Yang Shen's research while affiliated with Monash University (Australia) and other places

Glycoprotein Ib-IX-V (GPIb-IX-V) is a platelet adhesion receptor complex that initiates platelet aggregation. Glycoprotein Ibα (GPIbα) is the central component of the GPIb-IX-V complex, anchoring the complex to the cytoskeleton and harboring the binding site for von Willebrand factor (vWF). Previous studies suggest that the coagulation function in pigs differs from that in humans, especially with respect to the interaction between vWF and platelets. However, we have little knowledge about the function of porcine platelets, which is important with regard to studies of cardiovascular disease, clotting, and surgery that use pigs as animal models. To extend this information, we cloned and analyzed the porcine GPIbα sequence. Porcine GPIbα contains 1891 nucleotides and includes an open reading frame that encodes 627 amino acids. The nucleotide sequence showed 67% identity with human GPIbα, whereas the deduced amino acid sequences were 59% identical. The vWF binding domain shares the highest identity among different species, whereas the PEST domain shows variations. Evaluation of platelet function by using ristocetin-induced platelet aggregation revealed remarkably lower levels of aggregation in porcine than human platelets. According to the sequence analysis and platelet aggregation tests, we propose that the function of GPIbα, especially regarding the ristocetin-vWF-GPIbα interaction, differs between pigs and humans. This characterization of porcine GPIbα will enhance our knowledge of the porcine coagulation system.

Introduction: Ligands acting at the platelet collagen receptor, glycoprotein (GP)VI, induce intracellular FcRγ/Syk-dependent signaling pathways and Syk-dependent or Syk-independent generation of intracellular reactive oxygen species (ROS). Additional signaling-dependent or signaling-independent pathways lead to metalloproteinase-mediated shedding of GPVI. Aim: Analysis of platelet GPVI expression and signaling in a patient with a collagen-selective defect associated with myelodysplastic syndrome (MDS) uniquely demonstrates divergent pathways leading to ROS generation and Syk phosphorylation in human platelets. Methods: Surface expression of GPVI and ligand-induced ROS generation was quantitated by flow cytometry. GPVI shedding and Syk phosphorylation were analyzed by Western blot. Results: Despite platelet count/size and GPVI surface expression within normal ranges, platelet-rich plasma showed no aggregation in response to collagen or GPVI-selective agonist collagen-related peptide, but aggregated in response to other agonists, consistent with dysfunctional GPVI signaling. We observed rapid GPVI-dependent Syk-independent ROS generation and disulfide-dependent GPVI homodimerization, but not Syk-dependent ROS or ligand-induced shedding. Temporal analysis showed a gradual decline in platelet count and the appearance of ligand-induced phosphorylation of an ∼40-kDa Syk fragment. Conclusions: These studies show that GPVI ligation in platelets induces intracellular ROS production independent of either Syk activation or divergent pathways leading to platelet aggregation or ectodomain shedding.

Ischemic stroke induced by thrombosis may be triggered by atherosclerotic plaque rupture and collagen-induced platelet activation. Collagen induces glycoprotein VI shedding. We measured plasma-soluble glycoprotein VI (sGPVI) by enzyme-linked immunosorbent assay in 159 patients with acute (<7-day) ischemic stroke and age/sex-matched community-based control subjects. sGPVI was elevated in stroke compared with controls (P=0.0168). ORs were higher in Quartile 4 for stroke cases (P=0.0121), and sGPVI was significantly elevated in stroke associated with large artery disease across Quartiles 2 to 4 and small artery disease in Quartile 4. sGPVI decreased 3 to 6 months after antiplatelet treatment, consistent with elevated sGPVI due to platelet activation during the thrombotic event. sGPVI correlated with P-selectin (P=0.0007) and was higher in individuals with the GPVIa haplotype (P=0.024). Glycoprotein VI shedding is implicated in the pathology of acute ischemic stroke.

In the past 5 years, metalloproteinase-mediated ectodomain shedding of platelet receptors has emerged as a new mechanism for modulating platelet function. By regulating surface expression of the platelet-specific receptors, glycoprotein (GP)VI that binds collagen, and GPIbalpha (the major ligand-binding subunit of the GPIb-IX-V complex) that binds von Willebrand factor (VWF) and other procoagulant and proinflammatory ligands, shedding not only irreversibly downregulates GPVI/GPIbalpha function, but generates proteolytic fragments that might be unique biomarkers or modulators in plasma. This is potentially significant because GPVI and GPIbalpha are involved in initiating thrombotic diseases such as heart attack and stroke, as well as autoimmune diseases where anti-platelet antibodies result in thrombocytopenia. Altered expression levels of GPIbalpha/GPVI are associated with both thrombotic propensity and platelet aging, suggesting an additional role in platelet clearance. Although emerging data are elucidating molecular mechanisms underlying GPIbalpha/GPVI shedding, evidence for the functional consequences of shedding in vivo, either clinically or in animal models, is far more limited. Here we consider recent published evidence for GPVI or GPIbalpha shedding in humans, nonhuman primates and mice, and whether conservation of sheddase cleavage sites across species points to a functional role for metalloproteolytic shedding in vivo.

Platelet glycoprotein (GP)Ib-IX-V, which binds von Willebrand factor (VWF), and GPVI, which binds collagen, form an adhesion-signaling complex on platelets and mediate platelet adhesion in flowing blood. Platelet activation following engagement of GPIb-IX-V/GPVI by VWF/collagen is critical for initiation and development of a protective thrombus across a site of damaged or exposed endothelium. We examined platelet aggregation and signaling following selective engagement of platelet GPIbalpha (the major ligand-binding subunit of GPIb-IX-V) by a multivalent surface-expressed GPIbalpha-binding VWF-A1 domain on COS-7 cells. COS-7 cells expressing the VWF-A1 domain containing an R543W mutation (a gain-of-function mutation found in Type 2B von Willebrand's Disease) were used as a selective agonist for GPIb-IX-V. When incubated in a cell-to-platelet ratio of up to 1 : 1200, VWF-A1/R543W cells caused rapid, spontaneous aggregation of washed platelets that was GPIbalpha- and alpha(IIb)beta(3)-dependent (blocked by inhibitory anti-VWF-A1, anti-GPIbalpha and anti-alpha(IIb)beta(3) antibodies). Platelet aggregation was also sensitive to inhibitors of Src, phosphoinositide 3-kinase (PI3-kinase) or Syk, confirming a role for these proteins in GPIbalpha-mediated signal transduction. Platelet tyrosine phosphorylation patterns and specific tyrosine phosphorylation of Syk after GPIbalpha engagement by VWF-A1/R543W was comparable to that induced by engagement of GPVI by collagen or collagen-related peptide (CRP). These data indicate signaling events triggered by specific ligation of GPIbalpha can lead to robust platelet activation and help define GPIb-IX-V as both an adhesion and signaling receptor on platelets.

Adhesion of circulating platelets to the blood vessel wall initiates thrombus formation in haemostasis and thrombotic disease. The platelet collagen receptor, glycoprotein (GP) VI, is critical for thrombus formation at arterial shear rates and is a potential therapeutic target for anti-thrombotic drugs. In this study, we evaluate eight newly-derived, purified murine anti-human GPVI monoclonal antibodies (mAbs) for their effect on GPVI-dependent platelet aggregation and GPVI ectodomain shedding. All mAbs were raised against the ligand-binding GPVI ectodomain encompassing two immunoglobulin domains (residues 21-234, excluding the signal sequence) and recognized full-length GPVI in human platelet lysates by western blotting. The majority of antibodies induced aggregation in both human platelet-rich plasma (PRP) and washed platelets independently of the Fc receptor, FcgammaRIIa (not inhibited by the blocking anti-FcgammaRIIa mAb, IV.3), whereas one mAb (11A7) neither induced aggregation nor inhibited aggregation in response to GPVI ligands, collagen, and collagen-related peptide (CRP). In contrast, Fab fragments of mAb 12A5 strongly blocked collagen- and CRP-, but not convulxin-induced aggregation. In addition, it is shown for the first time in vitro that anti-GPVI mAbs can induce metalloproteinase-dependent ectodomain shedding of human GPVI, generating an approximately 10-kDa remnant that remained platelet-associated and an approximately 55-kDa soluble fragment. In conclusion, this analysis of anti-GPVI mAbs provides useful tools for studying the functional role of platelet GPVI.

Snake venom metalloproteinases represent unique probes for analyzing platelet adhesion receptors regulating hemostasis and thrombosis. Snake venom metalloproteinase-disintegrins consist of a propeptide domain, a catalytic domain containing a metal ion-coordination sequence (HEXXHXXGXXH), a disintegrin domain, and a Cys-rich domain. Here, we investigate whether metal ion-affinity chromatography may be used to fractionate venom metalloproteinases based on the metal ion-coordination motif. First, we showed that a purified cobra metalloproteinase, Nk, from Naja kaouthia bound Ni(2+)-agarose, and was eluted by approximately 10mM imidazole, confirming the validity of the approach. Nk cleaved the platelet von Willebrand factor (VWF) receptor, glycoprotein (GP)Ibalpha, with similar activity to the previously reported cobra metalloproteinase, mocarhagin, as shown by EDTA-inhibitable Nk-dependent proteolysis of a purified GPIbalpha extracellular fragment (glycocalicin), and inhibition of (125)I-VWF binding to GPIbalpha on washed human or canine platelets. Second, crude venom from the viper, Trimeresurus albolabris, was fractionated on Ni(2+)-agarose. Samples of flow-through, wash, and imidazole-eluted (0-30mM gradient) fractions were analyzed by (i) SDS-polyacrylamide gel electrophoresis, (ii) immunoblotting with a rabbit anti-mocarhagin antibody, and (iii) assessing metalloproteinase activity using human fibrinogen as substrate. The combined results support the general concept of using Ni(2+)-agarose to fractionate snake venom metalloproteinases.

Thrombus formation in hemostasis or thrombotic disease is initiated by adhesion of circulating platelets to damaged blood vessel walls. Exposed subendothelial collagen interacting with platelet glycoprotein (GP) VI leads to platelet activation and integrin alpha(IIb)beta(3)-mediated aggregation. We previously showed that ligand binding to GPVI also induces metalloproteinase-dependent shedding, generating an approximately 55-kDa soluble ectodomain fragment and an approximately 10-kDa membrane-associated remnant. Here, treatment of platelets with collagen or the GPVI-targeting rattlesnake toxin convulxin also induces rapid (10-30 s) formation of a high molecular weight GPVI complex (GPVIc) under nonreducing conditions, as detected by immunoblotting with anti-GPVI antibodies. The appearance of an approximately 20-kDa remnant detectable using a polyclonal antibody against the GPVI cytoplasmic tail under nonreducing, but not reducing, conditions after ectodomain shedding and nonreduced/reduced two-dimensional SDS-polyacrylamide gel analysis of biotinylated platelets confirmed that that GPVIc was a homodimer. Formation of disulfide-linked GPVIc was prolonged in the presence of metalloproteinase inhibitor GM6001 and was independent of GPVI signaling because it was unaffected by inhibitors of Src kinases, Syk, or phosphoinositide 3-kinase. To identify the thiol involved in disulfide bond formation, wild-type or mutant GPVI, where two available sulfhydryls (Cys-274 and Cys-338) were individually mutated to serine, was expressed in rat basophilic leukemia cells. Dimerization of wild-type and C274S GPVI, but not the C338S mutant, was observed after treating cells with convulxin. We conclude that (i) a subpopulation of GPVI forms a constitutive dimer on the platelet surface, facilitating rapid disulfide cross-linking, (ii) convulxin or other GPVI agonists induce disulfide-linked GPVI dimerization independent of GPVI signaling, and (iii) the penultimate residue of the GPVI cytoplasmic tail, Cys-338, mediates disulfide-dependent dimer formation.

The platelet collagen receptor, glycoprotein (GP)VI, of the immunoreceptor family forms a complex with the von Willebrand factor (VWF) receptor, GPIb-IX-V, critical for initiating thrombus formation. GPVI is co-associated with Fc receptor gamma-chain (FcRgamma), which contains a cytoplasmic immunoreceptor tyrosine-based activation motif domain, involved in activation of Syk, and a signalling cascade leading to (i) activation of alpha(IIb)beta(3), which binds VWF and fibrinogen and mediates platelet aggregation, and (ii) metalloproteinase-mediated shedding of the GPVI ectodomain (blocked by Syk inhibitors), a key mechanism for regulating GPVI surface expression. In this study, we report a familial case of abnormal platelet aggregation with dysfunctional signalling through GPVI that uniquely demonstrates divergent alpha(IIb)beta(3)-activating and GPVI-shedding pathways. The patient is a 60-year-old female with a history of immune disorders, excessive bleeding from childhood and a life-threatening haemorrhage post-trauma. Platelet aggregation to ADP, thrombin receptor-agonist peptide or ristocetin/VWF was normal (indicating normal expression and function of alpha(IIb)beta(3)), but platelet aggregation to GPVI agonists, collagen, collagen-related peptide, or convulxin, was defective. Both GPVI/FcRgamma expression and ligand-induced GPVI ectodomain shedding were normal, confirming expression of functional GPVI/FcRgamma, but suggesting a signalling defect downstream of Syk. A genetic defect in GPVI/Fcgamma signalling compromising platelet function is hypothesised in this family.