Biomedical subjects

V C Yang

Publications and source records attributed to V C Yang.

88 records · Page 5Linked to original sources

PubMedDate not supplied

A simple method for rapid and precise estimation of the protamine dose required for clinical heparin reversal.

A simple, colorimetric protamine titration method was developed for rapid and precise estimation of the protamine dose required for clinical heparin reversal. The method employs azure A dye as the titration indicator, and has replaced the time consuming clotting assay in the conventional protamine titration method with a rapid colorimetric assay. It offers the same accuracy in estimating the protamine dose as that of the conventional titration method, but allows the processing time to be shortened to within 5 min. In a similar manner, the colorimetric assay can also be employed with a heparin titration procedure to quickly assess the heparin dose required for protamine reversal. This would allow physicians to exercise a quick and accurate heparin back-titration to patients who are overdosed with protamine.

Cardiopulmonary Bypass↗

PubMedDate not supplied

A novel approach to anticoagulation control.

Heparin used in extracorporeal therapy often leads to bleeding complications. Protamine used for heparin reversal can cause adverse hemodynamic responses. To control both types of complications, a cellulosic hollow-fiber filter device containing immobilized protamine (defined as a protamine filter) was developed. In vivo experiments with dogs showed that the filter not only removed more than 80% of the anticoagulant activity of heparin, but also caused no clinically significant hemodynamic response. In addition, the protamine filter also significantly attenuated both the thrombocytopenic and granulocytopenic responses associated with the use of protamine. Moreover, the use of immobilized protamine considerably reduced activation of the blood complement system by free protamine.

Animals↗

PubMedDate not supplied

A disposable, coated wire heparin sensor.

The development of an ion-selective electrode heparin sensor consisting of a specially formulated polymer membrane doped with tridodecylmethylammonium chloride as the heparin complexing agent was recently reported. Because of the simple nature of the membrane technology used, the authors envisioned that the sensor could be configured as a disposable single-use device for rapid clinical or bedside measurement of heparin in a small, discrete sample. To explore this possibility, an inexpensive, disposable heparin sensor was created by dip-coating a copper wire with the specially formulated heparin-sensing polymeric membrane. Coated wire heparin sensors with a broad range of membrane thicknesses, prepared by repeatedly dipping the wire in the membrane solution for various times, were examined. Data show that increasing the membrane thickness of the sensor to a certain degree (more than 10 microns) enhanced the sensor's potentiometric response to heparin, although the time required to achieve 90% of the steady-state potential change was also prolonged. In addition, increasing membrane thickness also magnified the stirring effect on the sensor's response. In undiluted plasma samples, the coated-wire sensor with an optimized membrane thickness yielded a significant (5 to 30 mV) and reproducible response to heparin in a clinically relevant concentration range (0.5 to 12 units/ml, respectively). The clinical utility of the coated wire heparin sensor was shown using the sensor during protamine titration of heparinized plasma to assess the titration end-point. Preliminary results showed that the titration end-points determined by the heparin sensor strongly correlated with those determined by the activated partial thromboplastin time clotting assay. The overall time requirement to complete the titration process using a set of prefabricated coated wire heparin sensors, however, was less than 3 minutes. Further titration studies using undiluted clinical whole blood samples are in progress.

Biotechnology↗

PubMedDate not supplied

Optimization and assessment of the in vivo efficacy of a heparin removing bio-reactor using mathematical modeling.

The authors previously reported an approach that could be used to simultaneously control both heparin and protamine induced complications during extracorporeal perfusion. The approach consists of placing a hollow fiber based bio-reactor containing immobilized protamine (defined as the "protamine bio-reactor") at the distal end of the blood perfusion circuit for extracorporeal heparin removal. Preliminary in vitro and in vivo studies have successfully demonstrated the feasibility of the proposed approach. The authors present an in vivo theoretical model for this extracorporeal heparin removal approach. This model, which consists of a two compartment model for the metabolic clearance of heparin and a plug flow reactor model for the protamine bio-reactor, can be used to optimize and assess the required size of the bio-reactor for effective clinical heparin removal. To examine the utility of such a model, 14 female mongrel dogs (six dogs were used as controls and eight dogs were used to test the bio-reactor) were included in the in vivo study. The femoral artery and femoral vein of the dog were cannulated, and the bio-reactor was attached. Heparin was given intravenously at a dose of 150 IU/kg body weight, and its activity was measured using the aPTT assay. Preliminary studies show that the experimental data fits remarkably well with the theoretical model. The model indicates that the protamine bio-reactor can remove heparin efficiently, even in the presence of competitive binding between heparin and plasma proteins. Under clinical situations, such as hemodialysis and open heart operations, a protamine bio-reactor containing approximately 45,000 hollow fibers is necessary to reduce heparin to less than 10% of its initial concentration. The time required to saturate the protamine bio-reactor with heparin is dependent upon the blood flow rate which, as predicted by the model, is 10 min for open heart surgery (flow rate = 2,000 ml/min) and 60 min for hemodialysis (flow rate = 200 ml/min). A detailed description of the in vivo model, as well as future directions envisioned in the design of a clinically useful heparin removing system are discussed.

Animals↗

PubMedDate not supplied

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.

Hemofiltration↗

PubMedDate not supplied

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.

Hemorrhage↗

PubMedDate not supplied

Low molecular weight protamine: a potent but nontoxic antagonist to heparin/low molecular weight protamine.

To avoid bleeding complications, protamine is routinely used after cardiovascular surgery to neutralize the anticoagulant function of heparin. However, its clinical use is associated with adverse and sometimes fatal reactions. Based on literature review of the mechanism of heparin neutralization and protamine induced immunologic toxicity, we propose the following hypothesis: If a chain shortened low molecular weight protamine (LMWP) containing the heparin neutralizing domain could be derived from native protamine, it could be a potent and yet nontoxic heparin antagonist. In this study, we present results to validate this hypothesis. LMWP fragments containing an intact arginine sequence and an average molecular weight of approximately 1,100 daltons were successfully prepared by enzymatic digestion of protamine with thermolysin. In vitro studies show that such LMWP fragments completely neutralized the anticoagulant functions of heparin and LMWH, based on the anti-Xa chromogenic and aPTT clotting time assays. In vivo results reveal that although injection of protamine to mice led to obvious production of anti-protamine antibodies, injection of LMWP did not elicit any detectable immunogenic responses. In addition, these LMWP fragments exhibited a markedly reduced antigenicity and cross-reactivity toward the mice anti-protamine antibodies.

Animals↗

PubMedDate not supplied

A prodrug approach for delivery of t-PA: construction of the cationic t-PA prodrug by a recombinant method and preliminary in vitro evaluation of the construct.

Previously, we reported a novel prodrug approach, that could lead to targeted thrombolysis without the risk of bleeding. The approach consists of a protein conjugate made of two components: a fibrin targeting antibody (Ab) linked to an anionic heparin, and a plasminogen activator (PA) derivatized with cationic species. These two components are linked by means of an electrostatic interaction. Because the cationic species are small, the modified PA would retain its thrombolytic activity. However, this activity would be inhibited after binding to the counterpart due to the blockage of the PA active site by the appended macromolecules. Because protamine is a clinical antagonist to heparin, it can be used in humans to dissociate the modified PA from its counterpart. Thus, the approach would permit the administration of a fibrin targeting but inactive thrombolytic drug (thereby alleviating the bleeding risk by avoiding systemic generation of plasmin), and subsequently a triggered release of the active drug to the fibrin deposit. In our previous work, we demonstrated the feasibility of the approach by producing a positively charged PA by means of chemical conjugation of a cationic CRRRRRRR peptide with urokinase. In this study, we further extended our work and produced a similar cationic t-PA by means of a recombinant DNA approach; i.e., by fusion of a poly(Arg)7 peptide to the kringle-1 domain of t-PA. Results obtained from the restriction enzyme analysis and the Western blot yielded full identification of this recombinant protein. This recombinant poly(Arg)7-modified-t-PA protein conjugate (termed "rmt-PA" hereafter) completely retained the fibrinolytic activity of the original recombinant, unmodified t-PA (termed "rt-PA" hereafter), as measured by the chromogenic assay and fibrin agar lysis assay. The prodrug and triggered release features of the proposed approach were confirmed by partial inhibition of the plasminogen activating activity of this protein by heparin, and the partial reversal of such inhibition by protamine.

Base Sequence↗

PubMedDate not supplied

Protamine-coated Cuprophan. A potential nonthrombogenic hemodialysis membrane with improved blood compatibility.

A protamine-coated Cuprophan hemodialysis membrane possessing improved blood compatibility was developed. Protamine was covalently immobilized onto the inner walls of the Cuprophan hollow fibers, using the cyanogen bromide (CNBr) activation method. Studies conducted with aqueous solutions indicated that the immobilization process did not introduce deterioration of the membrane mechanical and mass transfer properties; the permeability of several compounds through the protamine-coated membrane was unchanged. The modified membrane was rendered nonthrombogenic by the adsorption of heparin to the protamine-bound surfaces. Heparin adsorbed on the immobilized protamine retained about 20% of its initial anticoagulant activity. Preliminary studies using the hemolytic complement assay indicated that complement activation by the protamine-coated membrane was statistically far less than that by the untreated membrane.

Cellulose↗

PubMedDate not supplied

A protamine-mediated heparin sensing device.

A convenient, protamine mediated strip assay for heparin was developed. The assay is established on the specific interaction between heparin and protamine, and the metachromatic color change of methylene blue dye from blue to purple in the presence of heparin. The device consists of a fibrin cup fabricated with a cellulose paper strip containing immobilized protamine. To analyze plasma heparin levels, a fixed volume of the test sample is placed in the fibrin cup and allowed to migrate through the strip in a descending manner until exhaustion. The region to which heparin binds is then visualized as purple in color by spraying methylene blue dye onto the strip. The heparin level in the sample is estimated from the length of the purple region using a preconstructed standard curve. This device proved to be capable of detecting and differentiating heparin in clinically relevant concentrations.

Heparin↗

PubMedDate not supplied

Rapid and precise whole blood protamine titration.

A rapid and precise whole blood protamine titration method was developed. The method uses azure A dye as the titration indicator and thereby replaces the tedious and time-consuming clotting assay with a facile colorimetric assay. The method provides the same accuracy in estimating the titration end-point, but allows the processing time to be shortened to that required by current clinical methods. The simplicity, flexibility, speed, and accuracy offered by the method, and the ability to use whole blood specimens for the measurements, should allow the method to be used by clinicians in the operating room or during a surgical procedure to estimate the adequate protamine dose required for heparin reversal.

Azure Stains↗

PubMedDate not supplied

A filter device for the prevention of both heparin- and protamine-induced complications associated with extracorporeal therapy.

When extracorporeal blood circulation (ECBC) is used, systemic heparinization is necessary to prevent clotting of the blood in the extracorporeal circuit. However, the high circulating heparin concentration needed often leads to bleeding complications. To avoid these, protamine, a heparin antagonist, is administered at the conclusion of the ECBC procedure to reverse the anticoagulant activity of heparin. Intravenous administration of protamine can cause hypotension and shock. To date, there has been no real alternative to control the bleeding risks associated with systemic use of heparin and the adverse effects resulting from heparin reversal with protamine. A novel approach that might control both the heparin- and the protamine-induced complications is suggested. It consists of placing a blood-compatible filter device containing immobilized protamine (a protamine filter) at the distal end of the ECBC apparatus. The filter removes heparin after heparin serves its anticoagulant purpose in the extracorporeal circuit and before blood is returned to the patient. The filter also allows for an external protamine treatment. Since protamine toxicity results from the direct contact of protamine with cells of the liver, lungs, and other organ tissues, the use of an external protamine treatment would minimize it. Protamine was covalently immobilized onto a cellulosic hollow fiber bundle obtained from a clinically used hemodialyzer. The bundle was accessed to the vascular system of a dog by femoral artery and vein cannulation. In in-vivo experiments the protamine-bound fiber bundle not only removed heparin from the extracorporeal circuit, but also caused no clinically significant hemodynamic change in the animal.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗

PubMedDate not supplied

A protamine filter for extracorporeal blood heparin removal.

Heparin employed in extracorporeal circuits often leads to hemorrhagic complications. Protamine employed for heparin neutralization can cause adverse hemodynamic responses. To control both types of complications, the authors propose an approach that consists of placing a filter device containing immobilized protamine (defined as a protamine filter) at the termination of the extracorporeal blood circulation (ECBC) procedure. This protamine filter would remove heparin from the extracorporeal circuit before heparin is returned to the patient. Meanwhile, the filter would also permit external protamine treatment. Since protamine toxicity generally results from the interaction of protamine with certain cells present in the liver, lungs, and tissues, the use of external protamine would minimize its potential adverse effects. Protamine was immobilized on a hollow fiber bundle obtained from a conventional hemodialyzer. Preliminary studies show that the protamine bound bundle is capable of neutralizing the anticoagulant activity of heparin both in vitro and in vivo. In addition, the protamine filter has abolished the hypotensive response normally associated with protamine reversal of heparin, as indicated by the insignificant changes in blood pressure, pulse rate, pulmonary artery systolic/diastolic pressures, and cardiac output. Further in vivo studies involving the use of dogs, as well as investigation of the activation of the complement system by the protamine filter, are currently being conducted.

Animals↗

PubMedDate not supplied

A novel electrochemical heparin sensor.

Heparin is one of the most important clinical drugs, and is employed universally during surgical procedures and extra-corporeal therapies to prevent blood from clotting. Its clinical use, however, is often associated with serious hemorrhagic complications. Because of this life threatening bleeding risk, there is a need for a simple sensing device that can rapidly and directly monitor heparin levels during extra-corporeal therapies to provide a safeguard during these procedures. Current heparin assays are all based on the measurement of blood clotting time, and none of them are suitable for direct and rapid determination of heparin. We describe applying conventional ion selective electrode (ISE) polymer membrane technology and using a specifically formulated membrane doped with tridodecylmethylammonium chloride (TDMAC) as the heparin complexing agent, to devise the first electrochemical sensor for heparin measurement. The sensor is capable of detecting directly and rather selectively the free heparin concentrations in both physiologic saline and undiluted plasma samples. In addition, the clinical utility of the sensor has been demonstrated by monitoring the levels of heparin in undiluted whole blood specimens obtained from patients undergoing open heart operations. It is envisioned that the sensor could be configured as an in-line device within extracorporeal blood loops to monitor current extracorporeal therapy, or as a convenient single use disposable device for rapid bedside or laboratory measurements of heparin in small discrete samples of undiluted whole blood. Preliminary studies concerning the feasibility of designing a mass fabricated, solid state, disposable heparin sensor also have been conducted.

Blood Chemical Analysis↗

PubMedDate not supplied

A protamine filter for extracorporeal heparin removal. Development, testing, blood compatibility evaluation, and future direction.

The authors previously developed a filter device containing immobilized protamine (termed "protamine filter") that could be used to remove heparin during extracorporeal perfusion. In vivo studies involving dogs showed that the protamine filter removed more than 50% of heparin from the animals' blood circuit in less than 20 min. In addition, the use of the protamine filter did not elicit statistically significant protamine induced hemodynamic and thrombocytopenic responses. Biocompatibility of the protamine filter was also evaluated, with the focus on its effect on the coagulation cascade, the complement system, and the blood antithrombin III levels. Results showed that heparin adsorbed to the protamine coated surface retained 20% of its original activated partial thromboplastin time activity, rendering the coated surface antithrombotic. Activation of the coagulation system by the protamine coated membrane and the untreated cellulose membrane, as measured by the elevation of prothrombin fragment F1 + 2 levels, was statistically identical. The CH50 hemolytic assay showed that the protamine coated membrane produced a reduction of 1.2 +/- 0.8% of the total complement levels, as compared to 9.4 +/- 1.6% by the untreated membrane. In addition, the change in C3a des Arginine levels after 30 min of circulation was 1.5 +/- 0.2 mg/ml by the protamine filter, as compared to 2.1 +/- 0.1 mg/ml by the untreated membrane. Unlike native heparin that would bind with antithrombin, heparin adsorbed on the protamine coated surface was devoid of such activity, and produced no depletion of circulating antithrombin. Because of the limited capacity of the protamine filter, the future system is envisioned to consist of two filters; while one filter is removing heparin the other will be regenerated. With a recently developed heparin sensor, it should be possible to design a sensor directed, biofeedback, two filter heparin removal system.

Adsorption↗

PubMedDate not supplied

Clinical application of disposable heparin sensors. Blood heparin measurements during open heart surgery.

The authors previously reported the development of an ion selective electrode type heparin sensor consisting of a specially formulated polymer membrane doped with tridodecylmethylammonium chloride as the heparin complexing agent. They also demonstrated the feasibility of measuring blood heparin levels by protamine titration, using a disposable copper wire sensor coated with the heparin sensing membrane to probe the titration end point. In this article, the results of further titration studies conducted on 44 clinical whole blood specimens obtained from 8 patients undergoing open heart surgery were reviewed. Samples were taken from patients at four different stages during the bypass surgery: 1) before heparin administration; 2) immediately after heparin administration; 3) within 30 min to 3 hr after heparin administration; and 4) within 30 min after protamine administration. Heparin anticoagulant activity in these samples was monitored by the activated clotting time assay, whereas heparin concentrations were measured by protamine titration using either the Hepcon HMS Titrator (Medtronic HemoTec Inc., Englewood, CO) or the coated wire heparin sensor to determine titration end points. Results indicate that heparin levels determined by the sensor method were in good agreement with those determined by the Hepcon HMS Titrator. When the heparin concentrations estimated by the two methods show significant discrepancy (> 1.0 unit/ml), the sensor method seems to provide more precise values, as verified by an additional chromogenic heparin assay. The overall time required to complete the titration process and heparin measurement with a pre made heparin sensor was less than 3 min. Clinically, the heparin sensor could be used as a safeguard to precisely monitor heparin levels during surgical procedures. Alternatively, the sensor could be used to assess the accurate protamine dose required for full heparin reversal.

Blood Chemical Analysis↗