H. C. Hemker

Fixed dosage of low-molecular-weight heparins causes large individual variation in coagulability, only partly correlated to body weight.

Summary.u2002 Backgrounds: 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 4.5u2003kD. Measurement of anti‐thrombin (aIIa) and antifactor Xa (aXa)‐activities and ETP at 11 time points over 24u2003h. 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, 2–5u2003h after a first injection.

Fibrin polymerization is crucial for thrombin generation in platelet-rich plasma in a VWF-GPIb-dependent process, defective in Bernard-Soulier syndrome.

Summary.u2002 Defective prothrombin consumption has been reported in the proband case of Bernard–Soulier syndrome (BSS). There is no consensus, however, on whether the formation of platelet procoagulant activity (PPA) is impaired in BSS and, if so, whether this is due to the lack of GPIb‐V‐IX‐dependent binding of thrombin or of von Willebrand factor (VWF). We show thrombin generation (TG) in platelet‐rich plasma of BSS (BSS‐PRP) to be defective provided that fibrin remains present in the reaction mixture and that the giant platelets are not damaged by frequent subsampling. In BSS‐PRP addition of (thrombin‐free) fibrin did not increase TG as in normal PRP, supporting our previous hypothesis that the interaction of fibrin, VWF and GPIb triggers PPA development. Fibrin formed during the lag phase of TG by a snake venom enzyme which only removed fibrinopeptide A induced an immediate burst of TG, that was inhibited by a monoclonal antibody against GPIb (6D1) that abolishes ristocetin‐induced binding of VWF to platelets. Inversely, inhibition of polymerization decreased TG and the residual activity was insensitive to 6D1. We conclude that polymerizing fibrin interacts with VWF so as to activate GPIb.

The inhibition of blood coagulation by heparins of different molecular weight is caused by a common functional motif—the C‐domain

Summary.u2002 Background:u2002Heparins in clinical use differ considerably as to mode of preparation, molecular weight distribution and pharmacodynamic properties. Objectives:u2002Find a common basis for their anticoagulant action. Methods:u2002In 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:u2002The 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 (>15u2003000) 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:u2002For 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.

Is there value in kinetic modeling of thrombin generation? No (unless…).

See also Stuijver DJF, Hooper JMW, Orme SM, van Zaane B, Squizzato A, Piantanida E, Hess K, Alzahrani S, Ajjan RA. Fibrin clot structure and fibrinolysis in hypothyroid individuals: the effects of normalising thyroid hormone levels. This issue, pp 1708–10.

Recollections on thrombin generation

Summary.u2002 Against an autobiographic background, a historical sketch is given of the development of the technique of thrombin generation, from subsampling to duly calibrated continuous measurement with fluorogenic substrates. Its application to various problems in the pathophysiology of hemostasis and thrombosis is discussed.

The balance of pro- and anticoagulant processes underlying thrombin generation

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.

Thrombin generation is extremely sensitive to preheating conditions

Bankhead C, Xu Y. Anticoagulation control and prediction of adverse events in patients with atrial fibrillation. A systematic review. Circ Cardiovasc Qual Outcomes 2008; 1: 84–91. 9 Morgan CL, McEwan P, Tukiendorf A, Robinson PA, Clemens A, Plumb JM. Warfarin treatment in patients with atrial fibrillation: observing outcomes associated with varying levels of INR control. Thromb Res 2009; 124: 37–41. 10 Barcellona D, Contu P, Margongiu F. Patient education and oral anticoagulation therapy. Haematologica 2002; 87: 1081–6. 11 Wofford JL, Wells MD, Singh S. Best strategies for patient education about anticoagulation with warfarin: a systematic review. BMCHealth Serv Res 2008; 8: 40.

The ionic contrast medium ioxaglate interferes with thrombin-mediated feedback activation of factor V, factor VIII and platelets

Summary.u2002 Clinical observation shows that radiographic contrast media (CM) may influence thrombus formation. In the search for the underlying mechanism, we have shown that the ionic CM ioxaglate is a potent inhibitor of thrombin generation in platelet‐poor and platelet‐rich plasma, whereas the influence of the non‐ionic contrast medium iodixanol is minimal. Ioxaglate boosts the inhibitory effect of the platelet GPIIb/IIIa antagonist abciximab and the effects of ioxaglate and heparin are additive. Ioxaglate inhibits the clotting of fibrinogen and the activation of factors V and VIII, and of platelets by thrombin. It does not inhibit hydrolysis of small chromogenic thrombin substrates, nor does it influence the heparin‐catalyzed inactivation of thrombin by antithrombin. We assume therefore that ioxaglate interferes with the binding of macromolecular substrates to the anionic exosite I of thrombin. The biological correlation to the observed antithrombotic effect of ioxaglate is then to be found in the inhibition of thrombin generation via inhibition of thrombin‐mediated feedback activations.

Conjectures and Refutations on the Mode of Action of Heparins

Low-molecular-weight heparins (LMWHs), like unfractionated heparin (UFH), exert their action primarily by accelerating the interaction between antithrombin (AT) and thrombin. At the levels of aXa activity that are attained in human pharmacology, it does not cause significant (>15%) inhibition of the clotting system. The essential differences between LMWHs and UFH are: (a) LMWHs attain higher plasma concentrations after subcutaneous injection (high bioavailability), and (b) in contrast to LMWHs, UFH contains very large heparin molecules with a putative hemorrhagic action. The reputedly higher aXa activity of LMWH can be shown to be largely due to the absence of Ca2+ using the current laboratory methods to estimate this activity. Via this artifact the apparently high aXa activity of LMWHs is correlated but not related to their favorable pharmacokinetic properties. Consequently dosage guidelines for the use of different LMWHs cannot be based upon their aXa activity. Until better laboratory methods are available, clinical results are the only reliable guideline to heparin dosage.

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.