Compositional Differences in Commercially Available Prothrombin Complex Concentrates
Authors: Sadeghi Nasir , Kahn Daniel , Cunanan Josephine , Only Arthur , Hoppensteadt Debra , Jeske Walter , Iqbal Omer , Harenberg Job , Fareed Jawed 
Published: October 2019

Abstract
Abstract 4391
Prothrombin complex concentrates (PCCs) are used in the management of bleeding complications with conventional oral anticoagulant drugs such as warfarin. These concentrates are also used in supportive therapy for hemostatic disorders. More recently these agents have been investigated for neutralization of the newer oral anticoagulant drugs such as the direct factor Xa and thrombin inhibitors. Since the activation of these complexes results in the generation of factor Xa and IIa, these agents may potentially neutralize both Xa and thrombin inhibitors. However, the potency of these agents is defined in units which represent the level of factor IX, other factors including factor II, VII, and X are also present, and in unspecified amounts. Moreover, other vitamin K-dependent proteins such as protein C, protein S, and protein Z may also be present. Varying amounts of albumin and other agent such as heparin and antithrombin may also be present as an additive. The purpose of this study is to compare the compositions of the currently available PCCs such as Profilnine®, Beriplex®, Cofact®, Octaplex®, Prothromplex®, and the older agents such as Konyne®, Preconetiv®, and Feiba®.

Materials and Methods:
Commercially available PCCs were obtained from various suppliers. Each of the individual vials of these concentrates was diluted with saline to obtain a 10 U/ml factor IX solution. Protein content was measured using Lowry's method. Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis was carried out by dilution of each concentrates to 2 U/ml. Western blot analysis was performed to determine presence of prothrombin, prothrombin-1, and thrombin using anti-human recombinant thrombin antibody capable recognizing these proteins. Tissue factor activation profiles of each PCCs was also studied using Innovin®. The protein composition of native and activated prothrombin complexes was also investigated using surface-enhanced laser desorption/ionization (SELDI) mass spectrometry utilizing the gold chip array (BioRad). Tissue factor mediated thrombin generation by each of the prothrombin complex was studied using a fluorometric method (Technoclone, Vienna, Austria).

Results:
The total protein content of these PCCs ranged from 18–106 mg/100U. Some of the products were found to contain varying amounts of albumin, antithrombin and heparin as evident in both SDS-PAGE and SELDI analysis. The SDS-PAGE profile of these complexes showed multiple protein bands ranging from 15 to 250 kDa. Beriplex and Profilnine showed fewer bands; Profilnine® mainly exhibited 250, 110, 75 kDa bands and Beriplex® mainly 75 and 66 kDa bands. The other products contain additional bands in the range of 15 to 66 kDa representing albumin and other products. In the SELDI analysis multiple peaks consistent with the SDS-PAGE profile were noted. The immunoblotting showed a major band 70–75 kDa (prothrombin) along with a 50 kDa band representing prethrombin-1. Prior to activation, Feiba® exhibited a distinct additional 37 kDa dense band, (thrombin). SELDI analysis also indicated variable amounts of prothrombin in the products. Upon activation all PCC's were capable of generating thrombin as measured by SELDI and immunoblotting. The prothrombin band completely disappears from all PCCs except Preconetiv®, the prevalent products being prethombin-1 and thrombin. The amount of the prethrombin-1 band varied widely among products; and nearly disappears from all as it is converted to thrombin with time of incubation. The amount of thrombin activity generated from each prothrombin complex was concentration dependent and ranged from 30–1044nm/1.25 units/ml. Octaplex and Cofact produced the strongest thrombin activity whereas Beriplex and Prothromplex produced the least thrombin activity.

Conclusions:
This study shows that despite standardization in factor IX units, at equivalent IX unit potency these agents widely vary in their composition. Beriplex® and Profilnine® represent purer preparations. Upon activation of prothrombin initiated by tissue factor each complex is capable of generating varying amounts of thrombin. Because of these wide variations in protease generation the relative neutralization potential of each of these PCCs may also differ widely. Thus each of these products should thus be considered as a distinct agent and their efficacies individually determined for a given indication.

Disclosures:
No relevant conflicts of interest to declare.