Stroke is the second most common cause of death globally and a common cause of reduced disability-adjusted life years . The majority of strokes are ischemic in nature, and are most commonly induced either by thromboembolic events or by local cerebral thrombosis. The development of new, effective and safe agents for the acute treatment of ischemic stroke is the greatest unmet need in the therapeutics of cerebrovascular thrombosis. Currently, administration of tissue-type plasminogen activator (tPA) is one of the very few approved therapies for acute ischemic stroke . However, given the narrow time window for its safe administration and the increased risk of symptomatic intracranial bleeding and downstream brain injury, its therapeutic use has major limitations [2,3].
In this issue of the Journal, Su et al.  demonstrate for the first time that Solulin, a soluble analogue of thrombomodulin (TM), is effective in a model of acute ischemic stroke, with data pointing to TM-like analogues as both a fruitful and safe strategy to pursue. TM is an integral membrane protein that is normally expressed on the surface of endothelial cells, where it acts as a receptor for thrombin. TM forms a 1:1 stoichiometric complex with thrombin that is responsible for the conversion of the plasma zymogen protein C to activated protein C (APC) . Interestingly, the thrombin/TM complex has a dual role in coagulation and fibrinolysis. On the one hand, this complex mediates APC generation, which is anticoagulant (and profibrinolytic), while on the other, it generates thrombin activatable fibrinolysis inhibitor (TAFI), which is antifibrinolytic .
The activation of protein C is strongly accelerated by several orders of magnitude by the thrombin/TM complex. This reaction normally occurs on the intact endothelium adjacent to the site of injury. The endothelial protein C receptor (EPCR) further augments this process . TM aligns the active site of thrombin to allow binding and cleavage/activation of protein C bound to EPCR. EPCR is primarily found on endothelial cells (particularly those of large vessels), promoting protein C activation in this location. APC has both anticoagulant and cytoprotective properties . Its anticoagulant function is well characterized and represents the major regulatory mechanism employed to inhibit thrombin generation. In conjunction with its non-enzymatic cofactor, protein S, APC downregulates thrombin generation through the proteolytic inactivation of activated procoagulant factors V (FVa) and VIII (FVIIIa) . Despite being primarily identified as an anticoagulant, over the past decade much attention has focused upon the cytoprotective effects of APC, which appear to be independent of its anticoagulant properties . The molecular mechanisms that enable this process depend on EPCR binding, which then enables cleavage of the integral membrane protein protease-activated receptor 1. The cytoprotective functions of APC, which have been explored by a number of groups, exert anti-inflammatory and anti-apoptotic properties as well as enhancing endothelial barrier integrity . These properties of APC may have profound therapeutic implications and the potential protective effects of APC have been shown in human sepsis  and in models of diabetic nephropathy , stroke , ischemic renal injury , cancer cell metastasis  and amyotrophic lateral sclerosis .
Su et al. elegantly tested the efficiency of Solulin in a photothrombotic model of stroke. The authors demonstrated that pretreatment with Solulin increases both the time to stable occlusion of the middle cerebral artery (MCA) and the reperfusion rate following occlusion. This is clearly an attractive outcome and, as the mode of action of Solulin dictates that the degree of anticoagulant function is dependent on the extent of endogenous thrombin generated, it might raise the potential for safe prophylactic therapy. Interestingly, Solulin successfully passed a phase I trial where it was shown to be a well-tolerated and safe anticoagulant with no evidence of bleeding risk, but with predictable antithrombotic effects . In their model of ischemic stroke, Su et al. further demonstrated that early (30 min) administration of Solulin following MCA occlusion increased vascular reperfusion while decreasing the extent of the infarcted penumbra. It is worth noting that while treatment with Solulin at 60 min following MCA occlusion had an initial impact on vascular reperfusion, this was not sustainable over time and had a lower effectiveness on infarct volume. If this is translated to ischemic stroke in a clinical setting, such narrow time windows are often missed due to delays in treatment that can arise due to both the patient and the medical system, and the need for extending the time window treatment after ischemic stroke is critical. A major limitation of tPA treatment is the 3 h window for its administration . If it is administered within 3 h of onset of ischemic stroke, it can improve the clinical outcome in patients by enhancing fibrinolysis, which in turn aids the restoration of blood flow to the ischemic region, while later administration is associated with increased risk of cerebral hemorrhage. Hemorrhagic complications are a major concern for the general use of anticoagulants for the treatment of acute stroke . In their study Su et al. showed that, in contrast to mice treated with heparin, Solulin-treated animals had no overt intracerebral hemorrhage, suggesting that it may be a safer anticoagulant treatment option.
Understanding the mechanism of action of Solulin in the ischemic stroke model used in this study may help in the design of effective and safe treatments. One may question why (particularly in previously healthy mice) the endogenous thrombin/TM complex is not sufficient to modulate the cerebral blood flow following an ischemic event, while the exogenously administrated TM analogue can promote reperfusion. This might be explained by the finding that the endogenous thrombin/TM complex is likely to balance both its anticoagulant and antifibrinolytic functions through APC and TAFI generation, respectively. In contrast, the administration of comparatively high amounts of Solulin (estimated > 200 nm) has been proposed to shift this balance towards APC generation . A further parameter worth considering is the site of action of thrombin/Solulin. TM is an endothelial receptor, which suggests that APC generation predominantly occurs on the endothelial wall. Conversely, as Solulin circulates in plasma, it can probably promote APC generation both in free circulation as well as on the surface of a thrombus (i.e. at any site at which it might encounter thrombin). It remains to be investigated whether the benefits of Solulin administration in this stroke model are merely due to the elevated protein C activating potential in plasma, or due to the ability of Solulin to promote APC generation at sites other than the endothelial surface such as the surface of a thrombus. However, the latter hypothesis may be favoured given that administration of APC is associated with increased risk of bleeding .
The authors further aimed to investigate the actions of Solulin in the factor V Leiden mutant mouse following occlusion of the MCA. In these mutant mice, although the thrombin/Solulin complex is expected to generate APC normally, its ability to inactivate FVa is reduced, which enabled an indirect assessment of the contribution of the cytoprotective properties of APC to the therapeutic efficacy in this model. In these mice, Solulin had no beneficial effect, suggesting that the beneficial effects are not attributable to cytoprotective mechanisms. In accordance with this, the authors also demonstrate that Solulin had no effect on cell death in the penumbra, while systemic administration of APC is associated with neuroprotective properties .
Whether the efficacy of Solulin is indeed attributable solely to the anticoagulant properties of APC remains to be confirmed. As the authors discuss, the activation of protein C results in downregulation of thrombin generation, which in turn reduces TAFI activation by free thrombin . Nevertheless, whether Solulin acts directly through APC anticoagulant functions or indirectly by promoting fibrinolysis by endogenous tPA through suppressed TAFI activation, or through both mechanisms, the potential use of Solulin in the treatment of stroke deserves further investigation.