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Hemostasis encompasses the tightly regulated processes of blood clotting, platelet activation, and vascular repair. After wounding, the hemostatic system engages a plethora of vascular and extravascular receptors that act in concert with blood components to seal off the damage inflicted to the vasculature and the surrounding tissue. The first important component that contributes to hemostasis is the coagulation system, while the second important component starts with platelet activation, which not only contributes to the hemostatic plug, but also accelerates the coagulation system. Eventually, coagulation and platelet activation are switched off by blood-borne inhibitors and proteolytic feedback loops. This review summarizes new concepts of activation of proteases that regulate coagulation and anticoagulation, to give rise to transient thrombin generation and fibrin clot formation. It further speculates on the (patho)physiological roles of intra- and extravascular receptors that operate in response to these proteases. Furthermore, this review provides a new framework for understanding how signaling and adhesive interactions between endothelial cells, leukocytes, and platelets can regulate thrombus formation and modulate the coagulation process. Now that the key molecular players of coagulation and platelet activation have become clear, and their complex interactions with the vessel wall have been mapped out, we can also better speculate on the causes of thrombosis-related angiopathies. [ABSTRACT FROM AUTHOR]

The article presents a study on the role of sodium (Na) and potassium (K) in enzyme function. It examines the diversity of monovalent cation-activated enzymes and discusses its theoretical and practical aspects. A derivation of the kinetic expressions for the analysis of cofactor (type I) and allosteric effector (type II) activation is offered. The article also discusses the implications on the molecular evolution of monovalent cation-activated enzymes in biochemistry and protein engineering.

Tissue factor (TF) is the most important trigger of blood coagulation in vascular pathology. Rabbit TF, with or without (ΔC) its COOH-terminal intracellular tail, has been conjugated to green fluorescent protein (GFP) to study subcellular localization and other functions of TF. TF-GFP and TFΔC-GFP are associated with Na2CO3-resistant buoyant fractions in KEK-293 cells (lipid rafts); there is no morphological difference in the surface distribution of these or other GFP-labeled membrane proteins present in or excluded from rafts (confocal microscopy, HEK-293 cells). Endogenous TF expressed by rabbit aortic smooth muscle cells (SMCs) is also raft associated. Membranes from HEK293 cells expressing recombinant TF-GFP or wild-type TF were equipotent to clot human plasma; however, TRΔC-GFP was ∼20-fold more active (per membrane weight). Immunoblot confirmed that the deletion mutant is more abundantly expressed, and confocal microscopy showed that it has preferential membrane localization, whereas TF-GFP is mainly intracellular (nuclear lining and multiple granules). With a similar half-life (lt;4 h), the two constructions differ by their intracellular retention, lower for TFΔC-GFP. In serum-starved SMCs, the expression of endogenous TF was upregulated by interleukin-1β and/or PBS treatment (immunoblot, immunofluorescence, clotting assay). However, TF secretion or surface expression was not regulated by stimuli of physiological intensity (such as stimulation of the coexpressed kinin B1 receptors), although a calcium ionophore was highly active in this respect. TF is a raft-associated molecule whose surface expression (secretion) is apparently retarded or impaired by structural determinant(s) located in its COOK-terminal tail. [ABSTRACT FROM AUTHOR]

Activation of any of the three known tachykinin receptors (NK1R, -2R, or -3R) can cause a rise in [Ca2+]i via a pertussis toxin-insensitive heterotrimeric G protein, Gq/G11, activation of phospholipase C (PLC), and a membrane depolarization. Tachykinins can depolarize neurons by two distinct mechanisms: 1) they reduce a resting K+ current in many neurons or 2) in parasympathetic and vagal primary sensory neurons, they activate a nonspecific cation current (Icat). Transient receptor potential channels (TRPC) are nonspecific cation channels that can be activated by a rise in [Ca2+]i in a PLC-dependent manner. The present work tests whether NK2R can signal TRPC. We applied standard whole cell patch-clamp recordings to HEK293 cells stably transfected with the human TRP3 channels (TRP3C), and transiently transfected with a functional NK2R-EGFP. Bath applied Substance P (SP, 1 μM) induced an Icat in the cells expressing both TRP3C and NK2R. Icat reached its peak value in approximately 3 min (195 ± 120.0 s, mean ± SE, n = 20), had a peak density of 11.3 ± 3.48 pA/pF (n = 24), and was blocked by an NK2R-specific antagonist (SR48968, 100 nM). The Erev value for the SP current was 6.8 ± 7.66 mV (n = 6), suggestive of a nonspecific cation channel. Icat was not measurable in TRP3C-expressing HEK293 cells without NK2R expression (n = 6) or in wild-type HEK293 cells with NK2R expression (n = 12). These data indicate that NK2R can be functionally coupled to TRP channels in HEK293 cells and suggest that SP-induced cation currents in vagal primary sensory neurons might be mediated by TRPC. [ABSTRACT FROM AUTHOR]

Recently, we have shown that a widely used antagonist of the human bradykinin B[sub 2] receptor (B2R) HOE 140 acts as a full agonist of the chicken ornithokinin receptor (B[sub o]R). To understand the molecular mechanisms underlying differential efficacy of HOE 140 for the various kinin receptors, we have constructed chimeric kinin receptors (CKR) in which the amino-terminal portion including the first two transmembrane regions and the first extracellular loop (CKR-2) or only the second transmembrane region and the first extracellular loop (CKR-1) of B[sub 2]R were substituted with the corresponding segments of B[sub o]R. Ligand efficacy of synthetic ligand HOE 140 decreased in the order B[sub o]R > CKR-2 > CKR1 > B[sub 2]R, whereas the efficacy of the endogenous kinin ligand was unchanged. Enhanced HOE 140 efficacy was not due to a structural change in the ligand binding site or to an enhanced receptor expression level. Rather, heterologous binding competition studies indicated that structural change(s) introduced into the engineered receptors caused a selective reduction in apparent affinity of HOE 140 for the uncoupled inactive receptor state R but not for the active G protein-coupled state R[sup *], thereby increasing the ratio of R[sup *] over R for a given ligand concentration. Our results may help explain the unusually broad efficacy spectrum of HOE 140, which varies from inverse to full agonism, depending on kinin receptor subtype, tissue origin, or species. [ABSTRACT FROM AUTHOR]