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.

WY-14643, a novel antiplatelet and antithrombotic agent targeting the GPIbα receptor.

BACKGROUND AND PURPOSE: Hypolipidemia and platelet activation play key roles in atherosclerotic diseases. Pirinixic acid (WY-14643) was originally developed as a lipid-lowering drug. Here we focused on its antiplatelet and antithrombotic abilities and the underlying mechanism. EXPERIMENTAL APPROACH: The effects of WY-14643 on platelet aggregation was measured using a lumi-aggregometer. Clot retraction and spreading on fibrinogen were also assayed. PPARα-/- platelets were used to identify the target of WY-14643. The interaction between WY-14643 and glycoprotein Ibα (GPIbα) was detected using cellular thermal shift assay (CETSA), surface plasmon resonance (SPR) spectroscopy and molecular docking. GPIbα downstream signaling was examined by Western blot. The antithrombotic effect was investigated using mouse mesenteric arteriole thrombosis model. Mouse tail bleeding model was used to study its effect on bleeding side effects. KEY RESULTS: WY-14643 concentration-dependently inhibits human washed platelet aggregation, clot retraction, and spreading. Significantly, WY-14643 inhibits thrombin-induced activation of human washed platelets with an IC50 of 7.026 µM. The antiplatelet effect of WY-14643 is mainly dependent of GPIbα. CESTA, SPR and molecular docking results indicate that WY-14643 directly interacts with GPIbα and acts as a GPIbα antagonist. WY-14643 also inhibits phosphorylation of PLCγ2, Akt, p38, and Erk1/2 induced by thrombin. Noteworthily, 20 mg/kg oral administration of WY-14643 inhibits FeCl3-induced thrombosis of mesenteric arteries in mice similarly to clopidogrel without increasing bleeding. CONCLUSION AND IMPLICATIONS: WY-14643 is not only a PPARα agonist with lipid-lowering effect, but also an antiplatelet agent as a GPIbα antagonist. It may have more significant therapeutic advantages than current antiplatelet agents for the treatment of atherosclerotic thrombosis, which have lipid-lowering effects without bleeding side effects.

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.

GPIbα-filamin A interaction regulates megakaryocyte localization and budding during platelet biogenesis.

ABSTRACT: Glycoprotein Ibα (GPIbα) is expressed on the surface of platelets and megakaryocytes (MKs) and anchored to the membrane skeleton by filamin A (flnA). Although GPIb and flnA have fundamental roles in platelet biogenesis, the nature of this interaction in megakaryocyte biology remains ill-defined. We generated a mouse model expressing either human wild-type (WT) GPIbα (hGPIbαWT) or a flnA-binding mutant (hGPIbαFW) and lacking endogenous mouse GPIbα. Mice expressing the mutant GPIbα transgene exhibited macrothrombocytopenia with preserved GPIb surface expression. Platelet clearance was normal and differentiation of MKs to proplatelets was unimpaired in hGPIbαFW mice. The most striking abnormalities in hGPIbαFW MKs were the defective formation of the demarcation membrane system (DMS) and the redistribution of flnA from the cytoplasm to the peripheral margin of MKs. These abnormalities led to disorganized internal MK membranes and the generation of enlarged megakaryocyte membrane buds. The defective flnA-GPIbα interaction also resulted in misdirected release of buds away from the vasculature into bone marrow interstitium. Restoring the linkage between flnA and GPIbα corrected the flnA redistribution within MKs and DMS ultrastructural defects as well as restored normal bud size and release into sinusoids. These studies define a new mechanism of macrothrombocytopenia resulting from dysregulated MK budding. The link between flnA and GPIbα is not essential for the MK budding process, however, it plays a major role in regulating the structure of the DMS, bud morphogenesis, and the localized release of buds into the circulation.

Antiplatelet Agents Inhibit Platelet Adhesion and Aggregation on Glass Surface Under Physiological Flow Conditions: Toward a Microfluidic Platelet Functional Assay Without Additional Adhesion Protein Modification.

ABSTRACT: As the pathogenesis of arterial thrombosis often includes platelet adhesion and aggregation, antiplatelet agents are commonly used to prevent thromboembolic events. Here, a new microfluidic method without additional adhesion protein modification was developed to quantify the inhibitory effect of antiplatelet drugs on the adhesion and aggregation behavior of platelets on glass surfaces under physiological flow conditions. Polydimethylsiloxane-glass microfluidic chips were fabricated by soft photolithography. Blood samples from healthy volunteers or patients before and after taking antiplatelet drugs flowed through the microchannels at wall shear rates of 300 and 1500 second -1 , respectively. The time to reach 2.5% platelet aggregation surface coverage (Ti), surface coverage (A 150s ), and mean fluorescence intensity (F 150s ) were used as quantitative indicators. Aspirin (80 µM) prolonged Ti and reduced F 150s . Alprostadil, ticagrelor, eptifibatide, and tirofiban prolonged Ti and reduced A 150s and F 150s in a concentration-dependent manner, whereas high concentrations of alprostadil did not completely inhibit platelet aggregation. Aspirin combined with ticagrelor synergistically inhibited platelet adhesion and aggregation; GPIb-IX-von Willebrand factor inhibitors partially inhibited platelet aggregation, and the inhibition was more pronounced at 1500 than at 300 second -1 . Patient administration of aspirin or (and) clopidogrel inhibited platelet adhesion and aggregation on the glass surface under flow conditions. This technology is capable of distinguishing the pharmacological effects of various antiplatelet drugs on inhibition of platelet adhesion aggregation on glass surface under physiological flow conditions, which providing a new way to develop microfluidic platelet function detection method without additional adhesive protein modification for determining the inhibitory effects of antiplatelet drugs in the clinical setting.

[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.

Inhibition of GPIb-α-mediated apoptosis signaling enables cold storage of platelets.

Cold storage of platelets has been suggested as an alternative approach to reduce the risk of bacterial contamination and to improve the cell quality as well as functionality compared to room temperature storage. However, cold-stored platelets (CSP) are rapidly cleared from the circulation. Among several possible mechanisms, apoptosis has been recently proposed to be responsible for the short half-life of refrigerated platelets. In the present study, we investigated the impact of apoptosis inhibition on the hemostatic functions and survival of CSP. We found that blocking the transduction of the apoptotic signal induced by glycoprotein Ib (GPIb)-α clustering or the activation of caspase 9 does not impair CSP functionality. In fact, the inhibition of GPIb-α clustering mediated-apoptotic signal by a RhoA inhibitor better conserved δ granule release, platelet aggregation, adhesion and the ability to form stable clots, compared to untreated CSP. In contrast, upregulation of the protein kinase A caused a drastic impairment of platelet functions and whole blood clot stability. More importantly, we observed a significant improvement of the half-life of CSP upon inhibition of the intracellular signal induced by GPIb-α clustering. In conclusion, our study provides novel insights on the in vitro hemostatic functions and half-life of CSP upon inhibition of the intracellular cold-induced apoptotic pathway. Our data suggest that the combination of cold storage and apoptosis inhibition might be a promising strategy to prolong the storage time without impairing hemostatic functions or survival of refrigerated platelets.

A novel role of acellular hemoglobin in hemolytic thrombosis.

BACKGROUND: Hemolytic thrombosis has been associated with acellular hemoglobin released from damaged red blood cells during hemolysis. However, the precise molecular mechanism underlying acellular hemoglobin-induced thrombosis remains arguable. In this study, we examined the interaction between hemoglobin and the A1 domain of von Willebrand factor (VWF), which is a critical mediator of platelet activation. METHODS: Previous studies have suggested that the interaction between hemoglobin and the A1 domain of VWF enhances VWF's hemostatic activity. We employed a multidisciplinary investigation to re-examine this interaction, and identified significant differences in binding affinity between the active and inactive forms of A1. RESULTS: We found that hemoglobin binds more strongly to the active A1 than the inactive form. Using hydrogen­deuterium exchange mass spectrometry, we identified the specific residues involved in this interaction, which are located on the α1-ß2 and ß3-α2 loops that are typically covered by the "autoinhibitory module" in the inactive A1. This observation provides a structural explanation for the differential binding affinity between the active and inactive forms of A1. We demonstrated that the binding of hemoglobin to A1 blocks the interaction between GPIbα and VWF, and inhibits VWF-mediated thrombosis in vivo. Furthermore, we found that administration of hemoglobin led to similar levels of thrombocytopenia and microthrombosis in both wildtype and VWF-deficient mice, indicating that the mechanism underlying acellular hemoglobin-induced thrombosis is VWF-independent. CONCLUSIONS: These findings challenge the previous theory that hemoglobin-induced thrombosis occurs solely through binding with VWF, and provide evidence supporting a novel role for hemoglobin in hemolytic thrombosis.

Synergy by Ristocetin and CXCL12 in Human Platelet Activation: Divergent Regulation by Rho/Rho-Kinase and Rac.

CXCL12, belonging to the CXC chemokine family, is a weak agonist of platelet aggregation. We previously reported that the combination of CXCL12 and collagen at low doses synergistically activates platelets via not CXCR7 but CXCR4, a specific receptor for CXCL12 on the plasma membrane. Recently, we reported that not Rho/Rho kinase, but Rac is involved in the platelet aggregation induced by this combination. Ristocetin is an activator of the von Willebrand factor that interacts with glycoprotein (GP) Ib/IX/V, which generates thromboxane A2 via phospholipase A2 activation, resulting in the release of the soluble CD40 ligand (sCD40L) from human platelets. In the present study, we investigated the effects of a combination of ristocetin and CXCL12 at low doses on human platelet activation and its underlying mechanisms. Simultaneous stimulation with ristocetin and CXCL12 at subthreshold doses synergistically induce platelet aggregation. A monoclonal antibody against not CXCR7 but CXCR4 suppressed platelet aggregation induced by the combination of ristocetin and CXCL12 at low doses. This combination induces a transient increase in the levels of both GTP-binding Rho and Rac, followed by an increase in phosphorylated cofilin. The ristocetin and CXCL12-induced platelet aggregation as well as the sCD40L release were remarkably enhanced by Y27362, an inhibitor of Rho-kinase, but reduced by NSC23766, an inhibitor of the Rac-guanine nucleotide exchange factor interaction. These results strongly suggest that the combination of ristocetin and CXCL12 at low doses synergistically induces human platelet activation via Rac and that this activation is negatively regulated by the simultaneous activation of Rho/Rho-kinase.

[The Effect of Ena/VASP Family on the Expression of GPIb-IX Complex in Human Megakaryoblastic Leukemia Dami Cells].

OBJECTIVE: To explore the effects of Ena/VASP gene family on the expression of glycoprotein (GP) Ib-IX complex in human megakaryoblastic leukemia Dami cells. METHODS: SiRNAs targeting Ena/VASP gene family were designed and synthesized to interfere Enah, EVL and VASP gene expression. When the siRNAs were transfected into Dami cells by using LipofectamineTM 2000 for 48 h, the expression of GPIb-IX complex was detected by quantitative real-time PCR, Western blot and flow cytometry. RESULTS: We successfully established siVASP , siEVL and si Enah Dami cell lines. And it was found that the expression of GPIb-IX complex had no evident reduction in siEVL or siVASP Dami cells at both mRNA and protein level, while the total protein and membrane protein of GPIb-IX complex were obviously reduced when Enah was knocked down. CONCLUSION: Enah could affect the expression of GPIb-IX complex in human megakaryoblastic leukemia Dami cells, but the underlying mechanism still needs to be further explored.

The Glycoprotein (GP)Ib-IX-V Complex on Platelets: GPIbα Protein Expression Is Reduced in HeartMate 3 Patients with Bleeding Complications within the First 3 Months.

Non-surgical bleeding (NSB) remains the most critical complication in patients under left ventricular assist device (LVAD) support. It is well known that blood exposed to high shear stress results in platelet dysfunction. Compared to patients without NSB, decreased surface expression of platelet receptor GPIbα was observed in LVAD patients with NSB. In this study, we aimed to compare the expression level of glycoprotein (GP)Ib-IX-V platelet receptor complex in HeartMate 3 (HM 3) patients with and without bleeding complications to investigate the alterations of the platelet transcriptomic profile on platelet damage and increased bleeding risk. Blood samples were obtained from HM 3 patients with NSB (bleeder group, n = 27) and without NSB (non-bleeder group, n = 55). The bleeder group was further divided into patients with early NSB (bleeder ≤ 3 mo, n = 19) and patients with late NSB (bleeder > 3 mo, n = 8). The mRNA and protein expression of GPIbα, GPIX and GPV were quantified for each patient. Non-bleeder, bleeder ≤ 3 mo and bleeder > 3 mo were comparable regarding the mRNA expression of GPIbα, GPIX and GPV (p > 0.05). The protein analysis revealed a significantly reduced expression level of the main receptor subunit GPIbα in bleeders ≤ 3 mo (p = 0.04). We suggest that the observed reduction of platelet receptor GPIbα protein expression in patients who experienced their first bleeding event within 3 months after LVAD implantation may influence platelet physiology. The alterations of functional GPIbα potentially reduce the platelet adhesion capacities, which may lead to an impaired hemostatic process and the elevated propensity of bleeding in HM 3 patients.

GPIbα shedding in platelets is controlled by strict intracellular containment of both enzyme and substrate.

BACKGROUND: A disintegrin and metalloprotease 17 (ADAM17) catalyzes platelet glycoprotein (GP) Ibα ectodomain shedding, thereby releasing glycocalicin in plasma. The spatiotemporal control over the enzyme-substrate interaction and the biological consequences of GPIbα shedding are poorly understood. OBJECTIVES: This study aimed to determine the spatiotemporal control over GPIbα shedding by ADAM17. METHODS: Transmission electron microscopy with immunogold staining, immunoprecipitation, and quantitative western blotting were used. RESULTS: Immunogold staining showed that all ADAM17 antigen is expressed intracellularly, irrespective of platelet activation. ADAM17 clustered in patches on a tortuous membrane system different from α- and dense granules. Mild activation by platelet adhesion to immobilized fibrinogen did not cause GPIbα shedding, whereas strong and sustained stimulation using thrombin and collagen (analogs) did. Glycocalicin release kinetics was considerably slower than typical hemostasis, starting at 20 minutes and reaching a plateau after 3 hours of strong stimulation. Inhibition of the ADAM17 scissile bond specifically in GPIbα receptors that reside on the platelet's extracellular surface did not prevent shedding, which is in line with the strict intracellular location of ADAM17. Instead, shedding was restricted to a large GPIbα subpopulation that is inaccessible on resting platelets but becomes partially accessible following platelet stimulation. Furthermore, the data show that proteinaceous, water-soluble ADAM17 inhibitors cannot inhibit GPIbα shedding, whereas membrane permeable small molecule ADAM inhibitors can. CONCLUSION: The data show that platelets harbor 2 distinct GPIbα subpopulations: one that presents at the platelet's surface known for its role in primary hemostasis and one that provides substrate for proteolysis by ADAM17 with kinetics that suggest a role beyond hemostasis.

Novel Antiplatelet Activity of Ginsenoside Re Through the Inhibition of High Shear Stress-Induced Platelet Aggregation.

ABSTRACT: Bleeding is one of the most serious side effects of antiplatelet drugs. Efforts have been made to find new antiplatelet agents without bleeding complications. Shear-induced platelet aggregation (SIPA) occurs only under pathological conditions and is a promising target for overcoming bleeding problems. This work demonstrates that the ginsenoside Re selectively inhibits platelet aggregation induced by high shear stress. Human platelets were exposed to high shear stress using microfluidic chip technology, and aggregation, activation, and phosphatidylserine (PS) exposure were measured. The Von Willebrand Ristocetin Cofactor (vWF:RCo) assay and western blot were used to evaluate the effect of the vWF-GPⅠb/PI3K/Akt signal pathway. The coagulation and bleeding risk were evaluated by measuring the coagulation parameters PT, APTT, TT, and thromboelastography. The 3-dimensional morphology of platelet aggregates was observed by a microscopic 3-dimensional imaging. Re was a potent inhibitor of SIPA, with an IC 50 of 0.071 mg/mL. It effectively blocked shear stress-induced platelet activation without any significant toxicity. It was highly selective against SIPA, effectively inhibiting vWF-GPIb and the downstream PI3K/Akt signaling pathway. Most importantly, Re did not affect normal blood coagulation and did not increase the risk of bleeding. In conclusion, Re inhibits platelet activation through the inhibition of the vWF-GPIb/PI3K/Akt pathway. Thus, it might be considered as a new antiplatelet drug in the prevention of thrombosis without increasing the risk of bleeding.

Rapid isolation of mature murine primary megakaryocytes by size exclusion via filtration.

Megakaryocytes (MKs), the largest and rarest cells of the hematopoietic system, differentiate by increasing their size, DNA and cytoplasmic contents during maturation in order to release high numbers of blood platelets into the circulation. The gold-standard to study these complex cells is the isolation of primary MKs from the native bone marrow (BM). This is typically achieved by using fluorescence- or magnetic-activated cell sorting. However, both methods are time-consuming and require a trained experimenter who is able to operate highly priced special equipment. Here, we demonstrate a simple and rapid alternative method to enrich mature MKs (≥16 N) from murine adult BM by size exclusion. The purity of the MK fraction reached 70-80% after isolation (100- to 250-fold enrichment). Reanalysis of isolated MKs by confocal microscopy revealed the expected expression of lineage-defining MK- and platelet-specific surface receptors, including CD42a/b/d and CD41/CD61. In addition, we detected a clear enrichment of MK-specific proteins/transcripts like ß1-tubulin, ß3-integrin, GPVI and GPIbα, whereas the neutrophil marker Ly6G was only detectable in the BM sample. Taken together, we demonstrate that the protocol proposed in this Technical Report is a compatible addition to established isolation methods.

Dual roles of fucoidan-GPIbα interaction in thrombosis and hemostasis: implications for drug development targeting GPIbα.

BACKGROUND: Platelet GPIbα-von Willebrand factor (VWF) interaction initiates platelet adhesion, activation, and thrombus growth, especially under high shear conditions. Therefore, the GPIb-VWF axis has been suggested as a promising target against arterial thrombosis. The polysaccharide fucoidan has been reported to have opposing prothrombotic and antithrombotic effects; however, its binding mechanism with platelets has not been adequately studied. OBJECTIVE: The objective of this study was to explore the mechanism of fucoidan and its hydrolyzed products in thrombosis and hemostasis. METHODS: Natural fucoidan was hydrolyzed by using hydrochloric acid and was characterized by using size-exclusion chromatography, UV-visible spectroscopy, and fluorometry techniques. The effects of natural and hydrolyzed fucoidan on platelet aggregation were examined by using platelets from wild-type, VWF and fibrinogen-deficient, GPIbα-deficient, and IL4Rα/GPIbα-transgenic and αIIb-deficient mice and from human beings. Platelet activation markers (P-selectin expression, PAC-1, and fibrinogen binding) and platelet-VWF A1 interaction were measured by using flow cytometry. GPIbα-VWF A1 interaction was evaluated by using enzyme-linked immunosorbent assay. GPIb-IX-induced signal transduction was detected by using western blot. Heparinized whole blood from healthy donors was used to test thrombus formation and growth in a perfusion chamber. RESULTS: We found that GPIbα is critical for fucoidan-induced platelet activation. Fucoidan interacted with the extracellular domain of GPIbα and blocked its interaction with VWF but itself could lead to GPIbα-mediated signal transduction and, subsequently, αIIbß3 activation and platelet aggregation. Conversely, low-molecular weight fucoidan inhibited GPIb-VWF-mediated platelet aggregation, spreading, and thrombus growth at high shear. CONCLUSION: Fucoidan-GPIbα interaction may have unique therapeutic potential against bleeding disorders in its high-molecular weight state and protection against arterial thrombosis by blocking GPIb-VWF interaction after fucoidan is hydrolyzed.

von Willebrand factor neutralizing and non-neutralizing alloantibodies in 213 subjects with type 3 von Willebrand disease enrolled in 3WINTERS-IPS.

BACKGROUND: Type 3 von Willebrand disease (VWD) is the most severe form of this disease owing to the almost complete deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived products containing VWF or recombinant VWF rarely cause the development of alloantibodies against VWF that may be accompanied by anaphylactic reactions. OBJECTIVE: The objective of this study was to assess the prevalence of anti-VWF alloantibodies in subjects with type 3 VWD enrolled in the 3WINTERS-IPS. METHODS: An indirect in-house enzyme-linked immunosorbent assay has been used to test all the alloantibodies against VWF. Neutralizing antibodies (inhibitors) have been tested with a Bethesda-based method by using a VWF collagen binding (VWF:CB) assay. Samples positive for anti-VWF antibodies were further tested with Bethesda-based methods by using the semiautomated gain-of-function glycoprotein-Ib binding (VWF:GPIbM) and a VWF antigen (VWF:Ag) enzyme-linked immunosorbent assay. RESULTS: In total, 18 of the 213 (8.4%) subjects tested positive for anti-VWF antibodies and 13 of 213 (6%) had VWF:CB inhibitors. These 13 were among the 18 with anti-VWF antibodies. Of the 5 without VWF:CB inhibitors, 3 had non-neutralizing antibodies, 1 only inhibitor against VWF:GPIbM, and one could not be tested further. Ten of the 13 subjects with VWF:CB inhibitors also had VWF:GPIbM inhibitors, 6 of whom also had VWF:Ag inhibitors. Subjects with inhibitors were homozygous for VWF null alleles (11/14), homozygous for a missense variant (1/14), or partially characterized (2/14). CONCLUSIONS: Anti-VWF antibodies were found in 8.4% of subjects with type 3 VWD, whereas neutralizing VWF inhibitors were found in 6%, mainly in subjects homozygous for VWF null alleles. Because inhibitors may be directed toward different VWF epitopes, their detection is dependent on the assay used.

Removal of the vicinal disulfide enhances the platelet-capturing function of von Willebrand factor.

A redox autoinhibitory mechanism has previously been proposed, in which the reduced state of the vicinal disulfide bond in the von Willebrand factor (VWF) A2 domain allows A2 to bind to A1 and inhibit platelet adhesion to the A1 domain. The VWF A1A2A3 tridomain was expressed with and without the vicinal disulfide in A2 (C1669S/C1670S) via the atomic replacement of sulfur for oxygen to test the relevance of the vicinal disulfide to the physiological platelet function of VWF under shear flow. A comparative study of the shear-dependent platelet translocation dynamics on these tridomain variants reveals that the reduction of the vicinal disulfide moderately increases the platelet-capturing function of A1, an observation counter to the proposed hypothesis. Surface plasmon resonance spectroscopy confirms that C1669S/C1670S slightly increases the affinity of A1A2A3 binding to glycoprotein Ibα (GPIbα). Differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry demonstrate that reduction of the vicinal disulfide destabilizes the A2 domain, which consequently disrupts interactions between the A1, A2, and A3 domains and enhances the conformational dynamics of A1-domain secondary structures known to regulate the strength of platelet adhesion to VWF. This study clarifies that the reduced state of the A2 vicinal disulfide is not inhibitory but rather slightly activating.

Biomechanical activation of blood platelets via adhesion to von Willebrand factor studied with mesoscopic simulations.

Platelet adhesion and activation are essential initial processes of arterial and microvascular hemostasis, where high hydrodynamic forces from the bloodflow impede coagulation. The process relies on von Willebrand factor (VWF)-a linear multimeric protein of blood plasma plays a pivotal role in mechanochemical regulation of shear-induced platelet aggregation (SIPA). Adhesive interactions between VWF and glycoprotein receptors GPIb are crucial for platelet recruitment under high shear stress in fluid. Recent advances in experimental studies revealed that mechanical tension on the extracellular part of GPIb may trigger a cascade of biochemical reactions in platelets leading to activation of integrins [Formula: see text] (also known as GPIIb/IIIa) and strengthening of the adhesion. The present paper is aimed at investigation of this process by three-dimensional computer simulations of platelet adhesion to surface-grafted VWF multimers in pressure-driven flow of platelet-rich plasma. The simulations demonstrate that GPIb-mediated mechanotransduction is a feasible way of platelet activation and stabilization of platelet aggregates under high shear stress. Quantitative understanding of mechanochemical processes involved in SIPA would potentially promote the discovery of new anti-platelet medication and the development of multiscale numerical models of platelet thrombosis and hemostasis.

Type 2B von Willebrand disease mutations differentially perturb autoinhibition of the A1 domain.

Type 2B von Willebrand disease (VWD) is an inherited bleeding disorder in which a subset of point mutations in the von Willebrand factor (VWF) A1 domain and recently identified autoinhibitory module (AIM) cause spontaneous binding to glycoprotein Ibα (GPIbα) on the platelet surface. All reported type 2B VWD mutations share this enhanced binding; however, type 2B VWD manifests as variable bleeding complications and platelet levels in patients, depending on the underlying mutation. Understanding how these mutations localizing to a similar region can result in such disparate patient outcomes is essential for detailing our understanding of VWF regulatory and activation mechanisms. In this study, we produced recombinant glycosylated AIM-A1 fragments bearing type 2B VWD mutations and examined how each mutation affects the A1 domain's thermodynamic stability, conformational dynamics, and biomechanical regulation of the AIM. We found that the A1 domain with mutations associated with severe bleeding occupy a higher affinity state correlating with enhanced flexibility in the secondary GPIbα-binding sites. Conversely, mutation P1266L, associated with normal platelet levels, has similar proportions of high-affinity molecules to wild-type (WT) but shares regions of solvent accessibility with both WT and other type 2B VWD mutations. V1316M exhibited exceptional instability and solvent exposure compared with all variants. Lastly, examination of the mechanical stability of each variant revealed variable AIM unfolding. Together, these studies illustrate that the heterogeneity among type 2B VWD mutations is evident in AIM-A1 fragments.