Neutralization of the Antiheparin Activity of Platelet Factor 4 by a Monoclonal Antibody

SummaryWe have produced a panel of monoclonal antibodies (mAbs) against rabbit platelet factor 4 (PF4). Two of these mAbs have been characterized in this study. In particular the antibody called 10B2, which also recognizes the human molecule, is able to block PF4’s ability to neutralize heparin in a modified Heparin-Factor Xa chromogenic assay. The inhibition appears to be more than 95% at 1:1 mAb/PF4 molar ratio both for purified rabbit and human PF4. Similar results were obtained using supernatants from stimulated human platelets (90% of inhibition at 1:1 mAb/ PF4 molar ratio) or using Fab fragments from 10B2. Studies to determine the antigenic determinant against which 10B2 is directed, show that this is an assembled epitope which involves disulfide bonds of the PF4.

The Effect of Lysed Platelets on Neutralization of Heparin In Vitro with Protamine as Measured by the Activated Coagulation Time (ACT)

SummaryThe effect of lysed platelets on the activated coagulation time (ACT) was studied in heparinized whole blood during titration with protamine. Frozen-thawed washed platelet suspension, or a chromatography fraction thereof, or autologous frozen-thawed platelet-rich plasma was added in various dilutions to freshly drawn blood anticoagulated with 3,000 USP units/1 heparin. After a 10 min incubation, the amount of protamine needed to restore the ACT to baseline ("protamine titration dose") was determined. We found that the protamine titration dose decreased in proportion to the amount of lysed platelet material added; expressed as a percentage of the total number of platelets present, each unit increase in lysed platelets produced a 1.7% ±0.8 (SD) reduction in the protamine dose needed to normalize the ACT. A heparin activity assay showed that this effect was not due to antiheparin activity of lysed platelets such as platelet factor 4 (PF4). Our data indicate that the procoagulant activity of platelet membranes reduced the sensitivity of the ACT to heparin. These findings suggest that membranous platelet microparticles may cause an inaccurate calculation, based on the ACT, of a protamine dose to reverse heparin anticoagulation in cardiopulmonary bypass procedures.

Effect of PGI2 Infusion During Long Term Extracorporeal Circulation with Membrane Lung in Sheep

PGI2 plus very low dose of heparin was infused in 6 lambs connected for long term extracorporeal circulation with a membrane lung. Hemodynamic and hemostasis parameters were compared to those of a control group treated only with standard doses of heparin. PGI2 efficacy in inhibiting platelet aggregation and platelet fall was confirmed. A small platelet release, measured as antiheparin activity, was observed during all the by-pass, but did not influence platelet capacity of recovering when PGI2 effect dissolved. Heparin needed resulted less than a quarter of the quantity used for the control group. In our experimental conditions the hemodynamic changes were mainly limited to a decrease in diastolic blood pressure.