Stoichiometry and architecture of the platelet membrane complex glycoprotein Ib-IX-V.

Glycoprotein (GP) Ib-IX-V is the second most abundant platelet receptor for thrombin and other ligands crucial for hemostasis and thrombosis. Its activity is involved in platelet adhesion to vascular injury sites and thrombin-induced platelet aggregation. GPIb-IX-V is a heteromeric complex composed of four subunits, GPIbα, GPIbß, GPV and GPIX, in a stoichiometric ratio that has been wildly debated. Despite its important physiological roles, the overall structure and molecular arrangement of GPIb-IX-V are not yet fully understood. Here, we purify stable and functional human GPIb-IX-V complex from reconstituted EXPi293F cells in high homogeneity, and perform biochemical and structural characterization of this complex. Single-particle cryo-electron microscopy structure of GPIb-IX-V is determined at ∼11 Å resolution, which unveils the architecture of GPIb-IX-V and its subunit organization. Size-exclusion chromatography-multi-angle static light scattering analysis reveals that GPIb-IX-V contains GPIb-IX and GPV at a 1:1 stoichiometric ratio and surface plasmon resonance assays show that association of GPV leads to slow kinetics of thrombin binding to GPIb-IX-V. Taken together, our results provide the first three-dimensional architecture of the intact GPIb-IX-V complex, which extends our understanding of the structure and functional mechanism of this complex in hemostasis and thrombosis.

Binding Mechanism between Platelet Glycoprotein and Cyclic Peptide Elucidated by McMD-Based Dynamic Docking.

The cyclic peptide OS1 (amino acid sequence: CTERMALHNLC), which has a disulfide bond between both termini cysteine residues, inhibits complex formation between the platelet glycoprotein Ibα (GPIbα) and the von Willebrand factor (vWF) by forming a complex with GPIbα. To study the binding mechanism between GPIbα and OS1 and, therefore, the inhibition mechanism of the protein-protein GPIbα-vWF complex, we have applied our multicanonical molecular dynamics (McMD)-based dynamic docking protocol starting from the unbound state of the peptide. Our simulations have reproduced the experimental complex structure, although the top-ranking structure was an intermediary one, where the peptide was bound in the same location as in the experimental structure; however, the ß-switch of GPIbα attained a different conformation. Our analysis showed that subsequent refolding of the ß-switch results in a more stable binding configuration, although the transition to the native configuration appears to take some time, during which OS1 could dissociate. Our results show that conformational changes in the ß-switch are crucial for successful binding of OS1. Furthermore, we identified several allosteric binding sites of GPIbα that might also interfere with vWF binding, and optimization of the peptide to target these allosteric sites might lead to a more effective inhibitor, as these are not dependent on the ß-switch conformation.

Structure of the platelet glycoprotein Ib receptor in complex with a novel antithrombotic agent.

Agkisacucetin, a snake C-type lectin-like protein isolated from the venom of Deinagkistrodon acutus (formerly Agkistrodon acutus), is a novel antithrombotic drug candidate in phase 2 clinical trials. Agkisacucetin specifically recognizes the platelet surface receptor glycoprotein Ib α chain (GPIbα) to block GPIb and von Willebrand factor (VWF). In this study, we solved the crystal structure of the GPIbα N-terminal domain (residues 1-305) in complex with agkisacucetin to understand their molecular recognition mechanism. The crystal structure showed that agkisacucetin primarily contacts GPIbα at the C-terminal part of the conserved leucine-rich repeat (LRR) domain (LRR-6 to LRR-8) and the previously described "ß-switch" region through the ß chain. In addition, we found that agkisacucetin α chain contacts part of the GPIbα C-terminal peptide after the LRR domain through complementary charge interactions. This C-terminal peptide plays a key role in GPIbα and thrombin recognition. Therefore, our structure revealed that agkisacucetin can sterically block the interaction between the GPIb receptor and VWF and thrombin proteins to inhibit platelet function. Our structural work provides key molecular insights into how an antithrombotic drug candidate recognizes the GPIb receptor to modulate platelet function to inhibit thrombosis.

[Molecular dynamics simulation of force-regulated interaction between glycoprotein Ib α and filamin].

In resting platelets, the 17 th domain of filamin a (FLNa17) constitutively binds to the platelet membrane glycoprotein Ibα (GPIbα) at its cytoplasmic tail (GPIbα-CT) and inhibits the downstream signal activation, while the binding of ligand and blood shear force can activate platelets. To imitate the pull force transmitted from the extracellular ligand of GPIbα and the lateral tension from platelet cytoskeleton deformation, two pulling modes were applied on the GPIbα-CT/FLNa17 complex, and the molecular dynamics simulation method was used to explore the mechanical regulation on the affinity and mechanical stability of the complex. In this study, at first, nine pairs of key hydrogen bonds on the interface between GPIbα-CT and FLNa17 were identified, which was the basis for maintaining the complex structural stability. Secondly, it was found that these hydrogen bonding networks would be broken down and lead to the dissociation of FLNa17 from GPIbα-CT only under the axial pull force; but, under the lateral tension, the secondary structures at both terminals of FLNa17 would unfold to protect the interface of the GPIbα-CT/FLNa17 complex from mechanical damage. In the range of 0~40 pN, the increase of pull force promoted outward-rotation of the nitrogen atom of the 563 rd phenylalanine (PHE 563-N) at GPIbα-CT and the dissociation of the complex. This study for the first time revealed that the extracellular ligand-transmitted axial force could more effectively relieve the inhibition of FLNa17 on the downstream signal of GPIbα than pure mechanical tension at the atomic level, and would be useful for further understanding the platelet intracellular force-regulated signal pathway.

Structure-Based Cyclic Glycoprotein Ibα-Derived Peptides Interfering with von Willebrand Factor-Binding, Affecting Platelet Aggregation under Shear.

The plasmatic von Willebrand factor (VWF) circulates in a compact form unable to bind platelets. Upon shear stress, the VWF A1 domain is exposed, allowing VWF-binding to platelet glycoprotein Ib-V-IX (GPIbα chain). For a better understanding of the role of this interaction in cardiovascular disease, molecules are needed to specifically interfere with the opened VWF A1 domain interaction with GPIbα. Therefore, we in silico designed and chemically synthetized stable cyclic peptides interfering with the platelet-binding of the VWF A1 domain per se or complexed with botrocetin. Selected peptides (26-34 amino acids) with the lowest-binding free energy were: the monocyclic mono- vOn Willebrand factoR-GPIbα InTerference (ORbIT) peptide and bicyclic bi-ORbIT peptide. Interference of the peptides in the binding of VWF to GPIb-V-IX interaction was retained by flow cytometry in comparison with the blocking of anti-VWF A1 domain antibody CLB-RAg35. In collagen and VWF-dependent whole-blood thrombus formation at a high shear rate, CLB-RAg35 suppressed stable platelet adhesion as well as the formation of multilayered thrombi. Both peptides phenotypically mimicked these changes, although they were less potent than CLB-RAg35. The second-round generation of an improved peptide, namely opt-mono-ORbIT (28 amino acids), showed an increased inhibitory activity under flow. Accordingly, our structure-based design of peptides resulted in physiologically effective peptide-based inhibitors, even for convoluted complexes such as GPIbα-VWF A1.

Oxidation shuts down an auto-inhibitory mechanism of von Willebrand factor.

The blood protein von Willebrand factor (VWF) is a key link between inflammation and pathological thrombus formation. In particular, oxidation of methionine residues in specific domains of VWF due to the release of oxidants in inflammatory conditions has been linked to an increased platelet-binding activity. However, the atomistic details of how methionine oxidation activates VWF have not been elucidated to date. Yet understanding the activation mechanism of VWF under oxidizing conditions can lead to the development of novel therapeutics that target VWF selectively under inflammatory conditions in order to reduce its thrombotic activity while maintaining its haemostatic function. In this manuscript, we used a combination of a dynamic flow assay and molecular dynamics (MD) simulations to investigate how methionine oxidation removes an auto-inhibitory mechanism of VWF. Results from the dynamic flow assay revealed that oxidation does not directly activate the A1 domain, which is the domain in VWF that contains the binding site to the platelet surface receptor glycoprotein Ibα (GpIbα), but rather removes the inhibitory function of the neighboring A2 and A3 domains. Furthermore, the MD simulations combined with free energy perturbation calculations suggested that methionine oxidation may destabilize the binding interface between the A1 and A2 domains leading to unmasking of the GpIbα-binding site in the A1 domain.

Molecular basis for unique specificity of human TRAF4 for platelets GPIbß and GPVI.

Tumor necrosis factor (TNF)-receptor associated factor 4 (TRAF4), an adaptor protein with E3-ligase activity, is involved in embryogenesis, cancer initiation and progression, and platelet receptor (GPIb-IX-V complex and GPVI)-mediated signaling for reactive oxygen species (ROS) production that initiates thrombosis at arterial shears. Disruption of platelet receptors and the TRAF4 interaction is a potential target for therapeutic intervention by antithrombotic drugs. Here, we report a crystal structure of TRAF4 (amino acid residues 290∼470) in complex with a peptide from the GPIbß receptor (amino acid residues 177∼181). The GPIbß peptide binds to a unique shallow surface composed of two hydrophobic pockets on TRAF4. Further studies revealed the TRAF4-binding motif Arg-Leu-X-Ala. The TRAF4-binding motif was present not only in platelet receptors but also in the TGF-ß receptor. The current structure will provide a template for furthering our understanding of the receptor-binding specificity of TRAF4, TRAF4-mediated signaling, and related diseases.

Force-Regulated Refolding of the Mechanosensory Domain in the Platelet Glycoprotein Ib-IX Complex.

In platelets, the glycoprotein (GP) Ib-IX receptor complex senses blood shear flow and transmits the mechanical signals into platelets. Recently, we have discovered a juxtamembrane mechanosensory domain (MSD) within the GPIbα subunit of GPIb-IX. Mechanical unfolding of the MSD activates GPIb-IX signaling into platelets, leading to their activation and clearance. Using optical tweezer-based single-molecule force measurement, we herein report a systematic biomechanical characterization of the MSD in its native, full-length receptor complex and a recombinant, unglycosylated MSD in isolation. The native MSD unfolds at a resting rate of 9 × 10-3 s-1. Upon exposure to pulling forces, MSD unfolding accelerates exponentially over a force scale of 2.0 pN. Importantly, the unfolded MSD can refold with or without applied forces. The unstressed refolding rate of MSD is ∼17 s-1 and slows exponentially over a force scale of 3.7 pN. Our measurements confirm that the MSD is relatively unstable, with a folding free energy of 7.5 kBT. Because MSD refolding may turn off GPIb-IX's mechanosensory signals, our results provide a mechanism for the requirement of a continuous pulling force of >15 pN to fully activate GPIb-IX.

Effects of Pro1266Leu mutation on structure and function of glycoprotein Ib binding domain of von Willebrand factor.

Glycoprotein Ibα (GpIbα) binding ability of A1 domain of von Willebrand factor (vWF) facilitates platelet adhesion that plays a crucial role in maintaining hemostasis and thrombosis at the site of vascular damage. There are both "loss as well as gain of function" mutations observed in this domain. Naturally occurring "gain of function" mutations leave self-activating impacts on the A1 domain which turns the normal binding to characteristic constitutive binding with GPIbα. These "gain of function" mutations are associated with the von Willebrand disease type 2B. In recent years, studies focused on understanding the mechanism and conformational patterns attached to these phenomena have been conducted, but the conformational pathways leading to such binding patterns are poorly understood as of now. To obtain a microscopic picture of such events for the better understanding of pathways, we used molecular dynamics (MD) simulations along with principal component analysis and normal mode analysis to study the effects of Pro1266Leu (Pro503Leu in structural context) mutation on the structure and function of A1 domain of vWF. MD simulations have provided atomic-level details of intermolecular motions as a function of time to understand the dynamic behavior of A1 domain of vWF. Comparative analysis of the trajectories obtained from MD simulations of both the wild type and Pro503Leu mutant suggesting appreciable conformational changes in the structure of mutant which might provide a basis for assuming the "gain of function" effects of these mutations on the A1 domain of vWF, resulting in the constitutive binding with GpIbα.

Cryopreserved platelets augment the inflammatory response: role of phosphatidylserine- and P-selectin-mediated platelet phagocytosis in macrophages.

BACKGROUND: Cryopreservation in dimethyl sulfoxide and storage at -80 °C extends the shelf life of platelets to at least 2 years, allowing greater availability in rural and military areas. While cryopreserved platelets (CPPs) have been extensively characterized for coagulation and thrombin generation, reports on the mechanism of adverse reactions to CPPs transfusion are scarce. Here, we tested the hypothesis that CPPs facilitate phagocytosis by Kupffer cells and subsequently promote the inflammatory response in Kupffer cells. STUDY DESIGN AND METHODS: P-selectin expression, glycoprotein Ibα clustering and phosphatidylserine (PS) surface exposure on platelets stored at 22 °C, 4 °C and - 80 °C for 3 days were examined by flow cytometry. The phagocytosis of mepacrine-labeled platelets coincubated with THP-1 cells was examined by flow cytometry and confocal microscopy, and the release of cytokines from THP-1 cells was measured by enzyme-linked immunosorbent assay. RESULTS: CPPs showed a marked enhancement of exposed PS but dramatically reduced glycoprotein Ibα expression and clustering compared with platelets stored at 4 °C. Activation of THP-1 cells was stronger by CPPs than by platelets stored at 22 °C and 4 °C. CPP interference tests using annexin V and anti-P-selectin showed that CPPs induced increases in PS- and P-selectin-mediated phagocytosis, as well as secretion of the proinflammatory cytokine tumor necrosis factor-α, and interleukins IL-1ß and IL-6, but a decrease in transforming growth factor-ß production in THP-1 cells. Surface-exposed PS was more effective than P-selectin for the activation of THP-1 cells. CONCLUSION: CPPs triggered PS and P-selectin-mediated phagocytosis by macrophages and stimulated the inflammatory response of macrophages.

Specific electrostatic interactions between charged amino acid residues regulate binding of von Willebrand factor to blood platelets.

The plasma protein von Willebrand factor (VWF) is essential for hemostasis initiation at sites of vascular injury. The platelet-binding A1 domain of VWF is connected to the VWF N-terminally located D'D3 domain through a relatively unstructured amino acid sequence, called here the N-terminal linker. This region has previously been shown to inhibit the binding of VWF to the platelet surface receptor glycoprotein Ibα (GpIbα). However, the molecular mechanism underlying the inhibitory function of the N-terminal linker has not been elucidated. Here, we show that an aspartate at position 1261 is the most critical residue of the N-terminal linker for inhibiting binding of the VWF A1 domain to GpIbα on platelets in blood flow. Through a combination of molecular dynamics simulations, mutagenesis, and A1-GpIbα binding experiments, we identified a network of salt bridges between Asp1261 and the rest of A1 that lock the N-terminal linker in place such that it reduces binding to GpIbα. Mutations aimed at disrupting any of these salt bridges activated binding unless the mutated residue also formed a salt bridge with GpIbα, in which case the mutations inhibited the binding. These results show that interactions between charged amino acid residues are important both to directly stabilize the A1-GpIbα complex and to indirectly destabilize the complex through the N-terminal linker.

A biophysical view on von Willebrand factor activation.

The process of hemostatic plug formation at sites of vascular injury crucially relies on the large multimeric plasma glycoprotein von Willebrand factor (VWF) and its ability to recruit platelets to the damaged vessel wall via interaction of its A1 domain with platelet GPIbα. Under normal blood flow conditions, VWF multimers exhibit a very low binding affinity for platelets. Only when subjected to increased hydrodynamic forces, which primarily occur in connection with vascular injury, VWF can efficiently bind to platelets. This force-regulation of VWF's hemostatic activity is not only highly intriguing from a biophysical perspective, but also of eminent physiological importance. On the one hand, it prevents undesired activity of VWF in intact vessels that could lead to thromboembolic complications and on the other hand, it enables efficient VWF-mediated platelet aggregation exactly where needed. Here, we review recent studies that mainly employed biophysical approaches in order to elucidate the molecular mechanisms underlying the complex mechano-regulation of the VWF-GPIbα interaction. Their results led to two main hypotheses: first, intramolecular shielding of the A1 domain is lifted upon force-induced elongation of VWF; second, force-induced conformational changes of A1 convert it from a low-affinity to a high-affinity state. We critically discuss these hypotheses and aim at bridging the gap between the large-scale behavior of VWF as a linear polymer in hydrodynamic flow and the detailed properties of the A1-GPIbα bond at the single-molecule level.

Refrigeration-Induced Binding of von Willebrand Factor Facilitates Fast Clearance of Refrigerated Platelets.

OBJECTIVE: Apheresis platelets for transfusion treatment are currently stored at room temperature because after refrigeration platelets are rapidly cleared on transfusion. In this study, the role of von Willebrand factor (VWF) in the clearance of refrigerated platelets is addressed. APPROACH AND RESULTS: Human and murine platelets were refrigerated in gas-permeable bags at 4°C for 24 hours. VWF binding, platelet signaling events, and platelet post-transfusion recovery and survival were measured. After refrigeration, the binding of plasma VWF to platelets was drastically increased, confirming earlier studies. The binding was blocked by peptide OS1 that bound specifically to platelet glycoprotein (GP)Ibα and was absent in VWF-/- plasma. Although surface expression of GPIbα was reduced after refrigeration, refrigeration-induced VWF binding under physiological shear induced unfolding of the GPIbα mechanosensory domain on the platelet, as evidenced by increased exposure of a linear epitope therein. Refrigeration and shear treatment also induced small elevation of intracellular Ca2+, phosphatidylserine exposure, and desialylation of platelets, which were absent in VWF-/- platelets or inhibited by OS1, which is a monomeric 11-residue peptide (CTERMALHNLC). Furthermore, refrigerated VWF-/- platelets displayed increased post-transfusion recovery and survival than wild-type ones. Similarly, adding OS1 to transgenic murine platelets expressing only human GPIbα during refrigeration improved their post-transfusion recovery and survival. CONCLUSIONS: Refrigeration-induced binding of VWF to platelets facilitates their rapid clearance by inducing GPIbα-mediated signaling. Our results suggest that inhibition of the VWF-GPIbα interaction may be a potential strategy to enable refrigeration of platelets for transfusion treatment.

A novel germline mutation in GP1BA gene N-terminal domain in monoallelic Bernard-Soulier syndrome.

Mutations in the GP1BA gene have been associated with platelet-type von Willebrand disease and Bernard-Soulier syndrome. Here, we report a novel GP1BA mutation in a family with autosomal dominant macrothrombocytopenia and mild bleeding. We performed analyses of seven family members. Using whole-exome sequencing of germline DNA samples, we identified a heterozygous single-nucleotide change in GP1BA (exone2:c.176T>G), encoding a p.Leu59Arg substitution in the N-terminal domain, segregating with macrothrombocytopenia. This variant has not been previously reported. We also analysed the structure of the detected sequence variant in silico. In particular, we used the crystal structure of the human platelet receptor GP Ibα N-terminal domain. Replacement of aliphatic amino-acid Leu 59 with charged, polar and larger arginine probably disrupts the protein structure. An autosomal dominant mode of inheritance, a family history of mild bleeding episodes, aggregation pattern in affected individuals together with evidence of mutation occurring in part of the GP1BA gene encoding the leucine-rich repeat region suggest a novel variant causing monoallelic Bernard-Soulier syndrome.

Two novel variants of uncertain significance in GP9 associated with Bernard-Soulier syndrome: Are they true mutations?

Bernard-Soulier syndrome (BSS) is an autosomal recessive major thrombocytopathy, the symptoms of which are mainly marked by mucocutaneous bleeding. This rare disease, initially described in the 1970s, is the result of an abnormal formation of the glycoprotein complex Ib-IX-V (GP Ib-IX-V), a platelet receptor of von Willebrand factor. A large number of mutations, sometimes involving the GP9 gene, have been described as possibly responsible for the disease. We report here the case of a BSS patient who presented with persistent thrombocytopenia (31x109/L) and decreased surface expression of GPIb-IX-V on large platelets with anisocytosis. Thorough molecular analyses disclosed two previously unreported GP9 variants, respectively c.230T>A (p.Leu77Gln) and c.255C>A (p.Asn85Lys). Both are likely to modify the conformation of GP-IX interactions with other glycoproteins of the Ib-IX-V complex and thus proper expression of this complex on the membrane of platelets.

Force-induced on-rate switching and modulation by mutations in gain-of-function von Willebrand diseases.

Mutations in the ultralong vascular protein von Willebrand factor (VWF) cause the common human bleeding disorder, von Willebrand disease (VWD). The A1 domain in VWF binds to glycoprotein Ibα (GPIbα) on platelets, in a reaction triggered, in part, by alterations in flow during bleeding. Gain-of-function mutations in A1 and GPIbα in VWD suggest conformational regulation. We report that force application switches A1 and/or GPIbα to a second state with faster on-rate, providing a mechanism for activating VWF binding to platelets. Switching occurs near 10 pN, a force that also induces a state of the receptor-ligand complex with slower off-rate. Force greatly increases the effects of VWD mutations, explaining pathophysiology. Conversion of single molecule kon (s(-1)) to bulk phase kon (s(-1)M(-1)) and the kon and koff values extrapolated to zero force for the low-force pathways show remarkably good agreement with bulk-phase measurements.

Mutational Constraints on Local Unfolding Inhibit the Rheological Adaptation of von Willebrand Factor.

Unusually large von Willebrand factor (VWF), the first responder to vascular injury in primary hemostasis, is designed to capture platelets under the high shear stress of rheological blood flow. In type 2M von Willebrand disease, two rare mutations (G1324A and G1324S) within the platelet GPIbα binding interface of the VWF A1 domain impair the hemostatic function of VWF. We investigate structural and conformational effects of these mutations on the A1 domain's efficacy to bind collagen and adhere platelets under shear flow. These mutations enhance the thermodynamic stability, reduce the rate of unfolding, and enhance the A1 domain's resistance to limited proteolysis. Collagen binding affinity is not significantly affected indicating that the primary stabilizing effect of these mutations is to diminish the platelet binding efficiency under shear flow. The enhanced stability stems from the steric consequences of adding a side chain (G1324A) and additionally a hydrogen bond (G1324S) to His(1322) across the ß2-ß3 hairpin in the GPIbα binding interface, which restrains the conformational degrees of freedom and the overall flexibility of the native state. These studies reveal a novel rheological strategy in which the incorporation of a single glycine within the GPIbα binding interface of normal VWF enhances the probability of local unfolding that enables the A1 domain to conformationally adapt to shear flow while maintaining its overall native structure.

Identification of a juxtamembrane mechanosensitive domain in the platelet mechanosensor glycoprotein Ib-IX complex.

How glycoprotein (GP)Ib-IX complex on the platelet surface senses the blood flow through its binding to the plasma protein von Willebrand factor (VWF) and transmits a signal into the platelet remains unclear. Here we show that optical tweezer-controlled pulling of the A1 domain of VWF (VWF-A1) on GPIb-IX captured by its cytoplasmic domain induced unfolding of a hitherto unidentified structural domain before the dissociation of VWF-A1 from GPIb-IX. Additional studies using recombinant proteins and mutant complexes confirmed its existence in GPIb-IX and enabled localization of this quasi-stable mechanosensitive domain of ∼60 residues between the macroglycopeptide region and the transmembrane helix of the GPIbα subunit. These results suggest that VWF-mediated pulling under fluid shear induces unfolding of the mechanosensitive domain in GPIb-IX, which may possibly contribute to platelet mechanosensing and/or shear resistance of VWF-platelet interaction. The identification of the mechanosensitive domain in GPIb-IX has significant implications for the pathogenesis and treatment of related blood diseases.

Exploiting the kinetic interplay between GPIbα-VWF binding interfaces to regulate hemostasis and thrombosis.

Platelet-von Willebrand factor (VWF) interactions must be tightly regulated in order to promote effective hemostasis and prevent occlusive thrombus formation. However, it is unclear what role the inherent properties of the bond formed between the platelet receptor glycoprotein Ibα and the A1 domain of VWF play in these processes. Using VWF-A1 knock-in mice with mutations that enhance (I1309V) or disrupt (R1326H) platelet receptor glycoprotein Ibα binding, we now demonstrate that the kinetic interplay between two distinct contact surfaces influences the site and extent to which platelets bind VWF. Incorporation of R1326H mutation into the major site shortened bond lifetime, yielding defects in hemostasis and thrombosis comparable to VWF-deficient animals. Similarly, disrupting this region of contact with an allosteric inhibitor impaired human platelet accrual in damaged arterioles. In contrast, the I1309V mutation near the minor site prolonged bond lifetime, which was essential for the development of a type 2B-like VWD phenotype. However, combining the R1326H and I1309V mutations normalized both bond kinetics and the hemostatic and thrombotic properties of VWF. These findings broaden our understanding of mechanisms governing platelet-VWF interactions in health and disease, and underscore the importance of combined biophysical and genetic approaches in identifying potential therapeutic avenues for treating bleeding and thrombotic disorders.

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.