Fibrinolytic Serine Proteases, Therapeutic Serpins and Inflammation: Fire Dancers and Firestorms

Abstract

The making and breaking of clots orchestrated by the thrombotic and thrombolytic serine protease cascades are critical determinants of morbidity and mortality during infection and with vascular or tissue injury. Both the clot forming (thrombotic) and the clot dissolving (thrombolytic or fibrinolytic) cascades are composed of a highly sensitive and complex relationship of sequentially activated serine proteases and their regulatory inhibitors in the circulating blood. The proteases and inhibitors interact continuously throughout all branches of the cardiovascular system in the human body, representing one of the most abundant groups of proteins in the blood. There is an intricate interaction of the coagulation cascades with endothelial cell surface receptors lining the vascular tree, circulating immune cells, platelets and connective tissue encasing the arterial layers. Beyond their role in control of bleeding and clotting, the thrombotic and thrombolytic cascades initiate immune cell responses, representing a front line, "off-the-shelf" system for inducing inflammatory responses. These hemostatic pathways are one of the first response systems after injury with the fibrinolytic cascade being one of the earliest to evolve in primordial immune responses. An equally important contributor and parallel ancient component of these thrombotic and thrombolytic serine protease cascades are the serine protease inhibitors, termed serpins. Serpins are metastable suicide inhibitors with ubiquitous roles in coagulation and fibrinolysis as well as multiple central regulatory pathways throughout the body. Serpins are now known to also modulate the immune response, either via control of thrombotic and thrombolytic cascades or via direct effects on cellular phenotypes, among many other functions. Here we review the co-evolution of the thrombolytic cascade and the immune response in disease and in treatment. We will focus on the relevance of these recent advances in the context of the ongoing COVID-19 pandemic. SARS-CoV-2 is a "respiratory" coronavirus that causes extensive cardiovascular pathogenesis, with microthrombi throughout the vascular tree, resulting in severe and potentially fatal coagulopathies.

Keywords: coagulation; fibrinolysis; infection; inflammation; serine protease; serpin; thrombolysis; virus.

Figure 1

The thrombotic and thrombolytic cascades and primordial immune response. The thrombotic pathways (intrinsic and extrinsic) and the thrombolytic (fibrinolysis) pathway involve a complex cascade of protease activation. Solid arrows indicate the conversion to an active protease, while dotted line arrows indicate the activity of the activating upstream protease. A variety of inhibitors are shown, with serpin inhibitors denoted by a serpin protein structural image. Examples of early primordial immune response origins are noted in context of the pathways. MYA, million years ago.

Figure 2

Serpin-dependent mechanism of protease inhibition. (A) Natively folded serpins present a reactive center loop (RCL) which acts as a bait for interaction with active serine proteases. Upon association, initiation of proteolytic activity by the protease triggers the formation of a Michaelis complex and covalent bonding of the serpin with the protease. (B) The primary outcome of Michaelis complex formation is reconfiguration of the serpin, with the RCL inserting as the third of five strands in the core beta sheet and permanent denaturation of the target serine protease. (C) The secondary and less frequent outcome is completion of proteolytic activity and dissociation of the active serine protease, while the RCL continues to insert as the third of five strands in the serpin core beta sheet.

Figure 3

Canonical signaling of the fibrinolysis pathway. Fibrinolysis is characterized by the degradation of a fibrin clot into degradation products by plasmin. Plasmin is generated from plasminogen by uPA and tPA. Several serpins and other inhibitors provide a tight regulation of this cascade. Substantial promiscuity exists across multiple elements of the pathway, providing redundant controls against inappropriate activation. AIIt and uPAR are shown as representative canonical fibrinolytic receptors for brevity. AIIt, Annexin II tetramer; PAI-1,2,3, Plasminogen Activator Inhibitor-1, 2, 3; PN-1, Protease Nexin-1; TAFI, Thrombin activatable fibrinolysis inhibitor; tPA, Tissue-type plasminogen activator; uPA, Urokinase-type plasminogen activator; uPAR, Urokinase-type plasminogen activator receptor.

Figure 4

Inhibition of SARS-CoV-2 infection and processing by fibrinolysis pathway-associated serpins. SARS-CoV-2 entry is dependent on proteolytic processing of the spike protein in order to engage the ACE2 receptor and internalize. Processing of the spike protein is performed primarily by TMPRSS2 and Furin. PAI-1 and A1AT are known inhibitors of TMPRSS2 and experimental evidence demonstrates that A1AT can inhibit SARS-CoV-2 infection in vitro. Based on similarities in inhibition properties, it may be predicted that Serp-1 will also inhibit TMPRSS2 with similar results. Furin has both an extracellular and intracellular role in the SARS-CoV-2 life cycle and there is evidence for extracellular furin inhibition by A1AT and A1AT variants (Portland and α-PDX) as well as endothelial PN-1, and for intracellular furin inhibition by PAI-1.