Search

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.

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.

A polymeric membrane electrode that exhibits significant and analytically useful potentiometric response to submicromolar levels of the heparin antagonist, protamine, is reported. The sensor is prepared by incorporating a lipophilic cation exchanger, potassium tetrakis(4-chlorophenyl)borate (KTpClPB) (at 1 wt%), within a specially formulated polymer membrane composed of 33 wt% 2-nitrophenyl octyl ether (2-NPOE), and 66 wt% poly(vinyl chloride) (PVC). When the polymer film is mounted in an appropriate electrode body, the resulting membrane electrode responds reproducibly to protamine via a nonequilibrium quasi-steady-state change in the phase boundary potential at the membrane/sample interface. Such response can be used to directly monitor, via classical potentiometric titrations, the binding between protamine and a variety of native (porcine and beef) as well as low-molecular-weight heparins. Scatchard analysis of the EMF titration data provides binding constants and stoichiometries for protamine-heparin interactions. The electrode can be further used to follow the enzymatic digestion of protamine by trypsin. In the presence of a given level of protamine, initial rates of potential decrease (-dE/dt) are shown to be linearly related to trypsin activity in solution over the range of 0-130 units/ml. The speed and simplicity of the protamine sensor make it an attractive alternative to classical methods for studying the interaction of protamine with other biologically important macromolecules as well as the proteolytic activity and reaction kinetics of trypsin.

The potentiometric response mechanism of a previously reported polymer membrane-based electrode sensitive to the polyanion heparin is established. Based on transport and extraction studies, the heparin response is attributed to a nonequilibrium change in the phase boundary potential at the sample/membrane interface. While true equilibrium polyion response, obtained for low heparin concentrations only after very long equilibration times (> 20 h), yields the expected Nernstian response slope of < 1 mV/decade, the observed large and reproducible EMF response to clinically relevant heparin concentrations (approximately 10(-7) M) during typical measurement periods (2-5 min) is ascribed to a steady-state kinetic process defined by the flux of the polyion both to the surface and into the bulk of the polymer membrane. A model describing this nonequilibrium response is presented. With this model, the uniqueness of the polymer membrane composition (e.g., very low plasticizer content, strictly controlled cationic site concentration, etc.) required to achieve analytically useful heparin response becomes clear. Practical working conditions and limitations of the sensor are discussed. To support the generality of the steady-state model proposed, corresponding EMF response data for a newly developed membrane electrode sensitive to a polycationic protein (protamine) are also presented. It is shown that the protamine-responsive membrane electrode appears to operate via the exact same kinetic mechanism as the heparin sensing system.

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.