A century of heparin: past, present and future.

Heparin was discovered around 1922 by Howell (Baltimore) and was further developed by the teams of Best (Toronto) and Jorpes (Stockholm). Kakkar (London) propagated its routine use for the prevention of postoperative thrombosis from 1971 onwards. The discovery of low molecular weight heparins (1976, Johnson, London) and their development in the subsequent years led to the present arsenal of clinically useful drugs. In 1976, three teams independently found that a specific structure in heparin binds tightly to antithrombin. This enabled the teams of Lindahl (Stockholm) and Casu (Milan) to determine the pentasaccharide structure responsible for this binding and Petitou, from the Choay team (Paris), to synthesize it (1983). It was found (Olson and others) that heparin facilitates the interaction between antithrombin and a clotting enzyme by allosteric changes in the antithrombin (important for factor Xa) and by facilitating the approach of the enzyme to antithrombin via its "sliding" along the heparin molecule (important for thrombin). Antithrombin action therefore requires a minimum length of seven sugar units next to the pentasaccharide whereas anti-factor Xa action does not. The effect of heparin is almost entirely due to anti-thrombin action (B≐guin), so anti-factor Xa activity does not reflect the concentration of anticoagulant heparin. The anticoagulant effect is poorly reflected by the activated partial thromboplastin time. Because present clinical use is based on the latter tests, it is not generally known that the individual response to heparin shows an extremely wide variation. Individualization of heparin dosage is likely to improve clinical results.

[Measuring thrombin formation]. / Het meten van trombinevorming.

Measurement of thrombin formation makes it possible to estimate the risk of haemorrhage or thrombosis much more accurately than by using clotting time. This new technique allows better monitoring of the effect of prophylactic and therapeutic anticoagulant therapy. Thrombin formation is, however, not yet routinely measured.

The balance of pro- and anticoagulant processes underlying thrombin generation.

BACKGROUND: The generation of thrombin in time is the combined effect of the processes of prothrombin conversion and thrombin inactivation. Measurement of prothrombin consumption used to provide valuable information on hemostatic disorders, but is no longer used, due to its elaborate nature. OBJECTIVES: Because thrombin generation (TG) curves are easily obtained with modern techniques, we developed a method to extract the prothrombin conversion curve from the TG curve, using a computational model for thrombin inactivation. METHODS: Thrombin inactivation was modelled computationally by a reaction scheme with antithrombin, α(2) Macroglobulin and fibrinogen, taking into account the presence of the thrombin substrate ZGGR-AMC used to obtain the experimental data. The model was validated by comparison with data obtained from plasma as well as from a reaction mixture containing the same reactants as plasma. RESULTS: The computational model fitted experimental data within the limits of experimental error. Thrombin inactivation curves were predicted within 2 SD in 96% of healthy subjects. Prothrombin conversion was calculated in 24 healthy subjects and validated by comparison with the experimental consumption of prothrombin during TG. The endogenous thrombin potential (ETP) mainly depends on the total amount of prothrombin converted and the thrombin decay capacity, and the peak height is determined by the maximum prothrombin conversion rate and the thrombin decay capacity. CONCLUSIONS: Thrombin inactivation can be accurately predicted by the proposed computational model and prothrombin conversion can be extracted from a TG curve using this computational prediction. This additional computational analysis of TG facilitates the analysis of the process of disturbed TG.

The effect of fibrin(ogen) on thrombin generation and decay.

Defibrination causes a ~30% decrease of thrombin generation (TG) which can be restored by adding native fibrinogen in its original concentration (3 mg/ml). The fibrinogen variant γA/γ', which binds thrombin with high affinity, is over four times more efficient in this respect than the more common γA/γA form. By using high tissue factor concentrations we accelerated prothrombin conversion so as to obtain a descending part of the TG curve that was governed by thrombin decay only. From that part we calculated the antithrombin (AT)- and α2-macroglobulin-dependent decay constants at a series of concentrations of native, γA/γA and γA/γ' fibrinogen. We found that the increase of TG in the presence of fibrinogen is primarily due to a dose-dependent decrease of thrombin inactivation by α2-macroglobulin, where the γA/γ' form is much more active than the γA/γA form. AT-dependent decay is somewhat decreased by γA/γ' fibrinogen but hardly by the γA/γA form. We assume that binding of thrombin to fibrin(ogen) interferes with its binding to inhibitors. Attenuation of decay only in part explains the stimulating effect of fibrinogen on TG, as fibrinogen stimulates prothrombin conversion, regardless of the fibrinogen variant.

A new regulatory function of activated factor V: inhibition of the activation by tissue factor/factor VII(a) of factor X.

BACKGROUND: We observed that minute amounts of thrombin or the enzyme Russell's viper venom activating factor V (RVV-V) added to plasma strongly diminish the potential of that plasma to generate thrombin after being triggered by tissue factor. OBJECTIVE: To find the mechanism behind this phenomenon. METHODS AND RESULTS: Thrombin generation (TG) initiated by tissue factor (TF) is strongly and dose-dependently inhibited by addition of activated factor V (FVa) or by addition of a factor V activator (thrombin or RVV-V). No inhibition is seen when TG is triggered via the intrinsic pathway or by direct activation of factor X. The effect is independent of proteins C and S and tissue factor pathway inhibitor (TFPI). In factor VII-deficient plasma the effect is seen when it is spiked with recombinant factor VII (FVII) and to a much lesser extent when spiked with recombinant FVIIa. In a purified system, FVa also dose-dependently inhibits the activation of FX by TF/FVII(a). The inhibitory effect is neutralized by antibodies against the light chain of FVa but not by antibodies against the heavy chain. CONCLUSIONS: Our observations can be explained by assuming that FVa, via its light chain, binds to the complex TF/FVII(a) and prevents it from activating FX. We assume that this mechanism reduces the possibility that thrombin and factor Xa escaping from a wound area into the circulation, together with blood-borne tissue factor, would trigger intravascular coagulation.

Thrombin generation assay using factor IXa as a trigger to quantify accurately factor VIII levels in haemophilia A.

BACKGROUND: The available methods for measuring factor VIII (FVIII) activity suffer reportedly from lack of sensitivity and precision in the < 1 IU dL(-1) range. This precludes correlation of clinical phenotype with FVIII levels. OBJECTIVES: To study a possible association between clinical phenotype in patients with FVIII levels < 1 IU dL(-1). METHODS/RESULTS: The FIXa-driven FVIII assay (FVIII-CAT) has a detection limit of 0.05 IU dL(-1). For the range of 0-2 IU dL(-1) FVIII, the intra-assay coefficient of variation (CV) is around 2% and the inter-assay CV is about 8%. We tested 30 hemophiliacs with FVIII:C between < 1 and 6 IU dL(-1) as measured in the one-stage clotting assay using the FVIII-CAT assay. For genetic defects related to moderate hemophilia, the FVIII-CAT test finds FVIII levels that are in good agreement with those determined with the one-stage assay. Of the 21 hemophilic patients with FVIII < 1 IU dL(-1), four patients exhibited a mild bleeding phenotype. When we applied TF-initiated thrombin generation, patients with a mild clinical phenotype showed significantly higher endogenous thrombin potentials. CONCLUSION: The novel developed FVIII assay measures accurately FVIII levels below 1 IU dL(-1). Its application demonstrated that the clinical heterogeneity in individuals with < 1 IU dL(-1) FVIII is not associated with their FVIII level.

The paradoxical stimulation by a reversible thrombin inhibitor of thrombin generation in plasma measured with thrombinography is caused by alpha-macroglobulin-thrombin.

BACKGROUND: Thrombin generation (TG) in plasma can be monitored continuously with a fluorogenic thrombin substrate using calibrated automated thrombinography (CAT). In the presence of low concentrations of a reversible direct thrombin inhibitor (DTI), CAT shows an unexpected effect: the endogenous thrombin potential (ETP) increases at low concentrations of the inhibitor to subsequently decrease concentration dependently at higher concentrations (> approximately 100 nm). OBJECTIVES: To find an explanation for this phenomenon, we measured the concentrations of free thrombin and alpha(2)-macroglobulin-thrombin complex (alpha(2)MT) with a sub-sampling technique in the presence of AR-H067637, a selective DTI. RESULTS: At all concentrations of the DTI there was a gradual dose-dependent decrease in the concentration of free, not-inhibited thrombin but a transient increase in free alpha(2)MT due to competition of thrombin and alpha(2)MT for the inhibitor. Because the CAT technique uses an algorithm to subtract alpha(2)MT activity from the total amidolytic activity, this transient increase in alpha(2)MT activity is not subtracted and erroneously attributed to thrombin itself. CONCLUSIONS: This study explains the spurious increase in ETP observed at low DTI concentrations. The results obtained in plasma were corroborated by observations in a thrombin generating system reconstituted with purified factors. In practise, the effect of DTIs on TG can be reliably evaluated from the area under the curve till time-to-peak.

Differential impact of conventional and low-dose oral hormone therapy (HT), tibolone and raloxifene on functionality of the activated protein C system.

Recent studies have shown that hormone therapy (HT) is associated with an acquired resistance to activated protein C (APC). The aims of the present study were to evaluate a possible dose-response relationship and differential effects of different HT regimens on functionality of the APC system. Two hundred two healthy women were randomly assigned to receive treatment for 12 weeks with tablets containing either low-dose HT containing 1 mg 17ss-oestradiol + 0.5 mg norethisterone acetate (NETA) (n = 50), conventional-dose HT containing 2 mg 17ss-oestradiol and 1 mg NETA (n = 50), 2.5 mg tibolone (n = 51), or 60 mg raloxifene (n = 51). Normalized APC system sensitivity ratios (nAPCsr) were determined in plasma collected at baseline and after 12 weeks using a thrombin generation-based APC resistance test probed with either recombinant APC (rAPC) or thrombomodulin (rTM). NAPCsr increased in both the conventional- and low-dose HT groups, consistent with reduced sensitivity to APC. The increase was slightly more pronounced in the conventional-dose group, but the difference between the two HT groups was not statistically significant. The sensitivity to APC was only marginally altered in those allocated to tibolone. Consequently, tibolone showed a different phenotype as compared with the low-dose HT group. A small increase in nAPCsr with both rAPC and rTM was seen in the raloxifene-group, but the increase was less than in the low-dose HT group. Our findings indicate that oestrogen-progestin therapy induces an APC resistant phenotype, which may be related to dose, whereas tibolone and raloxifene only marginally alter the sensitivity to APC.

Randomized, placebo-controlled trial of low molecular weight heparin in active ulcerative colitis.

BACKGROUND: In several open and 1 controlled trial, unfractionated heparin was effective in the treatment of active ulcerative colitis (UC). Low molecular weight heparin (LMWH) had a similar effect in several open studies. METHODS: We studied the efficacy, safety, and tolerability of LMWH in mild to moderately active UC in a randomized, double-blind, placebo-controlled trial. In all, 29 patients with a mild or moderate recurrence of UC during salicylate treatment were randomized to receive either reviparin 3,436 IU (n = 15) subcutaneously twice daily or placebo (n = 14). The study period was 8 weeks. Treatment was discontinued if there was no improvement at 4 weeks or at any disease progression. Primary outcome measure was clinical improvement at 8 weeks measured by the Colitis Activity Index (CAI) and the Clinical Symptoms Grading (CSG, based on the CAI). Endoscopic and histologic grading and quality of life as measured by the Inflammatory Bowel Disease Questionnaire (IBDQ) were secondary outcome measures. Patients were closely monitored for adverse events. RESULTS: Twenty of 29 patients finished the 8-week treatment period (reviparin versus placebo: 11 versus 9; P = 0.70). There was no difference in CSG, CAI, endoscopic and histologic grading, or IBDQ. Treatment was well tolerated and no serious adverse events occurred. CONCLUSION: In this study, treatment with LMWH showed no significant clinical advantage compared to placebo in mild to moderately active UC.