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 degrees C has not been determined. STUDY DESIGN AND METHODS: ADAMTS13 activity was measured in three types of plasma products and cryoprecipitate. Fresh-frozen plasma (FFP) aliquots and cryoprecipitate-poor plasma (CPP) products were produced from 10 whole-blood (WB) donations. Twenty-four-hour plasma products were manufactured from 10 additional WB donations. ADAMTS13 activity in these products at time of thaw and after 5 days of storage at 1 to 6 degrees C was measured with a modified version of the FRETS-VWF73 fluorogenic assay. ADAMTS13 activity at time of thaw was measured in 10 units of cryoprecipitate and five related CPP products. RESULTS: ADAMTS13 is present in similar amounts in FFP, CPP, and 24-hour plasma products. Storage at 1 to 6 degrees C for up to 5 days did not significantly diminish ADAMTS13 activity. The concentration of ADAMTS13 in cryoprecipitate was significantly higher than that observed in plasma products. CONCLUSION: FFP, CPP, and 24-hour plasma products should be equally effective for ADAMTS13 restoration through TPE and should remain so for the duration of the shelf life of the thawed products.     

Incidence of allergic reactions with fresh frozen plasma or cryo‐supernatant plasma in the treatment of thrombotic thrombocytopenic purpura

Plasma replacement for thrombotic thrombocytopenic purpura (TTP) is accomplished with various plasma products. This study sought to determine the incidence of allergic reactions with FFP or CPP as replacement in therapeutic plasma exchange (TPE). Forty‐one TTP patients were identified retrospectively who received TPE replacement with either FFP (n=21) or CPP (n=20). Anti‐histamine was administered prophylactically following the initial occurrence of an allergic reaction (urticaria, respiratory distress, or anaphylaxis with hypotension). Fifty‐one allergic reactions occurred in 65.8% of patients. Urticaria comprised 49 of 51 (96%) of reactions and respiratory distress the remaining 4%. No anaphylaxis occurred. Nineteen urticarial reactions occurred in 50% of CPP recipients compared to 71% of FFP recipients (P=0.28). Anti‐histamine breakthrough occurred in 36.3% of patients who experienced a previous allergic reaction with CPP and 37.5% with FFP (P=1.0). The overall risk of allergy per unit of plasma was 1.37% (1.23 % CPP, 1.48% FFP), comparable to estimates in non‐TTP recipients. The median number of donor exposures preceding the first allergic reaction was 35 and 32, CPP and FFP, respectively (P=0.63). The mean volume of plasma transfused prior to reaction was 9,883 mL for CPP and 9,348 mL for FFP (P=0.85). Neither product was advantageous in preventing allergic complications. Because of the large volume, the number of donor exposures, and prolonged duration of therapy, allergic reactions to plasma are common (65.8%) in the treatment of TTP. J. Clin. Apheresis. 16:134–138, 2001. © 2001 Wiley‐Liss, Inc.     

Comparison of von Willebrand factor antigen, von Willebrand factor-cleaving protease and protein S in blood components used for treatment of thrombotic thrombocytopenic purpura

Replacement of normal levels of von Willebrand factor-cleaving protease (VWF:CP, ADAMTS13) activity from infused plasma is important in plasma exchange (PEX) for the treatment of thrombotic thrombocytopenic purpura (TTP) patients. We have studied the VWF:CP activity, VWF multimer distribution, VWF:Ag, protein S (PS) activity and free PS antigen levels in fresh frozen plasma (FFP), cryosupernatant (CSP) and virally inactivated components treated with methylene blue/light (MB) or solvent detergent (SD) processes. VWF:CP activity was normal in all components tested and was retained following overnight storage at room temperature. CSP and SD plasma contained reduced levels of the highest molecular weight VWF multimers. Protein S activity was reduced below the normal range in SD plasma, but within the normal range for the other components tested. Virally inactivated SD- and MB-treated plasma may be an effective alternative to FFP and CSP in PEX for TTP. Reduced PS activity in SD plasma may predispose to venous thromboembolism, especially if infused in large volumes.     

The pathogenicity of von Willebrand factor in thrombotic thrombocytopenic purpura: reconsideration of treatment with cryopoor plasma

New developments in the understanding of thrombotic thrombocytopenic purpura (TTP) provide opportunities for improved patient care. A widely held historical model of TTP microvascular thrombosis implicated circulating ultra large von Willebrand factor (ULVWF) in causing spontaneous platelet (PLT) aggregation. From this pathogenic model, concerns about ULVWF in fresh-frozen plasma (FFP) used to treat patients led to widespread use of cryopoor plasma (CPP) as an alternative. There is scant evidence, however, that circulating ULVWF contributes to microvascular thrombosis in TTP. New evidence suggests that the formation of PLT aggregates in TTP may be mediated by VWF in the process of being released from endothelium. Moreover, clinical studies do not demonstrate superior efficacy of CPP compared to FFP in the treatment of TTP. Because CPP may have reduced concentrations of factors important in the treatment of TTP, including ADAMTS13 metalloprotease, a reappraisal of the use of CPP in the treatment of TTP is warranted.     

The use of 50% albumin/plasma replacement fluid in therapeutic plasma exchange for thrombotic thrombocytopenic purpura

Background: Acquired thrombotic thrombocytopenic purpura (TTP) is caused by a deficiency of von Willebrand factor‐cleaving protease (ADAMTS13) and is often associated with the presence of an antibody inhibiting the activity of the protease. Typically, 1–1.5 plasma volume exchanges are performed daily until symptoms have resolved and the platelet count exceeds 150,000/µl. Plasma is the usual replacement fluid as it provides a source of functional ADAMTS13, thus exposing patients to large volumes of plasma. Historically, Puget Sound Blood Center (PSBC) has performed therapeutic plasma exchange (TPEs) for TTP using 5% albumin for the first half of the procedure followed by plasma for the remainder. We sought to assess the efficacy of this approach. Study Design and Methods: All TPEs performed for the diagnosis of TTP by the PSBC apheresis service from January 1, 2004 through December 31, 2011 were reviewed. Response time, remission rates, relapses, and adverse events were evaluated for those patients with documented ADAMTS13 levels ≤10%. Comparisons were made with published data on TTP patients treated using 100% plasma replacement. Results: Twenty‐one patients required a median of 11 TPEs. Median time to response was 14 days. Ninety percent of patients responded to TPE. Among patients achieving remission, 53% relapsed. Out of 283 total procedures, there were 74 procedures with a documented adverse event (26%), mostly mild allergic reactions. Conclusions: TPE with an albumin/plasma replacement is safe and well‐tolerated. Remission and relapse rates were comparable to those reported using 100% plasma replacement. J. Clin. Apheresis, 28:416–421, 2013. © 2013 Wiley Periodicals, Inc.     

The active conformation of von Willebrand factor in patients with thrombotic thrombocytopenic purpura in remission

Summary.  Background:  Functional deficiency of ADAMTS13 in thrombotic thrombocytopenic purpura (TTP) patients is associated with circulating ultralarge von Willebrand factor (VWF) molecules that display spontaneous platelet-binding capacities. Upon remission, however, ADAMTS13 activity does not always return to baseline. Objective:  To study ADAMTS13 and VWF-related features in TTP patients in remission. Methods:  ADAMTS13 activity, anti-ADAMTS13 antibodies, VWF antigen, ultralarge VWF and levels of VWF that circulate in a glycoprotein Ibα-binding conformation were determined in plasma samples of 22 acquired TTP patients in remission between 1 month and 6 years after achieving remission. The composition of active multimers was investigated with a novel immunoprecipitation assay based on monoclonal antibody AU/VWF-a12, which specifically recognizes the active conformation of VWF. Results:  ADAMTS13 activity was undetectable in 23% of the patients, even years after they had achieved remission, and lack of ADAMTS13 activity was associated with increased active VWF levels and the presence of ultralarge VWF multimers. Active VWF levels and ultralarge VWF were also associated with blood groups. Results from immunoprecipitation experiments revealed the full range of multimers to be present. Conclusion : ADAMTS13 deficiency and the concurrent presence of ultralarge VWF and increased active VWF levels can be detected in TTP patients for years after they have achieved remission. Immunoprecipitation results suggest that the active conformation of VWF may be present in the lower molecular weight multimers, but future studies are necessary to confirm our findings.     

Rituximab and intermediate‐purity plasma‐derived factor VIII concentrate (Koate®) as adjuncts to therapeutic plasma exchange for thrombotic thrombocytopenic purpura in patients with an ADAMTS13 inhibitor

Thrombotic thrombocytopenic purpura (TTP) results from a congenital or acquired deficiency of the von Willebrand factor (vWF)‐cleaving protease ADAMTS13. The disease can be fatal and hence treatment should be initiated promptly. Therapeutic plasma exchange (TPE) remains the standard treatment along with adjunct therapies including steroids and immunosuppressive drugs. Addition of rituximab to TPE has been shown to be beneficial in refractory/relapsing TTP; however, TPE results in removal of rituximab from the circulation requiring more frequent dosing of rituximab to achieve a favorable outcome. The intermediate‐purity plasma‐derived Factor VIII concentrate (FVIII) Koate® contains the highest amount of ADAMTS13 activity yet reported and has been used successfully in treating congenital TTP. Here we report our experience with addition of this FVIII concentrate to rituximab, corticosteroids and TPE in three TTP patients with an ADAMTS13 inhibitor to permit withholding TPE for 48 h after rituximab infusion. J. Clin. Apheresis 30:50–54, 2015. © 2014 Wiley Periodicals, Inc.     

ADAMTS13: origins,applications, and prospects

ADAMTS13 is an enzyme that acts by cleaving prothrombotic von Willebrand factor (VWF) multimers from the vasculature in a highly regulated manner. In pathologic states such as thrombotic thrombocytopenic purpura (TTP) and other thrombotic microangiopathies (TMAs), VWF can bind to the endothelium and form large multimers. As the anchored VWF chains grow, they provide a greater surface area to bind circulating platelets (PLTs), generating unique thrombi that characterize TTP. This results in microvasculature thrombosis, obstruction of blood flow, and ultimately end‐organ damage. Initial presentations of TTP usually occur in an acute manner, typically developing due to an autoimmune response toward, or less commonly a congenital deficiency of, ADAMTS13. Triggers for TMAs that can be associated with ADAMTS13 deficiency, including TTP, have been linked to events that place a burden on hemostatic regulation, such as major trauma and pregnancy. The treatment plan for cases of suspected TTP consists of emergent therapeutic plasma exchange that is continued on a daily basis until normalization of PLT counts. However, a subset of these patients does not respond favorably to standard therapies. These patients necessitate a better understanding of their diseases for the advancement of future therapeutic options. Given ADAMTS13’s key role in the cleavage of VWF and the prevention of PLT‐rich thrombi within the microvasculature, future treatments may include anti‐VWF therapeutics, recombinant ADAMTS13 infusions, and ADAMTS13 expression via gene therapy.     

Emerging roles of adjunct therapies in acquired thrombotic thrombocytopenia purpura

• Acquired thrombotic thrombocytopenia purpura (aTTP) is caused by autoantibody‐mediated severe deficiency of the von Willebrand factor (vWF) cleaving protease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13), with subsequent accumulation of ultra‐large vWF‐multimers that spontaneously form platelet‐VWF complexes and microthrombi within the microcirculation.
• Therapeutic plasma exchange (TPE), by removing autoantibodies and excess ultra‐large vWF multimers and replenishing ADAMTS13 activity, remains the urgent primary initial treatment. Although heterogeneity in treatment exists, most centers add upfront immunosuppression with steroids, and many also add upfront rituximab. Refractoriness, exacerbation and relapse are commonly treated with adjunct rituximab.
• Despite adjunct steroids and rituximab, TTP refractoriness, exacerbation, relapse, morbidity, and mortality remain problematic. Newer adjunct therapies include suppression of ADAMTS13 autoantibody production via plasma cell depletion, inhibition of vWF‐platelet interaction, and replenishment of ADAMTS13 function with recombinant ADAMTS13 protein.

     

Platelet reactive conformation and multimeric pattern of von Willebrand factor in acquired thrombotic thrombocytopenic purpura during acute disease and remission

Summary. Background: Binding of von Willebrand factor (VWF) multimers of ultra‐large size to platelets is considered the triggering mechanism of microvascular thrombosis in thrombotic thrombocytopenic purpura (TTP). Objective: To assess the potential of VWF‐related measurements as markers of disease activity and severity in TTP. Methods: VWF antigen (VWF:Ag), platelet glycoprotein‐Ib‐α binding‐conformation (GPIb‐α/BC) and multimeric pattern were investigated in 74 patients with acquired TTP during acute disease, remission or both and 73 healthy controls. In patients with both acute and remission samples available, VWF ristocetin co‐factor activity (VWF:RCo) and collagen binding (VWF:CB) were also measured. The relationships of study measurements with the presence of acute disease and remission and with markers of disease severity were assessed. Results: VWF:Ag and VWF‐GPIb‐α/BC were higher in TTP patients than controls (P < 0.001 and 0.004). However, there was no statistically significant difference in VWF‐GPIb‐α/BC between samples obtained during acute TTP and remission. Larger VWF multimers were frequently lacking in acute TTP patients, who displayed ultra‐large multimers at remission. The degree of loss of larger VWF multimers correlated with the degree of abnormality of hemoglobin, platelet counts and serum lactate dehydrogenase (LDH) and was associated with low levels of both VWF:RCo/Ag and VWF:CB/Ag ratios. Conclusions: In TTP the platelet‐binding conformation of VWF is not exclusively present in acute disease, nor is it associated with its clinical and laboratory severity. The loss of larger VWF multimers, accompanied by low VWF:RCo/Ag and VWF:CB/Ag ratio values, represents an index of disease activity and severity of acute TTP in patients with severe ADAMTS‐13 deficiency.     

Quantitative and qualitative analysis of coagulation factors in cryoprecipitate prepared from fresh‐frozen plasma inactivated with amotosalen and ultraviolet A light

BACKGROUND: There were no previous studies about the quality of cryoprecipitate prepared from fresh‐frozen plasma (FFP) inactivated with amotosalen and ultraviolet A (UVA) light. The aim of this study was to analyze the quantity and quality of coagulation factors in cryoprecipitate prepared from FFP treated with amotosalen and UVA light. STUDY DESIGN AND METHODS: FFP was obtained from whole blood donations and inactivated with amotosalen and UVA light according to the manufacturer's instructions. Fibrinogen, factor VIII (FVIII), von Willebrand factor antigen (VWF : Ag) and activity (VWF : RCo), the von Willebrand factor cleavage protease activity (ADAMTS‐13), and the multimeric structure of VWF were analyzed. RESULTS: The content of fibrinogen, FVIII, and ADAMTS‐13 was lower in cryoprecipitates prepared from amotosalen‐treated plasma when compared with cryoprecipitates prepared from nontreated plasma (35, 40, and 18% loss, respectively). The quantity and quality of VWF as well as VWF multimer patterns were not affected by the inactivation method. CONCLUSION: Cryoprecipitates prepared from amotosalen‐treated FFP contained significantly reduced levels of fibrinogen, FVIII, and ADAMTS‐13. However, the VWF quantity and quality was well preserved.     

Disulfide bond reduction of von Willebrand factor by ADAMTS‐13

See also Lenting PJ, Rastegarlari G. ADAMTS‐13: double trouble for von Willebrand factor. This issue, pp 2775–7. Summary. Background: von Willebrand factor (VWF) released from endothelial cells is rich in ultra‐large (UL) multimers that are intrinsically active in binding platelets, whereas plasma‐type VWF multimers require shear stress to be activated. This functional difference may be attributed to thiols exposed on the surface of plasma‐type VWF multimers, but not on ULVWF multimers. Shear stress induces the exposed thiols to form disulfide bonds between laterally apposed plasma‐type VWF multimers, leading to enhanced VWF binding to platelets. Objectives: We tested a hypothesis that ADAMTS‐13 has a disulfide bond reducing activity that regulates shear‐induced thiol‐disulfide exchange of VWF. Methods: Thiol blocking agents and active thiol bead capturing were used to identify and locate this activity, along with truncated ADAMTS‐13 mutants. Results: ADAMTS‐13 contains a disulfide bond reducing activity that primarily targets disulfide bonds in plasma‐type VWF multimers induced by high shear stress or formed with thiol beads, but not disulfide bonds in native multimeric structures. Cysteine thiols targeted by this activity are in the VWF C‐domain and are known to participate in shear‐induced thiol‐disulfide exchange. ADAMTS‐13 contains cysteine thiols that remain exposed after being subjected to hydrodynamic forces. Blocking these active thiols eliminates this reducing activity and moderately decreases ADAMTS‐13 activity in cleaving ULVWF strings anchored to endothelial cells under flow conditions, but not under static conditions. This activity is located in this C‐terminal region of ADAMTS‐13. Conclusions: This novel disulfide‐bond‐reducing activity of ADAMTS‐13 may prevent covalent lateral association and increased platelet adherence of plasma‐type VWF multimers induced by high fluid shear stress.     

Coagulation factor levels in cryosupernatant prepared from plasma treated with amotosalen hydrochloride (S-59) and ultraviolet A light

BACKGROUND: The standard treatment for thrombotic thrombocytopenic purpura (TTP) is plasma exchange with fresh-frozen plasma (FFP). Exposure to large volumes of FFP increases the risk of transfusion-transmitted infections. Cryosupernatant (CSP) offers a theoretical advantage over FFP, because it lacks the large von Willebrand factor (VWF) forms implicated in the pathogenesis of TTP. This study compared the hemostatic variables of CSP prepared from FFP treated with a photochemical pathogen inactivation process to CSP prepared from conventional FFP. STUDY DESIGN AND METHODS: Forty CSP units were prepared from North American blood group A donor FFP. Twenty-one of the FFP units were individually treated with amotosalen hydrochloride (S-59) and ultraviolet A light (test, photochemically treated FFP), and 19 units were not treated (control, FFP). RESULTS: Hemostatic variables of test and control CSP were similar and within reported ranges for conventional FFP with the exception of those properties depleted in CSP. VWF-cleaving protease activity (VWF:CP) and protein S (PS) levels (total and free antigen and activity) were within the conventional FFP reference range for test and control CSP. There were statistical differences between test and control CSP for alpha(2)-antiplasmin, antithrombin, protein C, and VWF:CP on a per-volume basis, but all levels were within the reference range for FFP, and the differences were not significant when expressed per gram of CSP protein. CONCLUSION: S-59-treated CSP retained adequate levels of critical plasma proteins for plasma exchange therapy in acute TTP. The data indicate good preservation of hemostasis control proteins such as PS, alpha(2)-antiplasmin, and VWF:CP activity (ADAMTS13).     

A shear-based assay for assessing plasma ADAMTS13 activity and inhibitors in patients with thrombotic thrombocytopenic purpura

BACKGROUND: Severe deficiency of plasma ADAMTS13 activity is a frequent finding in patients with hereditary and acquired thrombotic thrombocytopenic purpura (TTP). To date, plasma ADAMTS13 activity is determined by cleavage of either predenatured von Willebrand factor (VWF) or small peptides derived from the VWF‐A2 domain. The physiologic relevance of the assay results is uncertain. STUDY DESIGN AND METHODS: We sought to develop a novel shear‐based assay to assess plasma ADAMTS13 activity and inhibitors. We also compared this assay with a fluorogenic peptide assay. RESULTS: We found that an incubation of purified plasma VWF with 0.5 to 1.0 µL of citrated plasma under constant vortexing at 2500 rpm for 60 minutes in the presence of 5 mmol/L CaCl₂ and 1.7 µmol/L ZnCl₂ and low concentration of NaCl resulted in the maximal cleavage of VWF. The cleavage product could be separated by a 2.5% agarose gel and detected by Western blotting. The assay revealed that plasma and recombinant ADAMTS13 are highly sensitive to inhibition by zinc and chloride ions. Under the optimal conditions, the shear‐based assay appeared to be more sensitive than the guanidine‐denaturization assay for determining plasma ADAMTS13 activity. CONCLUSIONS: Our fluid shear‐based assay may be useful for investigating basic biologic function and regulation of ADAMTS13 metalloprotease. It may also be applicable for assessing plasma ADAMTS13 activity and inhibitors in TTP patients.     

Platelet recovery rate during plasma exchange predicts early and late responses in patients with thrombotic thrombocytopenic purpura (CME)

BACKGROUND: Therapeutic plasma exchange (TPE) is the first‐line therapy for patients with thrombotic thrombocytopenic purpura (TTP). However, therapeutic response to TPE and late prognosis vary among different patients, and predictors of these outcomes may help customize treatments to individual patients. STUDY DESIGN AND METHODS: We retrospectively examined the platelet (PLT) recovery rate (PRR) in 64 consecutive patients with initial episode of TTP who received TPE in our institution between 2003 and 2010. PRRs were calculated by linear regression of the PLT counts at the start and during the first few days of TPE treatment. Its relationship with remission in response to TPE, exacerbation and relapse, and survival was analyzed by univariate and multivariate analysis. RESULTS: With multivariate analysis, which included ADAMTS13 activities, patients with a PRR by Day 3 (PRR3) of 5 × 10⁹/L per 24 hours or above were 18 times more likely to achieve remission in response to TPE than those with a lower PRR3 (p < 0.001). In addition, short‐term exacerbations and relapses beyond 1 month of remission occurred almost exclusively in patients with a PRR3 of 5 × 10⁹/L per 24 hours or above. Survival was significantly better in these patients than in patients with PRR3 below the cutoff (p < 0.001), and the hazard ratio adjusted for ADAMTS13 and age was 23.2 (p < 0.005). CONCLUSION: PRR3 with a cutoff of 5 × 10⁹/L per 24 hours provides a practical approach to risk stratify TTP patients receiving TPE early in their treatment and may guide the decision making both at initial encounters and during the long‐term follow‐up.     

Cryoprecipitate poor plasma does not improve early response in primary adult thrombotic thrombocytopenic purpura (TTP)

Thrombotic thrombocytopenic purpura (TTP) is a potentially fatal disease that is treated with plasma exchange and typically with replacement with fresh frozen plasma (FFP). This approach results in an approximate 50% response rate following 1 week of therapy and 80% survival. Cryoprecipitate poor plasma (CPP) is plasma from which the cryoprecipitate fraction is removed. CPP has been reported to be successful as salvage therapy in refractory TTP and has been suggested to be superior to FFP in retrospective studies. The present report compares initial therapy of TTP with exchange using replacement with either FFP or CPP in a multi-institutional prospective randomized study performed by the North American TTP Group (NATG Group) from 1993 to 1995. Initial therapy also included corticosteroids. Antiplatelet drugs or vinca alkaloids were not employed. A severity score index, response score, and individual clinical parameters (platelet count, LDH x upper limit of normal, hemoglobin level, and creatinine) were compared at their nadir or peak values, baseline, and days +6 and +13 of therapy. Thirteen patients were randomized to FFP exchange and 14 to CPP exchange. Results were equivalent for all parameters. Survival was equal with three deaths in each group. These data indicate that the efficacy of FFP and CPP are the same in the initial treatment of TTP in adults.     

Recombinant ADAMTS13 normalizes von Willebrand factor‐cleaving activity in plasma of acquired TTP patients by overriding inhibitory antibodies

Summary. Background: Severe deficiency of the von Willebrand factor (VWF)‐cleaving protease ADAMTS13 as observed in acquired thrombotic thrombocytopenic purpura (TTP) is caused by inhibitory and non‐inhibitory autoantibodies directed against the protease. Current treatment with plasma exchange is considered to remove circulating antibodies and to concurrently replenish the deficient enzyme. Objectives: To explore the use of recombinant ADAMTS13 (rADAMTS13) as a potential therapeutic agent in acquired TTP, we investigated its efficacy in normalizing VWF‐cleaving activity in the presence of ADAMTS13 inhibitors. Methods: Thirty‐six plasma samples from TTP patients were adjusted to predefined inhibitor titers, and recovery of ADAMTS13 activity was analyzed following supplementation with rADAMTS13. Results: We showed a linear relation between the inhibitor titer measured and effective rADAMTS13 concentration necessary for reconstitution of VWF‐cleaving activity in the presence of neutralizing autoantibodies. Conclusions: Our results support the further investigation of the potential therapeutic applicability of rADAMTS13 as an adjunctive therapy in acquired TTP.     

Comparison of ADAMTS13 and Von Willebrand factor levels and activities,and plasminogen levels,in plasma products currently available for the treatment of thrombotic thrombocytopenic purpura in South Africa

Objective

Thrombotic thrombocytopenic purpura (TTP) results from a deficiency in the Von Willebrand factor (VWF) cleaving protease, ADAMTS13. Treatment involves plasma exchange (PEX) therapy with either fresh frozen plasma (FFP), cryosupernatant (CSP) or solvent/detergent-treated plasma (SDP), available in South Africa as Bioplasma FDP. The aim of the study was to generate in vitro data on these products, and to explore possible differences between the products that may offer treatment advantages.

Therapeutic plasma exchange for thrombotic thrombocytopenic purpura with refractory thrombocytopenia

Thrombotic thrombocytopenic purpura (TTP) is an acute, life‐threatening illness with disseminated platelet‐rich thromboses of small vessels that variably presents with the classic clinical “pentad” of microangiopathic hemolytic anemia, thrombocytopenia, fever, altered mental status, and acute kidney injury. Most cases are caused by an acquired autoantibody to ADAMTS13, a metalloproteinase that cleaves large von Willebrand Factor (vWF) multimers. The mainstay of treatment is daily therapeutic plasma exchange (TPE), sometimes with adjunctive pharmacologic immunosuppression. TPE is generally continued until the platelet count is greater than 150 × 10³/µL and the lactate dehydrogenase is near normal for 2‐3 consecutive days. Unfortunately, there is no clear guidance for when thrombocytopenia is refractory for a prolonged period of time. The following case describes such a scenario in which consecutive ADAMTS13 activity and inhibitor levels were used to guide the decision to stop treatment with TPE in a patient who failed to recover their platelet count.     

Novel monoclonal antibody-based enzyme immunoassay for determining plasma levels of ADAMTS13 activity

BACKGROUND: ADAMTS13 specifically cleaves unusually large von Willebrand factor (VWF) multimers, which induce platelet thrombi formation under high shear stress. ADAMTS13 activity is deficient in patients with thrombotic thrombocytopenic purpura (TTP). The determination of plasma levels of ADAMTS13 activity is a prerequisite for a differential diagnosis of thrombotic microangiopathies. Here, a unique and highly sensitive enzyme immunoassay (EIA) of ADAMTS13 activity is described. STUDY DESIGN AND METHODS: ADAMTS13 hydrolyzes the peptide bond between Y1605 and M1606 of VWF. In this assay, a recombinant fusion protein (GST-VWF73-His) is used as a substrate. A panel of mouse monoclonal antibodies (MoAbs) that specifically recognizes Y1605, which is the C-terminal edge residue of the VWF-A2 domain and is generated by the enzymatic cleavage, has been produced. These antibodies were prepared with a synthetic decapeptide, termed N-10 (1596-DREQAPNLVY-1605), as the immunogen. Twenty-six clones specific to N10 were obtained, and one anti-N10 MoAb was used in this study. RESULTS: With horseradish peroxidase-conjugated anti-N10 MoAb, a standard enzyme assay was established. This assay was highly sensitive, and the detection limit was 0.5 percent of the normal. Further, an inhibitor of ADAMTS13 was measured to a level of 0.1 Bethesda units per mL. ADAMTS13 activity was measured in 20 patients with Upshaw-Schulman syndrome, a congenital TTP, and 61 acquired TTP patients. The activity measured by this assay and by the classic VWF multimer assay showed high correlation. CONCLUSION: A convenient and highly sensitive EIA for ADAMTS13 activity has been established. This assay can be introduced for routine laboratory work in transfusion medicine.