Thrombin interaction with a recombinant N-terminal extracellular domain of the thrombin receptor in an acellular system

Bouton, Marie‐Christine; Jandrot‐Perrus, Martine; Moog, Sylvie; Cazenave, Jean‐Pierre; Guillin, Marie‐Claude; Lanza, François

doi:10.1042/bj3050635

Cited by 37 publications

(31 citation statements)

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“…It was previously shown that GpIb␣ in solution does not alter the kinetic constants of hydrolysis of the PAR-1-(38 -60) peptide (7,26). This implies that membrane phenomena are responsible for the effect observed using intact platelets.…”

Section: Discussionmentioning

confidence: 90%

Binding of Thrombin to Glycoprotein Ib Accelerates the Hydrolysis of Par-1 on Intact Platelets

Candia¹,

Hall²,

Rutella³

et al. 2001

Journal of Biological Chemistry

212

176

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The activation of human platelets by ␣-thrombin is mediated at least in part by cleavage of protease-activated G-protein-coupled receptors, PAR-1 and PAR-4. Platelet glycoprotein Ib␣ also has a high affinity binding site for ␣-thrombin, and this interaction contributes to platelet activation through a still unknown mechanism. In the present study the hypothesis that GpIb␣ may contribute to platelet activation by modulating the hydrolysis of PAR-1 on the platelet membrane was investigated. Gel-filtered platelets from normal individuals were stimulated by ␣-thrombin, and the kinetics of PAR-1 hydrolysis by enzyme was followed with flow cytometry using an anti-PAR-1 monoclonal antibody (SPAN 12) that recognizes only intact PAR-1 molecules. This strategy allowed measurement of the apparent k cat /K m value for thrombin hydrolysis of PAR-1 on intact platelets, which was equal to 1.5 ؎ 0.1 ؋ Platelet activation by the coagulation protease ␣-thrombin plays a crucial role in physiologic hemostatic processes and in thrombotic diseases. The activation of human platelets by thrombin is mediated by at least two receptors belonging to the family of protease-activated receptors (PARs), 1 i.e. PAR-1 and PAR-4 (1-2). These receptors are activated upon cleavage by thrombin and mediate transmembrane signaling by coupling to G-proteins (1-2). ␣-Thrombin also binds with high affinity to the platelet glycoprotein Ib (GpIb) that belongs to the leucinerich repeat family of proteins (3). Thrombin binding to GpIb contributes to platelet activation by the enzyme, as demonstrated by the finding that Bernard-Soulier platelets, which lack the GpIb-IX-V complex, have a delayed response to thrombin stimulation (4). In addition, several in vitro studies have demonstrated that the inhibition of thrombin binding to GpIb by different strategies causes a reduction in thrombin-induced platelet activation (5-9).The mechanism by which the binding of ␣-thrombin to GpIb contributes to platelet activation is not clear. Numerous studies have shown that a proteolytic-active enzyme is required to activate platelets. PPACK-thrombin, which retains its ability to bind to GpIb, does not induce platelet aggregation (10). Moreover, GpIb does not undergo cleavage by thrombin. On the other hand, the cleavage of G-protein-coupled PARs seems to be essential in platelet activation and transmembrane signaling. The finding that thrombin binding to GpIb involves a distinct thrombin domain, the HBS, which is far from the thrombin catalytic site and the fibrinogen recognition site (7-9), would suggest that a ternary complex thrombin⅐GpIb⅐PAR-1 may form on the platelet membrane that could be responsible for optimal hydrolysis and signal transduction.In the present study, the hypothesis that thrombin binding to GpIb may affect the hydrolysis of PAR-1 by the enzyme on intact platelets was investigated. The hydrolysis of PAR-1 was evaluated as a paradigm to construct a model where GpIb acts as a cofactor for PAR(s) cleavage. EXPERIMENTAL PROCEDURESMaterials-Human ␣-thrombin w...

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Section: Discussionmentioning

confidence: 90%

Binding of Thrombin to Glycoprotein Ib Accelerates the Hydrolysis of Par-1 on Intact Platelets

Candia¹,

Hall²,

Rutella³

et al. 2001

Journal of Biological Chemistry

212

176

View full text Add to dashboard Cite

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“…These actions are mediated in part by its novel receptor, PAR1. Although this thrombin receptor was initially characterized on endothelium [11], subsequent studies have revealed that it is expressed on vascular smooth muscle cells [12], synovial cells [13] and neuronal cells [14]. The identification of PAR1 on various cell types has expanded our knowledge about the role of thrombin.…”

Section: Discussionmentioning

confidence: 99%

“…Thrombin has attracted much attention due its diverse array of actions on cells, including endothelial cells, vascular smooth muscle cells, synovial cells, and neuronal cells [11][12][13][14]. Thrombin may be generated in the brain parenchyma in various pathologic states, notably cerebrovascular disease, head trauma and organic brain disease [15][16][17].…”

Section: Introductionmentioning

confidence: 99%

Recombinant Thrombomodulin Inhibits Thrombin-Induced Vascular Endothelial Growth Factor Production in Neuronal Cells

Sarker

Yamahata²,

Nakata³

et al. 1999

Pathophysiol Haemos Thromb

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Thrombin is a serine protease which is generated from its precursor prothrombin by the activation of the blood coagulation cascade. Thrombin converts fibrinogen to fibrin, activates platelets and several coagulation factors, and plays a central role in thrombosis and hemostasis by regulating platelet aggregation and blood coagulation. Here, we show that thrombn enhanced vascular endothelial growth factor (VEGF) production in a dose- and time-dependent manner in the supernatant of cultured PC-12 cells, as determined by enzyme-linked immunosorbent assay (ELISA). Thrombin receptor agonist peptide (SFLLRNPNDKYEPF, TRAP) exerted an effect similar to thrombin on VEGF production. Thrombin-induced VEGF production was significantly attenuated by recombinant human thrombomodulin (rTM) and its minimal functional domain E456. Furthermore, the antioxidant N-acetyl-L-cysteine (NAC) markedly inhibited thrombin-induced VEGF production. Thus, rTM and NAC apparently inhibited the effect of thrombin on VEGF production in neuronal cells.

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“…Exosite II is known as a heparinbinding site and plays a role in platelet binding via GPIba and the direct recognition of some substrates such as F V and F VIII . Here, we described COMP binding to thrombin at an approximate K D value of 1.38 mM, which is similar to the binding affinity of PAR-1 31 and fibrinogen 32 to thrombin. In particular, the blockage of thrombin exosites with compounds specific for exosite I or II impaired the COMP-thrombin interaction, indicating that COMP binds to both exosites.…”

Section: Discussionmentioning

confidence: 66%

Cartilage oligomeric matrix protein is a natural inhibitor of thrombin

Liang

et al. 2015

Blood

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Key Points• COMP negatively regulates hemostasis and thrombosis.• COMP is a natural inhibitor of thrombin.Thrombin is an effector enzyme for hemostasis and thrombosis; however, endogenous regulators of thrombin remain elusive. Cartilage oligomeric matrix protein (COMP), a matricellular protein also known as thrombospondin-5, is essential for maintaining vascular homeostasis. Here, we asked whether COMP is involved in the process of blood coagulation. COMP deficiency shortened tail-bleeding and clotting time and accelerated ferric-chloride-induced thrombosis in mice. The absence of COMP had no effect on platelet count. In contrast, COMP specifically inhibited thrombin-induced platelet aggregation, activation, and retraction and the thrombin-mediated cleavage of fibrinogen. Furthermore, surface plasmon resonance analysis revealed direct thrombin-COMP binding (K D 5 1.38 6 0.24 mM). In particular, blockage of thrombin exosites with compounds specific for exosite I (hirudin and HD1 aptamer) or exosite II (heparin and HD22 aptamer) impaired the COMP-thrombin interaction, indicating a 2-site binding mechanism. Additionally, epidermal growth factor-like repeats (amino acids 84-261) were identified as a COMP binding site for thrombin. Moreover, COMP was expressed in and secreted by platelets. Using bone marrow transplantation and platelet transfusion to create chimeric mice, platelet-derived but not vessel-wall-derived COMP was demonstrated to inhibit coagulation. Taken together, COMP is an endogenous thrombin inhibitor and negative regulator of hemostasis and thrombosis. (Blood. 2015;126(7):905-914)

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Thrombin interaction with a recombinant N-terminal extracellular domain of the thrombin receptor in an acellular system

Cited by 37 publications

References 50 publications

Binding of Thrombin to Glycoprotein Ib Accelerates the Hydrolysis of Par-1 on Intact Platelets

Binding of Thrombin to Glycoprotein Ib Accelerates the Hydrolysis of Par-1 on Intact Platelets

Recombinant Thrombomodulin Inhibits Thrombin-Induced Vascular Endothelial Growth Factor Production in Neuronal Cells

Cartilage oligomeric matrix protein is a natural inhibitor of thrombin

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