Comparison of three commercially available buffy coat pooling sets for the preparation of platelet concentrates.

BACKGROUND: A disposable set for platelet concentrate (PC) preparation by the buffy coat method allows pooling of buffy coats, centrifugation and cell separation with in-line leucocyte filtration. This study compares three commercially available pooling sets in combination with INTERCEPT pathogen inactivation (PI). MATERIALS AND METHODS: Sets for pooling of buffy coats were from Fresenius Kabi (FRE), Macopharma (MAC) and Terumo BCT (TER). Platelet yield, recovery and concentration were compared before and after PI (n = 20). Platelet quality was assessed by annexin V binding, P-selectin expression and PAC1 binding. RESULTS: The TER pooling set had the highest platelet yield (5·39 ± 0·44 × 1011 ) compared with MAC (4·53 ± 0·77) and FRE (4·56 ± 0·51) prior to PI. This was the result of a significantly higher platelet concentration in the TER storage bag (1·41 ± 0·12 × 106 /µL) compared with MAC (1·18 ± 0·19) and FRE (1·28 ± 0·15). However, the TER platelet content decreased by 15·6% after PI, yielding 4·55 ± 0·47 × 1011 platelets compared with smaller reductions at 9·5% for MAC (4·10 ± 0·69) and 4·4% for FRE (4·36 ± 0·52). None of the individual PC contained >106 leucocytes. The pH in TER PC was lower compared with MAC and FRE caused by a higher lactic acid production rate. Consequently, PAC1 binding after TRAP activation was lowest for TER PC on day 6. P-selectin and annexin V were not different between suppliers. CONCLUSION: This study demonstrates the added value of evaluating the entire component production process when introducing a new consumable. This study helped to inform a decision on what pooling set is ideally suited for routine implementation taking into account PI.

High platelet content can increase storage lesion rates following Intercept pathogen inactivation primarily in platelet concentrates prepared by apheresis.

BACKGROUND: Pathogen inactivation methods for platelet concentrates are increasingly being used in blood banks worldwide. In vitro studies have demonstrated its effects on storage lesion, but little routine quality control data on blood banking outcomes have been reported. MATERIALS AND METHODS: Swirling of distributed products was monitored before and after implementation of Intercept pathogen inactivation. Metabolic parameters pH, glucose and lactic acid were determined in a random cohort of expired pathogen-inactivated products. Storage lesion indicators in apheresis concentrates with premature low swirling were compared to concentrates with normal swirling. RESULTS: During validation for implementing Intercept pathogen inactivation, pH and glucose levels decreased faster in apheresis platelet concentrates with high platelet content than with low platelet content or than in pathogen-inactivated pooled buffy coat-derived products. In routine products, glucose exhaustion was more often found in apheresis compared to buffy coat-derived platelet concentrates despite 3-7% more plasma carryover in the former. Annual incidence of premature low swirling increased significantly by 50% following implementation of pathogen inactivation implementation for apheresis but not for pooled buffy coat platelet concentrates. In addition, apheresis concentrates with premature low swirling had a significantly higher median platelet count (5·0 × 1011 ) than unaffected products (3·5 × 1011 ). CONCLUSION: The risk of increased storage lesion rates following Intercept pathogen inactivation is higher for apheresis than for buffy coat-derived platelet concentrates, especially when platelet contents are higher than 5·0 × 1011 .

Persistent aggregates in apheresis platelet concentrates are commonly collected from donors with a history of aggregate donation.

Platelet apheresis sometimes causes persistent aggregates (PA). This study (n = 211) shows that changing the apheresis settings to reach fixed product volumes instead of yields does not influence PA incidence, even though PA products on average contain more platelets than controls. Furthermore, logistic regression was used to model if PA can be predicted on the basis of certain predonation parameters. PA donation history was the only parameter retained, proving a strong determinant of predictability [AUC = 0.735 (SE = 0.022)]. Consequently, donations from a donor with previous PA history are 7.8 times more likely to contain PA than from a donor without preceding history.

The contribution of von Willebrand factor-GPIbα interactions to persistent aggregate formation in apheresis platelet concentrates.

BACKGROUND AND OBJECTIVES: Apheresis platelet concentrates sometimes contain persistent aggregates (PA). Because apheresis involves extracorporeal circulation, we hypothesized that interactions between GPIbα and von Willebrand factor (VWF) underlie their origin. MATERIALS AND METHODS: Platelets in donations with PA were compared to aggregate-free (AF) controls. Flow cytometry was used to determine platelet bound VWF. Degranulation was measured using P-selectin expression in flow cytometry and cytokine release using immunosorbent assays. Platelet adhesion to VWF was assessed in hydrodynamic flow and real-time video microscopy. RESULTS: Platelets in PA concentrates had significantly more (P = 0·009, n ≥ 8) bound VWF compared to AF platelets, but differences in VWF concentration, VWF collagen binding, activated VWF or GPIbα expression were not found. Degranulation was higher (P = 0·030, n = 7) in PA than AF concentrates on day 1 of storage, but adhesion to immobilized VWF under hydrodynamic flow conditions was normal at that moment. On day 6, however, significantly less VWF adhesion (P = 0·009, n ≥ 6) was found for PA platelets compared to AF, indicating accelerated storage lesion in PA products. In a model that mimicks PA formation by chemically induced binding of VWF to platelets, we found that degranulation, phosphatidylserine expression and metabolism did not differ with paired controls at any time during subsequent storage. CONCLUSION: Accelerated storage lesion is found in concentrates with PA, but this cannot be explained solely by increased platelet bound VWF following apheresis. Therefore, additional stressors are probably responsible for the increases observed in platelet degranulation and storage lesion in products with PA.

Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13.

BACKGROUND: Recently, conformational activation of ADAMTS-13 was identified. This mechanism showed the evolution from a condensed conformation, in which the proximal MDTCS and distal T2-CUB2 domains are in close contact with each other, to an activated, open structure due to binding with von Willebrand factor (VWF). OBJECTIVES: Identification of cryptic epitope/exosite exposure after conformational activation and of sites of flexibility in ADAMTS-13. METHODS: The activating effect of 25 anti-T2-CUB2 antibodies was studied in the FRETS-VWF73 and the vortex assay. Cryptic epitope/exosite exposure was determined with ELISA and VWF binding assay. The molecular basis for flexibility was hypothesized through rapid automatic detection and alignment of repeats (RADAR) analysis, tested with ELISA using deletion variants and visualized using electron microscopy. RESULTS: Eleven activating anti-ADAMTS-13 antibodies, directed against the T5-CUB2 domains, were identified in the FRETS-VWF73 assay. RADAR analysis identified three linker regions in the distal domains. Interestingly, identification of an antibody recognizing a cryptic epitope in the metalloprotease domain confirmed the contribution of these linker regions to conformational activation of the enzyme. The proof of flexibility around both the T2 and metalloprotease domains, as shown by by electron microscopy, further supported this contribution. In addition, cryptic epitope exposure was identified in the distal domains, because activating anti-T2-CUB2 antibodies increased the binding to folded VWF up to ~3-fold. CONCLUSION: Conformational activation of ADAMTS-13 leads to cryptic epitope/exosite exposure in both proximal and distal domains, subsequently inducing increased activity. Furthermore, three linker regions in the distal domains are responsible for flexibility and enable the interaction between the proximal and the T8-CUB2 domains.

Persistent aggregates in apheresis platelet concentrates.

BACKGROUND: Aggregates often appear during apheresis. Sometimes, these persist throughout storage, causing product wastage. This study assessed product quality of apheresis concentrates containing persistent aggregates (PA) and aimed to identify the factors that contribute to their formation. METHODS: Donation (n = 180) and platelet indices (n ≥ 10) from apheresis concentrates with PA were compared with aggregate-free products. RESULTS: The proportion of donors with at least one previous PA donation was twofold higher in the PA group (P < 0·0001) indicating a donor dependence. Significantly higher donor whole blood platelet counts (286 ± 50 vs. 266 ± 49 × 10(3) /µl, P < 0·0001) and higher apheresis yields (6·0 ± 1·6 vs. 5·4 ± 1·5 × 10(11) , P < 0·0001) were noted in the PA group. Haematocrit was also slightly higher, but age, gender and body mass were similar. The pH of PA products on day six postdonation was significantly lower (P < 0·001), in line with higher lactic acid concentrations. Flow cytometry showed no differences in GPIbα levels or phosphatidylserine exposure. However, there was slightly more integrin activation as well as increased degranulation measured by P-selectin expression. Cytokine concentrations were also significantly higher in PA concentrates. Aggregation was normal in response to SFLLRN peptide and collagen stimulation, but agglutination at low-dose ristocetin was significantly higher (P = 0.01) in PA products. Finally, PA were disintegrated by plasmin-mediated thrombolysis but not by integrin αIIb ß3 inhibition. CONCLUSION: Products with PA have acceptable quality parameters, but additional functional studies are warranted. Furthermore, PA are more likely to recur in certain donors who have higher platelet counts.

Riboflavin and amotosalen photochemical treatments of platelet concentrates reduce thrombus formation kinetics in vitro.

BACKGROUND: Photochemical treatment (PCT) of platelet concentrates using photosensitizers and ultraviolet light illumination reduces the proliferation potential of pathogens by damaging biomolecules. MATERIALS AND METHODS: The impact of riboflavin (RF-PRT)- and amotosalen (AS-PCT)-based pathogen inactivation on platelets was studied using microfluidic flow chambers on immobilized collagen using standard platelet concentrates prepared from buffy coats in additive solution. Flow cytometry, metabolic parameters and light transmission aggregometry with thrombin-related peptide, collagen and ristocetin were determined concurrently. RESULTS: Both PCTs significantly decreased the platelet surface coverage kinetics in flow chambers over the course of the 7-day study. Platelet aggregation was affected following RF-PRT in response to all agonists, while AS-PCT mainly impacted low-dose ristocetin agglutination. RF-PRT induces premature platelet activation because integrin αII b ß3 was spontaneously activated, and α-degranulation, phosphatidylserine/-ethanolamine exposure and anaerobic metabolism significantly increased following treatment, which was not the case for AS-PCT. On the other hand, AS-PCT significantly diminished thrombus growth onto von Willebrand factor under shear flow. This defect was caused by fewer integrin αII b ß3 interactions, not by defective GPIbα-VWF binding as shown by adhesion experiments in the presence of tirofiban. Moreover, integrin αII b ß3 activation was also affected following the activation of platelets via GPVI-FcγRIIa or PAR1. Finally, amotosalen illumination as such is sufficient to induce platelet damage, with no additional measurable effect of the chemical adsorption step. Gamma irradiation caused no significant difference compared to controls on any time-point or for any parameter. CONCLUSION: Both PCTs significantly reduce thrombus formation rate but by different biochemical mechanisms.

Oxygen removal during pathogen inactivation with riboflavin and UV light preserves protein function in plasma for transfusion.

BACKGROUND AND OBJECTIVE: Photochemical pathogen inactivation technologies (PCT) for individual transfusion products act by inhibition of replication through irreversibly damaging nucleic acids. Concern on the collateral impact of PCT on the blood component's integrity has caused reluctance to introduce this technology in routine practice. This work aims to uncover the mechanism of damage to plasma constituents by riboflavin pathogen reduction technology (RF-PRT). METHODS: Activity and antigen of plasma components were determined following RF-PRT in the presence or absence of dissolved molecular oxygen. RESULTS: Employing ADAMTS13 as a sentinel molecule in plasma, our data show that its activity and antigen are reduced by 23 ± 8% and 29 ± 9% (n = 24), respectively, which corroborates with a mean decrease of 25% observed for other coagulation factors. Western blotting of ADAMTS13 shows decreased molecular integrity, with no obvious indication of additional proteolysis nor is riboflavin able to directly inhibit the enzyme. However, physical removal of dissolved oxygen prior to RF-PRT protects ADAMTS13 as well as FVIII and fibrinogen from damage, indicating a direct role for reactive oxygen species. Redox dye measurements indicate that superoxide anions are specifically generated during RF-PRT. Protein carbonyl content as a marker of disseminated irreversible biomolecular damage was significantly increased (3·1 ± 0·8 vs. 1·6 ± 0·5 nmol/mg protein) following RF-PRT, but not in the absence of dissolved molecular oxygen (1·8 ± 0·4 nmol/mg). CONCLUSIONS: RF-PRT of single plasma units generates reactive oxygen species that adversely affect biomolecular integrity of relevant plasma constituents, a side-effect, which can be bypassed by applying hypoxic conditions during the pathogen inactivation process.

The distal carboxyterminal domains of murine ADAMTS13 influence proteolysis of platelet-decorated VWF strings in vivo.

BACKGROUND: The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentified. METHODS: Seven C-terminally truncated murine ADAMTS13 (mADAMTS13) mutants were constructed and characterized in vitro. Their ability to cleave VWF strings in vivo was studied in the ADAMTS13(-/-) mouse. RESULTS: Murine MDTCS (devoid of T2-8 and CUB domains) retained full enzyme activity in vitro towards FRETS-VWF73 and the C-terminal T6-8 (del(T6-CUB)) and CUB domains (delCUB) are dispensable under these assay conditions. In addition, mADAMTS13 fragments without the spacer domain (MDT and M) had reduced catalytic efficiencies. Our results hence indicate that similar domains in murine and human ADAMTS13 are required for activity in vitro, supporting the use of mouse models to study ADAMTS13 function in vivo. Interestingly, using intravital microscopy we show that removal of the CUB domains abolishes proteolysis of platelet-decorated VWF strings in vivo. In addition, whereas MDTCS is fully active in vivo, partial (del(T6-CUB)) or complete (delCUB) addition of the T2-8 domains gradually attenuates its activity. CONCLUSIONS: Our data demonstrate that the ADAMTS13 CUB and T2-8 domains influence proteolysis of platelet-decorated VWF strings in vivo.

Inactivation of ADAMTS13 by plasmin as a potential cause of thrombotic thrombocytopenic purpura.

BACKGROUND: ADAMTS13 deficiency causes accumulation of unusually large von Willebrand factor molecules, which cross-link platelets in the circulation or on the endothelial surface. This process of intravascular agglutination leads to the microangiopathy thrombotic thrombocytopenic purpura (TTP). Most TTP patients have acquired anti-ADAMTS13 autoantibodies that inhibit enzyme function and/or clear it from the circulation. However, the reason for ADAMTS13 deficiency is not always easily identified in a subset of patients. OBJECTIVES: To determine the origin of ADAMTS13 deficiency in a case of acquired TTP. METHODS: Western blotting of ADAMTS13 in plasmas from acute and remission phases was used. RESULTS: The ADAMTS13 deficiency was not caused by mutations or (detectable) autoantibodies; however, an abnormal ADAMTS13 truncated fragment (100 kDa) was found in acute-phase but not remission-phase plasma. This fragment resulted from enzymatic proteolysis, as recombinant ADAMTS13 was also cleaved when in the presence of acute-phase but not remission-phase plasma. Inhibitor screening showed that ADAMTS13 was cleaved by a serine protease that could be dose-dependently inhibited by addition of exogenous α2 -antiplasmin. Examination of the endogenous α2-antiplasmin antigen and activity confirmed deficiency of α2 -antiplasmin function in acute-phase but not remission-phase plasma. To investigate the possibility of ADAMTS13 cleavage by plasmin in plasma, urokinase-type plasminogen activator was added to an (unrelated) congenital α2 -antiplasmin-deficient plasma sample to activate plasminogen. This experiment confirmed cleavage of endogenous ADAMTS13 similar to that observed in our TTP patient. CONCLUSION: We report the first acquired TTP patient with cleaved ADAMTS13 and show that plasmin is involved.

Multi-step binding of ADAMTS-13 to von Willebrand factor.

BACKGROUND: ADAMTS-13 proteolytic activity is controlled by the conformation of its substrate, von Willebrand factor (VWF), and changes in the secondary structure of VWF are essential for efficient cleavage. Substrate recognition is mediated through several non-catalytic domains in ADAMTS-13 distant from the active site. OBJECTIVES: We hypothesized that not all binding sites for ADAMTS-13 in VWF are cryptic and analyzed binding of native VWF to ADAMTS-13. METHODS: Immunoprecipiation of VWF-ADAMTS-13 complexes using anti-VWF antibodies and magnetic beads was used. Binding was assessed by Western blotting and immunosorbent assays. RESULTS: Co-immunoprecipitation demonstrated that ADAMTS-13 binds to native multimeric VWF (K(d) of 79 +/- 11 nmol L(-1)) with no measurable proteolysis. Upon shear-induced unfolding of VWF, binding increased 3-fold and VWF was cleaved. Binding to native VWF was saturable, time dependent, reversible and did not vary with ionic strength (I of 50-200). Moreover, results with ADAMTS-13 deletion mutants indicated that binding to native VWF is mediated through domains distal to the ADAMTS-13 spacer, probably thrombospondin-1 repeats. Interestingly, this interaction occurs in normal human plasma with an ADAMTS-13 to VWF stoichiometry of 0.0040 +/- 0.0004 (mean +/- SEM, n = 10). CONCLUSIONS: ADAMTS-13 binds to circulating VWF and may therefore be incorporated into a platelet-rich thrombus, where it can immediately cleave VWF that is unfolded by fluid shear stress.

Development of monoclonal antibodies that inhibit platelet adhesion or aggregation as potential anti-thrombotic drugs.

Cardiovascular disease is the major cause of mortality in Western countries. Platelets play a crucial role in the development of arterial thrombosis and other pathophysiologies leading to clinical ischemic events. In the damaged vessel wall, platelets adhere to the subendothelium through an interaction with von Willebrand factor (VWF), which forms a bridge between subendothelial collagen and the platelet receptor glycoprotein (GP) Ib/IX/V. This reversible adhesion allows platelets to roll over the damaged area, decreasing their velocity and resulting in strong platelet activation. This leads to the conformational activation of the platelet GPIIb/IIIa receptor, fibrinogen binding and finally to platelet aggregation. As each interaction (collagen-VWF, VWF-GPIb and GPIIb/IIIa-fibrinogen) plays an essential role in primary haemostasis, loss of either of these interactions results in a bleeding diathesis, implying that interfering with these interactions might result in an anti-thrombotic effect. Whereas GPIIb/IIIa antagonists indeed are effective anti-thrombotics, it has been suggested that drugs which block the initial steps of thrombus formation (collagen-VWF or VWF-GPIb interaction) might have advantages over the ones that merely inhibit platelet aggregation. In this review we will discuss and compare the development of monoclonal antibodies (moAbs) that inhibit platelet adhesion or platelet aggregation. The effect of the moAbs in in vitro experiments, in in vivo models and in clinical trials will be described. Benefits, limitations, current applications and the future perspectives in the development of antibodies for each target will be discussed.

ADAMTS-13 plasma level determination uncovers antigen absence in acquired thrombotic thrombocytopenic purpura and ethnic differences.

BACKGROUND: The recently discovered plasma enzyme ADAMTS-13 cleaves the A2-domain of von Willebrand factor (VWF). A defective cleaving protease results in unusually large VWF multimers, which cause thrombotic thrombocytopenic purpura (TTP). AIM: Analysis of the ADAMTS-13 antigen levels in TTP patients compared with normal donors. METHODS: An antigen ELISA test was built, based on high affinity anti-ADAMTS-13 monoclonal antibodies, which were generated using genetic immunization. RESULTS: Specificity of the ADAMTS-13 antigen test was confirmed, as (i) plasma from a patient with acquired TTP but presenting without inhibitor did not contain antigen and (ii) the binding of recombinant ADAMTS-13 was inhibited by increasing amounts of normal plasma. The assay is sensitive as it can detect antigen levels as low as 1.6% of normal. The concentration in normal pooled human plasma was determined (1.03 +/- 0.15 microg mL(-1)) and arbitrarily set to 1 U mL(-1). The antigen levels in congenital TTP samples (34 +/- 21 mU mL(-1), n = 2), as well as in samples from patients with acquired TTP (231 +/- 287 mU mL(-1), n = 11), were clearly reduced when compared with normal Caucasian donors (951 +/- 206 mU mL(-1), n = 16). Remarkably, normal Chinese donors have a significantly lower antigen titer (601 +/- 129 mU mL(-1), n = 15), when compared with normal Caucasians. CONCLUSIONS: Our results show that acquired TTP patients suffer mainly from ADAMTS-13 antigen depletion, thereby indicating the importance of ADAMTS-13 antigen determination in diagnosis and patient follow-up.