membrane transbilayer phosphatidyl serine movement (flip-flop) can not be demonstrated in PRP as the activated platelets are caught in the emerging clot. In order to demonstrate flip-flop, we tried to investigate the influence of clots on washed platelets. However, contrary to platelets in a plasma milieu, isolated platelets are damaged by fibrin clots, especially in the presence of thrombin, as can be judged from the appearance of LDH. We conclude that, in PRP, clots induce the appearance of PCP from platelets by vesiculation, possibly accompanied by flip-flop and that thrombin accelerates the process but is not an absolute requirement.

From the saliva of the vampire bat Desmodus rotundus, we isolated an unknown anticoagulant protein which we have named draculin. Its molecular mass as determined by non-reduced SDS-PAGE is about 83 kDa. The reduced polypeptide shows a slower migration. HPLC in a molecular sieve matrix yields a single, symmetrical peak corresponding to 88.5 kDa. Isoelectric focusing shows an acidic protein with pI = 4.1-4.2. Aminoacid analysis is compatible with a single chain polypeptide of about 80 kDa. Cyanogen bromide cleavage yields a single 16-aminoacid peptide, corresponding to the amino-terminus of the native molecule. Draculin inhibits the activated form of coagulation factors IX and X. It does not act on thrombin, trypsin, chymotrypsin and does not express fibrinolytic activity. The inhibition is immediate and not readily reversible, with a stoichiometry of about two molecules of draculin per molecule of factor IXa or Xa. Surprisingly, the inhibitory activity against either factor is not affected by the presence of the other. Draculin binds quantitatively to either immobilised factor Xa or factor IXa. Our preliminary interpretation is that there are two forms of draculin that hardly differ in structure. Both bind to factor Xa and to factor IXa but one form inhibits factor Xa and the other inhibits factor IXa. When added to plasma, draculin increases the lag phase as well as the height of the peak of thrombin generation.

The decay rate of thrombin in plasma is shown to be linearly proportional to the concentration of antithrombin III (AT III), not only in the absence but also in the presence of heparin. This is a consequence of partitioning of heparin between AT III and other plasma proteins. In previous articles were calculated the prothrombin converting activity assuming a fixed concentration of AT III. Since AT III is consumed during the clotting process, prothrombinase activity is more accurately approximated using an algorithm that counts with the decrease of the AT III concentration. It is shown this leads to higher prothrombinase activities. The (absence of) inhibition of prothrombin conversion by prothrombinase in the presence of heparins found with the previous method is also found using the new algorithm. From the results presented it is evident that characteristic parameters of heparin action have to be normalised to the AT III concentration. On this basis we define a Standard Independent Unit of the antithrombin activity of heparin.

We report a study on the importance of factor IX activation in thromboplastin-dependent coagulation in plasma. Diluted, CaCl2-containing thromboplastin solutions at constant phospholipid concentration were used to trigger the coagulation in plasma from patients with congenital factor IX and factor VIII deficiency in the presence and absence of added factors IX and VIII, and the generation of thrombin activity was monitored. When coagulation is triggered with the high thromboplastin concentrations normally used in clinical routine tests, the generation of thrombin activity in plasma of patients with congenital factor IX deficiency before and after reconstitution with purified factor IX appears identical. When, however, coagulation is triggered with low thromboplastin concentrations, a clear dependency of the generation of thrombin activity on the concentration of factor IX becomes evident at factor IX concentrations lower than 30 nM (about 40% clotting factor activity). Factor VIII is a compulsory cofactor for this factor IX activity because the prothrombinase activity at optimal factor IX concentration is still critically dependent upon the amount of factor VIII present. The lower the amount of thromboplastin, the higher the importance of factor IX and factor VIII activation in thromboplastin-dependent coagulation. This suggests a role of this pathway in pathophysiological thrombin formation.