Downregulation of the Clotting Cascade by the Protein C
Total Page：16
File Type：pdf, Size：1020Kb
Downregulation of the clotting cascade by the protein C F. Stavenuiter, E.A.M. Bouwens, L.O. Mosnier I Simposio Conjunto EHA - SAH Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA HEMATOLOGÍA, Vol.17 Número Extraordinario XXI CONGRESO E-mail: [email protected] Octubre 2013 Abstract APC mutants, which provide unique insights into The protein C pathway provides important biologi- the relative contributions of APC’s anticoagulant or cal activities to maintain the fluidity of the circula- cytoprotective activities to the beneficial effects of tion, prevent thrombosis, and protect the integrity APC in various murine injury and disease models. of the vasculature in response to injury. Activated Because of its multiple physiological and pharmaco- protein C (APC), in concert with its co-factors and logical activities, the anticoagulant and cytoprotec- cell receptors, assembles in specific macromolecular tive protein C pathway have important implications complexes to provide efficient proteolysis of multiple for the (patho)physiology of vascular disease and substrates that result in anticoagulant and cytopro- for translational research exploring novel therapeu- tective activities. Numerous studies on APC’s struc- tic strategies to combat complex medical disorders ture-function relation with its co-factors, cell recep- such as thrombosis, inflammation, ischemic stroke tors, and substrates provide valuable insights into the and neurodegenerative disease. molecular mechanisms and presumed assembly of Learning goals the macromolecular complexes that are responsible At the conclusion of this activity, participants should for APC’s activities. These insights allow for molecu- know that: lar engineering approaches specifically targeting the - the protein C pathway provides multiple im- interaction of APC with one of its substrates or co- portant functions to maintain a regulated bal- factors. Thus far, these approaches resulted in several ance between hemostasis and host defense anticoagulant-selective and cytoprotective-selective systems; HEMATOLOGÍA • Volumen 17 Número Extraordinario: 11-25 • Octubre 2013 11 XXI ConRGESO ARgentino DE Hematología - APC’s anticoagulant activities prevent throm- different mechanisms. First, the γ-carboxyglutamic bosis whereas APC’s cytoprotective activities acid (Gla)-domain, common to most coagulation protect cells; factors, requires the presence of negatively charged - APC’s different activities require assembly of phosphatidylserine for calcium-dependent binding different macromolecular complexes with dif- to lipid surfaces.8 Thus, assembly of the tenase (acti- ferent cofactors that can be targeted by muta- vated factor IXa, X, and VIII (FIXa, FX and FVIIIa)) genesis to obtain activity-selective APC mu- and prothrombinase (FXa, FII, and FVa) complex is tants; limited by the presence of negatively charged lipid - anticoagulant-selective and cytoprotective-se- surfaces (such as on activated platelets).8,9 Second, lective APC mutants provide insights into the serine protease inhibitors (SERPINs) rapidly in- relative contributions of these APC activities hibit activated coagulation factors, thereby blunt- to beneficial effects in various murine injury ing coagulation and preventing the escape of active and disease models. coagulation factors in the circulation.10 Finally, the protein C pathway actively inhibits coagulation by Introduction proteolysis of the tenase and prothrombinase com- The hemostatic system safeguards the patency of the plex co-factors, FVa and FVIIIa, thereby providing a 11-15 vasculature. Platelet aggregation, coagulation, and dynamic regulation of coagulation. The focus of fibrinolysis operate in concert with the endothe- this review will be on the protein C pathway. Because lium and other vascular cells to arrest bleeding and activated protein C (APC) inactivates both FVa and prevent thrombosis. The coagulation pathway con- FVIIIa, it has important effects on the downregula- tributes to regulation of the hemostatic balance via tion of both primary and secondary thrombin forma- multiple mechanisms and pathways that ensure a tion that manifest as potent anti-thrombotic effects balanced and confined hemostatic response at the in vivo. Furthermore, APC’s relatively new activities site of injury. Despite these feedback mechanisms on cells provide physiological and pharmacological to fine-tune the hemostatic response, genetic and/or relevant protective effects on the endothelium and acquired defects often tilt the balance sufficiently to the vasculature. Thus APC conveys multiple activi- increase the risk for thrombophilia. In the current ties that require assembly of macromolecular com- model of coagulation,1,2 clot formation is initiated by plexes with different co-factors, cell receptors and the extrinsic pathway with little or no role for the substrates. Structure-function analysis of these APC contact system in the initiation of physiological co- complexes, discussed in the next sections, provides agulation, although platelet-derived polyphosphates a unique understanding of how a single enzyme can may provide an endogenous activation mechanism mediate multiple biologically and therapeutically for FXII.3,4 Thrombin formation occurs during two relevant activities. mechanistically different phases. In the first phase, referred to as primary thrombin formation, the ex- The protein C pathway trinsic pathway generates the clot initiated by the The protein C pathway provides important contri- tissue factor/activated factor VIIa (FVIIa) complex. butions to maintain the fluidity of the circulation, However, primary thrombin formation is short-lived prevent thrombosis, and protect the integrity of the due to rapid inactivation of the tissue factor/FVIIa vasculature in response to injury. The reactions of initiator complex by tissue factor pathway inhibitor the protein C pathway encompass protein C activa- (TFPI). In the second phase, when sufficient throm- tion on endothelial cells, the anticoagulant protein bin is generated to initiate FXI activation, secondary C pathway on activated platelets, the cytoprotective thrombin formation will continue inside the clot via protein C pathway on cell membranes, and inacti- thrombin-mediated FXI activation and amplification vation of APC by plasma serine protease inhibitors by the intrinsic pathway.5 This secondary thrombin (SERPINs) in the fluid phase (Figure 1). Each of these formation contributes to clot strength and conveys re- aspects of the protein C pathway will be discussed in sistance to fibrinolysis via the activation of FXIII and the sections below. The physiological importance of thrombin activatable fibrinolysis inhibitor (TAFI).6,7 the protein C system is best illustrated by the mani- Control of coagulation is generally provided by three festation of massive thrombotic complications in in- 12 HEMATOLOGÍA • Volumen 17 Número Extraordinario: 11-25 • Octubre 2013 XXI ConRGESO ARgentino DE Hematología fants with protein C deficiency.16,17 Neonatal purpura of oral anticoagulant therapy with coumarin deriva- fulminans, a rapidly progressing hemorrhagic ne- tives. This is due to a temporary functional protein crosis of the skin due to microvascular thrombosis, C deficiency caused by the shorter circulation half- inflammation, and disseminated intravascular co- life of protein C (8 h) compared to the other proco- agulation (DIC), is typically observed in severe pro- agulant coagulation factors (24-72 h).17,18,21 Acquired tein C deficiency, whereas heterozygous protein C protein C deficiency is also found in patients with deficiency in adults carries a significantly increased severe infection and sepsis, most likely due to con- risk for venous thrombosis.18-20 A rare complica- sumption and poor synthesis in the liver, and low tion referred to as warfarin- induced skin necrosis protein C levels correlate with poor clinical outcome with clinical symptoms similar to that of purpura and death.22 fulminans, may present within days after initiation Figure 1; Reactions of the protein C pathway. Schematic representation of the protein C pathway reactions, from left to right: protein C activation, the cytoprotective protein C pathway, the anticoagulant protein C pathway and the inactiva- tion of APC by serine protease inhibitors (SERPINs). Protein C activation: physiological activation of protein C (PC) by the thrombin (IIa)-thrombomodulin (TM) complex occurs on the surface of endothelial cell membranes when protein C is bound to the endothelial protein C receptor (EPCR). Since protein C and APC have a similar affinity for EPCR, after activation APC can remain bound to EPCR to initiate cytoprotective signaling. The cytoprotective protein C pathway: APC’s direct effects on endothelial cells require the cellular receptors EPCR and PAR1. These cellular activities of APC in- clude anti-apoptotic and anti-inflammatory activities, alteration of gene expression profiles, and protection of endothelial barrier functions and are collectively referred to as APC’s cytoprotective activities. The anticoagulant protein C pathway: APC anticoagulant activities involve proteolytic cleavages of FVa and FVIIIa. Different protein co-factors, such as protein S (PS), FV, and various lipid co-factors (e.g. phosphatidylserine, phosphatidylethanolamine cardiolipin, glucosylceramide, etc.), enhance the inactivation of FVa and FVIIIa by APC. APC inactivation: inactivation of APC in plasma by serine protease inhibitors (SERPINs)