PMC3870044_01
Factor V and TFPI in Patient Plasma. (A and B) Western blots of plasma for FV. Plasma samples were size-fractionated on 4-20% polyacrylamide SDS-gels, followed by western blotting with polyclonal goat-anti-human FV IgG. (A) Lanes were loaded with purified FV (FV); pooled normal plasma (PNP); patient plasma (Pt); patient plasma before (Pre), and 1, 2, or 3 hours after FFP infusion; and plasma from a patient with known severe FV deficiency (V-DP). (B) Lanes are loaded with purified FV (FV); pooled normal plasma (PNP); patient plasma before (Pre), and 3, 6, 24 and 48 hours after intravenous IgG; normal platelets; and patient platelets. For both panels, positions of molecular mass standards in kilo-Daltons are shown on the left, along with an indicator for the normal position of migration for human FV. (C) TFPI. Total TFPI and TFPIalpha in normal plasma, patient plasma from two separate draws, and plasma from two different FV deficient patients were measured by ELISA. Results are compared to those for a cohort of normal female donors (mean +- 1 S.D., n = 15). Patient Sample 1 corresponds to the "Pre" sample in panel A. Patient Sample 2 corresponds to the "Pre" sample in panel B.
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F1
ELISAs for anticardiolipin and anti-beta-2-glycoprotein antibodies (IgG, IgM) were unremarkable. ELISA and western blots (Figure 1A) demonstrated absence of FV antigen in the patient's plasma. However, there was no increase in plasma FV activity or antigen after infusion of three units of fresh frozen plasma (FFP) (Figure 1A), indicating that the FV deficiency was due, at least in part, to rapid selective removal from the circulation. We empirically treated the patient with intravenous immunoglobulin (IV IgG), which also had little effect (Figure 1B). On western blot (Figure 1B) and ELISA, FV antigen was detectable in the patient's platelets at a roughly comparable amount to FV in normal control platelets. As FV in human platelets appears to be primarily of plasma origin, the results in Figure 1B are consistent with the premise that our patient's liver makes FV, and suggest that the absence of bleeding symptoms may be related to a normal platelet FV pool. TFPIalpha in plasma from two patients with severe congenital FV deficiency (Figure 1C). TFPI is a Kunitz-type protease inhibitor that binds and inhibits the activities of factor Xa and the factor VIIa/tissue factor complex. TFPIalpha in our patient's plasma (Figure 1C) at two time points corresponding to the pre-plasma infusion (Sample 1) and pre-IVIG infusion (Sample 2) in Figures 1A and 1B, respectively. Total TFPI and TFPIalpha were below the normal range established with a gender-matched cohort, but were not as low as those observed in plasma from congenital FV deficient patients. These results are consistent with the reported effects of reduced plasma FV on TFPI, and indicate that acquired FV deficiency in this patient is unlikely to be due to an antibody to TFPI that clears the TFPI-FV complex.