PMC4147383_01
Plasma levels of factor VIII clotting activity (FVIII:C) and von Willebrand factor parameters (VWF:Ag and VWF:Ac) at presentation and during course of treatment. The first set of data points (d0) shows FVIII and VWF levels at initial presentation, while the second set of data points (d3) shows FVIII and VWF levels immediately before the administration of 2,000 IU of FVIII/VWF concentrate, as indicated by the dashed arrow. To assess incremental recovery and residence time of the infused FVIII/VWF, subsequent blood samples were drawn 15 min (d3 ), 60 min (d3  ), and 120 min (d3   ) after concentrate administration. One day later (d4), immunosuppressive therapy with prednisolone was started at a daily dose of 100 mg, which was gradually tapered to 20 mg on d31, as indicated by the solid arrows. One week later, there was a steep decline in FVIII and VWF, both of which readily responded to re-escalating the prednisolone dose to 50 mg per day.
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FVIII clotting activity (FVIII:C) was reduced to 2% (normal range, 60-160%) [Figure 1]. Consistently, VWF antigen (VWF:Ag) was <3% (normal range, 60-200%), and VWF activity (VWF:Ac), as determined by the INNOVANCE  GPIb binding assay (Siemens Healthcare), was 4% (normal range, 61-179%) [Figure 1]. Multimer analysis was carried out on a plasma sample obtained on day 3, on which VWF parameters had spontaneously increased to 15-25% [Figure 1], revealing the presence of ultralarge VWF plasma multimers and absence of the typical multimeric triplet structure [Figure 2B].The patient received 2,000 IU of a VWF-containing FVIII concentrate (Haemate  P, CSL Behring) on day 3. While there was only a marginal and short-lasting increment in FVIII:C, VWF:Ag and VWF:Ac increased to 114% and 86%, respectively, and temporarily steadied at these levels for 24 hrs before declining back to 34-41% on day 5 [Figure 1]. Thus the diagnosis of "late-onset SLE" was made based on the following clinical and laboratory criteria: skin rash, Coombs-positive hemolytic anemia, positive antiphospholipid antibodies and ANAs, lymphocytopenia, and C3 decrease.Immunosuppressive therapy with prednisolone was initiated on day 4 at a daily dose of 100 mg, which resulted in rapid elevations of VWF:Ag and VWF:Ac within four days and a more delayed recovery of FVIII:C to normal levels within 13 days into treatment [Figure 1]. Moreover, the anti-FVIII- and anti-VWF-IgG titers decreased to 0.21 OD and 0.8 OD, respectively, on day 31.The daily prednisolone dose was gradually tapered and reduced to 20 mg on day 31, which resulted in a sharp decline of VWF:Ag, VWF:Ac, and FVIII:C one week later [Figure 1] and was accompanied by a temporary increase in the anti-FVIII- and anti-VWF-IgG titers to 0.63 OD and 1.15 OD, respectively, on day 45.
Results of the in-house ELISA for the detection of anti-VWF-IgG and multimer analysis. (A) Immobilized recombinant human VWF derived from CHO cells was used to measure IgG autoantibodies to VWF. The titer was 2.6 OD at presentation (day 0) and decreased to 0.8 OD during effective prednisolone therapy (day 31). A steep decline in VWF on day 38 following reduction of the daily prednisolone dose to 20 mg a week earlier was accompanied by a temporary increase in the anti-VWF-IgG titer to 1.2 OD on day 45. After six months of follow-up, the titer had decreased to 0.6 OD, but was still above the reference range of the ELISA as indicated by the shaded red area. (B) Before the initiation of prednisolone, presence of unusually large VWF plasma multimers (arrows) and absence of typical VWF triplets (arrow heads) indicated decreased proteolysis of newly released, but rapidly cleared VWF (day 3). The multimeric pattern normalized during effective immunosuppressive therapy with prednisolone (day 24). A high-resolution 1.6% agarose gel (left four lanes) and a low-resolution 1.2% agarose gel (right two lanes), which allows for better migration of the larger multimers into the gel, are shown. Different dilutions of the patient plasma obtained on day 3 are indicated in the high-resolution gel. NHP denotes normal human plasma.
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IgG antibody of 2.6 OD (normal range, <0.4 OD) [Figure 2A]. Importantly, addition of rhVWF to the patient plasma completely blocked this reaction [data not shown], further confirming specificity of the ELISA. Multimer analysis was carried out on a plasma sample obtained on day 3, on which VWF parameters had spontaneously increased to 15-25% [Figure 1], revealing the presence of ultralarge VWF plasma multimers and absence of the typical multimeric triplet structure [Figure 2B].The patient received 2,000 IU of a VWF-containing FVIII concentrate (Haemate  P, CSL Behring) on day 3. Normalization of VWF parameters was accompanied by the loss of ultralarge plasma multimers and appearance of typical VWF triplets on day 24 [Figure 2B].