Abstract
Platelet interaction with collagens, via von Willebrand factor, is a potent trigger of shear-dependent thrombus formation mediated by subsequent engagement of the signaling collagen receptor glycoprotein (GP)VI, enforced by integrin α2β1. Protein tyrosine kinase Syk is central in the GPVI-induced signaling pathway, leading to elevated cytosolic Ca2+. We aimed to determine the Syk-mediated thrombogenic activity of several collagen peptides and (fibrillar) type I and III collagens. High-shear perfusion of blood over microspots of these substances resulted in thrombus formation, which was assessed by eight parameters and was indicative of platelet adhesion, activation, aggregation, and contraction, which were affected by the Syk inhibitor PRT-060318. In platelet suspensions, only collagen peptides containing the consensus GPVI-activating sequence (GPO)n and Horm-type collagen evoked Syk-dependent Ca2+ rises. In whole blood under flow, Syk inhibition suppressed platelet activation and aggregation parameters for the collagen peptides with or without a (GPO)n sequence and for all of the collagens. Prediction models based on a regression analysis indicated a mixed role of GPVI in thrombus formation on fibrillar collagens, which was abolished by Syk inhibition. Together, these findings indicate that GPVI-dependent signaling through Syk supports platelet activation in thrombus formation on collagen-like structures regardless of the presence of a (GPO)n sequence.
Highlights
Platelet interaction with subendothelial collagen is a crucial step in hemostasis or arterial thrombosis after vascular injury or rupture of an atherosclerotic plaque, respectively [1,2]
In blood flowing with high shear rates, the initial capture of platelets is mediated by the von Willebrand factor (VWF), which avidly binds to collagens and is a ligand for the glycoprotein (GP) complex GPIb–V–IX [3]
As a first estimation of the potency of distinct collagen peptides to act as ligands for platelet GPVI, we examined their ability to stimulate PLCγ2-mediated rises in cytosolic Ca2+ using Fura-2-loaded platelets
Summary
Platelet interaction with subendothelial collagen is a crucial step in hemostasis or arterial thrombosis after vascular injury or rupture of an atherosclerotic plaque, respectively [1,2]. The two platelet collagen receptors integrin α2β1 and GPVI ensure stable platelet adhesion and mediate platelet activation [4,5]. The collagen fibers immobilized in such devices, for instance those applied as microspots, bind plasma VWF and promote shear-dependent adhesion, activation, and aggregation of platelets [3,9]. The thrombi that formed on collagen fibers appeared to be heterogeneous in structure, with on the one hand patches of aggregated platelets with activated integrin αIIbβ binding fibrinogen and CD62P expression via α-granule secretion and on the other hand single procoagulant platelets, exposing phosphatidylserine (PS), which is required for coagulation factor binding [12]. Still unexplained is why other fibril-forming type I and III collagen preparations, binding with VWF, are less active in supporting thrombus formation under flow [9,13]
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