Eur. J. Biochem. 203, 121 - 125 (1992) 0FEBS 1992
Inhibition of factor X, factor V and prothrombin activation by the bis(1actobionic acid amide) LW 10082 Frederick A. OFOSU132,Jawed FAREED3, Lindsay M. SMITH ’, Noorildan ANVART’, Debra HOPPENSTEADT3 and Morris A. BLAJCHMAN‘.’ The Canadian Red Cross Society, Blood Transfusion Service, Hamilton, Canada Department of Pathology, McMaster University, Hamilton, Canada Departments of Pathology and Pharmacology, Loyola University, Maywood, Illinois, USA (Received July 26, 1991) - EJB 91 0995
The minimum concentrations of heparin, dermatan sulfate, hirudin, and ~-Phe-Pro-ArgCH,Cl required to delay the onset of prothrombin activation in contact-activated plasma also prolong the lag phases associated with both factor X and factor V activation. Heparin and dermatan sulfate prolong the lag phases associated with the activation of the three proteins by catalyzing the inhibition of endogenously generated thrombin. Thrombin usually activates factor V and factor VIII during coagulation. The smallest fragment of heparin able to catalyze thrombin inhibition by antithrombin I11 is an octadecasaccharide with high affinity for antithrombin 111. In contrast, a dermatan sulfate hexasaccharide with high affinity for heparin cofactor 11 can catalyze thrombin inhibition by heparin cofactor 11. A highly sulfated bis(1actobionic acid amide), LW10082 ( M , 2288), which catalyzes thrombin inhibition by heparin cofactor I1 and has both antithrombotic and anticoagulant activities, has been synthesized. In this study, we determined how the minimum concentration of LW10082 required to delay the onset of intrinsic prothrombin activation achieved this effect. We demonstrate that, like heparin and dermatan sulfate, LW10082 delays the onset of intrinsic prothrombin activation by prolonging the lag phase associated with both factor X and factor V activation. In addition, LW10082 is approximately 25% as effective as heparin and 10 times as effective as dermatan sulfate in its ability to delay the onset of prothrombin activation. The strong anticoagulant action of LW10082 is consistent with previous reports which show that the degree of sulfation is an important parameter for the catalytic effectiveness of sulfated polysaccharides on thrombin inhibition.
Anticoagulants which catalyze thrombin inhibition delay the onset of intrinsic prothrombin activation more effectively than extrinsic prothrombin activation [l - 31. Furthermore, the efficiency with which these anticoagulants inhibit prothrombin activation generally parallels their ability to catalyze thrombin inhibition in plasma [4, 51. Recent studies reported that several bis(1actobionic acid amide)s prolong the activated partial thromboplastin time and have antithrombotic activity in the rat [6, 71. This study determined whether the bis(lactobionic acid amide) LW10082, like heparin [3] and dermatan sulphate [8], inhibits prothrombin activation in vitro by delaying simultaneously the onset of factor X and factor V activation. This bis(1actobionic acid amide) catalyzes thrombin inhibition by heparin cofactor I1 161. We conclude from our results that, on a molar basis, this bis(1actobionic acid amide) is approximately 25% as effective as unfractionated heparin, and over ten times more effective than dermatan sulfate, in its ability to inhibit the onset of intrinsic activation of factor X, factor V and prothrombin. Thus, based Correspondence to F. A. Ofosu, Dept. of Pathology, McMaster ,University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 325 k z y r n e s . Thrombin (EC 3.4.21.5); coagulation factor Xa (EC 3.1.21.6); coagulation factor IXa (EC 3.4.21.22). Abbreviation. USP, United States Pharmacopea.
on its actions on in vitro coagulation [6, 71 and its antithrombotic activity in the rat [7],this bis(1actobionic acid amide) may be a useful antithrombotic agent in man.
MATERIALS AND METHODS Materials Activated partial thromboplastin time reagent used for contact-activation of plasma was obtained from Organon Teknika, Toronto, Canada. Rabbit brain tissue factor was a generous gift from Dr Leon Poller, Manchester, UK. D-PhePro-ArgCH2C1, a chloromethane inhibitor of thrombin, and dansyl-Glu-Gly-ArgCH2Cl, a chloromethane inhibitor of factor Xa and Factor IXa 19, 101, were obtained from Terachem, Toronto, Canada. Rabbit anti-(human prothrombin) serum and rabbit anti-(human antithrombin 111) serum were obtained from Behring Diagnostics, Montreal, Canada. Goat anti-(rabbit IgG) - alkaline-phosphatase conjugate was obtained from Dimension Laboratories, Toronto, Canada. The bis(1actobionic acid amide) LW10082 (Fig. 1) was generously provided by Dr W. Raake, Munich, FRG. When used at concentrations of up to 40pM, it did not catalyze the inhibition of factor Xa by purified antithrombin 111, using procedures described previously [8]. CNBr-activated Sepha-
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Fig. 1. The structure of LW10082 as reported in (71. R represents SO3 residues and n = 3 for LW10082.
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Fig. 2. Prolongation of the activated partial thromboplastin time of pooled normal plasma by increasing concentrations of LW10082.
rose 4B was obtained from Pharmacia Fine Chemicals, Montreal, Canada. p-Nitrophenyl phosphate and other reagentgrade chemicals were obtained from Sigma Chemical Company, St Louis, USA. Procedures for preparing human factor Xa have been previously described [ll]. Human a-thrombin, specific activity 2870 USP units/mg, prepared by the method of Fenton et al. [12], was generously provided by Dr J. W. Fenton 11, New York Department of Health, Albany, USA. Human antithrombin 111 was prepared by the methods of Miller-Andersson et al. [13]. '251-labeled human factor V was a gift from Dr M. E. Nesheim, Queen's University, Kingston, Canada. Antithrombin-111-depleted plasma was prepared by affinity chromatography on heparin-Sepharose 4B [14]. Effect on LW10082 on the thromboplastin and thrombin clotting times Pooled normal plasma was supplemented with LW10082 (to a final concentration of 0.04-40 pM in plasma) and the activated partial thromboplastin time and thrombin clotting time assays were performed of the plasmas determined according to manufacturers specifications. The structure of the bis(lactobionic acid amide) LW10082 is shown in Fig. 1. ELlSAs for quantitating prothrombin, thrombin - antithrombin-Ill, thrombin -heparin-cofactor-I1 and factor-Xa - antithrombin-111 Prothrombin consumption in plasma and the inhibition of exogenous thrombin by plasma antithrombin 111 and heparin cofactor 11, were quantitated using previously described ELISAs [8, 15, 161. The first antibody coated onto the microtiter plates for each quantitation was affinity-purified sheep anti-(human thrombin/prothrombin) IgG. The second IgG used to quantify prothrombin was that isolated from the rabbit anti-(human prothrombin) serum which reacted strongly with prothrombin but poorly with human thrombin [8, 15,161. The second antibodies used to measure the concentrations of thrombin - antithrombin-111 and thrombin - hep-
arin-cofactor-I1 were rabbit anti-(human antithrombin 111) serum and rabbit anti-(human heparin cofactor 11) serum, respectively [15]. Procedures for quantifying factor X activation as total factor-Xa - antithrombin-111 have been reported previously [8]. Effects of LW10082 on factor X and prothrombin activation Plasma (previously defibrillated with Arvin [15]) was subjected to contact activation by incubation for 5 min at 37°C with activated partial thromboplastin time reagent in a ratio of 2 vol. plasma to 1 vol. activated partial thromboplastin time reagent, which consisted of a suspension of micronized silica (as the contact activator) and rabbit brain coagulant phospholipids. Then 1 vol. 40 mM CaC12 (prewarmed to 37°C) was added to the contact-activated plasma to initiate f x t o r X and prothrombin activation. After incubations at 37 "C varying from 15 s to 90 s, 50-yl aliquots were withdrawn into 200 p1 of a buffer consisting of 5 mM EDTA, 0.036 M sodium barbiturate, 0.036 M acetic acid, 0.145 M NaC1,l mg/ ml bovine serum albumin, 1 pM ~-Phe-Pro-ArgCH~Cl and 5 units/ml heparin, pH 7.4. The EDTA stopped prothrombin and factor X activation. D-Phe-Pro-ArgCH2C1inactivated the thrombin present in each aliquot, thus minimizing competition between thrombin and factor Xa for antithrombin 111. Heparin in the buffer catalyzed the conversion of free factor Xa into factor-Xa - antithrombin-111. After a 10-min incubation at 3 7 T , the prothrombin was consumed and the concentration of total factor-Xa -antithrombin-I11 generated in each aliquot were quantitated using the ELISAs described above. Factor X and prothrombin activation were also initiated by substituting rabbit brain tissue factor for the activated partial thromboplastin tissue reagent. The effects of 0.04-40 pM LW10082 on factor X and prothrombin activation were also investigated. Activation of factor V Intrinsic and extrinsic pathway activation of factor V were initiated using procedures described above for initiating plasma factor X and prothrombin activation. For these experiments, '251-factor V was added to each plasma to a final concentration of 50 ng/ml prior to initiating factor V activation. The proteolysis of factor V was stopped at timed intervals by adding 1 vol. plasma to 4 vol. of an inhibitor buffer containing 1 pM ~-Phe-Pro-ArgCH~Cl, 1 pM dansylGlu-Gly-ArgCH,CI, 5 mM EDTA, 36 mM sodium barbiturate, 36 mM acetic acid, and 145 mM NaCI, pH 7.4. The final dilution of plasma in each case was 1:2. Aliquots (10 pl) of recalcified plasma were withdrawn at 15-s intervals and added to an equal volume of the inhibitor buffer. Electrophoretic sample buffer (80 pl) was then added. The resulting sample was boiled for 4 min, cooled to room temperature and a SO-pl aliquot was then subjected to electrophoresis in 5 - 15% gradient polyacrylamide gels containing 0.1 % SDS. The gels were dried under vacuum and exposed to X-ray films for 24 72 h to visualize factor V and its proteolytic derivatives.
RESULTS Effects of lactobionic acid on the clotting times of plasma As summarized in Fig. 2 , the activated partial thromboplastin time was prolonged in a dose-dependent manner by the bis(1actobionic acid amide). The plasma was essentially
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Fig. 3. Comparativeeffects of the bisOactobionic acid amide) LW10082 on the intrinsic (A) and the extrinsic (B) pathways of factor X activation in pooled normal plasma. Intrinsic factor X activation was initiated by adding CaCI, (prewarmed to 37 "C) to contact-activated plasma also prewdrmed to 37°C. Extrinsic factor X activation was initiated by adding CaCl, prewarmed to 37 "C to plasma pre-incubated with tissue factor at 37°C for 3 min. Factor X activation was quantitated as the concentration of total factor-Xa - antithrombin-111 obtained after timed aliquots had been added a buffer containing EDTA and heparin. Heparin was added to catalyze the conversion of free fator Xa in the plasma aliquot to factor-Xa - antithrombin-111. (0)Control plasma; ( A ) 0.4 pM LW10082; (m) 4 pM LW10082; ( 0 )40 pM LW10082.
unclottable when the concentration of the bis(1actobionic acid amide) exceeded 10 pM. In contrast, up to 40 pM LW10082 had no effect on the prothrombin time (not shown). The concentrations of this bis(1actobionic acid amide) required to double the thrombin clotting time initiated with 50 nM thrombin were 5 pM in the absence of CaCI,, and 10 pM in the presence of 10 mM CaCI,. Factor X activation
Nanomolar concentrations of factor Xa (as total factorXa - antithrombin-111) were first observed 45 s after CaC1, had been added to contact-activated plasma (Fig. 3A). The bis(1actobionic acid amide) effectively suppressed factor X activation, 0.4 pM LW10082 being the minimum concentration to delay the onset of intrinsic factor X activation for 30 s. Factor Xa could not be detected up to 90 s after CaCl, was added to contact-activated plasma which also contained 4 pM bis(1actobionic acid amide) (Fig. 3 A). As the bis(lactobionic acid amide) did not catalyze factor Xa inhibition in plasma (see above), its suppression of factor-Xaantithrombin-I11 generation demonstrates that this polyanion can directly inhibit intrinsic factor X activation. In contrast to its effect on intrinsic factor X activation, up to 40 pM LW3 0082 could neither delay the onset of nor totally suppress extrinsic factor X activation (Fig. 3B). Effect of LW10082 on intrinsic and extrinsic pathway activation of plasma factor V
When CaCl, was added to contact-activated plasma, production of factor Va heavy chain and the 230-kDa factor V fragment became evident 30 s later. After a 60-s incubation, factor Va light chain and factor V activation peptide (molecular mass M 150 kDa), were also generated (Fig. 4, lanes 1 3). When 0.4 pM LW10082 was added t o contact-activated plasma, conversion of plasma factor V into the above three
Fig. 4. The effect of LW10082 on intrinsic factor V activation. Factor V activation was initiated by adding CaC1, to contact-activated plasma. Aliquots were withdrawn at 15-s intervals and factor V activation achieved assessed by SDSjPAGE and autoradiography. Control contact-activated plasma was applied in lanes 1-4. LW10082 (0.4 pM) was added to contact-activated plasma in lanes 5 , 7 and 8. The incubation times were as follows: lanes 1 and 5, 30 s; lanes 2 and 7, 45 s; lanes 3 and 8, 60 s; lane 4, 0 s. Symbols used: V, factor V; H and L and factor Va heavy and light chains, respectively; A represents the 150-kDa factor V activation peptide. Lane 6 contains radioactive molecular mass markers with the stated molecular masses in kDa.
limit fragments was incomplete after a 60-s incubation (Fig.4, lane 8). When the concentration of the bis(1actobionic acid amide) was increased to 4 pM, proteolysis of factor V in contact-activated plasma was first detected in the 90-s aliquot (results not shown). The effects of 4.0 pM LW10082 on tissue-factor-dependent proteolysis of factor V were investigated and the results are shown in Fig. 5 (lanes 4, 5 and 8). This concentration of the bis(1actobionic acid amide) marginally suppressed tissuefactor-dependent formation of factor Va light chain (lanes 4 and 5) when compared to control plasma (lanes 1 - 3, in which the bands corresponding to factor Va heavy and light chains have similar intensity). A similar effect was obtained with 40 pM LW10082 (not shown). Thus, LW10082 could not inhibit tissue-factor-dependent factor V proteolysis as effectively as it could inhibit intrinsic factor V proteolysis. Prothrombin activation
Factor Xa and factor Va generation are both required for efficient prothrombin activation in plasma. Having established the effects of LW10082 on factor X and factor V ac& vation, we next investigated its effects on prothrombin activation. As summarized in Fig. 6A, the maximum rate of prothrombin activation (consumption) in contact-activated plasma was measured in the interval 30 - 45 s. The minimum concentration of LW10082 able to delay the onset of prothrombin activation in contact-activated plasma was 0.4 pM. In addition, 4 pM and 40 pM LW10082 could totally inhibit intrinsic prothrombin activation for at least 90 s. In contrast to its effect on intrinsic prothrombin activation, up to 40 pM
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Fig. 5. The effect of the bisOactobionic acid amide) LW10082 on the extrinsic activation of factor V. Factor V proteolysis was initiated by adding rabbit brain tissue Factor and CaClz to normal plasma. Aliquots were withdrawn at 10-s intervals and factor V proteolysis assessed after SDSjPAGE and autoradiography. The plasmas applied in lanes 4, 5 and 8 contained 4 pM lactobionic acid. Control plasma was applied in lanes 1, 2, 3 and 6. The incubation times were as follows: lanes 1 and 4, 20 s; lanes 2 and 5 , 40 s; lanes 3 and 8, 60 s; lane 6 , 0 s. Radioactive molecular mass markers were applied in lane 7 with the values given in kDa. Symbols: V, factor V ; H and L and A are factor Va heavy and light chains and factor V activation peptide, respectively.
could not delay the onset of extrinsic prothrombin activation (Fig. 6B). In addition, 40 pM LW10082 was the minimum concentration which could inhibit extrinsic prothrombin consumption. Catalysis of thrombin inhibition by bis(1actohionic acid amide) Thrombin (200nM) was added to an equal volume of pooled normal plasma and to antithrombin-111-depleted plasma. The effects of LW10082 on exogenous thrombin inhibition by antithrombin 111 and heparin cofactor I1 were assessed. As summarized in Fig. 7, this bis(1actobionic acid amide) accelerated thrombin - heparin-cofactor-I1 formation in normal plasma at the expense of thrombin- antithrombin111 formation. While the catalysis of thrombin -heparincofactor-I1 formation by LW10082 was readily apparent in antithrombin-111-depleted plasma, the rate of thrombin - heparin-cofactor-I1 formation in this plasma did not exceed that found in normal plasma.
DISCUSSION WE have demonstrated in this study that the bis(1actobionic acid amide) LW10082 effectively catalyses thrombin inhibition by heparin cofactor I1 in plasma. This action likely accounts for its ability to prolong the onset of intrinsic prothrombin activation. Heparin and dermatan sulfate prolong the lag phase associated with intrinsic prothrombin activation by delaying the onset Factor VII1 and fixtor V activation
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Fig. 6. The effect of the bis(1actohionic acid amide) LW10082 on intrinsic (A) and extrinsic (B) activation of prothrombin. Intrinsic prothrombin activation (consumption) was initiated by adding CaClz to contact-activated plasma. Extrinsic prothrombin activation was initiated by adding rabbit brain tissue Factor and CaCI, to plasma. Prothrombin consumption was quantitated by ELISA for prothrombin [I 51. (0)Control plasma; ( A ) plasma containing 0.4 pM LW10082; (B) plasma containing 4.0 pM LWZ0082; ( 0 )plasma containing 40 pM LW 10082.
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Fig. 7. The effect of LW10082 on thrombin- antithrombin-111 formation in normal plasma (A), on thrombin - heparin-cofactor-I1formation in normal plasma (B) and on thrombin heparin-cofactor-11 formation in antithrombin-Ill-depleted plasma (C). Thrombin (200 nM) was added to an equal volume of plasma and the concentration of thrombin-inhibitor complexes formed as a function of time determined by ELISAs. (0) Control plasma; ( A ) plasma containing 0.4 pM LW10082; ( W ) plasma containing 4.0 pM LWZ0082.
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usually catalyzed by the thrombin generated endogenously [l - 3, 8, 151. Heparin catalyzes endogenous thrombin antithrombin-I11 formation [5, 8, 15, 161, while dermatan sulfate catalyzes endogenous thrombin -heparin-cofactor-I1 formation [5, 81. The results of this study suggest that LW10082 inhibits intrinsic prothrombin activation by mechanisms similar to those of dermatan sulfate [8]. The minimum concentrations of LW10082 and dermatan sulfate able to delay the onset of intrinsic prothrombin activation were 0.4 pM and about 5 pM, respectively [8], were also the minimum concentrations to delay the onset of both factor X and factor V activation in contact-activated plasma. In contrast to its effects on intrinsic coagulation, up to 40 pM LW10082 could not delay the onset of tissue-factordependent Factor X and factor V activation. The ability of
125 LW10082 to inhibit intrinsic coagulation may be due to the mandatory role endogenous thrombin plays as the activator of factor VIII when this protein is complexed to von Willebrand factor [17]. If, like purified factor VIII, factor Xa is unable to activate plasma factor VIII complexed to von Willebrand factor [17], then thrombin, and not factor Xa, is the major enzyme which initiates Factor VIII activation during intrinsic coagulation. If thrombin is indeed the activator of factor VIII complexed to von Willebrand factor in plasma, then the availability of saturating concentrations of factor Xa in contact-activated plasma will be critically dependent on prior thrombin action on factor VIII. In contrast, activation of factor X by factor-VIIa - tissue-factor complex during extrinsic coagulation would make saturating concentrations of factor Xa available without prior thrombin action on factor VIII. Indeed, up to 40pM LW10082 could not delay the cleavage of factor V into factor Va heavy chain and the 230-kDa factor V intermediate during extrinsic coagulation. As production of these two fragments (by either thrombin or factor Xa) coincides with the availability of factor Va activity [3, 181, it was not surprising that up to 40pM LW10082 could not prolong the lag phase associated with tissue-factordependent activation of prothrombin. The inhibition of intrinsic factor X, factor V and prothrombin activation achieved with 0.4 pM LW10082 is similar to the inhibition achieved with about 0.1 pM heparin and about 5.0 pM dermatan sulfate [S]. Our results with LW10082 are consistent with those of Beguin et al. [6]. Our results also demonstrate that thrombin inhibition by heparin cofactor I1 can be readily catalyzed by a polyanion with M , comparable to those of highly sulfated pentasaccharides. In contrast, the minimum fragments of heparin which can catalyze thrombin inhibition by antithrombin III are octadecasaccharides 119, 201. Catalysis of thrombin inhibition by antithrombin 111can occur only if the glycosaminoglycan catalyst is able to bind antithrombin 111 and thrombin simultaneously [19]. A similar template mechanism has been proposed for heparin fragments to catalyze thrombin inhibition by heparin cofactor I1 [21]. Tollefsen et al. subjected dermatan sulfate to Smith degradation and isolated from the products an octasaccharide marginally able to catalyze thrombin - heparin-cofactor-I1 formation [21]. Maimone and Tollefsen [22] also subjected dermatan sulfate to hydrazinolysis and nitrous acid degradation, and subsequently isolated from the reaction products the hexasaccharide [IdoA(2-S04)GalN Ac(4-SO4)I3with high affinity for heparin cofactor I1 and M , comparable to that of LW10082 [22]. The two dermatan sulfate fragments are significantly less able to catalyze thrombin inhibition by heparin cofactor I1 than LW10082 [21, 221. Significantly, the dermatan sulfate hexasaccharide with high affinity for heparin cofactor I1 has only one sulfate group/monosaccharide [21]. In contrast, all hydroxyl groups of LW10082 are sulfated (Fig. 1). The resulting charge density of LW10082 is significantly higher than that of heparin, heparan sulfate and dermatan sulfate [23,24]. Our current results are consistent with previous observations that sulfate-dependent charge density contributes significantly to the Cdtdytk effectiveness of dermahn sulfate on thrombin inhibition by heparin cofactor I1 [4]. Sulfate-dependent charge
density is similarly important for the anticoagulant and antithrombotic activities of heparin and heparan sulfates [4, 23, 241. This study was funded in part by a Grant-In-Aid from the Ontario Heart and Stroke Foundation. The generous gifts of LW10082 by Dr W. Raake, human a-thrombin by Dr J . W. Fenton 11, and '251-human factor V by Dr M. E. Nesheim, and the assistance of Xianjun Yang are gratefully acknowledged.
REFERENCES 1. Ofosu, F. A., Modi, G. J., Hirsh, J., Buchanan, M. R. & Blajchman, M. A. (1986) Ann. N . Y . Arad. Sci. 485, 41 -55. 2. Beguin, S., Lindhout, T. & Hemker, H. C. (1 988) Thromb. Haemostasis 60, 457 - 462. 3. Yang, X.-J., Blajchman, M. A,, Craven, S., Smith, L. M., Anvari, N. & Ofosu, F. A. (1990) Biochem. J . 272,399-406. 4. Ofosu, F. A., Modi, G. J., Blajchman, M. A,, Buchanan, M. R. & Johnson, E. A. (1987) Biochem. J . 248, 889-896. 5. Ofosu, F. A., Buchanan, M. R., Anvari, N., Smith, L. M. & Blajchman, M. A. (1989) Ann. N . Y . Acad. Sci.556,123-131. 6. Beguin, S. S., Dol, F. & Hemker, H. C. (1991) Sem. Thromb. Hemostasis 17, Suppl. 1, 126- 128. 7. Raake, W., Klauser, R. J., Meintsberger, E., Zeller, P. & Elling, H. (1991) Sem. Thromh. Hemostasis 17, Suppl. I , 129-135. 8 Ofosu, F. A., Choay, J., Anvari, N., Smith, L. M. & Blajchman, M. A. (1990) Eur. J. Biochem. 193,485-493. 9 Kettner, C . & Shaw, E. (1979) Thromb. Res. 14,969-973. 10 Nesheim, M. E., Kettner, C., Shaw, E. & Mann, K. G . (1987) J . B i d . Chem. 256, 6537-6540. 11 Ofosu, F. A,, Blajchman, M. A. & Hirsh, J . (1980) Thromh. Rex 20, 391 -403. 12 Fenton, J. W., Fasco, M. J., Stackrow, A. B., Aronson, D., Young, A . M . & Finlayson, 3. (1977)J. B i d . Chem. 252,35873598. 13 Miller-Andersson, M., Borg, H. & Anderson, L.-0. (1974) Thromh. Res. 5,439 - 446. 14 Hoogendoorn, H., Cerskus, A,, Ofosu, F. A.. Blajchman, M. A. & Hirsh, J. (1980) Thromb. Res. 20, 77-83. 15 Ofosu, F. A,, Hirsh, J., Esmon, C. T., Modi, G. J., Smith, L. M., Anvari, N., Buchanan, M. R., Fenton, J. W. & Blajchman, M. A. (1989) Biochem. J . 257, 143- 150. 16. Ofosu, F. A,, Smith, L. M., Anvari, N. & Blajchman, M. A. (1988) Thromb. Haemostasis 60, 193-198. 17. Koedam, J. A., Hamer, R. J., Beeser-Visser, N. H., Bouma, B. N. & Sixma, J. J. (1990) Eur. J . Biochem. 189, 229-234. 18. Monkovic, D. D. &Tracy, P. B. (1990) Biochemistry 29, 11183 128. 19. Bjork, I., Olson, S. T. &Shore, J. D. (1989) in Heparin: chemical und biological properties (Lane, D. A. & Lindahl, U., eds) pp. 229-255, Edward Arnold, London. 20. Holmer, E., Soderberg, K., Bergqvist, D. & Lindahl, U. (1986) Haemostasis 16, Suppl. 2, 1 - 12. 21. Tollefsen, D . M., Peacock, M . E. & Monafo, W. J. (1986) J . Bid. Chem. 261,8854-8858. 22. Maimone, M. M. & Tollefsen, D. M. (1990) J . B i d . Chem. 265, 18263 - 18271. 23 Casu, B., Johnson, E. A,, Mantovani, M., Mulloy, B., Orestc, P., Pescador, R., Prino, G., Torr, G. & Zoppetti, G. (1983) Arzneim. Forsch. 33, 135 - 142. 24 Van Ryn-McKenna, J., Ofosu, F. A., Hirsh, J . & Buchanan, M. R. (1989) Br. J . Haematol. 71, 265-269.
Inhibition of factor X, factor V and prothrombin activation by the bis(1actobionic acid amide) LW 10082 Frederick A. OFOSU132,Jawed FAREED3, Lindsay M. SMITH ’, Noorildan ANVART’, Debra HOPPENSTEADT3 and Morris A. BLAJCHMAN‘.’ The Canadian Red Cross Society, Blood Transfusion Service, Hamilton, Canada Department of Pathology, McMaster University, Hamilton, Canada Departments of Pathology and Pharmacology, Loyola University, Maywood, Illinois, USA (Received July 26, 1991) - EJB 91 0995
The minimum concentrations of heparin, dermatan sulfate, hirudin, and ~-Phe-Pro-ArgCH,Cl required to delay the onset of prothrombin activation in contact-activated plasma also prolong the lag phases associated with both factor X and factor V activation. Heparin and dermatan sulfate prolong the lag phases associated with the activation of the three proteins by catalyzing the inhibition of endogenously generated thrombin. Thrombin usually activates factor V and factor VIII during coagulation. The smallest fragment of heparin able to catalyze thrombin inhibition by antithrombin I11 is an octadecasaccharide with high affinity for antithrombin 111. In contrast, a dermatan sulfate hexasaccharide with high affinity for heparin cofactor 11 can catalyze thrombin inhibition by heparin cofactor 11. A highly sulfated bis(1actobionic acid amide), LW10082 ( M , 2288), which catalyzes thrombin inhibition by heparin cofactor I1 and has both antithrombotic and anticoagulant activities, has been synthesized. In this study, we determined how the minimum concentration of LW10082 required to delay the onset of intrinsic prothrombin activation achieved this effect. We demonstrate that, like heparin and dermatan sulfate, LW10082 delays the onset of intrinsic prothrombin activation by prolonging the lag phase associated with both factor X and factor V activation. In addition, LW10082 is approximately 25% as effective as heparin and 10 times as effective as dermatan sulfate in its ability to delay the onset of prothrombin activation. The strong anticoagulant action of LW10082 is consistent with previous reports which show that the degree of sulfation is an important parameter for the catalytic effectiveness of sulfated polysaccharides on thrombin inhibition.
Anticoagulants which catalyze thrombin inhibition delay the onset of intrinsic prothrombin activation more effectively than extrinsic prothrombin activation [l - 31. Furthermore, the efficiency with which these anticoagulants inhibit prothrombin activation generally parallels their ability to catalyze thrombin inhibition in plasma [4, 51. Recent studies reported that several bis(1actobionic acid amide)s prolong the activated partial thromboplastin time and have antithrombotic activity in the rat [6, 71. This study determined whether the bis(lactobionic acid amide) LW10082, like heparin [3] and dermatan sulphate [8], inhibits prothrombin activation in vitro by delaying simultaneously the onset of factor X and factor V activation. This bis(1actobionic acid amide) catalyzes thrombin inhibition by heparin cofactor I1 161. We conclude from our results that, on a molar basis, this bis(1actobionic acid amide) is approximately 25% as effective as unfractionated heparin, and over ten times more effective than dermatan sulfate, in its ability to inhibit the onset of intrinsic activation of factor X, factor V and prothrombin. Thus, based Correspondence to F. A. Ofosu, Dept. of Pathology, McMaster ,University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 325 k z y r n e s . Thrombin (EC 3.4.21.5); coagulation factor Xa (EC 3.1.21.6); coagulation factor IXa (EC 3.4.21.22). Abbreviation. USP, United States Pharmacopea.
on its actions on in vitro coagulation [6, 71 and its antithrombotic activity in the rat [7],this bis(1actobionic acid amide) may be a useful antithrombotic agent in man.
MATERIALS AND METHODS Materials Activated partial thromboplastin time reagent used for contact-activation of plasma was obtained from Organon Teknika, Toronto, Canada. Rabbit brain tissue factor was a generous gift from Dr Leon Poller, Manchester, UK. D-PhePro-ArgCH2C1, a chloromethane inhibitor of thrombin, and dansyl-Glu-Gly-ArgCH2Cl, a chloromethane inhibitor of factor Xa and Factor IXa 19, 101, were obtained from Terachem, Toronto, Canada. Rabbit anti-(human prothrombin) serum and rabbit anti-(human antithrombin 111) serum were obtained from Behring Diagnostics, Montreal, Canada. Goat anti-(rabbit IgG) - alkaline-phosphatase conjugate was obtained from Dimension Laboratories, Toronto, Canada. The bis(1actobionic acid amide) LW10082 (Fig. 1) was generously provided by Dr W. Raake, Munich, FRG. When used at concentrations of up to 40pM, it did not catalyze the inhibition of factor Xa by purified antithrombin 111, using procedures described previously [8]. CNBr-activated Sepha-
122 RO RO
C-NH-(CH,),-NH-C
OR RO
.
OR
. OR
Fig. 1. The structure of LW10082 as reported in (71. R represents SO3 residues and n = 3 for LW10082.
0
0
I
I
I
I
10
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80
iLW100821 (pM)
Fig. 2. Prolongation of the activated partial thromboplastin time of pooled normal plasma by increasing concentrations of LW10082.
rose 4B was obtained from Pharmacia Fine Chemicals, Montreal, Canada. p-Nitrophenyl phosphate and other reagentgrade chemicals were obtained from Sigma Chemical Company, St Louis, USA. Procedures for preparing human factor Xa have been previously described [ll]. Human a-thrombin, specific activity 2870 USP units/mg, prepared by the method of Fenton et al. [12], was generously provided by Dr J. W. Fenton 11, New York Department of Health, Albany, USA. Human antithrombin 111 was prepared by the methods of Miller-Andersson et al. [13]. '251-labeled human factor V was a gift from Dr M. E. Nesheim, Queen's University, Kingston, Canada. Antithrombin-111-depleted plasma was prepared by affinity chromatography on heparin-Sepharose 4B [14]. Effect on LW10082 on the thromboplastin and thrombin clotting times Pooled normal plasma was supplemented with LW10082 (to a final concentration of 0.04-40 pM in plasma) and the activated partial thromboplastin time and thrombin clotting time assays were performed of the plasmas determined according to manufacturers specifications. The structure of the bis(lactobionic acid amide) LW10082 is shown in Fig. 1. ELlSAs for quantitating prothrombin, thrombin - antithrombin-Ill, thrombin -heparin-cofactor-I1 and factor-Xa - antithrombin-111 Prothrombin consumption in plasma and the inhibition of exogenous thrombin by plasma antithrombin 111 and heparin cofactor 11, were quantitated using previously described ELISAs [8, 15, 161. The first antibody coated onto the microtiter plates for each quantitation was affinity-purified sheep anti-(human thrombin/prothrombin) IgG. The second IgG used to quantify prothrombin was that isolated from the rabbit anti-(human prothrombin) serum which reacted strongly with prothrombin but poorly with human thrombin [8, 15,161. The second antibodies used to measure the concentrations of thrombin - antithrombin-111 and thrombin - hep-
arin-cofactor-I1 were rabbit anti-(human antithrombin 111) serum and rabbit anti-(human heparin cofactor 11) serum, respectively [15]. Procedures for quantifying factor X activation as total factor-Xa - antithrombin-111 have been reported previously [8]. Effects of LW10082 on factor X and prothrombin activation Plasma (previously defibrillated with Arvin [15]) was subjected to contact activation by incubation for 5 min at 37°C with activated partial thromboplastin time reagent in a ratio of 2 vol. plasma to 1 vol. activated partial thromboplastin time reagent, which consisted of a suspension of micronized silica (as the contact activator) and rabbit brain coagulant phospholipids. Then 1 vol. 40 mM CaC12 (prewarmed to 37°C) was added to the contact-activated plasma to initiate f x t o r X and prothrombin activation. After incubations at 37 "C varying from 15 s to 90 s, 50-yl aliquots were withdrawn into 200 p1 of a buffer consisting of 5 mM EDTA, 0.036 M sodium barbiturate, 0.036 M acetic acid, 0.145 M NaC1,l mg/ ml bovine serum albumin, 1 pM ~-Phe-Pro-ArgCH~Cl and 5 units/ml heparin, pH 7.4. The EDTA stopped prothrombin and factor X activation. D-Phe-Pro-ArgCH2C1inactivated the thrombin present in each aliquot, thus minimizing competition between thrombin and factor Xa for antithrombin 111. Heparin in the buffer catalyzed the conversion of free factor Xa into factor-Xa - antithrombin-111. After a 10-min incubation at 3 7 T , the prothrombin was consumed and the concentration of total factor-Xa -antithrombin-I11 generated in each aliquot were quantitated using the ELISAs described above. Factor X and prothrombin activation were also initiated by substituting rabbit brain tissue factor for the activated partial thromboplastin tissue reagent. The effects of 0.04-40 pM LW10082 on factor X and prothrombin activation were also investigated. Activation of factor V Intrinsic and extrinsic pathway activation of factor V were initiated using procedures described above for initiating plasma factor X and prothrombin activation. For these experiments, '251-factor V was added to each plasma to a final concentration of 50 ng/ml prior to initiating factor V activation. The proteolysis of factor V was stopped at timed intervals by adding 1 vol. plasma to 4 vol. of an inhibitor buffer containing 1 pM ~-Phe-Pro-ArgCH~Cl, 1 pM dansylGlu-Gly-ArgCH,CI, 5 mM EDTA, 36 mM sodium barbiturate, 36 mM acetic acid, and 145 mM NaCI, pH 7.4. The final dilution of plasma in each case was 1:2. Aliquots (10 pl) of recalcified plasma were withdrawn at 15-s intervals and added to an equal volume of the inhibitor buffer. Electrophoretic sample buffer (80 pl) was then added. The resulting sample was boiled for 4 min, cooled to room temperature and a SO-pl aliquot was then subjected to electrophoresis in 5 - 15% gradient polyacrylamide gels containing 0.1 % SDS. The gels were dried under vacuum and exposed to X-ray films for 24 72 h to visualize factor V and its proteolytic derivatives.
RESULTS Effects of lactobionic acid on the clotting times of plasma As summarized in Fig. 2 , the activated partial thromboplastin time was prolonged in a dose-dependent manner by the bis(1actobionic acid amide). The plasma was essentially
123
1
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1
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I
60 80 20 40 REACTION TIMES (s)
I
60
Fig. 3. Comparativeeffects of the bisOactobionic acid amide) LW10082 on the intrinsic (A) and the extrinsic (B) pathways of factor X activation in pooled normal plasma. Intrinsic factor X activation was initiated by adding CaCI, (prewarmed to 37 "C) to contact-activated plasma also prewdrmed to 37°C. Extrinsic factor X activation was initiated by adding CaCl, prewarmed to 37 "C to plasma pre-incubated with tissue factor at 37°C for 3 min. Factor X activation was quantitated as the concentration of total factor-Xa - antithrombin-111 obtained after timed aliquots had been added a buffer containing EDTA and heparin. Heparin was added to catalyze the conversion of free fator Xa in the plasma aliquot to factor-Xa - antithrombin-111. (0)Control plasma; ( A ) 0.4 pM LW10082; (m) 4 pM LW10082; ( 0 )40 pM LW10082.
unclottable when the concentration of the bis(1actobionic acid amide) exceeded 10 pM. In contrast, up to 40 pM LW10082 had no effect on the prothrombin time (not shown). The concentrations of this bis(1actobionic acid amide) required to double the thrombin clotting time initiated with 50 nM thrombin were 5 pM in the absence of CaCI,, and 10 pM in the presence of 10 mM CaCI,. Factor X activation
Nanomolar concentrations of factor Xa (as total factorXa - antithrombin-111) were first observed 45 s after CaC1, had been added to contact-activated plasma (Fig. 3A). The bis(1actobionic acid amide) effectively suppressed factor X activation, 0.4 pM LW10082 being the minimum concentration to delay the onset of intrinsic factor X activation for 30 s. Factor Xa could not be detected up to 90 s after CaCl, was added to contact-activated plasma which also contained 4 pM bis(1actobionic acid amide) (Fig. 3 A). As the bis(lactobionic acid amide) did not catalyze factor Xa inhibition in plasma (see above), its suppression of factor-Xaantithrombin-I11 generation demonstrates that this polyanion can directly inhibit intrinsic factor X activation. In contrast to its effect on intrinsic factor X activation, up to 40 pM LW3 0082 could neither delay the onset of nor totally suppress extrinsic factor X activation (Fig. 3B). Effect of LW10082 on intrinsic and extrinsic pathway activation of plasma factor V
When CaCl, was added to contact-activated plasma, production of factor Va heavy chain and the 230-kDa factor V fragment became evident 30 s later. After a 60-s incubation, factor Va light chain and factor V activation peptide (molecular mass M 150 kDa), were also generated (Fig. 4, lanes 1 3). When 0.4 pM LW10082 was added t o contact-activated plasma, conversion of plasma factor V into the above three
Fig. 4. The effect of LW10082 on intrinsic factor V activation. Factor V activation was initiated by adding CaC1, to contact-activated plasma. Aliquots were withdrawn at 15-s intervals and factor V activation achieved assessed by SDSjPAGE and autoradiography. Control contact-activated plasma was applied in lanes 1-4. LW10082 (0.4 pM) was added to contact-activated plasma in lanes 5 , 7 and 8. The incubation times were as follows: lanes 1 and 5, 30 s; lanes 2 and 7, 45 s; lanes 3 and 8, 60 s; lane 4, 0 s. Symbols used: V, factor V; H and L and factor Va heavy and light chains, respectively; A represents the 150-kDa factor V activation peptide. Lane 6 contains radioactive molecular mass markers with the stated molecular masses in kDa.
limit fragments was incomplete after a 60-s incubation (Fig.4, lane 8). When the concentration of the bis(1actobionic acid amide) was increased to 4 pM, proteolysis of factor V in contact-activated plasma was first detected in the 90-s aliquot (results not shown). The effects of 4.0 pM LW10082 on tissue-factor-dependent proteolysis of factor V were investigated and the results are shown in Fig. 5 (lanes 4, 5 and 8). This concentration of the bis(1actobionic acid amide) marginally suppressed tissuefactor-dependent formation of factor Va light chain (lanes 4 and 5) when compared to control plasma (lanes 1 - 3, in which the bands corresponding to factor Va heavy and light chains have similar intensity). A similar effect was obtained with 40 pM LW10082 (not shown). Thus, LW10082 could not inhibit tissue-factor-dependent factor V proteolysis as effectively as it could inhibit intrinsic factor V proteolysis. Prothrombin activation
Factor Xa and factor Va generation are both required for efficient prothrombin activation in plasma. Having established the effects of LW10082 on factor X and factor V ac& vation, we next investigated its effects on prothrombin activation. As summarized in Fig. 6A, the maximum rate of prothrombin activation (consumption) in contact-activated plasma was measured in the interval 30 - 45 s. The minimum concentration of LW10082 able to delay the onset of prothrombin activation in contact-activated plasma was 0.4 pM. In addition, 4 pM and 40 pM LW10082 could totally inhibit intrinsic prothrombin activation for at least 90 s. In contrast to its effect on intrinsic prothrombin activation, up to 40 pM
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Fig. 5. The effect of the bisOactobionic acid amide) LW10082 on the extrinsic activation of factor V. Factor V proteolysis was initiated by adding rabbit brain tissue Factor and CaClz to normal plasma. Aliquots were withdrawn at 10-s intervals and factor V proteolysis assessed after SDSjPAGE and autoradiography. The plasmas applied in lanes 4, 5 and 8 contained 4 pM lactobionic acid. Control plasma was applied in lanes 1, 2, 3 and 6. The incubation times were as follows: lanes 1 and 4, 20 s; lanes 2 and 5 , 40 s; lanes 3 and 8, 60 s; lane 6 , 0 s. Radioactive molecular mass markers were applied in lane 7 with the values given in kDa. Symbols: V, factor V ; H and L and A are factor Va heavy and light chains and factor V activation peptide, respectively.
could not delay the onset of extrinsic prothrombin activation (Fig. 6B). In addition, 40 pM LW10082 was the minimum concentration which could inhibit extrinsic prothrombin consumption. Catalysis of thrombin inhibition by bis(1actohionic acid amide) Thrombin (200nM) was added to an equal volume of pooled normal plasma and to antithrombin-111-depleted plasma. The effects of LW10082 on exogenous thrombin inhibition by antithrombin 111 and heparin cofactor I1 were assessed. As summarized in Fig. 7, this bis(1actobionic acid amide) accelerated thrombin - heparin-cofactor-I1 formation in normal plasma at the expense of thrombin- antithrombin111 formation. While the catalysis of thrombin -heparincofactor-I1 formation by LW10082 was readily apparent in antithrombin-111-depleted plasma, the rate of thrombin - heparin-cofactor-I1 formation in this plasma did not exceed that found in normal plasma.
DISCUSSION WE have demonstrated in this study that the bis(1actobionic acid amide) LW10082 effectively catalyses thrombin inhibition by heparin cofactor I1 in plasma. This action likely accounts for its ability to prolong the onset of intrinsic prothrombin activation. Heparin and dermatan sulfate prolong the lag phase associated with intrinsic prothrombin activation by delaying the onset Factor VII1 and fixtor V activation
40
20 40 60 80 REACTION TIMES (s)
60
Fig. 6. The effect of the bis(1actohionic acid amide) LW10082 on intrinsic (A) and extrinsic (B) activation of prothrombin. Intrinsic prothrombin activation (consumption) was initiated by adding CaClz to contact-activated plasma. Extrinsic prothrombin activation was initiated by adding rabbit brain tissue Factor and CaCI, to plasma. Prothrombin consumption was quantitated by ELISA for prothrombin [I 51. (0)Control plasma; ( A ) plasma containing 0.4 pM LW10082; (B) plasma containing 4.0 pM LWZ0082; ( 0 )plasma containing 40 pM LW 10082.
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Fig. 7. The effect of LW10082 on thrombin- antithrombin-111 formation in normal plasma (A), on thrombin - heparin-cofactor-I1formation in normal plasma (B) and on thrombin heparin-cofactor-11 formation in antithrombin-Ill-depleted plasma (C). Thrombin (200 nM) was added to an equal volume of plasma and the concentration of thrombin-inhibitor complexes formed as a function of time determined by ELISAs. (0) Control plasma; ( A ) plasma containing 0.4 pM LW10082; ( W ) plasma containing 4.0 pM LWZ0082.
-
usually catalyzed by the thrombin generated endogenously [l - 3, 8, 151. Heparin catalyzes endogenous thrombin antithrombin-I11 formation [5, 8, 15, 161, while dermatan sulfate catalyzes endogenous thrombin -heparin-cofactor-I1 formation [5, 81. The results of this study suggest that LW10082 inhibits intrinsic prothrombin activation by mechanisms similar to those of dermatan sulfate [8]. The minimum concentrations of LW10082 and dermatan sulfate able to delay the onset of intrinsic prothrombin activation were 0.4 pM and about 5 pM, respectively [8], were also the minimum concentrations to delay the onset of both factor X and factor V activation in contact-activated plasma. In contrast to its effects on intrinsic coagulation, up to 40 pM LW10082 could not delay the onset of tissue-factordependent Factor X and factor V activation. The ability of
125 LW10082 to inhibit intrinsic coagulation may be due to the mandatory role endogenous thrombin plays as the activator of factor VIII when this protein is complexed to von Willebrand factor [17]. If, like purified factor VIII, factor Xa is unable to activate plasma factor VIII complexed to von Willebrand factor [17], then thrombin, and not factor Xa, is the major enzyme which initiates Factor VIII activation during intrinsic coagulation. If thrombin is indeed the activator of factor VIII complexed to von Willebrand factor in plasma, then the availability of saturating concentrations of factor Xa in contact-activated plasma will be critically dependent on prior thrombin action on factor VIII. In contrast, activation of factor X by factor-VIIa - tissue-factor complex during extrinsic coagulation would make saturating concentrations of factor Xa available without prior thrombin action on factor VIII. Indeed, up to 40pM LW10082 could not delay the cleavage of factor V into factor Va heavy chain and the 230-kDa factor V intermediate during extrinsic coagulation. As production of these two fragments (by either thrombin or factor Xa) coincides with the availability of factor Va activity [3, 181, it was not surprising that up to 40pM LW10082 could not prolong the lag phase associated with tissue-factordependent activation of prothrombin. The inhibition of intrinsic factor X, factor V and prothrombin activation achieved with 0.4 pM LW10082 is similar to the inhibition achieved with about 0.1 pM heparin and about 5.0 pM dermatan sulfate [S]. Our results with LW10082 are consistent with those of Beguin et al. [6]. Our results also demonstrate that thrombin inhibition by heparin cofactor I1 can be readily catalyzed by a polyanion with M , comparable to those of highly sulfated pentasaccharides. In contrast, the minimum fragments of heparin which can catalyze thrombin inhibition by antithrombin III are octadecasaccharides 119, 201. Catalysis of thrombin inhibition by antithrombin 111can occur only if the glycosaminoglycan catalyst is able to bind antithrombin 111 and thrombin simultaneously [19]. A similar template mechanism has been proposed for heparin fragments to catalyze thrombin inhibition by heparin cofactor I1 [21]. Tollefsen et al. subjected dermatan sulfate to Smith degradation and isolated from the products an octasaccharide marginally able to catalyze thrombin - heparin-cofactor-I1 formation [21]. Maimone and Tollefsen [22] also subjected dermatan sulfate to hydrazinolysis and nitrous acid degradation, and subsequently isolated from the reaction products the hexasaccharide [IdoA(2-S04)GalN Ac(4-SO4)I3with high affinity for heparin cofactor I1 and M , comparable to that of LW10082 [22]. The two dermatan sulfate fragments are significantly less able to catalyze thrombin inhibition by heparin cofactor I1 than LW10082 [21, 221. Significantly, the dermatan sulfate hexasaccharide with high affinity for heparin cofactor I1 has only one sulfate group/monosaccharide [21]. In contrast, all hydroxyl groups of LW10082 are sulfated (Fig. 1). The resulting charge density of LW10082 is significantly higher than that of heparin, heparan sulfate and dermatan sulfate [23,24]. Our current results are consistent with previous observations that sulfate-dependent charge density contributes significantly to the Cdtdytk effectiveness of dermahn sulfate on thrombin inhibition by heparin cofactor I1 [4]. Sulfate-dependent charge
density is similarly important for the anticoagulant and antithrombotic activities of heparin and heparan sulfates [4, 23, 241. This study was funded in part by a Grant-In-Aid from the Ontario Heart and Stroke Foundation. The generous gifts of LW10082 by Dr W. Raake, human a-thrombin by Dr J . W. Fenton 11, and '251-human factor V by Dr M. E. Nesheim, and the assistance of Xianjun Yang are gratefully acknowledged.
REFERENCES 1. Ofosu, F. A., Modi, G. J., Hirsh, J., Buchanan, M. R. & Blajchman, M. A. (1986) Ann. N . Y . Arad. Sci. 485, 41 -55. 2. Beguin, S., Lindhout, T. & Hemker, H. C. (1 988) Thromb. Haemostasis 60, 457 - 462. 3. Yang, X.-J., Blajchman, M. A,, Craven, S., Smith, L. M., Anvari, N. & Ofosu, F. A. (1990) Biochem. J . 272,399-406. 4. Ofosu, F. A., Modi, G. J., Blajchman, M. A,, Buchanan, M. R. & Johnson, E. A. (1987) Biochem. J . 248, 889-896. 5. Ofosu, F. A., Buchanan, M. R., Anvari, N., Smith, L. M. & Blajchman, M. A. (1989) Ann. N . Y . Acad. Sci.556,123-131. 6. Beguin, S. S., Dol, F. & Hemker, H. C. (1991) Sem. Thromb. Hemostasis 17, Suppl. 1, 126- 128. 7. Raake, W., Klauser, R. J., Meintsberger, E., Zeller, P. & Elling, H. (1991) Sem. Thromh. Hemostasis 17, Suppl. I , 129-135. 8 Ofosu, F. A., Choay, J., Anvari, N., Smith, L. M. & Blajchman, M. A. (1990) Eur. J. Biochem. 193,485-493. 9 Kettner, C . & Shaw, E. (1979) Thromb. Res. 14,969-973. 10 Nesheim, M. E., Kettner, C., Shaw, E. & Mann, K. G . (1987) J . B i d . Chem. 256, 6537-6540. 11 Ofosu, F. A,, Blajchman, M. A. & Hirsh, J . (1980) Thromh. Rex 20, 391 -403. 12 Fenton, J. W., Fasco, M. J., Stackrow, A. B., Aronson, D., Young, A . M . & Finlayson, 3. (1977)J. B i d . Chem. 252,35873598. 13 Miller-Andersson, M., Borg, H. & Anderson, L.-0. (1974) Thromh. Res. 5,439 - 446. 14 Hoogendoorn, H., Cerskus, A,, Ofosu, F. A.. Blajchman, M. A. & Hirsh, J. (1980) Thromb. Res. 20, 77-83. 15 Ofosu, F. A,, Hirsh, J., Esmon, C. T., Modi, G. J., Smith, L. M., Anvari, N., Buchanan, M. R., Fenton, J. W. & Blajchman, M. A. (1989) Biochem. J . 257, 143- 150. 16. Ofosu, F. A,, Smith, L. M., Anvari, N. & Blajchman, M. A. (1988) Thromb. Haemostasis 60, 193-198. 17. Koedam, J. A., Hamer, R. J., Beeser-Visser, N. H., Bouma, B. N. & Sixma, J. J. (1990) Eur. J . Biochem. 189, 229-234. 18. Monkovic, D. D. &Tracy, P. B. (1990) Biochemistry 29, 11183 128. 19. Bjork, I., Olson, S. T. &Shore, J. D. (1989) in Heparin: chemical und biological properties (Lane, D. A. & Lindahl, U., eds) pp. 229-255, Edward Arnold, London. 20. Holmer, E., Soderberg, K., Bergqvist, D. & Lindahl, U. (1986) Haemostasis 16, Suppl. 2, 1 - 12. 21. Tollefsen, D . M., Peacock, M . E. & Monafo, W. J. (1986) J . Bid. Chem. 261,8854-8858. 22. Maimone, M. M. & Tollefsen, D. M. (1990) J . B i d . Chem. 265, 18263 - 18271. 23 Casu, B., Johnson, E. A,, Mantovani, M., Mulloy, B., Orestc, P., Pescador, R., Prino, G., Torr, G. & Zoppetti, G. (1983) Arzneim. Forsch. 33, 135 - 142. 24 Van Ryn-McKenna, J., Ofosu, F. A., Hirsh, J . & Buchanan, M. R. (1989) Br. J . Haematol. 71, 265-269.
