Platelet glycoprotein VI in the regulation of thrombus growth

Perrella, Gina ORCID: 0000-0002-0516-4261 (2022). Platelet glycoprotein VI in the regulation of thrombus growth. University of Birmingham. Ph.D.

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Abstract

Platelet glycoprotein VI (GPVI) is the principal signalling receptor for collagen, which is exposed upon damage to the extracellular matrix and at site of rupture of an atherosclerotic plaque. GPVI has recently been shown to be also a receptor for fibrinogen and fibrin, which mediate platelet aggregation and clot formation. In mice, GPVI deficiency protects form arterial thrombosis without causing excessive bleeding. Individuals with an inherited deficiency in GPVI have a mild bleeding diathesis. Together, these observations suggest a more important role of GPVI in thrombosis over haemostasis.

The research described in this thesis investigates the relative contribution of GPVI in adhesion and platelet aggregation under flow on collagen and fibrin(ogen). As overarching hypothesis, I propose that GVI is a relevant signalling receptor for fibrin and fibrinogen in supporting thrombus propagation and stability. The first part is a critical assessment of the evidence that fibrin and fibrinogen bind to monomeric or dimeric GPVI in view of the earlier existing controversies. This is followed by the exploration of the role of GPVI in platelets adhesion to collagen using blood from Chilean patients with a homozygous insertion mutation in the GP6 gene which causes lack of GPVI expression on the platelet surface. The results show a critical role for GPVI in supporting platelet aggregation and phosphtidylserine exposure on collagen and non-collagen surfaces, but not adhesion which, on collagen, is mediated by the integrin α2β1. Further, it is estimated that in Chile there are over 4,000 individuals GPVI-deficient, of whom only a handful are known to have a bleeding diathesis. The next study investigates the contribution of GPVI to platelet adhesion and thrombus growth on fibrin, fibrinogen, using the Syk inhibitor, PRT-060318, and an anti-GPVI Fab, 9O12. The results show that GPVI contributes to platelet activation in response to fibrin and fibrinogen, that induces platelet secretion but low level of Ca2+ rises and that it is not required for platelet adhesion. Further, they show that in response to fibrin, GPVI acts in concert with the integrin αIIbβ3 in a non-redundant way. Successively, the action of two small molecule inhibitors of GPVI, losartan and honokiol, on platelet activation is investigated. The results show that both compounds are not selective GPVI antagonists. The following study explores the contribution of GPVI and integrin αIIbβ3 to the stability of a preformed thrombus under flow conditions. A comparison is made between inhibitors of Syk, Src, Btk and those of secondary mediators, ADP and TxA2. The results demonstrate a critical role for Syk in supporting aggregate stability, likely through fibrinogen-induced activation of both GPVI and integrin αIIbβ3. In addition, Syk appears to act in synergy with ADP and TxA2 and to be independent of collagen-induced GVPI activation, as a blocking nanobody has minor effect.

Overall, the research in this thesis provides evidence that the role of GPVI extends beyond the onset of thrombus formation. GPVI activation in response to fibrin(ogen) contributes to platelet aggregation and it is key to thrombus stability. This conclusion further supports the argument that blocking GPVI may effectively prevent arterial thrombosis. Furthermore, this research highlights the importance of the integrin αIIbβ3 in supporting GPVI activation, suggesting that blocking signalling downstream of both receptors, can provide an alternative therapeutic strategy for the treatment of arterial thrombosis.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):

Supervisor(s)	Email	ORCID
Watson, Steve P.	UNSPECIFIED	UNSPECIFIED
Thomas, Mark	UNSPECIFIED	UNSPECIFIED
Heemskerk, Johan W.M.	UNSPECIFIED	UNSPECIFIED
Nagy, Magdolna	UNSPECIFIED	UNSPECIFIED