Constitutively active ADAMTS13: an emerging thrombolytic agent for acute ischemic stroke

1 ∣. INTRODUCTION

Acute ischemic stroke remains one of the leading causes of morbidity and disability worldwide. The underlying cause is arterial occlusion, which might be cardioembolic because of atrial fibrillation or valvular heart disease, or arterioembolic because of atherosclerosis in the carotid artery or cerebral artery. Currently, effective reperfusion treatment includes intravenous thrombolysis with recombinant tissue plasminogen activator (tPA), which degrades fibrin within the thrombus, or endovascular mechanical thrombectomy, which involves the removal of thrombus with stent-retriever devices with the help of advanced imaging techniques. While both treatments provide standard care to patients with thromboembolic stroke, each of them has strengths and limitations. For example, the strength of intravenous thrombolysis is that it can be applied to a wide spectrum of stroke patients. Unfortunately, however, it exhibits only a modest effectiveness [1]. Early arterial re-occlusion and unsatisfactory long-term outcomes were observed in approximately 30% of stroke patients after rtPA administration [2]. In addition, successful recanalization is less often achieved in patients with large vessel occlusion [3]. Other limitations include increase risk of hemorrhagic transformation and the narrow therapeutic window of up to 4.5 hours for starting t-PA reperfusion from the stroke onset. Beyond this timeframe, most admitted stroke patients are not suitable for reperfusion therapy, which substantially limits the eligible population [4]. On the other hand, while endovascular mechanical thrombectomy is much more efficacious, it can only be applied in a small subset of patients with a stroke caused by a large vessel occlusion [5]. Nearly 50% of treated acute stroke patients with large vessel occlusion still experience neurological deficits after thrombectomy [6]. Furthermore, some patients are not eligible for mechanical thrombectomy because of larger infarcts that cannot be salvaged [7]. Another constraint is that mechanical thrombectomy requires skilled interventional neurologists, not immediately available at all the hospitals. These shortcomings of rtPA and mechanical thrombectomy call for a compelling requirement to develop novel therapeutic interventions that can protect the stroke penumbra, reduce the size of infarct core and may offer better neurological outcomes if administered as early as possible following stroke onset.

2 ∣. ROLE FOR VWF/ADAMTS13 AXIS IN MODULATING THROMBO-INFLAMMATION

Von Willebrand factor (VWF) is a large multimeric glycoprotein that provides the initial adhesion of circulating platelets to the site of vascular injury by binding to GPIbα on platelets and collagen in the extracellular matrix. VWF is present in endothelial cells and megakaryocytes. It is stored as ultra-large VWF (ULVWF) multimers in endothelial Weibel-Palade bodies and platelet α-granules. Upon secretion in blood and under shear conditions, ULVWF multimers are cleaved rapidly by the protease ADAMTS13 into the less active VWF multimers that support normal hemostasis. The essential role of VWF and ADAMTS13 axis in hemostasis and thrombosis is illustrated by von Willebrand’s disease, a bleeding disorder associated with a functional deficiency in VWF, and thrombotic thrombocytopenic purpura, a thrombotic disorder linked to the deficiency of ADAMTS13 that could be either familial or acquired. Indeed, defects in hemostasis and thrombosis were observed in VWF-deficient mice [8, 9]. Further, follow-up studies in murine models suggested that VWF-deficient mice were less susceptible to thrombo-inflammation in experimental models of myocardial infarction [10], atherosclerosis [11-13], and acute ischemic stroke [14-17]. In contrast, ADAMTS13-deficient mice were prothrombotic, pro-inflammatory, and were susceptible to thrombotic thrombocytopenic purpura when challenged with Shiga toxin [18-21]. Additionally, ADAMTS13-deficient mice were more susceptible to thrombo-inflammation in experimental models of myocardial infarction [10, 22], atherosclerosis [23, 24] and stroke [14, 17, 25]. Together, these studies suggest that the VWF and ADAMTS13 axis contributes to thrombo-inflammation in experimental models. Indeed, high VWF levels and low ADAMTS13 levels are associated with an increased risk of ischemic stroke [26].

3 ∣. CONSTITUTIVELY ACTIVE ADAMTS13: AN EMERGING THERAPEUTIC AGENT FOR ACUTE ISCHEMIC STROKE

Previously it was demonstrated that recombinant wild-type ADAMTS13 (wtADAMTS13) inhibits experimental arterial thrombosis [19] and improves stroke outcome in wild-type mice [14]. In follow-up studies, wtADAMTS13 was demonstrated to facilitate efficient lysis of intracranial thrombi, resistant to t-PA treatment in acute ischemic stroke [27]. Physiologically, the wtADAMTS13 adopts a folded (dormant) conformation maintained by an auto-inhibitory interaction between its N-terminal spacer domain and its C-terminal CUB-domains. It restricts its proteolytic activity while maintaining its substrate specificity against VWF [28]. Upon binding with the D4-CK domain of the globular VWF, ADAMTS13 undergoes a conformational change, which causes an approximately 2.5-fold increase in its proteolytic activity against VWF [28]. Given the existence of wtADAMTS13 in a conformationally quiescent state that requires a substrate-dependent activation, it is effective only at supra-physiological concentrations. This puts a limitation on its application as a therapeutic agent for acute ischemic stroke. An alternate variant Gain of Function (GoF) ADAMTS13 was developed by inducing mutations to the key residues in the VWF-binding exosites of ADAMTS13. In contrast to the wtADAMTS13, the GoF ADAMTS13 adopted a preactivated confirmation that increased its ability to dissolve platelet aggregates in vitro [29]. Despite a pronounced protective effect of GoF ADAMTS13 in experimental stroke, its proteolytic activity was still regarded as insufficient [29].

Recently, South et al. developed several ADAMTS13 variants by introducing point mutations in the linker 3 region. One of the variants, containing a Ala1144Val substitution, was demonstrated to be a constitutively active form of ADAMTS13, which the authors referred to as caADAMTS13 [30]. Compared to the wtADAMTS13 or GoF ADAMTS13, caADAMTS13 at sub-physiological concentrations (<5 nM) completely inhibited VWF-mediated platelet adhesion under arterial shear conditions and enhanced t-PA/plasmin lysis of fibrin(ogen) in vitro. The high efficacy was attributed to the open conformation of caADAMTS13, in contrast to the substrate-activated wtADAMTS13 or preferentially preactivated conformation of GoF ADAMTS13 [30]. Next, the authors evaluated the therapeutic efficacy of caADAMTS13 in two models of ischemic stroke, the FeCl₃-induced distal middle cerebral artery occlusion (MCAO) model and transient MCAO with systemic inflammation and ischemia/reperfusion injury. In the FeCl₃-induced distal MCAO model, the authors demonstrated that infusion of caADAMTS13 in the wild-type mice significantly reduced VWF deposition, fibrin deposition, and platelet aggregate formation concomitant with a decreased neutrophil influx in the cerebral microvasculature compared to the vehicle-treated group. Together, these findings suggest that caADAMTS13 by degrading VWF multimers down-regulates thrombo-inflammation. In the transient MCAO model, the authors demonstrated that wild-type mice treated with caADAMTS13, 4 hours after reperfusion, exhibited reduced infarct size that was associated with fewer platelet aggregates, and reduction in tissue hypoperfusion. An important observation was the absence of hemorrhagic transformation in the caADAMTS13 treated group, although small bleeds within the infarct area of some animals were observed. Furthermore, no alteration in the tail bleeding time of caADAMTS13 treated animals was observed. These results are of considerable importance given the profound effect of caADAMTS13 on tissue reflow, along with its ability to retain its efficacy in an extended therapeutic time window to improve local cerebral blood flow restoration.

4 ∣. POTENTIAL IMPACT OF THESE FINDINGS

The early diagnosis and speed of treatment is critical factor that defines the outcome of thrombolytic intervention in the cases of acute ischemic stroke. Unfortunately, given its limitations, there has been a paucity in the generation of effective thrombolytics that can efficiently replace tPA as a front-line therapy to combat acute ischemic stroke. It is mainly because the effectiveness of a potential pharmacological candidate is subdued by multi-factorial processes that govern the pathophysiology of stroke. Stroke is an intricate conglomeration of thrombotic, immune, and inflammatory responses, which needs to be abrogated collectively to improve stroke outcomes without inducing any hemorrhagic risk. Over the past decade, ADAMTS13 has emerged as a promising candidate for developing into an effective therapeutic agent for acute ischemic stroke. It has been possible due to the flexibility with which ADAMTS13 can be engineered by incorporating point mutations to form a range of variants such as GoF ADAMTS13 (R568K/F592Y/R660K/Y661F/Y665F) [28] and Ala1144Val (caADAMTS13)[30]. The newly identified variant caADAMTS13 can be considered significant progress over its predecessors, given its exceptional thrombolytic ability and efficacy at a sub-physiological concentration (<5 nM) and absence of hemorrhagic transformation. Other than A1144V (caADAMTS13) discussed in this study, other variants (A1146V, P1180V, and P1182V) exhibited a strikingly comparable activity, which would be interesting to dissect further.

Despite the strengths, the study has few limitations. First, the authors have used healthy wild-type mice to test the efficacy of caADAMTS13 in acute ischemic stroke. Although several therapeutic agents have shown efficacy in preclinical studies in improving stroke outcomes, they have been unsuccessful in clinical studies. The lack of success of these agents from "bench to bedside" is likely because preclinical studies use healthy wild-type animals without pre-existing comorbidities. In contrast, human stroke usually occurs with pre-existing comorbidities. Second, reduced infarct core at day 1 or 2 in murine studies has not proven successful as the only end-point for clinical trials. It is essential to evaluate whether caADAMTS13 intervention improves long-term neurological outcomes. Future studies are warranted to determine whether caADAMTS13 improves stroke outcome in experimental stroke models with pre-existing comorbidities before evaluating efficacy and safety in human participants. In conclusion, given the extensive participation of VWF and ADAMTS13 in a variety of thrombotic and inflammatory pathologies, caADAMTS may have a broader implication than anticipated that may include myocardial infarction in addition to acute ischemic stroke.