Use of Batroxobin in Central and Peripheral Ischemic Vascular Diseases: A Systematic Review

Background and Purpose: The mechanism of action of Batroxobin included the decomposition of the fibrinogen to fibrin degradation products (FDPs) and D-dimer and mobilization of endothelial cells to release endogenous nt-PA and to promote thrombolysis. This review aims to summarize current study findings about batroxobin on correcting cerebral arterial, venous, and peripheral vascular diseases, to explore the mechanism of batroxobin on anti-thrombosis process. Methods: A thorough literature search was conducted utilizing the PubMed Central (PMC) and EMBASE databases to identify studies up to June 2021. Data from clinical studies and animal experiments about batroxobin were extracted, integrated and analyzed based on Cochrane handbook for systematic reviews of interventions approach and the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P), including the condition of subjects, the usage and dosage, research observation index and main findings. Results: A total of 62 studies were enrolled in this systematic review, including 26 clinical studies and 36 animal experiments. The 26 clinical studies involved 873 patients with arterial ischemic events, 92 cases with cerebral venous thrombosis, 13 cases with cerebral cortical vein thrombosis, and 1,049 cases with peripheral vascular diseases. These patients included 452 males and 392 females aged 65.6 ± 5.53 years. The results revealed that batroxobin had broad effects, including improving clinical prognosis (n = 12), preventing thrombosis (n = 7), promoting thrombolysis (n = 6), and improving vascular cognitive dysfunction (n = 1). The effects of batroxobin on reducing neuronal apoptosis (n = 8),relieving cellular edema (n = 4), improving spatial memory (n = 3), and promoting thrombolysis (n = 13) were concluded in animal experiments. The predominant mechanisms explored in animal experiments involved promoting depolymerization of fibrinogen polymers (n = 6), regulating the expression of related molecules (n = 9); such as intercellular adhesion molecule, heat shock proteins, tumor necrosis factor), reducing oxidative stress (n = 5), and reducing inflammation response (n = 4). Conclusion: Batroxobin can correct both arterial and venous ischemic diseases by promoting depolymerization of fibrinogen polymers, regulating the expression of related molecules, reducing oxidative stress, and reducing the inflammation response.


INTRODUCTION
Batroxobin, isolated from Bothrops atrox moojeni venom, is widely used in clinical such as postoperative hemostasis of surgery because of its hemostatic effect (1)(2)(3)(4). Batroxobin has also been investigated for the treatment of deep vein thrombosis and cerebral infarction as it promotes thrombolysis, prevents recurrence of thrombus, and provides neuroprotection (5)(6)(7)(8). In recent years, the role of Batroxobin in cerebral venous thrombotic diseases has attracted more attention with two clinical articles proposing to study the clinical value of Batroxobin in cerebral venous thrombosis (CVT) and cerebral venous sinus thrombosis (CVST), respectively (9,10). Batroxobin may promote venous sinus recanalization thrombosis recanalization, and is a potentially safe and effective adjunct therapeutic agent in patients with a high level of fibrinogen. Another small clinical study investigated the efficacy of Batroxobin in cerebral cortical vein thrombosis (CCVT). Batroxobin significantly improved the prognosis of patients with CCVT (11). All these studies prove that Batroxobin has a wide range of clinical applications. The mechanism of action of Batroxobin included the decomposition of the fibrinogen to fibrin degradation products (FDPs) and D-dimer (12,13) and mobilization of endothelial cells to release endogenous nt-PA and to promote thrombolysis (14,15). However, there is a lack of literature review that summarizes the clinical effects and related mechanisms of Batroxobin. Since there is a growing interest in studying Batroxobin as a treatment strategy in cerebral venous system diseases, our study aims to summarize the previous findings to provide a theoretical basis for the use of Batroxobin in cerebral venous system diseases and facilitate future research.
In this study, we review previous studies investigating Batroxobin in both clinical and experimental settings and summarize the most recent findings to provide a deep understanding of Batroxobin in treating thrombotic diseases. We also discuss the potential use of Batroxobin in the treatment of cerebral venous thrombotic diseases.

Search Strategy
A systematic review of the literature has been performed on PubMed Central (PMC) and EMBASE databases using the keywords "Batroxobin, " "animal study, " or "clinical study." Our review includes studies published till June 2021 that investigated Batroxobin. Cochrane handbook for systematic reviews of interventions approach and the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) was followed accordingly (Supplementary Table 1).

Study Selection
Clinical (prospective and retrospective) and experimental studies that evaluated the efficacy of Batroxobin were included. Studies not related to vascular system diseases and their complications were excluded. Conference abstracts, reviews, case reports, and letters were also not included in the analysis. If two or more studies had duplicate or overlapping data, then the study with the larger sample size and more detailed data was selected. Two reviewers (D-L and SY-S) independently performed the study selection and any disagreements were resolved by discussion (Figure 1).

Data Extraction
Two authors (D-L and SY-S) extracted data from the selected studies, which was evaluated by another author (BL-J). The data were further extracted and summarized as follows: the name of the first author, year of publication, country, study characteristics (sample size and research type), subject characteristics (population and animal status, comorbid status and animal model type), detailed information of Batroxobin use, primary outcome and other main findings. All disagreements were resolved by consensus.

Outcomes
The main outcomes of the clinical trials in this review were coagulation indicators, improvement of neurological function, and thrombus recanalization and recurrence. The main outcomes of animal experiments were histopathological indexes and blood factor indexes.

RESULTS
Sixty-two studies, including 26 clinical studies and 36 animal experiments, were selected for the systematic review. The specific screening process is shown in Figure 1 and detailed information about the selected studies is listed in Tables 1, 2.

Clinical Studies
Two clinical studies, including 31 and 61 subjects, evaluated the efficacy of the combination of Batroxobin and anticoagulation in cerebral venous thrombosis (CVT) and cerebral venous sinus thrombosis (CVST), respectively (9,10  [0(0, 4.25)−5 (2,11), p = 0.036] compared with the baseline in only one study (9). A clinical study with 13 patients evaluated the effectiveness of Batroxobin in acute cerebral cortical vein thrombosis (CCVT) (11). Compared with the non-Batroxobin group, the Batroxobin group achieved a significantly improved prognosis, evaluated by the global impression of change (PGIC) (p = 0.030) in patients.
One study investigated the effect of Batroxobin in improving vascular cognitive dysfunction (24). Significant differences were observed in Mini-mental state examination (MMSE) and activities of daily living (ADL) scores compared with baseline.

DISCUSSION
Our review for the first time summarizes the clinical applications and possible mechanisms of Batroxobin by systemically reviewing current clinical and experimental studies (Figure 2).

The Application of Batroxobin in Central Vascular Disease
The effectiveness of Batroxobin in promoting recanalization (9, 10) and preventing recurrence (7, 16) of thrombus in all patients with ischemic disease, including cerebral venous sinus thrombosis (CVST) or acute ischemic stroke (AIS), were supported by several studies. In addition to its benefit for recanalization and secondary stroke prevention, treatment with Batroxobin also improved the neurologic deficits which secondary to CVST or AIS (1, 8, 14-16, 23, 67). A case-control study showed that Batroxobin in combination with aspirin improved vascular cognitive impairment (VCI) (24). Batroxobin did not increase the relative risk of any adverse events, including intracranial bleeding (9), compared with the control group.

The Application of Batroxobin in Peripheral Vascular Diseases
Batroxobin treatment alone or in combination with other anticoagulant drugs could promote complete recanalization and prevent the incidence of postoperative deep venous thrombosis (DVT) without adverse events such as pulmonary embolism (PE) and hemorrhage (5,25,26,64). Also, injection of Batroxobin with long-term micropump may get a better efficacy for DVT (27). Batroxobin in combination with aspirin also prevented restenosis after arterial angioplasty which may be mediated by decreased regional inflammation (4,(28)(29)(30)32). In patients with atrial fibrillation (AF), Batroxobin improved blood rheology, decreased blood cell aggregation, and prevented left atrial thrombus formation (34).
In peripheral vascular-related animal models, Batroxobin improved hemostasis (56,57,67), and prevented thrombosis (54,58), accelerating tissue repair and vascular regeneration and stabilizing the atherosclerotic plaque (59,62). The effect of Batroxobin on fibrinogen metabolism played an important role in ameliorating the formation of disseminated intravascular coagulation (DIC) (60,61). As an adjunct, Batroxobin enhanced the thrombolytic effects of native tissue-type plasminogen activator (nt-PA) (55). The role of Batroxobin in inhibiting human vascular smooth muscle cell (SMC) migration may also play a clinical value in the future (63).

Cerebral Venous Sinus Thrombosis May Benefit More From Batroxobin
Timely diagnosis and treatment are essential for faster and more complete recanalization and better outcomes in patients with cerebral venous sinus thrombosis (68)(69)(70). However, the primary treatment of CVST is long-term oral anticoagulation. For acute and severe CVST, endovascular therapy is always used first (71). Whereas, venous recanalization is time consuming and there remains a risk of hemorrhagic transformation after anticoagulation. Further complications of endovascular interventions make these interventions a dilemma for most physicians. Therefore, exploration of optimized treatment strategies in CVST is necessary.
Hyperfibrinogenemia, decreased blood flow velocity, and increased viscosity of hyperfibrinogenemia are the three major factors that promote venous thrombosis (72). Batroxobin is a serine protease extracted from the venom of the snake Bothrops atrox moojeni, and it exerts defibrinogenating effects (13). Batroxobin reduces the concentration of fibrinogen in blood by degrading fibrinogen to fibrin degradation products (FDPs) and D-dimer (12,13). The defibrinogenating effect of batroxobin improves microcirculation by reducing vascular resistance and increasing blood flow velocity (30). Batroxobin can also mobilize endothelial cells to release endogenous t-PA, which indirectly promotes thrombolysis (12,13). Therefore, Batroxobin can play both preventative and therapeutic roles in pat without increasing the risk of bleeding events in patients with a high risk of CVST.
Despite the controversial effect of Batroxobin on coagulation status, the significant reduction of the amount of bleeding and the effect on hemostasis by Batroxobin was well studied. Batroxobin combined with anticoagulation can significantly promote the recanalization of CVST and cortical venous thrombosis (CCVT) without increasing the risk of bleeding (10,11). Venous stasis and the embolism from the venous sinus, especially the superior sagittal sinus, were the main risks CCVT in CVT patients (73)(74)(75). CCVT is often secondary to venous infarct and hemorrhagic transformation. A previous study reported that Batroxobin reduced the death/apoptosis of damaged neurons, the size of the ischemic infarct, and the risk of bleeding conversion (36). Therefore, CCVT patients are likely to benefit from Batroxobin treatment. CVST or venous infarct-induced cerebral edema resulted in a series of clinical symptoms of intracranial hypertension, which is often a predictor of poor prognosis (75,76). Previous studies showed that CVST patients benefit from decompressive craniotomy (77). However, decompressive craniotomy might be better suited for severe cerebral edema caused by large venous infarcts. For CVST patients with mild intracranial hypertension caused by edema, Batroxobin may be a better choice since it reduces tissue edema and inhibits cytotoxic damage, as demonstrated in previous studies (14,15,42,65).
CVST patients always showed good neurological and cognitive long-term outcomes (78). However, some patients also presented with significant neurological impairment or neuropsychological deficits due to the disruption of functional areas or conduction tracts when the cerebral cortex is infarcted because of CVST or thrombosis in the deep cerebral venous sinus (75,79). Cognitive dysfunction is an important factor affecting patients' quality of life and aggravating family burden. Therefore, in the acute stage of CVST or venous infarcts, intervention measures are needed to protect nerve cells in the damaged area to avoid or mitigate cognitive impairment as much as possible. Batroxobin improves free radical scavenging leading to neuroprotective function. A previous study reported that Batroxobin was effective in improving vascular cognitive impairment (VCI) caused by ischemic cerebrovascular disease after long-term treatment (24). Future studies are needed to investigate whether the cognitive dysfunction associated with CVST can benefit from the use of Batroxobin.
In summary, Batroxobin had broad clinical applications in both arterial and venous thrombosis, including promotion of thrombolysis, prevention of thrombotic formation, reduction of edema in infarcted areas, improvement of vascular cognitive dysfunction, and neuroprotection. The potential mechanisms include promotion of depolymerization of fibrinogen polymers, increase in the capacity of free radical scavenging, reduction of inflammation, and regulation of endogenous plasminogen activator expression. Batroxobin can also be therapeutic in CVST and their secondary diseases. However, the application of Batroxobin was still limited to clinical studies with small sample size. Future multi-centered studies with randomized design and larger sample size would provide more evidence on the potential effect of Batroxobin in cerebral vascular diseases.

CONCLUSION
Batroxobin could treat both arterial and venous ischemic diseases by promoting depolymerization of fibrinogen polymers, regulating the expression of related molecules, reducing oxidative stress, and reducing the inflammation response. However, current evidence of the beneficial effect of Batroxobin in cerebral vascular diseases was mostly from clinical and experimental studies with small sample size and high heterogeneity. Multicentered clinical trials with randomized design and larger sample size would be needed in the future.

AUTHOR CONTRIBUTIONS
DL and SYS: manuscript drafting and revision, study concept and design, collection, assembly, and interpretation of the data. BLJ: collection, assembly, and interpretation of the data. RM, YHL, and SYS: manuscript drafting and revision, study concept and design, deeply edited the revised version and contributed critical revision, and final approval of the manuscript.