John P. Sheehan, Department of Medicine/Hematology-Oncology, University of Wisconsin, 600 Highland Avenue, CSC K6/5 Mail Code 5669, Madison, WI 53792-5669, USA. Tel.: +1 608 262 1964; fax: +1 608 265 8133. E-mail: email@example.com
Summary. Background: Although heparin possesses multiple mechanisms of action, enhanced factor Xa inhibition by antithrombin is accepted as the predominant therapeutic mechanism. The contribution of FIXa inhibition to heparin activity in human plasma remains incompletely defined.
Objectives: To determine the relevance of FIXa as a therapeutic target for heparins, particularly serpin-independent inhibition of intrinsic tenase (FIXa–FVIIIa) activity.
Patients/Methods: Thrombin generation was detected by fluorogenic substrate cleavage. The inhibitory potencies (EC50s) of low molecular weight heparin (LMWH), super-sulfated LMWH (ssLMWH), fondaparinux and unfractionated heparin (UFH) were determined by plotting concentration vs. relative velocity index (ratio ± heparin). Inhibition was compared under FIX-dependent and FIX-independent conditions (0.2 or 4 pm tissue factor [TF], respectively) in normal plasma, and in mock-depleted or antithrombin/FIX-depleted plasma supplemented with recombinant FIX.
Results: UFH and fondaparinux demonstrated similar potency under FIX-dependent and FIX-independent conditions, whereas LMWH (2.9-fold) and ssLMWH (5.1-fold) demonstrated increased potency with limiting TF. UFH (62-fold) and fondaparinux (42-fold) demonstrated markedly increased EC50 values in antithrombin-depleted plasma, whereas LMWH (9.4-fold) and ssLMWH (two-fold) were less affected, with an EC50 within the therapeutic range for LMWH. The molecular target for LMWH/ssLMWH was confirmed by supplementing FIX/antithrombin-depleted plasma with 90 nm recombinant FIX possessing mutations in the heparin-binding exosite. Mutated FIX demonstrated resistance to inhibition of thrombin generation by LMWH and ssLMWH that paralleled the effect of these mutations on intrinsic tenase inhibition.
Conclusions: Therapeutic LMWH concentrations inhibit plasma thrombin generation via antithrombin-independent interaction with the FIXa heparin-binding exosite.
Unfractionated heparin (UFH), a mixture of polysaccharide chains (average molecular mass of 15–18 kDa) with broad polydispersity, has been a mainstay in the treatment of acute thrombosis since the late 1930s . UFH has multiple in vitro mechanisms of action, which include: antithrombin (AT)-dependent inhibition of thrombin, factor Xa, FIXa, FXIa, and FXIIa; heparin cofactor II (HCII)-dependent inhibition of thrombin; and serpin-independent inhibition of FX activation by the intrinsic tenase complex [2,3]. The AT-dependent activities of UFH require the high-affinity pentasaccharide sequence, which is present in fewer than one-third of heparin chains . Acceleration of thrombin inhibition additionally requires an oligosaccharide chain length of at least 18 saccharide units to act as a ‘template’ for the binding of both inhibitor and protease [2,5]. Low molecular weight heparin (LMWH), derived by partial chemical or enzymatic depolymerization of UFH (average molecular mass of 5 kDa), has lower inhibitory activity than thrombin relative to UFH, because of this reduced chain length. LMWH preferentially inhibits FXa, demonstrating anti-FXa/anti-FIIa activity ratios of 2 : 1 to 4 : 1 (vs. 1 : 1 for UFH). Similarly, fondaparinux is a semisynthetic form of the high-affinity pentasaccharide that accelerates protease inhibition solely by conformational activation of AT, and has trivial effects on thrombin inhibition . Thus, acceleration of FXa inhibition via conformational activation of AT is generally accepted as the predominant therapeutic mechanism for low molecular weight forms of heparin.
The equivalent clinical outcomes obtained with LMWH and UFH in the treatment of venous thromboembolism (VTE) argue that template-mediated thrombin inhibition is not critical to the efficacy of these heterogeneous preparations [6–8]. The efficacy of fondaparinux in the initial treatment of VTE likewise suggests that conformational activation of AT is sufficient for an antithrombotic effect [9,10]. In contrast, heparin depleted of high-affinity pentasaccharide (low-affinity heparin) retains in vivo antithrombotic efficacy in an animal model, suggesting that AT-independent mechanisms may also contribute to therapeutic efficacy . Therapeutic UFH and LMWH concentrations selectively inhibit intrinsic tenase activity via an AT-independent mechanism in a purified system; however, the complexity of protein–heparin interactions and simultaneous AT-dependent effects in human plasma obscure the potential contribution of this mechanism to antithrombotic effects [3,12,13]. Super-sulfated LMWH (ssLMWH), derived from LMWH by sodium periodate oxidation to reduce AT affinity followed by O-sulfation, is an AT-independent inhibitor of the assembled intrinsic tenase complex and, to a lesser extent, prothrombinase in a purified system . This AT-independent LMWH is a valuable tool for evaluating the importance of FIXa and the intrinsic tenase complex as a potential target for heparin in human plasma.
FX activation by the intrinsic tenase complex is the rate-limiting step for thrombin generation [15–17], and the interaction between FIXa and the FVIIIa A2 subunit is the critical protein–protein interaction for cofactor activation of the protease within this enzyme complex . Mutagenesis of human FIXa demonstrates an extensive overlap between the cofactor and heparin-binding sites on the protease domain [13,19,20]. Heparin oligosaccharides bound to this protease exosite specifically disrupt interaction with the cofactor A2 subunit, inhibiting FX activation by the intrinsic tenase complex . This protease exosite is a critical regulator of hemostasis, based on the ability of selected mutations to regulate the rate of plasma thrombin generation and formation of saphenous vein thrombi in response to FeCl3-induced injury in the mouse . To determine the physiologic relevance of serpin-independent inhibition of FIXa by therapeutic heparins, we employed a modified thrombin generation assay (TGA), immunodepleted plasmas and a panel of recombinant FIX proteins with mutations in the heparin-binding exosite to evaluate the contribution of: (i) the intrinsic tenase complex as a therapeutic target; (ii) AT-independent inhibition mechanisms; and (iii) the heparin binding exosite of FIXa as a molecular target. The results demonstrate that therapeutic concentrations of LMWH inhibit plasma thrombin generation in an AT-independent manner via interaction with the FIXa heparin-binding exosite.
Materials and methods
Human pooled plasma and FIX-deficient patient plasmas were purchased from George King (Overland Park, KS, USA). FIX-depleted and FIX/AT-double-depleted human plasmas derived from the same parent plasma and neutralizing sheep antibody against human HCII were purchased from Affinity Biologicals (Ancaster, ON, Canada). Corn trypsin inhibitor (CTI), human plasma-derived FIX, FIXa and thrombin were purchased from Enzyme Research (South Bend, IN, USA). Phosphatidylserine (PS) and phosphatidylcholine (PC) were purchased from Avanti Lipids (Alabaster, AL, USA). Cholesterol was purchased from Calbiochem (San Diego, CA, USA). PC:PS:cholesterol (molar ratio 75 : 25 : 1) phospholipid vesicles (PC:PS vesicles) were prepared by extrusion through a 100-nm polycarbonate filter .
The LMWH dalteparin was from Eisai (Woodcliff Lake, NJ, USA). Porcine intestinal UFH with a predominant Mr of 17 000–19 000 and bovine serum albumin (BSA) were purchased from Sigma (St Louis, MO, USA). Fondaparinux was from GlaxoSmithKline (Research Triangle Park, NC, USA). ssLMWH was generously provided by J. Weitz (McMaster University, Hamilton, ON, Canada). Dimethylsulfoxide (DMSO) was purchased from Mallinckrodt (St Louis, MO, USA). Lyophilized bovine thrombin–α2-macroglobulin complex was purchased from Diagnostica Stago (Parsippany, NJ, USA). Thromborel S, a human thromboplastin from Dade Behring (Deerfield, IL, USA), was used as the source of relipidated human tissue factor (TF). The fluorogenic substrate Z-Gly-Gly-Arg-AMC-HCl was obtained from Bachem (King of Prussia, PA, USA).
Expression and purification of recombinant FIX
A HEK 293 cell line stably transfected with human FIX R170A was provided by D. Stafford (University of North Carolina-Chapel Hill) . Stable HEK 293 cell lines expressing human FIX wild type (WT), K126A, K132A, R165A and R233A were constructed as described previously . Recombinant FIX proteins were purified to homogeneity from conditioned media, and quantified by absorbance at 280 nm.
Fluorogenic assay for detection of plasma thrombin generation
Thrombin generation in human plasma was detected by cleavage of the fluorogenic substrate Z-Gly-Gly-Arg-AMC as previously described, with a 360/40-nm excitation and 460/40-nm emission filter set in a Biotek Synergy HT fluorescent plate reader equipped with Gen 5 software (Biotek Instruments, Winooski, VT, USA) . The substrate Z-Gly-Gly-Arg-AMC-HCl was reconstituted at 100 mm in DMSO and stored at − 20 °C. Prior to each assay, a fresh fluorogenic substrate and calcium solution (Flu Ca substrate) was prepared by adding 100 μL of 1.0 m CaCl2 to 875 μL of 20 mm Hepes (pH 7.35) and 60 g L−1 BSA at 37 °C, and then 25 μL of 100 mm Z-Gly-Gly-Arg-AMC in DMSO with vigorous mixing, to yield a final concentration of 2.5 mm Z-Gly-Gly-Arg-AMC-HCl and 100 mm CaCl2. Plasmas were thawed sequentially on ice for 5 min, at room temperature for 5 min, and in a 37 °C water bath for 5 min. Calibration with bovine thrombin–α2-macroglobulin complex calibrator at final plasma concentrations of 500, 250, 50 and 5 nm was performed as described previously . The raw data for the first 10 min were imported into technothrombin tga evaluation software from Technoclone (Vienna, Austria) to construct calibration curves for each plasma.
The initiator solution was composed of 0.12 mg mL−1 CTI, 25 μm PC:PS vesicles, 0.6 or 12 pm TF and 0–40 μm heparins in TGA buffer. For comparison purposes, weight-based heparin concentrations were converted to approximate molarity by using average molecular masses of 18 000, 5000 and 1728 Da for UFH, LMWH/ssLMWH, and fondaparinux, respectively. Plasma (60 μL) and initiator solution (20 μL) were added to each well and preheated at 37 °C for 10 min. Flu Ca substrate (20 μL) at 37 °C was then added and mixed at medium intensity for 5 s, and readings were obtained at 30-s intervals for 1 h. Final concentrations (extrapolated to the 60-μL plasma volume) were 0.2 pm (limiting) or 4 pm (excess) TF, 8.3 μm PC:PS vesicles, 40 μg mL−1 CTI, heparin (0–10 or 0–40 μm), and 90 nm plasma-derived or recombinant FIX (if present). For experiments assessing the contribution of HCII to inhibition of thrombin generation by LMWH, FIX/AT-depleted plasma was preincubated with either neutralizing sheep anti-human HCII IgG or control sheep IgG at a 1 : 9 (v/v) ratio for 20 min at room temperature.
Fluorescent signal data were exported to technothrombin tga evaluation software, and thrombin generation over time was determined with the appropriate calibration curve for each plasma. The thrombin generation parameters lag time (start until first burst in thrombin formation), peak thrombin concentration, time to peak thrombin concentration and velocity index (slope between the end of lag time and peak thrombin concentration) were determined with the software. The potency of heparin inhibition of plasma thrombin generation was estimated by plotting inhibitor concentration vs. velocity index and fitting the data to the following equation to obtain the EC50:
B represents the fractional inhibition, I represents the concentration of heparin, m represents the response for an ‘infinite’ dose, the interval was set as [0,1], EC50 represents the concentration of heparin that that causes a 50% reduction in the velocity index for thrombin generation, and n represents the pseudo-Hill coefficient .
The time course of plasma thrombin generation as determined by western blotting
Thrombin generation was initiated in 1.5-mL Eppendorf tubes with 0.2 pm human TF, 8.3 μm PC:PS vesicles and 40 μg mL−1 CTI in mock-depleted plasma with or without the indicated concentrations of heparin at 37 °C. Individual reactions were quenched by addition of 100 μL of 2 × SDS-PAGE loading buffer containing 5 m urea at the indicated time points. Quenched samples were incubated at 37 °C for 5 min, boiled for 5 min, diluted 1 : 20 in non-reducing SDS-PAGE loading buffer, and subjected to 4–12% gradient SDS-PAGE, with overnight transfer to Immobilon-P (Millipore, Billerica, MA, USA). Membranes were blocked in 5% milk/1% BSA, and detected with polyclonal sheep anti-human thrombin antibody (Haematologic Technologies, Essex Junction, VT, USA) followed by peroxidase-conjugated affinity-purified donkey anti-sheep IgG (Jackson Immunoresearch, West Grove, PA, USA), as described previously . The washed Immobilon-P membrane was immersed in Supersignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA) for 5 min, and exposed to Kodak BioMax Xar film (Kodak, Rochester, NY, USA).
Inhibition of plasma thrombin generation by heparins in the presence of limiting or excess TF concentration
To evaluate the contribution of intrinsic tenase complex as a molecular target for heparin, thrombin generation was triggered by 0.2 pm or 4 pm TF in pooled normal human plasma in the presence of increasing concentrations of LMWH, ssLMWH, fondaparinux, and UFH (Fig. 1). As previously reported in our system, the magnitude of the thrombin response is highly dependent on FIX when triggered by limiting TF (0.2 pm), but is largely independent of FIX when triggered by excess TF (4 pm) . In the absence of TF, pooled plasma alone failed to generate detectable thrombin. Addition of 0.2 pm TF triggered the expected thrombin generation response (Fig. 1A,D,G,J) in the absence of heparin, whereas 4 pm TF triggered a response with a moderately enhanced peak thrombin concentration and a shortened lag phase (Fig. 1B,E,H,K). Representative dose responses for LMWH, ssLMWH, fondaparinux and UFH are shown for each TF condition (Fig. 1), and relative heparin potency is expressed as the mean EC50 value for reduction in the velocity index (slope) for thrombin generation (Table 1). Our analysis emphasizes the velocity index (slope), as opposed to the ‘endogenous thrombin potential’ (area under the curve), as the former parameter is defined earlier in the time course, and is consequently less affected by fluorogenic substrate or prothrombin depletion . LMWH demonstrated a dose-dependent decrease in velocity index (slope) and peak thrombin concentration under both TF conditions, with relatively less prolongation of the time to peak thrombin concentration in the presence of excess TF (Fig. 1A,B). Under limiting TF conditions, LMWH completely inhibited plasma thrombin generation, whereas in the presence of excess TF, modest (5–10%) activity remained at the highest concentration (10 μm; Fig. 1C). Increasing ssLMWH concentrations resulted in a similar pattern of inhibition, with dose-dependent reductions in velocity index and peak thrombin concentration, and minimal prolongation of the time to peak thrombin concentration in the presence of excess TF (Fig. 1D,E). Partial inhibition of thrombin generation was observed, with residual activity of approximately 5% and 35% for 0.2 pm and 4 pm TF, respectively (Fig. 1F). Finally, fondaparinux (Fig. 1G,H) and UFH (Fig. 1J,K) also demonstrated dose-dependent reductions in velocity index and peak thrombin concentration, with modest prolongation of the time to peak thrombin concentration in the presence of either 0.2 pm or 4 pm TF. Both of these heparins demonstrated complete or near complete inhibition (Fig. 1I,L), with almost identical EC50 values under both TF conditions (Table 1). In contrast, LMWH and ssLMWH demonstrated 2.9-fold and 5.1-fold lower EC50 values, respectively, in the presence of limiting TF (Table 1). The enhanced potency under FIX-dependent conditions suggests that inhibition of intrinsic tenase activity contributes to the mechanism of action for LMWH and ssLMWH in human plasma.
Table 1. EC50 values for inhibition of the mean velocity index for thrombin generation by heparin in pooled normal human plasma
0.2 pm TF EC50 (μm ± SEM)
4 pm TF EC50 (μm ± SEM)
LMWH, low molecular weight heparin; SEM, standard error of the mean; ssLMWH, super-sulfated low molecular weight heparin; TF, tissue factor; UFH, unfractionated heparin. Thrombin generation was initiated with 0.2 pm or 4 pm human TF, 8.3 μm phosphatidylcholine:phosphatidylserine vesicles and 40 μg mL−1 corn trypsin inhibitor (plasma concentrations) in pooled normal human plasma in the presence of increasing heparin concentrations (LMWH and fondaparinux, 0–10 μm; UFH, 0–1 μm). Duplicate wells were averaged to determine individual thrombin generation curves. The relative velocity index was plotted vs. heparin concentration for each curve, and the EC50 value eas determined as described in Materials and methods. The mean EC50 ± SEM was determined from three independent replicates (n = 3). Fold increase describes the ratio between EC50 values obtained in the presence of excess vs. limiting TF concentrations.
0.20 ± 0.02
0.57 ± 0.08
0.27 ± 0.03
1.4 ± 0.14
0.012 ± 0.002
0.010 ± 0.001
0.13 ± 0.003
0.12 ± 0.006
Effect of heparins on TF-triggered plasma thrombin generation as detected by western blotting
To further assess the relevant mechanism(s) for inhibition of plasma thrombin generation by each heparin, the time course of thrombin activation and inhibition products was monitored by western blotting. Thrombin generation was initiated with 0.2 pm TF in pooled normal human plasma in the absence or presence of each heparin preparation, present at the approximate EC50 value (Table 1). The reaction products were analyzed by SDS-PAGE under non-reducing conditions with a primary antibody that simultaneously detected prothrombin, thrombin-related cleavage products, and thrombin–AT complex (TAT) (Fig. 2). Pooled normal plasma without heparin demonstrated the initial appearance of free thrombin at 3 min, peak free thrombin at 6 min, initial TAT at 6 min with subsequent accumulation over time, and progressive depletion of the prothrombin/meizothrombin band (Fig. 2A). In the presence of 0.2 μm LMWH, peak thrombin concentration was delayed to 7.5 min, with reduced intensity of the TAT band and less depletion of the prothrombin/meizothrombin band relative to the absence of heparin (Fig. 2B). Similarly, addition of ssLMWH (0.25 μm) or fondaparinux (0.13 μm) resulted in a delay in the appearance of peak thrombin generation and TAT, and reduced prothrombin/meizothrombin depletion (Fig. 2C,D). In contrast, addition of UFH (0.012 μm) resulted in delayed peak thrombin concentration but early (3 min) and enhanced appearance of TAT, with depletion of the prothrombin/meizothrombin band that was similar to that in the absence of heparin (Fig. 2E). When UFH was increased to 0.31 μm (1.0 U mL−1), there was no discernible free thrombin, TAT accumulation, or prothrombin/meizothrombin depletion (Fig. 2F). The effect of heparins on the time course of plasma thrombin generation as determined with western blotting correlated well with results of the fluorogenic substrate assay. LMWH, ssLMWH and fondaparinux clearly reduced prothrombin/meizothrombin consumption, whereas UFH primarily accelerated inhibition of thrombin at their EC50 values in the TGA.
Effect of heparins on thrombin generation triggered by limiting TF in AT-depleted plasma
To evaluate the contribution of AT to the heparin inhibition mechanism, thrombin generation triggered by limiting TF (0.2 pm) was compared in FIX-depleted or FIX/AT-double-depleted plasma supplemented with 100% (90 nm) plasma-derived FIX (Fig. 3). These plasmas were subjected to identical immune depletion from the same parent plasma to minimize preanalytic variables in the comparison of control and AT-depleted plasma. In the absence of TF, no detectable thrombin was generated in either FIX-supplemented, immunodepleted plasma. In the presence of TF, the peak thrombin concentration in the absence of heparin was significantly enhanced in the AT-depleted plasma (Fig. 3B,E,H,K) relative to the AT-replete control plasma (Fig. 3A,D,G,J). The effect of LMWH on thrombin generation in control plasma was similar to that observed in pooled plasma, with dose-dependent reductions in velocity index and peak thrombin concentration, minimal prolongation of the time to peak thrombin concentration, and complete inhibition (Fig. 3A). In AT-depleted plasma, LMWH demonstrated more gradual reductions in velocity index and peak thrombin concentration, with progressive delay in the time to peak thrombin concentration (Fig. 3B). LMWH demonstrated partial inhibition in AT-depleted plasma, with residual activity appearing to reach a plateau beyond approximately 20 μm. AT depletion resulted in a 9.4-fold increase in the EC50 for inhibition of thrombin generation by LMWH (Fig. 3C; Table 2). The potential contribution of HCII to inhibition by LMWH was investigated by preincubation of the AT-depleted plasma with neutralizing antibodies against HCII. Neutralization of HCII resulted in a < 1.4-fold increase in the EC50 for LMWH, suggesting only a modest contribution of this serpin to the inhibition of thrombin generation in the absence of AT (data not shown). ssLMWH demonstrated dose-dependent reductions in velocity index and peak thrombin concentration, with progressive delays in the time to peak thrombin concentration in both plasmas (Fig. 3D,E). Nearly complete inhibition was observed for ssLMWH in both plasmas. AT depletion resulted in only a two-fold increase in the EC50 for thrombin inhibition by ssLMWH (Fig. 3F; Table 2). In contrast, fondaparinux demonstrated dose-dependent reductions in velocity index and peak thrombin concentration, with only a modest prolongation of the time to peak thrombin concentration, and complete inhibition in control plasma (Fig. 3G). This inhibition pattern was greatly blunted, with only partial inhibition (∼ 30% residual), in AT-depleted plasma (Fig. 3H), and there was only a modest delay in peak thrombin concentration in either plasma. AT depletion resulted in a 42-fold increase in the EC50 for thrombin inhibition by fondaparinux (Fig. 3I; Table 2). Similarly, UFH demonstrated complete inhibition in control plasma without a marked delay in the time to peak thrombin concentration (Fig. 3J), but only partial inhibition (∼ 25% residual) with a progressive delay to peak thrombin concentration in the AT-depleted plasma (Fig. 3K). AT depletion resulted in a 63-fold increase in the EC50 for thrombin inhibition by UFH (Fig. 3L; Table 2).
Table 2. EC50 values for heparin inhibition of the mean velocity index for thrombin generation in FIX-depleted or FIX/antithrombin (AT)-depleted plasma supplemented with plasma-derived FIX
FIX-depleted plasma EC50 (μm ± SEM)
FIX/AT-depleted plasma EC50 (μm ± SEM)
LMWH, low molecular weight heparin; NA, not applicable; SEM, standard error of the mean; ssLMWH, super-sulfated low molecular weight heparin; UFH, unfractionated heparin. Thrombin generation was initiated with 0.2 pm human tissue factor, 8.3 μm phosphatidylcholine:phosphatidylserine vesicles and 40 μg mL−1 corn trypsin inhibitor (plasma concentrations) in FIX-depleted or FIX/AT-depleted human plasma supplemented with 100% plasma-derived FIX in the presence of increasing heparin concentrations (LMWH and fondaparinux, 0–10 μm; UFH, 0–1 μm). Duplicate wells were averaged to determine individual thrombin generation curves. The relative velocity index was plotted vs. heparin concentration for each curve, and the EC50 value was determined as described in Materials and methods. The mean EC50 ± SEM was determined from three independent replicates (n = 3). Fold increase describes the ratio between EC50 values obtained in AT-depleted vs. control (mock depleted) plasma. *Therapeutic ranges for dalteparin and fondaparinux were obtained from the Micromedex and Physician’s Desk Reference, respectively (accessed online 15 January 2011).
Effect of mutations in the heparin-binding exosite of recombinant FIX on the ability of LMWH to inhibit TF-triggered plasma thrombin generation in the absence of AT
The molecular target for AT-independent inhibition of thrombin generation by LMWH was assessed by supplementing FIX/AT-depleted plasma with 90 nm recombinant FIX possessing mutations in the protease heparin-binding exosite [13,19]. When incorporated into the intrinsic tenase complex with purified protein components, the proteases derived from these zymogens showed increased resistance to inhibition by LMWH (R233A > R126A > R165A/K132A/R170A > WT) . In the absence of LMWH, these recombinant FIX mutants also showed different baseline thrombin generation in human plasma: peak thrombin concentrations were similar for FIX WT and R170A (or modestly enhanced), modestly reduced for FIX K126A and K132A, and significantly blunted and delayed for FIX R233A and R165A (Fig. 4), consistent with the effect of these mutations on the protease–cofactor interaction [13,19]. A minimal thrombin response was noted in the absence of FIX. Increasing LMWH concentrations resulted in a progressive reduction in velocity index (slope) and peak thrombin concentration, and prolongation of the time to peak thrombin concentration (Fig. 4). Similarly to plasma-derived FIX, LMWH partially inhibited thrombin generation in the presence of FIX WT, with residual activity (∼ 5–10%) even at 40 μm. Recombinant FIX with reduced heparin affinity showed relative resistance to inhibition of thrombin generation, similar to that observed for inhibition of the intrinsic tenase complex with the purified protease forms . In particular, FIX R233A was highly resistant to LMWH inhibition of thrombin generation (Fig. 4), with an 11-fold increase in EC50 value relative to FIX WT (Table 3). FIX K126A showed a 4.3-fold increase in EC50 for LMWH, with a marked increase in residual activity (∼ 45%) relative to wild-type protease at high concentrations (Fig. 4; Table 3). On the basis of both relative EC50 values for reduction in the velocity index and the maximal degree of inhibition, plasma supplemented with mutant FIX showed relative resistance to inhibition of thrombin generation by LMWH as follows: R233A > K126A/R165A/K132A > R170A > WT (Table 3).
Table 3. EC50 values for inhibition of the mean velocity index for thrombin generation by heparin in FIX/antithrombin (AT)-depleted plasma supplemented with 100% recombinant factor IX (rFIX)
LMWH (μm ± SEM)
Fold increase (relative to WT)
ssLMWH (μm ± SEM)
Fold increase (relative to WT)
LMWH, low molecular weight heparin; SEM, standard error of the mean; ssLMWH, super-sulfated low molecular weight heparin; WT, wild type. Thrombin generation was initiated with 0.2 pm human tissue factor, 8.3 μm phosphatidylcholine:phosphatidylserine vesicles and 40 μg mL−1 corn trypsin inhibitor (plasma concentrations) in FIX/AT-depleted plasma supplemented with 100% rFIX in the presence of increasing heparin concentrations (ssLMWH, 0–10 μm; LMWH, 0–40 μm). Duplicate wells were averaged to determine individual thrombin generation curves. The relative velocity index was plotted vs. heparin concentration for each curve, and the EC50 value was determined as described in Materials and methods. The mean EC50 ± SEM was determined from three independent replicates (n = 3).
1.00 ± 0.07
0.41 ± 0.03
11.4 ± 1.02
2.16 ± 0.12
4.34 ± 0.09
1.43 ± 0.05
5.80 ± 0.24
1.39 ± 0.09
2.26 ± 0.14
0.73 ± 0.06
4.08 ± 0.22
1.29 ± 0.14
Effect of mutations in the heparin-binding exosite of recombinant FIX on the ability of ssLMWH to inhibit TF-triggered plasma thrombin generation in the absence of AT
The molecular target for AT-independent inhibition of thrombin generation by ssLMWH was similarly assessed in FIX/AT-depleted plasma supplemented with 90 nm recombinant FIX (Fig. 5). Increasing ssLMWH concentrations resulted in progressive reductions in velocity index (slope) and peak thrombin concentration, and prolongation of the time to peak thrombin concentration similar to that seen with LMWH (Fig. 5). In contrast, ssLMWH demonstrated near complete inhibition of plasma thrombin generation in the presence of FIX WT, and enhanced potency relative to LMWH for all recombinant FIX proteins, based on relative EC50 values (Table 3). Recombinant FIX with reduced heparin affinity demonstrated relative resistance to inhibition of thrombin generation as compared with the wild-type protein, similar to that observed for inhibition of the intrinsic tenase complex with purified components . The magnitude of the differences between mutant and wild-type proteins was reduced for ssLMWH relative to LMWH (Table 3), with a less pronounced tendency for partial inhibition among the mutant FIX proteins (Fig. 5). However, the rank order of relative resistance to inhibition of thrombin generation based on the EC50 values for reduction in the velocity index for thrombin generation remained similar: R233A > K126A/R165A/K132A > R170A > WT (Table 3).
Heparin is a complex, heterogeneous mixture of oligosaccharide chains that is capable of inhibiting multiple steps in blood coagulation via both AT-dependent and AT-independent mechanisms. LMWH demonstrates more predictable pharmacokinetics and a reduced ability to accelerate thrombin inhibition via a ‘template’ mechanism, but retains the heterogeneity of UFH . Accelerated inhibition of FXa via conformational activation of AT is generally accepted as the predominant therapeutic mechanism for LMWH. We examined the potential contribution of AT-independent intrinsic tenase inhibition to the anticoagulant activity of LMWH in a physiologically relevant system. The biological significance of TF-triggered plasma thrombin generation measurements is supported by the association of enhanced thrombin generation with increased risk of recurrent VTE [26,27], and the ability to discern the clinical severity of bleeding phenotypes in hemophilia . Our results demonstrate the potential relevance of AT-independent inhibition of FIXa as a therapeutic target for LMWH. Whereas UFH and fondaparinux demonstrated similar potencies for inhibition of plasma thrombin generation under both FIX-dependent (limiting TF) and FIX-independent (excess TF) conditions , LMWH and ssLMWH demonstrated increased potency with limiting TF (Fig. 1; Table 1). Western blot analysis of thrombin generation suggests that ssLMWH, LMWH, fondaparinux and UFH (1.0 U mL−1) act predominantly to reduce prothrombin activation, rather than accelerating thrombin inhibition by AT (Fig. 2). Given the equivalent clinical outcomes for UFH and LMWH in the treatment of VTE [6–8], these results suggest that thrombin inhibition is not critical to the therapeutic action of these heterogeneous heparin preparations. In contrast, the enhanced potency of LMWH/ssLMWH under FIX-dependent conditions (Table 1) suggests that inhibition of the intrinsic tenase complex contributes to LMWH activity in human plasma.
The contribution of AT-independent mechanisms to the inhibition of thrombin generation by LMWH was demonstrated in AT-depleted plasma. Fondaparinux and UFH demonstrated markedly reduced potency (Fig. 3I,L), with EC50 values approximately three-fold to eight-fold higher than their therapeutic ranges (Table 2), confirming dependence on AT-dependent mechanisms. In contrast, LMWH and ssLMWH were substantially less affected by AT depletion (Fig. 3B,E; Table 2). Remarkably, the EC50 (1.13 ± 0.18 μm) for LMWH remained within the therapeutic range (0.76–2.31 μm or 0.5–1.5 U mL−1), suggesting that AT-independent mechanisms may contribute to LMWH activity in human plasma (Table 2). The LMWH dose response in AT-depleted plasma demonstrated partial inhibition of thrombin generation, with a plateau of approximately 8–10% starting activity (Fig. 3A–C), similar to inhibition of intrinsic tenase activity by LMWH with purified components . Likewise, recombinant FIX possessing mutations that reduce protease–heparin affinity (R233A, K126A, R165A, and K132A) and/or increase cofactor affinity (R170A) demonstrated resistance to LMWH inhibition of thrombin generation in AT-depleted plasma (Figs 4 and 5), with the rank order of EC50 values (Table 3) roughly correlating with the KI values for intrinsic tenase inhibition by LMWH with purified components [13,19,20]. These results suggest that therapeutic LMWH concentrations inhibit plasma thrombin generation via an AT-independent interaction with the FIXa heparin-binding exosite. Additionally, the dose response demonstrates partial inhibition at high LMWH concentrations, which may broaden the therapeutic range and limit the bleeding risk associated with this anticoagulant mechanism.
This mechanism for plasma thrombin inhibition is supported by studies performed with purified protein components and AT-independent glycosaminoglycans. These studies demonstrate that: (i) LMWH inhibits FX activation by FIXa (in the presence or absence of cofactor) via interaction with a protease exosite that overlaps with a critical FVIIIa-binding site [12,19,20]; (ii) mutations in the protease domain that reduce FIXa–heparin affinity result in resistance to LMWH inhibition of FX activation by FIXa–phospholipid or the intrinsic tenase complex [13,20]; (iii) LMWH and fucosylated chondroitin sulfate (also known as depolymerized holothurian glycosaminoglycan [DHG]) bind to overlapping sites on the FIXa protease domain; and (iv) protease mutations that reduce FIXa–heparin affinity also result in resistance to inhibition by DHG . Likewise, studies in human plasma (and the murine model of hemophilia B) demonstrate that: (i) mutations within the FIXa heparin-binding site regulate plasma thrombin generation and in vivo thrombosis ; (ii) DHG inhibits plasma thrombin generation via interaction with the FIXa heparin-binding exosite ; and (iii) LMWH inhibits plasma thrombin generation via an AT-independent interaction with the same protease exosite (this study). The purified system employs a two-step assay in which cofactor is activated by excess thrombin and neutralized by hirudin, and the thrombin-activated FVIIIa is immediately added to the tenase reaction at a 10-fold molar excess over FIXa. Under these conditions, a significant contribution from ‘feedback’ activation of FVIII by FXa is highly unlikely, given the near complete cofactor activation by excess thrombin in the first step, and extensive saturation of the protease with FVIIIa in the second step. Clearly, multiple mechanisms are possible in plasma, but the primacy of intrinsic tenase inhibition via interaction with the protease heparin-binding exosite is supported by the common AT-independent inhibition mechanism established for DHG and LMWH, the absence of a significant effect of LMWH on FIX activation (data not shown), and the dominant effect of mutations in the heparin-binding exosite of FIXa on the inhibition of thrombin generation. Addition of either 700 pm FVIII or thrombin-activated FVIIIa to FVIII-deficient plasma does not significantly affect inhibition of TF-triggered thrombin generation by DHG, indicating that effects on cofactor activation do not contribute significantly to this glycosaminoglycan-mediated, AT-independent inhibition mechanism . The inability to bypass this inhibition with activated cofactor (or activated protease), along with the dominant effect of the FIXa heparin-binding site mutations, provides strong support for direct interaction with the protease as the dominant inhibition mechanism.
The rationale for FIXa as an important antithrombotic target is provided by the markedly enhanced potency of this protease in triggering thrombin generation relative to FXa, and the significantly higher in vivo thrombogenicity of this protease than of both FXa and thrombin [15,30]. FIXa-induced thrombus formation also demonstrates enhanced sensitivity to heparin inhibition relative to FXa and thrombin . Targeting this rate-limiting step interferes with amplification of the coagulation response, and provides the broadest dynamic range of inhibition . Furthermore, an intact TF pathway facilitates the use of bypass agents (e.g. FVIIa) for bleeding or emergency procedures. Targeting intrinsic tenase activity with active site-blocked FIXa, mAb against the FIXa Gla domain or a fucosylated chondroitin sulfate (DHG) demonstrates reduced bleeding risk relative to equitherapeutic doses of UFH/LMWH in animal models of thrombosis or cardiac bypass [32–37]. Thus, both theoretical considerations and animal models suggest that selective FIXa inhibition will reduce the bleeding risk associated with antithrombotic therapy.
The need for antithrombotic agents with reduced bleeding risk is particularly important for patients with malignancy, because of their significantly higher rates of both VTE recurrence and bleeding . LMWH demonstrates superior efficacy compared to standard therapy with vitamin K antagonists in preventing VTE recurrence in this patient population . Furthermore, the anti-inflammatory, antimetastatic and antitumor effects of ‘non-anticoagulant’ heparins in animal models may help to explain the potential favorable impact of LMWH on overall mortality in cancer patients [40,41]. Although hypersulfation of LMWH enhances in vitro affinity for FIXa , the in vivo use of oversulfated glycosaminoglycans may be problematic, as exemplified by the life-threatening anaphylactic reactions triggered by oversulfated chondroitin sulfate contaminants in heparin preparations [42,43]. Furthermore, oversulfated glycosaminoglycans may demonstrate reduced potency and bioavailability, owing to increased non-specific binding interactions. For example, ssLMWH and depolymerized fucosylated chondroitin sulfate demonstrate significant disparity between their relative inhibitory potencies for in vitro intrinsic tenase activity (KI) and ex vivo plasma thrombin generation (EC50) relative to LMWH. Whereas > 300-fold differences in apparent inhibitor affinity for FIXa exist in the purified enzyme assays (DHG > ssLMWH > LMWH), the maximal differences are < 3-fold for inhibition of plasma thrombin generation [3,20,25,29]. The clearest comparison is between ssLMWH and LMWH, with the former demonstrating 45-fold higher potency in assays with purified proteins, but < 2.5-fold higher potency in AT-depleted plasma. These differences are probably attributable to increased plasma protein binding, as previously demonstrated in the comparison between UFH and LMWH . Thus, an LMWH retaining only the chain modifications required for FIXa binding should maximize tenase inhibitory potency by reducing plasma protein binding and AT-dependent activity (elimination of 3-O sulfation). Despite the favorable risk profile of AT-independent glycosaminoglycans in animal models of thrombosis [36,37], only very limited clinical evaluation of chemically modified LMWH has been reported . Chemoenzymatic synthesis of homogeneous LMWH preparations potentially allows for specific targeting of FIXa and/or other important therapeutic targets . Targeting of FIXa with a defined LMWH preparation would represent a novel antithrombotic approach for high-risk populations.
We would like to thank D. Stafford for providing the FIX R170A cell line, A. Mueller-Beckhaus of Bayer HealthCare, LLC for recombinant FVIII (Kogenate FS), J. Weitz for ssLMWH, H. Hoogendoorn of Affinity Biologicals for providing immunodepleted plasmas, and Technoclone, Ltd for the technothrombin tga software for analysis of plasma thrombin generation. This research was supported by National Institutes of Health grant HL080452 (J. P. Sheehan).
Disclosure of Conflict of Interests
The authors state that they have no conflict of interest.