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.     

Heparin-associated thrombocytopenia: low frequency in 104 patients treated with heparin of intestinal mucosal origin.

A prospective study of 104 patients receiving heparin obtained from porcine intestinal mucosa for 4 or more days was conducted to determine the frequency of associated significant thrombocytopenia (platelet count less than 100 x 10(9)/I on 2 consecutive days). No episodes of significant thrombocytopenia were identified in the 13 patients receiving heparin by continuous intravenous infusion for a mean of 8.0 days or in the 38 patients receiving heparin subcutaneously for a mean of 9.9 days. In 1 of the 26 patients receiving heparin as intermittent intravenous boluses for a mean of 8.2 days significant thrombocytopenia developed; this patient had laboratory evidence of disseminated intravascular coagulation. In none of the 17 patients receiving uninterrupted heparin therapy for 4 or more days by more than one route of administration but for less than 4 days by any single route did significant heparin-associated thrombocytopenia develop. Of the 104 patients 13 had one or more platelet counts of less than 150 x 10(9)/I, but in most it was not possible to definitely relate the thrombocytopenia to the heparin therapy. Platelets in normal platelet-rich plasma did not aggregate when heparin and serum from patients with thrombocytopenia were added. The frequency of heparin-associated thrombocytopenia noted in this study was considerably lower than that reported previously.     

Control of Heparin Therapy

Heparin therapy in 114 patients was controlled by daily blood tests—the whole blood coagulation time, kaolin-activated partial thromboplastin time of plasma, and plasma heparin assay. Bleeding episodes occurred in 7 out of 92 patients (7·6%) who had normal haemostatic mechanisms before therapy and in 11 out of 22 patients (50%) with defective haemostasis, mostly due to intravascular coagulation or renal failure. The dose of heparin ranged from 20,000 to 60,000 units in each 24-hour period. In some patients bleeding was related to overdosage, but in others the laboratory tests indicated satisfactory or suboptimal dosage at the time of bleeding. Though there were positive correlations between the results of the three tests, these were not close, and no one test was preferable. Hence laboratory control of heparin therapy is unsatisfactory and patients may bleed despite careful control of the dose by all three methods.     

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.     

Control of the therapeutic heparin dosage in platelet rich citrate plasma after protamine neutralization

A laboranalytical method for estimating the anticoagulative effect of therapeutic heparin doses based on coagulation physiology is presented. Corresponding to our expectation this method is overcome by the direct measuring heparin with chromogenic substrates. On the other hand there are some advantages against the often practically used prolongation of PTT. By neutralisation of the heparin influence with protamine sulphate it is possible to discriminate between the heparin effect and additional disturbances in consequence of deficit in a coagulation factor. An absence of the heparin effect should always lead to the use of an antithrombin III assay.     

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.     

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.     

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.     

Heparin conjugated polylactide as a blood compatible material

A heparin-conjugated biodegradable polymer (PLA-heparin) by the direct coupling of heparin to polylactide (PLA) was synthesized and characterized. The surface exposed heparin content associated PLA-heparin was measured to be 0.067 microg/cm2. PLA-heparin coated surface has shown higher hydrophilicity rather than control PLA surface. The clotting time of PLA-heparin conjugate measured by activated partial thromboplastin time (APTT) was significantly prolonged as compared to PLA. The bioactivity of bound heparin measured by APTT corresponds to 17.4% of free heparin. It has been also demonstrated that the conjugation of heparin suppresses the protein adsorption as well as the platelet adhesion. These results indicate that the unique property of bound heparin has an inhibiting influence on the coagulation, plasma protein adsorption, and subsequent platelet adhesion systems. This novel PLA-heparin conjugate could be applied as blood/tissue compatible biodegradable materials for implantable medical devices and tissue engineering.     

Fibrinolysis before and after gold therapy in rheumatoid arthritis]

In 12 patients with chronic rheumatoid arthritis the immunoglobulins IgG, IgA and IgM were determined according to Mancini and the fibrinolytic split products according to Nilehn before and after a gold treatment of 3 to 6 months. The IgG values were always increased and even after the treatment there was only little regression. The fibrinolytic split products found in 5 cases speak in favour of an enhanced intravasal coagulation, which increased still further after the gold therapy. In spite of clinical improvement the immunological processes and latent actions of coagulation seem to pass almost undiminished.     

Trousseau's syndrome.

We report 4 cases of Trousseau''s syndrome, in which spontaneous recurrent or migratory venous thromboses, arterial emboli caused by nonbacterial thrombotic endocarditis, or both, develop in a patient with a recognized or occult malignant tumor. The clinical course of 3 of the patients emphasizes a key point: The occurrence for no known reason of thromboses preventable by anticoagulation therapy with heparin but not with warfarin sodium should alert a physician to focus diagnostic efforts on uncovering an underlying malignant lesion. Thromboses may occur months to years before the tumor is discovered, and a thorough negative initial examination does not obviate the need for a continuing search. Patients with Trousseau''s syndrome have persistent low-grade intravascular coagulation, and therapy with heparin should be continued indefinitely. Stopping heparin therapy for even a day may permit a new thrombosis to develop. Immunostaining a biopsy specimen from 1 patient provided evidence that 2 properties of a neoplastic lesion are required for the syndrome to develop: The malignant cells express surface membrane tissue factor, and structural features of the tumor permit the malignant cells or vesicles it sheds to be exposed to circulating blood.     

Low-dose heparin in the management of acute myelocytic leukaemia.

The effectiveness of heparin in acute leukaemia complicated by disseminated intravascular coagulation (DIC) is still controversial. In this regard low-dose heparin was found to be therapeutically effective in three patients suffering from acute myelocytic leukaemia and DIC. With respect to the contraindication of high-dose heparin in these conditions the low dose regimen appears to be a valuable alternative.     

Circadian changes in anticoagulant effect of heparin infused at a constant rate.

Six patients with venous thromboembolism were treated with heparin, administered intravenously by a constant infusion pump. The initial daily dose of heparin was adjusted to keep the activated partial thromboplastin time, sampled at 0800, between 1.5 and 2.5 times the control level. Once that level was obtained, this dose was kept constant. Anticoagulation was thereafter measured, every four hours for 48 hours, by activated partial thromboplastin time, thrombin time, and coagulation factor Xa inhibition assay. The results of all three coagulation tests showed a circadian variation in the six patients. Maximum values were achieved at night and minimum values in the morning. These circadian variations were reproduced for two consecutive days. Differences between night and morning values reached almost 50% for activated partial thromboplastin time, 60% for thrombin time, and 40% for factor Xa inhibition assay. This circadian variation resulted from two rhythms, a circadian rhythm lasting 24 hours and an ultradian rhythm lasting 12 hours, which were detected by cosinor analysis for each coagulation test (p less than 0.01). A circadian rhythm was detected individually in most of the patients for each coagulation test (p less than 0.05). All patients had a nocturnal peak in activated partial thromboplastin time on both days. In four patients this peak exceeded the upper desired limit of activated partial thromboplastin time. These rhythms should be taken into account when evaluating the dosage of heparin to be administered.     

Neutralization and binding of heparin by S protein/vitronectin in the inhibition of factor Xa by antithrombin III. Involvement of an inducible heparin-binding domain of S protein/vitronectin

The interference of the heparin-neutralizing plasma component S protein (vitronectin) (Mr = 78,000) with heparin-catalyzed inhibition of coagulation factor Xa by antithrombin III was investigated in plasma and in a purified system. In plasma, S protein effectively counteracted the anticoagulant activity of heparin, since factor Xa inhibition was markedly reduced in comparison to heparinized plasma deficient in S protein. Using purified components in the presence of heparin, S protein induced a concentration-dependent reduction of the inhibition rate of factor Xa by antithrombin III. This resulted in a decrease of the apparent pseudo-first order rate constant by more than 10-fold at a physiological ratio of antithrombin III to S protein. S protein not only counteracted the anticoagulant activity of commercial heparin but also of low molecular weight forms of heparin (mean Mr of 4,500). The heparin-neutralizing activity of S protein was found to be mainly expressed in the range 0.2-10 micrograms/ml of high Mr as well as low Mr heparin. S protein and high affinity heparin reacted with apparent 1:1 stoichiometry to form a complex with a dissociation constant KD = 1 X 10(-8) M as determined by a functional assay. As deduced from dot-blot analysis, direct interaction of radiolabeled heparin with S protein revealed a dissociation constant KD = 4 X 10(-8) M. Heparin binding as well as heparin neutralization by S protein increased significantly when reduced/carboxymethylated or guanidine-treated S protein was employed indicating the existence of a partly buried heparin-binding domain in native S protein. Radiolabeled heparin bound to the native protein molecule as well as to a BrCN fragment (Mr = 12,000) containing the heparin-binding domain as demonstrated by direct binding on nitrocellulose replicas of sodium dodecyl sulfate-polyacrylamide gels. Kinetic analysis revealed that the heparin neutralization activity of S protein in the inhibition of factor Xa by antithrombin III could be mimicked by a synthetic tridecapeptide from the amino-terminal portion of the heparin-binding domain. These data provide evidence that the heparin-binding domain of S protein appears to be unique in binding to heparin and thereby neutralizing its anticoagulant activity in the inhibition of coagulation factors by antithrombin III. The induction of heparin binding and neutralization may be considered a possible physiological mechanism initiated by conformational alteration of the S protein molecule.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural features and anticoagulant activities of a novel natural low molecular weight heparin from the shrimp Penaeus brasiliensis.

A natural low molecular weight heparin (8.5 kDa), with an anticoagulant activity of 95 IU/mg by the USP assay, was isolated from the shrimp Penaeus brasiliensis. The crustacean heparin was susceptible to both heparinase and heparitinase II from Flavobacterium heparinum forming tri- and di-sulfated disaccharides as the mammalian heparins. (13)C and (1)H NMR spectroscopy revealed that the shrimp heparin was enriched in both glucuronic and non-sulfated iduronic acid residues. The in vitro anticlotting activities in different steps of the coagulation cascade have shown that its anticoagulant action is mainly exerted through the inhibition of factor Xa and heparin cofactor II-mediated inhibition of thrombin. The shrimp heparin has also a potent in vivo antithrombotic activity comparable to the mammalian low molecular weight heparins.     

Ribonuclease–gold Labels Chondroitin Sulphate in Guinea Pig Basophil Granules

Basophilic leucocytes are metachromatic granule-containing secretory granulocytes that contain a mixture of granular proteoglycans devoid of heparin. In guinea pigs, isolated basophilic leucocyte granules primarily contain chondroitin sulphate. We have recently demonstrated that an enzyme-affinity– gold technique to image RNA, using the reagent RNase gold, also binds specifically to heparin in human mast cell granules. Such binding is based on the known property of heparin as a competitive inhibitor of RNase. Using similar methods, we show here that RNase–gold binds to the chondroitin sulphate in the secretory granules of guinea pig basophils, thus broadening the applicability of this post-embedding affinity–gold method to studies that require imaging of chondroitin sulphate in routinely prepared electron microscopical samples.     

Mechanisms of Thrombocytopenia in Malignant Tertian Malaria

The mechanism of thrombocytopenia in six patients with falciparum malaria has been studied. All the patients recovered after antimalarial therapy, and cerebral malaria was not a feature. Radioactive-labelled platelets and fibrinogen were injected into the patients during the phase of thrombocytopenia. In all cases recovery of injected platelets was notably subnormal, indicating excessive splenic pooling of platelets. Platelet life span was moderately shortened in all patients, and platelet turnover increased approximately two-fold. Fibrinogen catabolism was moderately increased in all patients, but coagulation tests failed to reveal evidence of disseminated intravascular coagulation. The results suggest that in uncomplicated cases of malaria thrombocytopenia is the result of splenic pooling of platelets aggravated by a moderate decrease in platelet life span. In such cases thrombocytopenia is thus not the result of disseminated intravascular coagulation (D.I.C.), and heparin therapy is not indicated unless there is unequivocal ancillary evidence of D.I.C.     

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.     

Conformational changes in serpins: II. The mechanism of activation of antithrombin by heparindagger

Antithrombin, uniquely among plasma serpins acting as proteinase inhibitors in the control of the blood coagulation cascade, circulates in a relatively inactive form. Its activation by heparin, and specifically by a pentasaccharide core of heparin, has been shown to involve release of the peptide loop containing the reactive centre from partial insertion in the A sheet of the molecule. Here we compare the structures of the circulating inactive form of antithrombin with the activated structure in complex with heparin pentasaccharide. We show that the rearrangement of the reactive centre loop that occurs upon activation is part of a widespread conformational change involving a realignment of the two major domains of the molecule. We also examine natural mutants that possess high affinity for heparin pentasaccharide, and relate the kinetics of their interaction with heparin pentasaccharide to the structural transitions occuring in the activation process.     

The physiological inhibitors of blood coagulation. 1. Antithrombin III (AT III)

In a brief survey, recent data on the molecular structure and functions of antithrombin III as well as on the similarity of identity with antithrombin III of newborns and mammals are referred to. Moreover, the survival time in circulation and its broad inhibiting spectrum comprising not only all activating factors of coagulation, but also other proteases, is discussed. The accelerating effect of heparin on response and possible mechanisms of this acceleration are discussed. The various procedures of determination are briefly dealt with. Furthermore, the distinct tendency towards thromboembolic complications occurring in patients with only a slight reduction of antithrombin III level is dealt with, as it was described in inherited and acquired deficiency of antithrombin III. Finally, the possibilities of therapy for this deficiency are discussed and is underlined on the key position of antithrombin III for maintaining the balance of coagulation.