Antithrombin III

Heparin is a potent anticoagulant due to its ability to accelerate the rate at which antithrombin inhibits serine proteases in the coagulation cascade. Heparin and the structurally related Heparan Sulfate (HY-101916) are complex linear polymers composed of a mixture of chains of varying lengths with variable sequence. Heparin interacts most tightly with peptides containing complementary binding sites with high positive charge density. Heparin and Heparan Sulfate exhibit primarily a linear helical secondary structure in which sulfonic and carboxyl groups are displayed at defined intervals and in defined directions along the polysaccharide backbone. Heparin is similar to DNA in that both are highly charged linear polymers that function as polyelectrolytes. Heparin is believed to function as an anticoagulant primarily by interacting with AT III, enhancing AT-III-mediated inhibition of coagulation factors, including thrombin and factor Xa. Heparin binds to AT III and thrombin to form a ternary complex that increases the bimolecular rate constant for inhibition of thrombin by 2000-fold. Heparin is mainly located in the granules of tissue mast cells that are closely related to immune responses. Heparin makes extensive contacts with FGF-2 and FGFR-1, stabilizing FGF-FGFR binding. Heparin also contacts FGFR-1 in adjacent FGF-FGFR complexes and thus appears to promote FGFR dimerization [1].

Low-molecular-weight Heparin calcium (4 mg/kg; sc twice a day for 2 days) can reduce skeletal muscle damage and systemic inflammatory response in IRI SD rats [2].

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Ischemia-reperfusion injury (IRI) is a common postoperative complication of the tourniquet used surgery; low-molecular-weight heparin calcium (LMWH) is frequently used postoperatively to prevent the formation of deep venous thrombosis. However, subcutaneous hemorrhage can usually be seen in patients who underwent lower limb surgery, especially in total knee arthroplasty, the influence of LMWH on IRI remains controversial. In this experiment, we designed an animal model to observe the influence of LMWH on the skeletal muscle injury induced by tourniquets. Sprague-Dawley (SD) rats underwent either 2 h of unilateral hindlimb ischemia or anesthesia alone, at different time points of reperfusion interval, animals received either 4mg/kg LMWH or normal saline subcutaneously twice a day. The levels of inflammatory markers in serum, the expression of apoptosis proteins, as well as histological examination of skeletal muscles, were detected at 48-h reperfusion. We found that the injury of skeletal muscle and the systemic inflammatory response was less severe in LMWH-treated animals, indicating that LMWH could attenuate the tourniquet-induced IRI. In conclusion, LMWH given postoperatively after limb surgery may be clinically beneficial. [2]

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To our knowledge, little evidence on the influence of heparin calcium/LMWH on the IRI of skeletal muscle has been revealed. In this study, we constructed a rat model of skeletal muscle IRI induced by tourniquets, including 48 h of reperfusion after 2 h of ischemia. The damage of skeletal muscle was confirmed by checking morphological changes, the inflammatory response, and the expression of apoptosis proteins. To investigate the influence of LMWH on IRI, we designed two control groups that received either LMWH or saline but no hindlimb ischemia, the levels of inflammatory markers and the expression of apoptosis proteins in the two control groups were not significantly different, indicating that LMWH alone could not offer beneficial effects. In IR groups, the systemic inflammation and damage of gastrocnemius muscles in LMWH treated rats were significantly less severe than saline-treated rats, indicating that post hoc of LMWH attenuated the IR-induced damage to skeletal muscles and systemic inflammatory response.
We used 4mg/kg of heparin calcium/LWMH according to the findings of Abbruzzese et al, which was calculated by identifying the minimum concentration required for inhibition of Factor Xa. In clinical practice, LWMH is given at 4~6 hours after the tourniquets are removed and are maintained twice a day. The elimination half-life of LMWH is about 3.5 hours and it can still be detected after 24 hours according to the pharmacokinetics. In this study, LMWH was given at the 6 h of reperfusion interval, which was similar to the clinical therapeutic regimen. Abbruzzese found that a regimen of 4 mg/kg enoxaparin, twice a day was most relevant to the clinical application of LMWH. However, the dosing protocols may differ in rodent models and humans [2].

Biochemical analysis [2]
The concentrations of MDA and SOD in serum were measured to determine the oxidative stress response. In short, the samples were processed according to the instructions of MDA and SOD testing kits, and measured the absorbance by Microplate Reader. The concentration of TNF-α and IL-6 were measured using an immunosorbent assay (ELISA) kit to assess the inflammatory response, in detail, the samples were processed and incubated according to the instructions of the manufacturer, the absorbance at 430nm was measured, and the concentrations of TNF-α and IL-6 were calculated according to the stand curve.

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Caspase 3 immunohistochemistry [2]
To determine the degree of apoptosis in skeletal muscle, the expression of caspase 3 was evaluated by immunohistochemical examination. Slides were further processed and stained using the primary antibody anti-cleaved-caspase 3 and goat anti-rabbit antibody as the manufacturer protocol. The slides were observed at 40 × magnification. Twenty visual fields were captured in each slide, the image was examined by two blind pathologists to quantify the expression of cleaved caspase 3.

Histological assessment [2]
The gastrocnemius muscle was fixed in formalin for 72 h, rinsed in PBS (phosphate buffer saline) and dehydrated, processed by paraffin embedding, cut into sections (4-μm-thick) longitudinal sections, then deparaffinized and rehydrated. Five slides were cut for each gastrocnemius muscle sample, three of them were stained with hematoxylin‐eosin for morphology observation, two were further processed for IHC examination. Slides were checked under light microscopy at 20 × magnification. Twenty visual fields were captured in each slide, and the number of injured muscle fibers per sample was counted. The IRI of skeletal muscle was evaluated by two pathologists who were blinded to the experimental groups.

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Western blotting analysis of the apoptosis protein [2]
The gastrocnemius muscle was removed from -80°C and thawed at room temperature, cleaned by saline to remove the blood, dried by the filter paper, and weighed. The muscles were homogenized in RIPA buffer and PMSF (100:1), centrifugated at 20000 g for 20 min, the supernatant was aspirated, and processed by loading buffer, boiled at 100 °C for 5 minutes. Total concentrations of protein were determined by using the bicinchoninic acid (BCA) kit, the concentration of protein in each sample was calculated according to its standard curve. After the protein was separated, it was transferred to a PVDF membrane, and blocked with blocking buffer, then incubated overnight with the primary antibodies, including anti-cleaved-caspase 3 (1:1000), anti-caspase 9 (1:1000), anti-bax (1:2000), anti-bcl2 (1:2000), cleaned with TBST (tris-buffered saline and tween) and incubated by goat anti-rabbit antibody for 1 h. The same membrane was re-blotted with anti-β-actin (1:5000) for normalization. The image was exposed in the chemiluminescence imaging system.

Animal/Disease Models:Adult Sprague-Dawley rats (male, 200-300 g) with ischemia-injury (IR)[2]
Doses: 4 mg/kg
Route of Administration: S.c. twice daily for 2 days
Experimental Results: Could attenuated the tourniquet-induced IRI.

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Animals and grouping [2]
Forty adult Sprague-Dawley rats (male, 200–300 g) were used for this experiment, animals were randomly divided into four groups according to the ischemia-injury (IR) protocols and post hoc administration of heparin calcium/LMWH or saline: LMWH-treated IR group (IR-LMWH, n=10), saline-treated IR group (IR-saline, n=10), LMWH-treated control group (control-LMWH, n=10), and saline-treated control group (control-saline, n=10).

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Construction of the IR model and post hoc intervention [2]
Animals were anesthetized by pentobarbital sodium (50 mg/kg, intraperitoneal injection). A 37°C-heating plate was used to maintain body temperature during anesthesia. In IR groups (IR-LMWH and IR-saline), an orthodontic rubber band was located around the right upper thigh for 2 hours to induce acute unilateral hindlimb ischemia, which was identified by the absence of arterial pulse, cyanosis, and coldness of the limb (Fig. 1). Animals in control groups (control-LMWH and control-saline) were just anesthetized for the same time. Additional doses of pentobarbital sodium (10 mg/kg) were used to maintain anesthesia during the ischemia period, at the same time, animals received an intraperitoneal injection of 0.2 ml normal saline every 1 h to prevent dehydration. After recovering from anesthesia, animals were raised in an SPF laboratory with controlled temperature and humidity, water and mouse chow were provided unlimitedly. At 6 h, 18 h, 30 h, 42 h of reperfusion, animals received whether 4 mg/kg of heparin calcium/LMWH or the same dose of saline subcutaneously, which was similar to the twice-daily (Q12h) clinical use of anticoagulant postoperatively.

[1]. Capila I, et, al. Heparin-protein interactions. Angew Chem Int Ed Engl. 2002 Feb 1;41(3):391-412.

[2]. Low-molecular-weight heparin calcium attenuates the tourniquet-induced ischemia-reperfusion injury in rats. Injury. 2021 Aug;52(8):2068-2074.

The present study revealed that LMWH had anti-inflammatory effects, early use of LMWH after surgery was beneficial for attenuating tourniquets-induced IRI in the rat model, suggesting that it should not be considered as a relative contraindication even though subcutaneous hemorrhage is observed. Sine the LMWH is widely used in patients undergoing extremity surgery, it is worth investigating the dosing protocols in humans, if similar results were observed, LMWH might be considered a separate indication for clinical management of IRI. However, this study is mainly focused on animal experiments, the dosing protocol, and the translatability of rodent models must be considered in clinical practice, future studies should focus on the mechanisms.
There were several limitations in this study. First, the blood flow in the hind limb was not measured, which was useful in evaluating the situation of ischemia, however, the IR model of the hindlimb induced by rubber tourniquets has been demonstrated successful by many studies. Second, it is crucial to achieving the balance between the benefits in attenuating the IRI and the risk of hemorrhage, we did not observe subcutaneous hemorrhage in rats because they were put to death at 48 h reperfusion. Third, though the rodent models are widely used in the experimental study of IRI and are relatively translatable, they are inferior to primate models. Furthermore, it is difficult to set a control group that is absent from anticoagulants after lower limb surgery in clinical trials. However, the conclusion of the present study was consistent with previous studies that investigated the effect of LMWH on IRI in the brain and other organs. In conclusion, this basic experimental research demonstrated that post hoc of LMWH after tourniquet-induced IRI was beneficial, which may provide a reference to clinical management of IRI after tourniquet used limb surgery.[2]

C24H47CAN2O37P4S5

C, 26.58; H, 3.60; Ca, 3.41; N, 2.38; O, 50.38; S, 13.64

37270-89-6

9005-49-6 (free); 37270-89-6 (Ca)

168009871

Typically exists as solid at room temperature

2

9

21

89

1580

0

[CaH+].OC1C(OC2C(COS(=O)(=O)O)OC(OP)C(NS(=O)(=O)O)C2O)OC(C(=O)O)C(OP)C1O.OC1C(OC2C(OS(=O)(=O)O)C(O)C(OP)C(C(=O)O)O2)C(COS(=O)(=O)O)OC(OP)C1NS(=O)(=O)O

Heparin calcium; Nadroparin calcium (MW 15000-19000)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)