Tissue-engineered human arteries replicate primary and secondary haemostatic functions seen in vivo: An alternative to mouse thrombosis models?

Jacob Ranjbar¹, Ying Yang², Alan G.S. Harper¹

¹ School of Medicine, University of Keele, UK.
² School of Pharmacy and Bioengineering, University of Keele, UK.

E-mail of Presenting Author: j.a.ranjbar@keele.ac.uk

Background: Intravital microscopy in mice is widely used to study in vivo thrombus formation. Previously we have developed a 3D, tissue-engineered human arterial construct (TEAC) that replicates the primary haemostatic properties of the native artery [1].

Aims: To assess whether our TEAC can trigger the activation of the extrinsic coagulation cascade, and whether this can be used to generate a human arterial thrombosis model to replace current in vivo studies.

Methods: Medial and adventitial arterial layers were replicated by seeding human smooth muscle and adventitial cells into a collagen hydrogel. Prothrombin times were measured by exposing these constructs to Platelet-poor plasma (PPP) from healthy human volunteers. A novel fluorogenic tissue factor assay was performed to assess tissue factor activity by incubation with factor VII and SN17a. Platelet activation was assessed using physiological flow conditions in a 3D printed flow chamber.

Results: Both the medial and adventitial layer constructs are able to trigger rapid coagulation when exposed to PPP. Both constructs have measurable tissue factor activity. Tissue factor activity in the medial layer construct was enhanced by culturing these in the presence of ascorbic acid. Ascorbic acid supplementation of the medial construct also significantly increased platelet adhesion and aggregation on this construct under flow conditions.

Conclusions:  Our data demonstrates that our TEACs can replicate all of the major haemostatic processes of the native blood vessel and this was enhanced by supplementation of ascorbic acid into the culture media. The TEAC is a viable replacement to current murine thrombosis models.

This work is supported by an NC3R- and British Heart Foundation co-funded PhD studentship

Refs: [1] Musa et al. (2016) Tissue Eng Part C Methods 22, 691-699.

Platelet transcriptome yields progressive and predictive markers for subtype-specific risk in myeloproliferative neoplasms

Zhu Shen², Wenfei Du², Cecelia Perkins⁴, Lenn Fechter⁴, Vanita Natu³, Holden Maecker⁵, Jesse Rowley⁶, Jason Gotlib⁴, James Zehnder^1,4, and Anandi Krishnan¹.

¹ Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.
² Department of Statistics, Stanford University, Stanford, CA, USA.
³ Stanford Functional Genomics Facility, Stanford University, Stanford, CA, USA.
⁴ Division of Hematology, Department of Medicine, Stanford University School of Medicine and
Stanford Cancer Institute, Stanford, CA, USA.
⁵ Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
⁶ Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT, USA.

E-mail of Presenting Author: anandi.krishnan@stanford.edu

Background: Genotype-phenotype relationships in the pathogenesis of classic, Philadelphia chromosome (Ph-)-negative myeloproliferative neoplasms (MPNs) remain elusive beyond their key driver mutations (JAK2, CALR or MPL in over 90% of patients). Platelets hold vast amounts of biologic information reflecting the diversity in the genes that encode their canonical functions, but also disease- and treatment-related factors.

Aims: Here, we probe the platelet transcriptome in chronic progressive MPNs with the primary aim of defining subtype-specific molecular differences and predicting the advanced phenotype, myelofibrosis.

Methods: Using RNA sequencing (RNA-seq), we profile disease-relevant gene expression and alternative splicing in purified platelets from 120 peripheral blood samples that constitute two independently collected (mutually validating) cohorts of the three chronic clinical phenotypes of MPN patients – essential thrombocythemia, ET (n=24), polycythemia vera, PV (n=33), and primary or secondary myelofibrosis, MF (n=42) – and healthy donors (n=21).

Results: The MPN platelet transcriptome discriminates each clinical phenotype and reveals an incremental molecular reprogramming that is independent of driver mutation status or therapy. Leveraging this dataset, in particular the progressive expression gradient noted across MPN, and a novel application of established Lasso-penalized regression modeling with 10-fold cross-validation, we predict the advanced subtype MF at high accuracy (AUC-ROC of 0.95) and offer a robust core set of < 10 MPN progression markers.

Conclusion: Our platelet transcriptome snapshot of chronic MPNs establishes a methodological foundation for deciphering disease risk stratification and progression beyond genetic data alone, thus presenting an important avenue toward personalized medicine in a wide range of age-related disorders.

Elevated platelet-derived sGPVI is a biomarker of venous in-stent stenosis in patients with post-thrombotic syndrome

Adam M. Gwozdz¹, Prakash Saha¹, Stephen A. Black¹,Alexander P. Bye², Neline Kriek², Jonathan M. Gibbins², and Alberto Smith¹

¹ Academic Department of Vascular Surgery, Section of Vascular Risk and Surgery, School of Cardiovascular Medicine and Science, St Thomas’ Hospital, King’s College London, UK
² Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, UK

E-mail of Presenting Author: adam.gwozdz@kcl.ac.uk

Background: In-stent stenosis following intervention for post-thrombotic syndrome (PTS) occurs in ~30% of cases, despite therapeutic anticoagulation.

Aims: The aim of this study was to investigate whether platelets have a role in this process.

Methods: Blood samples were taken from case-matched patients before and after venous stent placement. Platelet activation markers (P-selectin and phosphatidylserine) and reactivity were determined by flow cytometry and plate-based aggregation, respectively. Soluble glycoprotein VI (sGPVI), shed during activation, was measured in plasma. Patients with in-stent stenosis requiring reintervention (>50% diameter reduction) were compared with those who did not during follow-up.

Results: Forty-five patients were recruited (median age: 43yrs, range: 33-55yrs; 65% female). Re-intervention was required in 19 patients (42%; median time: 3wks, range: 1day-3mths). There was no significant difference in platelet activation or reactivity after stenting, but P-selectin exposure pre-stent was significantly higher in patients who developed in-stent stenosis (2.7%±0.4 vs 1.7%±0.2; P<0.05). sGPVI levels before stent insertion were increased in patients who developed in-stent stenosis (18.9±3.6ng/mL vs. 7.4±0.9ng/mL; P<0.01). Platelet reactivity to collagen-related peptide, a GPVI-specific platelet agonist, was reduced in patients who developed in-stent stenosis (logEC₅₀ = -6.5M±0.3 vs -7.2M±0.2; P<0.05; n=33).

Conclusions: Venous stenting does not activate platelets or alter platelet function, but patients who developed in-stent stenosis exhibited greater levels of pre-stent platelet activation,  greater loss of platelet surface GPVI in the form of sGPVI and consequent reduction in reactivity to GPVI activation. sGPVI may have potential to risk-stratify patients undergoing deep venous reconstruction and predict who requires closer surveillance.

Investigating the role of the novel, monoallelic GPIba G138V variant in patients with inherited bleeding

J.I. Yule^1,2, I. Almazny³, C.W. Smith³, R.J. Stapley³, N.J. Jooss³,  J. Emsley^1,2,  N.V. Morgan³ and N.S. Poulter^1,3

¹ Centre of Membrane Proteins and Receptors, University of Birmingham, Edgbaston, Birmingham B15 2TT. The University of Nottingham, University Park, Nottingham, NG7 2RD
² Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham, NG7 2JE
^1,3 Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.

E-mail of Presenting Author: Jack.Yule@nottingham.ac.uk

Background: GPIbα-VWF binding is essential in tethering of platelets to a vascular injury site. The majority of GPIbα variants are linked with Bernard-Soulier syndrome and are homozygous. These typically present as macrothrombocytopenia with ablated GPIb-IX-V complex expression on the platelet surface.

Aims:  Through the Genotyping and Phenotyping of Platelets Project (GAPP), an atypical monoallelic GPIbα variant (G138V) was discovered in two siblings who exhibit a mild thrombocytopenia and bleeding symptoms. This work aims to characterise this variant and uncover the mechanism by which it contributes to bleeding.

Methods: Standard platelet function assays (e.g. FACS, aggregometry and spreading) were carried out using blood samples from one patient to assess the effect of the G138V variant on receptor levels and platelet activation. A whole blood flow adhesion assay over different substrates was also used to quantify the effect of G138V on thrombus formation and platelet activation. Microscopy was used to look at GPIbα localisation.

Results: The patient has normal platelet surface GPIbα, but exhibits reduced platelet activation   and aggregation responses to standard agonists. Flow adhesion showed impaired platelet deposition and activation on VWF, collagen and fibrinogen coated surfaces. Significantly more spread patient platelets contained a ‘centralised’ GPIbα structure compared to controls.

Conclusions:  We observe a platelet functional defect in a patient with a heterozygous G138V variant of GPIbα. Patient platelets show reduced activation, adhesion and thrombus formation under flow conditions, which is likely to contribute to the observed bleeding. Further work is now required to evaluate the extent to which this variant affects GPIbα-VWF binding capacity.

The importance of the GPIba intracellular tail in VWF-mediated platelet signalling events

Adela Constantinescu-Bercu¹, Yuxiao Alice Wang¹, Kevin J Woollard², Pierre Mangin³, Karen Vanhoorelbeke⁴, James TB Crawley¹ and Isabelle I. Salles-Crawley¹

¹ Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, UK
² Centre for Inflammatory Disease, Department of Immunology and Inflammation, Imperial College London, London, UK
³ Université de Strasbourg, INSERM, EFS Grand-Est, BPPS UMR-S 1255, FMTS, Strasbourg, France
⁴ Laboratory for Thrombosis Research, KU Leuven, Kortrijk, Belgium.

E-mail of Presenting Author: adela.constantinescu11@imperial.ac.uk

Background: The role of the VWF A1 domain interaction with platelet GPIba has classically been limited to platelet capture at sites of vessel injury. We recently demonstrated that human VWF-bound platelets are ‘primed’ under flow, recruiting neutrophils and inducing NETosis via activated a_IIbβ₃.

Aims: To evaluate the (patho)physiological importance of VWF/flow-dependent signalling through GPIba.

Methods: GPIba^Dsig/Dsig mice were generated via the CRISPR-Cas9 technology, by deleting the last 24 amino acids from the intracellular tail of GPIba. Platelet function was assessed ex vivo and in vivo.

Results: GpIba^Dsig/Dsig mice exhibited normal tail-bleeding responses, and unchanged platelet/fibrin accumulation in the laser-induced thrombosis model. GpIba^Dsig/Dsig platelets bound normally to VWF under flow, but had a reduced ability to form filopodia on VWF, in the presence of Botrocetin and GR144053, confirming that VWF-dependent signalling is impaired in these mice. GPIba^+/+ primed platelets recruited neutrophils under low shear, in a manner that could be inhibited by a_IIbβ₃ blockade, but not by β₂-integrin inhibition. Neutrophil recruitment was significantly increased in the absence of plasma. GPIba^Dsig/Dsig platelets exhibited a significantly reduced ability to recruit neutrophils compared to GPIba^+/+ platelets, confirming the importance of the intracellular tail of GPIba in mediating interactions between VWF-‘primed’ platelets and neutrophils under flow.

Summary/Conclusions: Our data suggest an important role for the GPIba intracellular tail to transduce signals downstream of the VWF A1-GPIbα interaction and mediate subsequent platelet-neutrophil interactions. Additional thrombosis models and platelet signalling assays are underway to fully appreciate the pathophysiological role of the GPIba-A1 signalling.