Endogenous Heparin Research Articles

Complexation with heparin prevents adhesion between fibrin-coated surfaces.

Heparin, in Langmuirian fashion, binds stoichiometrically with high affinity, Kd approximately 100 nM, to both fibrinogen and fibrin adsorbed as monomolecular films to lecithin-coated, microscopic, polystyrene-divinylbenzene beads. Complex formation inhibits aggregation of fibrin-coated beads, and it also results in dissociation of preformed aggregates of fibrin-coated beads. These phenomena are not caused by desorption of fibrin(ogen), indirect inhibition of thrombin activity, or mere electrostatic repulsion of charged particles. Instead, these data are consistent with the proposal that the complexed heparin interferes directly with dimer formation between fibrin molecules adsorbed to colliding beads. We describe these phenomena and their application to the development of sensitive analytical methods for quantitating heparin. Based on these observations, we also propose a role for endogenous heparin in the physiologic regulation of fibrin-mediated adhesion of surfaces.

Endogenous heparinase-sensitive anticoagulant activity in human plasma

In this paper we show that an anticoagulant activity, which we measure by thrombin time, appears in human plasma after its exhaustive proteolytic digestion. This activity is extremely heat stable, it is resistant to chondroitin ABC lyase (E.C.4.2.2.4) and heparan sulfate lyase (E.C.4.2.2.8), it is sensitive to heparin lyase (E.C. 4.2.2.7) and to nitrous acid treatment : we suggest that it can be identified as authentic heparin. The amount present in 1 ml of plasma of healthy subjects corresponds to 0.1-0.2 I.U. of standard heparin (150 I.U./mg). Proteolytically digested human plasma was submitted to ion-exchange chromatography on DEAE-Sephacel and the anticoagulant activity in the fractions eluted at the different molarities of NaCl was measured by thrombin time. This analysis shows that the anticoagulant activity elutes at very low ionic strength. The possibility that interactions of the endogenous heparin with proteins or protein fragments are responsible for the difficulty in isolating heparin from human plasma is discussed.

Role of protein conformational changes, surface approximation and protein cofactors in heparin-accelerated antithrombin-proteinase reactions.

Antithrombin functions as the principal plasma protein inhibitor of most blood coagulation proteinases.1,2 The essential role of this inhibitor in regulating the activity of these proteinases in vivo is indicated from the well-established link between inherited or acquired deficiencies of antithrombin and the tendency to develop thrombotic disease. Antithrombin is a member of the serpin superfamily of protein proteinase inhibitors and its main target enzymes include the blood coagulation factors IXa, Xa and thrombin. This and other serpins are distinguished from other family members in that their reactions with target enzymes are greatly accelerated by the binding of heparin or heparan sulfate glycosaminoglycans. This property is chiefly responsible for the anticoagulant activity of heparin and has suggested a role for endogenous heparin and heparan sulfate in the regulation of blood coagulation proteinases by antithrombin. In this article, we will review our present understanding of the relationship between antithrombin structure and function based on currently available evidence. Our discussion will focus principally on two areas: 1) the mechanism by which antithrombin and other serpins inhibit their target proteinases; and 2) the molecular basis of heparin’s accelerating effect on antithrombin-proteinase reactions.

Inhibition of coagulation and thrombin-induced platelet activities by a synthetic dodecapeptide modeled on the carboxy-terminus of hirudin

A synthetic, tyrosine-sulfated, dodecapeptide (BG8865) modeled on residues 53–64 of hirudin was found to elevate the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) of human plasma in a dose-dependent manner. The most sensitive assay was the TT, which was prolonged 2 and 3 times control values at 2.2 and 4.1 micrograms/mL hirudin peptide, respectively. The sulfated dodecapeptide exhibited no dependency on antithrombin III as monitored by the APTT in the presence of sheep anti-human antithrombin III antibodies, and its activity was not neutralized by platelet releasates or platelet factor 4. In studies of thrombin-induced platelet activation, the hirudin peptide was found to block aggregation, serotonin release and thromboxane A2 generation. At thrombin concentrations of 0.25 U/mL, the IC50 (concentration resulting in 50% inhibition) for inhibition of platelet aggregation was 0.72 micrograms/mL peptide. Inhibition of TXA2 generation and serotonin release correlated closely with inhibition of aggregation. Using platelets from patients with clinically documented heparin-induced thrombocytopenia anticoagulant doses of heparin were found to induce platelet aggregation and thromboxane A2 generation. In sharp contrast, anticoagulant-equivalent doses of hirudin peptide had no effect on patient platelets, as evidenced by a lack of platelet aggregation and thromboxane A2 generation. These data provide compelling in vitro evidence that the hirudin peptide has several potential advantages over heparin, namely effective inhibition of thrombin-induced platelet activities, co-factor independence, insensitivity to endogenous heparin- neutralizing factors, and an apparent lack of direct or immune-mediated platelet stimulating properties.

Heparin inhibits EcoRI endonuclease cleavage of DNA at certain EcoRI sites

Studies presented here demonstrate that heparin inhibits EcoRI endonuclease cleavage of DNA whereas related proteoglycans show no effect. The inhibition occurs at particular EcoRI sites that are near or overlap with palindromic sequences in the murine lambda 5 and Lyt-2 genes. Endogenous heparin from peritoneal mast cells co-isolates with DNA and inhibits digestion of peritoneal cell DNA at the inhibitable sites. Digestion of spleen DNA is inhibited at the same sites when commercial heparin is added prior to digestion. In both cases, the inhibition is abolished by pre-treating the DNA with heparinase. Thus, potential artifacts in restriction fragment length analyses could occur with DNA isolated either from cells that are naturally rich in heparin or from cells to which heparin has been added, e.g., as an anticoagulant.

Resistance of Heparinase-derived Heparin Fragments to Biotransformation

The biotransformation of heparinase-derived heparin fragments was examined via a combined approach using 35S-labeled heparin fragments as well as unlabeled chemically defined heparin fragments. Rats dosed with either [35S]di-, tetra-, hexa-, or octasaccharide fragments (2 mg/kg body weight, intravenously) excreted 63-69% of the injected radioactivity into the urine within 24 h with two-thirds being excreted during the first 6 h. Gel permeation chromatography of the urinary material shows that the tetra- and octasaccharides have undergone minor (approximately 5%) depolymerization whereas no change was observed for the di- and hexasaccharides. No N-desulfation was demonstrated for any of the substances. The hexa- and octasaccharide metabolites present in the urine 24 h after dosing exhibited the same antifactor Xa activity as that of the injected material. A chemically defined trisulfated disaccharide and a hexasulfated tetrasaccharide were prepared and dosed in a similar manner. Only one metabolite was recovered from animals dosed with disaccharide. This compound was characterized by anion exchange chromatography, proton nuclear magnetic resonance spectroscopy, Fourier transform infrared spectrometry, and mass spectrometry and shown to be identical to the injected disaccharide. Five metabolites were isolated from the urine of rats dosed with the hexasulfated tetrasaccharide. The major metabolite, consisting of at least 65% of the total, was characterized as described for the disaccharide and shown to be identical to the injected compound. The remaining material appeared to be disaccharides and, possibly, a tetrasaccharide conjugate. Taken together, our results show that the heparinase-derived heparin fragments are very resistant to biotransformation compared with heparin and endogenous heparin fragments. These fragments may therefore be useful in defining structure activity relationships in vivo.

Internalization and metabolism of endogenous heparin by cultured endothelial cells

We have studied the ability of bovine adrenal capillary cells cultured in vitro to uptake and metabolize heparin. We have previously demonstrated that endogenous heparin can be extracted from human plasma (Vannucchi, S. et al., (1985) Biochem. J. 227, 57-65), and here we show that also endothelial cells contain heparin. However, experiments with (35S)sodium sulfate labeling indicate that these cells do not synthesize de novo heparin, but they uptake it from culture serum. Bovine adrenal capillary endothelial cells are able to bind and uptake (3H)heparin added to culture medium and they also release its low molecular weight degradation products, thus indicating a metabolism of heparin. We discuss about the role of endothelial cell-mediated uptake and metabolism of endogenous heparin in relationship with circulating heparin. We also discuss about these events as related to some of the antithrombogenic properties of the endothelium.

Formation of the Fibrin Clot: the Balance of Procoagulant and Inhibitory Factors

Let us now briefly summarize some major known regulating mechanisms, most of which have already been discussed. A general regulating feature of the coagulation system is provided by the cofactors HMW-kininogen, tissue factor, factor V(a), factor VIII:C(a), protein S and thrombomodulin. Tissue factor and thrombomodulin, as cell membrane constituents, and the other cofactors, thanks to their affinity for certain surface sites, localize coagulation reactions and thus avoid generalized intravascular thrombosis when the clotting system is triggered. Thrombin activates factors V and VIII:C and activated protein C inactivates factors Va and VIII:Ca. Thrombin is regulated by AT III, alpha 2M and possibly heparin-cofactor II, whereby endothelial-cell-bound heparin-like molecules enhance thrombin neutralization. Moreover, binding of thrombin to thrombomodulin abolishes its clotting activity, at least in the case of rabbit thrombomodulin. Thrombin is able to cleave PT-fragment 1 from prothrombin, thus generating prethrombin 1, which lacks the gla-region and does not bind to phospholipids. The hypothesis that thrombin may regulate its own formation by this negative feedback, however, must probably be discarded, because no corresponding fragments are found after blood clotting in vitro (Aronson et al, 1977). Factor Xa and factor IXa are inhibited by AT III and endogenous heparin probably enhances their inactivation. However, phospholipid-bound factor Xa in the presence of factor Va (Marciniak, 1973) and phospholipid-bound factor IXa (Varadi and Elödi, 1982) are relatively protected from inhibition. Platelet-bound factor Xa is completely protected from AT III, even in the presence of heparin (Miletich et al, 1978). Thus, specific cell surface sites modulate the inhibition of proteases in situ. Factor XIa is inhibited by several protease inhibitors, the most important being alpha 1-AT. beta-factor XIIa is inhibited mainly by C1-inhibitor and kallikrein by both C1-inhibitor and alpha 2M. No serine protease inhibitor for factor VIIa is as yet known. However, after initial rapid activation by factor Xa, factor VIIa is subsequently proteolytically inactivated by factor Xa, resulting in a transient burst of factor Xa generation by factor VIIa (Morrison and Jesty, 1984). This proteolytic regulation of factor VIIa by factor Xa dampens factor IX or factor X activation via tissue factor-factor VIIa by feedback proteolytic inhibition and this may constitute a major regulatory mechanism for factor VIIa.(ABSTRACT TRUNCATED AT 400 WORDS)

Complexing of heparin with phosphatidylcholine. A possible supramolecular assembly of plasma heparin.

In a series of attempts to reveal plasma heparin, we found that high ionic strength and modification of protein amino groups were not effective in extracting endogenous heparin (or, indeed, a large percentage of exogenous labelled heparin), whereas delipidation in the presence of 4M-guanidinium chloride gave high yields, indicating that plasma heparin may be assembled with compounds other than proteins, in a form making it inaccessible to water and ions. During the extraction of lipids, a paradoxical entry of heparin into the organic phase was observed. Detergents, including sodium dodecyl sulphate, did not shift heparin into the aqueous phase, whereas repeated chloroform/methanol extraction did so. Using purified compounds we were able to reproduce in vitro both the scavenging of heparin from water as well as the formation of heparin-phosphatidylcholine complexes soluble in organic solvents. Evidence for complexing of heparin with phosphatidylcholine was also obtained by electrophoretic and ultracentrifugation assays. The quaternary-ammonium-containing phosphatidylcholine was the more effective phospholipid in binding heparin; anionic phospholipids did not bind. Only heparin-like glycosaminoglycans bound phosphatidylcholine, but less-sulphated compounds (heparan sulphate and dermatan sulphate) were weaker ligands. Gel-filtration experiments showed that heparin was not bound to liposome vesicles, but that a measurable percentage of the phospholipids was stripped off from vesicles and was found in the form of a complex separable from liposomes by gel filtration. The molecular basis as well as the biological role of the interaction of heparin with major membrane phospholipids are discussed.

A lethal syndrome in mice following administration of carbon tetrachloride and cycloheximide, and its prevention by heparin treatment

When a hepatotoxic dose of CCl 4 is followed in 6 h (but not in 18 h) by 30 μg per g body weight of cycloheximide, a lethal, shock-like state develops. This is prevented by heparin treatment. This lethal syndrome is compared with other, similar, induced lethal states in which cycloheximide plays an essential role, and in which heparin is life-saving. It is postulated that, after CCl 4, a phase of procoagulant activity occurs in the dying centrilobular zone hepatocytes, but that unimpaired protein synthesis permits responsive release of endogenous heparin and thereby prevents thrombosis in centrilobular sinusoids. Cycloheximide is thought to inhibit this heparin release and to allow a transient episode of occlusive centrilobular microthrombosis with consequent irreversible ischaemic damage to the mid-gut.

Cycloheximide challenge of mice bearing subcutaneous tumours, with observations on heparin treatment

Mice bearing large subcutaneous transplants of ovarian teratocarcinoma succumb when given large doses of cycloheximide, whereas animals with equally large subcutaneous Ehrlich tumours are resistant to this treatment. Adequate dosage of heparin is life-saving in cycloheximide-treated, teratoma-bearing mice. The results are discussed in the context of previous work, and one tentative suggestion made is that cycloheximide interferes with the mechanism for release of endogenous heparin which is an essential component of the host response to a growing teratocarcinoma.

The plasma contact system in atopic asthma

An in vitro test for the rate of appearance of kallikrein in plasma due to contact system activation by dextran sulfate at 0 °C was applied to plasmas of 19 atopic asthma patients and 19 age- and sex-matched controls without atopy. The average prekallikrein activation rate was markedly higher in the plasmas of the atopic patients. Mean endogenous heparin levels were also elevated.

Heparin Liberation in Urticaria Pigmentosa

Urticaria pigmentosa is often a benign and asymptomatic cutaneous disease. However, even minimal cutaneous lesions can be associated with acute systemic effects secondary to histamine liberation. Hemorrhagic manifestations associated with the release of heparin from mast cells in patients with mastocytosis have long been suspected but have been little reported in the literature.^1-4 We report herein the unusual complication of a paroxysmal coagulopathy, presumably caused by endogenous heparin liberation, in a child with urticaria pigmentosa. <h3>Report of a Case</h3> A 4-year-old boy was admitted to the hospital with the suspicion that he was a victim of the battered-child syndrome because of multiple ecchymoses that were located mainly on the face and buttocks. Despite these signs, the boy seemed to be in no distress. He was active, his behavior was normal, and his parents denied having struck him. Skin lesions had been present since he was 3 months old. The

Appearance of heparin antithrombin-active chains in vivo after injection of commercial heparin and in anaphylaxis

Recently it has been demonstrated that only one-third of commercial heparin binds with antithrombin III to enhance antithrombin activity, and that heparin is rapidly taken up by the RES. Commercial heparin was given to rabbits, dogs and men by subcutaneous and intravenous injection. Plasma samples were deproteinized with phenol, urine samples precipitated with benzidine, and the separated heparin measured for both antithrombin and metachromatic (dye-binding) activity compared to a reference heparin. Urinary excretion as heparin and uroheparin accounted for approximately 5% of the injected heparin. The extraction of heparin added to blood in vitro gave identical values in metachromatic and antithrombin assays, as did heparin from blood samples immediately after intravenous injection. However, later blood samples after injection gave much higher values in the test for antithrombic activity than in the metachromatic test. Subcutaneous injection of heparin subjected to acid inactivation in vitro, reducing its antithrombin activity, likewise resulted in heparin appearing in vivo with higher antithrombin activity. Endogenous heparin released to the circulation in canine anaphylaxis also gave much higher values in anti-thrombin assays. It is concluded that the difference in values is due to the conversion of antithrombin-inactive heparin chains to antithrombin-active heparin chains.

Analysis of the inhibitory effect of whole human red blood cells on plasma heparin activity

The accelerating effect of whole human red blood cells (RBC) on the thrombin time of plasma (deprothrombinized, platelet-deprived) is manifested regardless of the presence or absence of calcium ions or of preincubation of the RBC with plasma and it is not accompanied by a fall of the free heparin level in the plasma. Acceleration of the transformation of the plasma fibrinogen into fibrin under the influence of whole RBC is unconnected with inhibition of endogenous heparin but reflects the fibrinoplastic effect of the cell. The experimental results do not support the view expressed in the literature that whole erythrocytes have a regulating effect on the blood level of endogenous heparin.

Migraine therapy with heparin: pathophysiologic basis.

SYNOPSISDuring headache‐free intervals, migraine patients had in their blood fewer basophilic leukocytes, which also contained less than a normal amount of heparin, and excreted less uroheparin than did controls. During migraine attacks the levels of basophils and of uroheparin rose but did not reach normal values.Sixty patients with severe vascular headaches (fifty‐three migraine and seven cluster) were treated with heparin by intravenous injections or aerosol inhalations. After repeated treatments, basophilic leukocytes and uroheparin levels rose to normal values and remained normal for varying periods, up to 14 months, after the end of therapy.Of the two treatment forms, aerosol therapy was the more effective. It reduced the migraine index of all but two of thirty‐three patients by an average of 86 (75–100) percent, whereas after intravenous therapy eighteen of twenty‐seven patients showed a better than 75 percent improvement.Three to 14 months after the end of therapy, seven patients were entirely free from migraine headaches and three more reported an average improvement of 90 percent.The role of endogenous heparin in migraine and the fate of injected and inhaled heparin are reviewed.

Changes in blood clotting systems during migraine attacks.

SYNOPSISThe activity of various blood clotting systems was measured during attacks of migraine complicated by prolonged focal cerebral dysfunction and contrasted with those observed when the same and other patients had uncomplicated migraine. Results differed in the two groups.In patients with complicated migraine, plasma coagulability rose during both complicated and uncomplicated attacks. During a complicated migraine attack, platelet aggregability to ADP also rose in all but one patient. Platelet aggregability fell in the same patients when they had uncomplicated migraine. Plasma coagulability consistently increased more when the headache was associated with focal cerebral dysfunction.In patients with uncomplicated migraine, plasma coagulability dropped during the later phase of their attacks. This finding may reflect the release of endogenous heparin from mast cells and basophilic leucocytes. Platelet aggregability to ADP decreased in some but rose in others.The results support the hypothesis that prolonged focal cerebral dysfunction may reflect thromboembolic phenomena. The influence of estrogens and genetic factors on clotting systems is reviewed.

Demonstration of endogenous heparin in rat blood.

Dr. Lindahl has referred to my concept of macromolecular heparin as a multichain, whose structural integrity depends on a polysaccharide core. In 1971, I postulated that macromolecular heparin must be depolymerized to become biologically active (6). Later, I found that macromolecular heparin is a potent inhibitor of heart lipoprotein lipase (LPL) in vitro (7). Injected macromolecular heparin has a relatively low potency as an initiator of LPL activity in the blood. This activity appears in the blood rather slowly, compared with that of low-molecular-weight heparin (i.e., commercial heparin or enzymatically depolymerized macromolecular heparin) (7). These facts support the concept that depolymerization precedes the release of biologically active heparin from the mast cells. Endogenous heparin in the blood should therefore be of low molecular weight, and I would now like to demonstrate that this is so.

Demonstration of endogenous heparin in rat blood

Dr. Lindahl has referred to my concept of macromolecular heparin as a multichain, whose structural integrity depends on a polysaccharide core. In 1971, I postulated that macromolecular heparin must be depolymerized to become biologically active (6). Later, I found that macromolecular heparin is a potent inhibitor of heart lipoprotein lipase (LPL) in vitro (7). Injected macromolecular heparin has a relatively low potency as an initiator of LPL activity in the blood. This activity appears in the blood rather slowly, compared with that of low-molecular-weight heparin (i.e., commercial heparin or enzymatically depolymerized macromolecular heparin) (7). These facts support the concept that depolymerization precedes the release of biologically active heparin from the mast cells. Endogenous heparin in the blood should therefore be of low molecular weight, and I would now like to demonstrate that this is so.

Plasma factor X a-inhibitory activity in alcoholic liver disease and the effect of heparin

Factor X a-inhibitor (X a-I) activity was measured in twenty patients with alcoholic liver disease and found to be more than 3 standard deviations below normal in fourteen patients. When trace amounts of heparin were added to these plasmas, the X a-I activity was potentiated to the same level as normal plasma with heparin added. This suggests that X a-I is actually present in alcoholic liver disease, but has less than normal activity. Possible explanations such as the lack of endogenous heparin or the presence of an inhibitor are discussed.