https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1538-7836.2004.00665.x

The role of collagen in thrombosis and hemostasis.

Platelet Activation and Signal Transduction by Convulxin, a C-type Lectin from Crotalus durissus terrificus (Tropical Rattlesnake) Venom via the p62/GPVI Collagen Receptor

Glycoprotein VI Is a Major Collagen Receptor for Platelet Activation: It Recognizes the Platelet-Activating Quaternary Structure of Collagen, Whereas CD36, Glycoprotein IIb/IIIa, and von Willebrand Factor Do Not

Objective: Peptides consisting of a repeat Gly-Pro-Hyp sequence are potent platelet agonists. The aim of this study was: (1) to examine the specificity of this sequence for platelet activation; (2) to confirm its recognition by platelet glycoprotein VI; and (3) to assess with suitable peptides the relative importance of glycoprotein VI and integrin α2β1 in platelet activation by collagen. Methods: Peptides were synthesized by standard Fmoc chemistry and tested for their ability to support adhesion of human platelets and HT 1080 cells, induce platelet aggregation, bind integrin α2 subunit A-domain and to cause tyrosine phosphorylation of platelet proteins. Results: (1) Peptides consisting of a repeat Gly-Pro-Pro, Gly-Pro-Ala or Gly-Pro-Arg sequence exhibited little if any platelet-reactivity. (2) The platelet-reactive peptide consisting of a repeating Gly-Pro-Hyp sequence failed to induce tyrosine phosphorylation in glycoprotein VI-deficient platelets. Platelet adhesion to this peptide was inhibited by intact anti-glycoprotein VI antibody and its Fab fragment. The latter inhibited aggregation by the peptide and fibres of both collagens I and III. (3) A peptide containing a 15-mer α2β1-binding sequence in a repeat Gly-Pro-Pro structure supported α2β1-mediated platelet and HT 1080 cell adhesion and bound α2 A-domain, but failed to activate platelets or to induce tyrosine phosphorylation. Conversely, a peptide containing this sequence but with an essential Glu replaced by Ala and inserted in a repeat Gly-Pro-Hyp structure did not recognize α2β1, but was highly platelet activatory. Conclusions: Platelet activation by collagen involves the highly-specific recognition of the Gly-Pro-Hyp sequence by platelet glycoprotein VI. Recognition of α2β1 is insufficient to cause activation. Interaction between collagen and glycoprotein VI is unique since Gly-Pro-Hyp is common in collagens but occurs rarely in other proteins, and glycoprotein VI may be expressed solely by platelets. This sequence could provide a basis for a highly-specific anti-thrombotic reagent to control thrombosis associated with plaque rupture.

/pdf/collagen-platelet-interaction-gly-pro-hyp-is-uniquely-3pkfe51snd.pdf

Collagen-platelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen.

The platelet collagen receptor glycoprotein VI (GPVI) signals through a similar pathway to that used by immunoreceptors with pivotal roles for the Fc receptor (FcR) γ-chain and the tyrosine kinase Syk (28). Cross-linking of GPVI leads to tyrosine phosphorylation of the immunoreceptor tyrosine-based activation motif in the FcR γ-chain by a Src-like kinase, enabling the binding of Syk to the phosphorylated immunoreceptor tyrosine-based activation motif via its tandem SH2 domains (8, 13, 16). This leads to autophosphorylation and activation of Syk and regulation of downstream events that culminate in activation of phospholipase Cγ2 (PLCγ2). The activation of PLCγ2 is critical for the majority of functional responses to collagen, including aggregation and dense-granule secretion (38). Several proteins play important roles in the activation of PLCγ2 downstream of Syk in platelets. These include the adapter SLP-76, which is a substrate for Syk and is essential for tyrosine phosphorylation of PLCγ2 (12, 19); the tyrosine kinase Btk, which is implicated in the tyrosine phosphorylation of PLCγ2 (29); and the p85/110-kDa heterodimeric form of phosphatidylinositol 3-kinase (PI 3-kinase), which is required for regulation of PLCγ2 activity but not tyrosine phosphorylation (27).

Cross-linking of GPVI also leads to the tyrosine phosphorylation of a 36- to 38-kDa protein in platelets independent of PLCγ2, which is proposed to play a role in the regulation of the phospholipase (5). The 36- to 38-kDa protein has many of the characteristics of a protein with a similar molecular mass in T cells, including the ability to bind to the SH2 domains of PLCγ1 and Grb2 (31). The cloning of the 36- to 38-kDa protein from T cells, the linker for activation of T cells (LAT) (41), enabled the identification of the platelet protein as LAT (17). LAT was shown to associate with the p85α subunit of PI 3-kinase in platelets following cross-linking of GPVI by collagen or the snake venom convulxin, suggesting that it mediates recruitment to the membrane and activation of its associated p110 catalytic subunit (17). LAT has also been shown to be phosphorylated in thrombin-stimulated platelets (32).

LAT is a transmembrane protein which is found in glycolipid-enriched membrane domains (43). There are nine conserved tyrosine residues in the cytosolic domain in the murine and human sequences, five of which contain the optimal binding sequence for binding to the SH2 domain of Grb2. LAT is heavily phosphorylated on tyrosine residues upon T-cell receptor stimulation, most probably by ZAP-70 (41). Studies with a LAT-deficient Jurkat T-cell line, J.Cam2, have shown that LAT plays a critical role in the regulation of PLCγ1 and the mitogen-activated protein kinase pathway downstream of the T-cell antigen receptor (15, 42). LAT-deficient mice have no mature peripheral T cells, but they have a normal B-cell population, demonstrating that LAT is critical for T-cell but not B-cell development (42).

The present study was undertaken to investigate whether LAT is required for the activation of PLCγ2 and the onset of functional responses in platelets stimulated by cross-linking of GPVI by using platelets from mice engineered to lack the adapter (42). A collagen-related peptide (CRP) which does not bind to integrin α2β1 (25) was used rather than collagen to activate GPVI because it gives a more powerful and reproducible response between experiments and, unlike collagen, can be used in flow cytometry. Several studies have shown that activation of platelets by CRP mimics that by collagen (1, 2, 16). The results show that LAT plays a critical role in PLCγ2 tyrosine phosphorylation and activation and in subsequent functional events in response to GPVI activation. In contrast, the response to the G-protein-receptor-coupled agonist thrombin, which mediates activation through PLCβ isoforms (7) and induces minimal tyrosine phosphorylation of PLCγ2 (6), is not altered.

LAT Is Required for Tyrosine Phosphorylation of Phospholipase Cγ2 and Platelet Activation by the Collagen Receptor GPVI

Synthetic collagen-like peptides, of general structure [Gly-Pro-HyP]n, adopt the triple-helical structure which is essential for the platelet-reactivity of native collagens. These peptides are potent activators of platelets, stimulating platelet aggregation at much lower dose than collagen fibres, but, unlike collagen fibres, they are not recognised by the integrin alpha 2 beta 1. We have examined the ability of the synthetic peptides to activate the various signalling pathways which regulate human platelet function. The peptides are potent activators of Ca2+ mobilisation and of protein kinase C, and they stimulate tyrosine phosphorylation of some substrates preferentially. However, in contrast with native type I collagen fibres, they are unable to inhibit platelet adenylate cyclase. This suggests a mode of action for the synthetic peptides which substantially overlaps, but which is not entirely identical with, that of native collagen.

M J Barnes

Papers

Signals elicited from human platelets by synthetic, triple helical, collagen-like peptides.