Kenneth D. Friedman

Urgent reversal of warfarin with prothrombin complex concentrate

Summary.  Background: When life‐threatening bleeding occurs in patients on warfarin, timely reversal becomes imperative. In the USA, warfarin effect is commonly reversed with fresh frozen plasma (FFP). The use of FFP is complicated by delays in correction, volume overload and often, inadequate correction. Objective: Evaluate the feasibility and efficacy of a protocol for rapid administration of prothrombin complex concentrate (PCC) in the setting of the urgent need for reversal of warfarin. Methods/patients: We instituted a policy for rapid delivery and administration of PCC. Appropriate patients received 25–50 U kg−1 of PCC. The prothrombin time (PT)/International Normalized Ratios (INR) was recorded before and immediately after dosing, and 24 h postdosing. Patients requiring surgical interventions were cleared for the operating room (OR) immediately. Fifty‐eight patients were treated, with a median age of 75.5 years (range 26–92). Results: The median INR on presentation was 3.8 (1.4–52.8). Immediately following PCC administration the median INR was 1.3 (0.9–5.7), only two patients with INRs exceeding 2.0. The benefit was maintained at 24 h with a median INR of 1.5 (1.1–3.4). Four patients experienced thrombotic events during their hospitalization, (two deep vein thrombosis, two non‐q‐wave myocardial infarction) although none was attributed to PPC therapy. Conclusions: PCC administration is an effective treatment modality for the correction of warfarin anticoagulation in the urgent setting. Advantages over FFP include more timely correction, absence of volume overload and potentially more complete correction. Broader use of PCC in this setting appears to be appropriate.

A phase III trial of recombinant human erythropoietin therapy in nonanemic orthopedic patients subjected to aggressive removal of blood for autologous use: dose, response, toxicity, and efficacy

BACKGROUND: Previous clinical trials have shown that the use of recombinant human erythropoietin (EPO) can facilitate autologous blood donation and reduce allogeneic blood transfusions in autologous blood donors who are anemic at first donation. However, the role of EPO therapy in nonanemic patients remains undefined. To identify this role, a randomized, controlled, multicenter dose‐escalation trial was conducted in patients whose initial hematocrit was > 39 percent (0.39).

Measurement of von Willebrand factor cleaving protease (ADAMTS‐13): results of an international collaborative study involving 11 methods testing the same set of coded plasmas

Summary.  Background: ADAMTS‐13 is a von Willebrand factor (VFW)‐cleaving protease. Its congenital or acquired deficiency is associated with thrombotic thrombocytopenic purpura (TTP) and more rarely with the hemolytic uremic syndrome. We report on a survey evaluating 11 methods for ADAMTS‐13 measurement performed in different labs. Design: Two plasmas, one normal and one from a patient with familial TTP, were mixed at the co‐ordinating center to prepare 6 plasmas with 0%, 10%, 20%, 40%, 80% and 100% ADAMTS‐13 levels. Each plasma was aliquoted and assembled into sets of 60 (coded from 1 to 60), each containing 10 copies of the original 6 plasmas. Plasmas were frozen and shipped in dry ice to 10 labs with a common frozen reference plasma. Laboratories were asked to measure ADAMTS‐13 with their methods. Results were sent to the coordinating center for statistical analysis. Results: Of the 10 methods performed under static conditions 9 were quantitative and one was semiquantitative. One method performed under flow conditions evaluated the extent of cleavage of endothelial cell‐derived ultralarge VWF string‐like structures and expressed results as deficient, normal, or borderline. Linearity (expected‐vs‐observed levels), assessed as the squared correlation coefficient, ranged from 0.98 to 0.39. Reproducibility, expressed as the coefficient of variation for repeated measurements, ranged from < 10% to 83%. The majority of methods were able to discriminate between different ADAMTS‐13 levels. The majority were able to detect the plasma with 0% level and some of them to discriminate between 0% and 10%. Overall the best performance was observed for three methods measuring cleaved VWF by ristocetin cofactor, collagen binding, and immunoblotting of degraded multimers of VWF substrate, respectively. The poor interlaboratory agreement of results was hardly affected by the use of the common standard. The method performed under flow conditions identified the plasmas with 0%, 10%, 20% and 40% activity as deficient in 7, 5, 1 and 3 of the 10 replicate measurements. The plasmas with 80% and 100% were identified as normal in all of the 10 replicate measurements. Conclusions: The survey shows varied performance, but supports an optimistic view about the reliability of current methods for ADAMTS‐13.

Severe deficiency of VWF-cleaving protease (ADAMTS13) activity defines a distinct population of thrombotic microangiopathy patients

BACKGROUND: Severe deficiency of ADAMTS13 activity is a biologic risk factor for thrombotic microangiopathy (TMA). It was hypothesized that severe ADAMTS13 deficiency is associated with a distinct TMA subpopulation.

VWF mutations and new sequence variations identified in healthy controls are more frequent in the African-American population

Diagnosis and classification of VWD is aided by molecular analysis of the VWF gene. Because VWF polymorphisms have not been fully characterized, we performed VWF laboratory testing and gene sequencing of 184 healthy controls with a negative bleeding history. The controls included 66 (35.9%) African Americans (AAs). We identified 21 new sequence variations, 13 (62%) of which occurred exclusively in AAs and 2 (G967D, T2666M) that were found in 10%-15% of the AA samples, suggesting they are polymorphisms. We identified 14 sequence variations reported previously as VWF mutations, the majority of which were type 1 mutations. These controls had VWF Ag levels within the normal range, suggesting that these sequence variations might not always reduce plasma VWF levels. Eleven mutations were found in AAs, and the frequency of M740I, H817Q, and R2185Q was 15%-18%. Ten AA controls had the 2N mutation H817Q; 1 was homozygous. The average factor VIII level in this group was 99 IU/dL, suggesting that this variation may confer little or no clinical symptoms. This study emphasizes the importance of sequencing healthy controls to understand ethnic-specific sequence variations so that asymptomatic sequence variations are not misidentified as mutations in other ethnic or racial groups.

Gain-of-function GPIb ELISA assay for VWF activity in the Zimmerman Program for the Molecular and Clinical Biology of VWD

von Willebrand disease (VWD) is a common bleeding disorder, but diagnosis is sometimes challenging because of issues with the current von Willebrand factor (VWF) assays, VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo), used for diagnosis. We evaluated 113 healthy controls and 164 VWD subjects enrolled in the T.S. Zimmerman Program for the Molecular and Clinical Biology of VWD for VWF:Ag, VWF:RCo, and a new enzyme-linked immunosorbent assay (ELISA)-based assay of VWF-glycoprotein Ib (GPIb) interactions using a gain-of-function GPIb construct (tGPIbα(235Y;239V)) as a receptor to bind its ligand VWF in an assay independent of ristocetin (VWF:IbCo ELISA). Healthy controls, type 1, 2A, 2M, and 2N subjects had VWF:RCo/VWF:Ag ratios similar to the ratio obtained with VWF:IbCo ELISA/VWF:Ag. Type 2B VWD subjects, however, had elevated VWF:IbCo ELISA/VWF:Ag ratios. Type 3 VWD subjects had undetectable (< 1.6 U/dL) VWF:IbCo ELISA values. As previously reported, VWF:RCo/VWF:Ag ratio was decreased with a common A1 domain polymorphism, D1472H, as was direct binding to ristocetin for a 1472H A1 loop construct. The VWF:IbCo ELISA, however, was not affected by D1472H. The VWF:IbCo ELISA may be useful in testing VWF binding to GPIb, discrimination of type 2 variants, and in the diagnosis of VWD as it avoids some of the pitfalls of VWF:RCo assays.

Comparison and stability of ADAMTS13 activity in therapeutic plasma products

BACKGROUND: The von Willebrand factor (VWF)‐cleaving protease, ADAMTS13, is often deficient in cases of thrombotic thrombocytopenic purpura (TTP). The primary treatment of TTP is therapeutic plasma exchange (TPE) utilizing a variety of plasma products that help restore ADAMTS13 activity. However, multiple replacement products are available to choose from. Thawed plasma products have a variable refrigerated shelf life depending on the product type; stability of ADAMTS13 in thawed products stored at 1 to 6°C has not been determined.

Evaluation and comparison of coagulation factor activity in fresh-frozen plasma and 24-hour plasma at thaw and after 120 hours of 1 to 6°C storage.

BACKGROUND: Current transfusion‐related acute lung injury reduction strategies include avoiding transfusion of plasma products collected from female donors or female donors that have been pregnant to reduce transfusion of plasma‐containing HLA antibodies. Such a policy considerably decreases the number of donors available for generation of fresh‐frozen plasma (FFP). To increase the supply of FFP, substitution of 24‐hour plasma (FP24) and thawed plasma (TP) derived from either FFP or FP24 may be viable substitutes. To justify such a policy the coagulation factor content of FFP, FP24, and TP derived from both product types was assessed.

Limitations of the ristocetin cofactor assay in measurement of von Willebrand factor function.

Summary.  Background: Type 2M von Willebrand disease (VWD) is characterized by a qualitative defect in von Willebrand factor (VWF) and diagnosed by a disproportionate decrease in VWF ristocetin cofactor activity (VWF:RCo) as compared with VWF antigen (VWF:Ag). Objective: We report here on the spurious diagnosis of VWD in a patient with a sequence variation in the ristocetin‐binding domain of VWF. Patients/methods: The index case had a VWF:RCo of 11 IU dL−1, with VWF:RCo/VWF:Ag ratio of 0.09. DNA sequencing revealed a novel P1467S mutation in a known ristocetin‐binding region of the A1 domain. Because of the discrepancy between the laboratory findings, consistent with type 2M VWD, and the patient’s lack of bleeding symptoms, further studies were performed to determine whether this mutation affected VWF function or merely reduced its ability to interact with ristocetin. Results: Studies with recombinant VWF showed normal platelet binding with botrocetin, but a significant decrease in binding in response to ristocetin. Ristocetin‐induced binding to recombinant GPIb was also absent, but normal binding was seen when a gain‐of‐function GPIb construct was used in the absence of ristocetin. VWF function under shear stress was normal when analyzed with a cone and plate(let) analyzer. Conclusions: The decreased VWF:RCo seen with the P1467S sequence variation likely represents an artifact as a result of the use of ristocetin to measure VWF activity. The normal VWF function in other assays correlates with the lack of hemorrhagic symptoms, and suggests the need for more physiologically relevant assays of VWF function.

Absent Collagen Binding in a VWF A3 Domain Mutant: Utility of the VWF:CB in Diagnosis of VWD

Von Willebrand factor (VWF) tethers platelets to injured subendothelium through binding sites for collagen and platelet glycoprotein Ib (GPIb). The collagen binding site has been localized to the VWF A1 and A3 domains [1]. This interaction is measured in vitro by the VWF collagen binding assay, or VWF:CB [2,3]. VWD guidelines recently published by the NHLBI suggest restricting use of the VWF:CB to subjects who have abnormal initial screening results with VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo) [4]. The VWF:RCo assay measures VWF-platelet interactions, as induced by the antibiotic ristocetin, and therefore a defect exclusive to the VWF-collagen axis could potentially be missed by omitting the VWF:CB assay. We report here on a subject with type 1 VWD who was discovered to have abnormal collagen binding and a mutation in the VWF A3 domain. The index case and family members were enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWF after informed consent was obtained. VWF:Ag, VWF:RCo, VWF:CB, multimer distribution, and blood type analysis were performed in the clinical hemostasis laboratory of the BloodCenter of Wisconsin as previously described [5]. This subject had VWF:Ag of 41 IU/dL and VWF:RCo 44 IU/dL with normal multimer distribution, but VWF:CB was only 22 U/dL (figure 1A). Her history was significant for epistaxis requiring cautery and hemorrhage following tonsillectomy as a child. She also had prolonged bleeding following a dilation and curettage procedure. Her bleeding score, calculated using the scoring system from the European MCMDM-1 VWD study, was elevated at 8 [6]. DNA sequencing of the full length VWF coding sequence, performed on an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA), showed this subject was heterozygous for a mutation in exon 31 (5356C>G) which led to substitution of aspartic acid for the wild-type histidine at amino acid 1786 (H1786D). Figure 1 Panel A shows VWF levels for the H1786D proband and family members. VWF:Ag, VWF:RCo, and VWF:CB listed for each subject were performed in the clinical laboratory along with blood type and multimer analysis. The VWF:CB used type III collagen. In addition, ... Recombinant VWF containing the 1786D mutation was synthesized via site-directed mutagenesis using the Stratagene QuikChange kit (La Jolla, CA) and expressed in HEK293T cells. Additional constructs were synthesized to incorporate three previously reported mutations in the collagen binding domain, 1731T [7], 1745C, and 1783A [8]. For the research laboratory collagen binding assays, ELISA plates were coated with either type I human placental collagen (Sigma, St. Louis, MO) at 5 μg/mL or type III human placental collagen (Southern Biotech) at 1 μg/mL, diluted in carbonate coating buffer (15 mM sodium carbonate, 35 mM sodium bicarbonate, 3 mM sodium azide), and incubated at 4°C overnight. Either plasma or recombinant VWF diluted in phosphate-buffered saline with 1% BSA was added to each well and incubated at room temperature for 1 hour. VWF bound to collagen was detected using a biotin-conjugated polyclonal antibody to VWF (Dako, Carpinteria, CA) also diluted in phosphate-buffered saline with 1% BSA. Both the 1786D and the 1731T constructs had normal expression compared to wild-type (WT) VWD. Multimer analysis showed a full spectrum of multimers, with no decrease in high molecular weight multimers for the A3 domain mutants. Collagen binding studies were performed in our research laboratory using recombinant WT, 1786D, and 1731T VWF. Results are expressed as a ratio of VWF:CB to VWF:Ag. The previously described 1731T mutation showed a reduction in binding to both type I and type III collagen, at 45% and 50% of WT. In contrast, the 1786D mutant showed barely detectable binding to type I and type III collagen, at <1% of wild-type (figure 1B). Similar lack of binding was seen with the 1745C and 1783A constructs. The profound defect in collagen binding shown with the 1786D construct is supported by studies of the VWF A3 domain crystal structure, which shows H1786 at one of the interfaces with collagen [9]. Replacement of this histidine with alanine also abolished VWF-collagen binding [10]. The S1731T mutation previously reported by Ribba and colleagues led to a decrease in VWF:CB [7]. A recent report by Riddell and colleagues details two A3 domain mutations, W1745C and S1783A, both with a significant decrease in VWF:CB [8]. Three additional A3 domain mutations, Q1734H, I1741T, and Q1762R, have also been reported to be associated with decreased collagen binding although the affected subjects did not display profound bleeding symptoms [11]. Both the VWF A1 and A3 domains are capable of binding collagen [1]. The major binding site is thought to be in the A3 domain [12]. The in vitro collagen binding assays with 1786D VWF did not demonstrate any evidence of compensatory A1 domain binding. It is possible that in vivo the A1 domain binding site can adequately compensate for the H1786D mutation in the A3 domain when VWF is activated under flow conditions, which may decrease the symptoms seen in the patients with this defect. Previous work with recombinant H1786A VWF supports this hypothesis, as it has been demonstrated that under flow conditions, binding to collagen can occur through the VWF A1 domain [13]. It is also possible that the severity of the in vitro phenotype is partially alleviated by the presence of some normal VWF in the heterozygous patients since multimeric VWF would likely contain some monomers with the intact collagen binding site. The H1786D mutation fits best into the current VWD classification system as a type 2M variant, with abnormal VWF function despite normal multimer distribution. Not all 2M mutations, however, have abnormal collagen binding. Previous work from our laboratory demonstrated the A1 domain mutations F1369I and I1425F had normal binding to type III collagen, while an 11 amino acid deletion mutant, Δ1392–1492, did appear to affect collagen binding [14]. These patients, however, all presented with decreased VWF:RCo/VWF:Ag ratios, prompting their inclusion as 2M mutations. The H1786D mutation described here has a different effect on VWF function than those mutations considered as classic 2M. Defects in collagen binding may ultimately require an alternate classification scheme to distinguish them from platelet-binding defects. This case also suggests that the VWF:CB assay may have utility in diagnosis of variant VWD.