Understanding the concepts of Protease Activated Receptors

Platelets could be activated by thrombin, a serine peptidase which can adhere to and cleaves protease activated receptors ( PARs ) on thrombocytes. This cleavage is irreversible and reveals a new N-terminus that acts as a tethered ligand. This ligand binds to the receptor and initiates intracellular signaling tracts, which eventually result in thrombocyte form alterations, dense granule secernment and GPIIbIIIa receptor activation. { { 13 Dorsam, R.T. 2002 } }

Pars are G-protein conjugate receptors ( GPCRs ) on the surface of thrombocytes. There are four different isoforms of PARs characterized, from which PAR1 and PAR4 are present on the surface of human thrombocytes. Initially it was thought that the signaling pathways downstream of PAR1 and PAR4 twosome to the same set of heterotrimeric G-proteins. But now it is shown that these receptors signal differentially in human thrombocytes and other tissues. This likely consequences from differential G-protein yoke. Platelets are known to incorporate four different G-proteins involved in intracellular signaling tracts. These include Gs, Gi, Gq and G12/13. These G proteins have amino acerb sequence similarities and comparable maps. Both PAR1 and PAR4 twosome to Gq and G12/13 in human thrombocytes and cause factor I receptor activation upon activation by a ligand. The Gq tract involves phospholipase C activation and coincident intraplatelet Ca mobilisation and protein kinase C ( PKC ) activation. The G12/13 tract involves Rho/Rho kinase activation and actin remodeling, which causes thrombocyte form alteration. { { 1 Voss, B. 2007 } }

Another mechanism in which PAR1 and PAR4 differ in their signaling is that PAR1 mediates human thrombocyte activation at low thrombin concentrations, whereas PAR4 mediates thrombin-induced thrombocyte activation merely at high thrombin concentrations. { { 22 Stegner, D. 2011 } }

First, the G-proteins in common will be discussed, thenceforth the different G-proteins involved in thrombocyte signaling will be discussed.

G-proteins in common

When an extracellular signal molecule binds to a GPCR, the receptor undergoes a conformational alteration that enables it to trip a trimeric GTP-binding protein ( G protein ) . The G protein is connected to the cytoplasmatic face of the plasma membrane, where it functionally couples the receptor to enzymes or ion channels in the membrane. In some instances, the G protein is physically associated with the receptor before the receptor is activated, whereas in others it binds merely after receptor activation. As said before, there are assorted types of G proteins, each particular for a peculiar set of GPCRs and for a peculiar set of mark proteins in the plasma membrane. { { 11 Tfzira, T. 2008 } }

All G proteins are made up of three protein fractional monetary units, ? , ? and ? . In the unstimulated province, the ? fractional monetary unit has GDP edge and the G protein is inactive. When a ligand activates the GPCR, it induces a conformational alteration in the receptor that allows the receptor to map as a G nucleotide exchange factor ( GEF ) that induces the ? fractional monetary unit to let go of its edge GDP and adhere GTP in its topographic point. This exchange triggers, although non ever, the dissociation of the ? fractional monetary unit, edge to GTP, from the ?? dimer and the receptor. Both the GTP-bound ? fractional monetary unit and the ?? fractional monetary unit so trip different signaling Cascadess or 2nd courier tracts and effecter proteins. The receptor is able to trip another G protein. { { 11 Tfzira, T. 2008 } }

The ? fractional monetary unit of a G protein is a GTPase ; when it hydrolyzes its edge GTP to GDP it becomes inactive. The clip for which the G protein remains active depends on how rapidly the ? fractional monetary unit hydrolyzes its edge GTP. This clip is normally short because the GTPase activity is greatly enhanced by the binding of the ? fractional monetary unit to a 2nd protein, which can be either the mark protein or a specific regulator of G protein signaling ( RGS ) . RGS proteins act as ? fractional monetary unit specific GTPase-activating proteins ( GAPs ) , and they help close off G protein-mediated responses. { { 11 Tfzira, T. 2008 } }

There are besides “ little ” G proteins ( 20-25kDa ) that belong to the Ras superfamily of little GTPases. These proteins are homologous to the ? fractional monetary unit found in heterotrimeric G proteins, and are in fact monomeric. However, they besides bind GTP and GDP and are involved in signal transduction. These proteins will be discussed subsequently.

Gq? tract

Cleavage of PAR1 by thrombin and binding of the tethered ligand to the receptor stimulate Gq? to trip phospholipase C-? ( PLC? ) . { { 10 Carlile-Klusacek, M. 2007 } } Human thrombocytes contain four isoforms of PLC? that are activated by Gq? coupled receptors.

OF: As said in the chapter GPVI, PLC hydrolyzes PIP2 to bring forth the 2nd couriers IP3 and DAG. These 2nd courier molecules map in the mobilisation of Ca from intracellular shops ( via IP3 ) and activation of PKC ( via DAG ) , which lead to platelet secernment and collection. { { 25 Moraes, L.A. 2010 } }

Following to hydrolysis of PIP2, PIP2 becomes besides phosphorylated by PI3K? to bring forth phosphatidylinositol 3,4,5-triphosphate ( PIP3 ) . { { 8 Lian, L. 2005 } } PIP3 has several maps, among which the activation of PKB/Akt in thrombocytes. { { 9 Rittenhouse, S.E. 1996 } }

G12/13 tract

Following to the activation of Gq? , there is another G-protein stimulated after PAR1 stimulation. This is the G12/13 protein. G12/13? activates Rho/Rho kinase signaling, which in bend regulates the cytoskeleton. Active GTPase RhoA activates Rho kinase, which than activates and regulates assorted downstream mark proteins. One of them is ROCK ( regulator of G protein signaling ) , a serine/threonine kinase, which translocate from the cytosol to the PM in the presence of active RhoA. ROCK mediates cell contraction through phosphorylation and suppression of myosin visible radiation concatenation ( MLC ) phosphatase, therefore heightening MLC phosphorylation and MLC dependent contraction. It besides increases the serum response factor ( SRF ) -dependent cistron written text { { 13 Dorsam, R.T. 2002 } } { { 12 Siehler, S. 2009 } } The suppression of MLC phosphatase will ensue in a Ca independent thrombocyte form alteration. { { 13 Dorsam, R.T. 2002 } }

There is besides a Ca dependent thrombocyte form alteration after PAR1 activation. The Ca release after PAR1 activation has been shown to heighten MLC kinase activity, because MLC kinase is calcium/calmodulin-dependent. This enhancement consequences in increased MLC phosphorylation, the interaction of myosin with actin fibrils to organize emphasis fibres and therefore thrombocyte form alteration. { { 10 Carlile-Klusacek, M. 2007 } }

Platelet form alteration

A form alteration in thrombocytes is an highly rapid procedure based on the reorganisation of the cytoskeleton. During the form alteration, new actin fibrils are formed, taking to the formation of a submembranous actin fibril web and the extension of filopodia. Besides, actomyosin ( the contractile protein of thrombocytes ) -based contractile procedures are stimulated, which consequences in the centralisation of dense and ?-granules. Finally, the circumferential microtubule spiral depolymerizes, which allows the thrombocyte to alter from a discoid to a more spherical form. Platelet form alteration can be induced by agonist concentrations lower than those required for granule secernment and collection. Platelet form alteration is a requirement for efficient secernment of granule contents and greatly facilitate adhesion of thrombocytes to each other and to constituents of the ECM.

Actin assembly during platelet-shape alteration has besides been shown to be controlled by polyphosphoinositides such asPIP2. Formation of PIP2 by phosphatidylinositol 4-phosphate 5-kinase can be regulated by the little GTP-binding protein Rac, every bit good as by RhoA. Rac becomes activated in thrombocytes on receptor activation and can besides bring on actin polymerisation through the activation of the Arp2/3 composite mediated by WAVE proteins. However, in G q -deficient thrombocytes, activation of Rac by assorted agonists is abrogated while they are still able to bring on platelet-shape alteration, bespeaking that Rac activation is non required for agonist-induced platelet-shape alteration. Under these conditions, actin assembly and polymerisation may be induced through other RhoA-dependent tracts such as the activation of 4-phosphate 5-kinase or of formins, which promote additive elongation of actin fibrils.

Therefore, the G12/13 tract is required for thrombocyte form alterations, but it is non critical for integrin ?IIb?3 activation via PAR1 or PAR4. This is shown by add-on of a Rho-kinase inhibitor, which did non significantly affect integrin ?IIb?3 activation downstream of PAR1 or PAR4. Thus, Rho kinase is non needed downstream of either PAR1 or PAR4 for integrin ?IIb?3 activation. { { 1 Voss, B. 2007 } }

Gipathway

The Gi tract involves the suppression of the activity of adenylylcyclase by the ?-subunit of Gi and the activation of PI3K? by the ??-subunit of Gi. Inhibition of adenylyl cyclase consequences in the decrease of camp degrees, which regulate thrombocyte activity. { { 14 Swift, S. 2000 } } camp activates protein kinase A ( PKA ) , which in bend phosphorylates its downstream mark proteins, including PAR1, PAR3, PAR4, PC? , IP3 receptors and several others. So, in this manner cAMP inhibits thrombocyte activation. However, after thrombin stimulation, camp degrees are reduced, so the suppression of thrombocyte activity by camp will besides be reduced. { { 14 Swift, S. 2000 } }

PI3K

As said above, after stimulation of the GiPCR, the ?? fractional monetary unit of Gi activates phosphoinositide 3-kinase ( PI3K ) . PI3K, a plasma membrane edge enzyme, phosphorylates inositol phospholipids ( PI ) to bring forth phosphoinositides, including PIP, PIP2 and PIP3. { { 11 Tfzira, T. 2008 } }

PI3K is required downstream of PAR1, but non PAR4, for human thrombocyte integrin ?IIb?3 activation and human thrombocyte collection. PI3K plays a function in the addition in intraplatelet Ca concentration via Ca from the extracellular surroundings. Merely PAR1, non PAR4, straight activates PI3K and this consequences in the addition in the intraplatelet Ca concentration. { { 1 Voss, B. 2007 } }

Summary so far

Summarized, PAR1 activation leads to the activation of PLC, which in bend consequences in an addition in the intracellular Ca concentration via IP3 and the activation of PKC via DAG, activation of PI3K, suppression of adenylyl cyclase, and a calcium-dependent and -independent thrombocyte form alteration.