Articoli di riviste: "Heparin"

1

Zvibel, I., E. Halay e L. M. Reid. "Heparin and hormonal regulation of mRNA synthesis and abundance of autocrine growth factors: relevance to clonal growth of tumors". Molecular and Cellular Biology 11, n. 1 (gennaio 1991): 108–16. http://dx.doi.org/10.1128/mcb.11.1.108-116.1991.

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Highly sulfated, heparinlike species of heparan sulfate proteoglycans, with heparinlike glycosaminoglycan chains, are extracellular matrix components that are plasma membrane bound in growth-arrested liver cells. Heparins were found to inhibit the growth and lower the clonal growth efficiency of HepG2, a minimally deviant, human hepatoma cell line. Heparan sulfates, closely related glycosaminoglycans present in the extracellular matrix around growing liver cells, had no effect on the growth rate or clonal growth efficiency of HepG2 cells. Neither heparins nor heparan sulfates had any effect on the growth rate or clonal growth efficiency of two poorly differentiated, highly metastatic hepatoma cell lines, SK-Hep-1 and PLC/PRF/5. Heparin's inhibition of growth of HepG2 cells correlated with changes in the mRNA synthesis and abundance of insulinlike growth factor II (IGF II) and transforming growth factor beta (TGF beta). HepG2 cells expressed high basal levels of mRNAs encoding IGF II and TGF beta that were inducible, through transcriptional and posttranscriptional mechanisms, to higher levels by specific heparin-hormone combinations. For both IGF II and TGF beta, the regulation was multifactorial. Transcriptionally, IGF II was regulated by the additive effects of insulin, glucagon, and growth hormone in combination with heparin; TGF beta was regulated primarily by the synergistic effects of insulin and growth hormone in combination with heparin. Posttranscriptionally, the mRNA abundance of the IGF II 4.5- and 3.7-kb transcripts was affected by insulin. Heparin induction of all IGF II transcripts was also dependent on triiodotyronine and prolactin, but it is unknown whether their induction by heparin was via transcriptional or posttranscriptional mechanisms. Heparin-insulin combinations regulated TGF beta posttranscriptionally. The poorly differentiated hepatoma cell lines PLC/PRF/5 and SK-Hep-1 either did not express or constitutively expressed low basal levels of IGF I, IGF II, and TGF beta, whose mRNA synthesis and abundance showed no response to any heparin-hormone combination. We discuss the data as evidence that matrix chemistry is a variable determining the expression of autocrine growth factor genes and the biological responses to them.

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2

Zvibel, I., E. Halay e L. M. Reid. "Heparin and hormonal regulation of mRNA synthesis and abundance of autocrine growth factors: relevance to clonal growth of tumors." Molecular and Cellular Biology 11, n. 1 (gennaio 1991): 108–16. http://dx.doi.org/10.1128/mcb.11.1.108.

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Highly sulfated, heparinlike species of heparan sulfate proteoglycans, with heparinlike glycosaminoglycan chains, are extracellular matrix components that are plasma membrane bound in growth-arrested liver cells. Heparins were found to inhibit the growth and lower the clonal growth efficiency of HepG2, a minimally deviant, human hepatoma cell line. Heparan sulfates, closely related glycosaminoglycans present in the extracellular matrix around growing liver cells, had no effect on the growth rate or clonal growth efficiency of HepG2 cells. Neither heparins nor heparan sulfates had any effect on the growth rate or clonal growth efficiency of two poorly differentiated, highly metastatic hepatoma cell lines, SK-Hep-1 and PLC/PRF/5. Heparin's inhibition of growth of HepG2 cells correlated with changes in the mRNA synthesis and abundance of insulinlike growth factor II (IGF II) and transforming growth factor beta (TGF beta). HepG2 cells expressed high basal levels of mRNAs encoding IGF II and TGF beta that were inducible, through transcriptional and posttranscriptional mechanisms, to higher levels by specific heparin-hormone combinations. For both IGF II and TGF beta, the regulation was multifactorial. Transcriptionally, IGF II was regulated by the additive effects of insulin, glucagon, and growth hormone in combination with heparin; TGF beta was regulated primarily by the synergistic effects of insulin and growth hormone in combination with heparin. Posttranscriptionally, the mRNA abundance of the IGF II 4.5- and 3.7-kb transcripts was affected by insulin. Heparin induction of all IGF II transcripts was also dependent on triiodotyronine and prolactin, but it is unknown whether their induction by heparin was via transcriptional or posttranscriptional mechanisms. Heparin-insulin combinations regulated TGF beta posttranscriptionally. The poorly differentiated hepatoma cell lines PLC/PRF/5 and SK-Hep-1 either did not express or constitutively expressed low basal levels of IGF I, IGF II, and TGF beta, whose mRNA synthesis and abundance showed no response to any heparin-hormone combination. We discuss the data as evidence that matrix chemistry is a variable determining the expression of autocrine growth factor genes and the biological responses to them.

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3

Bar-Ner, M., A. Eldor, L. Wasserman, Y. Matzner, IR Cohen, Z. Fuks e I. Vlodavsky. "Inhibition of heparanase-mediated degradation of extracellular matrix heparan sulfate by non-anticoagulant heparin species". Blood 70, n. 2 (1 agosto 1987): 551–57. http://dx.doi.org/10.1182/blood.v70.2.551.551.

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Abstract Incubation of human platelets, human neutrophils, or highly metastatic mouse lymphoma cells with sulfate-labeled extracellular matrix (ECM) results in heparanase-mediated release of labeled heparan sulfate cleavage fragments (0.5 less than Kav less than 0.85 on Sepharose 6B). This degradation was inhibited by native heparin both when brought about by intact cells or their released heparanase activity. Degradation of heparan sulfate in ECM may facilitate invasion of normal and malignant cells through basement membranes. The present study tested the heparanase inhibitory effect of nonanticoagulant species of heparin that might be of potential use in preventing heparanase mediated extravasation of bloodborne cells. For this purpose, we prepared various species of low-sulfated or low-mol-wt heparins, all of which exhibited less than 7% of the anticoagulant activity of native heparin. N-sulfate groups of heparin are necessary for its heparanase inhibitory activity but can be substituted by an acetyl group provided that the O-sulfate groups are retained. O-sulfate groups could be removed provided that the N positions were resulfated. Total desulfation of heparin abolished its heparanase inhibitory activity. Heparan sulfate was a 25-fold less potent heparanase inhibitor than native heparin. Efficiency of low-mol-wt heparins to inhibit degradation of heparan sulfate in ECM decreased with their main molecular size, and a synthetic pentasaccharide, representing the binding site to antithrombin III, was devoid of inhibitory activity. Similar results were obtained with heparanase activities released from platelets, neutrophils, and lymphoma cells. We propose that heparanase inhibiting nonanticoagulant heparins may interfere with dissemination of bloodborne tumor cells and development of experimental autoimmune diseases.

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4

Bar-Ner, M., A. Eldor, L. Wasserman, Y. Matzner, IR Cohen, Z. Fuks e I. Vlodavsky. "Inhibition of heparanase-mediated degradation of extracellular matrix heparan sulfate by non-anticoagulant heparin species". Blood 70, n. 2 (1 agosto 1987): 551–57. http://dx.doi.org/10.1182/blood.v70.2.551.bloodjournal702551.

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Incubation of human platelets, human neutrophils, or highly metastatic mouse lymphoma cells with sulfate-labeled extracellular matrix (ECM) results in heparanase-mediated release of labeled heparan sulfate cleavage fragments (0.5 less than Kav less than 0.85 on Sepharose 6B). This degradation was inhibited by native heparin both when brought about by intact cells or their released heparanase activity. Degradation of heparan sulfate in ECM may facilitate invasion of normal and malignant cells through basement membranes. The present study tested the heparanase inhibitory effect of nonanticoagulant species of heparin that might be of potential use in preventing heparanase mediated extravasation of bloodborne cells. For this purpose, we prepared various species of low-sulfated or low-mol-wt heparins, all of which exhibited less than 7% of the anticoagulant activity of native heparin. N-sulfate groups of heparin are necessary for its heparanase inhibitory activity but can be substituted by an acetyl group provided that the O-sulfate groups are retained. O-sulfate groups could be removed provided that the N positions were resulfated. Total desulfation of heparin abolished its heparanase inhibitory activity. Heparan sulfate was a 25-fold less potent heparanase inhibitor than native heparin. Efficiency of low-mol-wt heparins to inhibit degradation of heparan sulfate in ECM decreased with their main molecular size, and a synthetic pentasaccharide, representing the binding site to antithrombin III, was devoid of inhibitory activity. Similar results were obtained with heparanase activities released from platelets, neutrophils, and lymphoma cells. We propose that heparanase inhibiting nonanticoagulant heparins may interfere with dissemination of bloodborne tumor cells and development of experimental autoimmune diseases.

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5

Fareed, Jawed, Adrian Sonevytsky, Omer Iqbal, Walter P. Jeske, Massimo Iacobelli e Debra Hoppensteadt. "Inhibition of Heparinase I by Defibrotide with Potential Clinical Implications." Blood 108, n. 11 (16 novembre 2006): 1626. http://dx.doi.org/10.1182/blood.v108.11.1626.1626.

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Abstract Defibrotide represents a poly-deoxyribonucleotide derived antischemic and antithrombotic agent. Currently this agent is used for the management of transplantation associated with vascular complications. Defibrotide is a polyanionic electrolyte capable of releasing endogenous antithrombotic mediators such as (tissue factor pathway inhibitor (TFPI) and heparans. Co-administration of defibrotide with heparin has been shown to produce an augmentation of the effect of heparin and an increase in the biologic half-life. This may be due to the drug interactions or inhibition of endogenous heparin digesting enzyme by defibrotide. To test the hypothesis that defibrotide may inhibit heparin digesting enzymes such as heparinases (I–III), the effects of defibrotide were investigated on the digestion of unfractionated heparin. Unfractionated heparin was subjected to bacterial heparinase I (flavobacterion heparinicum) in an isolated biochemical systems where unfractionated heparin was used as a substrate at a fixed concentration of 750 μg/ml. Graded amounts of defibrotide in the range of 6.25–250 μg/ml were supplemented to this mixture. The degree of heparinase digestion of unfractionated heparin was determined by using high performance liquid chromatographic methods and computation of oligosaccharide profiles. Heparinase I was found to digest heparin (MW=15.2kDa) converting it to low molecular mass heparins (MW=4.1kDa). At concentrations of >125 μg/ml defibrotide produced an almost complete inhibition of heparinase I digestion. This inhibition of heparinase digestion was dependent on defibrotide concentration and the IC50 was found to be 12.5 μg/ml. These results suggest that polyelectrolytes such as defibrotide are capable of inhibiting heparin/heparan digestion enzymes. The observed potentiation of anticoagulant effect of heparin in patients treated simultaneously with defibrotide may partially be due to the inhibition of endogenous heparin digesting enzymes. Subsequent studies in primates have revealed that the anticoagulant effects of both unfractionated heparin and low-molecular mass heparins are potentiated by defibrotide as determined by AUC measurements for the circulating level of these agents. Thus, these interactions should be taken into account to optimize anticoagulant management where these drugs are used in a combination regimen.

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6

Pinhal, Maria A. S., Isabel A. N. Santos, Irani F. Silva, Carl P. Dietrich e Helena B. Nader. "Minimum Fragments of the Heparin Molecule Able to Produce the Accumulation and Change of the Sulfation Pattern of an Antithrombotic Heparan Sulfate from Endothelial Cells". Thrombosis and Haemostasis 74, n. 04 (1995): 1169–74. http://dx.doi.org/10.1055/s-0038-1649898.

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SummaryHeparin and low molecular weight heparins stimulate two to three fold the accumulation of an antithrombotic heparan sulfate secreted by endothelial cells in culture. This led us to search for the minimum structural requirements of the heparin molecule able to elicit the enhancement of the heparan sulfate. Fragments were prepared from heparin by degradation with bacterial heparinase and heparitinases. A heparin pentasulfated tetrasaccharide was shown to be the minimum structural sequence able to enhance two to three fold the secretion of heparan sulfate by endothelial cells. The stimulation is specific for the endothelial cell, is concentration dependent and the effect is already noticed afterone hour of exposure of the cells to heparinand the tetrasaccharide. Degradation of the [35S]-heparan sulfate synthesized in the presence of heparin or the tetrasaccharide has shown a higher degree of sulfation of its iduronic acid residues.

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7

Hovingh, P., M. Piepkorn e A. Linker. "Biological implications of the structural, antithrombin affinity and anticoagulant activity relationships among vertebrate heparins and heparan sulphates". Biochemical Journal 237, n. 2 (15 luglio 1986): 573–81. http://dx.doi.org/10.1042/bj2370573.

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We analysed the distribution, structural characteristics, antithrombin-III-binding properties and anticoagulant activities of heparins and heparan sulphates isolated from the tissues of a wide range of vertebrates. Heparin has a curiously limited distribution, since it was absent from lower aquatic vertebrate species, present in only certain organs such as intestine in many higher vertebrates, and completely absent from the rabbit among mammals examined. The heparins were structurally diverse, and they exhibited a broad range of anticoagulant activities, from approx. 50% to 150% of average commercial heparins. Although there was a rough correlation between the anticoagulant potency of the starting isolate and the proportional content of material exhibiting high-affinity binding to the proteinase inhibitor antithrombin III, activities of high-affinity fractions from heparins low in activity overlapped those of low-affinity fractions from highly active heparins. Heparan sulphates, which in contrast were isolated from nearly all vertebrate organs, contained high-affinity subfractions constituting up to 5% of the starting material and possessing anticoagulant potencies of 2-30 units/mg. In consideration of the heparin data, we infer that its biological function is either species-specific or may be served by other molecular elements, and that there exists considerable diversity in the antithrombin-III-binding sequence of heparin. The more-generally distributed glycosaminoglycan heparan sulphate possesses within its variable structure a small high-affinity subfraction with low anticoagulant potency, whether isolated from aorta or other tissues. Although heparan sulphate appears to have an essential function at the cellular level, we suggest that this is probably not that of providing heparin-like antithrombotic effects on vascular surfaces.

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8

Sieme, Daniel, Christian Griesinger e Nasrollah Rezaei-Ghaleh. "Metal Binding to Sodium Heparin Monitored by Quadrupolar NMR". International Journal of Molecular Sciences 23, n. 21 (29 ottobre 2022): 13185. http://dx.doi.org/10.3390/ijms232113185.

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Heparins and heparan sulfate polysaccharides are negatively charged glycosaminoglycans and play important roles in cell-to-matrix and cell-to-cell signaling processes. Metal ion binding to heparins alters the conformation of heparins and influences their function. Various experimental techniques have been used to investigate metal ion-heparin interactions, frequently with inconsistent results. Exploiting the quadrupolar 23Na nucleus, we herein develop a 23Na NMR-based competition assay and monitor the binding of divalent Ca2+ and Mg2+ and trivalent Al3+ metal ions to sodium heparin and the consequent release of sodium ions from heparin. The 23Na spin relaxation rates and translational diffusion coefficients are utilized to quantify the metal ion-induced release of sodium ions from heparin. In the case of the Al3+ ion, the complementary approach of 27Al quadrupolar NMR is employed as a direct probe of ion binding to heparin. Our NMR results demonstrate at least two metal ion-binding sites with different affinities on heparin, potentially undergoing dynamic exchange. For the site with lower metal ion binding affinity, the order of Ca2+ > Mg2+ > Al3+ is obtained, in which even the weakly binding Al3+ ion is capable of displacing sodium ions from heparin. Overall, the multinuclear quadrupolar NMR approach employed here can monitor and quantify metal ion binding to heparin and capture different modes of metal ion-heparin binding.

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9

Diamond, M. S., R. Alon, C. A. Parkos, M. T. Quinn e T. A. Springer. "Heparin is an adhesive ligand for the leukocyte integrin Mac-1 (CD11b/CD1)." Journal of Cell Biology 130, n. 6 (15 settembre 1995): 1473–82. http://dx.doi.org/10.1083/jcb.130.6.1473.

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Previous studies have demonstrated that the leukocyte integrin Mac-1 adheres to several cell surface and soluble ligands including intercellular adhesion molecule-1, fibrinogen, iC3b, and factor X. However, experiments with Mac-1-expressing transfectants, purified Mac-1, and mAbs to Mac-1 indicate the existence of additional ligands. In this paper, we demonstrate a direct interaction between Mac-1 and heparan sulfate glycans. Heparin affinity resins immunoprecipitate Mac-1, and neutrophils and transfectant cells that express Mac-1 bind to heparin and heparan sulfate, but not to other sulfated glycosaminoglycans. Inhibition studies with mAbs and chemically modified forms of heparin suggest the I domain as a recognition site on Mac-1 for heparin, and suggest that either N- or O-sulfation is sufficient for heparin to bind efficiently to Mac-1. Under conditions of continuous flow in which heparins and E-selectin are cosubstrates, neutrophils tether to E-selectin and form firm adhesions through a Mac-1-heparin interaction.

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10

Basic-Micic, M., K. Krupinski, A. Thalhammer, C. Dechent, Ch Rauschenbach e H. K. Breddin. "Beeinflußt niedermolekulares Heparin die Thrombozytenfunktion?" Hämostaseologie 09, n. 05 (settembre 1989): 248–57. http://dx.doi.org/10.1055/s-0038-1655278.

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ZusammenfassungFolgende Thrombozytenfunktionen wurden unter dem Einfluß von unfraktioniertem Heparin und von drei niedermolekularen Heparinen untersucht:Die Thrombozytenzahl in Zitratblut und Zitratplasma, die spontane Aggregation (PAT III), die ADPund die adrenalininduzierte Aggregation, die Thrombozytenausbreitung und die Thrombozytenhaftung an silikonisiertem Glas und an boviner subendothelialer Matrix. Die Untersuchungen erfolgten an Zitratblut bzw. Zitratplasma, aber auch an Blutproben, die mit den verschiedenen Heparinen antikoaguliert waren.Im Zitrat-PRP findet sich eine Verminderung der Thrombozytenzahl unter dem Einfluß von UFH, die durch Bildung von kleinen Aggregaten bedingt ist. Niedermolekulare Heparine zeigen diese Wirkung nicht, sie haben auch in wesentlich höheren Konzentrationen als UFH einen steigernden Effekt auf die Spontanaggregation der Thrombozyten im Zitratblut. Niedermolekulare Heparine hemmen die Plättchenausbreitung gering und die Plättchenhaftung an silikonisiertem Glas sowie an endothelialer boviner Matrix deutlich.Die »aktivierenden« Effekte von unfraktioniertem Heparin waren in mit UFH antikoagulierten Blutproben nicht mehr vorhanden. Im PRP von mit niedermolekularen Heparinen gewonnenen Blutproben war die Plättchenhaftneigung an Glas und an subendothelialer Matrix stärker gehemmt als im PRP von mit UFH antikoagulierten Blutproben. Tauschversuche zeigten ein unterschiedliches Verhalten, je nachdem, ob ein niedermolekulares Heparin zu Zitrat oder umgekehrt Zitrat zu PRP zugegeben wurde, das aus den mit niedermolekularem Heparin antikoagulierten Blutproben gewonnen wurde. Zugabe von Zitrat zu dem mit niedermolekularem Heparin antikoagulierten Blut-PRP hob die Haftungshemmung auf, während die Hemmung der adrenalininduzierten Aggregation nicht aufgehoben werden konnte.Ein Teil der Effekte von Heparin und niedermolekularen Heparinen auf die Plättchenfunktion im Zitratblut ist wahrscheinlich auf die Abwesenheit von Kalziumionen zurückzuführen. Es wird postuliert, daß für die thrombosehemmende Wirkung von Heparin, besonders aber von niedermolekularen Heparinen, eine Hemmung der Plättchenhaftneigung an Endothelzelldefekten mitverantwortlich ist.

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11

Haas, P., e S. Haas. "Klinische Erfahrungen mit niedermolekularen Heparinen bei der primären Thromboembolieprophylaxe". Hämostaseologie 14, n. 01 (gennaio 1994): 25–32. http://dx.doi.org/10.1055/s-0038-1660340.

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ZusammenfassungFür die primäre Prophylaxe von thromboembolischen Komplikationen gelten Heparin und niedermolekulare Heparine als Mittel der Wahl, wobei im sogenannten Hochrisikobereich einmal täglich verabreichte pauschalierte Dosierungen von niedermolekularem Heparin sogar wirkungsvoller zu sein scheinen als die mehrfach täglich applizierte Low-dose-Heparinprophylaxe. Im Gegensatz zu herkömmlichem Heparin sollten aber niedermolekulare Heparine als individuelle Substanzen angesehen und nicht unter einem gemeinsamen Generikumsbegriff zusammengefaßt werden. Daraus folgt, daß für jedes Produkt der Wirksamkeits- und Verträglichkeitsnachweis separat erbracht werden muß. Durch die nur einmal täglich notwendige subkutane Injektion und die laborunabhängige Einsatzmöglichkeit hat die Prophylaxe mit niedermolekularen Heparinen auch erfolgreiche Anwendung im Rahmen der ambulanten Patientenversorgung gefunden.

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von Hugo, R. "Ist niedermolekulares Heparin plazentagängig?" Hämostaseologie 09, n. 05 (settembre 1989): 244–47. http://dx.doi.org/10.1055/s-0038-1655277.

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ZusammenfassungThromboembolische Komplikationen in der Schwangerschaft sind seltene, wegen des Risikos einer Lungenembolie jedoch lebensbedrohliche Krankheitszustände. Auch Langzeitschäden an den Venenklappen, die zu einem postthrombotischen Syndrom führen können, sind zu bedenken. Da in jedem Fall die Antikoagulation als erste therapeutische Maßnahme angezeigt ist, war es naheliegend, Heparin nicht nur wegen seiner akut einsetzenden Wirkung, sondern auch wegen fehlender Plazentagängigkeit einzusetzen. Hier liegt der Unterschied zu den Kumarinderivaten, die beim Fetus neben einer Kumarinembryopathie auch Blutungen auslösen können, so daß nur in zwei Drittel aller Schwangerschaften mit einem unkomplizierten Verlauf gerechnet werden kann.Ein wesentlicher Nachteil der Heparine ist, daß sie nur parenteral eingesetzt werden können und die Langzeitanwendung über 2-bis 3malige tägliche subkutane Injektionen erfolgen muß.Inzwischen sind Indikationen zur Prophylaxe thromboembolischer Erkrankungen in der Schwangerschaft etabliert. Liegt eine thromboembolisehe Erkrankung in der Anamnese vor, ist langdauernde Bettruhe mit oder ohne Tokolyse angezeigt und sind operative Eingriffe in der Schwangerschaft erforderlich, ist eine Prophylaxe mit niederdosiertem Heparin angezeigt. Insbesondere bei postthrombotischem Zustand kann es erforderlich sein, über viele Wochen subkutan Heparin zur Prophylaxe anzuwenden. Eine ähnliche Situation entsteht auch bei Patientinnen mit Herzklappenersatz, die dann allerdings therapeutisch antikoaguliert sind und Heparin, im allgemeinen subkutan über mehrere tägliche Dosen verteilt, erhalten. Relativ häufig ergeben sich Unverträglichkeitsreaktionen, die auf die häufigen Injektionen und die Antigenität des unfraktionierten Heparins zurückzuführen sind. Hier scheinen die niedermolekularen Heparine wegen ihres verzögerten Umsatzes und ihrer geringeren Antigenität ein alternatives Behandlungskonzept zur eröffnen.Ihre ausgeprägt anionische Ladungseigenschaft scheint in Übereinstimmung mit Befunden beim unfraktionierten Heparin für die fehlende Plazentapassage verantwortlich zu sein. In zahlreichen Tierversuchen konnte radioaktiv markiertes niedermolekulares Heparin beim Fetus nicht nachgewiesen werden und funktionelle Veränderungen (Anstieg der Anti-Xa-Aktivität bzw. eine Verlängerung der aPTT) waren nicht festzustellen.Erste Untersuchungen beim Menschen zeigten nach hochdosierter Anwendung niedermolekularen Heparins bei Müttern nach Schwangerschafts-abbrüchen im zweiten und dritten Schwangerschaftsdrittel keine Veränderung funktioneller Gerinnungsparameter beim Fetus.In einzelnen Fällen therapeutischer und prophylaktischer Antikoagulation bei Unverträglichkeitsreaktionen auf konventionelles Heparin wurde niedermolekulares Heparin erfolgreich über längere Zeiträume eingesetzt. Bei den Neugeborenen waren keine Gerinnungsveränderungen nachweisbar, die Mütter profitierten von geringeren Nebenwirkungen und längeren Inj ektionsin t ervallen.In einer eigenen prospektiven Studie wurde niedermolekulares Heparin bei einer Reihe von Patientinnen mit Unverträglicheitsreaktionen angewendet. In Übereinstimmung mit der bisher bekannten Literatur war ein Übertritt auf die Neugeborenen funktionell nicht nachweisbar. Weitere Fallbeobachtungen sollten angeschlossen werden, um die Vorteile niedermolekularen Heparins in der Schwangerschaft breiter nutzen zu können.

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Zhang, Fuming, Lanhong Zheng, Shuihong Cheng, Yanfei Peng, Li Fu, Xing Zhang e Robert Linhardt. "Comparison of the Interactions of Different Growth Factors and Glycosaminoglycans". Molecules 24, n. 18 (16 settembre 2019): 3360. http://dx.doi.org/10.3390/molecules24183360.

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Most growth factors are naturally occurring proteins, which are signaling molecules implicated in cellular multiple functions such as proliferation, migration and differentiation under patho/physiological conditions by interacting with cell surface receptors and other ligands in the extracellular microenvironment. Many of the growth factors are heparin-binding proteins (HBPs) that have a high affinity for cell surface heparan sulfate proteoglycans (HSPG). In the present study, we report the binding kinetics and affinity of heparin interacting with different growth factors, including fibroblast growth factor (FGF) 2,7,10, hepatocyte growth factor (HGF) and transforming growth factor (TGF β-1), using a heparin chip. Surface plasmon resonance studies revealed that all the tested growth factors bind to heparin with high affinity (with KD ranging from ~0.1 to 59 nM) and all the interactions are oligosaccharide size dependent except those involving TGF β-1. These heparin-binding growth factors also interact with other glycosaminoglycans (GAGs), as well as various chemically modified heparins. Other GAGs, including heparan sulfate, chondroitin sulfates A, B, C, D, E and keratan sulfate, showed different inhibition activities for the growth factor-heparin interactions. FGF2, FGF7, FGF10 and HGF bind heparin but the 2-O-sulfo and 6-O-sulfo groups on heparin have less impact on these interactions than do the N-sulfo groups. All the three sulfo groups (N-, 2-O and 6-O) on heparin are important for TGFβ-1-heparin interaction.

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Adler, S. "Inhibition of rat glomerular visceral epithelial cell growth by heparin". American Journal of Physiology-Renal Physiology 255, n. 4 (1 ottobre 1988): F781—F786. http://dx.doi.org/10.1152/ajprenal.1988.255.4.f781.

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The effect of several glycosaminoglycans and sulfated polysaccharides on the growth of cultured rat glomerular visceral epithelial cells (GEC) was studied in vitro. Heparin, one preparation of heparan sulfate proteoglycan, dextran sulfate, and pentosan polysulfate significantly inhibited the growth of several GEC clones studied (36.0-77.1% inhibition at 100 micrograms/ml). Other glycosaminoglycans studied did not affect GEC growth. Growth inhibition by heparin was dose related and did not appear to reflect cytotoxicity. Heparins with high or low affinity for antithrombin inhibited growth to similar degrees. When heparin was fractionated into high- and low-anticoagulant activity fractions by physicochemical means the high activity fraction displayed significantly greater growth inhibition. The degree of growth inhibition significantly correlated with serum concentration in the media (r = 0.64; P less than 0.001). Removal of heparin binding factors from serum resulted in a loss of this correlation as well as less overall growth inhibition. These experiments suggest that interactions of GEC with heparan sulfates and other heparin-like molecules in the extracellular matrix may be important in the control of GEC growth.

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Beurskens, Danielle M. H., Joram P. Huckriede, Roy Schrijver, H. Coenraad Hemker, Chris P. Reutelingsperger e Gerry A. F. Nicolaes. "The Anticoagulant and Nonanticoagulant Properties of Heparin". Thrombosis and Haemostasis 120, n. 10 (20 agosto 2020): 1371–83. http://dx.doi.org/10.1055/s-0040-1715460.

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AbstractHeparins represent one of the most frequently used pharmacotherapeutics. Discovered around 1926, routine clinical anticoagulant use of heparin was initiated only after the publication of several seminal papers in the early 1970s by the group of Kakkar. It was shown that heparin prevents venous thromboembolism and mortality from pulmonary embolism in patients after surgery. With the subsequent development of low-molecular-weight heparins and synthetic heparin derivatives, a family of related drugs was created that continues to prove its clinical value in thromboprophylaxis and in prevention of clotting in extracorporeal devices. Fundamental and applied research has revealed a complex pharmacodynamic profile of heparins that goes beyond its anticoagulant use. Recognition of the complex multifaceted beneficial effects of heparin underscores its therapeutic potential in various clinical situations. In this review we focus on the anticoagulant and nonanticoagulant activities of heparin and, where possible, discuss the underlying molecular mechanisms that explain the diversity of heparin's biological actions.

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Banik, Nipa, Seong-Bin Yang, Tae-Bong Kang, Ji-Hong Lim e Jooho Park. "Heparin and Its Derivatives: Challenges and Advances in Therapeutic Biomolecules". International Journal of Molecular Sciences 22, n. 19 (29 settembre 2021): 10524. http://dx.doi.org/10.3390/ijms221910524.

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Heparin has been extensively studied as a safe medicine and biomolecule over the past few decades. Heparin derivatives, including low-molecular-weight heparins (LMWH) and heparin pentasaccharide, are effective anticoagulants currently used in clinical settings. They have also been studied as functional biomolecules or biomaterials for various therapeutic uses to treat diseases. Heparin, which has a similar molecular structure to heparan sulfate, can be used as a remarkable biomedicine due to its uniquely high safety and biocompatibility. In particular, it has recently drawn attention for use in drug-delivery systems, biomaterial-based tissue engineering, nanoformulations, and new drug-development systems through molecular formulas. A variety of new heparin-based biomolecules and conjugates have been developed in recent years and are currently being evaluated for use in clinical applications. This article reviews heparin derivatives recently studied in the field of drug development for the treatment of various diseases.

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17

Shaughnessy, SG, E. Young, P. Deschamps e J. Hirsh. "The effects of low molecular weight and standard heparin on calcium loss from fetal rat calvaria". Blood 86, n. 4 (15 agosto 1995): 1368–73. http://dx.doi.org/10.1182/blood.v86.4.1368.bloodjournal8641368.

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Osteoporosis is a well-recognized complication of long-term heparin use. However, the mechanisms by which heparin can influence bone metabolism are unclear. We report here that unfractionated heparin stimulates the process of bone resorption and that the low molecular weight heparins (LMWHs), enoxaparin, fragmin, logiparin, and ardeparin produce significantly less calcium loss than unfractionated heparin. To assess calcium loss from bone, we quantified the release of 45Ca into the culture medium of fetal rat calvaria. 45Ca release was increased in a dose-dependent manner by the addition of either unfractionated heparin or the LMWHs; but more than 50-fold higher LMWH concentrations were required to obtain an equivalent effect to unfractionated heparin. Thus, at concentration > or = 2 micrograms/mL (0.35 anti-Xa units/mL), unfractionated heparin stimulated 45Ca release 1.53 +/- 0.06 fold. 45Ca release was increased to a similar extent by the addition of either 10(- 7) mol/L parathyroid hormone (PTH) or 10(-6) mol/L 1,25 dihydroxyvitamin D3 (1,25 Vit D3). In contrast to unfractionated heparin, LMWH concentrations > or = 100 micrograms/mL (> or = 14.0 anti- Xa units/mL) were required before maximum isotope release was observed. At concentrations well above therapeutic levels, the LMWHs stimulated 45Ca release by only 1.25 /+- 0.01-fold. Heparins with high and low antithrombin III affinities stimulated 45Ca release equally well. Both size and sulfation were found to be major determinants of heparin's ability to promote isotope release. Thus, the ability of defined heparin fragments to stimulate 45Ca release correlated with their molecular weight, and after N-desulfation the ability of heparin to induce isotope release was greatly diminished. Dermatan sulfate had no effect on 45Ca release. We conclude that size and sulfation are major determinants of heparin's ability to promote bone resorption and that the risk of heparin-induced osteoporosis may be reduced by the use of LMWH preparations.

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18

Malsch, R., J. Harenberg, D. L. Heene e L. Piazolo. "Pharmakodynamik von chemisch modifizierten niedermolekularen Heparinen". Hämostaseologie 16, n. 01 (gennaio 1996): 35–40. http://dx.doi.org/10.1055/s-0038-1656636.

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ZusammenfassungEine Verbesserung der pharmakologischen Eigenschaften von Heparinoiden und Heparinen kann durch gezielte chemische Modifikation erreicht werden. Ziel der vorliegenden Arbeit war es, durch die Kopplung von lipophilen Substanzen an niedermolekulares Heparin die intravenöse Pharmakodynamik zu verbessern. Die pharmakodynamischen Daten dieser Heparine wurden in vitro und ex vivo bestimmt. Cholesterinhemisuccinyl-LMM-Heparin hat eine In-vitro-Anti-Throm-binaktivität von 70 E/mg und eine Anti-Faktor-Xa-Aktivität von 140 E/mg. L-Ascor-binsäure-6-Palmitat-LMM-Heparin hat eine In-vitro-Anti-Thrombinaktivität von 34 E/mg und eine Anti-Faktor-Xa-Aktivität von 72 E/mg. Die modifizierten Heparine wurden mit der Pharmakodynamik von Fragmin® verglichen. Durch die Kopplung von Heparin an lipophile Substanzen wie Cholesterin und L-Ascorbinsäure-6-Palmitat wurden sowohl die Anti-Thrombin- als auch die Anti-Faktor-Xa-Halbwertszeiten signifikant verlängert, die totale Plasmaclearance war für die Anti-Faktor-Xa-Aktivität erniedrigt.

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19

Coltrini, D., M. Rusnati, G. Zoppetti, P. Oreste, G. Grazioli, A. Naggi e M. Presta. "Different effects of mucosal, bovine lung and chemically modified heparin on selected biological properties of basic fibroblast growth factor". Biochemical Journal 303, n. 2 (15 ottobre 1994): 583–90. http://dx.doi.org/10.1042/bj3030583.

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Heparins from bovine mucosa and lung, and chemically modified heparins were assayed for their capacity to: (i) protect human recombinant basic fibroblast growth factor (bFGF) from tryptic cleavage; (ii) prevent 125I-bFGF binding to heparan sulphate proteoglycans present in the extracellular matrix and on the cell surface of fetal bovine aortic endothelial GM 7373 cell cultures; (iii) affect 125I-bFGF binding to high-affinity tyrosine kinase FGF receptors present on the cell membrane of GM 7373 cells; (iv) inhibit the mitogenic activity exerted by bFGF in the same cells. The results demonstrate that the potency shown by mucosal heparins in the different assays is a direct function of size, very-low-molecular-mass heparin (2.0 kDa) being significantly less effective on a molar basis than unfractionated heparin (13.6 kDa). Increased flexibility of the backbone structure, as observed in reduced/oxidized heparins of different size, does not affect the capacity of the polysaccharide to interact with bFGF. In contrast, selective 2-O-desulphation, but not 6-O-desulphation, drastically reduced the capacity of heparin to protect bFGF from proteolytic cleavage, to affect its interaction with low- and high-affinity sites, and to inhibit its mitogenic activity. Two preparations of bovine lung heparin, differing in molecular mass, were as effective as mucosal heparin in the bFGF-tryptic-digestion assay and the endothelial-cell proteoglycan-binding assay, but they were highly inefficient at inhibiting the capacity of bFGF to interact with its tyrosine kinase receptors. Bovine lung heparins were also less effective than mucosal heparin as bFGF antagonists in GM 7373-cell-proliferation assays. N-Desulphated/N-acetylated bovine lung heparin retained only a significant capacity to protect bFGF from tryptic cleavage. The results demonstrate that different chemical features of the heparin molecule, including decrease in molecular mass, selective desulphation, disaccharide composition and clustering, affect differently the capacity of the glycosaminoglycan to interact with bFGF and to influence its biological behaviour in different assays in vitro and in endothelial cell cultures. Our findings should aid the design of synthetic oligosaccharides aimed at improving the bioavailability of bFGF when administered in vivo as a therapeutic agent.

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20

Pahl, M. V., N. D. Vaziri, F. Oveisi, J. Wang e Y. Ding. "Antithrombin III inhibits mesangial cell proliferation." Journal of the American Society of Nephrology 7, n. 10 (ottobre 1996): 2249–53. http://dx.doi.org/10.1681/asn.v7102249.

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Thrombin stimulates and heparin and heparan sulfate inhibit mesangial cell proliferation. In addition, heparin has been shown to inhibit thrombin-stimulated smooth muscle cell proliferation. The anticoagulant action of heparin is mediated by antithrombin III. This study investigated whether heparin's antiproliferative action is also mediated by antithrombin III. To this end, the effect of antithrombin III on thrombin-stimulated mesangial cell growth was examined. As expected, thrombin stimulated DNA synthesis and cell growth in cultured human mesangial cells. The effect of thrombin on DNA synthesis and mesangial cell proliferation was inhibited by standard heparin and antithrombin III, separately or together. The magnitude of the inhibitory action of antithrombin III was equal to that of equimolar concentrations of heparin and that observed with the combination of the two. Experiments carried out in serum (hence antithrombin III)-free medium revealed that heparin's inhibitory effects are independent of antithrombin III. It was concluded that antithrombin III, an endogenous inhibitor of thrombin's coagulant activity, is an equally effective inhibitor of thrombin's mitogenic action.

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21

Mycroft-West, Courtney J., Lynsay C. Cooper, Anthony J. Devlin, Patricia Procter, Scott E. Guimond, Marco Guerrini, David G. Fernig, Marcelo A. Lima, Edwin A. Yates e Mark A. Skidmore. "A Glycosaminoglycan Extract from Portunus pelagicus Inhibits BACE1, the β Secretase Implicated in Alzheimer’s Disease". Marine Drugs 17, n. 5 (16 maggio 2019): 293. http://dx.doi.org/10.3390/md17050293.

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Therapeutic options for Alzheimer’s disease, the most common form of dementia, are currently restricted to palliative treatments. The glycosaminoglycan heparin, widely used as a clinical anticoagulant, has previously been shown to inhibit the Alzheimer’s disease-relevant β-secretase 1 (BACE1). Despite this, the deployment of pharmaceutical heparin for the treatment of Alzheimer’s disease is largely precluded by its potent anticoagulant activity. Furthermore, ongoing concerns regarding the use of mammalian-sourced heparins, primarily due to prion diseases and religious beliefs hinder the deployment of alternative heparin-based therapeutics. A marine-derived, heparan sulphate-containing glycosaminoglycan extract, isolated from the crab Portunus pelagicus, was identified to inhibit human BACE1 with comparable bioactivity to that of mammalian heparin (IC50 = 1.85 μg mL−1 (R2 = 0.94) and 2.43 μg mL−1 (R2 = 0.93), respectively), while possessing highly attenuated anticoagulant activities. The results from several structural techniques suggest that the interactions between BACE1 and the extract from P. pelagicus are complex and distinct from those of heparin.

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22

Fareed, Jawed, Rakesh Wahi, Indermohan Thethi, Debra Hoppensteadt, Vinod Bansal, Jeanine M. Walenga e Harry L. Messmore. "Molecular Mimicry in the Adulteration of Heparins. Chemical Basis for the Development of Oversulfated Chondroitin Sulfate and Related Contaminants". Blood 118, n. 21 (18 novembre 2011): 4322. http://dx.doi.org/10.1182/blood.v118.21.4322.4322.

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Abstract Abstract 4322 The main contaminant in heparin and low molecular weight heparins reported during the heparin crisis (2007–8) was characterized to be oversulfated chondroitin sulfate (OSCS). However, several other sulfated glycosaminoglycans have also been reported to be present. Dermatan sulfate, heparan sulfate and chondroitin sulfate represent heparin byproducts and are invariably present in different proportions in heparin preparations. Hypersulfated derivatives of these are also found as contaminants. The chondroitin sulfate obtained from mammalian tissues and cartilage can be hypersulfated using simple chemical sulfonation techniques such as the chlorosulfonic acid to produce varying degrees of sulfation. The molecular profile of OSCS can be optimized utilizing various depolymerization methods to mimic the required molecular profile of heparin. Moreover, mixing of the molecular mass optimized OSCS to heparin exhibit the anticoagulant potencies in the pharmacopeial assays that are indistinguishable by the methods used prior to 2007. Initial studies carried out to obtain OSCS from bovine, porcine, squid and shark cartilage resulted in the generation of an almost perfect polymer mixture whose molecular profile was similar to heparin (14–16 Kda). The mixing of some of these OSCS preparation to heparin also showed either additive or synergistic effects in the anticoagulant assays. However, the OSCS preparations were resistant to heparinase digestion and some of the other chemical digestion processes. Several other heparinoids such as the galactans, sulfaminoheparosans and sulfated dextrans can also be used to contaminate heparins. Based on these observations, the development of OSCS contaminant was a result of a careful optimization of the molecular profile of non heparin GAG derived oversulfated derivatives and the knowledge of their biologic effects. Despite the implementation of sophisticated analytical techniques such as NMR and mass spectrometry along with the modified biochemical assays for heparin characterization, the heparin molecule renders it to additional approaches for adulteration utilizing chemically and biologically rational approaches. Disclosures: No relevant conflicts of interest to declare.

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23

Buddecke, E. "Non-anticoagulant functions of heparin and heparan sulfate". Hämostaseologie 16, n. 01 (gennaio 1996): 6–14. http://dx.doi.org/10.1055/s-0038-1656632.

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Abstract (sommario):

SummaryHeparin is known to bind a large number of proteins not involved in anticoagula-tion, such as growth factors, adhesive proteins of the extracellular matrix, viral coat proteins and other enzymes and proteins. In vivo predominantly heparan sulfate – the most ubiquitous cell surface glycosaminoglycan – takes the functional role of heparin. Structural features, sources and non-anticoagulant func-tions of heparin and heparan sulfate proteoglycan are described. The functional diversity of heparin and heparan sulfate is reviewed in the following sections: (I) heparin and heparan sulfate as partners in fibroblast growth factor action, (II) antiproliferative effects of heparan sulfate and heparin, (III) cell surface heparan sulfate as extracellular matrix receptor and coreceptor, (IV) proteoheparan sulfate in central and peripheral nervous system, (V) role of proteoheparan sulfate in binding and uptake of lipoproteins, (VI) virus and spirochete binding to heparin and heparan sulfate.

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24

Chao, Yapeng, Shaoxiang Xiong, Xiulan Cheng e Shijun Qian. "Mass Spectrometric Evidence of Heparin Disaccharides for the Catalytic Characterization of a Novel Endolytic Heparinase". Acta Biochimica et Biophysica Sinica 36, n. 12 (1 dicembre 2004): 840–44. http://dx.doi.org/10.1093/abbs/36.12.840.

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Abstract (sommario):

Abstract Heparinase from different sources can eliminate heparin or/and heparan sulfate into various low-molecular weight heparins with different characteristics. Porcine intestinal mucosa heparin was degraded into a series of oligosaccharides by a novel heparinase from the species Sphingobacterium. Disaccharide components from the digests were separated and purified by ultrafiltration and HPLC. Five major peaks appeared as three types according to their retention time. The mass spectrometry of peak I mainly gave the non-sulfated disaccharide with the mass of 379 Da. Peak II and III were indicated as two major monosulfated disaccharides with molecular mass of 417 and 459 Da respectively. Moreover, the peak III represented an Nacetyl disaccharide. Both peak IV and V showed the same mass of 496 Da, hinting that they were disulfate-substituted disaccharides. No trisulfate-substituted disaccharides were detected in the mixture of the heparin digest though they were abundant in the heparin structure. The results revealed that the heparinase might specifically cut the sites with low sulfated domain in heparin.

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25

Yanaka, Kiyoyuki, Stephen R. Spellman, James B. McCarthy, Theodore R. Oegema, Walter C. Low e Paul J. Camarata. "Reduction of brain injury using heparin to inhibit leukocyte accumulation in a rat model of transient focal cerebral ischemia. I. Protective mechanism". Journal of Neurosurgery 85, n. 6 (dicembre 1996): 1102–7. http://dx.doi.org/10.3171/jns.1996.85.6.1102.

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Abstract (sommario):

✓ Heparin has long been established as an anticoagulant. Although heparin has been demonstrated to reduce brain injury after ischemia and reperfusion, its mechanism of action remains unknown. Recent investigations reveal that it can modulate biological processes such as binding to adhesion receptors on endothelial cells and leukocytes. The authors hypothesized that heparin's protective effect is closely related to its antileukocyte adherence property. They evaluated the efficacy of sulfated polysaccharides (unfractionated heparin, low-molecular-weight heparin, heparan sulfate, chondroitin sulfate C, and dextran sulfate) on leukocyte accumulation, infarction size, and neurological outcome after transient focal cerebral ischemia in rats subjected to 1 hour of ischemia and 48 hours of reperfusion. Forty-nine animals were included in the study. The animals receiving unfractionated heparin or dextran sulfate showed a significant reduction in leukocyte accumulation, infarct size, and neurological dysfunction 48 hours after reperfusion (p < 0.05) when compared to untreated animals. The animals receiving unfractionated heparin also showed significantly better results than the animals receiving an equivalent anticoagulant dose of low-molecular-weight heparin. These data indicate that heparin's antileukocyte property plays a more important role than its anticoagulant ability in neuronal protection. The relative potency of the sulfated polysaccharides tested in leukocyte depletion was closely related to their degree of sulfation. Thus, in addition to demonstrating the potential efficacy of heparin as a therapeutic agent for ischemia and reperfusion injury by the prevention of leukocyte accumulation, the results also serve as a basis for studying important cellular and molecular events that contribute to tissue damage.

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26

Jaques, L. B. "Heparin". Hämostaseologie 05, n. 04 (luglio 1985): 154–59. http://dx.doi.org/10.1055/s-0038-1655119.

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ZusammenfassungDie Verteilung des Heparins ist von der Zahl der betroffenen RES-Zellen abhängig, die nach der Art der Heparinanwendung variiert. Intravenöses Heparin (besonders dauerinfundiertes und »Ultra-low dose«-Heparin) ist hauptsächlich auf das kardiovaskuläre Endothel beschränkt. Bei intramuskulärer, subkutaner und intrapulmonaler Applikation wird es auf dem Lymphweg ab transportiert, ist im lokalen Kreislauf mit einer zunehmenden Zahl von RES-Zellen konfrontiert und tritt daher im Venensystem verzögert auf.Inhalation oder Instillation in die Lunge ist eine neue Form der Heparinanwendung, die das Heparin einem sehr großen Zellpool zuleitet, mit dem Ergebnis einer langsamen Freisetzung des Heparins in den Kreislauf und das Gefäßendothel, was aber den Streß der wiederholten Venenpunktionen bzw. Injektionen vermeidbar macht. Das offensichtliche Fehlen einer Nachweisbarkeit des Heparins in der allgemeinen Zirkulation bei oraler Anwendung ist wahrscheinlich nicht die Folge einer fehlenden Resorption, sondern dadurch bewirkt, daß das Heparin dabei zuerst einen Großteil des RES und des Endothels passieren muß.Die Schwierigkeiten, Heparin zu standardisieren, erklären sich aus seiner einzigartigen chemischen Natur sowie aus der daraus resultierenden Pharmakokinetik und aus der klinischen Wirkung.Es ist zu vermuten, daß diese Schwierigkeiten durch eine Reihe von physikochemischen Spezifikationen überwindbar wären ausgehend von einer Anzahl von klinisch effektiven Handelsheparinpräparaten.Die mangelnde Korrelation von Thromboseverhütungspotenz und Antikoagulansaktivität in vitro läßt vermuten, daß ein Heparin mit geringer Gerinnungsaktivität klinisch wirksam sein könnte. Blutungen bei der Anwendung von Antikoagulantien kommen durch eine Kombination von Ursachen, meist von Streß und Antikoagulation, zustande. Die Schutzwirkungen des Heparins und der Heparinoide bedürfen weiterer klinischer Erforschung, da sie vielleicht einen bedeutenden Faktor der Heparinwirkung darstellen.

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Badillo-Samapayo, Jessica, e Jesús Aguilar-Castro. "Trombocitopenia como efecto secundario por la administración de heparinas en los pacientes de COVID-19. Una revisión sistemática". Casos y Revisiones de Salud 4, n. 1 (1 luglio 2022): 72–88. http://dx.doi.org/10.22201/fesz.26831422e.2022.4.1.7.

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Abstract (sommario):

Introducción. En la actualidad existen algunas publicaciones que describen alteraciones de la coagulación y complicaciones trombóticas arteriales y venosas principalmente en pacientes ingresados en unidades de cuidados intensivos por COVID-19. Sin embargo, no se ha publicado una revisión sistemática sobre dicha alteración. Objetivo. Presentar una síntesis del conocimiento sobre la frecuencia de la trombocitopenia como efecto secundario por la administración de heparinas en pacientes por COVID-19. Métodos. Se llevó a cabo una búsqueda en las plataformas de PubMed, SCOPUS, Biblioteca Cochrane, LILACS, Web of Science y literatura gris como TESIUNAM desde el 10 diciembre de 2021 hasta el 13 de enero de 2022, para identificar estudios que reportaran casos con HIT con pruebas confirmatorias que demuestren la activación plaquetaria de los Abs anti-PF4 / H en presencia de heparina, se utilizaron las siguientes palabras clave y estrategia “COVID-19" OR "SARS-CoV-2” OR "Coronavirus Infections” AND "low-molecular-weight heparin” OR “LMWH” OR “heparin” AND “unfractionated heparin" OR "UFH" AND “Thrombocytopenia” OR “TIH” OR “anti-H/PF4” OR “factor 4-heparin” OR “HPIA”. Resultados. Se identificaron 861 artículos, se revisaron 26 artículos de texto completo, se realizó un análisis cualitativo de 12 estudios que cumplían con los criterios de elegibilidad. Se encontró dentro de cuatro estudios de cohorte una incidencia de trombocitopenia de 0.16% a 8%. De los 12 casos clínicos, con un promedio de edad de 61.3 años. Al 50% de los pacientes de las cohortes confirmados con trombocitopenia se le administró Heparina fraccionada y a los pacientes reportados en los casos clínicos se le administró al 43%. Conclusión. Los resultados sugieren que el efecto secundario de la administración de heparinas podría aumentarse en personas mayores en estado crítico que reciben heparina fraccionada en dosis terapéuticas, sin embargo, es necesario realizar más estudios para confirmar los hallazgos. Palabras clave: COVID-19, SARS-CoV-2, heparina, Trombocitopenia

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Shi, Deling, Changkai Bu, Peng He, Yuefan Song, Jonathan S. Dordick, Robert J. Linhardt, Lianli Chi e Fuming Zhang. "Structural Characteristics of Heparin Binding to SARS-CoV-2 Spike Protein RBD of Omicron Sub-Lineages BA.2.12.1, BA.4 and BA.5". Viruses 14, n. 12 (1 dicembre 2022): 2696. http://dx.doi.org/10.3390/v14122696.

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Abstract (sommario):

The now prevalent Omicron variant and its subvariants/sub-lineages have led to a significant increase in COVID-19 cases and raised serious concerns about increased risk of infectivity, immune evasion, and reinfection. Heparan sulfate (HS), located on the surface of host cells, plays an important role as a co-receptor for virus–host cell interaction. The ability of heparin and HS to compete for binding of the SARS-CoV-2 spike (S) protein to cell surface HS illustrates the therapeutic potential of agents targeting protein–glycan interactions. In the current study, phylogenetic tree of variants and mutations in S protein receptor-binding domain (RBD) of Omicron BA.2.12.1, BA.4 and BA.5 were described. The binding affinity of Omicron S protein RBD to heparin was further investigated by surface plasmon resonance (SPR). Solution competition studies on the inhibitory activity of heparin oligosaccharides and desulfated heparins at different sites on S protein RBD–heparin interactions revealed that different sub-lineages tend to bind heparin with different chain lengths and sulfation patterns. Furthermore, blind docking experiments showed the contribution of basic amino acid residues in RBD and sulfo groups and carboxyl groups on heparin to the interaction. Finally, pentosan polysulfate and mucopolysaccharide polysulfate were evaluated for inhibition on the interaction of heparin and S protein RBD of Omicron BA.2.12.1, BA.4/BA.5, and both showed much stronger inhibition than heparin.

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Kouta, Ahmed, Walter Jeske, Debra Hoppensteadt, Omer Iqbal, Yiming Yao e Jawed Fareed. "Comparative Pharmacological Profiles of Various Bovine, Ovine, and Porcine Heparins". Clinical and Applied Thrombosis/Hemostasis 25 (1 gennaio 2019): 107602961988940. http://dx.doi.org/10.1177/1076029619889406.

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Abstract (sommario):

Unfractionated heparin is the first anticoagulant drug and has been successfully used clinically for over 80 years. Heparin and its analogues are used during surgery and dialysis and are often used to coat indwelling catheters and other devices where the vascular system is exposed. Most of the heparins used clinically are derived from porcine intestinal mucosa. However, heparins have also been manufactured from tissues of other mammalian species such as cows and sheep. Recently there have been attempts to generate bioengineered heparin in order to overcome contamination and antigenicity problems. Currently there are some concerns about the shortage of the porcine heparins as they are widely used in the manufacturing of the low-molecular-weight heparins. Moreover, due to cultural and religious reasons in some countries, alternative sources of heparins are needed. The Food and Drug Administration and other regulatory agencies have considered alternative sourcing of heparin for potential substitution of porcine heparin and are currently reviewing this matter. Numerous studies are ongoing to understand the structure-activity relationships of these various heparins. In this article, heparins from different animal sources were studied to determine the extent of biosimilarity between them. For these investigations, 10 batches each of bovine mucosal heparin (BMH), ovine mucosal heparin (OMH), and porcine mucosal heparin (PMH) were studied. These studies have demonstrated that OMH and PMH have comparable anticoagulant and antiproteases activities. However, BMH exhibited somewhat a lower potency compared to OMH and PMH in functional assays.

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Hoppensteadt, Debra, Jeanine M. Walenga, Jawed Fareed e Rodger L. Bick. "Heparin, low–molecular-weight heparins, and heparin pentasaccharide". Hematology/Oncology Clinics of North America 17, n. 1 (febbraio 2003): 313–41. http://dx.doi.org/10.1016/s0889-8588(02)00091-6.

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31

Biasiutti, F., B. Lämmle e W. A. Wuillemin. "Therapie der akuten tiefen Beinvenenthrombose mit niedermolekularen Heparinen". Hämostaseologie 18, n. 01 (gennaio 1998): 27–30. http://dx.doi.org/10.1055/s-0038-1655325.

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Abstract (sommario):

ZusammenfassungDie akute tiefe Beinvenenthrombose ist eine häufige Erkrankung. Die klassische Behandlung besteht aus einer initialen Phase mit Heparin sowie einer Langzeittherapie mit oralen Antikoagulanzien. Die Therapie mit Heparin muß rasch begonnen werden, innerhalb von 24 Stunden im therapeutischen Bereich liegen und über 5 bis 7 Tage weitergeführt werden, bis die orale Antikoagulation ihrerseits im therapeutischen Bereich liegt. Zahlreiche Studien zeigten, daß die Therapie mit subkutan applizierten niedermolekularen Heparinen mindestens so wirksam und sicher ist wie die herkömmliche parenterale und labormäßig kontrollierte Gabe von unfraktioniertem Heparin. Die niedermolekularen Heparine haben jedoch den Vorteil der einfacheren Anwendung und der geringeren Rate von immunologisch-bedingter Thrombozytopenie. Sie stellen heute die Therapie der ersten Wahl dar bei der initialen Behandlung der akuten tiefen Beinvenenthrombose. Sie bieten außerdem die Möglichkeit der ambulanten bzw. der verkürzten stationären Therapie.Eine Thrombolyse sollte nur in Ausnahmefällen durchgeführt werden, da sie mit einem höheren Blutungsrisiko verbunden ist und bezüglich klinisch relevanter Endpunkte der Therapie mit Heparinen nicht überlegen ist.

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Greinacher, A., I. Michels e C. Mueller-Eckhardt. "Heparin-Associated Thrombocytopenia: The Antibody Is Not Heparin Specific". Thrombosis and Haemostasis 67, n. 05 (1992): 545–49. http://dx.doi.org/10.1055/s-0038-1648491.

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Abstract (sommario):

SummaryIn this study the hypothesis was assessed whether heparin-associated thrombocytopenia (HAT) may be caused by an antibody dependent on polysulfated oligosaccharide epitopes, present not only on heparin but also on different polysulfated substances such as dextran sulfate and pentosan polysulfate. We found that the major factor for eliciting platelet activation with sera of HAT type II patients is neither the structure nor the AT III binding capacity of an oligosaccharide, but rather its grade of sulfation. This was shown by in vitro crossreactivity studies with 40 sera of HAT type II patients using unfractionated heparins, LMW heparins (Fragmin, Fraxiparin), enoxaparin, LMW heparinoid (Org 10172 and its subfractions), de-N-sulfated heparin, dermatan sulfate, dextran sulfate, pentosan polysulfate and dextran. Platelet activation was measured by the heparin induced platelet activation (HIPA) assay and the serotonin release assay (SRA). The platelet activating factor was isolated with the IgG fraction, but did not bind to heparin and dextran sulfate fixed to a solid phase. By isoimmune fixation electrophoresis a monoclonal gammopathy was ruled out in the three sera assessed. The in vivo effect of different LMW heparins and the heparinoid Org 10172 was observed in 10 patients with HAT type II. In a prospective study, a compatible heparin-like anticoagulant was selected for 10 HAT patients for whom further parenteral anticoagulation was required. The only substance that showed no crossreactivity in vitro was the LMW heparinoid Org 10172, which differs from heparin and LMW heparins by its low-grade sulfation. Upon treatment with the heparinoid, all 10 patients had a good clinical outcome, even if they had previously developed thromboembolic complications under LMW heparin administration. As Org 10172 contains a small amount of a LMW heparin-like substance (3%) this heparinoid should not be used in HAT patients without prior in vitro testing. We conclude that heparin-associated thrombocytopenia is not caused by a heparin-specific antibody and that a major factor contributing to the pathomechanism is the high grade of sulfation present in a variety of polysulfated oligosaccharides.

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33

Olson, Guy, Walter Jeske, Omer Iqbal, Emily Krupa, Amber Farooqui, Fakiha Siddiqui, Debra Hoppensteadt, Ahmed Kouta e Jawed Fareed. "Potency Adjusted Blended Heparin of Bovine, Ovine, and Porcine Heparin Exhibit Comparable Biologic Effects to Referenced Single-Sourced Porcine Heparin". Clinical and Applied Thrombosis/Hemostasis 29 (gennaio 2023): 107602962311632. http://dx.doi.org/10.1177/10760296231163251.

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Abstract (sommario):

Introduction: Bovine and ovine mucosa represent alternate anticoagulants to porcine mucosa for production of unfractionated heparin (UFH). Standardized heparins from various sources can be blended and potency adjusted, blended heparins exhibit comparable effects as single-sourced porcine UFH. This study evaluated the pharmacologic profile of blended heparin and compared their activities to that of single sourced porcine, ovine, and bovine heparins. Methods: The anticoagulant effects of gravimetric and potency-adjusted heparins were evaluated with aPTT, TT, anti-Xa, anti-IIa, ACT, and TGA studies. Protamine sulfate studies were used for neutralization potential of each of the individual heparins. Results: The potency-adjusted heparins demonstrated comparable aPTT, TT, anti-Xa, anti-IIa, and ACT values at all concentrations (U/mL). However, in gravimetric studies, bovine heparin consistently showed lower values with the exception of thrombin generation inhibition studies. The protamine sulfate neutralization studies demonstrated complete neutralization at all concentrations for the potency-adjusted heparins. However, at gravimetric concentrations, minor differences were noted in the neutralization profile in each of these heparins. Conclusion: These studies support the hypothesis that blended heparin from bovine, ovine, and porcine tissue, when standardized in unit-equivalent proportions, exhibits a comparable anticoagulant profile to the single species derived heparins.

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Scully, M. F., V. Ellis, N. Shah e V. Kakkar. "Effect of a heparan sulphate with high affinity for antithrombin III upon inactivation of thrombin and coagulation factor Xa". Biochemical Journal 262, n. 2 (1 settembre 1989): 651–58. http://dx.doi.org/10.1042/bj2620651.

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The kinetics of inhibition of human alpha-thrombin and coagulation Factor Xa by antithrombin III were examined under pseudo-first-order reaction conditions as a function of the concentration of heparan sulphate with high affinity for antithrombin III. The maximum observed second-order rate constant was, for the antithrombin III-thrombin reaction, 1.2 x 10(9) M-1.min-1 compared with 2.4 x 10(9) M-1.min-1 in the presence of high-affinity heparin. However, the maximum rate was catalysed by much higher concentrations of heparan sulphate (1.3 microM) than of heparin (0.025 microM). Differences were also observed in the maximal acceleration of the antithrombin III-Factor Xa interaction: 1.2 x 10(9) M-1.min-1 at 0.2 microM-heparin sulphate compared with 2.2 x 10(9) M-1.min-1 at 0.04 microM-heparin. The differences in properties of heparan sulphate and heparin were analysed by using the random bi-reactant model of heparin action [Griffith (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5460-5464]. It was observed that the apparent binding affinity for thrombin was higher for heparan sulphate (180 nM) than for heparin (14 nM). The rate constant for transformation of the antithrombin III-Factor Xa complex into irreversible product differed between heparan sulphate (96 min-1) and heparin (429 min-1). These properties of the high-affinity heparan sulphate may be of importance in consideration of a putative role in the control of intravascular haemostasis.

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Su, Dunhao, Yong Li, Edwin A. Yates, Mark A. Skidmore, Marcelo A. Lima e David G. Fernig. "Analysis of protein-heparin interactions using a portable SPR instrument". PeerJ Analytical Chemistry 4 (8 aprile 2022): e15. http://dx.doi.org/10.7717/peerj-achem.15.

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Abstract (sommario):

Optical biosensors such as those based on surface plasmon resonance (SPR) are a key analytical tool for understanding biomolecular interactions and function as well as the quantitative analysis of analytes in a wide variety of settings. The advent of portable SPR instruments enables analyses in the field. A critical step in method development is the passivation and functionalisation of the sensor surface. We describe the assembly of a surface of thiolated oleyl ethylene glycol/biotin oleyl ethylene glycol and its functionalisation with streptavidin and reducing end biotinylated heparin for a portable SPR instrument. Such surfaces can be batch prepared and stored. Two examples of the analysis of heparin-binding proteins are presented. The binding of fibroblast growth factor 2 and competition for the binding of a heparan sulfate sulfotransferase by a library of selectively modified heparins and suramin, which identify the selectivity of the enzyme for sulfated structures in the polysaccharide and demonstrate suramin as a competitor for the enzyme’s sugar acceptor site. Heparin functionalised surfaces should have a wide applicability, since this polysaccharide is a close structural analogue of the host cell surface polysaccharide, heparan sulfate, a receptor for many endogenous proteins and viruses.

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Lindsay, Susan L., Rebecca Sherrard Smith, Edwin A. Yates, Colin Cartwright, Bryan E. Thacker, Jeremy E. Turnbull, Charles A. Glass e Susan C. Barnett. "Validation of Recombinant Heparan Sulphate Reagents for CNS Repair". Biology 12, n. 3 (4 marzo 2023): 407. http://dx.doi.org/10.3390/biology12030407.

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Therapies that target the multicellular pathology of central nervous system (CNS) disease/injury are urgently required. Modified non-anticoagulant heparins mimic the heparan sulphate (HS) glycan family and have been proposed as therapeutics for CNS repair since they are effective regulators of numerous cellular processes. Our in vitro studies have demonstrated that low-sulphated modified heparan sulphate mimetics (LS-mHeps) drive CNS repair. However, LS-mHeps are derived from pharmaceutical heparin purified from pig intestines, in a supply chain at risk of shortages and contamination. Alternatively, cellular synthesis of heparin and HS can be achieved using mammalian cell multiplex genome engineering, providing an alternative source of recombinant HS mimetics (rHS). TEGA Therapeutics (San Diego) have manufactured rHS reagents with varying degrees of sulphation and we have validated their ability to promote repair in vitro using models that mimic CNS injury, making comparisons to LS-mHep7, a previous lead compound. We have shown that like LS-mHep7, low-sulphated rHS compounds promote remyelination and reduce features of astrocytosis, and in contrast, highly sulphated rHS drive neurite outgrowth. Cellular production of heparin mimetics may, therefore, offer potential clinical benefits for CNS repair.

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Baig, Nausheen, Ahmed Kouta, Walter Jeske, Debra Hoppensteadt, Jeanine Walenga, Omer Iqbal, Fakiha Siddiqui, Mamdouh Bakhos e Jawed Fareed. "Validation of the Bioequivalence of USP Potency Adjusted Porcine, Ovine, and Bovine Heparins". Blood 136, Supplement 1 (5 novembre 2020): 6. http://dx.doi.org/10.1182/blood-2020-142861.

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Abstract (sommario):

Introduction: Currently, there is a shortage of porcine heparin due to several factors such as limited availability of porcine mucosa, supply chain issues, and increased usage due to COVID-19. This has warranted the development of heparin from alternate sources such as bovine and ovine mucosa which is abundantly available for this purpose. On a mass basis, commercially available porcine heparins exhibit a similar potency (200 units/mg) to their ovine counterpart (190 units/mg) and a higher potency in contrast to their bovine counterpart (130-150 units/mg). Therefore, at gravimetric levels, the porcine heparins exhibit stronger biochemical and pharmacological effects in various laboratory assays in comparison to bovine heparin and similar effects in comparison to ovine heparins. Since heparin is standardized in biologic units and cross referenced against USP or EP Standard, it is hypothesized that potency equated porcine, ovine, and bovine heparin will exhibit similar biologic activities in laboratory assays carried out in the in vitro setting. The purpose of this study is to compare the biologic properties of the porcine, ovine, and bovine heparin at USP potency equated levels in standardized laboratory assays. Materials and Methods: Active pharmaceutical ingredients (API) of porcine mucosal heparin (200 units/mg) of U.S. origin was commercially obtained from Medefil Inc. (Glendale Heights, IL). Ovine heparin was obtained from Ronnsi Pharmaceutical (Jiangsu, China). Bovine heparin (140 units/mg) was obtained from Kin Master Pharmaceuticals (Posso Fundo, Brazil). All heparins were diluted at a concentration of 100 units/mL in saline. The anticoagulant effect of all heparins were evaluated using the whole blood clotting assays such as the ACT and thromboelastographic methods. Heparins were diluted in citrated human plasma yielding a final concentration range of 0-1 unit/mL. Clot based assays such as aPTT, TT, and prothrombinase induced clotting time (PiCT) were measured. Thrombin generation inhibition assay was carried out using a kinetic assay (CAT system, Diagnositca Stago, Paris, France). Protamine and heparinase neutralization profiles of these agents were also investigated in the plasma-based systems. These assays were then repeated at gravimetric dosages at final concentrations of 0-10 ug/mL. The results collected from these trials were then mathematically converted to units and compared to the results collected from the potency adjusted trials. All results were tabulated and compared, and applicable statistical methods were applied. Results: The USP potency adjusted heparin exhibited comparable anticoagulant effects in both the ACT and TEG assays. At equigravimetric levels porcine and ovine heparins produced comparable anticoagulant effects and bovine heparin produced weaker anticoagulant effect in both assays. In the citrated plasma supplementation studies, all drugs produced similar anticoagulant effects at potency adjusted dosages. In the chromogenic anti-Xa and anti-IIa assays, the behaviors of the agents were also comparable. In the thrombin generation assays, in terms of peak thrombin generation, area under the curve, and lag time, the porcine, ovine, and bovine heparins showed comparable effects. The protamine neutralization profiles of the porcine, ovine, and bovine heparin exhibited variable assay dependent results. Potency adjusted bovine heparin required higher amount of protamine for the complete neutralization of the biologic effects in comparison to the porcine heparin. At gravimetric concentrations, bovine heparins exhibited lower potencies than both the porcine and ovine heparins, which produced similar results. Summary and Conclusion: These results show that at potency adjusted concentrations, the porcine, ovine, and bovine heparin exhibit comparable biochemical and anticoagulant responses in the plasma-based systems. Therefore, the hypothesis that potency equated porcine, ovine, and bovine heparins exhibit comparable biochemical and anticoagulant activities is validated. Thus, the proposed approach to standardize heparins against a common standard in a biologic assay such as the USP method is valid. Furthermore, these results warrant regulatory considerations to fast track the review process for the re-introduction of bovine heparin and approval of bovine heparin as a biosimilar anticoagulant to porcine heparin. Disclosures No relevant conflicts of interest to declare.

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Vairo, Bruno, Leonardo Cinelli, Gustavo Santos, Roberto Fonseca, Rafael Aquino, Mariana Pereira e Paulo Mourão. "Heparins from porcine and bovine intestinal mucosa: Are they similar drugs?" Thrombosis and Haemostasis 103, n. 05 (2010): 1005–15. http://dx.doi.org/10.1160/th09-11-0761.

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Abstract (sommario):

SummaryIncreasing reports of bleeding and peri- or post-operative blood dyscrasias in Brazil were possibly associated with the use of heparin from bovine instead of porcine intestine. These two pharmaceutical grade heparins were analysed for potential differences. NMR analyses confirmed that porcine heparin is composed of mainly trisulfated disaccharides →4-α-IdoA2S-1→4-α-GlcNS6S-1→. Heparin from bovine intestine is also composed of highly 2-sulfated α-iduronic acid residues, but the sulfation of the α-glucosamine units vary significantly: ~50% are 6-and N-disulfated, as in porcine heparin, while ~36% are 6-desulfated and ~14% N-acetylated. These heparins differ significantly in their effects on coagulation, thrombosis and bleeding. Bovine heparin acts mostly through factor Xa. Compared to porcine heparin on a weight basis, bovine heparin exhibited approximately half of the anticoagulant and antithrombotic effects, but similar effect on bleeding. These two heparins also differ in their protamine neutralisation curves. The doses of heparin from bovine intestine required for effective antithrombotic protection and the production of adverse bleeding effects are closer than those for porcine heparin. This observation may explain the increasing bleeding observed among Brazilian patients. Our results suggest that these two types of heparin are not equivalent drugs.

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Patton, W. A., C. A. Granzow, L. A. Getts, S. C. Thomas, L. M. Zotter, K. A. Gunzel e L. J. Lowe-Krentz. "Identification of a heparin-binding protein using monoclonal antibodies that block heparin binding to porcine aortic endothelial cells". Biochemical Journal 311, n. 2 (15 ottobre 1995): 461–69. http://dx.doi.org/10.1042/bj3110461.

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Abstract (sommario):

The binding of heparin or heparan sulphate to a variety of cell types results in specific changes in cell function. Endothelial cells treated with heparin alter their synthesis of heparan sulphate proteoglycans and extracellular matrix proteins. In order to identify a putative endothelial cell heparin receptor that could be involved in heparin signalling, anti-(endothelial cell) monoclonal antibodies that significantly inhibit heparin binding to endothelial cells were prepared. Four of these antibodies were employed in affinity-chromatographic isolation of a heparin-binding protein from detergent-solubilized endothelial cells. The heparin-binding protein isolated from porcine aortic endothelial cells using four different monoclonal antibodies has an M(r) of 45,000 assessed by SDS/PAGE. The 45,000-M(r) heparin-binding polypeptide is isolated as a multimer. The antibody-isolated protein binds to heparin-affinity columns as does the pure 45,000-M(r) polypeptide, consistent with its identification as a putative endothelial heparin receptor.

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Visentin, Gian Paolo. "Heparin-Induced Thrombocytopenia: Molecular Pathogenesis". Thrombosis and Haemostasis 82, n. 08 (1999): 448–56. http://dx.doi.org/10.1055/s-0037-1615865.

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Abstract (sommario):

IntroductionHeparin-induced thrombocytopenia (HIT), a relatively common complication of heparin therapy, is characterized by an unexpected fall in platelet count occurring 5 days or more after the initiation of treatment.1,2 Patients who manifest only the thrombocytopenia rarely experience severe bleeding or other major adverse effects.3 Often, however, the thrombocytopenia is accompanied by arterial and/or venous thrombosis and thromboembolism, a complication which can be devastating.4 HIT, with or without thrombosis, is probably the most common cause of immunologically-mediated, drug-induced thrombocytopenia. HIT usually occurs in patients given standard therapeutic doses of heparin, especially of bovine origin.5 However, it has been reported following low-dose, subcutaneous heparin6 and in patients exposed to heparin “flushes” used to maintain the patency of intravenous lines.7 Even minute quantities of heparin released from heparin-bonded catheters appear to be capable of causing the disorder in previously sensitized patients.8 HIT has been induced by low molecular weight heparin (LMWH)9,10 and other heparin-like polysaccharides.11,12 HIT differs from most other forms of drug-induced immune thrombocytopenia in that the responsible antibodies activate platelets in the presence of pharmacologic doses of heparin (0.1-1 U/ml), rather than merely binding to platelets to promote their destruction in the reticuloendothelial system.3,13,14 The observation that heparin-induced platelet activation can be blocked in vitro by monoclonal antibodies specific for the platelet Fc receptor (FcγRIIA)15 suggests that antibodies associated with HIT somehow interact with heparin to form platelet-activating immune complexes. Attempts to demonstrate such complexes and their binding to intact platelets, however, have generally yielded negative or equivocal results.14,16,17 The association of HIT with thrombosis and disseminated intravascular coagulation (DIC) suggests that heparin-induced antibodies might react with endothelial cells (EC). Binding of IgG from the serum of patients with HIT to cultured human umbilical vein endothelial cells (HUVEC) observed in one study,18 but not confirmed in another,19 left open the question of whether immune-mediated vascular damage is important in pathogenesis.In 1992, Amiral and coworkers obtained evidence that antibodies associated with HIT are specific for complexes of heparin and platelet factor 4 (PF4), a heparin-binding protein stored in platelet α granules.20 These findings were subsequently confirmed by several groups, including ours.21-23 We also provided the first direct evidence that HIT antibodies bind to HUVEC in the presence of PF4, but not in its absence, thus, suggesting that the antibodies also recognize PF4 complexed with the heparan sulfate molecules displayed on HUVEC.21 Furthermore, Greinacher et al22 showed, by adsorption/elution of total IgG derived from HIT patients on PF4-coated EC, that the eluted antibodies also caused heparin-dependent platelet activation, thus, indicating that they recognize epitope(s) present on both PF4-heparin and PF4-heparan complexes.

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Khiella, Marco, Walter Jeske, Jeanine Walenga, Omer Iqbal, Rajan Laddu, Fakiha Siddiqui e Mamdouh Bakhos. "Bovine Heparin Compared to Porcine Heparin to Demonstrate Bioequivalence". Blood 132, Supplement 1 (29 novembre 2018): 1253. http://dx.doi.org/10.1182/blood-2018-99-118809.

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Abstract (sommario):

Abstract Introduction: Heparin prevents blood clots from forming in patients undergoing heart surgeries, dialysis, multiple other procedures, and for medical treatment of thrombosis such as associated with cancer. Currently, all heparin products in the U.S. are derived from the intestinal mucosa of pigs. Seventy-five percent of the crude porcine heparin used to make the active pharmaceutical ingredient (API) comes from outside the U.S., with a majority originating from China. Reintroduction of bovine heparin into the U.S. market would expand sources for this critical drug, thus addressing concerns about potential shortages and product adulteration. This study was undertaken to determine the bioequivalence of bovine and porcine heparins using assays relevant to the clinical setting. The assays used in this study were chosen because they overcome limitations of the conventional APTT for heparin monitoring. Selected TEG and ACT assays use whole blood which better simulates physiologic conditions and assesses the full in vitro anticoagulation potential of heparin; these assays are also routinely used clinically for heparin monitoring. The thrombin generation assay is state-of-the-art for hemostatic clinical lab testing, and it provides a more sensitive endpoint of the final generation of thrombin once coagulation is activated. Heparins were studied at concentrations used clinically and tested by assays sensitive to these concentrations. Methods: Bovine heparin API (BH; 7 lots) and US clinical grade porcine heparin (PH: 3 lots) were tested in parallel using recalcified whole blood thromboelastography (TEG; Haemonetics), celite activated clotting time (ACT; Hemochron), and thrombin generation (TGA; Diapharma). Fresh blood was obtained from healthy volunteers under an IRB approved protocol (n=6 per group). Previous work from our lab (Jeske W, Thrombosis & Hemostasis Societies of North America, P57, 2018) identified a weaker potency of BH than PH when compared on an equigravimetric basis in pharmacopeial assays; however, equivalent potency could be obtained when BH was standardized against PH on a unitage basis. In this study, heparins were studied on both a gravimetric and a unitage basis for a comprehensive evaluation. The potency of the BH was determined by pharmacopeial compliant anti-Xa and anti-IIa chromogenic assays cross-referenced to the porcine USP Heparin Reference Standard. Results: All results are depicted in Table 1. For the TEG, the R value, time of latency from start to initial fibrin formation, and the K value, time to achieve a defined clot strength due to thrombin generation and platelet activation, bovine heparin and porcine heparin did not demonstrate a significant difference in anticoagulant activity. The TEG angle, a measure of the speed of fibrin build-up and cross-linking (clot strengthening or rate of clot formation), and the maximum amplitude (MA) value, a function of the maximum of fibrin and platelet binding representing the strongest point of fibrin clot formation, also revealed no significant difference between bovine and porcine heparin anticoagulant activity. For the ACT, at concentrations of 10 µg/mL and 25 µg/mL, there were no statistically significant differences between BH and PH. For the TGA, measuring the time delay until the initiation of thrombin generation following tissue factor (TF) activation, the highest amount of thrombin generated after TF activation, and the total amount of thrombin generated after TF activation (AUC), showed a trend that PH was more potent than BH, but wide variation in the results did not allow for statistical differences to be identified. Conclusion: The results of this investigation demonstrate that bovine heparin produces an equivalent anticoagulant activity as porcine heparin in the TEG, ACT, and TGA assay systems. The equivalent ACT results, in particular, were unexpected since gravimetric amounts of heparins were evaluated. As for all heparins, standardization of bovine heparin in accordance with the process used for porcine heparin will assure equivalent anticoagulant activity among bovine and porcine heparins in whole blood, plasma-based, and pharmacopeial assays. This study further demonstrates that the current assays used to monitor porcine heparin can be similarly used to monitor bovine heparin. Disclosures Walenga: Eurofarma: Research Funding.

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Weiss, Ryan J., Philipp N. Spahn, Alejandro Gómez Toledo, Austin W. T. Chiang, Benjamin P. Kellman, Jing Li, Christopher Benner et al. "ZNF263 is a transcriptional regulator of heparin and heparan sulfate biosynthesis". Proceedings of the National Academy of Sciences 117, n. 17 (10 aprile 2020): 9311–17. http://dx.doi.org/10.1073/pnas.1920880117.

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Abstract (sommario):

Heparin is the most widely prescribed biopharmaceutical in production globally. Its potent anticoagulant activity and safety makes it the drug of choice for preventing deep vein thrombosis and pulmonary embolism. In 2008, adulterated material was introduced into the heparin supply chain, resulting in several hundred deaths and demonstrating the need for alternate sources of heparin. Heparin is a fractionated form of heparan sulfate derived from animal sources, predominantly from connective tissue mast cells in pig mucosa. While the enzymes involved in heparin biosynthesis are identical to those for heparan sulfate, the factors regulating these enzymes are not understood. Examination of the promoter regions of all genes involved in heparin/heparan sulfate assembly uncovered a transcription factor-binding motif for ZNF263, a C2H2 zinc finger protein. CRISPR-mediated targeting and siRNA knockdown of ZNF263 in mammalian cell lines and human primary cells led to dramatically increased expression levels of HS3ST1, a key enzyme involved in imparting anticoagulant activity to heparin, and HS3ST3A1, another glucosaminyl 3-O-sulfotransferase expressed in cells. Enhanced 3-O-sulfation increased binding to antithrombin, which enhanced Factor Xa inhibition, and binding of neuropilin-1. Analysis of transcriptomics data showed distinctively low expression of ZNF263 in mast cells compared with other (non–heparin-producing) immune cells. These findings demonstrate a novel regulatory factor in heparan sulfate modification that could further advance the possibility of bioengineering anticoagulant heparin in cultured cells.

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Sung, Michelle, Jeanine Walenga, Walter Jeske, Omer Iqbal e Mamdouh Bakhos. "Comparing a New Bovine Source Heparin to the Clinically Used Porcine Heparin for Platelet Function Effects and Heparin-Induced Thrombocytopenia Potential". Blood 132, Supplement 1 (29 novembre 2018): 2540. http://dx.doi.org/10.1182/blood-2018-99-118781.

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Abstract (sommario):

Abstract Background Heparin is a sulfated polysaccharide obtained from intestinal mucosa with anticoagulant properties that is widely used as a standard clinical therapeutic agent to treat and prevent thrombosis. Heparin is known to affect platelet function, and among its side effects is heparin-induced thrombocytopenia (HIT) that can occur in about 1% of patients exposed to heparin. Presently, only porcine source heparin is approved for use in the United States. The aims of this study were to determine if platelet activation by physiological agonists and platelet aggregation induced by HIT antibodies would be equivalent in the presence of bovine source heparin and porcine source heparin. Materials and Methods Seven lots of bovine heparin from Eurofarma and 3 lots of commercial clinical grade porcine heparin (Pfizer/Hospira) were evaluated. The USP Reference Standard for porcine heparin was used to determine anti-Xa and anti-IIa potencies of the bovine heparins. For each study, blood was collected from healthy volunteers (n=5 per test group), anticoagulated with sodium citrate, and centrifuged to obtain platelet rich plasma (PRP). Platelet aggregation responses were assessed using the BioData PAP-8 platelet aggregometer. For the first aim to evaluate platelet function, PRP was combined with heparin at final concentrations of 10.0, 1.0, and 0.1 µg/mL, covering both therapeutic and prophylactic ranges. Platelet agonists included adenosine diphosphate (ADP), collagen, epinephrine, arachidonic acid, and thrombin receptor agonist peptide (TRAP). The aggregation response was quantitated in terms of primary slope (PS), area under the curve (AUC), maximum aggregation (MA), and final aggregation (FA). For the second aim to evaluate the HIT potential, antibodies to the complex of heparin-platelet factor 4 (H-PF4) from banked HIT patient apheresis fluid were combined with donor PRP and heparin. Heparins were tested at final concentrations of 0.1, 0.4, 0.8, 1, and 100 U/mL. PS and FA results were recorded. For all data, comparisons were analyzed with 2-Way ANOVA using SigmaPlot software. Results In the presence of either bovine (BMH) or porcine heparin (PMH), the normal platelet aggregation response of all donors was not altered from that obtained with saline (see representative aggregation tracing in the image below). All heparin concentrations produced the same response. There were no significant differences between the bovine and porcine heparins for each of the 4 platelet aggregation parameters for ADP, arachidonic acid, collagen, epinephrine, and TRAP. Variation in the PS for arachidonic acid and collagen need to be assessed in a larger pool of donors to assure the lack of significant difference. Platelet activation to H-PF4 antibodies was strong at 0.1 to 1 U/mL concentrations with the expected inhibition observed when using 100 U/mL heparin. The HIT potential between bovine heparin and porcine heparin demonstrated no significant difference between the heparins (see MA responses in the image below). There were no lot to lot differences for the bovine heparins or the porcine heparins in either the platelet aggregation studies or the assessment for HIT. Conclusion In these studies of platelet function, the bovine and porcine source heparins were comparable with regards to their effects on platelet aggregation induced by multiple different agonists and their HIT potential. Figure. Figure. Disclosures Walenga: Eurofarma: Research Funding.

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Carlsson, Pernilla, Jenny Presto, Dorothe Spillmann, Ulf Lindahl e Lena Kjellén. "Heparin/Heparan Sulfate Biosynthesis". Journal of Biological Chemistry 283, n. 29 (16 maggio 2008): 20008–14. http://dx.doi.org/10.1074/jbc.m801652200.

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45

Walsh, R. L., T. J. Dillon, R. Scicchitano e G. McLennan. "Heparin and heparan sulphate are inhibitors of human leucocyte elastase". Clinical Science 81, n. 3 (1 settembre 1991): 341–46. http://dx.doi.org/10.1042/cs0810341.

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Abstract (sommario):

1. Heparin and heparan sulphate strongly inhibited human leucocyte elastase activity in an automated assay using the soluble substrate, n-succinyl-(l-alanine)3-p-nitroanilide (50% inhibition of 250 μl of 10 μg of human leucocyte elastase/ml was obtained with 80 μl of 2.8 μg of heparin/ml and 8 μg of heparan sulphate/ml). Less significant inhibition at the same concentrations was seen with the other glycosaminoglycans tested: hyaluronic acid and chondroitin sulphates A–C. 2. Heparin and heparan sulphate also strongly inhibited human leucocyte elastase activity towards insoluble human lung elastin, as determined by an e.l.i.s.a. for soluble elastin-derived peptides released by elastolytic activity on the elastin. This inhibition was shown not to be due to a direct interference of the glycosaminoglycans in the e.l.i.s.a. nor to the inhibition causing a change in the size of the elastin-derived peptides. However, unlike the chromogenic assay with n-succinyl-(l-alanine)3-p-nitroanilide as substrate, where heparin was the more effective inhibitor, in this assay system heparan sulphate was the more effective inhibitor (50% inhibition of 100 μl of 50 ng of human leucocyte elastase/ml was obtained with 100 μl of 4.5 μg of heparin/ml and 0.8 μg of heparan sulphate/ml). These results suggest that heparin and heparan sulphate, as components of cellular and basement membranes, are likely to have a role in protecting structural proteins, such as elastin, from the proteolytic activity of human leucocyte elastase. 3. The degree of inhibition by heparin was independent of pH within the range of pH studied (pH 6–9) and was almost immediate in the automated chromogenic assay system where a 10 s preincubation step was used. The inhibitory effect of heparin could be prevented by the addition of protamine sulphate or by the removal of heparin by an ion-exchange resin. 4. Low Mr preparations of heparin were also found to inhibit human leucocyte elastase, although preparations with Mr of 2000 or less did not inhibit the enzyme to the same extent as commercial preparations. 5. Heparin at the same concentrations did not inhibit other leucocyte enzymes, such as cathepsin G and myeloperoxidase, nor the pancreatic enzymes, pancreatic elastase, trypsin and chymotrypsin.

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Ljungberg, B., e H. Johnsson. "In Vivo Effects of a Low Molecular Weight Heparin Fragment on Platelet Aggregation and Platelet Dependent Hemostasis in Dogs". Thrombosis and Haemostasis 60, n. 02 (1988): 232–35. http://dx.doi.org/10.1055/s-0038-1647036.

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SummaryPlasma defibrinogenated dogs were used to study the influence of conventional heparin and a low molecular weight heparin fragment (Fragmin®, mean MW 5,000 d) on platelet dependent hemostasis. The heparins were given intravenously in gravimetri- cafly equal doses. The bleeding from standardized skin flap wounds and platelet aggregation (ADP and thrombin) was studied. In comparison, higher doses of the fragment than of heparin were required to increase the bleeding. ADP-induced aggregation in defibrinogenated platelet rich plasma (after addition of normal dog plasma) was potentiated by both heparins. After injection of heparin or the fragment, ADP induced platelet aggregation without prior addition of normal plasma to the testtube.In conclusion the heparin fragment affected bleeding to a less extent than conventional heparin. One explanation might be a weaker inhibition of thrombin-induced platelet aggregation.

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47

Walenga, Jeanine, Walter Jeske, Sabrina Bertini, Giulia Risi, Michelle Sung, Ambar Farooqui, Cassie Bacher et al. "Bovine Heparin Demonstrates the Same Interaction with HIT Antibodies As Porcine Heparin". Blood 134, Supplement_1 (13 novembre 2019): 2351. http://dx.doi.org/10.1182/blood-2019-127304.

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Heparin, an anticoagulant widely used in numerous medical applications, is considered an essential medicine by the WHO. Due to its high volume use and that it is the parent material for low molecular weight heparins, there is potential for the raw material to be in short supply. The African swine fever epidemic in China, ongoing since August 2018, has added further restraints on heparin source material supply. At present medical grade heparin in the US is only derived from porcine intestinal mucosa; however, there are explorations into using bovine, ovine, and other sources. Bovine heparin, once common place in the US pharmaceutical sector, is again under consideration by the US FDA. This study focused on the primary immunogenic activity associated with heparin, that is heparin-induced thrombocytopenia (HIT), and how the interaction of bovine heparin with functional HIT antibodies compares to that of porcine heparin. Materials and Methods Bovine unfractionated heparin from multiple manufacturers was compared to commercial medical grade porcine heparin obtained from US pharmacies. The US Pharmacopeia porcine heparin standard was used to determine potency equivalence. Antibodies to the complex of platelet factor 4/heparin (PF4/heparin) from banked clinically confirmed HIT patient apheresis fluids were combined with heparin and donor platelet rich plasma (PRP; blood collected in citrate from volunteers after signing a consent document). Heparins were tested at final concentrations of 0.1, 0.4, 0.8, 1, and 100 U/mL. The platelet activation response was determined on the BioData PAP-8 Platelet Aggregometer and quantitated in terms of primary slope (PS), area under the curve (AUC), maximum aggregation (MA), and final aggregation (FA). Characterization of the biophysical interaction between varying molar ratios of human PF4 and heparin was performed using photon correlation spectroscopy (PCS) and zeta potential (Zp) measurements of PF4/heparin complexes using Zetasizer Nano ZS instrumentation and software. Differences between bovine and porcine heparin were assessed by t-test or Mann-Whitney test. Concentration-response curves were analyzed by two-way ANOVA followed by the Holm-Sidak multiple comparison test using SigmaPlot software. Results Platelet activation to PF4/heparin antibodies at bovine and porcine heparin concentrations of 0.1 U/mL (56 ± 9 % vs. 54 ± 11 % MA) and 0.4 U/mL (59 ± 10 % vs. 65 ± 8 % MA) were the same with the expected inhibition (9 ± 4% MA) at the supra-therapeutic concentration of 100 U/mL. Consistent responses were obtained across 21 lots of bovine heparin, 30 lots of porcine heparin, and 38 platelet-HIT antibody combinations. The HIT potential of bovine heparin and porcine heparin was not statistically different (p>0.05). At higher medical use doses, the platelet aggregation response in the presence of HIT antibodies was actually lower for bovine heparin than porcine heparin (0.8 U/mL, 49 ± 10 % vs. 64 ± 9 % MA, p<0.05; and 1.0 U/mL, 45 ± 11 % vs. 62 ± 9 % MA, p<0.05). By PCS, it was observed that the maximal aggregation between PF4 and either porcine or bovine heparin occurred at comparable molar ratios (7.3 ± 1.5 vs. 6.4 ± 0). Although the porcine and bovine heparins exhibited comparable molecular weights (16,333 ± 153 vs. 16,790 ± 230 Da) and polydispersities (1.19 ± 0.02 vs. 1.15 ± 0.01), porcine heparin formed somewhat larger complexes with PF4 (1113 ± 65 nm) than did bovine heparin (863 ± 68 nm). The molar ratios of PF4 to heparin at which the charge of the complex was fully neutralized (Zp = 0) was comparable for porcine and bovine heparin (9.04 ± 0.19 vs. 9.97 ± 0.65). Consistent responses were obtained across 4 lots of bovine heparin and 3 lots of porcine heparin. Conclusions Bovine heparin and porcine heparin had the same in vitro functional platelet activation response in the presence of HIT antibodies, the same potential to form complexes with human PF4, and the same associated features that make PF4 immunogenic. This investigation demonstrates that bovine heparins should have a similar immunogenic response as porcine heparin at equi-unit dosing. Current refinements in the manufacturing process for bovine and porcine heparins have led to well-characterized and purified products which is reflected in their comparable biological behavior. Disclosures No relevant conflicts of interest to declare.

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Sandoval, Daniel R., Alejandro Gomez Toledo, Chelsea D. Painter, Ember M. Tota, M. Osman Sheikh, Alan M. V. West, Martin M. Frank et al. "Proteomics-based screening of the endothelial heparan sulfate interactome reveals that C-type lectin 14a (CLEC14A) is a heparin-binding protein". Journal of Biological Chemistry 295, n. 9 (21 gennaio 2020): 2804–21. http://dx.doi.org/10.1074/jbc.ra119.011639.

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Animal cells express heparan sulfate proteoglycans that perform many important cellular functions by way of heparan sulfate–protein interactions. The identification of membrane heparan sulfate–binding proteins is challenging because of their low abundance and the need for extensive enrichment. Here, we report a proteomics workflow for the identification and characterization of membrane-anchored and extracellular proteins that bind heparan sulfate. The technique is based on limited proteolysis of live cells in the absence of denaturation and fixation, heparin-affinity chromatography, and high-resolution LC-MS/MS, and we designate it LPHAMS. Application of LPHAMS to U937 monocytic and primary murine and human endothelial cells identified 55 plasma membrane, extracellular matrix, and soluble secreted proteins, including many previously unidentified heparin-binding proteins. The method also facilitated the mapping of the heparin-binding domains, making it possible to predict the location of the heparin-binding site. To validate the discovery feature of LPHAMS, we characterized one of the newly-discovered heparin-binding proteins, C-type lectin 14a (CLEC14A), a member of the C-type lectin family that modulates angiogenesis. We found that the C-type lectin domain of CLEC14A binds one-to-one to heparin with nanomolar affinity, and using molecular modeling and mutagenesis, we mapped its heparin-binding site. CLEC14A physically interacted with other glycosaminoglycans, including endothelial heparan sulfate and chondroitin sulfate E, but not with neutral or sialylated oligosaccharides. The LPHAMS technique should be applicable to other cells and glycans and provides a way to expand the repertoire of glycan-binding proteins for further study.

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VILAR, Rolando E., Dineshchandra GHAEL, Min LI, Devan D. BHAGAT, Lisa M. ARRIGO, Mary K. COWMAN, Harry S. DWECK e Louis ROSENFELD. "Nitric oxide degradation of heparin and heparan sulphate". Biochemical Journal 324, n. 2 (1 giugno 1997): 473–79. http://dx.doi.org/10.1042/bj3240473.

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NO is a bioactive free radical produced by NO synthase in various tissues including vascular endothelium. One of the degradation products of NO is HNO2, an agent known to degrade heparin and heparan sulphate. This report documents degradation of heparin by cultured endothelial-cell-derived as well as exogenous NO. An exogenous narrow molecular-mass preparation of heparin was recovered from the medium of cultured endothelial cells using strong-anion exchange. In addition, another narrow molecular-mass preparation of heparin was gassed with exogenous NO under argon. Degradation was evaluated by gel-filtration chromatography. Since HNO2 degrades heparin under acidic conditions, the reaction with NO gas was studied under various pH conditions. The results show that the degradation of exogenous heparin by endothelial cells is inhibited by NO synthase inhibitors. Exogenous NO gas at concentrations as low as 400 p.p.m. degrades heparin and heparan sulphate. Exogenous NO degrades heparin at neutral as well as acidic pH. Endothelial-cell-derived NO, as well as exogenous NO gas, did not degrade hyaluronan, an unrelated glycosaminoglycan that resists HNO2 degradation. Peroxynitrite, a metabolic product of the reaction of NO with superoxide, is an agent that degrades hyaluronan; however, peroxynitrite did not degrade heparin. Thus endothelial-cell-derived NO is capable of degrading heparin and heparan sulphate via HNO2 rather than peroxynitrite. These observations may be relevant to various pathophysiological processes in which extracellular matrix is degraded, such as bone development, apoptosis, tissue damage from inflammatory responses and possible release of growth factors and cytokines.

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Ahmed, Tahir, Jaime Ungo, Min Zhou e Carlos Campo. "Inhibition of allergic late airway responses by inhaled heparin-derived oligosaccharides". Journal of Applied Physiology 88, n. 5 (1 maggio 2000): 1721–29. http://dx.doi.org/10.1152/jappl.2000.88.5.1721.

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Inhaled heparin has been shown to inhibit allergic bronchoconstriction in sheep that develop only acute responses to antigen (acute responders) but was ineffective in sheep that develop both acute and late airway responses (LAR) (dual responders). Because the antiallergic activity of heparin is molecular-weight dependent, we hypothesized that heparin-derived oligosaccharides (<2,500) with potential anti-inflammatory activity may attenuate the LAR in the dual-responder sheep. Specific lung resistance was measured in 24 dual-responder sheep before and serially for 8 h after challenge with Ascaris suum antigen for demonstration of early airway response (EAR) and LAR, without and after treatment with inhaled medium-, low-, and ultralow-molecular-weight (ULMW) heparins and “non-anticoagulant” fractions (NAF) of heparin. Airway responsiveness was estimated before and 24 h postantigen as the cumulative provocating dose of carbachol that increased specific lung resistance by 400%. Only ULMW heparins caused a dose-dependent inhibition of antigen-induced EAR and LAR and postantigen airway hyperresponsiveness (AHR), whereas low- and medium-molecular-weight heparins were ineffective. The effects of ULMW heparin and ULMW NAF-heparin were comparable and inhibited the LAR and AHR even when administered “after” the antigen challenge. The ULMW NAF-heparin failed to inhibit the bronchoconstrictor response to histamine, carbachol, and leukotriene D4, excluding a direct effect on airway smooth muscle. In six sheep, segmental antigen challenge caused a marked increase in bronchoalveolar lavage histamine, which was not prevented by inhaled ULMW NAF-heparin. The results of this study in the dual-responder sheep demonstrate that 1) the antiallergic activity of inhaled “fractionated” heparins is molecular-weight dependent, 2) only ULMW heparins inhibit the antigen-induced EAR and LAR and postantigen AHR, and 3) the antiallergic activity is mediated by nonanticoagulant fractions and resides in the ULMW chains of <2,500.

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