[Frontiers in Bioscience, Landmark, 21, 1372-1392, June 1, 2016]

Heparin and anticoagulation

Akihiro Onishi1, Kalib St Ange1, Jonathan S. Dordick1, Robert J. Linhardt1

1
Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th St, Troy,
NY 12180 USA

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Detailed description of coagulation pathway
3.1. Primary hemostasis – platelets
3.2. Secondary hemostasis – biochemical cascade reactions
3.3. Endothelium
4. Diseases requiring anticoagulants
4.1. Venous thromboembolism
4.2. Venous thromboembolism treatments
4.3. Extracorporeal circuit
4.4. Disseminated intravascular coagulation
5. Anticoagulants
5.1. Classes of anticoagulants
5.2. Heparins
5.3. Vitamin K antagonists
5.4. Direct inhibitors
5.5. Others
5.6. Comparison of anticoagulants
5.7. Recent topics
6. Classes of heparin
6.1. Heparin
6.2. Heparan sulfate
6.3. Low molecular weight heparin
6.4. Ultra low molecular weight heparin
7. Heparin’s biological roles
7.1. Inflammation and angiogenesis
7.2. Growth factor signaling
7.3. Developmental process
7.4. Various disease processes
8. Heparin’s therapeutic application
8.1. Heparin’s anti-thrombic and anti-embolitic therapeutic applications
8.2. Heparin’s anti-inflammatory therapeutic applications
9. Adverse effects of heparin
9.1. Hemorrhage
9.2. Heparin-induced-thrombocytopenia
9.3. Osteoporosis
9.4. Others
10. Future prospects
10.1. Risks associated with porcine and bovine derived heparins
10.2. Comparison of porcine and bovine derived heparins
10.3. Bioengineered heparin
10.4. Synthetic heparin oligosaccharides
11. Conclusions
12. Acknowledgements
13. References

1372
Heparin and anticoagulation

1. ABSTRACT 3. DETALIED DESCRIPTION OF

COAGULATION PATHWAY
Heparin, a sulfated polysaccharide, has been
used as a clinical anticoagulant for over 90 years. Newer Blood in a healthy individual freely circulates
anticoagulants, introduced for certain specialized through arteries and veins. The normal vascular
applications, have not significantly displaced heparin endothelium acts as an antithrombotic surface. However,
and newer heparin-based anticoagulants in most when the hemostatic system is triggered it becomes
medical procedures. This chapter, while reviewing active instantly due to the cascade reactions. When
anticoagulation and these newer anticoagulants, the blood vessel wall becomes damaged, platelets and
focuses on heparin-based anticoagulants, including fibrin aggregate to prevent hemorrhage. Although a rapid
unfractionated heparin, low molecular weight heparins hemostasis is required to prevent the loss of blood, an
and ultra-low molecular weight heparins. Heparin’s excessive amount clotting can lead serious thrombotic
structures and its biological and therapeutic roles are complications.
discussed. Particular emphasis is placed on heparin’s
therapeutic application and its adverse effects. The The primary source of hemostasis is through
future prospects are excellent for new heparins platelet aggregation and adhesion to a damaged vessel.
and new heparin-based therapeutics with improved Secondary hemostasis is mediated by plasma-based
properties. coagulation factors, which undergo a biochemical
cascade resulting in platelet-fibrin clots (8, 9). The
2. INTRODUCTION heparan sulfate proteoglycan (HSPG) on the surface of
the endothelium lining the lumen of the vessel also plays
Heparin is an essential drug and is the most a role in controlling coagulation.
widely used clinical anticoagulant worldwide (1).
However, in the past decade there is a rising concern 3.1. Primary hemostasis – platelets
about the safety and supply security of heparin. An Following vascular injury, platelets adhere to
international heparin contamination crisis, which exposed endothelial collagen forming a ‘platelet plug’
occurred in 2007 to 2008, was associated with more resulting in primary hemostasis (Figure 1A). Von Willebrand
than 80 fatalities in the U.S. alone (2). Moreover, factor (vWF) bridges between endothelial collagen and
most of the world’s supply of heparin comes from platelet surface receptors, mainly glycoprotein (GP)
a single animal species, hogs, and is collected in receptor Ib, promoting platelet adhesion and aggregation.
a single country, China. This review presents the An activated platelet degranulates to release various
current fundamental knowledge of anticoagulants, factors including serotonin, adenosine diphosphate
including an overview of coagulation pathway, major (ADP), and thromboxane A2 (TXA2). Serotonin and TXA2
diseases and conditions requiring anticoagulants, have vasoconstrictive effect. ADP and TXA2 stimulate
and the various classes of anticoagulant drugs. The further platelet aggregation. In addition, activated platelet
focus of this review is on heparin and heparin-based exposes fibrinogen binding site, thus, fibrinogen bridges
anticoagulants, including their biochemistry, chemical platelets to form a platelet plug. The generated platelet
structure and their wide range of biological activities. plug helps activated coagulation factors assemble on
The therapeutic roles of heparin-based drugs, including its surface and the secondary hemostasis, involving the
unfractionated heparin (UFH), low molecular weight plasma-based coagulation cascade follows.
heparin (LMWH) (3), and synthetic heparins (4) are
discussed. These heparin-based drugs are essential 3.2. Secondary hemostasis – biochemical
in the medical treatment of thrombosis, embolisms cascade reactions
and in the prevention of clotting in surgery. As with Secondary hemostasis involves a cascade
any class of drugs, heparin-based therapeutics result of biochemical reactions (Figure 1C). This cascade
in side-effects and complications. The redesign, is comprised of inactive zymogens (or pro-enzyme)
re-formulation and appropriate application of these called blood coagulation factors being activated to
agents can reduce the risks associated with heparin- serine proteases (i.e. factor X to factor Xa) that can
based therapy. However, contamination/impurities and then go on to activate subsequent coagulation factors
bovine/porcine risks that have received much attention (i.e., factor Xa activates factor II to form factor IIa) that
since the 2007-2008 contamination crisis are currently ultimately convert the soluble plasma protein fibrinogen,
being addressed by considering the re-introduction to the insoluble plasma protein, fibrin (comprising a clot).
of bovine heparin (5), through the introduction of There are two traditional major secondary coagulation
bioengineered heparin (6), and in the improvement cascade pathways, the intrinsic pathway and the
of synthetic heparin oligosaccharides (7). This review extrinsic pathway. The intrinsic pathway, which is also
focuses on advances that have been made in the field called contact pathway, is triggered by factors XII and XI.
over the past decade. When factor XII contacts the negatively charged surface

1373 © 1996-2016
Heparin and anticoagulation

Figure 1. Coagulation pathway. A) Molecular mechanism of a platelet adhesion and aggregation involving von Willibrand factor (vWF) and glycoproteins
(GPs), releasing adenosine diphosphate (ADP) and thromboxane A2 (TXA2). B) Endothelial heparan sulfate proteoglycan (HSPG) interaction with
antithrombin (AT) resulting in the inactivation of thrombin and factor Xa (FXa) to control coagulation. C) Main reactions of the coagulation cascade after
activation through vascular injury or by tissue factor (TF) and major clinical anticoagulants including direct factor Xa inhibitor (DXI), direct thrombin
inhibitor (DTI) and vitamin K antagonists (VKAs).

(phospholipids exposed at a vascular injury site), it of factor IXa, factor VIIIa, calcium and phospholipids,
causes a local increase in its concentration, which then generates. This tenase complex activates factor X. After
autoactivates to factor XIIa. Factor XIIa then catalyzes the formation of the tenase complex, the prothrombinase
the conversion of prekallekerin to kininogen and factor XI complex, which consists of factor Xa, factor Va, calcium
to XIa (9, 10). Consequently, these activations lead to the and phospholipids generates. Although factor Xa alone
formation of factor IXa. can catalyze prothrombin (factor II) into thrombin
(factor IIa), this activation is greatly accelerated by factor
The extrinsic pathway, which is also called Va and the complex. Thrombin, the final serine protease
tissue factor pathway, is the initial step in plasma- formed in the coagulation cascade has various roles in
mediated hemostasis. The contact of the membrane- clotting (9). Thrombin activates various components of
bound protein tissue factor (TF) with plasma containing coagulation pathway, such as platelets, factors V, VIII and
factor VII triggers the extrinsic pathway, forming TF-VIIa IX, protein C and thrombin-activatable fibrinolysis inhibitor
complex. Alternatively the extrinsic pathway can also to amplify the coagulation cascade. Most importantly,
be initiated if monocytes and smooth muscle cells are thrombin converts fibrinogen to fibrin, ultimately forming
exposed to cytokines or other inflammatory mediators. a clot.
This also causes the release of tissue factor (9, 10).
Once the TF-VIIa complex forms, it converts factor IX and The conversion from soluble fibrinogen to
factor X to factor IXa and factor Xa, respectively. insoluble fibrin is the final step of the coagulation process.
Factor XIIIa leads fibrin-monomer cross-linking to form
Once factor IXa is formed either by the intrinsic a stabilized fibrin clot. In parallel, fibrinolytic system is
or extrinsic pathway, the ‘tenase’ complex, consisting activated to control the size of fibrin clots. Fibrinolysis

1374 © 1996-2016
Heparin and anticoagulation

cleaves insoluble fibrin into fibrin degradation products, pulmonary embolism (13). Deep vein thrombosis results
and rapidly clears it out to maintain hemostatic balance. from clot formation in a major vein, normally one in the
Plasmin, which circulates as an inactive zymogen lower extremities. Unlike clots in superficial veins that
plasminogen, is the enzyme responsible for fibrinolysis. stay in place, these clots can dislodge and, thus, are very
dangerous. Clots that dislodge from the vein can travel into
Antithrombin (AT), previously known as the lungs or other organs causing permanent damage by
antithrombin III, is a serine protease inhibitor that way of tissue death or loss of oxygen. There are several
inactivates various activated coagulation serine proteases, types of embolism that include a venous and arterial
including factors IXa, Xa, TF-VIIa complex and thrombin. embolism and these can be anterograde and retrograde,
AT covalently binds to serine residue of serine proteases depending on whether the embolus travels with or
causing their inactivation. However, in the presence of against the blood flow. There are several environmental
heparin or heparan sulfate (HS), the ability of AT to inhibit and hereditary causes of deep vein thrombosis including
serine proteases is markedly enhanced and in the case of factor deficiencies, hormone treatments, stress, diabetes,
thrombin forming heparin-AT-thrombin ternary complex. chronic inflammatory disorder, vein damage during
surgery, broken bones, physical trauma, slow blood flow
The intrinsic pathway and the extrinsic pathway from lack of movement, pregnancy, venous catheters, old
were previously considered as independent pathways of age, obesity and smoking (13-15). These clots can block
factor X activation. However, it is now thought that the blood flow leading to swelling, pain and death. Venous
extrinsic pathway initiates thrombin generation (initiation thromboembolism affects up to 5% of the population
phase) and the intrinsic pathway augments thrombin during their lifetime and approximately 20% of patients
generation (propagation phase) (9, 10). with pulmonary embolism die before diagnosis or within a
day after diagnosis. Patients who survive past that period
3.3. Endothelium face an 11% mortality rate in the first 3 months, even with
Thrombin promotes coagulation by catalyzing adequate therapy (14).
the conversion of fibrinogen to fibrin. In the healthy intact
vessel, AT bound to the endothelial HSPG can inactivate 4.2. Venous thromboembolism treatments
thrombin (Figure 1B). AT-HSPG complex can also The initial treatment venous thromboembolism
inactivate factor Xa through formation of AT-Xa complex. relies on is heparin and LMWH anticoagulants. These
Since the healthy endothelium of an undamaged vessel acute treatments lower risk and costs associated and
wall is non-thrombogenic, clotting on healthy vessels is reduce risk of recurrence. Anticoagulant treatment is
prevented. In contrast, endothelial damage in a wounded effective in thinning the blood preventing clot growth. While
vessel, results in the loss of AT-HSPG, allowing thrombin heparin does not remove the clot, it is slowly dissolved
to promote clot formation and coagulation (11). The over the course of about 3 months. LMWH can only be at
endothelium is also responsible for the active transport reduced doses with careful observation on patients with
and regulation of extravasation of fluid, solutes, renal failure since LMWH is cleared through the kidney as
hormones, macromolecules platelets and blood cells. described in Chapter 6.3. Vitamin K antagonists (VKAs),
This is accomplished through the use of smooth such as warfarin, used alone are not recommended for
muscle cells, interendothelial junctions, vasodilation, the initial treatment since a randomized trial demonstrated
and vasoconstriction. Dysfunction in the endothelium, more recurrent symptomatic and asymptomatic events
i.e., failure to maintain blood fluidity, the appropriate in patients treated with VKAs alone. However, after the
concentration of factors, or inflammation, can result in initial use of heparin or LMWH, VKAs can be used to
catastrophic changes in coagulation (12). continue treatment. This improves its prospects as a long
term treatment since VKAs are inexpensive. Conversely,
Blood coagulation should occur rapidly but rivaroxaban a direct oral factor Xa inhibitor (DXI) is
only if necessary. Otherwise, bleeding or thrombosis will easily administered but costly. Rivaroxaban treatment,
occur. To achieve expediency the successive activation like LMWH treatment, cannot be used in patients with
of pro-enzymes to enzymes, known as cascade reaction, renal failure. Thrombolytics, such as tissue plasminogen
triggers procoagulant activity explosively by producing activator (t-PA), are used to dissolve the clot directly,
an exponential increase in the number of these enzyme however, due to the high bleeding risks, such agents
molecules. In contrast, under normal physiological are generally only used in emergency situations. Vena
conditions, inhibitors of coagulation (such as AT) limit the cava filters are another form of treatment primarily used
clot formation to avoid the thrombus formation. to prevent the dislodging clots from migrating to vital
organs by physically removing them. This treatment is
4. DISEASES AND ABNORMALITIES preferred if the patient cannot take anticoagulants or in
addition to thrombolytic treatment. Vena cava filters have
4.1. Venous thromboembolism the drawback of potentially causing thrombi formation at
Venous thromboembolism is a coagulation the insertion site but if the patient can take anticoagulants
disease that involves deep vein thrombosis and then this drawback can be remedied (13, 14).

1375 © 1996-2016
Heparin and anticoagulation

4.3. Extracorporeal circuit related marker, are conducted. DIC treatment should be
Clotting abnormalities can also be caused by based on the proper remedy of the underlying disorder.
non-biological processes. Many medical procedures But sometimes, supportive treatment by anticoagulants is
require the separation, purification, or oxygenation of also required. UFH and LMWH are used for the treatment
blood and the instruments used in this process need to and prophylaxis of thrombosis and embolic complications
be used in tandem with anticoagulants to prevent clot associated with DIC. Concentrated platelets or fresh
formation. An extracorporeal circuit or extracorporeal frozen plasma are transfused to replenish consumed
membrane oxygenator takes the patient’s blood outside platelets or clotting factors.
his or her body using plastic tubing, a hemodialysis
machine, a dialyzer, or an oxygenator (16). There are two Recently, a recombinant thrombomodulin
different types of extracorporeal membrane oxygenators was approved for DIC. The International Society on
(ECMOs), Veno-arterial and veno-venous oxygenerator. Thrombosis and Haemostasis harmonized the guideline
Veno-arterial ECMO is used for heart failure providing for diagnosis and treatment of DIC in 2013 (19). An
both gas exchange and hemodynamic support for a failing observational study conducted in 2014 showed the gradual
cardiopulmonary system. This means deoxygenated improved in-hospital mortality of DIC patients associated
venous blood is drained into an oxygenator, oxygenated, with infectious diseases and this study assumed that
and is returned into circulation. Veno-venous ECMO recombinant thrombomodulin and the new practice
involves only gas exchange without the use of clinical guideline contributed this improvement (18).
hemodynamic support and is used for refractory hypoxic
respiratory failure with preserved cardiac function. 5. ANTICOAGULANTS
Deoxygenated venous blood is drained into an oxygenator
and hyperoxygenated arterial blood is returned to venous 5.1. Classes of anticoagulants
circulation. The blood interacts with the nonendothelial Anticoagulant drugs are categorized into four
surfaces of the ECMO causing widespread inflammatory broad types: heparins, direct inhibitors, VKAs, and others.
and prothrombotic response. Within minutes of initiation Heparins include UFH, LMWH and ultra-low-molecular
there is a widespread activation of the clotting cascade weight heparin (ULMWH). UFH acts with AT and
and also a dilution of coagulation factors in the blood. inactivates both FXa and thrombin. LMWH acts with AT to
Platelets adhere to surface fibrinogen, causing platelet inactivate FXa and to a lesser degree thrombin (FIIa) and
activation and platelet aggregation, resulting in platelet ULMWH acts with AT to exclusively inactivate thrombin.
loss, thrombocytopenia. These complications require In contrast, direct inhibitors work independently of AT and
the use of anticoagulants. Heparin is used in the coating inhibit either FXa or thrombin (Figure 2). Warfarin is the
of these circuits to free tissue factor pathway inhibitor, most commonly used of the VKAs. Other anticoagulants
and augment AT-dependent inhibition, freeing factors include several peptide and small molecules with a
Xa, XIa, and VIIa/TF. AT has been shown to decrease variety of mechanisms of actions.
with the initiation of the extracorporeal circuit leading to a
procoagulative statedecreased heparin responsiveness. 5.2. Heparins
This means that AT should also be monitored and kept Heparin is a highly sulfated glycosaminoglycan.
above 60% to prevent venous thrombosis. Understanding UFH is extracted and purified from animal tissues
of the extracorporeal circuit and what coagulation factors including porcine intestine and bovine lung and
need to be monitored when blood is pumped outside intestine. LMWH is produced through the controlled
the body is important when performing cardiopulmonary depolymerization of UFH. ULMWH is a synthetic specific
bypass, hemofilitration, dialysis, and surgery. pentasaccharide, which is similar to a pentasaccharide
sequence found within UFH and LWMH. Heparin was
4.4. Disseminated intravascular coagulation discovered in 1916 by medical student Jay McLean (20).
Disseminated intravascular coagulation (DIC) is The clinical use of UFH started in 1930s. LWMHs have
an acquired syndrome characterized by an unregulated been clinically used since 1980s. ULMWH is the most
systemic activation of coagulation, leading to excessive recent subtype of heparin, which was approved by US
formation of microthrombi, microcirculation obstruction, Food and Drug Administration (FDA) in 2000s. Heparins
resulting in organ dysfunction and death (17, 18). In work by primarily inhibiting thrombin (FIIa) and/or FXa.
addition, the extravagant consumption of platelets The ratios of anti-Xa activity to anti-IIa activity of different
and coagulation factors cause serious hemorrhagic heparins differ. Shorter heparin chains having a low
complications, which is called consumption coagulopathy. averaged-molecular weight display higher anti-Xa/anti-
Therefore, a DIC patient can present thrombotic and IIa ratios.
bleeding symptoms concomitantly. DIC is commonly
caused by sepsis, malignancy, pregnancy complications 5.3. Vitamin K antagonists
and massive inflammation. Regarding DIC diagnosis, a Coumarins work as VKAs acting as
combination of laboratory tests such as prothrombin time, anticoagulants by inhibiting the biosynthesis of several
activated partial thromboplastin time, platelet count, fibrin vitamin K-dependent clotting factors, including Factor

1376 © 1996-2016
Heparin and anticoagulation

and the cofactor through which heparin exerts its activity.

Thus, concentrated AT can be used as an anticoagulant.
Nafamostat and gabexate are the synthetic serine
protease inhibitors. These drugs are competitive inhibitors
and do not require AT for their mechanism of action.
Thrombomodulin is an about 75 kDa integral membrane
protein. Thrombomodulin has functions of both in the
inhibition of thrombin and in the conversion of protein
C into activated protein C, which exerts anticoagulant
activity.

In addition to anticoagulants, many hemostatics

and antithrombolytics are also available. Major types of
hemostatics are capillary stabilizers (carbazochrome,
vitamin C, etc.), coagulation accelerator (vitamin K, etc.),
and antiplasmimn (tranexamic acid, etc.).
Antithrombolytics include antiplatelet drugs (ticlopidine,
aspirin, beraprost, etc.).

5.6. Comparison of anticoagulants

When selecting anticoagulant drugs, the cost,
availability of antidotes, route of administration, safety and
efficacy are important factors. Two other critical factors
are their therapeutic indications and contraindications.

Figure 2. Schematic representation of direct inhibitors, including direct Heparins and warfarin are much less expensive
thrombin (FIIa) inhibitors (DTIs), Direct factor Xa inhibitors (DXIs) and
indirect inhibitors, antithrombin (AT) with unfractionated heparin (UFH), than direct inhibitors. Protamine neutralizes heparins,
low molecular weight heparin (LMWH) and ultra low molecular weight and vitamin K is an antidote for VKAs, but there is
heparin (ULMWH), of factor IIa and factor Xa. The heparin sequence currently no antidote for direct inhibitors. While warfarin
displayed in this figure corresponds to the pentasaccharide binding
site (containing N-acetylglucosamine, glucuronic acid, glucosamine, and the direct inhibitors are oral drugs, heparins are
idouronic acid, and glucosamine residues, respectively) and repeating used intravenously (UFH) or subcutaneously (LMWH
idouronic acid, and glucosamine residues. Sulfo groups are not shown for and ULMWH). Direct inhibitors do not have yet sufficient
simplicity. The symbols used are defined in Figure 5.
safety and efficacy data for long-term use, patients
during pregnancy, patients with mechanical heart valves,
VIIa, IXa, Xa and thrombin. Warfarin, a specific etc. Thus, heparins and warfarin remain the agents of
member of the coumarin family, has been widely used choice. The period of administration is another important
as a clinical anticoagulant for more than half a century. factor to consider when selecting an anticoagulant. In the
However, its major shortcomings are the drug-food treatment of venous thromboembolism there are three
interactions and need for regular monitoring of the blood phases of treatment: acute, long-term and extended.
drug concentration. In addition, a drug interaction is Heparins are generally used in acute phase treatment
another issue because warfarin is mainly metabolized while VKAs are used for both the long-term and the
by CYP2C9. Until recently, warfarin had been the only extended phase. A number of phase III studies are
clinically used oral anticoagulant. ongoing to evaluate direct inhibitors for the treatment of
venous thromboembolism (22).
5.4. Direct inhibitors
Direct inhibitors are the newest class of oral 5.7. Recent topics
anticoagulants. FDA-approved DXIs include rivaroxaban, As of 2013, the annual sales worldwide of
apixaban and edoxaban. FDA-approved direct thrombin LMWH, VKAs, and direct inhibitors were, US$ 6.5 billion,
inhibitors (DTIs) include dabigatran (21). These new 0.6 billion, 4.7 billion, respectively (Figure 3). It is
direct inhibitors provide wider therapeutic options than estimated that the sales of LMWH and VKAs in 2018
before. At the same time, their high cost, lack of antidotes will decrease by 27% and 18% from 2013, respectively.
and limited information on their long-term use represent The sales of direct inhibitors are expected to increase by
serious drawbacks. 181% during this period (23).

5.5. Others Recently, direct inhibitors have attracted

Other anticoagulants include AT, synthetic much attention. Several new direct inhibitors and those
serine protease inhibitor, and thrombomodulin. AT is a antidotes are in development (22). For example, phase III
glycoprotein and the major inhibitor of clotting factors clinical trials of a candidate antidote are ongoing (25, 26).

1377 © 1996-2016
Heparin and anticoagulation

the body through its rapid metabolism in endothelial cells,

by the liver, and also through slower renal clearance.
Endothelial metabolism is through a zero order
mechanism as heparin is bound by surface receptors
on endothelial cells and macrophages, internalized and
depolymerized into smaller oligosaccharides and renal
clearance is first order mechanism. The molecular weight
of a heparin, i.e., UFH, LMWH or ULMWH, impacts
its route of clearance and can preclude the use of a
particular heparin in patients with renal failure. Heparin
is administrated by intravenously or subcutaneously
(subcutaneous bioavailability depends on a heparin’s
molecular weight) and the level of heparin in the circulation
is monitored by the activated partial thromboplastin time
assay.

6.2. Heparan sulfate

HS is a heparin-related polysaccharide localized
Figure 3. Anticoagulant drug market sales in 2013 are estimated based
on the surface of cells, including endothelial cells, which
on (23, 24). The market share of low molecular weight heparins (LMWHs), can contain and AT pentasaccharide binding site and
direct factor Xa inhibitors (DXIs), direct thrombin inhibitors (DTIs), vitamin K exhibit anticoagulant properties. HS differs from heparin
antagonists (VKAs), and unfractionated heparin (UFH) are shown.
with respect to its level of sulfation and fine structure.
HS contains about 0.6. sulfo groups per disaccharide
Comparison of direct inhibitors and existing anticoagulants repeating unit compared to heparins 2.6. sulfo groups.
is also under intensive study. A meta-analysis of direct Less than 40% of the uronic acid residues in HS are
inhibitors suggested that both apixaban and rivaroxaban iduronic compared to up to 90% in heparin. HS is also
are associated with lower acute coronary events than more polydisperse than heparin with molecular weights
dabigatran (27). ranging from 10-70 kDa. HS generally has only about
10% of heparins anticoagulant activity. HS, found on
6. CLASSES OF HEPARIN the surface of endothelial cells throughout the body, is
responsible for a variety of functions from inflammatory
6.1. Heparin responses, regulating levels of anticoagulant activity
Heparin is a highly sulfated polysaccharide within blood vessels, and in cell-cell communication (33).
that is used as a major clinical anticoagulant (1, 28, 29).
Heparin is also a linear glycosaminoglycan (GAG) with 6.3. Low molecular weight heparin
an average molecular weight of between 15 and19 kDa LMWH consists of smaller fragmented heparin
(1, 30). It is made up of trisulfated disaccharide molecules prepared through the controlled chemical or
glucosamine and uronic acid repeating units, together with enzymatic depolymerization of UFH (Figure 4B). The
less sulfated and variably sulfated domains (Figure 4A). depolymerization method in the production process
Heparin is biosynthesized in the endoplasmic reticulum affects generated LMWH’s properties. Commonly used
and the Golgi of mast cells that are present in larger methods include oxidation, deamination and β-elimination.
numbers in the liver, intestines, and lungs (Figure 5). It Oxidation processes generate polysaccharide molecules
is extracted from food animal sources including cows with both even and odd numbers of residues. Deaminative
and pigs, with porcine intestinal mucosa being the methods produce terminal anhydromannitol residues
standard species and tissue source (1, 30). Heparin’s at the reducing end. Elimination methods result in the
anticoagulant activity is due to its ability to inhibit formation of an unsaturated uronic residue at the non-
multiple factors in the coagulation cascade. Heparin reducing end. More than ten LMWHs have been clinically
binds to AT, as serine protease inhibitor, and targets used and they display similar biological properties.
coagulation proteins including factor Xa, and factor IIa Because of their subtle structural differences, LMWHs
(thrombin). AT binds a variably sulfated pentasaccharide are not clinically interchangeable. Most LMWHs have
sequence having a central 3-O-sulfoglucosamine an average molecular weight between 4-5 kDa (1, 29),
residue. Only about 30% of the heparin chains contain a longer plasma half-life, better bioavailability at low
this sequence. AT bound to heparin undergoes a doses, as well as a more predictable dose response
conformational change, exposing a reactive loop that characteristic than UFH. This allows outpatient
is acted upon and by factor Xa and thrombin catalyzing subcutaneous treatment with LMWH instead of inpatient
their inactivation. A heparin polysacaride of at least 18 intravenous administration of UFH. LMWHs have a
saccharide residues is required to bind thrombin and AT reduced ability to inactivate thrombin as only 25% to
in a ternary complex (31, 32). Heparin is cleared from 50% of LMWH species contain the 18 saccharide units

1378 © 1996-2016
Heparin and anticoagulation

Figure 4. Structure of unfractionated heparin (UFH), low molecular weight heparin (LMWH) and ultra low molecular weight heparin (ULMWH). The
brackets shown indicate multiple copies of the domains. A) Major domains in UFH are labeled. There are typically a combined number of 20 to 50 copies
of both trisulfated and disulfated domains in UFH. B) LMWHs synthesized using different methods. Three major methods and typical structures with
reducing and non-reducing ends are shown. Approximately 7-10 of the domains, shown in brackets, are present in each LMWH. C) AT binding domain of
the ULMWH, fondaparinux. The symbols used are defined in Figure 5.

Figure 5. Biosynthetic pathway of heparin and heparan sulfate proteoglycan (HSPG). The glycosaminoglycan-protein linkage region is assembled
by several glycosyltransferases. Repeating disaccharide unit is then elongated by GlcA and GlcN transferases. Next, various modifications, such as
N-deacetylation and N-sulfonation of glucosamine, conversion of GlcA to IdoA, and O-sulfonations, take place through the actions of the specific enzymes
shown.

1379 © 1996-2016
Heparin and anticoagulation

Table 1. Some of heparin’s biological roles and neutralize UFH, does not completely neutralize LMWHs
related heparin binding proteins heparins, so bleeding effects are much more likely. While
the shorter sized LMWH chains pose a problem for using
Biological Binding protein References protamine as an antidote, they offer an advantage by
role reducing LMWH binding to platelet factor 4 reducing the
Coagulation Factors IIa (thrombin), IXa, and Xa (45, 46, 49) risk of the HIT side effect (39).
pathway
Antithrombin (AT)
6.4. Ultra low molecular weight heparin
Protein C inhibitor ULMWHs, such as fondaparinux (Figure 4C), are
Inflammation Platelet growth factor 4 even smaller heparin chains, many being homogenous
and compounds, ranging in size from 1.5.-3.5. kDa (40, 41).
Interleukin 8
angiogenesis The advantages of ULMHs include a higher degree of
Stromal‑derived factor 1a bioavailability, longer plasma half-lives, lower bleeding
Neutrophil elastase
risk, lower risk of osteoporosis, and penetration of the
blood brain barrier (40, 42).
Growth factor Fibroblast growth factors (FGFs)
signaling
Fibroblast growth factor receptors ULMWHs are pure Factro Xa inhibitors, having
(FGFRs) high anti-Xa activity but no anti-IIa activity. A phase I
study implied that larger safety margins with respect to
Endothelial growth factors (EGFs)
bleeding risk (43). Although these ULMWHs have some
Platelet derived growth factors (PDGFs) significant benefits such as no substantial binding to
Pathogen Human immunodeficiency virus (HIV)‑1
PF4, their drawbacks include high cost and inability to be
proteins envelop protein
removed by other means than renal clearance.

Herpes simplex virus (HSV) envelop 7. HEPARIN’S BIOLOGICAL ROLES

proteins

Hepatitis viruses (B, C and E) Heparin’s major biological role is the regulation
of coagulation system. Moreover, heparin and HS have
a wide range of biological roles related to inflammation,
required to form a ternary complex with AT and thrombin. angiogenesis, growth factors, developmental process,
Despite this limitation, LMWHs still inactivate factor Xa as and various disease processes (44-46) (Table 1).
well as heparin, since factor Xa inactivation only requires Heparin is found in the intracellular vesicles in mast cells
a pentasaccharide. LMWHs also show low non-specific while the less sulfated HS, is ubiquitously distributed on
binding to macrophages, endothelial cells, platelets, various cell surfaces and in the extracellular matrix of
osteoblasts, platelet factor 4 (PF4), and nonspecific most animal tissues. Heparin-protein interactions have
binding to plasma proteins ((1, 29, 34-36) reducing many been energetically investigated over the past 25 years.
of the problems associated with heparin like shorter More than 400 of heparin/HS-binding-proteins are known
plasma half-lives, heparin induced thrombocytopenia and this accumulated knowledge and computational
(HIT), and osteoporosis ((1, 29, 37). technology opened up systematic investigations into the
HS-protein interactome (47, 48).
Since LMWHs contains chains smaller than
UFHs and larger than ULMWHs it shows intermediate 7.1. Inflammation and angiogenesis
activities. The longer chains in a LMWH can capitalize Heparin and HS can bind to chemokines,
on UFH characteristics, binding to stabilin-2 a scavenger which are a group of cytokine-like proteins involved with
receptor on liver endothelial cells is responsible for inflammation and angiogenesis (44). Chemokines have
internalizing UFHs, or shorter chains can capitalize on various functions including leucocyte degranulation
ULMWHs characteristics, having excellent subcutaneous and migration, selective recruitment and activation of
bioavailability. In the case of kidney failure, renal clearance cells, and angiogenesis promotion. On the surface of
is blocked or greatly reduced so the alternative routes for endothelial cells, HS enhances the local accumulation of
heparin clearance become more important. The liver plays chemokines and chemokine binding to G-protein-coupled
a critical role in heparin clearance (7, 38). Liver clearance receptors (44). In inflammation, PF4 is also associated
is believed to involve the stabilin-2-receptor (7). This with heparin and HS. One of the most serious adverse
receptor requires GAG chains larger than decasaccharides effects of clinically administered heparin is a rapid loss of
in length for binding and clearance. In UFH, most of the platelets, resulting in HIT.
chains satisfy this chain size requirement In LMWH many
of chains are of insufficient size for liver clearance and in 7.2. Growth factor signaling
ULMWH none of the chains are sufficient in size for liver Heparin binds to multiple families of growth
clearance. Protamine, an FDA-approved drug used to factors, including fibroblast growth factors (FGFs),

1380 © 1996-2016
Heparin and anticoagulation

endothelial growth factors (EGFs), and platelet Heparin and HS also function as pathogen
derived growth factors (PDGFs). The 23 members receptors (49). Since HS is found on the external surface
of the FGF family are involved in developmental and of the cells, a viral coat protein may bind HS and help the
physiological processes, including cellular proliferation virus invade. The interaction of heparin or HS with human
and differentiation, morphogenesis, and angiogenesis. immunodeficiency virus (HIV) and herpes simplex virus
Heparin and HS activate signal transduction. HS (HSV) have been attracted much attention.
proteoglycans on the cell’s surface are required for
the high affinity binding of FGFs to their family of 8. HEPARIN’S THERAPEUTIC APPLICATIONS
seven fibroblast growth factor receptors (FGFRs). HS
mediates the activity of FGF by inducing the dimerization Heparin is a complicated drug to use
FGF·FGFR, resulting in a signal transduction (50). The therapeutically. Its primary use is as an essential
crystal structure revealed how heparin intimately binds component of extracorporeal therapy to maintain blood
to both FGF and FGFR (51, 52). Recent study showed flow in kidney dialysis and heart-lung oxygenation. It has
that HS proteoglycan is responsible for accumulation of its uses as a drug that can be used to treat and/or prevent
Factor XIIa, which acts as a growth factor by expressing deep vein thrombosis, pulmonary embolism, ischemic
pro-mitogenic activities (53). complications of unstable angina and other diseases
related to anticoagulation. It is also used in general
7.3. Developmental process medical procedures like surgery and implantation.
HS participates in developmental processes. Furthermore, heparins’ anti-inflammatory effect has been
Studies in Drosophila indicated that HS proteoglycan is investigated to treat allergic asthma, allergic rhinitis and
absolutely necessary in morphogen signaling pathway similar diseases.
involving Wnt and Hedgehog (54-57). In mice, several
studies demonstrated the importance of heparin/HS UFH is mainly administrated intravenously
biosynthetic enzymes in development. A mutation in while LMWH and ULMWH are mainly administrated
the sulfotransferase, 2-O-sulfotransferase (OST), leads subcutaneously. Other routes of heparin administration
to multiple abnormalities (58), while a deficiency in the that have been explored include oral, intranasal,
glycosyltransferases Ext1 or Ext2 show embryonic inhalation and even transdermal but its low bioavailability
lethality or exostoses, respectively (59, 60). Deficiencies by these routes generally precludes these routes.
in the secreted 6-O-endosulfatases, Sulf1 and Sulf2,
display that these enzymes, while redundant, are 8.1. Heparin’s anti-thrombotic and anti-
essential for the survival of neonatal mice (61). embolytic therapeutic applications
With deep vein thrombosis and pulmonary
7.4. Various disease processes embolism there are various dosing requirements
Heparin and HS are related to a variety of depending on the severity of illness, the heparin being
disease processes. Thrombin, the key factor in the administered, and other preexisting medical problems.
coagulation pathway, plays a role in cancer progression. For the initial treatment of if there is intermediate risk of
Thus, the antitumor effects of anticoagulants have pulmonary embolism, intravenous or subcutaneous UFH,
been investigated (62). A meta-analysis of clinical trials or subcutaneous LMWH heparin is administered over the
of heparin’s antitumor activity has shown that among first 5 to 10 days (14, 65, 66). The dosing is generally
anticoagulants, LMWH in particular, improved the 170-200 IU/kg subcutaneously for LMWH and 230-
survival period of cancer patients, but increased the risks 300 IU/kg for UFH (14, 66, 67). These can be given as
for bleeding complications (63). The remaining important one dose or split into two smaller doses twice daily. After
questions for exploiting heparin as a cancer therapeutic this initial period the patient can be transitioned to VKAs
includes the optimal types of cancer for its use, the use of or a newer oral anticoagulant. This treatment normally
non-anticoagulant heparin, the safety of heparin’s long- continues for three months or longer to ensure that the
term use, the influence of cancer stage, and duration risk has been reduced (14, 65). Risk is determined based
of heparin treatment. In addition, the complicated on an individual patient’s chance of recurrence and their
mechanism of heparin’s antitumor activity remains to be bleeding risk. In high-risk situations where pulmonary
elucidated. embolism has been triggered by shock or hypertension
the treatment parameters can be different. The initial
Heparin and HS have also been proposed treatment is an immediate intravenous bolus of UFH,
as therapeutic agents for the treatment of then with thrombolytic therapy, surgical or catheter
Alzheimer’s disease. The precise pathological role pulmonary embolectomy, followed by the same three-
of HS-proteoglycans in Alzheimer’s disease remains month treatment used in lower risk patients (66).
elusive, however, possible protective mechanisms
include reduction of amyloid β-peptide (Aβ) generation, Special cases of thrombolic pulmonary
prevention of Aβ aggregation and deposition, attenuation embolism, such as in patients with cancer or pregnant,
of Aβ’s toxic effects and acceleration of Aβ removal (64). have to be treated differently (14, 65, 66). LMWHs are

1381 © 1996-2016
Heparin and anticoagulation

highly recommended in pregnant patients as they can be the clotting parameters were unchanged (76), reducing
subcutaneously administered twice a day, forgoing the the risk of adverse side effects like bleeding. Similarly
use of VKAs. The twice-daily administration of LMWHs heparin is much milder than steroids potentially making
balances the peaks and dips in the plasma levels of it a viable alternative to current asthma treatment (74).
LMWH (14). The use of VKAs are contraindicated by
pregnancy as there are side effects including teratogenic 9. ADVERSE REACTIONS
effects in the first trimester, and fetal or neonatal intracranial
bleeding in the third trimester since VKAs can cross the Heparin’s most well-known adverse reactions are
blood brain barrier (14, 66). VKAs can be used after hemorrhage, HIT, osteoporosis, general hypersensitivity
pregnancy even to breast feeding mothers (66). In cancer reactions, and elevations of aminotransferase
patients the initial treatment time is extended 3-6 months levels (78-80).
with LMWH or VKAs but can continue indefinitely if the
cancer is not cured since venous thromboembolism is 9.1. Hemorrhage
4-times more likely in cancer patients. If the cancer is Since heparins work as anticoagulants, bleeding
cured or is in remission treatment is normally continued or hemorrhage complication is expected. Bleeding, or
for 6 months. Heparin has also been shown to exhibit hemorrhage is one of the major adverse reactions (>2%)
anti-neoplastic properties (68). This is largely due to of heparins. Hemorrhage site includes adrenal gland,
heparins anti-angiogenic properties, which help to inhibit ovary, retroperitoneal area, but hemorrhage can occur
tumor formation, since tumor growth beyond a size of virtually anywhere in the patient. The highest risk of
1 mm3 is dependent on angiogenesis (68). bleeding reported for UFH is in women over 60 years of
age (78) and for patients with cardiovascular, hematologic
Ischemic complications of acute coronary and gastrointestinal diseases and those with a hereditary
syndrome, which includes myocardial infarction and AT deficiency.
unstable angina, can be prevented with heparin. Heparin
is not the primary source of treatment and is normally Neurological impairment resulted from spinal or
used in conjunction with aspirin to inhibit platelet activation epidural hematomas may occur. Indeed, one ULMWH
and prevent the growth of plaques or a vasodialator, like medical drug label states that “Epidural or spinal
nitroglycerine, to widen the blood vessels. Both heparin hematomas may occur in patients who are anticoagulated
and LMWH have been shown to reduce the recurrence with LMWH, heparinoids, or fondaparinux sodium and
rate of angina and myocardial infarction (69, 70). Despite are receiving neuraxial anesthesia or undergoing spinal
the minor advantages in reduced angina recurrence puncture. These hematomas may result in long-term or
with UFH, compared to LMWH, UFH results in a greater permanent paralysis” (80).
increase in the risk of HIT (71). Heparin also dampens
the coagulation reactions to levels similar to patients Clinical trials of the LMWH Lovenox®
with stable coronary artery disease (70). Similarly the (enoxaparin sodium) showed that both Lovenox and
thromboembolic complications associated with atrial UFH have the similar rate of major bleeding events (79).
fibrillation can also be treated and prevented with heparin, In addition, another set of clinical trials of the ULMWH,
reducing the risk of stroke (72, 73). Arixtra® (fondaparinux sodium), reported that the rates
of major and minor bleeding between the ULMWH Arixtra
8.2. Heparin’s anti-inflammatory therapeutic and LMWHs (enoxaparin sodium or dalteparin sodium)
applications are similar (80).
In addition to the intravenous and subcutaneous
routes of heparin administration, heparin has also been 9.2. Heparin-induced thrombocytopenia
used intranasaly. The intranasal effects of heparin, while HIT is a serious antibody-associated reaction
not related to anticoagulation, are still important. Heparin resulting in abnormal and irreversible aggregation
administered intranasaly has been shown to reduce of platelets, leading to thromboembolic events and
inflammation (74-77). Heparin inhibits changes in nasal potential death. HIT can occur several weeks after the
airway pressure, leukocyte infiltration, eosinophil cell discontinuation of heparin treatment. The antibody that
migration and eosinophil cationic proteins (75, 77). These triggers HIT reacts with a complex formed between
properties along with inhibition of mast cell-endothelial UFH (or LMWH) and a partially unfolded conformation
cell interaction and the reduction of methacholine of the chemokine PF4 (39). Interaction with the platelet
hypersensitivity also show potential for heparin to treat monocyte Fc receptors leads to pro-coagulant factor
diseases related to allergic responses like asthma, release and thrombin generation (81). However, the
allergic rhinitis and many more (75, 77). The dose for precise mechanistic details of developing the heparin-PF4
this type of anti-inflammatory response can be as much immune response and subsequent HIT remain elusive.
as 1000 IU/kg with lower doses 300 IU/kg not exhibiting
the same airway response for asthma patients (74, 75). Platelet count and pre-test clinical scoring
Despite the uses of large doses and repeated doses systems are used in the diagnosis of HIT. Recently, the

1382 © 1996-2016
Heparin and anticoagulation

HIT expert probability score was developed (82). This 9.4. Others
score focuses on 8 clinical features and each feature Both UFH and LMWH can cause an increase
has a point range from -3 to +3. Laboratory tests can in aspartate (AST (SGOT)) and alanine (ALT (SGPT))
then be used to confirm the presence of PF4/heparin aminotransferase levels, but to levels expected to be
antibodies. asymptomatic. The rates of these elevations are reported
that 5.9.% or 6.1. % of patients with UFH or Lovenox,
According to a meta-analysis, LMWH had respectively (79). Other adverse reactions of heparin
statistically significant lower risk of HIT over UFH include local irritation, hypersensitivity and delayed
(P <.001), and the absolute risk for HIT with UFH and transient alopecia.
LMWH, determined by inverse variance weighted
average, are 2.6.% and 0.2.%, respectively (83). In 10. FUTURE PROSPECTS
this meta-analysis, HIT was defined as a “decrease in
platelets greater than 50% or to less than 100 x 109/L For the foreseeable future heparins will continue
and positive laboratory HIT assay”. It is noteworthy to serve crucial roles in anticoagulant therapy needed in
that while patients treated with heparin frequently modern medicine and remain largely unthreatened from
demonstrated thrombocytopenia, HIT accounts for only competition from other types of anticoagulants. Thus,
a small fraction of these cases. The same meta-analysis the future of heparins relies less on the improvement of
showed that the total events of HIT for UFH and LMWH their properties and competition than on the heparin’s
are 31/1255 patients and 1/1255 patients, respectively, continued availability in sufficient quantities in high
while the total events of thrombocytopenia for UFH and quality at reasonable costs. Currently, most of the
LMWH are 238/3758 and 152/3758, respectively (83). heparin approved for use in the U.S. and worldwide is
prepared from the intestinal mucosa of Chinese pigs.
UFH and LMWH exhibit the similar risk Most of the pigs raised worldwide are already used in the
of thrombocytopenia. In clinical trials, moderate production of heparin. As modern medical procedures
thrombocytopenia (platelet counts between 50,000 to (i.e., hemodialysis, open-heart surgery, etc.,) become
100,000/mm3) occurred at 1.2. % in UFH, at 1.3. % in increasingly needed and increasingly available in
LMWH and at 0.7.% in placebo. Severe thrombocytopenia second-world and third-world countries the demand for
(platelet counts less than 50,000/mm3) occurred at 0.2. % heparin-based products will outstrip their supply.
in UFH, at 0.1. % in LMWH and at 0.4.% in placebo (79).
As for ULMWH, another set of clinical trials reported that 10.1. Risks associated with porcine and bovine
the rate of moderate thrombocytopenia in patients given derived heparins
Arixtra® occurred 0.5.% and severe thrombocytopenia Due to the pivotal role of heparin and LMWH
occurred at 0.0.4% (80). in the anticoagulant market there are concerns about
sourcing, processing and the quality of heparin active
The definition of HIT or thrombocytopenia pharmaceutical ingredient (API). Porcine intestinal
varies in clinical studies, and sometimes is not clearly heparin produced in China accounts for more than 50% of
delineated. Another limitation is that many studies the heparin API worldwide (5). This single source raises
evaluate the risk of thrombocytopenia but not the risk of major concerns (5, 30, 87). First, sourcing of heparin API
HIT. It is often difficult to compare the rates of adverse from a single species could lead to shortages if a disease
reactions for different drugs since clinical trials are often (i.e., porcine reproductive and respiratory disease
conducted under different conditions. syndrome (also called as pig blue ear disease) (88) or
porcine epidemic diarrhea virus (89), etc.,) decreases
9.3. Osteoporosis the number of animals available from which to prepare
Long-term use of heparin can cause heparin API (87). Second, without a domestic supply,
osteoporosis and increases fracture risk. This is because there could be a severe shortage of heparin API in the
long-term use leads to reductions in bone-mineral US since China controls a majority of the worldwide
density. The reason of UFH-induced osteoporosis is that market. Moreover, low regulatory control in China’s
UFH inhibits osteoblast differentiation and its function, food and drug industry is, although the heparin supply
which prevents bone formation (84). In addition, UFH chain has been safe over the years, believed to be partly
accelerates bone resorption by reducing osteoclast responsible for the 2007 contamination crisis involving
differentiation controlling factor. The risk of osteoporosis the adulteration of Chinese porcine intestinal heparin
associated with LMWH’s has not yet been evaluated well with oversulfated chondroitin sulfate leading to deaths in
because there is a scarcity of long-term data. It is possible the Americas, Europe, and Asia (2, 5, 30). Oversulfated
to speculate that ULMWH may be better than UFH and chondroitin sulfate tightly binds to FXIIa enhancing the
show a reduced risk of osteoporosis, as fondaparinux production of vasoactive bradykinin causing severe
does not inhibit human osteoblast cell proliferation hypotensive effects (2). One solution to these problems
in vitro ((85, 86). However, appropriate clinical studies is the introduction of new sources of heparin API. The
are needed to verify this hypothesis. FDA has shown interest in reintroducing bovine heparin

1383 © 1996-2016
Heparin and anticoagulation

to diversify the market but there would need to be a current issues surrounding the supply and quality of
method of mitigating the risk of contamination (5). Thus, heparin API. Bioengineered heparin is a synthetic
further investigation is required to determine differences heparin, relying on chemoenzymatic synthesis, and
in structure, composition, activity and risks associated designed to be equivalent (a generic version) to animal-
with cows or other animal sources. based heparin API (91, 92). Despite the clear advantages
of a bioengineered heparin API (i.e. elimination of virus/
10.2. Comparison of porcine and bovine prion impurities, better controlled process, independence
derived heparins from sourcing from a single species or country) the
The structure and activity of both bovine challenges are numerous. These include: a complicated
intestinal and bovine lung heparins have been under multi-step process to match heparin’s complex structure
intensive investigation and these bovine heparins and heterogeneity in chain length and sulfation patterns;
exhibit different structure and activity than porcine the large (multi-ton) quantities of heparin required;
intestinal heparin. Bovine intestinal heparin is generally the relatively low cost of heparin API ($15-20/g); and
less sulfated and more heterogeneous than porcine development costs and regulatory hurdles.
heparin (90). Furthermore bovine intestinal heparin has
a lower molecular weight and is more polydisperse than The scheme currently proposed for the
porcine heparin (90). These results show a larger inherent preparation of bioengineered heparin can be divided
variability in the bovine intestinal heparin physically and into three parts, up-stream, mid-stream and down-
chemically. Porcine intestinal heparin displays a lower stream (91, 92). The upstream portion of the scheme
glucuronic acid content and higher GlcNS3S6S than uses a fermentation of E. coli K5 strain to prepare the
bovine intestinal heparin suggesting that they undergo heparin’s polysaccharide backbone, heparosan. The
different levels of biosynthetic modification (90). Porcine midstream portion of the scheme involves the chemical
intestinal heparin also shows significantly higher activity conversions of N-acetyl heparosan into an N-acetyl,
than bovine intestinal heparin (37, 90). Bovine intestinal N-sulfo heparosan of the appropriate molecular weight
heparin requires twice the dose of porcine intestinal and composition. The downstream portion of the
heparin to obtain the same antithrombotic effect, however, scheme involves a group of enzymatic modifications,
the bleeding risks between the two are comparable at C5-epimerization, 2-O-sulfation, 6-O-sulfation, and
similar doses (90). Bovine intestinal heparin also requires 3-O-sulfation to afford a heparin’s chemical structure
higher doses of the antidote, protamine, in order to be (Figure 6). These reactions mimic heparin’s biosynthetic
neutralized (90). Bovine lung heparin is also distinctly pathway occurring within the Golgi but without using any
different from porcine intestinal heparin, having higher animal sourced materials.
levels of N-sulfo and O-sulfo groups, a lower average
molecular weight and reduced anticoagulant activity (30). Our laboratory is actively developing a process
While comparison studies on heparins derived from to prepare bioengineered heparin. Improvements in
different animal species and tissues continue, it is becoming the up-stream portion of the scheme include increase
clear that these are not equivalent drugs and will require yields of crude heparosan of up to 17 g/L (93, 94).
different monographs, and will not be easily interchanged Metabolic engineering is also being investigated to
by physicians administering these anticoagulants. enhance heparosan biosynthesis (94, 95). The mid-
stream chemical process step parameters have been
Different production issues also come into statistically examined, using response surface method,
play for heparin API derived from different species and to enhance the control of the reaction conditions to obtain
tissues (91). Since the processes used for isolation and the N-acetyl, N-sulfo heparosan intermediate having the
purification are different, process impurities might be desired structural characteristics (96). The down-stream
encountered. Moreover, cows are susceptible to “mad portion of the scheme has been improved through the
cow disease” or bovine spongiform encephalopathy high-cell density cultivation of the E. coli to express
(BSE) that can cause Creutzfeldt-Jakob disease (CJD) larger amounts of the biosynthetic enzymes (97-99), the
in humans (5). Bovine lung heparin was once used in removal of endotoxins, associated with E. coli produced
the U.S. but was voluntarily withdrawn from the U.S. heparosan and biosynthetic enzymes have been
market following an outbreak BSE and CJD in Europe in examined (100), as have the covalent immobilization
the 1990s (5). New requirements for the slaughtering of of these enzymes (101), and the preliminary study of
cattle may be required if a bovine sourced heparin API reduction of enzymatic process steps (102).
were to be reintroduced into the U.S. market. Other food
animal sources might also possible but each new source Future challenges of bioengineered heparin
will undoubtedly encounter similar problems. include successful synthesis of bioengineered heparin
which is physicochemically and biologically equivalent
10.3. Bioengineered heparin to heparin API produced from porcine intestine, process
Bioengineered heparin, made from non-animal development to produce bioengineered heparin with a
source materials, has been proposed to address the commercially feasible method and affordable cost, and

1384 © 1996-2016
Heparin and anticoagulation

Figure 6. Chemoenzymatic synthesis of heparin. A) Homogenous low molecular weight heparin. B) Enzymatic portion of the bioengineered heparin
scheme. C) Cofactor recycling where 3’-phosphoadenosine-5’-phosphosulfate (PAPS) is generated from 3’-phosphoadenosine-5’-phosphate (PAP)
using aryl sulfotransferase IV and p-nitrophennol sulfate (PNPS) as a sacrificial sulfo group donor affording p-nitrophennol (PNP). The symbols used are
defined in Figure 5.

a scale-up to more than kilogram batch with high quality dodecasaccharide, prepared in 22 steps in an overall
(e.g. purity > 99.5% and no unknown impurity exceeding yield of 10%, showed protamine reversible activity. In
0.1.% (103)). The regeneration of 3’-phosphoadenosine- addition, this compound has been metabolized in liver
5’-phosphosulfate (PAPS), which is an expensive sulfo in mouse model. In a final example, highly purified
donor for the enzymatic O-sulfation, is a typical approach heparin-oligosaccharides having up to 21 saccharide
to reduce the cost of material (Figure 6) (91). residues have been synthesized (106). Interestingly, this
study suggests that the minimum length for a heparin
10.4. Synthetic heparin oligosaccharides to possess anti-IIa activity is 19 saccharide residues
Heparin oligosaccharide synthesis has also (molecular weight ~ 3850).
received significant attention. By using a chemoenzymatic
approach, more than 30 heparin/HS oligosaccharide Purely chemical approaches have also been
with various chain lengths and sulfation patterns have demonstrated to synthesize heparin oligosaccharides.
been synthesized (104). Three recent examples are These long and elaborate chemical syntheses require
described here. In the first example, a homogenous highly specialized techniques, making the resulting
heptasaccharide was synthesized in 10 enzymatic products very expensive. It is possible to synthesize
steps starting from a simple disaccharide with 43% heparin oligosacchardies containing tailor-made unnatural
recovery yield (105). The heptasaccharide had a similar saccharide residues, which are difficult to synthesize with
in vitro anti-Xa activity and pharmacokinetic profiles in an enzymatic approach due to a high level of enzyme
rabbits to that of fondaparinux (ULMWH), a chemically substrate specificity. A recent study, for example, utilized
synthesized homogenous pentasaccharide, which is iterative combination of three tetrasaccharide modules
currently a clinically used drug. In the second study, a to chemically synthesize a dodecasaccharide heparin-
new LMWH, a homogenous dodecasaccharide, with like molecule (107). Study of heparin oligosaccharide
better pharmaceutical profiles than current clinically synthesis providing the tools to develop new heparin-
used LMWH, was chemoenzymatically synthesized (7) based oligosaccharide therapeutics and improves our
(Figure 6). Current clinically used LMWHs have a understanding of the structure and function relationships
number of major drawbacks, as stated above. These are of heparin.
not completely neutralized with FDA-approved antidote,
protamine, while UFH can be completely neutralized and 11. CONCLUSIONS
these LMWHs can only be at reduced doses in renal-
impaired patients, since LMWH is partially excreted Heparin-based anticoagulants are an essential
through kidney. The chemoenzymatically synthesized component of modern medicine. Despite the longevity

1385 © 1996-2016
Heparin and anticoagulation

of heparin as a drug, the prospects for its future use 7. Y. Xu, C. Cai, K. Chandarajoti, P. H. Hsieh,
are quite good. Some improvements of heparin-based L. Li, T. Q. Pham, E. M. Sparkenbaugh, J.
therapeutics are still needed and will undoubtedly Sheng, N. S. Key, R. Pawlinski, E. N. Harris,
transpire in the next decade. A more immediate concern R. J. Linhardt and J. Liu: Homogeneous low-
is meeting the world’s needs for safe, high quality and
molecular-weight heparins with reversible
relatively inexpensive sources of this critical life-saving
anticoagulant activity. Nature Chemical
drug.
Biology 10, 248-250 (2014)
12. ACKNOWLEDGEMENTS DOI: 10.1038/nchembio.1459
8. J. W. Heemskerk, E. M. Bevers and T.
Akihiro Onishi, Kalib St. Ange equally Lindhout: Platelet activation and blood
contributed to this paper.The authors acknowledge the
coagulation. Thrombosis and haemostasis
funding of their work on heparin in the form of grants
from the National Institutes of Health (grants HL125371,
88, 186-193 (2002)
HL094463, GM102137, HL62244, HL096972) and from (doi not found)
the Heparin Consortium. 9. S. Palta, R. Saroa and A. Palta: Overview
of the coagulation system. Indian journal of
13. REFERENCES anaesthesia 58, 515-523 (2014)
DOI: 10.4103/0019-5049.144643
1. R. J. Linhardt: Heparin: structure and
10. L. A. Norris: Blood coagulation. Best Practice
activity. Journal of medicinal chemistry 46,
& Research Clinical Obstetrics & Gynaecology
2551-2564 (2003)
17, 369-383 (2003)
DOI: 10.1021/jm030176m
DOI: 10.1016/S1521-6934(03)00014-2
2. H. Liu, Z. Zhang and R. J. Linhardt: Lessons
11. E. M. Munoz and R. J. Linhardt: Heparin-
learned from the contamination of heparin.
binding domains in vascular biology.
Natural product reports 26, 313-321 (2009)
Arteriosclerosis, thrombosis, and vascular
DOI: 10.1039/b819896a
biology 24, 1549-1557 (2004)
3. R. J. Linhardt and N. S. Gunay: Production DOI: 10.1161/01.ATV.0000137189.22999.3f
and chemical processing of low molecular
12. V. van Hinsbergh: Endothelium-role in
weight heparins. Seminars in Thrombosis and
regulation of coagulation and inflammation.
Hemostasis 25 Suppl 3, 5-16 (1999)
Seminars in Immunopathology 34,
(doi not found)
93-106 (2012)
4. R. J. Linhardt and J. Liu: Synthetic heparin. DOI: 10.1007/s00281-011-0285-5
Current opinion in pharmacology 12,
13. G. Piazza and P. M. Ridker: Is venous
217-219 (2012)
thromboembolism a chronic inflammatory
DOI: 10.1016/j.coph.2011.12.002
disease? Clinical chemistry 61, 313-316 (2015)
5. J. Woodcock: Introduction of Proposal for DOI: 10.1373/clinchem.2014.234088
Reintroduction of Bovine Heparin to the US
Market. Science Board to the Food and Drug 14. P. S. Wells, M. A. Forgie and M. A. Rodger:
Administration, June 4, 2014, accessed on Treatment of venous thromboembolism.
June 18, 2015, Drug Administration, June JAMA 311, 717-728 (2014)
4, 2014, accessed on June 18, 2015, DOI: DOI: 10.1001/jama.2014.65
downloads/Advisory Committees/Committees 15. Pubmed Health: Deep Vein Thrombosis, June
Meeting Materials/Science Board to the Food 11, 2014, accessed on June 18, 2015, DOI:
and Drug Administration/UCM399418.pdf pubmedhealth/PMH0062951/
(doi not found) (doi not found)
6. Z. Wang, B. Yang, Z. Zhang, M. Ly, M. 16. F. Fransson, T. Kyrk, M. Skagerlind and
Takieddin, S. Mousa, J. Liu, J. S. Dordick B. Stegmayr: Rinsing the extra corporeal
and R. J. Linhardt: Control of the heparosan circuit with a heparin and albumin solution
N-deacetylation leads to an improved reduces the need for systemic anticoagulant
bioengineered heparin. Applied microbiology in hemodialysis. The International Journal of
and biotechnology 91, 91-99 (2011) Artificial Organs 36, 725-729 (2013)
DOI: 10.1007/s00253-011-3231-5 DOI: 10.5301/ijao.5000253

1386 © 1996-2016
Heparin and anticoagulation

17. F. B. Taylor, Jr., C. H. Toh, W. K. Hoots, H. 26. A. Enriquez, G. Y. Lip and A. Baranchuk:
Wada and M. Levi: Towards definition, Anticoagulation reversal in the era of the non-
clinical and laboratory criteria, and a scoring vitamin K oral anticoagulants. Europace In
system for disseminated intravascular press (2015)
coagulation. Thrombosis and haemostasis DOI: 10.1093/europace/euv030
86, 1327-1330 (2001) 27. Y. K. Loke, S. Pradhan, J. K. Yeong and C.
(doi not found) S. Kwok: Comparative coronary risks of
18. A. Murata, K. Okamoto, T. Mayumi, K. apixaban, rivaroxaban and dabigatran: a meta-
Muramatsu and S. Matsuda: The recent analysis and adjusted indirect comparison.
time trend of outcomes of disseminated British journal of clinical pharmacology 78,
intravascular coagulation in Japan: an 707-717 (2014)
observational study based on a national DOI: 10.1111/bcp.12376
administrative database. Journal of thrombosis 28. J. Hirsh, T. E. Warkentin, R. Raschke, C.
and thrombolysis 38, 364-371 (2014) Granger, E. M. Ohman and J. E. Dalen:
DOI: 10.1007/s11239-014-1068-3 Heparin and low-molecular-weight heparin:
19. H. Wada, J. Thachil, M. Di Nisio, P. Mathew, mechanisms of action, pharmacokinetics,
S. Kurosawa, S. Gando, H. K. Kim, J. D. dosing considerations, monitoring, efficacy,
Nielsen, C. E. Dempfle, M. Levi and C. H. Toh: and safety. Chest 114, 489S-510S (1998)
Guidance for diagnosis and treatment of DIC DOI: 10.1378/chest.114.5_Supplement.489S
from harmonization of the recommendations 29. J. Hirsh, S. S. Anand, J. L. Halperin, V.
from three guidelines. Journal of thrombosis Fuster and A. H. Association: Guide to
and haemostasis 11, 761-767 (2013) anticoagulant therapy: Heparin: a statement
DOI: 10.1111/jth.12155 for healthcare professionals from the
20. J. McLean: The thromboplastic action of American Heart Association. Circulation 103,
cephalin. Am j Physiol 41, 250 (1916) 2994-3018 (2001)
(doi not found) DOI: 10.1161/01.CIR.103.24.2994

21. W. I. Gonsalves, R. K. Pruthi and M. M. 30. L. Fu, G. Li, B. Yang, A. Onishi, L. Li, P.
Patnaik: The new oral anticoagulants in Sun, F. Zhang and R. J. Linhardt: Structural
clinical practice. Mayo Clinic proceedings 88, characterization of pharmaceutical heparins
495-511 (2013) prepared from different animal tissues.
DOI: 10.1016/j.mayocp.2013.03.006 Journal of pharmaceutical sciences 102,
1447-1457 (2013)
22. C. Becattini, M. C. Vedovati and G. Agnelli: DOI: 10.1002/jps.23501
Old and new oral anticoagulants for venous
31. J. D. D A Lane, A M Flynn, L Thunberg, and
thromboembolism and atrial fibrillation: a
U Lindahl: Anticoagulant activities of heparin
review of the literature. Thrombosis research
oligosaccharides and their neutralization
129, 392-400 (2012)
by platelet factor 4. Biochem J 218,
DOI: 10.1016/j.thromres.2011.12.014
725-732 (1984)
23. K. Chaudhari, B. Hamad and B. A. Syed: DOI: 10.1042/bj2180725
Antithrombotic drugs market. Nature reviews.
32. S. Jonas and D. Quartermain: Low molecular
Drug discovery 13, 571-572 (2014)
weight heparin and the treatment of ischemic
DOI: 10.1038/nrd4365
stroke. Animal results, the reasons for failure
24. Bayer Investor Presentation, Meet in human stroke trials, mechanisms of action,
Management, Page 52, March 2013, accessed and the possibilities for future use in stroke.
on June 12, 2015, http:DOI: securedl/1994 Annals of the New York Academy of Sciences
(doi not found) 939, 268-70 (2001)
25. J. Costin, J. Ansell, B. Laulicht, S. Bakhru and DOI: 10.1111/j.1749-6632.2001.tb03634.x
S. Steiner: Reversal agents in development 33. J. Liu and L. C. Pedersen: Anticoagulant
for the new oral anticoagulants. Postgraduate heparan sulfate: structural specificity and
medicine 126, 19-24 (2014) biosynthesis. Applied microbiology and
DOI: 10.3810/pgm.2014.11.2829 biotechnology 74, 263-272 (2007)

1387 © 1996-2016
Heparin and anticoagulation

DOI: 10.1007/s00253-006-0722-x 41. M. R. Lassen, O. E. Dahl, P. Mismetti, D.

34. J. Hirsh, T. E. Warkentin, S. G. Shaughnessy, Destree and A. G. Turpie: AVE5026, a
S. S. Anand, J. L. Halperin, R. Raschke, C. new hemisynthetic ultra-low-molecular-
Granger, E. M. Ohman and J. E. Dalen: weight heparin for the prevention of venous
Heparin and low-molecular-weight heparin: thromboembolism in patients after total knee
mechanisms of action, pharmacokinetics, replacement surgery--TREK: a dose-ranging
dosing, monitoring, efficacy, and safety. Chest study. Journal of thrombosis and haemostasis
119, 64S-94S (2001) 7, 566-572 (2009)
DOI: 10.1378/chest.119.1_suppl.64S DOI: 10.1111/j.1538-7836.2009.03301.x
35. C. Wang, Z. Zhai, Y. Yang, Z. Cheng, K. Ying, 42. L. Hao, Q. Zhang, T. Yu, L. Yu and Y. Cheng:
L. Liang, H. Dai, K. Huang, W. Lu, Z. Zhang, Modulation of ultra-low-molecular-weight
X. Cheng, Y. H. Shen, B. L. Davidson and heparin on (Ca2+)i in nervous cells. Brain
C. N. V. T. V. S. Group: Inverse relationship Research Bulletin 86, 355-359 (2011)
of bleeding risk with clot burden during DOI: 10.1016/j.brainresbull.2011.08.018
pulmonary embolism treatment with LMW 43. S. Rico, R. M. Antonijoan, I. Gich, M. Borrell,
heparin. The Clinical Respiratory Journal n/a- J. Fontcuberta, M. Monreal, J. Martinez-
n/a (2015) Gonzalez and M. J. Barbanoj: Safety
(doi not found) assessment and pharmacodynamics of a
36. M. Guerrini, P. A. Mourier, G. Torri and C. Viskov: novel ultra low molecular weight heparin
Antithrombin-binding oligosaccharides: (RO-14) in healthy volunteers--a first-time-
structural diversities in a unique function? in-human single ascending dose study.
Glycoconjugate Journal 31, 409-416 (2014) Thrombosis research 127, 292-298 (2011)
DOI: 10.1007/s10719-014-9543-9 DOI: 10.1016/j.thromres.2010.12.009
37. A. M. Tovar, L. A. Teixeira, S. M. Rembold, 44. R. J. Linhardt and T. Toida: Role
M. Leite, Jr., J. R. Lugon and P. A. Mourao: of glycosaminoglycans in cellular
Bovine and porcine heparins: different drugs communication. Accounts of chemical
with similar effects on human haemodialysis. research 37, 431-438 (2004)
BMC research notes 230 (2013) DOI: 10.1021/ar030138x
DOI: 10.1186/1756-0500-6-230 45. I. Capila and R. J. Linhardt: Heparin-protein
38. C. I. Oie, R. Olsen, B. Smedsrod and J. interactions. Angewandte Chemie 41,
B. Hansen: Liver sinusoidal endothelial 391-412 (2002)
cells are the principal site for elimination DOI: 10.1002/1521-3773(20020201)41:3
of unfractionated heparin from the <390: AID-ANIE390>3.0.CO;2-B
circulation. American journal of physiology.
46. D. L. Rabenstein: Heparin and heparan
Gastrointestinal and liver physiology 294,
sulfate: structure and function. Natural product
G520-8 (2008)
reports 19, 312-331 (2002)
DOI: 10.1152/ajpgi.00489.2007
DOI: 10.1039/b100916h
39. D. Mikhailov, H. C. Young, R. J. Linhardt
47. A. Ori, M. C. Wilkinson and D. G. Fernig:
and K. H. Mayo: Heparin dodecasaccharide
A systems biology approach for the
binding to platelet factor-4 and growth-related
investigation of the heparin/heparan sulfate
protein-alpha. Induction of a partially folded
interactome. The Journal of biological
state and implications for heparin-induced
chemistry 286, 19892-19904 (2011)
thrombocytopenia. The Journal of biological
DOI: 10.1074/jbc.M111.228114
chemistry 274, 25317-25329 (1999)
DOI: 10.1074/jbc.274.36.25317 48. F. Peysselon and S. Ricard-Blum: Heparin-
40. Z. Liu, S. Ji, J. Sheng and F. Wang: protein interactions: from affinity and kinetics
Pharmacological effects and clinical to biological roles. Application to an interaction
applications of ultra low molecular weight network regulating angiogenesis. Matrix
heparins. Drug Discoveries & Therapeutics 8, biology 35, 73-81 (2014)
1-10 (2014) DOI: 10.1016/j.matbio.2013.11.001
DOI: 10.5582/ddt.8.1 49. E. Kamhi, E. J. Joo, J. S. Dordick and R. J.

1388 © 1996-2016
Heparin and anticoagulation

Linhardt: Glycosaminoglycans in infectious 58. S. L. Bullock, J. M. Fletcher, R. S. Beddington

disease. Biological reviews of the Cambridge and V. A. Wilson: Renal agenesis in mice
Philosophical Society 88, 928-943 (2013) homozygous for a gene trap mutation
DOI: 10.1111/brv.12034 in the gene encoding heparan sulfate
50. K. Sugahara and H. Kitagawa: Heparin and 2-sulfotransferase. Genes & development 12,
Heparan Sulfate Biosynthesis. IUBMB Life 1894-1906 (1998)
54, 163-175 (2002) DOI: 10.1101/gad.12.12.1894
DOI: 10.1080/15216540214928 59. X. Lin, G. Wei, Z. Shi, L. Dryer, J. D. Esko,
51. J. Schlessinger, A. N. Plotnikov, O. A. Ibrahimi, D. E. Wells and M. M. Matzuk: Disruption of
A. V. Eliseenkova, B. K. Yeh, A. Yayon, R. gastrulation and heparan sulfate biosynthesis
J. Linhardt and M. Mohammadi: Crystal in EXT1-deficient mice. Developmental
structure of a ternary FGF-FGFR-heparin biology 224, 299-311 (2000)
complex reveals a dual role for heparin in DOI: 10.1006/dbio.2000.9798
FGFR binding and dimerization. Molecular 60. D. Stickens, B. M. Zak, N. Rougier, J. D.
cell 6, 743-750 (2000) Esko and Z. Werb: Mice deficient in Ext2
DOI: 10.1016/S1097-2765(00)00073-3 lack heparan sulfate and develop exostoses.
52. L. Pellegrini, D. F. Burke, F. von Delft, B. Development 132, 5055-5068 (2005)
Mulloy and T. L. Blundell: Crystal structure of DOI: 10.1242/dev.02088
fibroblast growth factor receptor ectodomain 61. C. R. Holst, H. Bou-Reslan, B. B. Gore, K.
bound to ligand and heparin. Nature 407, Wong, D. Grant, S. Chalasani, R. A. Carano,
1029-1034 (2000) G. D. Frantz, M. Tessier-Lavigne, B. Bolon,
DOI: 10.1038/35039551
D. M. French and A. Ashkenazi: Secreted
53. L. Wujak, M. Didiasova, D. Zakrzewicz, H. sulfatases Sulf1 and Sulf2 have overlapping
Frey, L. Schaefer and M. Wygrecka: Heparan yet essential roles in mouse neonatal survival.
Sulfate Proteoglycans Mediate Factor XIIa PLoS ONE 2, e575 (2007)
Binding to the Cell Surface. Journal of DOI: 10.1371/journal.pone.0000575
Biological Chemistry 290, 7027-7039 (2015)
62. R. Castelli, F. Porro and P. Tarsia: The
DOI: 10.1074/jbc.M114.606343
heparins and cancer: review of clinical trials
54. F. Zhang, J. S. McLellan, A. M. Ayala, D. and biological properties. Vascular medicine
J. Leahy and R. J. Linhardt: Kinetic and 9, 205-213 (2004)
structural studies on interactions between DOI: 10.1191/1358863x04vm566ra
heparin or heparan sulfate and proteins of the
hedgehog signaling pathway. Biochemistry 63. N. M. Kuderer, A. A. Khorana, G. H. Lyman
46, 3933-3941 (2007) and C. W. Francis: A meta-analysis and
DOI: 10.1021/bi6025424 systematic review of the efficacy and safety of
anticoagulants as cancer treatment: impact on
55. R. C. Binari, B. E. Staveley, W. A. Johnson, survival and bleeding complications. Cancer
R. Godavarti, R. Sasisekharan and A. S. 110, 1149-1161 (2007)
Manoukian: Genetic evidence that heparin- DOI: 10.1002/cncr.22892
like glycosaminoglycans are involved in
wingless signaling. Development 124, 64. L. Bergamaschini, E. Rossi, C. Vergani
2623-2632 (1997) and M. G. De Simoni: Alzheimer’s disease:
(doi not found) another target for heparin therapy.
TheScientificWorldJournal 9, 891-908 (2009)
56. T. E. Haerry, T. R. Heslip, J. L. Marsh and M. B.
DOI: 10.1100/tsw.2009.100
O’Connor: Defects in glucuronate biosynthesis
disrupt Wingless signaling in Drosophila. 65. A. T. Cohen, M. Dobromirski and M. M. P.
Development 124, 3055-3064 (1997) Gurwith: Managing pulmonary embolism
(doi not found) from presentation to extended treatment.
57. X. Lin and N. Perrimon: Dally cooperates with Thrombosis Research 133, 139-148 (2014)
Drosophila Frizzled 2 to transduce Wingless DOI: 10.1016/j.thromres.2013.09.040
signalling. Nature 400, 281-284 (1999) 66. S. V. Konstantinides: 2014 ESC Guidelines
DOI: 10.1038/22343 on the diagnosis and management of acute

1389 © 1996-2016
Heparin and anticoagulation

pulmonary embolism. European heart journal 75. Z. Diamant and C. P. Page: Heparin and related
35, 3145-3146 (2014) molecules as a new treatment for asthma.
(doi not found) Pulmonary pharmacology & therapeutics 13,
67. C. Kearon, L. Harrison, M. Crowther and J. S. 1-4 (2000)
Ginsberg: Optimal Dosing of Subcutaneous DOI: 10.1006/pupt.1999.0222
Unfractionated Heparin for the Treatment of 76. H. W. C. Richard J. Martin, Joyce M. Honour,
Deep Vein Thrombosis. Thrombosis Research and Ronald J. Harbeck: Airway Inflammation
97, 395-403 (2000) and Bronchial Hyperresponsiveness after
DOI: 10.1016/S0049-3848(99)00189-9 Mycoplasma pneumoniae Infection in a Murine
68. N. K.: Low-molecular-weight heparins and Model. American Journal of Respiratory Cell
angiogenesis. APMIS: acta pathologica, and Molecular Biology 24, 249-254 (2001)
microbiologica, et immunologica Scandinavica (doi not found)
114, 79-102 (2006) 77. Y. M. Al Suleimani, Y. Dong and M. J. Walker:
DOI: 10.1111/j.1600-0463.2006.apm_235.x Differential responses to various classes of
drugs in a model of allergic rhinitis in guinea
69. H. S. Amane and N. P. Burte: A comparative
pigs. Pulmonary pharmacology & therapeutics
study of dalteparin and unfractionated
21, 340-348 (2008)
heparin in patients with unstable angina
DOI: 10.1016/j.pupt.2007.08.004
pectoris. Indian journal of pharmacology 43,
703-706 (2011) 78. Pfizer Inc.: Heparin Sodium Injection Official
(doi not found) Label, Revised July, 2011
(doi not found)
70. A. Undas, K. Szułdrzyński, K. E. Brummel-
Ziedins, W. Tracz, K. Zmudka and K. G. 79. Sanofi-aventis: Lovenox (enoxaparin sodium
Mann: Systemic blood coagulation activation injection) Official Label, Revised October,
in acute coronary syndromes. Blood 113, 2013
2070-2078 (2009) (doi not found)
DOI: 10.1182/blood-2008-07-167411 80. ASPEN NDB: Arixtra (fondaparinux sodium)
71. J. D. Rich, J. M. Maraganore, E. Young, Official Label, Revised July, 2014
R.-M. Lidon, B. Adelman, P. Bourdon, S. (doi not found)
Charenkavanich, J. Hirsh, P. Theroux and 81. G. M. Lee and G. M. Arepally: Heparin-
C. P. Cannon: Heparin resistance in acute induced thrombocytopenia. Hematology/the
coronary syndromes. Journal of Thrombosis Education Program of the American Society of
and Thrombolysis 23, 93-100 (2007) Hematology. American Society of Hematology.
DOI: 10.1007/s11239-006-9049-9 Education Program 668-674 (2013)
72. D. E. Singer, G. W. Albers, J. E. Dalen, A. (doi not found)
S. Go, J. L. Halperin and W. J. Manning: 82. A. Cuker, G. Arepally, M. A. Crowther, L. Rice,
Antithrombotic therapy in atrial fibrillation: the F. Datko, K. Hook, K. J. Propert, D. J. Kuter,
Seventh ACCP Conference on Antithrombotic T. L. Ortel, B. A. Konkle and D. B. Cines:
and Thrombolytic Therapy. Chest 126, The HIT Expert Probability (HEP) Score: a
429S-456S (2004) novel pre-test probability model for heparin-
DOI: 10.1378/chest.126.3_suppl.429S induced thrombocytopenia based on broad
73. C. A. Ciervo, C. B. Granger and F. A. expert opinion. Journal of thrombosis and
Schaller: Stroke prevention in patients with haemostasis 8, 2642-2650 (2010)
atrial fibrillation: disease burden and unmet DOI: 10.1111/j.1538-7836.2010.04059.x
medical needs. The Journal of the American 83. N. Martel, J. Lee and P. S. Wells: Risk for
Osteopathic Association 112, eS2-8 (2012) heparin-induced thrombocytopenia with
(doi not found) unfractionated and low-molecular-weight
74. K. E. B. A. J. I. JENSEN: Inhaled heparin heparin thromboprophylaxis: a meta-analysis.
is effective in exacerbations of asthma. Blood 106, 2710-2715 (2005)
Respiratory Medicine 94, 174-175 (2000) DOI: 10.1182/blood-2005-04-1546
DOI: 10.1053/rmed.1999.0677 84. K. Panday, A. Gona and M. B. Humphrey:

1390 © 1996-2016
Heparin and anticoagulation

Medication-induced osteoporosis: screening synthesis of heparin for a safer bioengineered

and treatment strategies. Therapeutic alternative.” PhD dissertation, Rensselaer
advances in musculoskeletal disease 6, Polytechnic Institute.
185-202 (2014) (doi not found)
DOI: 10.1177/1759720X14546350 93. Z. Wang, M. Ly, F. Zhang, W. Zhong, A. Suen,
85. G. Matziolis, C. Perka, A. Disch and H. A. M. Hickey, J. S. Dordick and R. J. Linhardt:
Zippel: Effects of fondaparinux compared with E. coli K5 fermentation and the preparation
dalteparin, enoxaparin and unfractionated of heparosan, a bioengineered heparin
heparin on human osteoblasts. Calcified precursor. Biotechnology and bioengineering
tissue international 73, 370-379 (2003) 107, 964-973 (2010)
DOI: 10.1007/s00223-002-2091-5 DOI: 10.1002/bit.22898
86. A. E. Handschin, O. A. Trentz, S. P. Hoerstrup, 94. Z. Wang, J. S. Dordick and R. J. Linhardt:
H. J. Kock, G. A. Wanner and O. Trentz: Effect Escherichia coli K5 heparosan fermentation
of low molecular weight heparin (dalteparin) and improvement by genetic engineering.
and fondaparinux (Arixtra) on human Bioengineered bugs 2, 63-67 (2011)
osteoblasts in vitro. The British journal of DOI: 10.4161/bbug.2.1.14201
surgery 92, 177-183 (2005) DOI: 10.1128/genomea.00049-13
DOI: 10.1002/bjs.4809 95. B. F. Cress, Z. R. Greene, R. J. Linhardt and
87. D. Barboza: Virus Spreading Alarm and Pig M. A. Koffas: Draft Genome Sequence of
Disease in China. New York Times, August Escherichia coli Strain ATCC 23506 (Serovar
16, 2007, accessed on June 19, 2015. DOI: O10:K5:H4). Genome announcements, 1,
2007/08/16/business/worldbusiness/16pigs. e0004913 (2013)
html?pagewanted=all DOI: 10.1128/genomea.00049-13
(doi not found) 96. Z. Wang, J. Li, S. Cheong, U. Bhaskar, O.
88. J. E. Butler, K. M. Lager, W. Golde, K. S. Akihiro, F. Zhang, J. S. Dordick and R. J.
Faaberg, M. Sinkora, C. Loving and Y. I. Linhardt: Response surface optimization
Zhang: Porcine reproductive and respiratory of the heparosan N-deacetylation in
syndrome (PRRS): an immune dysregulatory producing bioengineered heparin. Journal of
pandemic. Immunologic research 59, biotechnology 156, 188-196 (2011)
81-108 (2014) DOI: 10.1016/j.jbiotec.2011.08.013
DOI: 10.1007/s12026-014-8549-5 97. O. F. Restaino, U. Bhaskar, P. Paul, L.
Li, M. De Rosa, J. S. Dordick and R. J.
89. K. Jung and L. J. Saif: Porcine epidemic
Linhardt: High cell density cultivation of a
diarrhea virus infection: Etiology, epidemiology,
recombinant E. coli strain expressing a key
pathogenesis and immunoprophylaxis.
enzyme in bioengineered heparin production.
Veterinary journal 204, 134-143 (2015)
Applied microbiology and biotechnology 97,
DOI: 10.1016/j.tvjl.2015.02.017
3893-3900 (2013)
90. G. R. Santos, A. M. Tovar, N. V. Capille, M. DOI: 10.1007/s00253-012-4682-z
S. Pereira, V. H. Pomin and P. A. Mourao:
98. J. Zhang, M. Suflita, C. M. Fiaschetti, G. Li, L.
Structural and functional analyses of bovine
Li, F. Zhang, J. S. Dordick and R. J. Linhardt:
and porcine intestinal heparins confirm they
High cell density cultivation of a recombinant
are different drugs. Drug discovery today 19,
Escherichia coli strain expressing a
1801-1807 (2014)
6-O-sulfotransferase for the production of
DOI: 10.1016/j.drudis.2014.07.004
bioengineered heparin. Journal of applied
91. U. Bhaskar, E. Sterner, A. M. Hickey, A. Onishi, microbiology 118, 92-98 (2015)
F. Zhang, J. S. Dordick and R. J. Linhardt: DOI: 10.1111/jam.12684
Engineering of routes to heparin and related 99. J. Zhang, M. Suflita, G. Li, W. Zhong, L. Li, J. S.
polysaccharides. Applied microbiology and Dordick, R. J. Linhardt and F. Zhang: High cell
biotechnology 93, 1-16 (2012) density cultivation of recombinant Escherichia
DOI: 10.1007/s00253-011-3641-4 coli strains expressing 2-O-sulfotransferase
92. U. Bhaskar. 2014. “Chemoenzymatic and C5-epimerase for the production of

1391 © 1996-2016
Heparin and anticoagulation

bioengineered heparin. Applied Biochemistry Abbreviations: UFH: unfractionated heparin;

and Biotechnology 175, 2986-2995 (2015) LMWH: low-molecular-weight heparin; HSPG:
DOI: 10.1007/s12010-014-1466-1 heparan sulfate proteoglycan; vWF: Von
100. J. Suwan, A. Torelli, A. Onishi, J. S. Dordick and Willebrand factor; GP: glycoprotein; ADP:
R. J. Linhardt: Addressing endotoxin issues in adenosine diphosphate; TXA2: thromboxane A2;
bioengineered heparin. Biotechnology and TF: tissue factor; AT: antithrombin; HS: heparan
applied biochemistry 59, 420-428 (2012) sulfate; DXI: direct factor Xa inhibitor; DTI: direct
DOI: 10.1002/bab.1042 thrombin inhibitor; VKA: vitamin K antagonist;
t-PA: tissue plasminogen activator; ECMO:
101. J. Xiong, U. Bhaskar, G. Li, L. Fu, L. Li, F. extracorporeal membrane oxygenators; DIC:
Zhang, J. S. Dordick and R. J. Linhardt: disseminated intravascular coagulation; ULMWH:
Immobilized enzymes to convert N-sulfo, ultra-low-molecular weight heparin; FDA: Food and
N-acetyl heparosan to a critical intermediate Drug Administration; GAG: glycosaminoglycan;
in the production of bioengineered heparin. PF4: platelet factor 4; HIT: heparin induced
Journal of biotechnology 167, 241-247 (2013) thrombocytopenia; FGF: fibroblast growth factor;
DOI: 10.1016/j.jbiotec.2013.06.018 EGF: endothelial growth factor; PDGF: platelet
102. U. Bhaskar, G. Li, L. Fu, A. Onishi, M. Suflita, derived growth factors; FGFR: fibroblast growth
J. S. Dordick and R. J. Linhardt: Combinatorial factor receptors; OST: O-sulfotransferase; HIV:
one-pot chemoenzymatic synthesis of heparin. human immunodeficiency virus; HSV: herpes
Carbohydrate Polymers 122, 399-407 (2014) simplex virus; API: active pharmaceutical ingredient;
DOI: 10.1016/j.carbpol.2014.10.054 BSE: bovine spongiform encephalopathy; CJD:
103. P. A. Driguez, P. Potier and P. Trouilleux: Creutzfeldt-Jakob disease
Synthetic oligosaccharides as active
Key Words: Heparin, Anticoagulation,
pharmaceutical ingredients: lessons learned
Anticoagulants, Bovine Heparin, Bioengineered
from the full synthesis of one heparin derivative
Heparin, Review
on a large scale. Natural product reports 31,
980-989 (2014) Send correspondence to: Robert J. Linhardt,
DOI: 10.1039/C4NP00012A Center for Biotechnology and Interdisciplinary
104. J. Liu and R. J. Linhardt: Chemoenzymatic Studies, Rensselaer Polytechnic Institute,
synthesis of heparan sulfate and heparin. 110 8th St, Troy, NY 12180, USA, Tel: 518-276-3404,
Natural product reports 31, 1676-1685 (2014) Fax: 518-276-3405, E-mail: linhar@rpi.edu
DOI: 10.1039/C4NP00076E
105. Y. Xu, S. Masuko, M. Takieddin, H. Xu, R. Liu,
J. Jing, S. A. Mousa, R. J. Linhardt and J. Liu:
Chemoenzymatic synthesis of homogeneous
ultralow molecular weight heparins. Science
334, 498-501 (2011)
DOI: 10.1126/science.1207478
106. Y. Xu, E. H. Pempe and J. Liu: Chemoenzymatic
synthesis of heparin oligosaccharides
with both anti-factor Xa and anti-factor IIa
activities. The Journal of biological chemistry
287, 29054-29061 (2012)
DOI: 10.1074/jbc.M112.358523
107. S. U. Hansen, G. J. Miller, C. Cole, G.
Rushton, E. Avizienyte, G. C. Jayson and
J. M. Gardiner: Tetrasaccharide iteration
synthesis of a heparin-like dodecasaccharide
and radiolabelling for in vivo tissue distribution
studies. Nature Communications, 4 (2013)
DOI: 10.1038/ncomms3016

1392 © 1996-2016