R. Al Dieri

Thrombin generation for the control of heparin treatment, comparison with the activated partial thromboplastin time

Summary.  Heparin can be quantified with antifactor Xa and IIa tests (aXa, aIIa) but the anticoagulant power of heparin depends upon plasma properties as well as upon heparin concentrations and thus differs between subjects. Measuring the effect, as with the activated partial thromboplastin time (APTT) therefore is clinically more relevant. Here we investigate the use of the endogenous thrombin potential (ETP) for this purpose. In 12 volunteers 9000 IU of four heparins of different mol. wt distributions were injected. Samples were taken at 11 time points between 0 and 24 h. With the exception of the 0 and 24‐h time points, heparin could be demonstrated by its aIIa and aXa activity in virtually all samples. The APTT showed the effect of this heparin in 34% of the samples; the ETP in 80%. This is partly due to the wide margins of the normal values, caused by large interindividual variation [coefficient of variation (CV) approximately 12% for the APTT, approximately 17% for the ETP]. The intraindividual variation is much smaller (CV approximately 4% for the APTT, approximately 5% for the ETP). Relative to the baseline value of the individual, the heparin effect was recognized by the APTT in 55% of the cases and by the ETP in 98%. There were no large differences between the different types of heparin.

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

Summary.  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 4.5 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.

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

Summary.  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.  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.

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

Summary.  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.

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

We observed that minute amounts of thrombin or the enzyme Russells 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.

Monitoring new oral antithrombotics: what we should know before we can decide

1 White RH. The epidemiology of venous thromboembolism. Circulation 2003; 107: I4–8. 2 Le Gal G, Righini M, Roy PM, Meyer G, Aujesky D, Perrier A, Bounameaux H. Differential value of risk factors and clinical signs for diagnosing pulmonary embolism according to age. J Thromb Haemost 2005; 3: 2457–64. 3 Röck F, Barsan N, Weimar U. Electronic nose: current status and future trends. Chem Rev 2008; 108: 705–25. 4 Dragonieri S, Schot R, Mertens BJ, Le CS, Gauw SA, Spanevello A, Resta O, Willard NP, Vink TJ, Rabe KF, Bel EH, Sterk PJ. An electronic nose in the discrimination of patients with asthma and controls. J Allergy Clin Immunol 2007; 120: 856–62. 5 Phillips M, Altorki N, Austin JH, Cameron RB, Cataneo RN, Kloss R, Maxfield RA, Munawar MI, Pass HI, Rashid A, Rom WN, Schmitt P, Wai J. Detection of lung cancer using weighted digital analysis of breath biomarkers. Clin Chim Acta 2008; 393: 76–84. 6 Fens N, Zwinderman AH, van der Schee MP, de Nijs SB, Dijkers E, Roldaan AC, Cheung D, Bel EH, Sterk PJ. Exhaled breath profiling enables discrimination of chronic obstructive pulmonary disease and asthma. Am J Respir Crit Care Med 2009; 180: 1076–82. 7 van Belle A, Buller HR, Huisman MV, Huisman PM, Kaasjager K, Kamphuisen PW, Kramer MH, Kruip MJ, Kwakkel-van Erp JM, Leebeek FW, Nijkeuter M, Prins MH, Sohne M, Tick LW. Effectiveness of managing suspected pulmonary embolism using an algorithm combining clinical probability, D-dimer testing, and computed tomography. JAMA 2006; 295: 172–9. 8 Greiter MB, Keck L, Siegmund T, Hoeschen C, Oeh U, Paretzke HG. Differences in exhaled gas profiles between patients with type 2 diabetes and healthy controls. Diabetes Technol Ther 2010; 12: 455–63. 9 PengG, HakimM, Broza YY, Billan S, Abdah-Bortnyak R, Kuten A, Tisch U, Haick H. Detection of lung, breast, colorectal, and prostate cancers from exhaled breath using a single array of nanosensors. Br J Cancer 2010; 103: 542–51. 10 Voss A, Baier V, Reisch R, von Roda K, Elsner P, Ahlers H, Stein G. Smelling renal dysfunction via electronic nose. Ann Biomed Eng 2005; 33: 656–60. 11 PhillipsM, CataneoRN,Greenberg J, GrodmanR, SalazarM. Breath markers of oxidative stress in patients with unstable angina.Heart Dis 2003; 5: 95–9. 12 Huisman MV, Klok FA. Diagnostic management of clinically suspected acute pulmonary embolism. J Thromb Haemost 2009; 7(Suppl. 1): 312–17. 13 Lewis NS. Comparisons between mammalian and artificial olfaction based on arrays of carbon black–polymer composite vapor detectors. Acc Chem Res 2004; 37: 663–72. 14 Bossuyt PM, Reitsma JB, Bruns DE, Gatsonis CA, Glasziou PP, Irwig LM, Moher D, Rennie D, de Vet HC, Lijmer JG. The STARD statement for reporting studies of diagnostic accuracy: explanation and elaboration. Ann Intern Med 2003; 138: W1–12. 15 Broadhurst DI, Kell DB. Statistical strategies for avoiding false discoveries in metabolomics and related experiments.Metabolomics 2006; 2: 171–96.

The contribution of α2-macroglobulin thrombin to the endogenous thrombin potential

Ignjatovic et al (2007) were right to stress the importance of a2-macroglobulin-bound thrombin (a2M-thrombin) in the interpretation of thrombin generation curves, particularly under conditions where a2M concentrations are likely to differ widely from those in the normal adult population, such as in neonates. When fluorogenic methods are used, such as that of Varadi et al (2003), that do not correct for substrate consumption and for the quenching of fluorescence by formed product (the inner filter effect) no steady end-level amidolytic activity because of formed a2M-thrombin can be calculated. Consequently, no correction is possible for the contribution of that complex to the calculated thrombin concentrations. Under those circumstances the a2M content of the plasma contributes significantly to the outcome of the measured parameters. However, using the calibrated automated thrombin (CAT) method (Hemker et al, 2003), the a2M-thrombin end level can be, and is, calculated. The algorithm that is used to calculate the curve of free thrombin (and hence also the endogenous thrombin potential; ETP) does not assume a ‘likely contribution of a2M-thrombin to the ETP’, as stated by Ignjatovic et al (2007), but is assessed from the above mentioned steady end-level of amidolytic activity. At high plasmatic a2M concentrations a high end level is found and more amidolytic activity is attributed to the a2M-thrombin complex than at low plasmatic a2M levels. a2M-thrombin levels are subtracted from the calculated thrombin concentrations and hence do not contribute to the ETP and peak value of thrombin. The measured ETP therefore is independent of the plasma a2M level, except in so far as a2M contributes to the over-all anti-thrombin action of the plasma. For the underlying assumptions and the mathematical details the reader is referred to our articles as presented by Ignjatovic et al (2007). In fact, the algorithm is the same in the fluorogenic CAT method as in the subsampling methods and continuous chromogenic methods that we used and validated earlier (Hemker & Beguin, 1995). As an aside, we would like to note that fibrinogen strongly interferes with the reaction between a2M and thrombin (Hemker et al, 2003). Because the experiments reported by Ignjatovic et al (2007) were performed with defibrinated plasma, it is likely that their experimental contributions of a2M-thrombin were 3–5 times higher than in their patients.

Procoagulant effect of vitamin K antagonists

Strachan DP, Tang W, O Donnell CJ, Smith NL, de Maat MP. Effect of genetic variations in syntaxin-binding protein-5 and syntaxin-2 on von Willebrand factor concentration and cardiovascular risk. Circ Cardiovasc Genet 2010; 3: 507–12. 8 Eriksson N, Macpherson JM, Tung JY, Hon LS, Naughton B, Saxonov S, Avey L, Wojcicki A, Pe er I, Mountain J. Web-based, participant-driven studies yield novel genetic associations for common traits. PLoS Genet 2010; 6: e1000993. 9 Klinge U, Binnebosel M,Mertens PR. Are collagens the culprits in the development of incisional and inguinal hernia disease?Hernia 2006; 10: 472–7. 10 Liem EB, Lin CM, Suleman MI, Doufas AG, Gregg RG, Veauthier JM, Loyd G, Sessler DI. Anesthetic requirement is increased in redheads. Anesthesiology 2004; 101: 279–83.