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Sensing active heparin by counting aggregated quantum dots at single-particle level Suli Dong, Xiaojun Liu, Qingquan Zhang, Wenfeng Zhao, Chenghua Zong, Aiye Liang, and Hongwei Gai ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.6b00528 • Publication Date (Web): 23 Dec 2016 Downloaded from http://pubs.acs.org on December 26, 2016

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Sensing active heparin by counting aggregated quantum dots at single-particle level

Suli Dong a, Xiaojun Liu a, Qingquan Zhang a, Wenfeng Zhao a, Chenghua Zong a, Aiye Liang b, Hongwei Gai a * a, Jiangsu Key Laboratory of Green Synthesis for Functional Materials, School of Chemistry and Chemical Engineering, Jiangsu Normal University, Xuzhou, Jiangsu, 221116, China. b, Department of Physical Sciences, Charleston Southern University, Charleston, South Carolina, USA

*Corresponding author. Email: [email protected] Fax: 86-516-83536972

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Abstract Developing highly sensitive and highly selective assays for monitoring heparin levels in blood is required during and after surgery. In previous studies, electrostatic interactions are exploited to recognize heparin and changes in light signal intensity are used to sense heparin. In the present study, we developed a quantum dot (QD) aggregation-based detection strategy to quantify heparin. When cationic micelles and fluorescence QDs modified with anti-thrombin III (AT III) are added into heparin sample solution, the AT III-QDs, which specifically bind with heparin, aggregate around the micelles. The aggregated QDs are recorded by spectral imaging fluorescence microscopy and differentiated from single QDs based on the asynchronous process of blue shift and photobleaching . The ratio of aggregated QD spots to all counted QD spots is linearly related to the amount of heparin in the range U/mL to 0.023 U/mL. The limit of detection is 9.3×10 -5 U/mL (~0.1 nM), and the recovery of the spiked heparin at 0.00465 U/mL (~5 nM) in 0.1% human plasma is acceptable. of 4.65×10

-4

Key words: heparin; spectral imaging microscopy; single particle; quantum dot; anti-thrombin III; aggregation


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Heparin is a linear heterogeneous polysaccharide that carries the strongest negative charges in the biological system due to its high degree of sulfate substitution. Heparin has been used as the dominant choice for anticoagulation during cardiac surgery because of its effectiveness, low cost and easy removal 1. Heparin specifically binds to antithrombin III (AT III) at a stoichiometric ratio of 1:1 through a pentasaccharide sequence. The complex enhances the inhibitory activity of AT III by up to 4500-fold against several key coagulation factors in the blood, such as thrombin, factor Xa and IXa 2, 3. Monitoring and quantification of heparin levels in the blood are critical issues in preventing clot formation during surgery and avoiding serious complications and bleeding after surgery. The current practice of heparin monitoring during surgery is primarily based on various clotting time assays, such as activated clotting time and activated partial thromboplastin time 4, 5. However, the coagulation cascade is a complicated process involving many coagulation factors, coagulation enzymes, and their interactions. Therefore, clotting time is affected by numerous variables. Blood clotting time is related to the anticoagulant activity of heparin, but the relationship is not a simple one. Assays based on clotting time provide the extrapolated heparin response time but not the exact amount of heparin. The results obtained from different instruments may vary considerably and are hardly interchangeable. Consequently, rapid, precise, and easy-to-operate heparin sensors should be developed for clinical detection. Bromfield et al. published a comprehensive review on heparin sensing in 2013 6. Heparin sensors can be classified into three types, namely, turn-on, turn-off and ratiometric (wavelength shift) sensors. For the turn-on sensors, two ways are usually adopted to switch on the optical signals. In the first method, the quenched fluorescence or the inhibited scattering light is recovered by adding heparin , which binds and removes the quencher or inhibitor. The recovered light signal intensity is related to the amount of heparin added. Typical quencher-removed sensors for the detection of heparin include Ru complex quench CdTe quantum dots (QDs) 7, graphene oxide quench rhodamine B modified polyethyleneimine (PEI) 8, protamine quench fluorescein-AuNPs 9, dendrimer quench graphene QDs 10, and gold nanoparticle quench gold nanocluster 11. In addition to fluorescence recovery, resonance Rayleigh scattering can be activated after heparin is added into a solution of phloxine B with PEI 12. The second method for constructing turn-on sensors employs the aggregation-induced emission (AIE) dye. Tetra-phenylethylene is a typical AIE dye introduced onto the positively charged molecules of organoplatinum (II) metallacycle. Metallacycle molecules assemble on heparin when negatively charged heparin is added due to electrostatic interactions, which results in remarkable fluorescence enhancement 13. For the turn-off type sensor, fluorescence, phosphorescence and surface enhanced


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Raman spectroscopy of the delicately designed probes are quenched by adding heparin. Probes of the polyadenosine–coralyne complex 14, CuInS2 QDs modiﬁed by L–cysteine 15, Mn-doped ZnS QDs 16, Au@Ag core-shell nanoparticles 17, silicon quantum dots 18 and upconversion nanoparticles 19 have been synthesized to detect heparin. To reduce the measured errors from a single detection channel, ratiometric sensors are developed. These sensors take advantages of the light intensity ratio at two detection windows. In general, one signal increases with a concomitant decreasing signal at another detection wavelength, upon which target molecules are sensed. In particular, excimer-monomer probes are typically used 20-23. In addition to the simultaneous use of double detection wavelengths to sense heparin, shifts of single-response wavelength are also useful in heparin sensing. By adding heparin, the color of positively charged gold nanoparticles or soluble cationic polythiophene changes as a result of assembling the nanoparticles or the polymer 24, 25. A lot of heparin detection methods have been developed and significant progress has been made in recent years. However, after summarizing recently published literature, we noticed that almost all heparin sensors were based on electrostatic interactions for recognizing heparin. Whether electrostatic interactions are sufficient to ensure specific heparin sensing while other anion molecules demonstrate slight interference in blood samples can be a particularly contentious issue. Moreover, methods based on electrostatic interactions quantify the overall amount of heparin without differentiating the active and inactive parts. Consequently, the amount of heparin is overestimated, which may cause overdose of the neutralizer protamine after surgery. Protamine can cause several adverse effects 26. To avoid the limitations arising from heparin sensing by electrostatic interaction, we propose a novel active heparin-sensing platform. AT III labeled with QD specifically binds to active heparin molecules and forms the QDs-AT III-heparin complex. The complexes are then induced to aggregate upon addition of cationic surfactant micelles. The QDs attach onto the surface of micelles because of the electrostatic attractions between heparin and micelles. QD aggregations are observed and recognized based on their spectral images, which are recorded using a single-molecule spectral imaging fluorescence microscope. The ratio of QD aggregation spots to all counted fluorescence spots is proportional to heparin concentration under optimal conditions. Active heparin concentrations are then quantified in aqueous solution and 1000-fold diluted human blood plasma. We name this method as counting aggregated nanoparticles by spectral imaging microscopy (CANSIM). CANSIM demonstrates the following advantages. i) The affinity interaction of AT III and heparin is employed instead of electrostatic attractions to improve specific binding. CTAB was only used


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as a bridge to form the QD-ATIII-heparin complex aggregates. It plays the role of signal amplifier rather than recognition. However, electrostatic interactions are used in previous publications to recognize and bind heparin, which is non-specific interaction. ii) Active components of heparin are quantified instead of the overall amount because AT III binds to the pentasaccharide sequence, the active region of heparin. iii) The single-particle-level sensor significantly decreases the detectable concentration of heparin to 0.1 nM. The complicated sample can then be diluted continuously until the background exerts a minimal effect on the measurement. iv) Finally, the quantification of the sensor is not dependent on the change in fluorescence intensity but on the aggregation ratio, which is helpful in minimizing the errors from the intensity fluctuation. EXPERIMENTAL SECTION Chemicals and Materials Carboxyl Qdots 655 was purchased from Invitrogen/Molecular Probes (Eugene, OR). Antithrombin (AT III) was obtained from Dongfeng Biotechnology (Shanghai, China). Heparin (150 U/mg) was purchased from BioSharp (Hefei, China). 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) was obtained from Sigma- Aldrich Corporation. Hexadecyltrimethylammonium bromide (CTAB) was supplied by Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Human serum and plasma samples were obtained from a local hospital. Microscope coverslips (0.17 mm thick, 22 mm × 22 mm) and glass slides (25 mm × 75 mm × 1.0 mm) were purchased from Fisher Scientific (Hanover Park, IL, USA). All other chemical reagents were of analytical grade and purchased from local reagent suppliers. All aqueous solutions were prepared with ultrapure water (18.2 MΩ·cm -1) through a Milli-Q purification system (Millipore, USA). Characterization of QD and QDs aggregation An aliquot of 1.4 µL of the QD solution was deposited on a glass slide and immediately covered with a coverslip. An inverted fluorescent microscope (Olympus IX71) equipped with a 100× oil immersion objective (numerical aperture of 1.45, UPLSAPO, Olympus, Japan) and an electron-multiplied charge-coupled device (EMCCD; Evolve512, Photometrics, USA) was used to observe QDs and QDs aggregation. A transmission grating with 70 lines/mm (Edmund Scientific, Barrington, NJ) and a long-pass filter (510 to 800 nm, Semrock, Rochester, USA) were placed before the EMCCD to obtain the zeroth-order spot and the first-order streak of QDs and QD aggregation. Image J and Origin software were used to process the data. Dynamic light scattering (DLS) and zeta potential measurements were performed using a Zeta sizer ZEN3600 (Malvern Instruments, England). Transmission electron microscopy (TEM) images of QDs were captured using a FEI Tecnai G2 T12 (FEI


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company, USA). Preparation of AT III modified with QDs The procedure of the QD capped with AT III (QD-ATIII) was achieved according to the manufacturing. In brief, 1.25 µL of 8µM carboxyl QD stock solution and 10µL AT-III stock solution (100 IU) were mixed together in 485.75 µL borate buffer solution (10 mM, pH 7.4). Then 3µL of EDC solution (10 mg/mL) was added. EDC solution was prepared just before use. The mixture was reacted overnight at 4 °C with gentle stirring. The mixture was then purified using an ultrafiltration unit (Amicon Ultra device, 100 kDa cutoff) by centrifugation at 14,000 g/min for 5 minutes and washed more than five times sequentially by using 50 mM borate buffer solution (pH 8.3) to remove excess AT III. Finally, the conjugate solution was filtered through 0.22 µm polyethersulfone syringe filter after centrifugation. The final concentration of QD-AT III was 20 nM. Determining the critical micelle concentration (CMC) in borate buffer The CMC of CTAB was determined by UV-visible spectroscopy (TU-1900, Beijing General Instrument Co., Ltd., China). A series of different concentrations of CTAB (10, 20, 30, 40, 50, 60, 70, 80, 90, 100, and 200 µM) was prepared in borate buffer solution (50 mM, pH 8.3) and scanned by UV-visible spectroscopy Formation of QD-AT III @ CTAB aggregates To obtain different ratios of aggregation of QD-ATIII@CTAB, QD-AT III (2.5 µL, 20 nM) was initially reacted with heparin of various concentrations in borate buffer solution (50 mM, pH, 8.3) for 35 min at 39 °C. Subsequently 10 µL of CTAB (2 mM) was added into the above solution (90 µL) before spectral imaging. The final concentration of QD was 0.5 nM. Heparin detection in human plasma samples The human plasma used in this study did not contain heparin. The plasma was diluted 10-, 25-, 50-, 200-, 500- and 1000-fold with borate buffer. QD-AT III and CTAB were added into the diluted plasma. The final concentrations of QD-AT III and CATB were 0.5 nM and 200µM, respectively. The plasma samples were then spiked with 5 nM heparin. RESULTS AND DISCUSSION Zeta potentials of different samples in Table S1 demonstrate that QD covalently binds with AT III molecules. The QDs modified with AT III are still monodispersed in solution. Even when the heparin molecules are added, the QD-AT III-heparin complex remains monodispersed because one heparin molecule only binds with one AT III molecule (Figure 3, column 1 and 4). When the micelles of the cationic surfactant (CTAB) are formed in the solution, the QD-AT III-heparin complexes attach to the micellar surfaces through electrostatic interactions between the negative charge on


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heparin and the positive charge on the hydrophilic head of the surfactant. Therefore, QDs assemble onto the micellar surface, and aggregations are formed (Figure 1A). TEM photographs of QD-AT III-heparin in the absence and presence of CTAB show the monodispersed and aggregated QDs (Figures 1B and 1C). The sizes of QD 655 based on TEM are 7.38 ± 0.96 nm and 3.60 ± 0.33 nm in the longitudinal and lateral directions, respectively, which are comparable with the dimensions of AT III

27

.

Therefore, the heterogeneity of QD size should not significantly affect the number of AT III and heparin bond to the QD. Notably, although the micelles attract QDs via charge interaction, the electrostatic interaction actually played a role in transducing the signal from single QD to aggregated QDs instead of recognizing heparin.

Figure 1. A) Scheme of aggregated QD formation. B) TEM of QD-AT III-heparin without CTAB. C) TEM of QD aggregation with CTAB. To observe and differentiate the aggregated QDs from the monodispersed QDs, we developed a spectral imaging microscopy technique by inserting a transmission grating before EMCCD

28, 29

. Grating divides the fluorescence into the zero-order

(spot shape) and the first-order (streak shape) as shown in Figure 2A. The distance between the two orders reflects the QD fluorescence spectrum. The processes of blue shift and photobleaching of QDs can be recorded based on the distance of the two orders and the disappearance of the spots. Photobleaching and blue shift among the QDs are asynchronous and the degree of aggregation can be documented in accordance with the splitting times of the overlapped first-order streaks 30 as shown in Figure 2B. A series of fluorescence images of the zeroth-order and the first-order from the single QD and aggregated QDs under continuous illumination is presented in


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Figure 2A and 2B, respectively. The first-order streak of the single QD remains constantly intact as it moves to the zero-order spot until disappears. By contrast, the first-order streak of the aggregated QDs splits as it moves to the zero-order spot. Regardless of the number contained in the aggregated QDs, an aggregation is considered when the first-order streak is found split (Figure 2B), that is different from our published work

29

. The aggregation ratio is then calculated by the proportion of

the number of aggregated QD spots to all counted QD spots.

Figure 2. Comparison of a single QD with aggregated QDs under optical spectral imaging microscopy. (A) Blue shift of the single QD illuminated by a mercury lamp; (B) blue shift of QD aggregation; the spots in the left parts of (A), (B) correspond to the zeroth-order. The streaks in the right portions of (A), (B) belong to the first-order spectra. Figure 3 shows various control experiments to confirm the validity of the proposed sensing mechanism. The blank of QD-AT III (0.5 nM) is monodispersed except for approximately 7%-9% occasional aggregation (column 1). The occasional aggregation ratio decreases with the decrease in concentration. The exclusive addition of surfactant molecules to the blank does not affect the aggregated ratio regardless of whether micelles are formed (columns 2 and 3), which suggests that QDs-AT III does not attach onto the surface of micelles in the absence of heparin. Heparin itself also exerts no influence on the aggregated ratio of the blank without the presence of


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micelles (column 4). The QD-aggregated

ratio obviously increases once a cationic

surfactant is added into the QD-AT III-heparin complex solution, particularly at surfactant concentration beyond the CMC (columns 5, 6, and 7). The CMC of CTAB in borate buffer was determined as 45µM by UV-vis spectroscopy as described in the literature 31. Further details are provided in Figure S1. Although the CTAB concentration of 20 µM (column 5) is lower than CMC, which resulting in the absence of micelle formation, the ratio of aggregation remains higher than that of the blank in

this case because the microviscosity is higher than that of free solution and

lower than that of micelle solution in a wide range of concentrations less than CMC 32. The high viscosity offers the opportunity for multiplex QD assembling and contributes to the aggregation ratio. After the micelles are formed, the aggregated ratio is significantly enhanced with increasing surfactant concentration (column 7) because the micelle number increases as well as the micelle size. The aggregation cannot be formed if QDs are not linked with AT III (column 8). We also tested several other cationic surfactants, such as cetyldimethyl benzylammonium chloride, tetradecyldimethyl benzylammonium chloride, and cetylpyridinium bromide. All these surfactants initiate the aggregation of QD-AT III in the presence of heparin. The results of the control experiments confirm the validity of the proposed sensing mechanism.

Figure 3. Aggregated ratio of QD under different experimental conditions. The counted spots in each experiment are not less than 100.


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The optimized experimental conditions, including incubation temperature, incubation pH and CTAB concentration, are shown in Figure 4. We chose those experimental conditions which present the maximum QD aggregation ratio. Another reason for selecting 200µM CTAB to form the aggregation is that CTAB molecules accelerate the QD photobleaching process (Figure 4D) although the mechanism is unknown. Under continuous illumination, the quantity of QDs in the field of view under a microscope gradually decreases following the equation of consecutive elementary reaction

33-35

. When CTAB is added, the photobleaching profiles are obviously

changed. High CTAB concentration significantly reduces the number of QDs in the field of view in a very short duration. An appropriate concentration of CTAB contributes in accelerating the process of QDs first-order spectra splitting but not exceedingly fast to follow the split.

Figure 4. Results of optimization of reaction conditions to obtain the highest aggregated ratio, including incubation temperature (A), incubation pH (B), and CTAB concentration (C); (D) effects of CTAB concentration on the photobleaching process of QDs. Under optimal conditions, the ratio of aggregated QDs is related to the concentration of heparin. Figure 5 shows the relationship between the heparin concentration and the ratio of aggregated QDs. The inset of Figure 5 presents the linear range of 4.65 × 10 -4 U/mL to 0.023 U/mL (0.5 to 25 nM, assuming that the average molecular weight of heparin is 10,000). The linear regression equation is y=0.12+18.52x, and the limit of detection (LOD) is 9.3 × 10

-5

U/mL (~0.1 nM). We also attempted to use dynamic


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laser scattering to calibrate the standard curve for heparin detection, but failed to quantify heparin because of the poor linear relationship and substantial measurement errors (Figure S2). The table of polydispersity index inserted in Figure S2 shows that when heparin and CTAB are added into QD-AT III solution, the distribution type is polydisperse. The LOD and detection range of this proposed method are far below the recommended therapeutic ranges of heparin levels (2 to 8 U/mL, 17 to 67 µM) during cardiovascular surgery and (0.2 to 1.2 U/mL, 1.7 to 10 µM) during postoperative and long-term care

36, 37

. For comparison, Table 1 lists the recently published methods for

detecting heparin including the detection ranges and LODs. Such excellent performance in our method might benefit heparin sensing in blood plasma. Theoretically, heparin can still be detected even if plasma is diluted 10,000 times. As such, sample preparation will be eased by continuous dilution. The developed detection method is then applied to

dilute human plasma to demonstrate its

applicability in real-sample analyses. In our experiments, the human plasma does not contain heparin. Table 2 lists the ratios of aggregated QDs and recoveries of spiked heparin in the diluted human plasma. When the dilution is

less than 1000 fold, the

aggregation of QDs in plasma without heparin is significant, and the recoveries are poor. The recovery becomes excellent when the plasma is diluted 1000 fold. This finding illustrates the effectiveness of the proposed sensing platform, considering the complexity of blood plasma. We are not surprised that the plasma interferes the detection results, which may be the reason lots of literature reports the sensing of heparin in buffer or serum instead of plasma or whole blood. However, the interference is negligible after the plasma is 1000-fold diluted. The spiked 5 nM heparin is detected with an acceptable recovery, which meets the requirement of clinical medicine since the original plasma has a heparin level of 5000 nM.

Figure 5. Relationships between aggregation ratios and heparin concentrations.


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Table 1 Summary of typical heparin sensors. Mechanism

Response range

Limit of Detection

Ref

1

Ratiometric fluorescence

0.048 - 0.42 U/mL

6 mU/mL

23

(0.4–3.5 µM)

(50 nM)

2

Turn-off phosphorescence

0.05 - 1.4 U/mL

0.021 U/mL

16

3

Turn-on SERS

6.5 - 104 U/mL

5.69 U/mL

17

4


0 - 1000 nM

1 nM

22

5


0 – 0.9 nM

0.036 nM

20

6

Turn-off upconverson nanoparticle

0.002 - 2.0 µg/mL

0.7 ng/mL

19

(0.12 – 121.2 nM)

(0.0424 nM)

7

Turn-on fluorescence

0.09–0.9 U/mL

0.00132 U/mL −4

8

Turn-on scattering, fluorescence

0.001-0.15 U/mL

5.0 × 10 U/mL

and absorption

(8.3×10-3-1.25 µM)

(4.2×10-3 µM)

9

Turn-on quantum dots

0.04–1.6 µg/mL

0.02 µg/mL

10

Aggregation induced emission

0 − 28000 nM

Not available

13

11


50 - 15000 nM

12.46 nM.

15

12

Ratiometric Detection

0 - 0.05 nM

0.003 nM

21

13

Turn-off fluorescence

10 -1000 nM

4 nM

14

14


21–77 nM

0.38 nM

7

15

Turn-on gold nanocluster

0.1 - 4.0 µg/mL

0.05 µg/mL

11

(6.06 – 242.4 nM)

(3.03 nM)

16

Turn-off fluorescence

0.004 - 1.6 µg/mL

0.0013 µg/mL

0.24 – 96.96 nM)

0.08 nM

8

12 10

9

17


40 - 80000 nM

40 nM

38

18

Voltammetric detection

100 - 8000 nM

100 nM

39

19


0 – 700 nM

30 nM

40

20

Turn-off gold nanorod

0.02–0.28 µg/mL

5 ng/mL

41

(0.012 – 16.97 nM)

(0.303 nM)

21

Potentiometric

0.01–0.4 U/mL

0.005 U/mL

42

22

Turn-on

1000 - 4000 nM

50 nM

43

23

Turn-on fluorescence

0 - 1.76 U/mL

0.046 U/mL

44

24

Color change

0 - 6.7 U/mL

0.01 U/mL

25

25

Pulsed chronopotentiometry

1 - 20 U/mL

NA

45

26


1000 - 10000 nM

23 nM

46

27

Turn-off Si-QDs

0.002 – 1.4 µg/mL

0.67 ng/mL

27

Counting aggregated QD

Phosphorescence

4.65×10-4 U/mL- 0.023 U/mL

9.3×10-5 U/mL

(0.5 nM - 25 nM)

(0.1 nM)

18 This work

Table 2 Determination of heparin in human plasma Diluted

Aggregated ratio

Aggregated ratio with spiked heparin

Times

without heparin

of 0.00465 U/mL (~5 nM)

10

0.520 ± 0.008

25

0.379 ± 0.043

0.463 ± 0.022

50

0.260 ± 0.049

0.397 ± 0.037

20.9 ± 9.3

100

0.160 ± 0.021

0.391 ± 0.019

129.6 ± 4.8


Recovery (%)

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500

0.113 ± 0.039

0.381 ± 0.049

172.3 ± 12.8

1000

0.101 ± 0.005

0.304 ± 0.016

97.2 ± 5.3

CONCLUSION In summary, we developed a novel strategy to detect heparin in solution and in a diluted human plasma sample at the single-nanoparticle level by fluorescence microscope with an EMCCD. The proposed method demonstrated two main differences from previously reported sensors. First, we adopted AT III for the specific recognition of heparin instead of electrostatic interactions. Second, we counted the aggregated spots of QDs to quantify heparin instead of the changes in optical signal intensity. These two improvements conferred the developed method with an extremely low LOD and the capacity to sense active heparin. Although the equipment is high-end and a little expensive, it is easy to use and had a potential application in medical field with the development of miniaturized microscopy and low-cost high sensitivity camera detector. ASSOCIATED CONTENT Supporting Information Available: The following file is available free of charge on the ACS Publications website at DOI:, in which additional experimental results of Zeta potential, data of determining CMC of CTAB in borate buffer, and hydrodynamic size of the complex of heparin-AT III-QD-CTAB are included. Acknowledgements The authors are grateful to the NSFC (21575053, 21505058, 21505059, 21405064) , Natural Science Foundation of the Higher Education Institutions of Jiangsu Province (16KJA150006) and Priority Academic Program Development of Jiangsu Higher Education Institutions. References (1) Wardrop, D.; Keeling, D. The story of the discovery of heparin and warfarin. Brit.

J. Haematol. 2008, 141, 757-763. (2) Olson, S.T.; Richard, B.; Izaguirre, G.; Schedin-Weiss, S.; Gettins, P. G. W. Molecular mechanisms of antithrombin-heparin regulation of blood clotting proteinases. A paradigm for understanding proteinase regulation by serpin family protein proteinase inhibitors. Biochimie 2010, 92, 1587-1596. (3) Jordan, R. E.; Oosta, G. M.; Gardner, W. T.; Rosenberg, R. D. The kinetics of hemostatic enzyme-antithrombin interactions in the presence of low-molecular weight heparin. J. Biol. Chem. 1980, 255, 81-90.


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