Inhibition of platelet-surface-bound proteins during coagulation under flow II: Antithrombin and heparin

Blood coagulation is a self-repair process regulated by activated platelet surfaces, clotting factors, and inhibitors. Antithrombin (AT) is one such inhibitor that impedes coagulation by targeting and inactivating several key coagulation enzymes. The effect of AT is greatly enhanced in the presence of heparin, a common anticoagulant drug. When heparin binds to AT, it either bridges with the target enzyme or induces allosteric changes in AT leading to more favorable binding with the target enzyme. AT inhibition of fluid-phase enzymes caused little suppression of thrombin generation in our previous mathematical models of blood coagulation under flow. This is because in that model, flow itself was a greater inhibitor of the fluid-phase enzymes than AT. From clinical observations, it is clear that AT and heparin should have strong inhibitory effects on thrombin generation, and thus we hypothesized that AT could be inhibiting enzymes bound to activated platelet surfaces that are not subject to being washed away by flow. We extended our mathematical model to include the relevant reactions of AT inhibition at the activated platelet surfaces as well as those for unfractionated heparin and a low molecular weight heparin. Our results show that AT alone is only an effective inhibitor at low tissue factor densities, but in the presence of heparin, it can greatly alter, and in some cases shut down, thrombin generation. Additionally, we studied each target enzyme separately and found that inactivation of no single enzyme could substantially suppress thrombin generation.     

Interaction of pregnancy-associated plasma protein-A (PAPP-A) with coagulation: a bioassay for PAPP-A

The observation that pregnancy-associated plasma protein A (PAPP-A) concentrations are higher in plasma compared to serum obtained from the same patient, together with fact that PAPP-A binds to heparin, prompted us to study the interaction between PAPP-A and the clotting system. It was determined that pure PAPP-A inhibits thrombin-induced coagulation of citrated plasma. The presence of antithrombin III (AT III) was necessary since PAPP-A had no inhibitory effect on coagulation of AT III-depleted plasma. The effect of PAPP-A is thus similar to that of heparin. This property of PAPP-A was used to develop a bioassay. Thrombin-induced polymerization of purified fibrinogen was measured in a spectrophotometer. AT III is a weak inhibitor of polymerization, but its effect is magnified in the presence of PAPP-A or heparin. The residual thrombin activity, when plotted against the concentration of PAPP-A, gives a linear relationship. The assay conditions developed allow maximal sensitivity and reproducibility. The kinetics of inhibition due to PAPP-A and heparin was first order. With this bioassay, activities of PAPP-A molecules isolated by the same technique from different fetomaternal compartments were compared.     

Mechanism of prolonged hypocoagulation appearing after intratracheal administration of heparin]

The comparative study of intratracheal and intravenous effect of administration of heparin on blood clotting and mast cell population condition was carried out in experiments. Unlike intravenous bolus injection of heparin, which induced fast short-time inactivation of all enzyme clotting factors, a single intratracheal injection inactivated "internal" rout of thrombin production. It was shown, that long-term hypocoagulability effect and inhibition of factors of blood coagulation after intratracheal administration of heparin correlated with accumulation of heparin in mast cell.     

Review: heparin. 1. Methods of determination and inhibitory mechanisms of some coagulation factors]

In a brief survey four groups of determining methods are reported for the purpose of monitoring the heparin therapy, with their advantages and disadvantages being assessed simultaneously. Emphasis is placed on the heterogeneity of heparin with approximately 120 fractions which are the cause of different manners of response and an impediment to the development of a rapid, reliable and economically advantageous method. Finally, all heparin factors in the plasma are enumerated and their mechanisms of action on individual coagulation factors are referred to.     

Antithrombin-S195A factor Xa-heparin structure reveals the allosteric mechanism of antithrombin activation

Regulation of blood coagulation is critical for maintaining blood flow, while preventing excessive bleeding or thrombosis. One of the principal regulatory mechanisms involves heparin activation of the serpin antithrombin (AT). Inhibition of several coagulation proteases is accelerated by up to 10,000-fold by heparin, either through bridging AT and the protease or by inducing allosteric changes in the properties of AT. The anticoagulant effect of short heparin chains, including the minimal AT-specific pentasaccharide, is mediated exclusively through the allosteric activation of AT towards efficient inhibition of coagulation factors (f) IXa and Xa. Here we present the crystallographic structure of the recognition (Michaelis) complex between heparin-activated AT and S195A fXa, revealing the extensive exosite contacts that confer specificity. The heparin-induced conformational change in AT is required to allow simultaneous contacts within the active site and two distinct exosites of fXa (36-loop and the autolysis loop). This structure explains the molecular basis of protease recognition by AT, and the mechanism of action of the important therapeutic low-molecular-weight heparins.     

Heparin treatment in abruptio placentae

Two cases of abruptio placentae with disseminated intravascular coagulation (DIC) were treated with heparin, and coagulation was monitored by thromboelastography as well as the usual hematology tests. The cases demonstrated the vagaries of DIC and both showed decreased overt hemorrhage after heparin treatment was started. Heparin may be indicated for the management of abruptio placentae where delivery is not imminent, where significant disseminated intravascular coagulation exists, and when adequate serial coagulation studies are available.     

Heparin: role in protein purification and substitution with animal-component free material

Heparin is a highly sulfated polysaccharide which belongs to the family of glycosaminoglycans. It is involved in various important biological activities. The major biological purpose is the inhibition of the coagulation cascade to maintain the blood flow in the vasculature. These properties are employed in several therapeutic drugs. Heparin’s activities are associated with its interaction to various proteins. To date, the structural heparin-protein interactions are not completely understood. This review gives a general overview of specific patterns and functional groups which are involved in the heparin-protein binding. An understanding of the heparin-protein interactions at the molecular level is not only advantageous in the therapeutic application but also in biotechnological application of heparin for downstreaming. This review focuses on the heparin affinity chromatography. Diverse recombinant proteins can be successfully purified by this method. While effective, it is disadvantageous that heparin is an animal-derived material. Animal-based components carry the risk of contamination. Therefore, they are liable to strict quality controls and the validation of effective good manufacturing practice (GMP) implementation. Hence, adequate alternatives to animal-derived components are needed. This review examines strategies to avoid these disadvantages. Thereby, alternatives for the provision of heparin such as chemical synthesized heparin, chemoenzymatic heparin, and bioengineered heparin are discussed. Moreover, the usage of other chromatographic systems mimetic the heparin effect is reviewed.

     

Mechanism of the anticoagulant action of heparin

Summary The anticoagulant effect of heparin, a sulfated glycosaminoglycan produced by mast cells, requires the participation of the plasma protease inhibitor antithrombin, also called heparin cofactor. Antithrombin inhibits coagulation proteases by forming equimolar, stable complexes with the enzymes. The formation of these complexes involves the attack by the enzyme of a specific Arg-Ser bond in the carboxy-terminal region of the inhibitor. The complexes so formed are not dissociated by denaturing solvents, which indicates that a covalent bond may contribute to their stability. This bond may be an acyl bond between the active-site serine of the enzyme and the arginine of the cleaved reactive bond of the inhibitor. However, the native complexes dissociate slowly at near-neutral pH into free enzyme and a modified inhibitor, cleaved at the reactive bond. So, antithrombin apparently functions as a pseudo-substrate that traps the enzyme in a kinetically stable complex.The reactions between antithrombin and coagulation proteases are slow in the absence of heparin. However, optimal amounts of heparin accelerate these reactions up to 2 000-fold, thereby efficiently preventing the formation of fibrin in blood. The accelerating effect, and thus the anticoagulant activity, is shown by only about one-third of the molecules in all heparin preparations, while the remaining molecules are almost inactive. The highly active molecules bind tightly to antithrombin, i.e. with a binding constant of slightly below 10⁸ M^–1 at physiological ionic strength, while the relatively inactive molecules bind about a thousand-fold more weakly. The binding of the high-affinity heparin to antithrombin is accompanied by a conformational change in the inhibitor that is detectable by spectroscopic and kinetic methods. This conformational change follows an initial, weak binding of heparin to antithrombin and causes the tight interaction between polysaccharide and inhibitor that is prerequisite to heparin anticoagulant activity. It has also been postulated that the conformational change leads to a more favourable exposure of the reactive site of antithrombin, thereby allowing the rapid interaction with the proteases.Heparin also binds to the coagulation proteases. Recent studies indicate that this binding is weaker and less specific that the binding to antithrombin. Nevertheless, for some enzymes, thrombin, Factor IX_a and Factor XI_a, an interaction between heparin and the protease, in addition to that between the polysaccharide and antithrombin; apparently is involved in the accelerated inhibition of the enzymes. The effect of this interaction may be to approximate enzyme with inhibitor in an appropriate manner. However, the bulk of the evidence available indicates that binding of heparin to the protease alone cannot be responsible for the accelerating effect of the polysaccharide on the antithrombin-protease reaction.Heparin acts as a catalyst in the antithrombin-protease reaction, i.e. it accelerates the reaction in non-stoichiometric amounts and is not consumed during the reaction. This ability can be explained by heparin being released from the antithrombin-protease complex for renewed binding to antithrombin, once the complex has been formed. Such a decresed affinity of heparin for the antithrombin complex, compared to the affinity for antithrombin alone, has been demonstrated.The structure of the antithrombin-binding region in heparin has been investigated following the isolation of oligosaccharides with high affinity for antithrombin. The smallest such oligosaccharide, an octasaccharide, obtained after partial random depolymerization of heparin with nitrous acid, was found to contain a unique glucosamine-3-O-sulfate group, which could not be detected in other portions of the high affinity heparin molecule and which was absent in heparin with low affinity for antithrombin. The actual antithrombin-binding region within this octasaccharide molecule has been identified as a pentasaccharide sequence with he predominant structure: N-acetyl-D-glucosamine(6-O-SO₃)D-glucoronic acidD-glucosamine(N-SO₃;3,6-di-O-SO₃)L-iduronic acid(2-O-SO₃)D-glucosamine(N-SO₃;6-O-SO₃). In addition to the 3-O-sulfate group, both N-sulfate groups as well as the 6-O-sulfate group of the N-acetylated glucosamine unit appear to be essential for the interaction with antithrombin. The remarkably constant structure of this sequence, as compared to other regions of the heparin molecule, suggests a strictly regulated mechanism of biosynthesis.The ability of heparin to potentiate the inhibition of blood coagulation by antithrombin generally decreases with decreasing molecular weight of the polysaccharide. However, individual coagulation enzymes differ markedly with regard to this molecular-weight dependence. Oligosaccharides in the extreme low-molecular weight range, i.e. octa- to dodecasaccharides, with high affinity for antithrombin have high anti-Factor X_a-activity but are virtually unable to potentiate the inhibition of thrombin. Furthermore, such oligosaccharides are ineffective in preventing experimentally induced venous thrombosis in rabbits. Slightly larger oligosaccharides, containing 16 to 18 monosaccharide residues, show significant anti-thrombin as well as antithrombotic activities, yet have little effect on overall blood coagulation. These findings indicate that the affinity of a heparin fragment for antithrombin is not in itself a measure of the ability to prevent venous thrombo-genesis, and that the anti-Factor X_a activity of heparin is only a partial expression of its therapeutic potential as an antithrombotic agent.The biological role of the interaction between heparin and antithrombin is unclear. In addition to a possible function in the regulation of hemostasis, endogenous heparin may serve as a regulator of extravascular serine proteinases. Mouse peritoneal macrophages have been found to synthesize all the enzymes that constitute the extrinsic pathway of coagulation. Moreover, tissue thromboplastin is produced by these cells in response to a functional interaction with activated T-lymphocytes. The inhibition of this extravascular coagulation system by heparin, released from mast cells, may be potentially important in modulating inflammatory reactions.     

Heparin and antiheparin in childhood. 3. Heparin level measurements and their importance in heparin monitoring

Measurements of the heparin level were made under continuous anticoagulation in a total of 7 patients. For the purpose of monitoring heparin the coagulation time values were determined parallelly. Except a patient with a sepsis and a 7 days old newborn baby the desired prolongation for the partial thromboplastin time and the reaction time of thrombelastogram resulted from heparin titres lying within the range of 0.2-0.7 U/ml of plasma. Even after applying depot preparations there was a relatively good correspondance of heparin level curves and coagulation parameters. In childhood the partial thromboplastin time is primarily suitable for monitoring the heparin therapy. Heparin half-life times calculated during the transumbilical exchange transfusion in 7 children amounted to values ranging between 40-110 minutes. In addition to checking low dose heparinizing, measurements of the level are suitable for deriving dosage standards for neutralizing heparin effects by protamine sulfate.     

Anti-heparin activity of plasma with various low density lipoprotein content]

Anti-heparin activity correlated with LDL concentration in the plasma. Blood plasma of women in labour is characterized by the high antiheparin activity and low LDL levels. Anti-heparin activity is low and LDL levels are low in blood plasma in childhood. An effect of other factors neutralizing heparin (e.g. fibrinogen, platelet factor 4, acid alpha 1-glycoprotein, globulins, basic proteins) and differences of anti-thrombin III on plasma anti-heparin activity has been excluded. Neutralization of heparin anticoagulation activity by LDL is of clinical value. Blood LDL level should be considered, while heparin therapeutical doses are under scrutiny.     

Low-molecular-weight heparin and prevention of postoperative deep vein thrombosis.

The efficacy of low-molecular-weight heparin as a prophylactic agent was assessed in 150 consecutive patients over the age of 40 undergoing major abdominal surgery. Fifty of these patients received 1250 activated partial thromboplastin time (APTT) units of low-molecular-weight heparin every 12 hours: three developed isotopic deep vein thrombosis, which was confirmed by phlebography in two cases. The other 100 patients received a single injection of 1850 APTT units of low-molecular-weight heparin. Three of them developed isotopic deep vein thrombosis; phlebography failed to confirm the presence of thrombi in each case. None of the 150 patients studied died from fatal or contributory pulmonary emboli. Low-molecular-weight heparin was not associated with any increase in preoperative or postoperative bleeding. The effect of equal amounts of postoperative bleeding. The effect of equal amounts of low-molecular-weight heparin and unfractionated heparin on the coagulation mechanism during surgery was investigated in another 30 patients. The clotting assays and results of in-vivo platelet function tests indicated that both preparations produced similar effect. Intragroup comparisons, however, showed significant differences in the anti-factor Xa activity, lipoprotein lipase release, and plasma prekallikrein concentrations. A single injection of low-molecular-weight heparin daily is a convenient way of preventing deep vein thrombosis in high-risk patients undergoing major abdominal surgery.     

Determination of galactosamine impurities in heparin sodium using fluorescent labeling and conventional high-performance liquid chromatography

Heparin is a sulfated glycosaminoglycan (GAG), which contains N-acetylated or N-sulfated glucosamine (GlcN). Heparin, which is generally obtained from the healthy porcine intestines, is widely used as an anticoagulant during dialysis and treatments of thrombosis such as disseminated intravascular coagulation. Dermatan sulfate (DS) and chondroitin sulfate (CS), which are galactosamine (GalN)-containing GAGs, are major process-related impurities of heparin products. The varying DS and CS contents between heparin products can be responsible for the different anticoagulant activities of heparin. Therefore, a test to determine the concentrations of GalN-containing GAG is essential to ensure the quality and safety of heparin products. In this study, we developed a method for determination of relative content of GalN from GalN-containing GAG in heparin active pharmaceutical ingredients (APIs). The method validation and collaborative study with heparin manufacturers and suppliers showed that our method has enough specificity, sensitivity, linearity, repeatability, reproducibility, and recovery as the limiting test for GalN from GalN-containing GAGs. We believe that our method will be useful for ensuring quality, efficacy, and safety of pharmaceutical heparins. On July 30, 2010, the GalN limiting test based on our method was adopted in the heparin sodium monograph in the Japanese Pharmacopoeia.     

Lymphocytosis produced by heparin and other sulphated polysaccharides in mice and rats

Lymphocytosis has been produced in mice and rats using heparin and other sulphated polysaccharides. Two hours after heparin (50 mg/kg ip) the concentration of lymphocytes in mouse blood increased threefold; it fell to control levels after 9 hr. The height of the lymphocytosis was related to the dose of heparin injected. After intravenous heparin in rats there was a comparable lymphocytosis maximal 1 hr after injection. In mice other negatively charged sulphated polysaccharides also caused lymphocytoses, which were greater and occurred later with increase in molecular weight of the substance injected. Results in rats were similar. No lymphocytosis followed the injection of negatively charged phosphated dextrans, positively charged DEAE dextran, or neutral dextran. There was no correlation between the effect of these substances on lymphocytes and their effect on coagulation of blood, hepatic phagocytosis, or the immune response to sheep red blood cells.     

凝血酶原复合物中肝素含量的测定

凝血酶原复合物按标示量加蒸馏水溶解成１０ＰＥ／ｍｌ。通过加入适量的硫酸鱼精蛋白中和凝血酶原复合物样品中肝素。取中和后的样品加入缺血小板血浆、脑磷脂和氯化钙,记录凝固时间,挑选凝固时间最短的样品管计算肝素含量。该方法误差２０％。以５家血制品生产单位生产的２４批ＰＣＣ肝素含量的测定,按ＰＣＣ效价标示量小于等于１３０％计算,不合格６批占２５％。     

Effect of heparin on the activity of liver microsome oxidative enzyme systems in healthy rats and rats with experimental glomerulonephritis]

In experiments on mongrel rats it was found that heparin in doses of 2, 5, and 10 mg/kg had an inducing effect on the oxidative systems of hepatic microsomes: hexenal test period was shortened, cytochrome P450 content increased, relative liver weight rose; the activity of histochemically-detectable NAD-enzymes of hepatocytes became greater. Heparin was capable of considerable (2-3-fold) stimulation of antitoxic activity of the liver reduced in experimental glomerulonephritis. The effect of heparin on the antitoxic function of the liver did not correlate with its effect, on the blood coagulation system.     

Heparin and antiheparin in young children. 1. Report: development of a method for determining heparin and antiheparin in the antithrombin-thrombin system

A new method of determining the biological activity of heparin and antiheparin is described. The principle is based on measuring the coagulation time in the antithrombin-thrombin system. By using a partially purified antithrombin III preparation and after coagulating the plasma samples with small amounts of thrombin the measuring system proves to be mostly independent on the inhibitory content of the test plasmas to be investigated. Heat defibrinated plasma cannot be used because it has essential properties with a close affinity to heparin. Additions of sodium and calcium ions will make the system more sensitive. Criteria of reaction kinetics are used for standardizing the antithrombin-thrombin system. The heparin level of up to 0.01 U/ml and the antiheparin titre of up to 0.005 U/ml can be covered by the procedure presented. Thus, it has a high sensitivity. The quality controls which were performed give evidence of the high precision of this method.     

Modulatory effects on proteinase kinetics caused by association of both enzyme and substrate to heparin

Heparin is shown to produce modulatory effects on the amidolytic activity of trypsin, thrombin and plasmin with various synthetic peptide substrates. Simple Michaelis-Menten kinetics are observed in the absence of heparin. In its presence an enhancement effect is observed at low substrate concentrations, and an inhibitory effect is observed at high substrate concentrations. Other polyanions like dextran sulphate, phosvitin and inositol hexakisphosphate produces a similar effect. The modulatory effect of heparin is abolished when it binds cations. Co-binding of both substrate and enzyme to heparin seems to be a necessary requirement for the effect to occur. A model is proposed which can account semiquantitatively for the kinetics observed. It is suggested that the mechanism, which involves co-binding of substrate and enzyme in an competitive manner to a macromolecular structure, may be of primary importance as a regulatory mechanism in blood coagulation and fibrinolysis.     

SEASONAL VARIATION IN THE EFFECT OF A FIXED DOSE OF HEPARIN ON ACTIVATED CLOTTING TIME IN PATIENTS PREPARED FOR OPEN-HEART SURGERY *

We investigated the effect of an injected bolus of 5 mg kg^- heparin at one circadian stage (08:30 to 11:00) on blood coagulation during different months of the year. Activated clotting times (ACTs) were assessed before and 5 min after heparin dosing to ensure extracorporeal circulation during open-heart surgery. The ACT data of 1083 presumably day-active Turkish patients (816 men and 267 women, mostly older than 46 years) who underwent coronary bypass surgery between 08:30 and 11:00 in the years from 1994 to 1997 were analyzed for annual rhythmicity. The ACT values obtained just before and 5 min after heparinization were subjected to cosinor analysis using a 365.25-day period to assess seasonality in basal ACT level and heparin effect. A small-amplitude annual rhythm with a wintertime peak was documented in the morning ACT in the group of 1083 patients. Rhythms of similar magnitude and staging were also detected in heparin effect on ACT in the 1083 patients and in subgroups categorized by gender. Circannual rhythmicity in the heparin effect on ACT was also documented in the elderly (≥ 45 years old), but not young (18-45 years old) patients. The annual mean effect of heparin on the ACT was statistically significantly greater in younger than older patients. The relatively low-amplitude circannual rhythm in heparin effect on ACT (∼10% of the annual mean) is not viewed as being meaningful in patient preparation for bypass surgery for the 5 mg kg^-1 level of heparin dosing. (Chronobiology International, 18(5), 865-873, 2001)     

Behavior of the heparinocytes after gold therapy]

A reduction of the total leukocytes as well as a significant decrease of heparinocytes and BAI (basophilic age index) can be observed in rheumatoid arthritis in the course of a gold therapy. As some coagulation parameters simultaneously speak in favour of an enhancement of the intravasal coagulation, a partial blocking of the endogenous heparin caused by gold may be supposed. A combined heparin or heparinoid therapy in a low dosage is being recommended for risk patients of the vascular and coagulation system.     

Affinity chromatography of hyaluronate on glutaraldehyde-fixed SV-3T3 cells

A simple, rapid colorimetric assay for plasma heparin is presented. The assay employs the metachromasia of azure A when heparin is added. It is useful for 0 to 10 units/ml and does not depend on heparin's anticoagulant activity. Heparin concentrations determined with this assay are not exactly the same as those determined with coagulation assays. This is probably because azure A determines chemical heparin, not anticoagulant active heparin.