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Of HIT, Scissors and Rocks - Mechanistic basis of GPVI shedding in heparin-induced thrombocytopenia

Lee, Christine

Description

The most famous role for platelets is in thrombosis and haemostasis. Glycoprotein (GP) VI is a platelet-specific collagen and fibrin receptor which controls thrombus formation and growth. Human GPVI is stable on resting platelets but is rapidly metalloproteolysed by A-Disintegrin-And-Metalloproteinase (ADAM) 10 on activated platelets to release soluble GPVI (sGPVI). Regulation of platelet ADAM10 metalloproteolytic activity remains unclear. Active ADAM10 are detectable on resting human platelets...[Show more]

dc.contributor.authorLee, Christine
dc.date.accessioned2019-12-03T22:28:21Z
dc.date.available2019-12-03T22:28:21Z
dc.identifier.urihttp://hdl.handle.net/1885/187245
dc.description.abstractThe most famous role for platelets is in thrombosis and haemostasis. Glycoprotein (GP) VI is a platelet-specific collagen and fibrin receptor which controls thrombus formation and growth. Human GPVI is stable on resting platelets but is rapidly metalloproteolysed by A-Disintegrin-And-Metalloproteinase (ADAM) 10 on activated platelets to release soluble GPVI (sGPVI). Regulation of platelet ADAM10 metalloproteolytic activity remains unclear. Active ADAM10 are detectable on resting human platelets but GPVI levels remain stable implying that there are mechanisms that control GPVI cleavage. Four tissue-inhibitor-of-metalloproteinases (TIMPs) regulate vascular metalloproteinase activity, but a role in controlling platelet ADAMs activity has not been described. Heparin-induced thrombocytopenia (HIT) is a drug-induced, autoimmune disorder with potentially fatal outcomes. HIT occurs in a subset of patients exposed to heparin due to platelet-activating antibodies binding an antigenic complex of heparin and platelet factor 4 (PF4). This immune complex engages platelet FcgRIIa and triggers signalling via immunoreceptor tyrosine-activation motifs (ITAMs) to induce platelet activation, aggregation and clearance. Engagement of FcgRIIa also induces ectodomain shedding of platelet GPVI. However, the mechanistic basis of GPVI shedding induced by HIT immune complex remains to be explored. Identifying patients at risk of developing HIT remains clinically challenging because not all patients with detectable anti-PF4/heparin antibody develop HIT. Plasma sGPVI levels may indicate the presence of an activating anti-PF4/heparin antibody. Polymorphisms of FcgRIIa may influence receptor function and platelet reactivity, and potentially modulate the risk of HIT. This study aimed to (i) characterise changes in platelet surface GPVI and sGPVI levels upon engagement of FcgRIIa by HIT immune complexes; (ii) assess the utility of sGPVI to inform on pathological PF4/heparin autoantibodies in a prospective clinical cohort at risk of HIT; (iii) evaluate the haplotype-based association between FcgRIIa polymorphisms, Q27W and H131R, and HIT susceptibility using a long-read sequencing technology; and (iv) explore levels of TIMPs on platelets and the potential for TIMPs to regulate platelet ADAM10 activity. Engagement of healthy donor platelet FcgRIIa by HIT patient immunoglobulins (Ig) induced cleavage of GPVI that was dependent on heparin, divalent cations, ITAM signalling and FcgRIIa, as measured by flow cytometry and ELISA. Inclusion of patient plasma sGPVI improved the overall accuracy, specificity and positive predictive value when evaluating risk of developing HIT. Higher plasma sGPVI was observed among individuals carrying at least one R allele at position 131. The haplotype combination of FCGR2A at positions 27 and 131 was not significantly associated with HIT susceptibility. TIMPs were detected on circulating platelets and in plasma, and surface levels increased upon platelet activation. TIMP1 and TIMP3 reduced resting platelet ADAM10 activity while only TIMP3 significantly reduced ADAM10 activity when platelets were stimulated. Patient sGPVI levels may provide patient-specific information on platelet-activating HIT antibodies while the FCGR2A genotype may determine the state of activity of platelet in response to activation. This is the first study to assess the polymorphisms of FCGR2A in-phase. Dynamic platelet-associated TIMP levels may modulate ADAM10 activity and maintain platelet patency by stabilising GPVI levels. The findings in this thesis extended our knowledge of the molecular pathway involved in HIT Ig-mediated GPVI shedding and presented a new mechanism for regulating platelet ADAM10 activity.
dc.language.isoen_AU
dc.titleOf HIT, Scissors and Rocks - Mechanistic basis of GPVI shedding in heparin-induced thrombocytopenia
dc.typeThesis (PhD)
local.contributor.supervisorGardiner, Elizabeth
local.contributor.supervisorcontactu1023050@anu.edu.au
dc.date.issued2019
local.identifier.doi10.25911/5f58b07f58ea7
local.identifier.proquestNo
local.identifier.researcherIDW-9387-2019
local.thesisANUonly.authore4e9068d-8c95-4fde-aef5-72dec3ece8f4
local.thesisANUonly.title000000012997_TC_1
local.thesisANUonly.keyb968ab19-7255-e3c8-731f-23680b51ecfc
local.mintdoimint
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