Lung Heparin Research Articles

Low Dose Protamine for Heparin Reversal in 40 Consecutive Cardiopulmonary Bypass Patients

(J. Extra-Corpor. Technol. 19[3] p. 348-351 Fall 1987, 13 ref.) Reversal of systemic heparinization in cardiac surgery has been a subject of controversy for some time. For reversal, protamine sulfate (PS) is given in as high a dose as 2:1. We heparinize our patients with 2.0 mg./kg. of beef lung heparin and reverse this with 70% of the total heparin dose. The mean preoperative activated clotting time (ACT) was 148 seconds. The mean ACT on cardiopulmonary bypass (CPB) was 446 seconds. After successfully weaning the patients from CPB at a bladder temperature of 37°C or greater, the 70% dose of PS was given resulting in a mean ACT of 134 seconds post bypass. The mean post-operative blood loss was 695cc (230cc-3120cc) with only 32% (13 patients) requiring platelet administration and 22% (9 patients) requiring fresh frozen plasma (FFP). No patient required re-operation for hemorrhage. There were 33 coronary artery bypass grafts, all of which were on aspirin and persantine, four aortic valve replacements and three atrial septal defects. These data strongly suggest that heparin reversal can be obtained with low dose PS when these patients are weaned from CPB at 37°C bladder temperature.

Isolation and characterization of four heparin-binding cyanogen bromide peptides of human plasma apolipoprotein B

Apolipoprotein B-100 (apoB-100) is the major protein constituent of human plasma low-density lipoproteins (LDL). On the basis of its amino acid sequence [Chen, S.-H., Yang, C.-Y., Chen, P.-F., Setzer, D., Tanimura, M., Li, W.-H., Gotto, A. M., Jr., & Chan, L. (1986) J. Biol. Chem. 261, 12918-12921], apo B-100 is one of the largest monomeric proteins known with a calculated molecular weight of 512937. Heparin binds to the LDL surface by interacting with positively charged amino acid residues of apoB-100, forming soluble complexes in the absence of divalent metals and insoluble complexes in their presence. The purpose of this study was to isolate and characterize the heparin-binding domain(s) of apoB-100. Human plasma LDL were fragmented with cyanogen bromide (CNBr). After delipidation and reduction-carboxymethylation, the CNBr peptides were fractionated by sequential chromatography on DEAE-Sephacel, Mono S, and high reactive heparin (HRH) AffiGel-10; HRH was purified by chromatography of crude bovine lung heparin on LDL AffiGel-10. Heparin-binding peptides were further purified by reverse-phase high-performance liquid chromatography. Heparin-binding activity was monitored by a dot-blot assay with 125I-HRH. The amino-terminal sequences of four CNBr heparin-binding peptides (CNBr-I-IV) were determined. CNBr-I-IV correspond to residues 2016-2151, 3109-3240, 3308-3394, and 3570-3719, respectively, of the amino acid sequence of apoB-100. Each CNBr peptide contains a domain(s) of basic amino acid residues which we suggest accounts for their heparin-binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Physical-chemical interaction of heparin and human plasma low-density lipoproteins.

This study characterizes the physical-chemical interactions of heparin with human plasma low-density lipoproteins (LDL). A high reactive heparin (HRH) specific for the surface determinants of LDL was isolated by chromatography of commercial bovine lung heparin on LDL immobilized to AffiGel-10. HRH was derivatized with fluoresceinamine and repurified by affinity chromatography, and its interaction with LDL in solution was monitored by steady-state fluorescence polarization. Binding of LDL to fluoresceinamine-labeled HRH (FL . HRH) was saturable, reversible, and specific; HRH stoichiometrically displaced FL . HRH from the soluble complex, and acetylation of lysine residues on LDL blocked heparin binding. Titration of FL.HRH with excess LDL yielded soluble complexes with two LDL molecules per heparin chain (Mr 13,000) characterized by an apparent Kd of 1 microM. Titration of LDL with excess HRH resulted in two classes of heparin binding with two and five heparin molecules bound per LDL and apparent Kd values of 1 and 10 microM, respectively. At physiological pH and ionic strength, the soluble HRH-LDL complexes were maximally precipitated with 20-50 mM Ca2+. Insoluble complexes contained 2-10 HRH molecules per LDL with the final product stoichiometry dependent on the ratio of the reactants. The affinity of HRH for LDL in the insoluble complexes was estimated between 1 and 10 microM. Insoluble LDL-heparin complexes were readily dissociated with 1.0 M NaCl, and their formation was prevented by acetylation of the lysine residues on LDL.

Binding of magnesium and lanthanum ions to heparin in the presence of sodium ions. A relationship between 13C chemical shift displacements and counterion condensation theory

Interactions between beef lung heparin and Mg2+ and La3+ counterions, in the presence of sodium ions, have been investigated by 13C-nmr spectroscopy. The chemical shift displacements observed for the carbons of the main disaccharide unit of heparin, when analysed on the basis of counterion condensation theory, indicate that the association between the heparin polyanion and Mg2+ is a delocalized process. This is analogous to the Ca2+/Na+/heparin system, despite the fact that the binding affinity of heparin for Ca2+ is stronger than that for Mg2+. By contrast, the interaction with La3+ is in excess of the theoretical predicted, which suggests that there is at least a partial site-binding of this counterion. In Ca2+/M+/heparin systems in which M+ = Na+ or K+, the selective binding preference of heparin for the univalent cations may be related to the order of their crystallographic ionic radii. A critical assessment is offered of the validity of this nmr spectroscopic approach to the study of counterion binding to heparin.

Conformational equilibria of α-L-iduronate residues in disaccharides derived from heparin

The disaccharides IdoA(2SO3)-anManOH(6SO3) and IdoA-anManOH (where IdoA represents alpha-L-iduronate, anManOH represents 2,5-anhydro-D-mannitol and SO3 represents sulphate ester) were prepared from bovine lung heparin using HNO2 depolymerization, borohydride reduction and desulphation, and were examined by 400 MHz 1H-n.m.r. spectroscopy. Three-bond proton-proton coupling constants around the IdoA ring were determined under a range of experimental conditions. For unsulphated IdoA all four proton-proton coupling constants varied markedly as a function of temperature, pH and solvent, providing clear evidence for a rapid conformational equilibrium. These data were analysed in terms of the three most energetically stable IdoA conformers: 1C4, 4C1, and 2S0. Predicted coupling constants for these conformers were determined using a modified Karplus-type relationship. For unsulphated IdoA in dimethyl sulphoxide the equilibrium was provoked strongly in favour of a slightly distorted 4C1 'chair' IdoA conformer for which coupling constants have not previously been reported. For sulphated IdoA in aqueous conditions and at low pH the equilibrium is strongly in favour of the alternative 1C4 chair conformer. Under many conditions, however, significant contributions from all three conformers occur for the non-reducing terminal IdoA in these disaccharides.

Heparin-associated thrombocytopenia and thrombosis: a serious clinical problem and potential solution.

Heparin-associated thrombocytopenia and thrombosis (HATT) is an unusual but serious complication of heparin therapy. Twenty-five patients (13 men and 12 women) had thrombocytopenia and arterial or venous thrombosis 1 to 10 days (mean, 6.3 days) after the start of heparin administration. The vessels in the affected extremity had been entered for catheterization, arteriography, or passage of a balloon counterpulsation device in 19 of the 25 patients. In vitro platelet aggregation with heparin was seen in all patients. Additional studies were performed to see whether other lots or sources of heparin also produced in vitro aggregation. Four separate lots of beef lung heparin, commercial heparin from porcine intestinal mucosa, and two types of low molecular weight heparin were all highly stimulatory in this system. However, Org 10172, a heparinoid, did not induce aggregation in any of 13 patient plasmas tested. Inhibition of platelet aggregation by aspirin was also examined. Aspirin abolished in vitro aggregation in 9 of 16 cases and decreased the degree of aggregation from 85% to 55% (p = 0.02) in the remaining seven cases. We conclude that in patients with HATT platelet aggregation is equally induced by beef lung, porcine intestinal, and some forms of low molecular weight heparin. Org 10172 does not stimulate platelet aggregation in plasma from these patients in vitro. Finally, there may be a role for aspirin in treating patients with HATT.

Heparin-associated thrombocytopenia and thrombosis: A serious clinical problem and potential solution

Heparin-associated thrombocytopenia and thrombosis (HATT) is an unusual but serious complication of heparin therapy. Twenty-five patients (13 men and 12 women) had thrombocytopenia and arterial or venous thrombosis 1 to 10 days (mean, 6.3 days) after the start of heparin administration. The vessels in the affected extremity had been entered for catheterization, arteriography, or passage of a balloon conterpulsation device in 19 of the 25 patients. In vitro platelet aggregation with heparin was seen in all patients. Additional studies were performed to see whether other lots or sources of heparin also produced in vitro aggregation. Four separate lots of beef lung heparin, commercial heparin from porcine intestinal mucosa, and two types of low molecular weight heparin were all highly stimulatory in this system. However, Org 10172, a heparinoid, did not induce aggregation in any of 13 patient plasmas tested. Inhibition of platelet aggregation by aspirin was also examined. Aspirin abolished in vitro aggregation in 9 of 16 cases and decreased the degree of aggregation from 85% to 55% (p = 0.02) in the remaining seven cases. We conclude that in patients with HATT platelet aggregation is equally induced by beef lung, porcine intestinal, and some forms of low molecular weight heparin. Org 10172 does not stimulate platelet aggregation in plasma from these patients in vitro. Finally, there may be a role for aspirin in treating patients with HATT.

N.M.R. studies of oligosaccharides obtained by degradation of bovine lung heparin with nitrous acid

The oligosaccharides IdoA(2SO 3)-[GlcNSO 3(6SO 3)-IdoA(2SO 3)] n -anManOH(6SO 3) ( n = 1–4) have been prepared from bovine lung heparin by treatment with nitrous acid followed by borohydride reduction and then fractionation by gel filtration. The major resonances of the 1H- (400 MHz) and 13C-n.m.r. (100 MHz) spectra for each oligosaccharide fraction have been assigned using two-dimensional COSY, NOESY, and 13C- 1H correlation experiments. The 1H resonances, representing the three distinct molecular environments of IdoA(2SO) 3), namely, at the non-reducing terminus, adjacent to the reducing terminal anManOH(6SO 3), and in the [GlcNSO 3(6SO 3)-IdoA(2SO 3)] repeat sequence, have been assigned.

Distribution of glucuronic and iduronic acid units in heparin chains.

The distribution of glucuronic and iduronic acid within the chains of anticoagulantly active and inactive beef lung heparin was investigated. A fraction with an average molecular weight of 19,500 was isolated from the heterodisperse mixture and then separated into active and inactive components by affinity chromatography. Each sample was linked through its reducing terminus to tyramine, reduced with sodium borotritide, and bound covalently to Sepharose via an azo bridge. The bound reduced heparin was treated with a limited amount of HNO2 and the degraded fragments were removed. The sections of the chain contiguous with the original reducing terminus were then detached from the insoluble matrix by reaction with sodium dithionite. The recovered polysaccharide was fractionated according to size on Sephadex G-200 and the amount of each uronic acid in the individual fractions was determined. Inactive heparin showed a constant percentage of glucuronic acid in all fragments, i.e. about 8.9% of the total uronic acid. With active heparin the percentage of glucuronic acid increased with the distance from the reducing terminus of the polysaccharide chain, ranging from 9.5 to 20% of the uronic acids. These results suggest that the biosynthesis of active heparin involves unique reactions or specific processing of the macromolecule.

N.M.R. studies of the disulphated disaccharide obtained by degradation of bovine lung heparin with nitrous acid

The disulphated disaccharide IdoA(2SO 3)—anManOH(6SO 3) was prepared from bovine lung heparin by treatment with nitrous acid followed by borohydride reduction. The 1H- (400 MHz) and 13C-n.m.r. (100 MHz) spectra of this disaccharide derivative have been assigned completely using homonuclear spin-decoupling experiments, 13C 1H correlations, and a COSY-45 two-dimensional homonuclear correlation experiment. The 3J H,H values show that the IdoA(2SO 3) residue exists in a single conformation throughout the temperature range 20–90°.

Cardiovascular effects of protamine sulfate are dependent on the presence and type of circulating heparin

Man's response to clinical doses of protamine is highly variable. We investigated the influence of circulating heparin in nine swine (mean age 6 weeks, weight 10 kg). Through a sternotomy we implanted an electromagnetic flow probe around the pulmonary artery for cardiac output determination and catheters into the ascending aorta, pulmonary artery, right atrium, and left atrium for pressure monitoring. Each animal was allowed to recover and was studied awake on 3 consecutive days. Protamine, 3 mg/kg, beef lung heparin, 300 U/kg, and pork mucosal heparin, 300 U/kg, followed by protamine, were given in rotation by intravenous bolus. Protamine alone had no effect. Beef lung heparin followed by protamine induced a marked increase in pulmonary artery pressure (mean 38 +/- 3 to 51 +/- 5 mm Hg in 3 minutes). Pulmonary vascular resistance doubled (mean 0.12 +/- 0.01 to 0.23 +/- 0.04 R within 4 minutes), returning to normal within 15 minutes. Cardiac index and aortic pressure changed minimally. Pork mucosal heparin followed by protamine induced a similar but greater increase in mean pulmonary arterial pressure; however, cardiac index fell significantly (p less than 0.05, 207 +/- 16 to 117 +/- 16 ml/kg/min-1 at 1 minute) despite a regular rhythm and adequate left atrial filling pressure. Thus cardiac contractility was depressed. Systemic hypotension occurred in three of nine pigs. Both mean pulmonary vascular resistance and systemic vascular resistance increased (0.12 +/- 0.01 to 0.67 +/- 0.25 R and 0.40 +/- 0.04 to 1.09 +/- 0.25 R, respectively), significantly (p less than 0.05) more with pork than beef heparin. These data demonstrate that cardiovascular response to protamine neutralization varies significantly in regard to the type of heparin used. Furthermore, circulating heparin is required to produce those effects previously attributed to protamine alone.

Heterogeneity of heparin lots associated with thrombocytopenia and thromboembolism

Thromboembolic complications may develop in patients with heparin-associated thrombocytopenia, presumably due to the formation of platelet aggregates. An unexpectedly high incidence of pulmonary embolism following coronary artery bypass surgery occurred during a brief period of time at a single institution, and all of these cases were found to be associated with thrombocytopenia. All patients tested during thrombocytopenia (five of five) had an increase in platelet-associated antibody. Serum samples from all five patients tested caused normal platelets to aggregate in vitro in the presence of one specific lot of beef lung heparin, which was in use in the operating room at the time; none of six other lots of beef lung heparin mediated in vitro platelet aggregation. Heparinase digestion of the heparin abolished the aggregating activity. It is concluded that thrombocytopenia and platelet activation caused by heparin may vary greatly even among different lots of heparin prepared from the same source.

Toxicity of heparin in isolated rat hepatocytes

The effect of heparin on isolated rat hepatocytes in monolayer culture was assessed to investigate the observed increase in serum aminotransferase activity in patients treated with heparin for thromboembolic disorders. Cells were treated with porcine intestinal mucosal heparin or beef lung heparin in concentrations ranging from 0.01 to 100 units/ml. Toxicity was evaluated based on cell damage or death measured by LDH release into the culture media as a fraction of total system LDH (LDH index). Toxicity appeared at concentrations between 1 and 10 units/ml (P < 0.05). The uptake and binding of heparin by the hepatocyte were evaluated by addition of tritium-labeled heparin to the cultures. Sucrose gradient centrifugation with isolation of the liver plasma membranes (LPM) showed little membrane binding of heparin. The majority of intracellular heparin was located in the cytosol fraction. Heparin gains access to hepatocytes and causes a dose-related toxic effect resulting in cell damage and death. This investigation indicates that the increased serum aminotransferase concentrations seen with heparin treatment may be due to a direct hepatotoxic effect of heparin.

Heparin-Associated Thrombocytopenia

Two hundred eleven consecutive patients treated for acute thromboembolic disease were evaluated prospectively for the incidence of thrombocytopenia while receiving heparin treatment. One hundred patients received beef lung heparin and 111 patients received porcine intestinal mucosal heparin. All patients received a minimum of 4 consecutive days of continuous intravenous heparin, and platelet counts were determined prior to, at least twice weekly, and at the cessation of heparin therapy. Heparin-associated thrombocytopenia was defined as a decline from a normal platelet count (100,000-400,000/mm3) to less than 100,000/mm3 with a return to above 100,000/mm3 after the discontinuation of heparin. Heparin-associated thrombocytopenia developed in 11 patients (5.2% incidence). Ten of the thrombocytopenic patients had received beef lung heparin and one received porcine mucosal heparin. Chi-square analysis of these data was significant (p = 0.007). Plasma from seven of nine thrombocytopenic patients demonstrated a plasma factor compatible with a heparin-sensitive antiplatelet antibody. Heparin-associated thrombocytopenia appeared on days 2 to 10 of therapy. Cessation of heparin resulted in remission of thrombocytopenia within 4 days in all patients. Serial quantitative platelet count determinations are indicated in all patients receiving therapeutic heparinization for the early recognition and resolution of heparin-associated thrombocytopenia.

A Randomized Study of Beef Lung and Pork Mucosal Heparin in Cardiac Surgery

Beef lung heparin had substantially greater anticoagulant activity than pork mucosal heparin during a preoperative heparin tolerance test and also during cardiopulmonary bypass (CFB) in 100 randomized patients. Supplemental heparin was needed during CPB in many more of the patients receiving pork mucosal heparin. Heparin rebound was detected in 16 patients; this low incidence may result from a relatively high protamine: heparin ratio. There was notably less postoperative bleeding in those who received beef lung heparin. Platelet counts were not altered by either type of heparin. It is surprising that the preoperative tolerance curve only predicted heparin sensitivity during CPB in one-half of the patients. Blood activated coagulation time levels were increased markedly by hemodilution and further raised by hypothermia. These data demonstrate that beef lung heparin is better than pork mucosal heparin for CPB.

Fibrinogen binding on human platelets. Influence of different heparins and of pentosane polysulfate

Unfractionated heparin increased the binding of fibrinogen on ADP-treated platelets. The results varied according to both, the platelets of the donor and the kind of heparin preparation used. Beef lung heparin was more active than porcine intestinal mucosa heparin (p < 0.02). A fraction of low molecular weight low sulfated heparin, did not significantly increase the binding of fibrinogen, except in one case for which the binding of fibrinogen to platelets was largely increased by standard heparin. On the contrary, pentosane polysulfate, a sulfated polysaccharide (of low molecular weight) significantly increased the binding of fibrinogen (p < 0.01).

Simple electrophoresis of glycosaminoglycuronans and the distinction of lung heparins from mucosal heparins

Simple electrophoresis of glycosaminoglycuronans and the distinction of lung heparins from mucosal heparins

The Effect of Bovine Lung Heparin on Normal Human Platelets as Measured by Electronic Particle Size Analysis

The response of normal human platelets to treatment with bovine lung heparin was evaluated using electronic particle size analysis and aggregometry. Samples for electronic particle size analysis were obtained both before and after the addition of heparin to platelet-rich plasma (PRP). The number of single platelets decreased significantly after the addition of heparin to PRP obtained from 22 individuals. This decrease averaged 18% in 19 samples which did not show a significant increase in transmittance and was at least 70% in 3 samples which showed a significant increase in transmittance. The size distribution of single platelets was not significantly altered. In addition, electron microscopic examination revealed that platelets treated with heparin are activated.

Potentiation of endoperoxide analog-induced platelet aggregation by heparin

The mechanism of heparin-induced platelet aggregation potentiation is much debated. We examined the effects of heparin on platelet aggregation induced by cyclic endoperoxide analog U46,619. Heparin enhanced platelet aggregation in a dose-related fashion. Potentiation of platelet aggregation was also found to be related to the incubation time of heparin and platelet rich plasma. Porcine intestinal heparin and bovine lung heparin exhibited similar patterns. To test the hypothesis of increased platelet thromboxane A 2 production under the influence of heparin, we measured U46,619-induced thromboxane A 2 generation. Heparin was found not to affect platelet thromboxane A 2 generation over a wide range of concentrations. The platelet aggregation potentiating actions of heparin are thus unrelated to thromboxane A 2 pathway.

Heparin enhances fibrinolysis in B16 mouse melanoma cells

The fibrinolytic activity of two tumorigenic B16 mouse melanoma lines was stimulated by exogenous hog mucosal or beef lung heparin. In contrast, the activity of two normal fibroblast lines was unaffected. The degradation of 125l-fibrin was increased up to 3.6-fold by the addition of heparin. Chondroitin-4-sulfate or dextran sulfate did not change the fibrinolytic activity of three of the cell lines, but, at concentrations where enhancement by heparin was much reduced, the activity of one of the B16 melanoma lines was somewhat elevated. Antithrombin III did not alter the plasminogen activator activity of the B16 cell lines, but, in the presence of exogenous heparin, the enhancement of fibrinolysis was greatly reduced. The polymers were not cytotoxic during the assay period, and, had little affect on the plating efficiencies of the lines.