which has become near to legendary in the medical world in our country. ... problems that lasts for a lifetime, even if, like me, one hardly ever treated a ... and a technician. ..... stopping times on a computer via pushbuttons on the pipettes.
Journal of Thrombosis and Haemostasis, 6: 219–226
HISTORICAL SKETCH
Recollections on thrombin generation H. C. HEMKER Synapse BV, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, the Netherlands
To cite this article: Hemker HC. Recollections on thrombin generation. J Thromb Haemost 2008; 6: 219–26.
Stage one: one stage Summary. 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. Keywords: antithrombotic treatment, heparins, hypercoagulability, memoirs, screening test, thrombin generation. Introduction Thrombin generation is a subject with about equal parts of biochemistry, physiology and medicine. So, with hindsight, once put on the trail of hemostasis and thrombosis, there was hardly a chance that I could have escaped it. When I entered medical school, in Amsterdam in 1951, I was not aware that I was to be brought up by a faculty, the fame of which has become near to legendary in the medical world in our country. Professors like Borst (internal medicine) and Boerema (surgery) gave us an understanding of medical problems that lasts for a lifetime, even if, like me, one hardly ever treated a patient afterwards. My medical career is limited to an unfinished training as a pediatrician, with Van Creveld, but not in his hemophilia laboratory or clinics. I soon realized that I preferred science to medical practise and did a PhD in enzymology (with E. C. Slater). I could not betray my medical roots completely, however, so I was happy to find a position at the interface of medicine and science, in the department of Fredi Loeliger, Haemostasis and Thrombosis, at the University of Leiden. There, in 1962, I started research in blood coagulation in my own laboratory: 10 sq. m, a water bath and a technician. And a thrombelastograph, left over from FrediÕs clinical laboratory because thrombelastography had proved not to live up to its promises (an experience that seems to require repetition every generation). Correspondence: Prof. H. Coenraad Hemker, Synapse B.V. CARIM, PO Box 616, 6200MD Maastricht, the Netherlands. Tel.: +31 43 3881675; fax: +31 43 3670916; e-mail: hc.hemker@ thrombin.com Received 12 November 2007, accepted 26 November 2007 2007 International Society on Thrombosis and Haemostasis
In the early 1960s, with a few notable exceptions (Franc¸ois Duckert, Peter Esnouf), most research in the field was done by MDs. In most ingenious ways they used observations on patients, experiments of nature, to solve the riddles of the coagulation system. Thirteen factors had been recognized, mostly by astute combinations of measuring clotting times. The general atmosphere was perhaps best characterized by Rosemary Biggs when she said ÔAfter all it is more like cooking than like anything elseÕ (and in the meantime enriched the field with one finding after the other). Thus it is perhaps because of my hobby in gourmet cooking that I immediately felt at home in the subject. Indeed, hardly a better subject could be found to try and apply biochemistry to medical problems. My apprenticeship was as thorough as it was pleasant. Until this day I wonder how Fredi managed to teach me so much, while at the same time leaving me complete freedom in my research - as long as I prepared enough fraction 1.0 for his hemophiliacs. Every morning at 8.30, in his office, with his staff, he discussed the patients seen the day before and gave the backgrounds from his vast knowledge of the field. Every week or so he improvized (?) an in-depth discourse on a badly understood phenomenon, that might be worthwhile to investigate further. One of these was the discrepancy, in oral anticoagulation, between the prolongation of the Quick time and the level of the individual factors. As reported elsewhere [1], this soon led to the recognition of the abnormal (as we later learned noncarboxylated) form of prothrombin (PIVKA) [2]. I finished my training with half a year in the laboratory of Jean-Pierre Soulier in Paris, which started a life-long friendship with Franc¸ois Josso and Suzette Be´guin; and with a few weeks in Oxford, in the laboratories of Gwyn Macfarlane, Rosemary Biggs and Peter Esnouf, where I did my first thrombin generation curve. Fredi Loeliger himself had learned the trade in the department of Fritz Koller, where he had been instrumental in the discovery of factor (F) VII. In the Basel–Leiden tradition, thrombin generation did not play a dominant role [3,4]; we mostly used one stage clotting times (Fig. 1). In fact it was not until much later, when I came to spend a sabbatical with Suzette Be´guin in Paris, that my interest in thrombin generation started in earnest.
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Fig. 1. Stage one: one stage. Leiden 1973.
Why Paris? In Leiden my laboratory had steadily expanded. Of the work we did there, I only mention that, in collaboration with the Oxford group, we were the first to describe (and coined the words) prothrombinase- and the tenase-complexes [5,6]. Then, in 1973, I was asked to join a group of seven MDs that was to start a medical school in the city of Maastricht, by a quirk of history belonging to the Netherlands and not to Belgium. It was the crystallization nucleus for the full-blown university with 10 000 students that it is today. In the new school I could establish my own department (1975) and select a staff of excellent biochemists. This account is on thrombin generation so I am not supposed to dwell on the beautiful work that they did over the years. Yet I cannot help mentioning the discovery of platelet transbilayer movement by Bevers and Zwaal [7], work on the unraveling of prothrombinase and tenase kinetics by Rosing, Tans and Van Dieijen [8,9], on the discovery of annexin V by Reutelingsperger and Hornstra [10], on the role of diffusion in prothrombinase kinetics by Lindhout [11,12], on vitamin K by Soute and Vermeer [13] or on the development of ellipsometry as a tool for studying reactions at interfaces by Hermens and Cuypers [14]. In those starting years (problem based) teaching was considered the core business of our new university [15]. Research was, at best, tolerated. Mainly to establish an equilibrium between the two I agreed to become Ôrector magnificusÕ and officially responsible for both. This, together with budget discussions with the government, the foundation of the faculties of Law and Economics and a thousand other things that come with the running of a university, kept me occupied for 3 years (1982–84). It taught me how much I preferred the adventure of research to the power of management. At the end I was so homesick for science that I politely declined an offer for an even higher administrative function and returned to the laboratory.
biochemical research in my department reached an increasingly higher level. I also noticed how this brought an increasingly further distance from the patient. If I still had a role to play, it was to try and bridge that gap. I therefore decided to spend the sabbatical leave, which my administrative period had earned me, in a laboratory that still centred on pathophysiology: the coagulation laboratory of the C.H.U. Necker-Enfants Malades in Paris. It had been founded by Franc¸ois Josso, who had become a close personal friend over the years. He combined the humanity of an old fashioned family doctor with a gift for brilliant and original research. His laboratory was the first to find that FIX was activated by TF-FVII [16], that thrombin generated within seconds in a bleeding wound [17] and that leucocytes carry tissue factor [18]. Alas he did not feel the urge to publish and several of his findings had to be rediscovered by others to become known. When to our great dismay, he passed away in 1981, aged only 55, he left a wealth of original research plans and a void never to be filled. Although all over the world biochemistry with purified clotting factors and upcoming molecular biology were hailed as the bringers of breakthrough – which they often were – in the laboratory of Franc¸ois and Suzette thrombin generation in plasma had remained an important tool of investigation, among others for the study of antithrombin deficiencies and abnormal prothrombins [19] (Fig. 2). Suzette Be´guin, who had worked with him for some 20 years, desperately tried to keep his spirit alive in the laboratory and, apart from refreshing my experience in pathophysiology, I thought that I might be helpful in the enterprise. The problem that we attacked was the mode of action of heparins, a timely subject because of the upcoming low molecular weight heparins. As everybody knows, diminishing
Stage two: two stage During 3 years I had witnessed – with relief, gratitude and a slight pinch to my ego – how, in my virtual absence,
Fig. 2. Thrombin generation anno 1975. Suzette Be´guin, Paris. 2007 International Society on Thrombosis and Haemostasis
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the molecular weight of a heparin goes hand in hand with a decrease of its capacity to catalyze the binding between antithrombin and thrombin, whereas it maintains its capacity to foster the inactivation of FXa. Following an old idea of Yin and Wessler [20], it was thought that the favourable properties of LMWHs were due to the fact that they inhibit prothrombin conversion rather than inactivating the product. That it was more efficient to close the tap than to mop the floor. On the other hand, it had been shown [21] that the FXa in prothrombinase was well-nigh immune to antithrombin. The anti-Xa activity of heparins is measured on the free enzyme; would this reflect the effect of heparin on FXa in prothrombinase? Was it the free enzyme or the bound one that determined prothrombinase action in clotting plasma? This could only be determined in clotting plasma itself, so we decided to study the effect of heparins on thrombin generation. We reasoned that the net velocity of thrombin formation, as measured in a thrombin generation experiment, must be the velocity of prothrombin conversion minus that of thrombin inactivation. The latter can be calculated independently from the decay constant of thrombin in plasma and the concentration of thrombin. Thus from thrombin generation curves we could determine the influence of heparin on prothrombin conversion. To our surprise heparins, be it unfractionated or low molecular weight, hardly inhibited prothrombin conversion at all [22,23]. Only very low molecular weight heparins, like Enoxaparin, have a demonstrable effect on prothrombin conversion, which was still largely outweighed by its antithrombin action [24]. This is confirmed beyond any reasonable doubt when one compares the anti-Xa activity of equally effective concentrations of heparins and synthetic pentasaccharide (Fig. 3). (Note: prothrombin conversion is inhibited by heparin as soon as thrombin dependent FVIII activation becomes rate determining, as in the APTT. FV can be activated by meizothrombin, which is heparin insensitive [25]). Despite the fact that we demonstrated the irrelevance of antiXa action in a series of publications from 1987 onwards [22–
Fig. 4. Stage two: two stage. S. Be´guin and the author, Paris 1985.
24,26–28], up to this day clinical dosage is done on the basis of anti-Xa units. It makes one wonder what (a lifetime spent in) research can in fact be good for. To borrow an argot expression from the French: we might as well have peed in a violin. Anyhow, these studies showed us how useful it could be to investigate the physiological mechanism of thrombin generation by assessing its overall function in the Ôisolated organÕ plasma instead of taking it apart and studying isolated reactions. It also made it painfully clear how tedious it is to determine thrombin generation curves by hand (Fig. 4). Imagine the experiment in Fig. 3: thrombin generation is to be measured for 15 min, so 120 (hand-)numbered tubes, filled with buffer and chromogenic substrate, are waiting in the water bath. In four Eppendorf tubes thrombin generation is started. Every half minute, one person (e.g. Suzette), at exactly 5-s intervals, puts a 10-lL sample from one of the four into a tube with chromogenic substrate. Another person, usually me, stops the chromogenic reaction after precisely 2 min by adding acid. Then the OD is read in the 120 tubes and the thrombin concentration in the subsamples calculated. Together with the filling and the numbering of the tubes the experiment thus takes about half a day. Continuous thrombin generation measurement
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My congenital laziness forced me to find a less wearisome way to get the same information. Already in 1980 Suzette had done experiments in which she added chromogenic thrombin substrates directly to plasma (Fig. 5). The developing thrombin indeed converted the substrate; in fact it converted all the substrate long before thrombin generation was over and only the very beginning of thrombin generation was visualized. In fact it severely disturbed thrombin generation because, at the required concentration, S2238 acts as a thrombin inhibitor of about the strength of a dose of Melagatran. So the thrombinrelated feedback reactions are killed and the relation of the results to physiology was questionable, to say the least. But then it is a venerable tradition in medical science not to ask for relations but to stick with correlations. Spider naevi or the sedimentation rate have been, and still are, useful for recognizing liver cirrhosis or infection. Doctors do not
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Fig. 5. An early continuous chromogenic TG curve. From the notebook of S. Be´guin, Paris 1980.
necessarily care why, as long as it helps in the diagnosis. Similarly, clotting blood (-plasma), can be made to yield signals related to viscosity, tensile strength, specific gravity, turbidity, light dispersion, conductivity, electric capacitance and probably more. Each of them will vary during the clotting process and vary differently when coagulation is abnormal. So they can all be correlated with disease and used for diagnostic purposes. This is the poor doctorÕs substitute to the understanding, which comes from measuring physiological function. Lungs are there to provide oxygen to the blood, so, no matter how impressive a stridor or blue the patient, the situation can be judged quantitatively only by measuring air flow and oxygen saturation; likewise diabetes is better judged from a blood sugar curve than from polyuria and furuncles, etc. We aimed for accurate measurement of thrombin generation (i.e. of the physiological function) as close as possible to what occurs in a patient. So we did not develop OD-measurement with a conventional substrate any further (and we wonder why some people do). If a ÔgoodÕ thrombin substrate cannot be used because it binds too strongly to thrombin (low Km) and is consumed too fast (high kcat), it is natural to look for thrombin substrates that bind loosely (high Km) and are not consumed during the process (low kcat). We reasoned that such substrates
should be available among the stuff shelved as failures by chemists that were looking for good thrombin substrates. So we paid a visit to Serbio (now Stago), where Jean-Luc Martinoli scraped the bottom of his drawers and found SQ68 and some other substrates that were specific for thrombin but with low affinity and slow to be converted. Such substrates leave enough free thrombin for the clotting mechanism to function normally [29] and only a small fraction of the substrate is consumed. Later, Dirk Rijkers, first in the organic chemistry department of Nijmegen University, later in ours, made many more such substrates [30,31]. It should be realized that addition of a substrate always influences the clotting system. Any thrombin substrate will interfere with the action of the physiological antithrombins. The thrombin concentrations measured in the presence of a substrate are therefore always higher than those found in subsampling experiments and by a well-defined factor that depends on the substrate concentration and the Km of the substrate [32] [33]). Some substrates cannot be used because they inhibit, for example, FXa; such effects have to be carefully excluded before a substrate can be accepted as useful. The slow substrates allowed us to do large numbers of thrombin generation curves in a centrifugal analyzer [29]. Together with the team of Vijay Kakkar in London, we thus showed for the first time that thrombin generation is enhanced in venous thrombosis as well as in coronary infarction patients; that it is inhibited by oral anticoagulation and heparins alike and can be used to measure the combined effect of these treatments [34]. Continuous chromogenic measurement made life much easier, but at a price: OD readings require optically clear solutions so clotting cannot be tolerated. Polymerization inhibitors disturb the thrombin generation mechanism [32], so we preferred to remove the fibrinogen with a venom enzyme (see below). Thrombin generation and platelets The necessity to remove fibrinogen in the chromogenic method raised the question of what the influence of that component might be. We realized that, in vivo, the bulk of thrombin is generated in the maces of the fibrin web, to which the platelets stick, under conditions that are very different from the wellstirred solutions dear to the heart of the (bio-)chemist. This opened two new lines of research: (i) Does fibrin–platelet interaction play a role in thrombin generation? (ii) What is the role of diffusion in thrombin generation in clotted plasma? We had started our work on TG in platelet-rich plasma (PRP) already in 1986, in the department of ÔtheÕ platelet specialist in Paris: Jacques Caen. There, in the friendly atmosphere of the laboratory of Claudine Soria, we saw for the first time how platelets strongly boost the effect of minute amounts of tissue factor [35] and how amounts of heparin that inhibit TG in PPP almost entirely, are hardly inhibitory in PRP and only prolong the lag-time [35].
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The year 1987 was decisive for the further developments in that Suzette decided to accept my invitation and continue her work on thrombin generation in Maastricht. Not without some reluctance, however, because she doubted what her place could be, as a biologist amidst our sophisticated biochemistry. At that point my biochemists agreed and it took some time before thrombin generation was accepted as more than of historical interest. Without SuzetteÕs enthusiasm and never failing perseverance we might well have dropped the subject. Apart from her work on heparins she became fascinated by the role of the platelet in thrombin generation. Years earlier, Bevers and Zwaal [7] had shown that platelets, upon activation, provide the procoagulant surface for prothrombinase and tenase formation by scrambling the lipids in their cell membrane. As physiological triggers they had identified thrombin and collagen, together many times more potent than each of them alone. Yet in clotting plasma platelets develop their full procoagulant potential in the absence of collagen. Suzette thought that fibrin might take its place and could indeed prove that it did. The experiment is too amusing not to recall it here. We used the clotting properties of a reptilase-like snake venom enzyme, which clots fibrinogen without any other demonstrable effect on plasma or platelets, in a concentration that clots PPP in 2 min. Carefully prepared PRP takes several minutes to clot after recalcification (t = 0). At t 2 we added so much venom as to obtain clotting at t4. To our surprise thrombin-independent clotting immediately provoked thrombin generation (Fig. 6) [36,37]! We could show that polymerizing fibrin activates the procoagulant capacities of platelets via a mechanism that requires von Willebrand factor, a minimum of sheer and the receptor GP1b. In Bernard–SoulierÕs thrombopathy the phenomenon can be shown to be absent [38]. This solved the old enigma of anomalous prothrombin consumption mentioned in the original description of that disease [39]. A parallel pathway for developing procoagulant activity is the interaction of GPIIb/IIIa with fibrinogen. That is why in GlanzmannÕs thrombopathy TG is defective and why abciximab inhibits thrombin generation [40]. One realizes that the old story of first
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platelets and then thrombin (i.e. the paradigm of primary and secondary hemostasis) urgently requires revision. Because defibrination automatically removes all platelets, the experiments on PRP had to be done by subsampling, which we had somewhat automated by recording sampling and stopping times on a computer via pushbuttons on the pipettes. The results obtained with platelets and antiplatelet drugs reinforced our suspicions that TG might develop into an all round function test of the hemostatic system. It also made it abundantly clear that it never could be used as such, unless we found a form that did not require defibrination and still allowed high throughput. Calibrated automated fluorogenic thrombinography At that moment, in Cambridge, we encountered Manosch Ramjee, who suggested that we should try fluorogenic substrates and use a 96-well plate fluorometer. This proved to be golden advice. The first few experiments we did together in his laboratory. The turbidity caused by the clot did not disturb the measurements in the least, so fibrin could stay in. For a moment we thought that red cells could stay in as well but, although from time to time we got a useful curve, it proved impossible to make TG in whole blood sufficiently reproducible. With platelet-poor plasma reproducible curves could be readily obtained and – once Suzette had found out that one should not shake between readings – PRP worked all right as well. It took a while before we realized that the difficult part was not getting a signal out of clotting plasma but getting thrombin concentrations out of the signal. In our initial enthusiasm we thought that, like in OD measurements, we could simply take the first derivative of fluorescence as a good approximation of the thrombin generation curve; that it only had to be multiplied by an independently determined calibration factor to convert reaction rates in thrombin concentrations [41]. We soon found out that the very nature of fluorescence measurement made it exasperatingly difficult to obtain exact values for the thrombin concentrations. Optical densities may be pretty well linear with the concentration of the product, fluorescence intensities are not; so constant product formation causes a steadily decreasing velocity of fluorescence increase. This effect is reinforced by the consumption of substrate (for 30%) during the experiment. Finally, and most important: the magnitude of a fluorescent signal depends upon the quenching properties of the medium, and plasma samples appeared to differ widely in this respect, even samples of the same person obtained on the same day! In short, the calibration factor necessary to obtain the thrombin concentration from the velocity of fluorescence increase changes from sample to sample and with the level of fluorescence attained. The first derivative of a fluorescence signal can naively been mistaken for a thrombin generation curve but it is not more than its distorted shadow. The solution was continuous individual calibration [42], that is, a fixed enzymatic activity, the calibrator, is added to a parallel sample of plasma, in which no TG is triggered. From
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Fig. 7. A fluorometric TG curve. Upper frame: normal plasma. Lower frame, plasma from a patient treated with acenocoumarol; INR = 3.1. Blue curves, thrombin as a function of time; upper blue curves, no addition; lower blue curves, 15 nM of soluble thrombomodulin. Green curves, fluorescence (AU) caused by the calibrator (i.e. 200 lM a2macroglobulinthrombin). It is seen that OAC diminishes thrombin generation but induces almost complete resistance to thrombomodulin. Experiment of R. Al Dieri.
the resulting trace the calibration factor is determined at all relevant fluorescence levels. Thrombin cannot be used as a calibrator, because its activity rapidly vanishes in plasma. Rob Wagenvoord suggested using the a2macroglobulin-thrombin complex, which indeed proved to serve the purpose perfectly. Peter Giesen developed a computer program to compare the two signals. The result was a method that allows monitoring the exact concentration of thrombin in up to 24 parallel samples in real time. The price to pay is measuring in two wells for each sample (Fig. 7). Finally, after some 15 years we thus had a method that could be used beyond the limited circle of specialized laboratories. Recent developments At present, the most interesting development is precisely this spread of TG determination beyond the coagulation laboratory per se, to pharmacological, epidemiological and clinical surroundings.
In pharmacology it is increasingly recognized that all compounds that inhibit thrombin generation in one way or another have an antithrombotic action. Reportedly ÔnonanticoagulantÕ antithrombotic drugs like dermatansulfate or aggregation inhibitors do not prolong clotting times but they do inhibit TG [43]. In fact it does not matter how TG is inhibited as long as it is inhibited. One therefore can screen for new antithrombotics simply by measuring their effect on thrombin generation. It is an educated guess that any compound that can inhibit TG for 40–60% is an efficient antithrombotic that does not cause bleeding. The rest is pharmacokinetics and the absence of side effects. TG cannot replace thrombosis models but it offers a convenient possibility to zoom in on the interesting compounds in the interesting concentration range and thus diminishes the use of experimental animals. Likewise it can be a guide in dose-finding and monitoring when the drug is tested in humans. Our studies on heparin pharmacodynamics in volunteers showed that the same dose of heparin (unfractionated and low molecular weight alike!) can easily inhibit thrombin generation twice as much in one healthy person than in another (of similar weight) [44]. In fact we saw this with any antithrombotic drug that we tested. TG allows antithrombotic treatment to be tailored to individual needs and we surmize that this could significantly ameliorate clinical results. Standard dosage is a strong selling argument, however, and research in this field is largely industry driven. Another fascinating aspect of TG is that it allows us to quantify the concept of ÔhypercoagulabilityÕ. The normal mean ETP is around 1000 nM min (after correction for the augmenting effect of the added thrombin substrate [29,33]). There is a large and apparently normally distributed spread around the mean (SD 150 nM min). People with TG higher than the average are at a much higher risk of recurrence of venous thrombosis than those below [45]. It remains for the epidemiologists to show this for the normal population. Anyhow, all conditions that increase thrombin generation, create a tendency to venous thrombosis: deficiencies of AT, proteins C and S, FVLeiden, use of oral contraceptives (see [46] for a review). The link between TG and arterial thrombosis is less clear but far from being inconceivable. After all, anticoagulant prophylaxis does decrease the recurrence of infarction (see [46] for references). Possibly the risk of arterial thrombosis is more clearly reflected by thrombin generation in the presence of platelets. In clinical research TG is being applied to a number of problems that have been resistant to other approaches. It has been shown to be an exquisite means to demonstrate APC and thrombomodulin resistance (e.g. due to oral contraceptives [47] or the lupus anticoagulant) [48]. Several studies suggest that bleeding tendency in hemophilia and other congenital clotting factor deficiencies is better indicated by TG than by factor levels [49,50]; and possibly better by TG in PRP than in PPP [51]. TG reflects the effect of inhibitor bypassing therapy in hemophiliacs [52,53] and has been used as a guidance for prophylaxis during operations [54]. 2007 International Society on Thrombosis and Haemostasis
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As mentioned above, the main clinical application probably will be the control of antithrombotic therapy, both for new and existing drugs and maybe also in preoperative screening. The future The data available at the moment support the Ôfirst law of hemostasis and thrombosisÕ that I formulated some 10 years ago (and that my friends mockingly call ÔHemkerÕs lawÕ): ÔThe more thrombin the more thrombosis but the less bleeding, the less thrombin the less thrombosis but the more bleedingÕ. If it is to be refuted, it will be by TG. As long as it is not, TG will be the method par excellence to assess the role of the blood in bleeding and thrombosis. To end with a personal note: where purified enzymes reign, plasma is considered as a soup. When molecular biology is the word of the day chemical physiology stands no chance of being taken seriously. For some 15 years Suzette Be´guin and myself have worked on thrombin generation in surroundings where it was tolerated but neither esteemed nor supported; rather considered as a hobby of the elderly with nostalgia for the times when clotting was still like cooking. One can only be grateful when, in the Indian summer of a career, this hobby now appears as being of some utility. Disclosure of Conflict of Interests The author states that he has no conflict of interest. References 1 Hemker HC. The initiation phase – a review of old (clotting-) times. Thromb Haemost 2007; 98: 20–3. 2 Hemker HC, Veltkamp JJ, Hensen A, Loeliger EA. Nature of prothrombin biosynthesis: preprothrombinaemia in vitamin K-deficiency. Nature 1963; 200: 589–90. 3 Hemker HC, Muller AD, Loeliger EA. Two types of prothrombin in vitamin K deficiency. Thromb Diath Haemorrh 1970; 23: 633–7. 4 Hemker HC, Hemker PW, Torren Kvd, Devilee PP, Hermens WT, Loeliger EA. The evaluation of the two-stage prothrombin assay. Thromb Diath Haemorrh 1971; 25: 545–54. 5 Hemker HC, Esnouf MP, Hemker PW, Swart AC, Macfarlane RG. Formation of prothrombin converting activity. Nature 1967; 215: 248– 51. 6 Hemker HC, Kahn MJ. Reaction sequence of blood coagulation. Nature 1967; 215: 1201–2. 7 Bevers EM, Comfurius P, van Rijn JL, Hemker HC, Zwaal RF. Generation of prothrombin-converting activity and the exposure of phosphatidylserine at the outer surface of platelets. Eur J Biochem 1982; 122: 429–36. 8 Rosing J, Tans G, Govers-Riemslag JW, Zwaal RF, Hemker HC. The role of phospholipids and factor Va in the prothrombinase complex. J Biol Chem 1980; 255: 274–83. 9 van Dieijen G, Tans G, Rosing J, Hemker HC. The role of phospholipid and factor VIIIa in the activation of bovine factor X. J Biol Chem 1981; 256: 3433–42. 10 Reutelingsperger CP, Hornstra G, Hemker HC. Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord. Eur J Biochem 1985; 151: 625–9.
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11 Billy D, Briede J, Heemskerk JW, Hemker HC, Lindhout T. Prothrombin conversion under flow conditions by prothrombinase assembled on adherent platelets. Blood Coagul Fibrinolysis 1997; 8: 168–74. 12 Billy D, Willems GM, Hemker HC, Lindhout T. Prothrombin contributes to the assembly of the factor Va–factor Xa complex at phosphatidylserine-containing phospholipid membranes. J Biol Chem 1995; 270: 26883–9. 13 Morris DP, Soute BA, Vermeer C, Stafford DW. Characterization of the purified vitamin K-dependent gamma-glutamyl carboxylase. J Biol Chem 1993; 268: 8735–42. 14 Cuypers PA, Corsel JW, Janssen MP, Kop JM, Hermens WT, Hemker HC. The adsorption of prothrombin to phosphatidylserine multilayers quantitated by ellipsometry. J Biol Chem 1983; 258: 2426–31. 15 Hemker HC. Critical perceptions on problem-based learning. Adv Health Sci Educ Theory Pract 1998; 3: 71–6. 16 Josso F, Prou-Wartelle O. Interaction of tissue factor and factor VII at the earliest phase of coagulation. Thromb Diath Haemorr Suppl 1965; 17: 35–44. 17 Jensen AH, Beguin S, Josso F. Factor V and VIII activation ‘‘in vivo’’ during bleeding. Evidence of thrombin formation at the early stage of hemostasis. Pathol Biol (Paris) 1976; 24 (Suppl.): 6–10. 18 Beguin S, Goutner A, Josso F. Tissue factor activity of rabbit peritoneal macrophages: influence of immune stimulation. Ann Immunol (Paris) 1977; 128C: 785–98. 19 Josso F, Rio Y, Beguin S. A new variant of human prothrombin: prothrombin Metz, demonstration in a family showing double heterozygosity for congenital hypoprothrombinemia and dysprothrombinemia. Haemostasis 1982; 12: 309–16. 20 Yin ET, Wessler S, Stoll PJ. Biological properties of the naturally occurring plasma inhibitor to activated factor X. J Biol Chem 1971; 246: 3703–11. 21 Marciniak E. Factor-Xa inactivation by antithrombin. 3. Evidence for biological stabilization of factor Xa by factor V–phospholipid complex. Br J Haematol 1973; 24: 391–400. 22 Hemker HC, Beguin S. The generation of thrombin in whole plasma. Biochemical possibilities and physiological realities. Verh K Acad Geneeskd Belg 1985; 47: 321–39. 23 Beguin S, Lindhout T, Hemker HC. The mode of action of heparin in plasma. Thromb Haemost 1988; 60: 457–62. 24 Beguin S, Hemker HC. Mode of action of enoxaparin in plasma. Acta Chir Scand Suppl 1990; 556: 51–6. 25 Tans G, Janssen-Claessen T, Hemker HC, Zwaal RF, Rosing J. Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma. J Biol Chem 1991; 266: 21864–73. 26 Beguin S, Wielders S, Lormeau JC, Hemker HC. The mode of action of CY216 and CY222 in plasma. Thromb Haemost 1992; 67: 33– 41. 27 Hemker HC, Beguin S. Mode of action of unfractionated and low molecular weight heparins on the generation of thrombin in plasma. Haemostasis 1990; 20: 81–92. 28 Beguin S, Mardiguian J, Lindhout T, Hemker HC. The mode of action of low molecular weight heparin preparation (PK10169) and two of its major components on thrombin generation in plasma. Thromb Haemost 1989; 61: 30–4. 29 Hemker HC, Wielders S, Kessels H, Beguin S. Continuous registration of thrombin generation in plasma, its use for the determination of the thrombin potential. Thromb Haemost 1993; 70: 617–24. 30 Rijkers DT, Wielders SJ, Tesser GI, Hemker HC. Design and synthesis of thrombin substrates with modified kinetic parameters. Thromb Res 1995; 79: 491–9. 31 Rijkers DT, Hemker HC, Tesser GI. Synthesis of peptide p-nitroanilides mimicking fibrinogen- and hirudin- binding to thrombin. Design of slow reacting thrombin substrates. Int J Pept Protein Res 1996; 48: 182–93.
226 H. C. Hemker 32 Lau A, Berry LR, Mitchell LG, Chan AK. Effect of substrate and fibrin polymerization inhibitor on determination of plasma thrombin generation in vitro. Thromb Res 2007; 119: 667–77. 33 Hemker HC, de Smedt E. Caution in the interpretation of continuous thrombin generation assays. A rebuttal. J Thromb Haemost 2007; 5: 1085–7. 34 Wielders S, Mukherjee M, Michiels J, Rijkers DT, Cambus JP, Knebel RW, Kakkar V, Hemker HC, Beguin S. The routine determination of the endogenous thrombin potential, first results in different forms of hyper- and hypocoagulability. Thromb Haemost 1997; 77: 629–36. 35 Beguin S, Lindhout T, Hemker HC. The effect of trace amounts of tissue factor on thrombin generation in platelet rich plasma, its inhibition by heparin. Thromb Haemost 1989; 61: 25–9. 36 Kumar R, Beguin S, Hemker HC. The effect of fibrin clots and clotbound thrombin on the development of platelet procoagulant activity. Thromb Haemost 1995; 74: 962–8. 37 Beguin S, Kumar R, Keularts I, Seligsohn U, Coller BS, Hemker HC. Fibrin-dependent platelet procoagulant activity requires GPIb receptors and von Willebrand factor [In Process Citation]. Blood 1999; 93: 564–70. 38 Beguin S, Keularts I, Al Dieri R, Bellucci S, Caen J, Hemker HC. Fibrin polymerization is crucial for thrombin generation in plateletrich plasma in a VWF-GPIb-dependent process, defective in Bernard– Soulier syndrome. J Thromb Haemost 2004; 2: 170–6. 39 Bernard J, Soulier J-P. Sur une nouvelle varie´te´ de dystrophy thrombocytaire he´morrhagipare conge´nitale. Semin Hop PAris 1948; 24: 3217–9. 40 Reverter JC, Beguin S, Kessels H, Kumar R, Hemker HC, Coller BS. Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody. Potential implications for the effect of c7E3 Fab treatment on acute thrombosis and ‘‘clinical restenosis’’. J Clin Invest 1996; 98: 863–74. 41 Hemker HC, Giesen PL, Ramjee M, Wagenvoord R, Beguin S. The thrombogram: monitoring thrombin generation in platelet-rich plasma. Thromb Haemost 2000; 83: 589–91. 42 Hemker HC, Giesen P, Al Dieri R, Regnault V, De Smedt E, Wagenvoord R, Lecompte T, Beguin S. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003; 33: 4–15. 43 Beguin SS, Dol F, Hemker HC. Influence of lactobionic acid on the kinetics of thrombin in human plasma. Semin Thromb Hemost 1991; 17: 126–8.
44 Al Dieri R, Alban S, Beguin S, Hemker HC. Fixed dosage of lowmolecular-weight heparins causes large individual variation in coagulability, only partly correlated to body weight. J Thromb Haemost 2006; 4: 83–9. 45 Hron G, Kollars M, Binder BR, Eichinger S, Kyrle PA. Identification of patients at low risk for recurrent venous thromboembolism by measuring thrombin generation. JAMA 2006; 296: 397–402. 46 Hemker HC, Al Dieri R, Beguin S. Thrombin generation assays: accruing clinical relevance. Curr Opin Hematol 2004; 11: 170–5. 47 Nicolaes GA, Thomassen MC, Tans G, Rosing J, Hemker HC. Effect of activated protein C on thrombin generation and on the thrombin potential in plasma of normal and APC-resistant individuals. Blood Coagul Fibrinolysis 1997; 8: 28–38. 48 Lecompte T, Wahl D, Perret-Guillaume C, Hemker HC, Lacolley P, Regnault V. Hypercoagulability resulting from opposite effects of lupus anticoagulants is associated strongly with thrombotic risk. Haematologica 2007; 92: 714–5. 49 Al Dieri R, Peyvandi F, Santagostino E, Giansily M, Mannucci PM, Schved JF, Beguin S, Hemker HC. The thrombogram in rare inherited coagulation disorders: its relation to clinical bleeding. Thromb Haemost 2002; 88: 576–82. 50 Dargaud Y, Beguin S, Lienhart A, Al Dieri R, Trzeciak C, Bordet JC, Hemker HC, Negrier C. Evaluation of thrombin generating capacity in plasma from patients with haemophilia A and B. Thromb Haemost 2005; 93: 475–80. 51 Siegemund T, Petros S, Siegemund A, Scholz U, Engelmann L. Thrombin generation in severe haemophilia A and B: the endogenous thrombin potential in platelet-rich plasma. Thromb Haemost 2003; 90: 781–6. 52 Hedner U. Recombinant factor VIIa (NovoSeven) as a hemostatic agent. Dis Mon 2003; 49: 39–48. 53 Turecek PL, Varadi K, Keil B, Negrier C, Berntorp E, Astermark J, Bordet JC, Morfini M, Linari S, Schwarz HP. Factor VIII inhibitorbypassing agents act by inducing thrombin generation and can be monitored by a thrombin generation assay. Pathophysiol Haemost Thromb 2003; 33: 16–22. 54 Dargaud Y, Lienhart A, Meunier S, Hequet O, Chavanne H, Chamouard V, Marin S, Negrier C. Major surgery in a severe haemophilia A patient with high titre inhibitor: use of the thrombin generation test in the therapeutic decision. Haemophilia 2005; 11: 552–8.
2007 International Society on Thrombosis and Haemostasis
