Novel approach for optimizing the capacity and efficacy of a protamine filter for clinical extracorporeal heparin removal

The authors previously reported the development of a blood filter device containing immobilized protamine (termed "protamine filter") that could be used at the conclusion of an extracorporeal blood procedure to prevent heparin and protamine induced complications. In vitro and in vivo experiments have fully demonstrated the feasibility and utility of the approach. The bottleneck limitations of this approach, however, lie in the lack of efficacy and capacity of the filter device. In this article, the authors describe a method to improve the efficacy in heparin adsorption, by incorporating a poly(ethylene glycol) spacer arm between the immobilized protamine and the fiber surface to enhance its freedom to dynamic motion. The authors also describe a method to increase the capacity of the filter, by using a poly-L-lysine based amplification method to augment protamine loading on the fiber, and to create multiple layers of immobilized protamine for heparin adsorption. Results show that with a poly(ethylene glycol) spacer arm of 3,400 Da, heparin adsorption on the protamine-poly(ethylene glycol) fibers was increased dramatically from a value of 9.1 mg heparin per gram of fibers in the control (i.e., without the poly[ethylene glycol] spacer) to 60 mg heparin/g fiber. The use of the amplification method with 110 kDa poly-L-lysine also yielded a threefold increase in protamine loading, and, consequently, an approximately fourfold enhancement in heparin adsorption (from 9.1 to 38.0 mg heparin/g fiber). A combination of these two methods would yield an optimized protamine filter that could meet all types of clinical needs in heparin removal. As assessed from the in vivo theoretical model reported previously for the protamine filter, a 95% heparin removal under cardiopulmonary bypass conditions could be achieved with a single optimized protamine filter with a size smaller than a hemodialyzer cartridge.     

Quality assessment of intraoperative blood salvage and autotransfusion

Intraoperative blood salvage and autotransfusion are commonly used to minimize exposure to banked blood. Although this technique has been used widely for years, data vary regarding the quality of autotransfused blood. Salvaged blood may contain plasma, residual heparin, and free hemoglobin released from damaged cells. All of these factors may contribute to the adverse sequelae sometimes seen with autotransfusion. For these reasons, we have monitored autotransfused blood to assess its quality. Intraoperative blood salvage was used during most cardiac procedures and at the discretion of the surgeon in other specialties. Blood was collected through a double lumen catheter that was anticoagulated with heparin, filtered, centrifuged, and washed with saline. A sample of the blood was removed for analysis, which included hematocrit, heparin assay, fibrinogen, and free hemoglobin levels. Over a 6-year period, 1593 patients had intraoperative blood salvage with quality assessment. The majority of patients underwent cardiac operations (941 patients, 59%), whereas 243 had orthopedic (15%) and 208 had vascular (13%) procedures. Additionally, there were 127 pediatric patients (8%) and 74 miscellaneous procedures (5%). The highest average yield of salvaged blood was during vascular procedures (1073 +/- 76 mL), whereas orthopedic cases had the lowest yield (378 +/- 19 mL) and hematocrit (39%). There was minimal residual heparin activity, even in patients requiring systemic anticoagulation (0.3 to 0.5 units/mL). Patients undergoing pediatric procedures had the lowest concentration of free hemoglobin (476 mg/L), whereas all adult patients had higher free hemoglobin levels, especially vascular operations (990 mg/L). Intraoperative salvaged blood has minimal heparin activity, even in procedures requiring systemic anticoagulation. Fibrinogen, a marker of residual plasma, was undetectable in the majority of cases. These data indicate that intraoperative blood salvage generally results in a high-quality product (good hematocrit, low heparin, minimal plasma), although there are significant differences in free hemoglobin levels depending on the operative procedure.     

Removal of heparin and protamine from plasma

A simple chromatographic technique for rapid adsorption of heparin and protamine from plasma samples is described, allowing accurate interpretation of coagulation screening tests and specific clotting factor assays. With the use of columns of ECTEOLA-cellulose, up to 300 U. of heparin could be completely adsorbed from a 1 ml. plasma sample. When citrated nonheparinized plasma was passed over the ECTEOLA-cellulose columns, the thrombin, prothrombin, and partial thromboplastin times were unaffected. Levels of fibrinogen, prothrombin, and factors V, VII, VIII, IX, and XI average within 90 per cent of control, nonchromatographed samples. When heparinized plasma samples (0.1 and 1.0 U. per milliliter) were passed over columns, heparin was completely removed and the results of the screening tests and the specific factor assays were the same as for the chromatographed nonheparinized samples. In addition, heparinized samples with decreased factor VIII activity maintained their pretreatment factor VII activities after heparin removal. Blood samples containing heparin were obtained from two patients during open-heart surgery. Following heparin adsorption on ECTEOLA-cellulose columns, factor VIII activity levels remained above 60 per cent during cardiopulmonary by-pass. The presence of protamine sulfate in plasma samples prolonged the prothrombin and partial thromboplastin times while slightly shortening the thrombin time. The protamine effect persisted after ECTEOLA-cellulose, but could be removed by a similar column of carboxymethyl-cellulose. The latter resin had no effect on screening tests or on assays of factors VIII or IX activity. The combination of the two resins was then used to remove the separate inhibitory effects from heparinized plasma samples to which protamine had been added.     

Experiences and consequences of pain in persons with post-polio syndrome

An improved protamine-sensitive electrode based on a polymeric membrane doped with the charged ion exchanger dinonylnaphthalenesulfonate (DNNS) is used for monitoring heparin concentrations in whole blood. The electrode exhibits significant nonequilibrium potentiometric response to polycationic protamine over the concentration range of 0.5-20 mg/L in undiluted whole-blood samples. The sensor can serve as a simple end point detector for the determination of heparin via potentiometric titrations with protamine. Whole-blood heparin concentrations determined by the electrode method (n > or = 157) correlate well with other protamine titration-based methods, including the commercial Hepcon HMS assay (r = 0.934) and a previously reported potentiometric heparin sensor-based method (r = 0.973). Reasonable correlation was also found with a commercial chromogenic anti-Xa heparin assay (r = 0.891) with corresponding plasma samples and appropriate correction for whole-blood hematocrit levels. Whereas a significant positive bias (0.62 kU/L; P < 0.001) is observed between the anti-Xa assay and the protamine sensor methods, insignificant bias is observed between the protamine sensor and the Hepcon HMS tests (0.08 kU/L; P = 0.02). The possibility of fully automating these titrations offers a potentially simple, inexpensive, and accurate method for monitoring heparin concentrations in whole blood.     

Delivery system for targeted thrombolysis without the risk of hemorrhage

Cardiovascular diseases that result from thrombosis of critically situated blood vessels remain the leading cause of death in industrialized countries. One primary clinical treatment is dissolution of the thrombus with thrombolytic agents, plasminogen activators (PA). Activation of plasminogen by a PA agent produces plasmin that degrades fibrin. However, plasmin also degrades other circulating clotting factors. Therefore, thrombolytic therapy, which introduces systemic generation of excess plasmin, carries the risk of hemorrhage. We propose a novel approach that could lead to targeted thrombolysis without bleeding risk. The system is comprised of a protein conjugate made of two parts: a fibrin-targeting antibody (Ab) linked with anionic heparin; and a PA derivatized with cationic species. These two parts are linked via an electrostatic interaction. Because the cationic species are relatively small, the derivatized PA would retain its thrombolytic activity, but this activity would be inhibited after binding with the Ab-heparin counterpart because of the blockage of the PA's active site by these appended macromolecules. Because protamine is a clinical heparin antagonist with a much stronger affinity for heparin than the incorporated cations, it can be used safely to dissociate the modified PA from the Ab-heparin counterpart. Therefore, this approach would permit administration of a fibrin-targeting but inactive thrombolytic, and subsequently, a triggered release of the active modified PA drug in close proximity to the fibrin deposit. These features would enhance the potency and the specificity of the thrombolytic agent and alleviate the bleeding risk by avoiding systemic generation of excess plasmin. In this report, we present preliminary results demonstrating the feasibility of the approach. A cationic octapeptide, (Arg)7-Cys, was linked to urokinase (UK) using the N-succinimidyl-3-[2-pyridylidithio]propionate activation method. This UK peptide retained a significant amount of its catalytic activity, as measured by the S-2251 chromogenic assay. However, this activity was almost completely inhibited (approximately 99%) after the addition of heparin, but was fully reversed (100%) after the addition of protamine.     

Optical detection of macromolecular heparin via selective coextraction into thin polymeric films

Thin plasticized polymer films, poly(vinyl chloride) doped with a specific ion pairing quaternary ammonium compound, tridodecylmethylammonium chloride, and a lipophilic pH indicator, 3-hydroxy-4-(4-nitrophenylazo)phenyl octadeconate, are shown to exhibit significant and analytically useful optical response toward macromolecular heparin. The response mechanism is based on favorable extraction of heparin into the bulk organic film, owing to the specific ion-pairing complexation reaction between the quaternary ammonium species and the polyanion. A simultaneous coextraction of hydrogen ions results in protonation of the pH chromophore and hence a change in the optical absorbance of the polymeric film. When used in a limited volume/fixed exposure (10 min) detection mode, film absorbances change as a function of the initial heparin concentration in the range of 0.2-3.0 units/mL (1.2-18 micrograms/mL). The practical measurement response time is controlled by heparin diffusion through the stagnant diffusion layer adjacent to the surface of the film as well as within the bulk of the polymer film and is shown to increase with the molecular weight of the heparin species tested. No optical response to heparin is observed when a strong heparin complexing agent (e.g., protamine) is present in the test solution, suggesting that the polymer film can be used to conveniently monitor heparin-protamine (or other antagonist) titrations. The theory relating to the operation of the sensing film in either the equilibrium or the kinetic mode and the selectivity of the optimized film to heparin relative to small anions are presented.     

Surface plasmon resonance sensor for heparin measurements in blood plasma

Surface plasmon resonance (SPR) was used as an affinity biosensor to determine absolute heparin concentrations in human blood plasma samples. Protamine and polyethylene imine (PEI) were evaluated as heparin affinity surfaces. Heparin adsorption onto protamine in blood plasma was specific with a lowest detection limit of 0.2 U/ml and a linear window of 0.2-2 U/ml. Although heparin adsorption onto PEI in buffer solution had indicated superior sensitivity to that on protamine, in blood plasma it was not specific for heparin and adsorbed plasma species to a steady-state equilibrium. By reducing the incubation time and diluting the plasma samples with buffer to 50%, the non-specific adsorption of plasma could be controlled and a PEI pre-treated with blood plasma could be used successfully for heparin determination. Heparin adsorption in 50% plasma was linear between 0.05 and 1 U/ml so that heparin plasma levels of 0.1-2 U/ml could be determined within a relative error of 11% and an accuracy of 0.05 U/ml.     

Yeast artificial chromosome cloning and chromosomal localization of the abundant odontogenic keratocyst protein elafin

Heparin has been shown to decrease total vascular resistance while protamine stimulates endothelium-dependent vasodilation. This study was undertaken to determine whether heparin and/or protamine could enhance endothelium-derived relaxing factor (EDRF), as determined by nitric oxide (NO) production. Porcine carotid artery endothelial cells (PAECs) were seeded on multiwell plates, grown to confluence, and exposed to heparin (1-20 U/ml) or protamine (50-200 microg/ml) for 24 hours. With the addition of the NO synthase inhibitor, N(G)-monomethyl-L-arginine (NMMA), to heparin and/or protamine, the medium samples were collected in one hour. In a parallel clinical study, plasma samples were collected from patients undergoing cardiopulmonary bypass (CPB). The NO production was measured as reflected by the formation of nitrite (NO2-) and nitrate (NO3-), the stable end-metabolites of NO. NO production by PAECs was significantly increased by heparin > or = 5 U/ml or protamine > or = 50 microg/ml in a concentration-dependent manner. The increase of NO production was prevented by the addition of NMMA. In CPB patients, plasma NO2-/NO3- concentration was significantly increased after heparin administration compared to the preoperative value, at which time the mean plasma heparin level was 4.9+/-0.5 U/ml. Following slow protamine infusion, there was no significant difference in plasma NO2-/NO3- concentration compared to preoperative value. In conclusion NO production increases following exposure of PAECs to heparin and/or protamine. In patients, NO concentration significantly increased after heparin administration by IV bolus, but not with a slow infusion of protamine after CPB.     

The role of urinary potassium in the pathogenesis and diagnosis of interstitial cystitis

PURPOSE: We determined whether intravesical potassium absorption in normal bladders correlates with increased sensory urgency, and corroborated the hypothesis that mucus is important in the regulation of epithelial permeability. We compared sensory nerve provocative ability of sodium versus potassium, and determined whether intravesical potassium sensitivity discriminates patients with interstitial cystitis from normal subjects and those with other sensory disorders of the bladder. MATERIALS AND METHODS: A total of 231 patients with interstitial cystitis and 41 normal subjects underwent intravesical challenge with 40 ml. water and then 40 ml. of 40 mEq./100 ml. potassium chloride. Subjective responses of urgency or pain stimulation were recorded on a scale of 0 to 5. In 19 normal subjects potassium absorption was measured at baseline, after injury of the bladder mucus with protamine, after heparin treatment to reverse mucus damage and then for a final time. These subjects simultaneously recorded the symptoms of sensory urgency and pain at baseline, after protamine and after heparin. Another group of normal volunteers underwent a challenge with sodium versus potassium to determine which cation was more provocative. Patients with bladder outlet obstruction secondary to benign prostatic hyperplasia (BPH), detrusor instability, and acute and chronic urinary tract infection but no current infection were also evaluated for potassium sensitivity. RESULTS: Neither normal subjects nor patients with interstitial cystitis reacted to water administered intravesically. There was marked sensitivity to intravesical potassium in 75% of patients with interstitial cystitis versus 4% of controls (p <0.01). Only 1 patient with BPH responded to potassium and none of the 5 with chronic urinary tract infection responded. All 4 patients (100%) with a current acute urinary tract infection reacted positively to the potassium challenge. Of 16 patients with detrusor instability 25% responded. Normal subjects had minimal sensitivity to potassium before (11%) and markedly increased sensitivity after (79%) protamine treatment, and these symptoms were reversed by heparin in 42%. Potassium absorption directly correlated with symptoms (0.4, 3.0 and 1.3 mEq. before and after protamine, and after heparin reversal, respectively). In regard to sodium versus potassium provocation, potassium was far more provocative for causing urgency after protamine (10 versus 90%). Neither group underwent provocation before protamine. CONCLUSIONS: Chronic diffusion of urinary potassium into the bladder interstitium may induce sensory symptoms, damage tissue and be a major toxic factor in the pathogenesis of interstitial cystitis. Intravesical potassium sensitivity is a reliable method for detecting abnormal epithelial permeability. It discriminates between patients with interstitial cystitis and normal subjects with intact epithelial function, and it is a useful diagnostic test for interstitial cystitis. Potassium sensitivity correlates with increased potassium absorption in normal subjects, and potassium is far more provocative than sodium. Potassium sensitivity is also present in acute urinary tract infection and occasionally detrusor instability but not in BPH or chronic urinary tract infections.     

Individual responses to heparinization for extracorporeal circulation

It has been proposed that wide variations in individual response to heparin lead to deficiencies in popular heparinization protocols for extracorporeal circulation (ECC). Thirty-nine patients undergoing open cardiac operations with ECC were anticoagulated with the heparinization protocol in use at St. Thomas' Hospital. The coagulation state was monitored with the blood activated recalcification time (BART) test. Wide variations in heparin dose-response and heparin activity-decay curves were observed. No patient was underheparinized, but many had markedly prolonged BART's. The total dose of heparin declined because BART monitoring allowed elimination of incremental heparin doses up to 180 minutes of ECC. Adequate reversal with protamine was achieved in all patients regardless of response to herparin. Alternative approaches for heparinization for ECC can be developed with the aid of rapid tests of the intraoperative coagulation state.     

Heparin coating reduces blood cell adhesion to arterial filters during coronary bypass: a clinical study

Deleterious effects of cardiopulmonary bypass (CPB) are partly sequelae of blood-foreign surface reactions. Coating the inner surfaces of CPB sets with heparin has been shown to decrease activation of humoral cascade systems. The aim of this study was to investigate whether the heparin-coated CPB sets influence adhesion of blood cells to surfaces of arterial filters during CPB. Thirty-one patients undergoing coronary artery bypass grafting were studied. In the control group (C) standard CPB sets and standard doses of heparin (300 IU/kg) were employed; in the HC (heparin-coated) group, heparin-coated CPB sets and reduced heparin doses (range, 150 to 225 IU/kg) were used. Two additional groups were also studied; group FC (coated filter), with standard CPB sets and heparin-coated arterial filters (heparin dose, 300 IU/kg), and group OC (uncoated filter), with heparin-coated CPB sets and standard arterial filters (heparin dose, 300 IU/kg). The inner surfaces of the arterial filters were examined after CPB with scanning electron microscopy. Scanning electron microscopy demonstrated almost clean surfaces in heparin-coated filters even when other parts of the circuit were uncoated. Using an arbitrary adhesion score, significant differences between the groups were noted in the adhesion grade; it was lowest in group HC (2.2 +/- 0.27 [mean +/- standard error of the mean]) versus group C (5.4 +/- 0.53; p < 0.001). In group FC it was marginally higher than in group HC but almost significantly lower than in group OC (2.6 +/- 0.68 versus 5.4 +/- 0.81; p = 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracorporeal heparin adsorption following cardiopulmonary bypass with a heparin removal device--an alternative to protamine

OBJECTIVES: To evaluate the therapeutic efficacy and applicability of a heparin removal device (HRD) based on plasma separation and poly-L-lysine (PLL) affinity adsorption as an alternative to protamine in reversing systemic heparinization following cardiopulmonary bypass (CPB). DESIGN: A prospective study. SETTING: University research laboratory. SUBJECTS: Adult female swine (n=7). INTERVENTIONS: Female Yorkshire swine (n=7, 67.3+/-3.5 [SEM] kg) were subjected to 60 mins of right atrium-to-aortic, hypothermic (28 degrees C) CPB. After weaning from CPB, the right atrium was recannulated with a two-stage, dual-lumen cannula which was connected to an HRD via extracorporeal circulation. Blood flow was drained at 1431.2+/-25.4 mL/min from the inferior vena cava, through the plasma separation chamber of the HRD (where heparin was bound to PLL), and reinfused into the right atrium. The HRD run time was determined by a previously established mathematical model of first-order exponential depletion. MEASUREMENTS AND MAIN RESULTS: Heart rate, mean arterial pressure, pulmonary arterial pressure, central venous pressure, kaolin and celite activated clotting time (ACT), activated partial thromboplastin time (APTT), heparin concentration, and plasma free hemoglobin were obtained before, during, and after the use of the HRD. Pre-CPB ACT was 167+/-89 secs (kaolin) and 99+/-7 secs (celite), and APTT was 34+/-5 secs. The HRD run time averaged 27.4 +/-1.5 mins targeted to remove 90% total body heparin. Use of the HRD was not associated with any adverse hemodynamic reactions or increases in plasma free hemoglobin. The heparin concentration immediately following CPB was 4.85+/-0.24 units/mL, with ACT >1000 secs and APTT >150 secs in all animals. During heparin removal, total body heparin content followed first-order exponential depletion kinetics. At the end of the HRD run, heparin concentration decreased to 0.51+/-0.09 units/mL, with kaolin ACT returning to 177+/-22 secs, celite ACT returning to 179+/-17 secs, and APTT returning to 27+/-3 secs (p > .05 vs. pre-CPB baseline for all variables). CONCLUSIONS: The HRD is capable of reversal of anticoagulation following CPB without significant blood cell damage or changes in hemodynamics. The HRD, therefore, can serve as an alternative to achieve heparin clearance in clinical situations where use of protamine may be contraindicated.     

Inactivation of leukocytes in platelet concentrates by photochemical treatment with psoralen plus UVA

A photochemical treatment (PCT) process using a novel psoralen and long wavelength ultraviolet light (UVA, 320-400 nm) has been developed to inactivate bacteria and viruses in platelet concentrates. This study evaluated the efficacy of PCT for inactivation of leukocytes that contaminate platelet preparations. Three psoralens, 8-methoxypsoralen (8-MOP), 4'-aminomethyl 4,5', 8-trimethylpsoralen (AMT), and the novel psoralen S-59, were compared using the following four independent but complementary biological and molecular assays. (1) T-cell viability: Treatment with 150 mumol/L S-59 and 1.0 to 3.0 Joules/cm2 UVA inactivated >5.4 +/- 0.3 log10 of T cells in full-sized single-donor plateletpheresis units. Using 1.0 Joule/cm2 UVA, the lowest dose of S-59, AMT and 8-MOP required to reduce the number of T cells to the limit of detection was 0.05 micromol/L, 1.0 micromol/L, and 10.0 micromol/L, respectively. (2) Cytokine synthesis: Treatment with 1.9 Joules/cm2 UVA and 150 micromol/L S-59 or AMT completely inhibited synthesis of the cytokine IL-8 by contaminating leukocytes during 5 days of platelet storage. After treatment with 75 micromol/L 8-MOP and 1.9 Joules/cm2 UVA, only low levels of IL-8 were detected. (3) Psoralen-DNA adduct formation: The combination of 1.9 Joules/cm2 UVA and 150 micromol/L S-59, AMT, or 8-MOP induced 12.0 +/- 3.0, 6.0 +/- 0. 9, and 0.7 psoralen adducts per 1,000 bp DNA, respectively. (4) Replication competence: Polymerase chain reaction (PCR) amplification of small genomic DNA sequences (242-439 bp) after PCT was inhibited. The degree of PCR amplification inhibition correlated with the level of adduct formation (S-59 > AMT > 8-MOP). In contrast, 2,500 cGy gamma radiation, a dose that inactivates >5 log10 of T cells in blood products, had minimal effect on cytokine synthesis and did not induce sufficient DNA strand breaks to inhibit PCR amplification of the same small DNA sequences. These results demonstrate that leukocytes are sensitive to PCT with psoralens and among the psoralens tested S-59 is the most effective. Therefore, PCT has the potential to reduce the incidence of leukocyte-mediated adverse immune reactions associated with platelet transfusion.     

Management of postoperative heparin rebound following cardiopulmonary bypass

Postoperative heparin rebound was investigated in 50 adult patients undergoing cardiopulmonary bypass with the use of the Hepcon heparin analyzer. Prior to bypass each patient received 2 mg/kg of heparin. During bypass, the activated clotting time (ACT) was utilized to assess the need for additional heparin to maintain the value between 300 and 400 seconds. The average amount of heparin given was 160 mg. Once cardiopulmonary bypass was terminated the Hepcon unit was employed to determine the actual amount of active circulating heparin and to calculate the dose of protamine sulfate. The average amount of protamine administered intraoperatively was 200 mg. The overall mean ratio of protamine-to-heparin was 1.25 : 1. Once hemostasis was achieved, no circulating heparin was measured with the Hepcon unit, and the ACT value had returned to its baseline, the incisions were closed and the patients were transferred to the intensive care unit. One hour later a blood sample was obtained and analyzed by the Hepcon unit for any heparin rebound. We found that 26 patients (52%) had circulating heparin and required an additional dose of protamine, averaging 70 mg. Drainage from the thoracotomy tubes averaged 400 cc in the first 24 hours, and a mean of 2 units of packed cells was infused. Three patients (6%) did not require any blood transfusions. The use of the Hepcon unit has produced a safe and expedient method of analyzing and neutralizing active circulating heparin in patients following cardiopulmonary bypass. It is a useful adjunct in blood conservation because it reduces excessive postoperative blood loss associated with heparin rebound.     

A comparison of thromboelastography with heparinase or protamine sulfate added in vitro during heparinized cardiopulmonary bypass

Thromboelastography (TEG) has been used after cardiopulmonary bypass (CPB) to diagnose excessive postoperative hemorrhage. Conventional TEG during CPB is not possible due to the sensitivity of the TEG to even small amounts of heparin, which produces a nondiagnostic tracing. The purpose of this study was to compare heparin neutralization using heparinase or protamine in TEG blood samples obtained during CPB. TEG testing was performed on 48 patients before, during and after CPB. Tissue plasminogen activator activity and antigen were measured on a subset of 32 patients. We found: 1) heparinase neutralized at least 10 IU/ml heparin while 1.6 ug/ml protamine neutralized up to 7 IU/ml heparin, 2) in samples with complete heparin neutralization by both methods, there was no significant difference in the R values, 3) while there was good correlation for other TEG parameters between heparinase and protamine treated samples, heparinase treatment produced shorter K values and higher angle, MA and A60, 4) while fibrinolysis was detected using both methods, heparinase treatment suppressed fibrinolysis in the TEG in both samples from patients and after in vitro addition of tissue plasminogen activator, 5) TEG was not a sensitive indicator of t-PA activity, detecting only 21% of samples with increased t-PA activity during bypass, and 5) heparinase was at least 100 times more expensive than protamine. We conclude that while both heparinase and protamine can be used to neutralize heparin in TEG samples obtained during CPB, protamine neutralization is more sensitive to fibrinolysis and less expensive, but the protamine dose must be carefully selected to match the heparin level used at individual institutions.     

Low dialysate [K+] decreases efficiency of hemodialysis and increases urea rebound

In a previous study, it was reported that hemodialysis with dialysate [K+] (KD) of 1.0 or 2.0 mmol/L caused an increase in BP shortly after completion of treatment due to arteriolar constriction. With this background, it was hypothesized that a low KD might decrease dialysis efficiency by a similar mechanism. To evaluate this hypothesis, paired observations of two consecutive 3-h treatments, with KD of 1.0 or 3.0 mmol/L, were performed in 14 stable end-stage renal disease patients. A KD of 1.0 mmol/L resulted in lower values for both urea reduction ratio and Kt/V evaluated at completion of dialysis and 1 h thereafter. Values at equilibrium were urea reduction ratio 42+/-1% versus 47+/-2% (P < 0.02), Kt/V 0.65+/-0.03 versus 0.73+/-0.03 (P < 0.02) for KD 1.0 or 3.0 mmol/L, respectively. The mechanisms responsible for the observed differences in dialysis efficiency were examined using a urea kinetics model that predicts urea sequestration caused by impaired blood flow to urea-rich tissues. For this purpose, urea rebound and its effect on Kt/V (by means of deltaKt/V, calculated as equilibrated minus single pool value) with KD 1.0 and 3.0 mmol/L were assessed. Greater urea rebound, 12.8+/-1.6% versus 8.6+/-1.4% (P < 0.001), and larger deltaKt/V, 0.12+/-0.01 versus 0.10+/-0.02 (P < 0.02), were observed with KD 1.0 mmol/L compared with 3.0 mmol/L. The theoretical model accurately predicted the deltaKt/V observed with KD 1.0 mmol/L. It is concluded that a low KD decreases dialysis efficiency. This effect is likely caused by reduced blood perfusion to nonvisceral organs, largely skeletal muscle. Conversely, hemodialysis with KD 3.0 mmol/L facilitates tissue perfusion, minimizes urea trapping in poorly perfused areas, and improves the efficiency of this treatment modality.     

The effects of heparinase 1 and protamine on platelet reactivity

BACKGROUND: Protamine is currently the most widely used drug for the reversal of heparin anticoagulation. Heparinase 1 (heparinase) is being evaluated as a possible alternative to protamine for the reversal of heparin anticoagulation. The authors evaluated the effects of equivalent doses of heparinase and protamine on platelet reactivity by measuring agonist-induced P-selectin expression. METHODS: After Institutional Review Board (IRB) approval, informed consent was obtained from 12 healthy volunteers and 8 patients undergoing surgery requiring cardiopulmonary bypass (CPB). Twenty-four ml of blood was obtained from each volunteer; 10 ml of blood was obtained from each patient before the CPB, and another 10 ml was obtained after CPB. Heparin was neutralized using heparinase or protamine. Platelet reactivity was assessed by measuring the expression of P-selectin after stimulation of platelets with increasing concentrations of a thrombin receptor agonist peptide (TRAP). Data were analyzed using analysis of variance. P < 0.05 was considered significant. RESULTS: For the healthy volunteers, the activated coagulation times (ACTs) of the heparinized samples returned to baseline values with heparinase (12.5 U/ml) or protamine (32.5 microg/ml). For the 8 patients, the ACTs returned to baseline with heparinase (20 U/ml) or protamine (50 microg/ml). The authors found no difference in the expression of P-selectin in samples neutralized with heparinase, but samples neutralized with protamine showed a significant decrease in the expression of P-selectin when compared with heparinized samples. CONCLUSIONS: At dosages that reverse the anticoagulant effects of heparin, heparinase has minimal effects on platelets, whereas platelet reactivity was markedly inhibited by protamine.     

More effective suppression of hemostatic system activation in patients undergoing cardiac surgery by heparin dosing based on heparin blood concentrations rather than ACT

This study was designed to determine whether the maintenance of higher than usual patient-specific heparin concentrations during cardiopulmonary bypass (CPB) was associated with more effective suppression of hemostasis system activation. Thirty-one patients scheduled for repeat cardiac surgery or combined procedures (i.e., coronary revascularization + valve repair/replacement) were consented and enrolled in this study. All patients received porcine heparin and protamine and were randomly assigned to monitoring of anticoagulation by either celite ACT alone (Control, n = 16) or by kaolin ACT combined with on-site measurements of whole blood heparin concentration (Intervention, n = 15). Blood specimens collected before administration of heparin, before weaning from CPB and after administration of protamine were analyzed with a battery of coagulation assays. Patients in the intervention cohort received appreciably greater heparin doses than control patients, resulting in higher anti-Xa heparin levels at the end of CPB. Fibrinopeptide A and D-dimer levels were higher in the control group before discontinuation of CPB. Percent decrease during CPB were greater in the control group for factors V and VIII, fibrinogen and antithrombin III. Percent decrease in complement 3 was greater in the control group after protamine and bleeding times measured in the Intensive Care Unit were significantly more prolonged in this group. Maintenance of higher patient-specific heparin concentrations during CPB more effectively suppresses excessive hemostatic system activation than do standard heparin doses chosen based on measurement of ACT. These findings may explain, at least in part, the significant reduction in perioperative blood loss and blood product use when higher heparin concentrations are maintained.     

Heparin inhibits leukocyte rolling in pial vessels and attenuates inflammatory changes in a rat model of experimental bacterial meningitis

Heparin is a natural proteoglycan that was first described in 1916. In addition to its well characterized effect on blood coagulation, it is becoming clear that heparin also modulates inflammatory processes on several levels, including the interference with leukocyte-endothelium interaction. Anecdotal observations suggest a better clinical outcome of heparin-treated patients with bacterial meningitis. The authors demonstrate that heparin, a glycosaminoglycan, inhibits significantly in the early phase of experimental pneumococcal meningitis the increase of 1) regional cerebral blood flow (125 +/- 18 versus 247 +/- 42%), 2) intracranial pressure (4.5 +/- 2.0 versus 12.1 +/- 2.2 mm Hg), 3) brain edema (brain water content: 78.23 +/- 0.33 versus 79.49 +/- 0.46%), and 4) influx of leukocytes (571 +/- 397 versus 2400 +/- 875 cells/microL) to the cerebrospinal fluid compared with untreated rats. To elucidate the possible mechanism of this observation, the authors investigated for the first time leukocyte rolling in an inflammatory model in brain venules by confocal laser scanning microscopy in vivo. Heparin significantly attenuates leukocyte rolling at 2, 3, and 4 hours (2.8 +/- 1.3 versus 7.9 +/- 3.2/100 microm/min), as well as leukocyte sticking at 4 hours (2.1 +/- 0.4 versus 3.5 +/- 1.0/100 microm/min) after meningitis induction compared with untreated animals. The authors conclude that heparin can modulate acute central nervous system inflammation and, in particular, leukocyte-endothelium interaction, a key process in the cascade of injury in bacterial meningitis. They propose to evaluate further the potential of heparin in central nervous system inflammation in basic and clinical studies.     

On the value of the activated partial thrombo-plastin time (aptt) in monitoring heparin therapy

Of 1068 blood samples referred for evaluation of the haemostatic mechanism 135 contained heparin as judged by the polybrene titration method. Of the 57 samples with a heparin concentration in the defined therapeutic range, 65 per cent had APTT values in the "therapeutic range". Of the samples from patients who had not received any anticoagulant, 213 (24 per cent) had prolonged APTT, and 48 of these samples had APTT values in the "therapeutic range" for heparin therapy. It is suggested that the APTT test is of limited value in monitoring heparin therapy.