Heparin was discovered byJay McLean andWilliam Henry Howell in 1916, although it did not enter clinical trials until 1935.[15] It was originally isolated from dogliver cells, hence its name (ἧπαρhēpar is Greek for 'liver';hepar +-in).
McLean was a second-year medical student atJohns Hopkins University, and was working under the guidance of Howell investigating pro-coagulant preparations when he isolated a fat-soluble phosphatide anticoagulant in canine liver tissue.[16] In 1918, Howell coined the term 'heparin' for this type of fat-soluble anticoagulant. In the early 1920s, Howell isolated a water-solublepolysaccharide anticoagulant, which he also termed 'heparin', although it was different from the previously discovered phosphatide preparations.[17][18] McLean's work as a surgeon probably changed the focus of the Howell group to look for anticoagulants, which eventually led to the polysaccharide discovery.
It had at first been accepted that it was Howell who discovered heparin. However, in the 1940s, Jay McLean became unhappy that he had not received appropriate recognition for what he saw as his discovery. Though relatively discreet about his claim and not wanting to upset his former chief, he gave lectures and wrote letters claiming that the discovery was his. This gradually became accepted as fact, and indeed after he died in 1959, his obituary credited him as being the true discoverer of heparin. This was elegantly restated in 1963 in a plaque unveiled at Johns Hopkins to commemorate the major contribution (of McLean) to the discovery of heparin in 1916 in collaboration with Professor William Henry Howell.[19]
In the 1930s, several researchers were investigating heparin.Erik Jorpes atKarolinska Institutet published his research on the structure of heparin in 1935,[20] which made it possible for the Swedish companyVitrum AB to launch the first heparin product forintravenous use in 1936. Between 1933 and 1936,Connaught Medical Research Laboratories, then a part of the University of Toronto, perfected a technique for producing safe, nontoxic heparin that could be administered to patients, in a saline solution. The first human trials of heparin began in May 1935, and, by 1937, it was clear that Connaught's heparin was safe, easily available, and effective as a blood anticoagulant. Before 1933, heparin was available in small amounts, was extremely expensive and toxic, and, as a consequence, of no medical value.[21]
Heparin production experienced a break in the 1990s. Until then, heparin was mainly obtained from cattle tissue, which was a by-product of themeat industry, especially in North America. With the rapid spread ofBSE, more and more manufacturers abandoned this source of supply. As a result, global heparin production became increasingly concentrated in China, where the substance was now procured from the expanding industry of breeding and slaughtering hogs. The dependence of medical care on the meat industry assumed threatening proportions in the wake of theCOVID-19 pandemic. In 2020, several studies demonstrated the efficacy of heparin in mitigating severe disease progression, as its anticoagulant effect counteracted the formation ofimmunothrombosis. However, the availability of heparin on the world market was decreased, because concurrently a renewedswine flu epidemic had reduced significant portions of the Chinese hog population. The situation was further exacerbated by the fact that mass slaughterhouses around the world became coronavirus hotspots themselves and were forced to close temporarily. In less affluent countries, the resulting heparin shortage also led to worsened health care beyond the treatment of COVID-19, for example through the cancellation ofcardiac surgeries.[22]
Heparin acts as an anticoagulant, preventing the formation of clots and extension of existing clots within the blood. Heparin itself does not break down clots that have already formed, instead, it prevents clot formation by inhibiting thrombin and other procoagulant serine proteases. Heparin is generally used for anticoagulation for the following conditions:[23]
Heparin and its low-molecular-weight derivatives (e.g.,enoxaparin,dalteparin,tinzaparin) are effective in preventing deep vein thromboses and pulmonary emboli in people at risk,[24][25] but no evidence indicates any one is more effective than the other in preventing mortality.[26]
Inangiography, 2 to 5 units/mL of unfractionated heparin saline flush is used as alocking solution to prevent the clotting of blood in guidewires, sheaths, and catheters, thus preventing thrombus from dislodging from these devices into the circulatory system .[27][28]
Unfractionated heparin is used inhemodialysis. Compared tolow-molecular-weight heparin, unfractionated heparin does not have prolonged anticoagulation action after dialysis and is low cost. However, the short duration of action for heparin would require it to maintain continuous infusion to maintain its action. Meanwhile, unfractionated heparin has higher risk ofheparin-induced thrombocytopenia.[29]
A serious side-effect of heparin isheparin-induced thrombocytopenia (HIT), caused by an immunological reaction that makesplatelets a target of immunological response, resulting in the degradation of platelets, which causes thrombocytopenia.[30] This condition is usually reversed on discontinuation, and in general can be avoided with the use of synthetic heparins. Not all patients with heparin antibodies will develop thrombocytopenia. Also, a benign form of thrombocytopenia is associated with early heparin use, which resolves without stopping heparin. Approximately one-third of patients with diagnosed heparin-induced thrombocytopenia will ultimately develop thrombotic complications.[31]
Two non-hemorrhagic side effects of heparin treatment are known. The first is an elevation of serumaminotransferase levels, which has been reported in as many as 80% of patients receiving heparin. This abnormality is not associated with liver dysfunction, and it disappears after the drug is discontinued. The other complication ishyperkalemia, which occurs in 5 to 10% of patients receiving heparin, and is the result of heparin-induced aldosterone suppression. The hyperkalemia can appear within a few days after the onset of heparin therapy. More rarely, the side-effectsalopecia andosteoporosis can occur with chronic use.[23]
As with many drugs, overdoses of heparin can be fatal. In September 2006, heparin received worldwide publicity when three prematurely born infants died after they were mistakenly given overdoses of heparin at an Indianapolis hospital.[32]
Heparin is contraindicated in those with risk of bleeding (especially in people with uncontrolled blood pressure, liver disease, and stroke), severe liver disease, or severe hypertension.[33]
Protamine sulfate has been given to counteract the anticoagulant effect of heparin (1 mg per 100 units of heparin that had been given over the past 6 hours).[34] It may be used in those who overdose on heparin or to reverse heparin's effect when it is no longer needed.[35]
Heparin's normal role in the body is unclear. Heparin is usually stored within the secretory granules ofmast cells and released only into thevasculature at sites of tissue injury. It has been proposed that rather than anticoagulation, the main purpose of heparin is defense at such sites against invading bacteria and other foreign materials.[36] In addition, it is observed across many widely different species, including some invertebrates that do not have a similar blood coagulation system. It is a highly sulfated glycosaminoglycan and has the highest negativecharge density of any knownbiological molecule.[37]
In addition to the bovine and porcine tissue from which pharmaceutical-grade heparin is commonly extracted, it has also been extracted and characterized from:
The biological activity of heparin within species 6–11 is unclear and further supports the idea that the main physiological role of heparin is not anticoagulation. These species do not possess any blood coagulation system similar to that present within the species listed 1–5. The above list also demonstrates how heparin has been highlyevolutionarily conserved, with molecules of a similar structure being produced by a broad range of organisms belonging to many differentphyla.[citation needed]
Innature, heparin is apolymer of varying chain size. Unfractionated heparin (UFH) as a pharmaceutical is heparin that has not beenfractionated to sequester the fraction of molecules with lowmolecular weight. In contrast,low-molecular-weight heparin (LMWH) has undergone fractionation to make itspharmacodynamics more predictable. Often either UFH or LMWH can be used; in some situations one or the other is preferable.[51]
Heparin binds to the enzyme inhibitorantithrombin III (AT), causing a conformational change that results in its activation through an increase in the flexibility of its reactive site loop.[52] The activated AT then inactivatesthrombin,factor Xa and other proteases. The rate of inactivation of these proteases by AT can increase by up to 1000-fold due to the binding of heparin.[53] Heparin binds to AT via a specific pentasaccharide sulfation sequence contained within the heparin polymer:
The conformational change in AT on heparin-binding mediates its inhibition of factor Xa. For thrombin inhibition, however, thrombin must also bind to the heparin polymer at a site proximal to the pentasaccharide. The highly negative charge density of heparin contributes to its very strongelectrostatic interaction withthrombin.[37] The formation of aternary complex between AT, thrombin, and heparin results in the inactivation of thrombin. For this reason, heparin's activity against thrombin is size-dependent, with the ternary complex requiring at least 18 saccharide units for efficient formation.[54] In contrast, antifactor Xa activity via AT requires only the pentasaccharide-binding site.
This size difference has led to the development oflow-molecular-weight heparins (LMWHs) andfondaparinux as anticoagulants. Fondaparinux targets anti-factor Xa activity rather than inhibiting thrombin activity, to facilitate a more subtle regulation of coagulation and an improved therapeutic index. It is a synthetic pentasaccharide, whose chemical structure is almost identical to the AT binding pentasaccharide sequence that can be found within polymeric heparin andheparan sulfate.
Danaparoid, a mixture of heparan sulfate,dermatan sulfate, andchondroitin sulfate can be used as an anticoagulant in patients having developed HIT. Because danaparoid does not contain heparin or heparin fragments, cross-reactivity of danaparoid with heparin-induced antibodies is reported as less than 10%.[55]
The effects of heparin are measured in the lab by the partial thromboplastin time (aPTT), one of the measures of the time it takes theblood plasma to clot. Partial thromboplastin time should not be confused withprothrombin time, or PT, which measures blood clotting time through a different pathway of thecoagulation cascade.
Heparin is givenparenterally because it is not absorbed from the gut, due to its high negative charge and large size. It can be injected intravenously or subcutaneously (under the skin); intramuscular injections (into muscle) are avoided because of the potential for forminghematomas. Because of its short biologichalf-life of about one hour, heparin must be given frequently or as a continuousinfusion.Unfractionated heparin has a half-life of about one to two hours after infusion,[56] whereas LMWH has a half-life of four to five hours.[57] The use of LMWH has allowed once-daily dosing, thus not requiring a continuous infusion of the drug. If long-term anticoagulation is required, heparin is often used only to commence anticoagulation therapy until an oral anticoagulant e.g.warfarin takes effect.
Unfractionated heparin has a half-life of about one to two hours after infusion,[56] whereaslow-molecular-weight heparin's half-life is about four times longer. Lower doses of heparin have a much shorter half-life than larger ones. Heparin binding tomacrophage cells is internalized and depolymerized by the macrophages. It also rapidly binds toendothelial cells, which precludes the binding to antithrombin that results in anticoagulant action. For higher doses of heparin, endothelial cell binding will be saturated, such that clearance of heparin from the bloodstream by the kidneys will be a slower process.[59]
Native heparin is a polymer with amolecular weight ranging from 3 to 30kDa, although the average molecular weight of most commercial heparin preparations is in the range of 12 to 15 kDa.[60] Heparin is a member of theglycosaminoglycan family ofcarbohydrates (which includes the closely related moleculeheparan sulfate) and consists of a variably sulfated repeatingdisaccharide unit.[61]The main disaccharide units that occur in heparin are shown below. The most common disaccharide unit* (see below) is composed of a 2-O-sulfatediduronic acid and 6-O-sulfated, N-sulfated glucosamine, IdoA(2S)-GlcNS(6S). For example, this makes up 85% of heparins from beef lung and about 75% of those from porcine intestinal mucosa.[62]
Not shown below are the rare disaccharides containing a 3-O-sulfated glucosamine (GlcNS(3S,6S)) or a free amine group (GlcNH3+). Under physiological conditions, theester andamide sulfate groups are deprotonated and attract positively charged counterions to form a heparin salt. Heparin is usually administered in this form as an anticoagulant.
One unit of heparin (the "Howell unit") is an amount approximately equivalent to 0.002 mg of pure heparin, which is the quantity required to keep 1 ml of cat's blood fluid for 24 hours at 0 °C.[63]
The three-dimensional structure of heparin is complicated becauseiduronic acid may be present in either of two low-energy conformations when internally positioned within an oligosaccharide. The conformational equilibrium is influenced by the sulfation state of adjacent glucosamine sugars.[64] Nevertheless, the solution structure of a heparin dodecasaccharide composed solely of six GlcNS(6S)-IdoA(2S) repeat units has been determined using a combination of NMR spectroscopy and molecular modeling techniques.[65] Two models were constructed, one in which all IdoA(2S) were in the2S0 conformation (A andB below), and one in which they are in the1C4 conformation (C andD below). However, no evidence suggests that changes between these conformations occur in a concerted fashion. These models correspond to the protein data bank code 1HPN.[66]
Two different structures of heparin
In the image above:
A = 1HPN (all IdoA(2S) residues in2S0 conformation)Jmol viewer
C = 1HPN (all IdoA(2S) residues in1C4 conformation)Jmol viewer
D = van der Waals radius space-filling model ofC
In these models, heparin adopts a helical conformation, the rotation of which places clusters of sulfate groups at regular intervals of about 17 angstroms (1.7 nm) on either side of the helical axis.
Either chemical or enzymatic depolymerization techniques or a combination of the two underlie the vast majority of analyses carried out on the structure and function of heparin and heparan sulfate (HS).
The enzymes traditionally used to digest heparin or HS are naturally produced by the soil bacteriumPedobacter heparinus (formerly namedFlavobacterium heparinum).[67] This bacterium is capable of using either heparin or HS as its sole carbon and nitrogen source. To do so, it produces a range of enzymes such aslyases,glucuronidases,sulfoesterases, andsulfamidases.[68] The lyases have mainly been used in heparin/HS studies. The bacterium produces three lyases, heparinases I (EC4.2.2.7), II (noEC number assigned) and III (EC4.2.2.8) and each has distinct substrate specificities as detailed below.[69][70]
GlcNS/Ac(±6S)-GlcA/IdoA (with a preference for GlcA)
UA(2S)-GlcNS(6S)
The lyases cleave heparin/HS by abeta elimination mechanism. This action generates an unsaturated double bond between C4 and C5 of the uronate residue.[71][72] The C4-C5 unsaturated uronate is termed ΔUA or UA. It is a sensitive UVchromophore (max absorption at 232 nm) and allows the rate of an enzyme digest to be followed, as well as providing a convenient method for detecting the fragments produced by enzyme digestion.
Nitrous acid can be used to chemically depolymerize heparin/HS. Nitrous acid can be used at pH 1.5 or a higher pH of 4. Under both conditions, nitrous acid affects deaminative cleavage of the chain.[73]
IdoA(2S)-aMan: The anhydromannose can be reduced to an anhydromannitol
At both 'high' (4) and 'low' (1.5) pH, deaminative cleavage occurs between GlcNS-GlcA and GlcNS-IdoA, albeit at a slower rate at the higher pH. The deamination reaction, and therefore chain cleavage, is regardless of O-sulfation carried by either monosaccharide unit.
At low pH, deaminative cleavage results in the release of inorganic SO4, and the conversion of GlcNS intoanhydromannose (aMan). Low-pH nitrous acid treatment is an excellent method to distinguish N-sulfated polysaccharides such as heparin and HS from non N-sulfated polysaccharides such aschondroitin sulfate anddermatan sulfate, chondroitin sulfate and dermatan sulfate not being susceptible to nitrous acid cleavage.
Current clinical laboratory assays for heparin rely on an indirect measurement of the effect of the drug, rather than on a direct measure of its chemical presence. These includeactivated partial thromboplastin time (APTT) and antifactor Xa activity. The specimen of choice is usually fresh, nonhemolyzed plasma from blood that has been anticoagulated with citrate, fluoride, or oxalate.[74][75]
Blood specimen test tubes,vacutainers, andcapillary tubes that use thelithium salt of heparin (lithium heparin) as an anticoagulant are usually marked with green stickers and green tops. Heparin has the advantage overEDTA of not affecting levels of mostions. However, the concentration of ionized calcium may be decreased if the concentration of heparin in the blood specimen is too high.[76] Heparin can interfere with someimmunoassays, however. As lithium heparin is usually used, a person's lithium levels cannot be obtained from these tubes; for this purpose, royal-blue-topped (and dark green-topped) vacutainers containingsodium heparin are used.
Heparin-coated blood oxygenators are available for use in heart-lung machines. Among other things, these specialized oxygenators are thought to improve overallbiocompatibility and host homeostasis by providing characteristics similar to those of native endothelium.
The DNA binding sites onRNA polymerase can be occupied by heparin, preventing the polymerase from binding to promoter DNA. This property is exploited in a range of molecular biological assays.
Common diagnostic procedures requirePCR amplification of a patient's DNA, which is easily extracted from white blood cells treated with heparin. This poses a potential problem, since heparin may be extracted along with the DNA, and it has been found to interfere with the PCR reaction at levels as low as 0.002 U in a 50 μL reaction mixture.[77]
Heparin is being trialed in a nasal spray form as prophylaxis againstCOVID-19 infection.[85] Furthermore, its reported from trials that due to anti-viral, anti-inflammatory and its anti-clotting effects its inhalation could improve at a 70% rate on patients that were actively struck by a COVID-19 infection.[86]
Considering the animal source of pharmaceutical heparin, the number of potential impurities is relatively large compared with a wholly synthetic therapeutic agent. The range of possible biological contaminants includes viruses, bacterial endotoxins, transmissible spongiform encephalopathy (TSE) agents, lipids, proteins, and DNA. During the preparation of pharmaceutical-grade heparin from animal tissues, impurities such as solvents, heavy metals, and extraneous cations can be introduced. However, the methods employed to minimize the occurrence and to identify and/or eliminate these contaminants are well established and listed in guidelines and pharmacopeias. The major challenge in the analysis of heparin impurities is the detection and identification of structurally related impurities. The most prevalent impurity in heparin is dermatan sulfate (DS), also known as chondroitin sulfate B. The building block of DS is a disaccharide composed of 1,3-linked N-acetyl galactosamine (GalN) and a uronic acid residue, connected via 1,4 linkages to form the polymer. DS is composed of three possible uronic acids (GlcA, IdoA, or IdoA2S) and four possible hexosamine (GalNAc, Gal- NAc4S, GalNAc6S, or GalNAc4S6S) building blocks. The presence of iduronic acid in DS distinguishes it from chondroitin sulfate A and C and likens it to heparin and HS. DS has a lower negative charge density overall compared to heparin. A common natural contaminant, DS is present at levels of 1–7% in heparin API but has no proven biological activity that influences the anticoagulation effect of heparin.[87]
In December 2007, theUS Food and Drug Administration (FDA) recalled a shipment of heparin because of bacterial growth (Serratia marcescens) in several unopened syringes of this product.S. marcescens can lead to life-threatening injuries and/or death.[88]
2008 recall due to adulteration in drug from China
In March 2008, majorrecalls of heparin were announced by the FDA due to contamination of the raw heparin stock imported from China.[89][90] According to the FDA, the adulterated heparin killed nearly 80 people in the United States.[91] The adulterant was identified as an "over-sulphated" derivative ofchondroitin sulfate, a popular shellfish-derived supplement often used forarthritis, which was intended to substitute for actual heparin in potency tests.[92]
According to theNew York Times: "Problems with heparin reported to the agency include difficulty breathing, nausea, vomiting, excessive sweating and rapidly falling blood pressure that in some cases led to life-threatening shock".
In 2006,Petr Zelenka, a nurse in theCzech Republic, deliberately administered large doses to patients, killing seven, and attempting to kill ten others.[93]
In 2007, a nurse atCedars-Sinai Medical Center mistakenly gave the 12-day-old twins of actorDennis Quaid a dose of heparin that was 1,000 times the recommended dose for infants.[94] The overdose allegedly arose because the labeling and design of the adult and infant versions of the product were similar. The Quaid family subsequently sued the manufacturer,Baxter Healthcare Corp.,[95][96] and settled with the hospital for $750,000.[97] Prior to the Quaid accident, six newborn babies at Methodist Hospital in Indianapolis, Indiana, were given an overdose. Three of the babies died after the mistake.[98]
In July 2008, another set of twins born at Christus Spohn Hospital South, inCorpus Christi, Texas, died after an accidentally administered overdose of the drug. The overdose was due to a mixing error at the hospital pharmacy and was unrelated to the product's packaging or labeling.[99] As of July 2008[update], the exact cause of the twins' death was under investigation.[100][101]
In March 2010, a two-year-old transplant patient from Texas was given a lethal dose of heparin at theUniversity of Nebraska Medical Center. The exact circumstances surrounding her death are still under investigation.[102]
Pharmaceutical-grade heparin is derived frommucosal tissues ofslaughtered meat animals such asporcine (pig) intestines orbovine (cattle) lungs.[103] Advances to produce heparin synthetically have been made in 2003 and 2008.[104] In 2011, a chemoenzymatic process of synthesizing low molecular weight heparins from simple disaccharides was reported.[105]
As detailed in the table below, the potential is great for the development of heparin-like structures asdrugs to treat a wide range ofdiseases, in addition to their current use as anticoagulants.[106][107]
As a result of heparin's effect on such a wide variety of disease states, a number of drugs are indeed in development whose molecular structures are identical or similar to those found within parts of the polymeric heparin chain.[106]
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