Using thrombin time to evaluate the ecacy of anticoagulant therapy for acute cerebral infarction

Although intravenous thrombolysis therapy has been considered as a signicant progress in the treatment of acute ischemic stroke, there are limited effective treatments for patients with onset ischemic symptom that beyond six hours during acute ischemic stroke. In this study, we investigated the effectiveness of the intravenous argatroban therapy in acute ischemic stroke patients who cannot be treated with intravenous thrombolysis due to the limited time window.


Abstract Background
Although intravenous thrombolysis therapy has been considered as a signi cant progress in the treatment of acute ischemic stroke, there are limited effective treatments for patients with onset ischemic symptom that beyond six hours during acute ischemic stroke. In this study, we investigated the effectiveness of the intravenous argatroban therapy in acute ischemic stroke patients who cannot be treated with intravenous thrombolysis due to the limited time window.

Methods
One hundred and eighty patients with acute ischemic stroke that had beyond six hours ischemic symptom were admitted to our hospital and were analyzed retrospectively. Levels of activated partial thromboplastin time (aPTT), prothrombin time (PT) and thrombin time (TT) in peripheral blood of these patients were measured by ELISA at 24 hours post initial therapy.

Results
We found that plasma TT was signi cantly prolonged after 24 hours of argatroban treatment. aPTT showed slightly increased prolongation after 24 hours of argatroban treatment. PT also showed slightly prolonged after treatment, however, there was no difference from the basal line. We further investigated the relationship between the level of TT and the clinical effectiveness and safety of intravenous argatroban therapy. We found that when TT was between 40 and 80 seconds, intravenous argatroban effectively promoted the complete recovery rates without increasing the risk of hemorrhage.

Conclusion
Our study implies that TT assay might be useful for guiding regular dose of agratroban for therapy.

Background
Acute ischemic stroke (AIS) is the second most common cause of death and the leading cause of disability in China. Intravenous (IV) recombinant tissue plasminogen activator and urokinase thrombolysis have shown to be effective for patients with AIS within six hours of onset [1][2][3]. However, there are currently no effective and safe treatments for patients with AIS after 6 hours of the onset of ischemic symptoms.
Argatroban is a synthetic direct thrombin inhibitor derived from L-arginine. Argatroban exerts its anticoagulant effects by selectively binding to the activation site of thrombin, which leads to the inhibition of thrombin-induced brin formation and platelet aggregation [4,5]. Previous studies have demonstrated that argatroban could improve regional blood ow and ameliorate neurological de cits without increasing intracranial hemorrhage [6][7][8][9][10]. Compared with heparin, argatroban has a short elimination half-life of approximately 39-51 minutes. Moreover, argatroban has a predictable anticoagulant effect that does not potentiate heparin-induced thrombocytopenia; thus, it causes less bleeding with the same anticoagulant effect [3, 10- However, there have been few studies on the application of coagulation markers, including TT for assessing the anticoagulant pharmacological effects in patients with acute cerebral infarction. This study aimed to assess the e cacy and safety of IV argatroban in patients with AIS at > 6 h after the onset of ischemic symptoms. Moreover, it aimed to evaluate the relationship between TT levels and the clinical effectiveness and safety of IV argatroban therapy.

Patients
We retrospectively analyzed 180 patients (102 males, 78 females; age <80 years) with AIS who received IV argatroban treatment between February 2019 and December 2019. These patients were admitted within 48 hours of the onset of symptoms with an NIHSS score of 4-22. All the patients met the diagnostic criteria of cerebral infarction formulated by the Fourth National Cerebrovascular Disease conference in 1995. All the patients suffered from mild to severe neurological symptoms, including hemiparesis, hemi-sensory disturbance, dysarthria, hemianopsia, diplopia, and vertigo. The major risk factors for AIS, including hypertension, diabetes mellitus, cigarette smoking, severe alcohol drinking, hyperlipidemia, previous history of stroke, as well as underlying cardiac diseases e.g. atrial brillation, were evaluated. All the patients underwent CT or MRI brain scanning before any treatments.

IV argatroban
For the rst two days, argatroban (Novastan, Mitsubishi Pharma Corporation, Osaka, Japan) was administered at a loading dose of 60 mg/day by continuous IV infusion. This was followed by IV infusion of 20 mg/day in two divided doses over three hours for the subsequent ve days. All the patients received oral antiplatelets and IV edaravone along with argatroban treatment. The patients underwent routine CT examinations after the termination of IV argatroban therapy. In the case of neurological deterioration, emergent CT was performed to detect any hemorrhage or progression of infarcted area.

Clinical assessment
Clinical assessment included vital signs, blood glucose titration, complete blood counts, coagulation pro le, NIHSS score, and CT/MRI scanning. A stroke team member obtained the NIHSS scores at admission, at 7 days after initial argatroban treatment, and on the discharge day. The initial stroke severity was analyzed and divided into three categories based on the baseline NIHSS score as follows: mild (0-6), moderate (7-15), and severe (>16). Among the included patients, 46 (25.6%), 112 (62.2%), and 22 (12.2%) patients were diagnosed with mild, moderate, and severe stroke, respectively. The NIHSS score at admission was compared to the score at 7 days after treatment. The levels of PT, aPTT and TT were measured by enzyme-linked immunosorbent assay.
The criterion of therapeutic effect According to the evaluation criteria for the patients with acute cerebral infarction, the therapeutic effect was classi ed as complete recovery (function defect score decreased by 91%-100%), remarkable progress (function defect score decreased by 46%-90%), progress (function defect score decreased by 18%-45%), no change (function defect score decreased to < 17%), and deterioration (function defect score increased by > 17%).

Statistical analysis
The NIHSS, aPTT, PT, and TT values were expressed as mean ± SD. Pre-and posttreatment values were compared using the unpaired ttest. Between-group comparisons were performed using the chi-square test. Statistical signi cance was set at p < 0.05. All statistical analyses were performed using the StatView software (Macintosh version 5.0, SAS Institute).

Results
We enrolled 180 patients who were treated for deteriorating acute subcortical ischemic stroke. Table 1 presents the baseline characteristics of the patients. The average age of the patients was 63.13 ± 8.44 years. Moreover, 43.89% of the patients were ≥ 65 years old while 56.67% of the patients were male. The median baseline National Institutes of Health Stroke Scale (NIHSS) score was 10 ± 4. The median time from onset to IV argatroban was 16 ± 8 hours with 44% of the patients receiving IV argatroban within 12 hours.

Discussion
Most patients with AIS cannot reach the hospital within six hours of symptom onset, and therefore there is a speci c need for an effective and safe treatment for these patients, which is yet to be established. Argatroban, which is a synthetic peptidomimetic antithrombin agent, is the rst clinical anticoagulant to exclusively target thrombin. The present study showed that IV argatroban therapy could prevent recurrence and progression of thrombosis in the infarcted area. A previous study showed that argatroban allowed safe anticoagulation in patients with AIS at 12 hours after the onset of symptoms without increasing intracranial hematoma [12]. In this study, we observed that IV argatroban treatment achieved good results and had a very low hemorrhagic risk (1/180 patient, 0.56%). The median NIHSS score at admission (10 ± 4) decreased to 6.98 ± 3.88 after 7 days of therapy. Appropriate supportive care, including blood pressure, glucose, and lipid control, was paramount to achieving these results. Clinical symptom deterioration occurred in patients with severe stenosis of the middle or basal cerebral artery (10/15 patients, 66.67%).
Monitoring the status of anticoagulation is an important clinical parameter in patients with acute cerebral infarction during IV anticoagulant therapy. Although argatroban is commonly used in patients with AIS, there have only been a few studies on the relationship between the anticoagulation degree and clinical outcomes. A previous study recommended the analysis of the aPTT for argatroban. A steady-state aPTT was achieved after several hours of treatment, which should be approximately 1.5-3.0 fold increase of the aPTT baseline; however, it should not exceed 100 seconds (Kawai et al., 1996). In North America, a randomized, double-blinded, placebo-controlled study on direct thrombin inhibition in AIS showed that argatroban at each dose prolonged the aPTT without increasing symptomatic intracranial hemorrhage or major bleeding [12]. We measured the values of aPTT, PT, and TT in the peripheral blood of patients who received initial therapy for 24 hours. Further, we analyzed the relationship between blood coagulation biomarkers (APTT, PT, TT) and the therapeutic effect. Consistent with previous ndings, we found a slightly increased aPTT prolongation after 24 hours of argatroban treatment; however, the value was only about 44% greater than the pretreatment level. Notably, plasma TT was signi cantly prolonged after 24 hours of argatroban treatment.
In our study, the patients were allocated to three groups according to the TT value, which indicated the anticoagulation degree. There was no signi cant inter-group difference in the total effective rate after IV argatroban. We observed high rates of positive outcomes in all three groups. However, the proportion of patients who completely recovered in group b and group c was signi cantly higher than that in group a. Given the hemorrhagic risk, we observed that when the TT value was 40-80 seconds, IV argatroban effectively improved the complete recovery rate without increasing the hemorrhage risk. In addition, we found that TT prolongation did not increase the total effective rate; however, it increased the chance of complete recovery and reduced the risk of worsening. Moreover, our results indicated that therapy with argatroban should be monitored using TT. TT assessment at 24 hours after initial therapy for con rming whether the desired therapeutic range and dose adjustment was achieved may be required to achieve the target TT. However, given the effect of individual variations on anticoagulant activity, it is di cult to determine the optimal argatroban dose to avoid hemorrhagic complications. Therefore, there is a need for large-scale randomized controlled trials to con rm our ndings.

Conclusion
We found that IV argatroban could be an effective and safe treatment for patients with AIS at > 6 h after the onset of ischemic symptoms. Moreover, our ndings suggest that TT could be used as an indicator for the evaluation of argatroban e cacy.
QQL was responsible for the study design, investigation, writing-original draft, conceptualization, and methodology. TJL mainly contributed to formal analysis, data curation, review writing, and editing. SSY revised and edited the manuscript for important intellectual content, as well as project administration and funding acquisition. All the authors approved the nal version of the manuscript and agreed to be accountable for all study aspects.