a-uniform-shear-assay-for-human-platelet-cell-surface-receptors-via

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

Children's Healthcare of Atlanta

Emory University School of Medicine

The glycoprotein (GP) Ib-IX-V complex, the von Willebrand factor receptor on the platelet surface, is critically involved in hemostasis and thrombosis. The GPV subunit interacts with GPIb-IX to form the GPIb-IX-V complex, but the underlying molecular basis remains unclear. It was observed earlier that efficient expression of GPV in the plasma membrane requires co-expression of GPIb-IX.

Transmembrane domains are critical to the interaction between platelet glycoprotein V and glycoprotein Ib-IX complex.

The glycoprotein (GP)Ib-IX-V complex is the platelet receptor for von Willebrand factor and many other molecules that are critically involved in hemostasis and thrombosis. The lack of functional GPIb-IX-V complexes on the platelet surface is the cause of Bernard-Soulier syndrome, a rare hereditary bleeding disorder that is also associated with macrothrombocytopenia. GPIb-IX-V contains GPIbα, GPIbβ, GPIX and GPV subunits, all of which are type I transmembrane proteins containing leucine-rich repeat domains. Although all of the subunits were identified decades ago, not until recently did the mechanism of complex assembly begin to emerge from a systematic characterization of inter-subunit interactions. This review summarizes the forces driving the assembly of GPIb-IX-V, discusses their implications for the pathogenesis of Bernard-Soulier syndrome, and identifies questions that remain about the structure and organization of GPIb-IX-V.

The organizing principle of the platelet glycoprotein Ib-IX-V complex.

Semaphorin 4D (Sema4D) is a transmembrane protein that supports contact-dependent amplification of platelet activation by collagen before being gradually cleaved by the metalloprotease ADAM17, as we have previously shown. Cleavage releases a soluble 120-kDa exodomain fragment for which receptors exist on platelets and endothelial cells. Here we have examined the mechanism that regulates Sema4D exodomain cleavage. The results show that the membrane-proximal cytoplasmic domain of Sema4D contains a binding site for calmodulin within the polybasic region Arg762-Lys779. Coprecipitation studies show that Sema4D and calmodulin are associated in resting platelets, forming a complex that dissociates upon platelet activation by the agonists that trigger Sema4D cleavage. Inhibiting calmodulin with W7 or introducing a membrane-permeable peptide corresponding to the calmodulin-binding site is sufficient to trigger the dissociation of Sema4D from calmodulin and initiate cleavage. Conversely, deletion of the calmodulin-binding site causes constitutive shedding of Sema4D. These results show that (1) Sema4D is a calmodulin-binding protein with a site of interaction in its membrane-proximal cytoplasmic domain, (2) platelet agonists cause dissociation of the calmodulin-Sema4D complex, and (3) dissociation of the complex is sufficient to trigger ADAM17-dependent cleavage of Sema4D, releasing a bioactive fragment.

Identification of a calmodulin-binding domain in Sema4D that regulates its exodomain shedding in platelets.

Ectodomain shedding of glycoprotein Ibα (GPIbα), a proteolytic event in which metalloprotease ADAM17 cleaves the Gly464-Val465 bond and releases glycocalicin to the plasma, is considered a critical step in mediating clearance of stored platelets. Supporting evidence has largely come from studies using ADAM17 inhibitors. However, the definitive proof is lacking due to the broad substrate specificity of ADAM17.

Specific inhibition of ectodomain shedding of glycoprotein Ibα by targeting its juxtamembrane shedding cleavage site.

The development of anti-factor VIII antibodies (inhibitors) is a significant complication in the management of patients with hemophilia A, leading to significant increases in morbidity and treatment cost. Using a panel of mAbs against different epitopes on FVIII, we have recently shown that epitope specificity, inhibitor kinetics and time to maximum inhibition are more important than inhibitor titer in predicting responses to FVIII and the combination of FVIII and recombinant FVIIa. In particular, a subset of high-titer inhibitors responded to high-dose FVIII, which would not be predicted on the basis of their inhibitor titer alone. Thus, the ability to quickly map the epitope spectrum of patient plasma with a clinically feasible assay may fundamentally change how clinicians approach the treatment of high-titer inhibitor patients.

Epitope mapping of inhibitory antibodies targeting the C2 domain of coagulation factor VIII by hydrogen-deuterium exchange mass spectrometry.

Intrinsically disordered protein regions are widely distributed in the cytoplasmic domains of many transmembrane receptors. The cytoplasmic domain of a disintegrin and metalloprotease (ADAM)10, a transmembrane metalloprotease mediating ectodomain shedding of diverse membrane proteins, was recently suggested to mediate the homodimerization of ADAM10. Here we show that a recombinant cytoplasmic domain of ADAM10 (A10Cp) is unstructured as judged by its susceptibility to limited trypsin digestion and its circular dichroism spectrum. In comparison, recombinant transmembrane-cytoplasmic domain of ADAM10 (A10TmCp) reconstituted in dodecylphosphocholine (DPC) micelles exhibits much greater resistance to trypsin digestion, with its cytoplasmic domain taking on a significant ordered structure. FRET analysis demonstrates that, although A10Cp remains monomeric, A10TmCp forms a tight homodimer (K(d) ∼ 7 nM) in DPC micelles. Phospholipid-conjugated A10Cp dose-dependently inhibits formation of A10TmCp homodimer, whereas A10Cp achieves only limited inhibition. Placing the transmembrane and cytoplasmic domains of ADAM10, but not the transmembrane domain alone, in their native orientation in the inner membrane of Escherichia coli produces specific and strong dimerization signal in the AraC-based transcriptional reporter assay. A chimeric construct containing the otherwise monomeric transmembrane domain of L-selectin and the cytoplasmic domain of ADAM10 produces a similar dimerization signal. Overall, these results demonstrate that a transmembrane domain imparts a stable structure to the adjacent and intrinsically disordered cytoplasmic domain of ADAM10 to form a homodimer in the membrane. This finding advances our understanding of the regulatory mechanism of ADAMs and has general implications for membrane-protein interactions in the process of transmembrane signaling.

Membrane-enabled dimerization of the intrinsically disordered cytoplasmic domain of ADAM10.

The mechanism of action of the widely used in vivo ferric chloride (FeCl3) thrombosis model remains poorly understood; although endothelial cell denudation is historically cited, a recent study refutes this and implicates a role for erythrocytes. Given the complexity of the in vivo environment, an in vitro reductionist approach is required to systematically isolate and analyze the biochemical, mass transfer, and biological phenomena that govern the system. To this end, we designed an "endothelial-ized" microfluidic device to introduce controlled FeCl3 concentrations to the molecular and cellular components of blood and vasculature. FeCl3 induces aggregation of all plasma proteins and blood cells, independent of endothelial cells, by colloidal chemistry principles: initial aggregation is due to binding of negatively charged blood components to positively charged iron, independent of biological receptor/ligand interactions. Full occlusion of the microchannel proceeds by conventional pathways, and can be attenuated by antithrombotic agents and loss-of-function proteins (as in IL4-R/Iba mice). As elevated FeCl3 concentrations overcome protective effects, the overlap between charge-based aggregation and clotting is a function of mass transfer. Our physiologically relevant in vitro system allows us to discern the multifaceted mechanism of FeCl3-induced thrombosis, thereby reconciling literature findings and cautioning researchers in using the FeCl3 model. 

Resolving the multifaceted mechanisms of the ferric chloride thrombosis model using an interdisciplinary microfluidic approach.

Inhibitor formation in hemophilia A is the most feared treatment-related complication of factor VIII (fVIII) therapy. Most inhibitor patients with hemophilia A develop antibodies against the fVIII A2 and C2 domains. Recent evidence demonstrates that the C1 domain contributes to the inhibitor response. Inhibitory anti-C1 monoclonal antibodies (mAbs) have been identified that bind to putative phospholipid and von Willebrand factor (VWF) binding epitopes and block endocytosis of fVIII by antigen presenting cells. We now demonstrate by competitive enzyme-linked immunosorbent assay and hydrogen-deuterium exchange mass spectrometry that 7 of 9 anti-human C1 mAbs tested recognize an epitope distinct from the C1 phospholipid binding site. These mAbs, designated group A, display high binding affinities for fVIII, weakly inhibit fVIII procoagulant activity, poorly inhibit fVIII binding to phospholipid, and exhibit heterogeneity with respect to blocking fVIII binding to VWF. Another mAb, designated group B, inhibits fVIII procoagulant activity, fVIII binding to VWF and phospholipid, fVIIIa incorporation into the intrinsic Xase complex, thrombin generation in plasma, and fVIII uptake by dendritic cells. Group A and B epitopes are distinct from the epitope recognized by the canonical, human-derived inhibitory anti-C1 mAb, KM33, whose epitope overlaps both groups A and B. Antibodies recognizing group A and B epitopes are present in inhibitor plasmas from patients with hemophilia A. Additionally, group A and B mAbs increase fVIII clearance and are pathogenic in a hemophilia A mouse tail snip bleeding model. Group A anti-C1 mAbs represent the first identification of pathogenic, weakly inhibitory antibodies that increase fVIII clearance.

High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors.

The platelet storage lesion accelerates platelet clearance after transfusion, but the underlying molecular mechanism remains elusive. Although inhibiting sheddase activity hampers clearance of platelets with storage lesion, the target platelet protein responsible for ectodomain shedding-induced clearance is not definitively identified. Monoclonal antibody 5G6 was developed recently to bind specifically human platelet receptor glycoprotein (GP)Ibα and inhibit its shedding but not shedding of other receptors. Here, the role of GPIbα shedding in platelet clearance after transfusion was addressed.

Inhibiting GPIbα Shedding Preserves Post-Transfusion Recovery and Hemostatic Function of Platelets After Prolonged Storage.

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.

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

Immune thrombocytopenia (ITP) is a prevalent autoimmune disease characterized by autoantibody-induced platelet clearance. Some ITP patients are refractory to standard immunosuppressive treatments such as intravenous immunoglobulin (IVIg). These patients often have autoantibodies that target the ligand-binding domain (LBD) of glycoprotein Ibα (GPIbα), a major subunit of the platelet mechanoreceptor complex GPIb-IX. However, the molecular mechanism of this Fc-independent platelet clearance is not clear. Here, we report that many anti-LBD monoclonal antibodies such as 6B4, but not AK2, activated GPIb-IX in a shear-dependent manner and induced IVIg-resistant platelet clearance in mice. Single-molecule optical tweezer measurements of antibodies pulling on full-length GPIb-IX demonstrated that the unbinding force needed to dissociate 6B4 from the LBD far exceeds the force required to unfold the juxtamembrane mechanosensory domain (MSD) in GPIbα, unlike the AK2-LBD unbinding force. Binding of 6B4, not AK2, induced shear-dependent unfolding of the MSD on the platelet, as evidenced by increased exposure of a linear sequence therein. Imaging flow cytometry and aggregometry measurements of platelets and LBD-coated platelet-mimetic beads revealed that 6B4 can sustain crosslinking of platelets under shear, whereas 6B4 Fab and AK2 cannot. These results suggest a novel mechanism by which anti-LBD antibodies can exert a pulling force on GPIb-IX via platelet crosslinking, activating GPIb-IX by unfolding its MSD and inducing Fc-independent platelet clearance.

Fc-independent immune thrombocytopenia via mechanomolecular signaling in platelets.

Hundreds of billions of platelets are cleared daily from circulation via efficient and highly regulated mechanisms. These mechanisms may be stimulated by exogenous reagents or environmental changes to accelerate platelet clearance, leading to thrombocytopenia. The interplay between antiapoptotic Bcl-x and proapoptotic molecules Bax and Bak sets an internal clock for the platelet lifespan, and BH3-only proteins, mitochondrial permeabilization, and phosphatidylserine (PS) exposure may also contribute to apoptosis-induced platelet clearance. Binding of plasma von Willebrand factor or antibodies to the ligand-binding domain of glycoprotein Ibα (GPIbα) on platelets can activate GPIb-IX in a shear-dependent manner by inducing unfolding of the mechanosensory domain therein, and trigger downstream signaling in the platelet including desialylation and PS exposure. Deglycosylated platelets are recognized by the Ashwell-Morell receptor and potentially other scavenger receptors, and are rapidly cleared by hepatocytes and/or macrophages. Inhibitors of platelet clearance pathways, including inhibitors of GPIbα shedding, neuraminidases, and platelet signaling, are efficacious at preserving the viability of platelets during storage and improving their recovery and survival in vivo. Overall, common mechanisms of platelet clearance have begun to emerge, suggesting potential strategies to extend the shelf-life of platelets stored at room temperature or to enable refrigerated storage.

Mechanisms of platelet clearance and translation to improve platelet storage.

Platelet glycoprotein Ibα (GPIbα) extracellular domain, which is part of the receptor complex GPIb-IX-V, plays an important role in tumor metastasis. However, the mechanism through which GPIbα participates in the metastatic process remains unclear. In addition, potential bleeding complication remains an obstacle for the clinical use of anti-platelet agents in cancer therapy.

Novel antibodies against GPIbα inhibit pulmonary metastasis by affecting vWF-GPIbα interaction.

FAM-tastic phospho-regulation of von Willebrand factor activity.

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 s. 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 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 kT. 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.

Article	Year
Transmembrane domains are critical to the interaction between platelet glycoprotein V and glycoprotein Ib-IX complex. Journal of thrombosis and haemostasis : JTH\| PubMed ID: 22759073	2012
The organizing principle of the platelet glycoprotein Ib-IX-V complex. Journal of thrombosis and haemostasis : JTH\| PubMed ID: 23336709	2013
Identification of a calmodulin-binding domain in Sema4D that regulates its exodomain shedding in platelets. Blood\| PubMed ID: 23564909	2013
Specific inhibition of ectodomain shedding of glycoprotein Ibα by targeting its juxtamembrane shedding cleavage site. Journal of thrombosis and haemostasis : JTH\| PubMed ID: 24119228	2013
Epitope mapping of inhibitory antibodies targeting the C2 domain of coagulation factor VIII by hydrogen-deuterium exchange mass spectrometry. Journal of thrombosis and haemostasis : JTH\| PubMed ID: 24152306	2013
Membrane-enabled dimerization of the intrinsically disordered cytoplasmic domain of ADAM10. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 25349418	2014
Resolving the multifaceted mechanisms of the ferric chloride thrombosis model using an interdisciplinary microfluidic approach. Blood\| PubMed ID: 25931587	2015
High-affinity, noninhibitory pathogenic C1 domain antibodies are present in patients with hemophilia A and inhibitors. Blood\| PubMed ID: 27381905	2016
Inhibiting GPIbα Shedding Preserves Post-Transfusion Recovery and Hemostatic Function of Platelets After Prolonged Storage. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 27417583	2016
Refrigeration-Induced Binding of von Willebrand Factor Facilitates Fast Clearance of Refrigerated Platelets. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 29097365	2017
Fc-independent immune thrombocytopenia via mechanomolecular signaling in platelets. Blood\| PubMed ID: 29203584	2018
Mechanisms of platelet clearance and translation to improve platelet storage. Blood\| PubMed ID: 29475962	2018
Novel antibodies against GPIbα inhibit pulmonary metastasis by affecting vWF-GPIbα interaction. Journal of hematology & oncology\| PubMed ID: 30223883	2018
FAM-tastic phospho-regulation of von Willebrand factor activity. Journal of thrombosis and haemostasis : JTH\| PubMed ID: 30985992	2019
Force-Regulated Refolding of the Mechanosensory Domain in the Platelet Glycoprotein Ib-IX Complex. Biophysical journal\| PubMed ID: 31030883	2019

Renhao Li

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