High Molecular Weight Heparin Research Articles

Generation of Anaphylatoxins by Human β-Tryptase from C3, C4, and C5

Both mast cells and complement participate in innate and acquired immunity. The current study examines whether beta-tryptase, the major protease of human mast cells, can directly generate bioactive complement anaphylatoxins. Important variables included pH, monomeric vs tetrameric forms of beta-tryptase, and the beta-tryptase-activating polyanion. The B12 mAb was used to stabilize beta-tryptase in its monomeric form. C3a and C4a were best generated from C3 and C4, respectively, by monomeric beta-tryptase in the presence of low molecular weight dextran sulfate or heparin at acidic pH. High molecular weight polyanions increased degradation of these anaphylatoxins. C5a was optimally generated from C5 at acidic pH by beta-tryptase monomers in the presence of high molecular weight dextran sulfate and heparin polyanions, but also was produced by beta-tryptase tetramers under these conditions. Mass spectrometry verified that the molecular mass of each anaphylatoxin was correct. Both beta-tryptase-generated C5a and C3a (but not C4a) were potent activators of human skin mast cells. These complement anaphylatoxins also could be generated by beta-tryptase in releasates of activated skin mast cells. Of further biologic interest, beta-tryptase also generated C3a from C3 in human plasma at acidic pH. These results suggest beta-tryptase might generate complement anaphylatoxins in vivo at sites of inflammation, such as the airway of active asthma patients where the pH is acidic and where elevated levels of beta-tryptase and complement anaphylatoxins are detected.

Different anticoagulation strategies in off-pump coronary artery bypass operations: a European survey

In order to determine anticoagulation strategies in OPCAB a questionnaire survey among 750 European cardio-thoracic surgeons was performed. Questions addressed volume of OPCAB procedures performed, intra- and perioperative heparinization and antiplatelet therapy, as well as perioperative management. A total of 325 (43.7%) questionnaires were returned and validated. Perioperative protocols for administration of antiplatelets differed among the respondent surgeons. Perioperative prophylaxis of thrombosis (low or high molecular weight heparin) is performed by 78%. Intraoperative heparin dosage range between 70 U/kg to 500 U/kg, 60% of respondents prefer a low-dose regimen (< or = 150 U/kg). Correspondingly, the lowest activated clotting time (ACT) during surgery is accepted to be 200 s by 24%, 250 s by 18% and 300 s by 26% of surgeons. Protamine is used by 91% of respondents, while 52% perform a 1:1 reversal. A cell-saver and antifibrinolytics are used by 70% and 40%, respectively. Interestingly, 56% of respondents think bleeding in OPCAB patients is not reduced when compared to on-pump CABG. In addition, 34% of respondents believe there is an increased risk of early graft occlusion following OPCAB. This survey demonstrates widely different intra- and perioperative anticoagulation strategies for OPCAB procedures among European surgeons.

The role of heparin self-association in the gelation of heparin-functionalized polymers

The extensive use of the glycosaminoglycan (GAG) heparin in the design of emerging biomaterials has made the physical characterization of this heterogeneous biomacromolecule increasingly important. In this work, heparin solutions are characterized via dynamic light scattering to investigate heparin's self-association, since this behavior was recently hypothesized to play a role in the gelation of heparin-functionalized polymer hydrogels. Samples of either low molecular weight heparin or high molecular weight heparin were filtered using membranes with 100, 220, or 450 nm average pore sizes. The 100 and 220 nm filters produce a single population of monomers with a diameter range of 3–10 nm in the intensity-weighted size distribution. However, the 450 nm filters reveal a second population of associated heparin. Increasing the solution concentration of high molecular weight heparin (HMWH) from 2.5 to 10 wt% causes the magnitude of the smaller population to decrease, while the diameter of the larger associated species approximately doubles. HMWH from different manufacturers displays varying degrees of association. Therefore, weaker associating HMWH can potentially be identified to control heparin self-interactions. Finally, fractionated, N-deacetylated low molecular weight heparin (LMWH) is compared to unmodified LMWH. The chemically modified heparin exhibits a heightened degree of association, suggesting an enhanced self-interaction. The increased negative charge of LMWH in the fractionated sample likely enhances polyelectrolyte interactions proposed to drive the association of these similarly charged polysaccharides. A more detailed understanding of heparin–heparin interactions will assist in the design of new scaffold materials with controlled release profiles, in the clinical use of heparin as an anticoagulant, and in investigations of interactions of other like-charged biomacromolecules.

Comparative study of hemostatic properties of the complex and mixture of high molecular weight heparin and adenosine triphosphate

Anticoagulant and nonenzymatic fibrinolytic activities increased in blood plasma of 6-7-month-old rats after repeated intramuscular administration of the heparin-adenosine triphosphate complex (G-ATP). The mixture of heparin and ATP had no fibrin depolymerizing activity in vitro. Repeated intramuscular administration of the mixture had anticoagulant effect although it was 1.5-1.6 times less pronounced compared to the complex. A higher anticoagulant and fibrinolytic efficiency of the G-ATP complex compared to the mixture is concluded.

Production of heparin-functionalized hydrogels for the development of responsive and controlled growth factor delivery systems

Methods to assemble polymeric hydrogels on the basis of noncovalent protein–glycosaminoglycan interactions have been previously demonstrated by us and others and hold promise in the development of receptor-responsive hydrogel materials; improvements in the mechanical properties of such systems would broaden their utility. Thus, in situ crosslinkable and degradable heparin-containing hydrogels were designed for the binding and controlled release of growth factors. Specifically, maleimide-functionalized high molecular weight heparin (HMWH) was synthesized via straightforward chemical methods that permitted facile and controllable modification of carboxylates in HMWH with maleimide groups via control of catalyst and reaction conditions, as assessed via 1H NMR spectroscopy. These modified heparins were crosslinked into hydrogels via reaction with various thiol-functionalized PEGs. The gelation times and elastic moduli of the gels, as assessed through oscillatory rheometry, could be tuned by controlling the functionality of HMWH, the concentration of the hydrogel, the identity of the PEG-based crosslinker, as well as the molar ratio between maleimide and thiol groups. The capability of the hydrogels to bind to growth factors was investigated with immunochemical assays. Preliminary studies indicate the controlled release of basic fibroblast growth factor (bFGF) from these materials and suggest their broader use in the design of responsive materials.

Comparative analysis of complex formation of magnesium and calcium ions with low-molecular-weight and unfractionated heparin

The acid-base and complexing properties of Logiparin (a low-molecular-weight variety (LMH) of heparin (H4L) with magnesium and calcium ions were studied using mathematical modeling of chemical equilibria in aqueous solutions of Logiparin and its solutions with magnesium and calcium ions and pH titration. The heparin concentration was set equal to the concentration of its disaccharide units. The protonated heparin form HL3− and the most significant heparin complexes of Mg2+ and Ca2+ were identified; relevant formation constants were estimated. Comparative analysis of the results is carried out against available data on ion complexing with unfractionated (high-molecular-weight) heparin (HMH). The Logiparin complexes of calcium and magnesium ions are inferior to the HMH complexes in both their formation constants in solution and their effect on the decrease in the equilibrium concentration of free calcium ions, a participant of all blood coagulation processes. This distinction creates prerequisites for the lower blood-coagulating activity of Logiparin compared to that of HMH, which is for the first time confirmed in biological experiments with the in vitro administration of the same concentration of HMH or LMH into the blood plasma of laboratory rats.

Eczematous infiltrated plaques to subcutaneous heparin: a type IV allergic reaction

Two patients are described who had delayed reactions with eczematous plaques to subcutaneous injections of heparin. Subcutaneous challenge with high and low molecular weight heparin compounds were positive in both patients. Patch and intradermal tests with heparin were positive in one subject. Histological and immunohistochemical studies indicated that it was a type IV allergic reaction.

Transdermal Delivery of Heparin Using Pulsed Current Iontophoresis

In clinical practice heparin has to be administered by injection with obvious disadvantages; thus, transdermal delivery by electrically assisted methods have been studied. In this study we evaluated the efficacy of a Food and Drug Administration-approved pulsed current iontophoresis system in delivering heparin through living rat skin. Fluorescent and radioactive heparin as well as a commercial heparin preparation were delivered through rat skin via a pulsed current iontophoresis system. Pulsed current iontophoresis allowed fluorescent heparin to cross the stratum corneum localizing in epidermis and dermis. Unfractionated, high-, and low molecular weight fraction pools, obtained by fractionating [35S]-unfractionated heparin on a molecular weight sieve, were then separately tested. Pulsed current iontophoresis elicited the transdermal delivery of low molecular weight heparin, but not that of high molecular weight heparin. Finally, pulsed current iontophoresis of an unfractionated pharmaceutical heparin preparation significantly decreased plasmatic factor Xa activity. We hypothesize that this technique could be used to administer low molecular weight heparin in a cost-efficient and safe manner without the need for syringes and needles.

Pathogenic Role of Surface Platelet Factor 4 Complexes in Heparin-Induced Thrombocytopenia: Diagnostic and Therapeutic Implications.

Heparin-induced thrombocytopenia (HIT) is caused by antibodies that recognize complexes between high molecular weight heparin and Platelet Factor 4 (PF4). Current therapy with direct thrombin inhibitors is not effective in all cases, likely because it acts downstream of antibody-induced platelet activation. More directed therapies to the underlying pathology in HIT may be more effective. Heparin and PF4 only bind HIT antibodies over a narrow molar ratio of reactants at which ultralarge soluble complexes are formed. We asked whether similar complexes form between PF4 and endogenous platelet glycosaminoglycans (GAG) and their pathogenic role in experimental HIT. Platelet surface GAG:PF4 complexes are indeed antigenic over a narrow molar range of reactants. Heparin is not required for either HIT-IgG or a HIT-like monoclonal antibody KKO to bind to PF4 on human or mouse platelet surfaces in vitro, but enhances antigenicity when very high levels of surface PF4 are present. Antigenicity is maximal at a PF4 concentration of 50 μg/mL (well within the range that can be achieved within a thrombus) and ~25 μg/mL heparin (~0.5 U/mL, which is within the therapeutic range) optimally enhances antigenicity when surface PF4 levels were increased 4-fold. Using transgenic mice lines each with platelets expressing a different level of hPF4, ranging from 0.5 – 6 X's human platelet levels and all expressing FcRγIIA, were given KKO. The different lines developed thrombocytopenia proportional in severity and duration to hPF4 expression. A standard subcutaneous (sq) heparininzing dose (20 U/kg, sq daily) prolonged the duration of severe thrombocytopenia in high hPF4 expressing mice. We reasoned that altering the ratio of PF4 to GAG in either direction would alter antigenicity and could block the development of thrombocytopenia. In accordance with this concept, both high concentrations of anionic heparin (100 U/kg, sq daily) and cationic protamine sulfate (2 mg/kg, sq daily) decreased KKO binding in vitro and prevented KKO-induced thrombocytopenia in vivo as a demonstration of successful therapeutic intervention. These studies affirm a central role of surface GAG:PF4 complexes in the development of HIT, suggest ways to identify patients at high risk to develop HIT even prior to heparin exposure, and offers a new and rationale therapeutic paradigm based on disrupting surface antigen formation.

Structural Basis of Citrate-dependent and Heparan Sulfate-mediated Cell Surface Retention of Cobra Cardiotoxin A3

Anionic citrate is a major component of venom, but the role of venom citrate in toxicity other than its inhibitory effect on the cation-dependent action of venom toxins is poorly understood. By immobilizing Chinese hamster ovary cells in microcapillary tubes and heparin on sensor chips, we demonstrated that heparan sulfate-mediated cell retention of the major cardiotoxin (CTX) from the Taiwan cobra, CTX A3, near membrane surfaces is citrate-dependent. X-ray determination of a CTX A3-heparin hexasaccharide complex structure at 2.4 A resolution revealed a molecular mechanism for toxin retention in which heparin-induced conformational changes of CTX A3 lead to citrate-mediated dimerization. A citrate ion bound to Lys-23 and Lys-31 near the tip of loop II stabilizes hydrophobic contact of the CTX A3 homodimer at the functionally important loop I and II regions. Additionally, the heparin hexasaccharide interacts with five CTX A3 molecules in the crystal structure, providing another mechanism whereby the toxin establishes a complex network of interactions that result in a strong interaction with cell surfaces presenting heparan sulfate. Our results suggest a novel role for venom citrate in biological activity and reveal a structural model that explains cell retention of cobra CTX A3 through heparan sulfate-CTX interactions.

Effect of heparin and antivenom on skeletal muscle damage produced by Bothrops jararacussu venom.

We examined the effect of treatment with heparin and polyvalent antivenom on mice muscle Extensor digitorum longus (EDL) regeneration, after damage induced by injection of Bothrops jararacussu crude venom over the muscle of the right posterior limb. The mice were separated into groups and each group received treatment, by intravenous route with either high molecular weight heparin (H), low molecular weight heparin (LMWH), polyvalent antivenom (PAV) or with the combination of PAV plus H or PAV plus LMWH at 15 minutes and 4 hours after the injection of the venom. Myotoxicity was measured by the increase in plasma creatine kinase (CK) activity at two hours after the injection of the venom. The histological changes in EDL at 1, 3, 7 and 21 days after the injection of the venom were analyzed by light microscopy. In each group the normal and regenerated muscle fibers were quantified using Scion Image computer program. We also evaluated in vitro, the influence of these substances in the proteolytic and phospholipase activities of the venom. Heparins decreased the proteolytic activity of the venom but did not affect its phospholipase activity. However the PAV antagonized both activities. PAV and its combinations showed antimyotoxic activity, according to the magnitude of CK plasma levels. At 21 days the regeneration was observed in all animals, also in those that received only the venom. All treatments, except LMWH, promote a significant increase in the number of muscle fibers.

Structures of heparin-derived disaccharide bound to cobra cardiotoxins: context-dependent conformational change of heparin upon binding to the rigid core of the three-fingered toxin.

Glycosaminoglycans (GAGs) have been suggested to be a potential target for cobra cardiotoxin (CTX) with high affinity and specificity via a cationic belt at the concave surface of the polypeptide. The interaction of GAGs, such as high-molecular weight heparin, with CTXs not only can induce aggregation of CTX molecules but also can enhance their penetration into membranes. The binding of short chain heparin, such as a heparin-derived disaccharide [DeltaUA2S(1-->4)-alpha-D-GlcNS6S], to CTX A3 from Taiwan cobra (Naja atra), however, will not induce aggregation and was, therefore, investigated by high-resolution (1)H NMR. A novel heparin binding site on the convex side of the CTX, near the rigid disulfide bond-tightened core region of Cys38, was identified due to the observation of intermolecular NOEs between the protein and carbohydrate. The derived carbohydrate conformation using complete relaxation and conformational exchange matrix analysis (CORCEMA) of NOEs indicated that the glycosidic linkage conformation and the ring conformation of the unsaturated uronic acid in the bound state depended significantly on the charge context of CTX molecules near the binding site. Specifically, comparative binding studies of several heparin disaccharide homologues with two CTX homologues (CTX Tgamma from Naja nigricollis and CTX A3) indicated that the electrostatic interaction of N-sulfate of glucosamine with NH(3)(+)zeta of Lys12 and of the 2-O-sulfate of the unsaturated uronic acid with NH(3)(+)zeta of Lys5 played an important role. These results also suggest a model on how the CTX-heparin interaction may regulate heparin-induced aggregation of the toxin via the second heparin binding site.

Heparina de alto peso molecular. Uma alternativa nas operações com circulação extracorpórea: estudo experimental

Introduction: In surgical procedures, with cardiopulmonary bypass, hemorrhagic syndromes during and after pump constitute a major concern and a great number of cases they are heparin-related, a substance still without substitute. Most authors point out the anticoagulant action of heparin as the main problem bleeding situations and research is developing on antifibrinolytic or platelet-like- drugs to try to substitute for usual heparin. Experience with low-molecular weight heparin, without anticoagulant properties, cardiopulmonary bypass, was disastrous. High dosage was accompanied by high tube drainage suggesting that postoperative bleeding does not happen just because of the anticoagulant effect of heparin. Material and Methods: Believing the vascular component of hemostasis and that low-molecular weight heparin non-neutralized by protamin is responsible for the paralysis of small vessels during and after cardiopulmonary bypass surgery, we isolated a high-molecular weight heparin ( modal weight of 25.000 Daltons) to be tested in vitro and in vivo. Results: Its specific anticoagulant activity, by mass, was superior to usual heparin ( modal weight of 15.000 Daltons) in vitro (273 ui/mg against 181 ui/mg, respectively) as in vivo, dogs, utilizing cardiopulmonary bypass and measuring its activity by activated clotting time, APTT and heparin blood levels. In the experimental laboratory the half-life of usual heparin was of 60 minutes, while for high-molecular weight heparin was above 90 minutes. Conclusion: We believe that this unprecedented experience will lead to its future use anima nobile to further test its neutralization by protamin as well as to confirm the decreased prevalence of bleeding phenomena with its use.

Coimmobilized native macromolecular heparin proteoglycans strongly inhibit platelet-collagen interactions in flowing blood.

We coimmobilized mast cell-derived heparin proteoglycans (HEP-PGs) of very high molecular weight (750 kDa) or unfractionated heparin (UFH) on coverslips together with collagen without altering the amount of immobilized collagen. Subsequently, platelet-collagen interactions were studied under both flowing and static conditions in D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone-anticoagulated blood and platelet-rich plasma (PRP), respectively. At a high shear rate (1600 1/s), the mean platelet deposition (PD) on collagen monomers was 7.5+/-6.1x10(6)/cm(2) (n=5). When the monomers were coimmobilized with UFH, PD was inhibited by 73% (2.0+/-1.2x10(6)/cm(2)), whereas HEP-PG completely blocked it (0. 42+/-0.38x10(6)/cm(2); P<0.05). Also, when collagen fibrils were used for coating, HEP-PG significantly inhibited PD. At a low shear rate (200 1/s) and under static conditions in PRP, the inhibitory effect of HEP-PG on PD was less marked. Inhibition of glycoprotein IIb/IIIa did not affect PD on coimmobilized HEP-PG in contrast to coimmobilized UFH or collagen alone. As a sign of inactivation, platelets adhering to the HEP-PG surface released considerably less beta-thromboglobulin than did those adhering to pure collagen. In summary, immobilized HEP-PG strongly inhibited PD on collagen by attenuating adhesion-induced platelet activation. The stronger effect on collagen monomers suggests the inhibition of glycoprotein Ia/IIa-mediated activation.

Heparin proteoglycans released from rat serosal mast cells inhibit proliferation of rat aortic smooth muscle cells in culture.

-Mast cells are present in the human arterial intima. To study whether mast-cell degranulation influences the rate of proliferation of smooth muscle cells, we cocultured sensitized (IgE-bearing) rat serosal mast cells and rat aortic smooth muscle cells (SMCs). When sensitized mast cells were stimulated to degranulate with antigen, the rate of proliferation of the cocultured SMCs decreased sharply. This inhibitory effect was found to be due mainly to the very high molecular weight (Mr) heparin proteoglycans (average Mr 750 000) released from the stimulated mast cells. When the heparin proteoglycans were purified from mast-cell granule remnants and added to the SMC culture, they were found to block the cell cycle at the G0-->S transition and the exit from the G2/M phase, their inhibitory effect resembling that of commercial heparin. However, in contrast to the reported dependence of the inhibitory effect of commercial heparin on the release of transforming growth factor-beta from serum, the inhibitory effect of the mast cell-derived heparin proteoglycans in the presence of serum was not transforming growth factor-beta dependent. Moreover, the effect of the mast cell-derived heparin proteoglycans was more efficient than that of commercial heparins of high (average Mr 15 000) and low (average Mr 5000) molecular weight. We also purified heparin glycosaminoglycans (average Mr 75 000) from the mast cell-derived heparin proteoglycans and found that they also inhibited SMC growth efficiently, although less strongly than their parent heparin proteoglycans. These results reveal, for the first time, that mast cells are able to regulate SMC growth. Thus, activated mast cells, by releasing heparin proteoglycans, possibly participate in the regulation of SMC growth in the human arterial intima, the site of atherogenesis.

A fraction of high molecular weight heparin has high affinity for vitronectin

A fraction of high molecular weight heparin has high affinity for vitronectin

Influence of Molecular Weight, Protein Core and Charge of Native Heparin Fractions on Pulmonary Artery Smooth Muscle Cell Proliferation

Heparin macromolecules have been shown to inhibit cultured pulmonary artery smooth muscle cell proliferation in vitro and prevent hypoxic vascular remodeling in vivo. In an attempt to understand the structural determinants of heparin's antiproliferative properties, we have fractionated an antiproliferative preparation of commercial heparin into low and high molecular weight fractions. Then the high molecular weight heparin fraction was further fractionated on a DEAE-cellulose column by charge density eluting with 0 - 1 M NaCl linear gradient. The heparin protein peptides were both removed and isolated. These heparin fractions were assayed for antiproliferative effects on cultured bovine pulmonary artery smooth muscle cells. No appreciable differences were found among high and low molecular weight heparin fractions The core peptides showed no antiproliferative activity. However, higher charge density fraction was less antiproliferative.

The Mechanism by Which Heparin Promotes the Inhibition of Coagulation Factor XIa by Protease Nexin-2

Previous kinetic studies have shown that protease nexin-2 is a potent, reversible, and competitive inhibitor of factor XIa. Here we show that high molecular weight heparin potentiates the ability of protease nexin-2 to inhibit factor XIa with a parabolic concentration dependence, predominantly because of an increase of the association rate constant with little perturbation of the dissociation rate constant. No effect on factor XIa inhibition by protease nexin-2 was observed with heparin preparations of 6-22 saccharide units (0.1 nM-10 microM), whereas heparin preparations with 32-64 saccharide units potentiated factor XIa inhibition by protease nexin-2 in a size- and concentration-dependent manner. We propose a model wherein heparin exerts this effect by providing a template for the assembly of factor XIa-protease nexin-2 complexes, and only heparin polymers consisting of greater than 32 saccharide units (Mr approximately 10,000) are sufficiently long to provide a template to which factor XIa and protease nexin-2 molecules can bind simultaneously. Heparin-mediated enhancement of factor XIa inhibition by protease nexin-2 was partially abrogated by high molecular weight kininogen, suggesting that high molecular weight kininogen may play a role in regulating factor XIa activity.

Long-term administration of heparin and heparin fractions and osteoporosis in experimental animals

To test whether heparin-induced osteoporosis is influenced by the molecular weight of heparin, 24 male rabbits received single daily subcutaneous injections of either physiological saline (controls, n = 5), low molecular weight heparin (LMWH, n = 7), conventional heparin (UFH, n = 7) or high molecular weight heparin (HMWH, n = 6). Heparin was administered in supratherapeutic daily dosages for 120 days (750 anti-FXa units/kg for 90 days and 1500 anti-FXa units/kg for another 30 days). Studied variables were: serial analysis of serum calcium, albumin, phosphate and alkaline phosphatase, measurement of the cortical thickness of the femur (radiographically), tibial and trabecular bone density (both by cross-sectional analysis) and femoral fragility. Observed changes in blood biochemistry associated with bone metabolism were not correlated to any of the treatments. Compared with the controls, a reduction in cortical and trabecular bone density was seen with UFH (P < 0.05) and HMWH (P < 0.01) but not with LMWH. Femoral fragility was also significantly increased (P < 0.002) by HMWH. In conclusion, LMWH did not cause toxic skeletal effects as opposed to HMWH which clearly did, and UFH which induced some osteoporotic changes.

Effects of low-molecular weight and high-molecular weight heparin on the intrinsic and extrinsic endogenous fibrinolytic system

The aim of the study was to elucidate the mechanisms, how heparin enhances thrombolytic therapy. In a randomized, placebo controlled, open — labelled, three armed, double cross — over study in healthy volunteers, we investigated the effects of 2500 IU LMW and 7500 IU HMW heparin on the fibrinolytic system within 120 min after application. Heparin doses were selected as to achieve equal anti-Xa activities. During observation time heparin-cofactor II and antithrombin III, and plasminogen and α 2-antiplasmin levels remained constant. As demonstrated by the lapses of the prekallikrein and F XII activities by the α-F XIIa- and β-F XIIa inhibition capacities any direct short-term effect of heparin on the intrinsic endogenous fibrinolytic system (contact-phase) was ruled out. By virtue of the fate of t-PA, PAI-1, Protein C and Protein S levels, a significant direct effect on the extrinsic endogenous fibrinolytic system was also excluded. Thus, our results are not in accordance with a direct, short-term (120 min) pro-fibrinolytic potential of heparin. Therefore an indirect pro-fibrinolytic effect by suppression of the circulating pro-coagulant activities seems the most likely mechanism, by which heparin achieves a potentative effect in thrombolytic therapy.