Factors affecting the relative distribution of high and low molecular weight forms of factor VIII

Abstract

We have recently shown that in heparinized plasma, approximately 50% of the Factor VIII procoagulant activity has a very low molecular weight (VLMW) of approximately 20,000. The balance of the procoagulant activity is in a high molecular weight form (HMW) which is associated with the carrier protein (VIII:RAG). On the other hand, Factor VIII which has been purified from citrated plasma is known to have a molecular weight of about 10 6 daltons and can be separated after CaCl 2 dissociation into a high molecular weight carrier (VIII:RAG, molecular weight 10 6) and a low molecular weight procoagulant component (LMW, molecular weight 150,000). We have now studied the factors which influence the aggregation of the native VLMW form of Factor VIII. The ratio of VLMW:HMW in heparinized plasma is 1:1 and decreases to 1:5 when CPD is used as anticoagulant. VLMW is not detectable in EDTA plasma. The collection of blood into heparin and graduated amounts of CPD results in an increased proportion of VLMW as the CPD concentration is reduced. The ratio of VLMW:HMW remains constant at 1:1 during storage of the heparinized plasma but decreases to almost 0:1 after 24 hours in citrated plasma. When separated from HMW, the VLMW alone aggregates on storage at −80°C for 72 hours producing forms of intermediate molecular weight although the majority of the activity is in a high molecular weight form. This aggregated VLMW material is devoid of VIII:RAG and dissociates into low and high molecular weight peaks following chromatography with buffer containing 0.25 M CaCl 2. Both cryoprecipitation and PEG precipitation of heparinized plasma shift the VLMW:HMW ratio from 1:1 to 0:1. These results show that the usual conditions of storage and processing of plasma result in the aggregation of the normal VLMW to high molecular weight forms which are artifacts of the isolation process, and are further evidence against the “one molecule” theory of Factor VIII structure.