Coagulation and Transfusion Medicine / PARTIAL FUNCTIONALITY OF GPIb/IX/V IN INFUSIBLE PLATELET MEMBRANES

Infusible platelet membranes (IPMs) prepared from fresh or outdated human platelets have been shown to correct prolonged bleeding times in thrombocytopenic rabbits. In previous trials, IPMs did not seem to be immunogenic and lacked dose-limiting toxicity. The present study was undertaken to explore whether the platelet glycoprotein (GP) Ib/IX/V complex might retain functionality in the IPM preparation. IPMs did not spontaneously bind von Willebrand factor (vWF), but saturable binding could be induced by ristocetin, with a dissociation constant (Kd) of 0.31 ± 0.03 μg/mL at 1.0 mg/mL of ristocetin. Of 4 anti–GPIb-alpha monoclonal antibodies tested, AN-51 inhibited vWF binding 67.8% ± 5.8%, whereas AS-2, AS-7, and SZ-2 were ineffective. Maximal vWF binding induced by botrocetin was only 10% to 15% of that observed with ristocetin. Retention of partial functionality of the GPIb/IX/V receptor allowing vWF binding in a modulated manner seems to represent a critical mechanism by which IPMs may provide hemostatic efficacy. Infusible platelet membranes (IPMs) are one of several new agents undergoing evaluation as possible adjuncts to traditional platelet transfusion therapy.1 IPMs (Cyplex, Cypress Bioscience, San Diego, CA) are 0.3to 1.1-μm diameter particles prepared from 10to 19-day-old blood bank human platelets by lysis and differential centrifugation and treatment to inactivate blood-borne viruses.2 IPMs, when administered to thrombocytopenic rabbits, demonstrate hemostatic activity by producing shortening of prolonged bleeding times without causing a clinically significant level of thrombogenicity.2 Since IPMs recently have been shown to bind to subendothelial surfaces under high shear conditions,3 the present study was undertaken to determine whether IPM might retain any functionality of the glycoprotein (GP) Ib/IX/V receptor, which mediates binding of von Willebrand factor (vWF) by platelets under conditions of high shear.4 Materials and Methods IPMs were reconstituted to 2 mg/mL with distilled water. Unfiltered IPMs were washed twice and resuspended in Hanks buffered salt solution (Gibco BRL, Grand Island, NY) containing 1% bovine serum albumin and a 10-mmol/L concentration of EDTA. For study of vWF binding, 100 μL of IPM was added to tubes containing 25 μL of purified human vWF,5 in the presence of ristocetin (Helena Laboratories, Beaumont, TX ) or botrocetin (Centerchem, Stanford, CT) as a modulator. After 45 minutes’ incubation at 22°C, the IPMs were washed and incubated for 30 minutes with rabbit antihuman vWF (DAKO, Carpinteria, CA). The IPMs were washed again and incubated for 30 minutes with fluorescein isothiocyanate–conjugated goat antirabbit IgG (Sigma Coagulation and Transfusion Medicine / ORIGINAL ARTICLE Am J Clin Pathol 2001;115:144-147 145 © American Society of Clinical Pathologists Chemical, St Louis, MO). After incubation, the IPMs were washed once more and fixed with 1.0% paraformaldehyde before analysis. Ten thousand events from each specimen were analyzed for fluorescein isothiocyanate fluorescence on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The settings for the flow cytometer were forward scatter (FSC), log; side scatter (SSC), log, photomultiplier tube (PMT) voltage, 288; and fluorescence channel 1, log, PMT voltage, 487. The instrument was calibrated daily, and, for the entire study period, machine drift was less than 5%. A forward light scatter threshold setting of 112 permitted measurement of IPMs or similarly sized latex particles, but background noise was excluded effectively when IPM-free control buffer was sampled. For data analysis, the gate was drawn around the main population on an FSC vs SSC dot blot. This gate included particles having low FSC and SSC distribution, but it excluded particles with high FSC and SSC distribution representing larger particles. The particles included in this analysis represented more than 95% of the entire population collected. Studies also were undertaken with murine monoclonal antibodies (Mabs) directed against epitopes within platelet GPIb-alpha. Commercially available Mabs SZ-2 (Immunotech, Marseilles, France) and AN-51 (DAKO) or the AS-2 and AS-7 Mabs developed in our laboratory,6 were added at 10 μg/mL or 20 μg/mL. Following a 30-minute preincubation of IPMs with Mabs or phosphate-buffered saline (PBS), vWF and modulator were added, and analysis of binding was performed as described in the preceding text. Statistical significance was determined by paired t-test of the values obtained with each antibody, as compared with PBS.