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LMWH – New mechanisms of action
Thrombosis Research (2009) 123 Suppl. 3, S1–S4
intl.elsevierhealth.com/journals/thre
LMWH
New mechanisms of action
J. Harenberg* Clinical Pharmacology Mannheim, Faculty of Medicine Mannheim, Ruprecht-Karls-University Heidelberg, Germany
Biochemical aspects In recent years, considerable interest has arisen in the new anticoagulant and non-anticoagulant actions of heparins and other glycosaminoglycans (GAGs) [1]. In addition, oral direct factor (F) Xa inhibitors are being developed as an alternative treatment for thromboembolic disorders. Great progress has been made on the enzymatic synthesis of GAG heparin. Heparin and its low molecular weight heparin (LMWH) derivatives, are acidic polysaccharide members of a family of biomacromolecules called GAGs. Heparin and the related heparan sulfate (HS) are biosynthesized in the Golgi apparatus of eukaryotic cells. Heparin is a polycomponent drug that is currently prepared for clinical use by extraction from animal tissues. A heparin pentasaccharide, fondaparinux, has also been prepared through chemical synthesis for use as a homogenous anticoagulant drug. Recent enabling technologies suggest that it may now be possible to synthesize heparin and its derivatives enzymatically. Moreover, new technologies including advances in synthetic carbohydrate synthesis, enzyme-based GAG synthesis, rapid on-line structural analysis, and microarray/ microfluidic technologies might be applied to the enzymatic synthesis of heparins with defined structures and exhibiting selected activities [2]. The advent of these new technologies also makes it possible to consider the construction of artificial Golgi to increase our understanding of * Correspondence: J. Harenberg. Clinical Pharmacology Mannheim, Faculty of Medicine Mannheim, Ruprecht-Karls-University Heidelberg, Maybachstrasse 14, D-68169 Mannheim, Germany. Tel.: +49 621 383 9623; fax: +49 621 383 9622. E-mail address: [email protected] (J. Harenberg).
the cellular control of GAG biosyntheses in this organelle. A new type of LMWH has been developed by a depolymerization process involving the cleavage of glycosidic bonds, leading to natural terminal reducing end residues, mainly represented by N-sulfated glucosamine. Natural uronic acids, especially the 2-O-sulfate iduronic acid, are also present as reducing residues. With respect to the original heparin, this LMWH is characterized by a lower number of nonsulfated uronic acid residues, and as a consequence, by a lower degree of structural heterogeneity than LMWHs prepared with other procedures [3]. The structural differentiation of LMWHs is achieved by bidimensional NMR spectroscopy. Individual LMWHs exhibit distinct pharmacological and biochemical profiles because of manufacturing differences. Correlation of biological properties with particular structural motifs is a major challenge in the design of new LMWHs as well as in the development of generic versions of proprietary LMWHs. Structural characterization of three commercially available LMWHs (enoxaparin, dalteparin, and tinzaparin) showed distinct molecular profiles [4]. The variability of heparins and LMWHs has been characterised by typical disaccharide sequences, sulfation degree, and biological activities of unfractionated heparins (UFHs) and LMWHs obtained by different depolymerization processes. The heterogeneity of different LMWHs stems from different manufacturing processes and particular specifications of pharmacopoeias. Most of the variability is derived from the parent UFH. In contrast, fingerprint groups and residues are specific to the depolymerization process and their extent can be roughly controlled through the process parameters [5].
0049-3848/ $ – see front matter © 2009 Elsevier Ltd. All rights reserved.
S2 Monitoring of anticoagulant effects The determination of antithrombin-dependent FXa inhibitors as well as of thrombin inhibitors can now be performed also using the method of prothrombin-induced clotting time (PiCT). At present, this is the only method that measures the effects of all of these inhibitors, in contrast to the prothrombin time, activated partial thromboplastin time (aPTT), Heptest, ecarin clotting time, and the chromogenic assays. The antithrombin-dependent direct FXa inhibitors fondaparinux and idraparinux were compared with the LMWH dalteparin on PiCT, aPTT, Heptest, and chromogenic anti-FXa assays in pooled human normal plasma samples. Fondaparinux and idraparinux influenced the PiCT, Heptest, and chromogenic FXa assays in a dose-dependent manner, in contrast to the aPTT [6]. PiCT is a suitable assay to determine the anticoagulant effects of these two new FXa inhibitors in patients receiving treatment with these compounds. The thrombin-generating capacity of blood and plasma improved the diagnostics of a hyper- or hypocoagulable state. The first attempt to solve this problem was the method of endogenous thrombin potential (ETP) by Hemker. In ETP, activators and a chromogenic substrate are added to diluted plasma samples and the thrombin generation is measured. By analysis of acquired data, three characteristics of ETP are seen: lag phase, peak thrombin, and velocity index. ETP is not suited for exact determination of maximum activated thrombin. Therefore, a new method was developed: the thrombin generation assay (THROGA). With the use of THROGA, the maximum generated thrombin in a blood or plasma sample can be measured easily. The background of the method is the addition of a certain amount of recombinant hirudin (r-hirudin) to the blood or plasma sample. After activation, the generated thrombin is bound quantitatively and neutralized by r-hirudin so that at the end of the activation phase the amount of generated thrombin can be determined easily and exactly by measurement of residual r-hirudin in the sample [7]. Heparin and inflammation Emerging links between thrombosis, angiogenesis, and inflammation are currently of high interest for heparin and heparin derivatives [8]. The key reason behind the success of heparin in thrombosis and beyond is its polypharmacological sites of action for the prevention and treatment of multifactorial diseases that will only benefit
J. Harenberg slightly from agents based on a single pharmacological mechanism. Thromboembolic disorders are driven by hypercoagulable, hyperactive platelet, proinflammatory, endothelial dysfunction, and proangiogenesis states. Heparin can effectively modulate all of those multifactorial components, as well as the interface among those components. In this context, the selectins play a major role as link between the anticoagulant and non-anticoagulant actions of heparins. P- and L-selectins are adhesion receptors that participate in inflammation and tumor cell metastasis. The anti-inflammatory and antimetastatic activities of heparins have been related partly to their ability to interact with P- and L-selectin. The recent findings that various heparins differ in antimetastatic activity were explained by differences in their Pand L-selectin binding ability. To obtain data to illustrate the binding characteristics, the binding kinetics and affinity of the two LMWHs, enoxaparin and nadroparin, and of the unfractionated heparin to P- and L-selectin were assessed using a quartz crystal microbalance biosensor. The differences are caused by a higher association rate compared with that of the LMWHs [9]. These data support recent findings of antimetastatic activities, but illustrate that the intrinsic selectin binding does not entirely reflect the antimetastatic activities in vivo. Heparin derivatives and cancer Heparin was demonstrated in several retrospective and prospective clinical trials to have an effect on cancer survival. Experimental evidence from animal models consistently demonstrates that heparin is an efficient inhibitor of metastasis. To clarify the mechanism of heparin antimetastatic activity, several biological effects are being investigated. Cancer progression and metastasis are associated with enhanced expression of heparanase, which is inhibited efficiently by heparin. Heparin is also a potent inhibitor of selectin-mediated interactions. P- and L-selectin were shown to contribute to the early stages of metastasis, which is associated with platelet tumor cell thrombi formation. To delineate the biological activities of heparin contributing to metastasis inhibition, modified heparins with specific activities were evaluated. Low anticoagulant heparin preparations still inhibited metastasis efficiently, indicating that anticoagulation is not a necessary component for heparin attenuation of metastasis. Modified heparins characterized for heparanase inhibitory activity are also potential
LMWH
New mechanisms of action
inhibitors of selectins [10]. Selectin inhibition is a clear component of heparin inhibition of metastasis. The contribution to selectin or heparanase inhibition by heparin can provide evidence about its antimetastatic activity. O-sulfated bacterial polysaccharides with low anticoagulant activity on inhibition of metastases have been described [11]. Heparin-like polysaccharides possess the capacity to inhibit cancer cell proliferation, angiogenesis, heparanase-mediated cancer cell invasion, and cancer cell adhesion to vascular endothelia via adhesion receptors, such as selectins. The clinical applicability of the antitumor effect of such polysaccharides, however, is compromised by their anticoagulant activity. Chemically O-sulfated and N,O-sulfated bacterial polysaccharide (capsular polysaccharide from E. coli K5 [K5PS]) species inhibited metastasis of mouse B16-BL6 melanoma cells and human MDA-MB-231 breast cancer cells in two in vivo models. O-sulfated K5PS was a potent inhibitor of metastasis. Reducing the molecular weight of the polysaccharide, however, resulted in lower antimetastatic capacity. Furthermore, O-sulfated K5PS efficiently inhibited the invasion of B16-BL6 cells through Matrigel and also inhibited the in vitro activity of heparanase. Moreover, treatment with O-sulfated K5PS lowered the ability of B16-BL6 cells to adhere to endothelial cells, intercellular adhesion molecule-1, and P-selectin, but not to E-selectin. Importantly, O-sulfated K5PSs were largely devoid of anticoagulant activity. These findings indicate that O-sulfated K5PS polysaccharide should be considered as a potential antimetastatic agent. PI-88 is another new inhibitor of tumor growth and metastasis, expected to commence phase III clinical evaluation as an adjuvant therapy for post-resection hepatocellular carcinoma [12]. Its anticancer properties are attributed to inhibition of angiogenesis via antagonism of the interactions of angiogenic growth factors and their receptors with HS. It is also a potent inhibitor of heparanase, an enzyme that plays a key role in both metastasis and angiogenesis. A series of PI-88 analogs have been prepared with enhanced chemical and biological properties. The new compounds consist of single, defined oligosaccharides with specific modifications designed to improve their pharmacokinetic properties. These analogs all inhibit heparanase and bind to the angiogenic fibroblast growth factor 1 (FGF-1), FGF-2, and vascular endothelial growth factor with similar affinity to PI-88. However, compared with PI-88, some of the newly designed compounds are more
S3 potent inhibitors of growth factor-induced endothelial cell proliferation and of endothelial tube formation on Matrigel. Representative compounds were also tested for antiangiogenic activity in vivo and were found to reduce significantly blood vessel formation. Moreover, the pharmacokinetic profile of several analogs was also improved, as evidenced primarily by lower clearance in comparison with PI-88. The current data support the development of HS mimetics as potent antiangiogenic anticancer agents. Future perspectives Indirect and oral direct factor Xa and oral direct factor IIa inhibitors with improved pharmacological profiles compared to heparins and vitamin K antagonists are currently in clinical development. The development focuses on the indirect antithrombin-dependent pentasaccharide derivatives, and the oral direct inhibitors to factor Xa (rivaroxaban and apixaban) and IIa (dabigatran). If these compounds will be available for long-term use for patients with a life-long need of oral anticoagulation, the non-anticoagulant actions of heparins and their derivatives will become more important compared to the current use for anticoagulation. The present overview gives some insight into this development. Conflicts of interest: The author has no conflicts of interest to declare. References [1] Harenberg J, Casu B. Preface. Semin Thromb Hemost 2007;33:449 53. [2] Linhardt RJ, Dordick JS, Deangelis PL, Liu J. Enzymatic synthesis of glycosaminoglycan heparin. Semin Thromb Hemost 2007;33:453 65. [3] Vismara E, Pierini M, Guglieri S, Liverani L, Mascellani G, Torri G. Structural modification induced in heparin by a Fenton-type depolymerization process. Semin Thromb Hemost 2007;33:466 77. [4] Guerrini M, Guglieri S, Naggi A, Sasisekharan R, Torri G. Low molecular weight heparins: structural differentiation by bidimensional nuclear magnetic resonance spectroscopy. Semin Thromb Hemost 2007;33: 478 87. [5] Bianchini P, Liverani L, Spelta F, Mascellani G, Parma B.Variability of heparins and heterogeneity of low molecular weight heparins. Semin Thromb Hemost 2007;33:496 502. [6] Harenberg J, Giese C, Hagedorn A, Traeger I, Fenyvesi T. Determination of antithrombin-dependent factor Xa inhibitors by prothrombin-induced clotting time. Semin Thromb Hemost 2007;33:503 7. [7] Nowak G, Lange U, Wiesenburg A, Bucha E. Measurement of maximum thrombin generation capacity in blood and
S4 plasma using the thrombin generation assay (THROGA). Semin Thromb Hemost 2007;33:508 14. [8] Mousa SA. Heparin, low molecular weight heparin, and derivatives in thrombosis, angiogenesis, and inflammation: emerging links. Semin Thromb Hemost 2007;33:524 33. [9] Simonis D, Christ K, Alban S, Bendas G. Affinity and kinetics of different heparins binding to P- and L-selectin. Semin Thromb Hemost 2007;33:534 9. [10] Borsig L. Antimetastatic activities of modified heparins:
J. Harenberg selectin inhibition by heparin attenuates metastasis. Semin Thromb Hemost 2007;33:540 6. [11] Borgenstr¨ om M, W¨ arri A, Hiilesvuo K, K¨ ak¨ onen R, K¨ ak¨ onen S, Nissinen L, et al. O-sulfated bacterial polysaccharides with low anticoagulant activity inhibit metastasis. Semin Thromb Hemost 2007;33:547 56. [12] Ferro V, Dredge K, Liu L, Hammond E, Bytheway I, Li C, et al. PI-88 and novel heparan sulfate mimetics inhibit angiogenesis. Semin Thromb Hemost 2007;33:557 68.
intl.elsevierhealth.com/journals/thre
LMWH
New mechanisms of action
J. Harenberg* Clinical Pharmacology Mannheim, Faculty of Medicine Mannheim, Ruprecht-Karls-University Heidelberg, Germany
Biochemical aspects In recent years, considerable interest has arisen in the new anticoagulant and non-anticoagulant actions of heparins and other glycosaminoglycans (GAGs) [1]. In addition, oral direct factor (F) Xa inhibitors are being developed as an alternative treatment for thromboembolic disorders. Great progress has been made on the enzymatic synthesis of GAG heparin. Heparin and its low molecular weight heparin (LMWH) derivatives, are acidic polysaccharide members of a family of biomacromolecules called GAGs. Heparin and the related heparan sulfate (HS) are biosynthesized in the Golgi apparatus of eukaryotic cells. Heparin is a polycomponent drug that is currently prepared for clinical use by extraction from animal tissues. A heparin pentasaccharide, fondaparinux, has also been prepared through chemical synthesis for use as a homogenous anticoagulant drug. Recent enabling technologies suggest that it may now be possible to synthesize heparin and its derivatives enzymatically. Moreover, new technologies including advances in synthetic carbohydrate synthesis, enzyme-based GAG synthesis, rapid on-line structural analysis, and microarray/ microfluidic technologies might be applied to the enzymatic synthesis of heparins with defined structures and exhibiting selected activities [2]. The advent of these new technologies also makes it possible to consider the construction of artificial Golgi to increase our understanding of * Correspondence: J. Harenberg. Clinical Pharmacology Mannheim, Faculty of Medicine Mannheim, Ruprecht-Karls-University Heidelberg, Maybachstrasse 14, D-68169 Mannheim, Germany. Tel.: +49 621 383 9623; fax: +49 621 383 9622. E-mail address: [email protected] (J. Harenberg).
the cellular control of GAG biosyntheses in this organelle. A new type of LMWH has been developed by a depolymerization process involving the cleavage of glycosidic bonds, leading to natural terminal reducing end residues, mainly represented by N-sulfated glucosamine. Natural uronic acids, especially the 2-O-sulfate iduronic acid, are also present as reducing residues. With respect to the original heparin, this LMWH is characterized by a lower number of nonsulfated uronic acid residues, and as a consequence, by a lower degree of structural heterogeneity than LMWHs prepared with other procedures [3]. The structural differentiation of LMWHs is achieved by bidimensional NMR spectroscopy. Individual LMWHs exhibit distinct pharmacological and biochemical profiles because of manufacturing differences. Correlation of biological properties with particular structural motifs is a major challenge in the design of new LMWHs as well as in the development of generic versions of proprietary LMWHs. Structural characterization of three commercially available LMWHs (enoxaparin, dalteparin, and tinzaparin) showed distinct molecular profiles [4]. The variability of heparins and LMWHs has been characterised by typical disaccharide sequences, sulfation degree, and biological activities of unfractionated heparins (UFHs) and LMWHs obtained by different depolymerization processes. The heterogeneity of different LMWHs stems from different manufacturing processes and particular specifications of pharmacopoeias. Most of the variability is derived from the parent UFH. In contrast, fingerprint groups and residues are specific to the depolymerization process and their extent can be roughly controlled through the process parameters [5].
0049-3848/ $ – see front matter © 2009 Elsevier Ltd. All rights reserved.
S2 Monitoring of anticoagulant effects The determination of antithrombin-dependent FXa inhibitors as well as of thrombin inhibitors can now be performed also using the method of prothrombin-induced clotting time (PiCT). At present, this is the only method that measures the effects of all of these inhibitors, in contrast to the prothrombin time, activated partial thromboplastin time (aPTT), Heptest, ecarin clotting time, and the chromogenic assays. The antithrombin-dependent direct FXa inhibitors fondaparinux and idraparinux were compared with the LMWH dalteparin on PiCT, aPTT, Heptest, and chromogenic anti-FXa assays in pooled human normal plasma samples. Fondaparinux and idraparinux influenced the PiCT, Heptest, and chromogenic FXa assays in a dose-dependent manner, in contrast to the aPTT [6]. PiCT is a suitable assay to determine the anticoagulant effects of these two new FXa inhibitors in patients receiving treatment with these compounds. The thrombin-generating capacity of blood and plasma improved the diagnostics of a hyper- or hypocoagulable state. The first attempt to solve this problem was the method of endogenous thrombin potential (ETP) by Hemker. In ETP, activators and a chromogenic substrate are added to diluted plasma samples and the thrombin generation is measured. By analysis of acquired data, three characteristics of ETP are seen: lag phase, peak thrombin, and velocity index. ETP is not suited for exact determination of maximum activated thrombin. Therefore, a new method was developed: the thrombin generation assay (THROGA). With the use of THROGA, the maximum generated thrombin in a blood or plasma sample can be measured easily. The background of the method is the addition of a certain amount of recombinant hirudin (r-hirudin) to the blood or plasma sample. After activation, the generated thrombin is bound quantitatively and neutralized by r-hirudin so that at the end of the activation phase the amount of generated thrombin can be determined easily and exactly by measurement of residual r-hirudin in the sample [7]. Heparin and inflammation Emerging links between thrombosis, angiogenesis, and inflammation are currently of high interest for heparin and heparin derivatives [8]. The key reason behind the success of heparin in thrombosis and beyond is its polypharmacological sites of action for the prevention and treatment of multifactorial diseases that will only benefit
J. Harenberg slightly from agents based on a single pharmacological mechanism. Thromboembolic disorders are driven by hypercoagulable, hyperactive platelet, proinflammatory, endothelial dysfunction, and proangiogenesis states. Heparin can effectively modulate all of those multifactorial components, as well as the interface among those components. In this context, the selectins play a major role as link between the anticoagulant and non-anticoagulant actions of heparins. P- and L-selectins are adhesion receptors that participate in inflammation and tumor cell metastasis. The anti-inflammatory and antimetastatic activities of heparins have been related partly to their ability to interact with P- and L-selectin. The recent findings that various heparins differ in antimetastatic activity were explained by differences in their Pand L-selectin binding ability. To obtain data to illustrate the binding characteristics, the binding kinetics and affinity of the two LMWHs, enoxaparin and nadroparin, and of the unfractionated heparin to P- and L-selectin were assessed using a quartz crystal microbalance biosensor. The differences are caused by a higher association rate compared with that of the LMWHs [9]. These data support recent findings of antimetastatic activities, but illustrate that the intrinsic selectin binding does not entirely reflect the antimetastatic activities in vivo. Heparin derivatives and cancer Heparin was demonstrated in several retrospective and prospective clinical trials to have an effect on cancer survival. Experimental evidence from animal models consistently demonstrates that heparin is an efficient inhibitor of metastasis. To clarify the mechanism of heparin antimetastatic activity, several biological effects are being investigated. Cancer progression and metastasis are associated with enhanced expression of heparanase, which is inhibited efficiently by heparin. Heparin is also a potent inhibitor of selectin-mediated interactions. P- and L-selectin were shown to contribute to the early stages of metastasis, which is associated with platelet tumor cell thrombi formation. To delineate the biological activities of heparin contributing to metastasis inhibition, modified heparins with specific activities were evaluated. Low anticoagulant heparin preparations still inhibited metastasis efficiently, indicating that anticoagulation is not a necessary component for heparin attenuation of metastasis. Modified heparins characterized for heparanase inhibitory activity are also potential
LMWH
New mechanisms of action
inhibitors of selectins [10]. Selectin inhibition is a clear component of heparin inhibition of metastasis. The contribution to selectin or heparanase inhibition by heparin can provide evidence about its antimetastatic activity. O-sulfated bacterial polysaccharides with low anticoagulant activity on inhibition of metastases have been described [11]. Heparin-like polysaccharides possess the capacity to inhibit cancer cell proliferation, angiogenesis, heparanase-mediated cancer cell invasion, and cancer cell adhesion to vascular endothelia via adhesion receptors, such as selectins. The clinical applicability of the antitumor effect of such polysaccharides, however, is compromised by their anticoagulant activity. Chemically O-sulfated and N,O-sulfated bacterial polysaccharide (capsular polysaccharide from E. coli K5 [K5PS]) species inhibited metastasis of mouse B16-BL6 melanoma cells and human MDA-MB-231 breast cancer cells in two in vivo models. O-sulfated K5PS was a potent inhibitor of metastasis. Reducing the molecular weight of the polysaccharide, however, resulted in lower antimetastatic capacity. Furthermore, O-sulfated K5PS efficiently inhibited the invasion of B16-BL6 cells through Matrigel and also inhibited the in vitro activity of heparanase. Moreover, treatment with O-sulfated K5PS lowered the ability of B16-BL6 cells to adhere to endothelial cells, intercellular adhesion molecule-1, and P-selectin, but not to E-selectin. Importantly, O-sulfated K5PSs were largely devoid of anticoagulant activity. These findings indicate that O-sulfated K5PS polysaccharide should be considered as a potential antimetastatic agent. PI-88 is another new inhibitor of tumor growth and metastasis, expected to commence phase III clinical evaluation as an adjuvant therapy for post-resection hepatocellular carcinoma [12]. Its anticancer properties are attributed to inhibition of angiogenesis via antagonism of the interactions of angiogenic growth factors and their receptors with HS. It is also a potent inhibitor of heparanase, an enzyme that plays a key role in both metastasis and angiogenesis. A series of PI-88 analogs have been prepared with enhanced chemical and biological properties. The new compounds consist of single, defined oligosaccharides with specific modifications designed to improve their pharmacokinetic properties. These analogs all inhibit heparanase and bind to the angiogenic fibroblast growth factor 1 (FGF-1), FGF-2, and vascular endothelial growth factor with similar affinity to PI-88. However, compared with PI-88, some of the newly designed compounds are more
S3 potent inhibitors of growth factor-induced endothelial cell proliferation and of endothelial tube formation on Matrigel. Representative compounds were also tested for antiangiogenic activity in vivo and were found to reduce significantly blood vessel formation. Moreover, the pharmacokinetic profile of several analogs was also improved, as evidenced primarily by lower clearance in comparison with PI-88. The current data support the development of HS mimetics as potent antiangiogenic anticancer agents. Future perspectives Indirect and oral direct factor Xa and oral direct factor IIa inhibitors with improved pharmacological profiles compared to heparins and vitamin K antagonists are currently in clinical development. The development focuses on the indirect antithrombin-dependent pentasaccharide derivatives, and the oral direct inhibitors to factor Xa (rivaroxaban and apixaban) and IIa (dabigatran). If these compounds will be available for long-term use for patients with a life-long need of oral anticoagulation, the non-anticoagulant actions of heparins and their derivatives will become more important compared to the current use for anticoagulation. The present overview gives some insight into this development. Conflicts of interest: The author has no conflicts of interest to declare. References [1] Harenberg J, Casu B. Preface. Semin Thromb Hemost 2007;33:449 53. [2] Linhardt RJ, Dordick JS, Deangelis PL, Liu J. Enzymatic synthesis of glycosaminoglycan heparin. Semin Thromb Hemost 2007;33:453 65. [3] Vismara E, Pierini M, Guglieri S, Liverani L, Mascellani G, Torri G. Structural modification induced in heparin by a Fenton-type depolymerization process. Semin Thromb Hemost 2007;33:466 77. [4] Guerrini M, Guglieri S, Naggi A, Sasisekharan R, Torri G. Low molecular weight heparins: structural differentiation by bidimensional nuclear magnetic resonance spectroscopy. Semin Thromb Hemost 2007;33: 478 87. [5] Bianchini P, Liverani L, Spelta F, Mascellani G, Parma B.Variability of heparins and heterogeneity of low molecular weight heparins. Semin Thromb Hemost 2007;33:496 502. [6] Harenberg J, Giese C, Hagedorn A, Traeger I, Fenyvesi T. Determination of antithrombin-dependent factor Xa inhibitors by prothrombin-induced clotting time. Semin Thromb Hemost 2007;33:503 7. [7] Nowak G, Lange U, Wiesenburg A, Bucha E. Measurement of maximum thrombin generation capacity in blood and
S4 plasma using the thrombin generation assay (THROGA). Semin Thromb Hemost 2007;33:508 14. [8] Mousa SA. Heparin, low molecular weight heparin, and derivatives in thrombosis, angiogenesis, and inflammation: emerging links. Semin Thromb Hemost 2007;33:524 33. [9] Simonis D, Christ K, Alban S, Bendas G. Affinity and kinetics of different heparins binding to P- and L-selectin. Semin Thromb Hemost 2007;33:534 9. [10] Borsig L. Antimetastatic activities of modified heparins:
J. Harenberg selectin inhibition by heparin attenuates metastasis. Semin Thromb Hemost 2007;33:540 6. [11] Borgenstr¨ om M, W¨ arri A, Hiilesvuo K, K¨ ak¨ onen R, K¨ ak¨ onen S, Nissinen L, et al. O-sulfated bacterial polysaccharides with low anticoagulant activity inhibit metastasis. Semin Thromb Hemost 2007;33:547 56. [12] Ferro V, Dredge K, Liu L, Hammond E, Bytheway I, Li C, et al. PI-88 and novel heparan sulfate mimetics inhibit angiogenesis. Semin Thromb Hemost 2007;33:557 68.