Wasp-stung rat model translationally expresses the coagulopathy manifestations of human wasp patients

Abstract

Two over 80 wasp stings male victims appeared severe abnormal coagulation were consecutively examined by thromboelastography (TEG) guided with heparinase during hospitalization. However, the cause of coagulopathy remains unsolved. Rats were applied to establish a wasp-stung animal model highly resembled the manifestations of wasp-stung patients. According body surface area conversion, Sprague-Dawley rats were stung based on wasp sting numbers (0, 4, 8, 12 stings; n = 6 each) with various exposure times (0, 1, 3, 6 h) to determine the simulation of coagulopathy. The blood R, K values, and angle degree of wasp-stung rats were measured by TEG. The TEG profiles of stung rats were found to be concomitant with that of wasp-stung patients. Data showed that the endogenous heparinization of rats was time-dependent. Compared to the TEG profile of eight stings given rat, the coagulation time of 2 mm clot formation at 3 h (R value) was longer than that at 0 h. The coagulation time was prolonged with increasing sting numbers when compared to the various stings at 1, 3, and 6 h exposed. Interestingly, there was observed the peak coagulation at 3 h of eight stings. The Ck-standard and Ck-heparinase at 3 h after 8 stings given were R: 9.6–4.4 min; K: 3.8–1.8 min; angle degree: 49.8–68.0, respectively. The original data of R, K values and angle degree in two wasp-stung victims were 11.7–13.6 min, 4.3–5.5 min, and 41.2-32.8° in CK-standard, respectively; whereas those of the CK-heparinase groups were 5.6–6.7 min, 2.4–2.5 min, and 59.5–58.8°, correspondingly. Conclusively, this massive wasp-stung animal model can be applied to the investigations of pathogenesis and provides a clinical strategy or guideline for clinical intervention.

Bullet points

• The mechanical manifestations of coagulopathy in wasp-stung victim remains uncertain.

• Manipulating sting numbers with exposure times in rats are simulated wasp-stung human victims.

• Coagulopathy of rats mimicking human victims is confirmed by TEG with heparinase.

• Wasp-stung rats may be employed to investigate pathogenesis and clinical protocol for intervention

Introduction

In clinical reports, the most common human wounds are caused by Apoidea, including stings by wasps, bumblebees, or bees, and 49% of these stings injury is usually only in local. ¹ However, along increasing expansion of urban to rural areas and the seasonal operation of agriculture, the proportion of systemic symptoms caused by mass and severe wasp-stung is gradually increased, especially in summer and autumn season.^2,3 In rural areas of New World and Africa, massive stings and envenomations by Apoidea may lead hundreds or more casualties but there are insufficient clinical records to be accessed for deep insight in these areas. Of all Apoidea venoms, the envenomation by wasp-stung is the most clinically threatening that causes kidney and other organ failure and death in 20–200 stings, and this stung events typically occurred in subtropical and tropical Asia. ⁴ According to species, the stings of various Hymenoptera venoms show different clinical manifestations, among which local signs or symptoms broadly comprise pain, redness, wound infection, etc. Remarkably, 20 hours after severe wasp-stung envenomation, some patients showed vomiting, abdominal pain, and lower gastrointestinal bleeding. ⁵ Mesenteric ischemia, rhabdomyolysis, acute renal failure, and hepatotoxicity have been diagnosed as delayed toxin-mediated systemic reactions of wasp venoms.^6,7,8 Severe systemic reactions by wasp venoms manifested as hemolytic activity and rhabdomyolysis, leading to diffuse alveolar hemorrhage, adult respiratory distress syndrome, coagulation dysfunction and multiple organ failure.^3,9 There were no thrombi or emboli, but there was an abruptly tapering of mesenteric arteries. It has been strongly suggested non-occlusive mesenteric ischemia. ² From a retrospective investigation of 1091 multiple wasp stings hospitalized patients in the multicenter of China, the analytical results have exhibited 21.0% AKI, 24.1% rhabdomyolysis, 19.2% hemolysis, 30.1% liver injury, and 22.5% coagulopathy, respectively. ¹⁰ One case report presented a patient with coagulation abnormalities induced by wasp-stung anaphylaxis with a prolongation on the activated partial thromboplastin time (APTT) and significant increase the anti-Xa activity (equivalent to a therapeutic heparin range), but the medical records of this patient showed without any heparin treatment. ¹¹ Moreover, the examinations of the blood and urine samples from 121 hospitalized wasp-stung patients are found that patients with acute kidney injury (AKI) had longer APTT (median 70.0 vs 42.5 s) than those of patients did not show the AKI. ⁶ Another study revealed the patients with more than 20 stings massive wasp-stung may be prone to induce disseminated intravascular coagulation (DIC). ¹²

On review of the literature, only one report showed multiple wasp stings induced an acute renal injury that inferred endogenous heparinization in patients, ¹¹ while the other studies in wasp venoms induced organs failure did not have in-depth etiological analysis on the relationship of pathogenesis and underlined mechanisms between wasp venoms and coagulation abnormalities,^12-16 and that illustrate the earliest screen on the coagulation of massive wasp-stung patients can be applied to eliminate serious complications. Thereby in clinical Medicare of wasp-stung patients, an early examination of laboratory data and coagulation phenomenon can assist to monitor DIC and early exclude serious complications.

In our hospital, there were two male victims attacked by Hymenoptera wasp (Vespa affinis, Xiamen common species, Fujian, China) with multiple stings (86, 121), who showed the typical manifestations of wasp stings and further confirmed the coagulopathy without having received any heparin treatment in clinical practice. Based on the heparinase-coated thromboelastography (TEG) test results, the coagulopathy of multiple wasp-stung patients was observed to be induced by endogenous heparin effect. ¹⁷ The coagulopathy is a clinical symptom and a critical risk factor for the management of massive wasp stings, but the relevance between coagulation abnormality and wasp envenomation remains unclear.^10,18 In this study, rats were given various numbers of wasp stings to highly resemble the coagulopathy phenomenon of human mass wasp envenomation as a rodent model, particular in the analysis of the alterations of endogenous heparinization. Furthermore, this wasp-stung animal model was employed to analyze its heparinization and to explore the episode of mass wasp envenomation for the investigation of the occurrence of DIC that defies the Medicare strategy or guideline.

Materials and Methods

Two patients provided a written consent letter allowing the case to be publically published. Ethics approval for the collection and analysis of all data from patients was permitted by the Ethics Committee of the Second Affiliated Hospital of Medical College (IRB registration number 2,017,015). All the experimental procedures were done in accordance with the guidelines published by the National Institutes of Health (Guide for the Care and Use of Laboratory Animals, eighth edition) and they fulfilled the ARRIVE guidelines. Animal experiments were approved by the Animal Ethics Committee of Medical College, and the “Guide for the Care and Use of Laboratory Animals” of Xiamen Medical College was followed (Approved protocol ID SYXK 2018-0010). 8-weeks-old male Sprague-Dawley rats (250–300 g Bwt) were obtained from Wu’s animal laboratory (Fujian, China) and kept under controlled environmental conditions at room temperature of 22 ± 2°C and humidity of 50 ± 10%. The 12/12 h light/dark (6 am–6 pm) cycle was sustained throughout the entire study. The rats had free access to feed (a standard laboratory diet) and water. Animal welfare was evaluated according to Laboratory animal-Guideline for ethical review of animal welfare (GB/T 35,892-2018).

Wasp injured model

Wasp comb (Vespa affinis, Xiamen common species, Fujian) was collected from the suburban of Xiamen City by a firefighter. The wasp hive was kept at ambient temperature at 10°C to reduce the activity of the wasps. Wings of the individual wasp were cut off by using scissors for the experimental use immediately. The Sprague-Dawley rats were anesthetized by 5% isoflurane gas in the inhale chamber with vaporizer (AS-01, Northern Vaporiser Ltd. Skipton, UK) and retained in 2% isoflurane during the entire experimental procedure. The anesthetizing protocol was followed in McGill Standard Operating Procedure (SOP) #111 that describes the methods for rat anesthesia.

In fact, the incidence of wasp-stung usually occurred outdoors, the amount of toxic substance is impossible to determine and consequently only the sting numbers are available for counting in the clinical setting.^5-7,10 Thereby, the experimental design of this study was followed: all treatments were allotted into four groups (n = 6) with various numbers of stings (0, 4, 8, 12 stings administered on the animals’ back) and duration (0, 1, 3, 6 h) of exposure. One mL of blood was obtained from the left ventricle of the anesthetized rats by cardiac puncture. Each 340 μL for heparinase-coated cup and standard assay were performed in TEG (YZ5000, Yuzeyi Medical Tech. Company, Shanxi, China). The results of the TEG are recorded as shown in Figure 1. The TEG scheme is divided into two phases: coagulation and fibrinolysis. In this study, the coagulation phase was mainly focused on the R and K values in particular setting. After treatments, tested rats were kept in individual ventilation cages (IVC) for monitoring their condition. At the end of the experiments, all rats were sacrificed followed the procedure of euthanasia via CO₂.

Figure 1.

The profile of thromboelastography.

Statistical analysis

Data were expressed as means ± SD for all results. The results of the time course experiment were compared with paired t-test. Statistical comparisons of the different stings results were carried out using unpaired sample test with two ways (sting numbers and exposure time) analysis of variance (ANOVA). Means within each column followed by the different letters are significantly different at p < 0.05 using the post-hoc Tukey’s test.

Results

Two male victims who cut grass field in outdoor of Xiamen city (Fujian) accidentally disturbed a hive and were attacked by wasps. From the physical examinations, one of the patients had 121 stings while the other one had 86 stings. In our report, two patients who were stung over 80 stings that resulted in severe coagulation abnormality were consecutively examined by TEG guided with heparinase during their hospitalization. The TEG test showed there was coagulation dysfunction without using heparin-like substances in clinical practice. Numerous tests were also conducted to confirm whether the coagulation abnormality caused by the wasp-stung attributed to hyper-endogenous heparinization and allergic reactions, rhabdomyolysis, and vascular endothelial cell injury, that might interfere with the production of endogenous heparin. The original data of R, K values and angle degree in the two wasp-stung patients were 11.7–13.6 min, 4.3–5.5 min, and 41.2–32.8°, respectively; whereas those of the CK-heparinase-groups were 5.6–6.7 min, 2.4–2.5 min, and 59.5–58.8°, correspondingly (Figure 2). In the clinical data showed that two patients had persistently low coagulation. The lower coagulation caused by hyper-endogenous heparinization was concomitant with the binding of anti-thrombin and the activation of fibrinolysis. Obviously, this coagulopathy was attenuated by the intervention with protamine to neutralize the hyper-heparinization.

Figure 2.

The TEG profile of two wasp-stung victims. The black curve represents CK-standard test. The purple curve presents CK-heparinase test. (a) CK-standard, (b) CK-heparinase, and (C) Merge.

Firstly, for the calibration of the variables R, K, α-angle, and MA in TEG assay, the mean values were found not statistically different when citrated blood was used in CK-standard or CK-heparinase assays. Next, to investigate the coagulopathy of rats mimicking that of human wasp-stung victims, heparinized blood assayed in standard cups displayed a significant increase in R and K values, and significantly decrease in α-angle and MA when compared to heparinized blood assayed in heparinase-coated cups. According to the body surface of human which was converted to that of rat, the rats were given 4, 8, and 12 stings as over 80-200 stings in human victim. Compared to the TEG profile of eight stings given rat, the coagulation time of 2 mm clot formation at 3 h (R value) was greater than that of 0 h. The R, K values and angle degree of the control rats were 1.0 min, 0.8 min, and 80.4°, respectively (Figure 3, Table 1). The data of 1 h after wasp-stung revealed that R, K values, and angle degree of standard cup were 9.6 min, 3.8 min, and 49.8°, respectively. On the other hand, the R, K values, and angle degree of heparinase-coated cup were 4.4 min, 1.8 min, and 68° (Figure 3). The results indicate that abnormal blood coagulation is caused by endogenous heparin.

Figure 3.

The TEG profile of given eight stings rat. (a) pre-sting (0 h), (b) post-sting (3 h), (c) CK-standard test, and (d) CK-heparinase test.

Table 1.

Summary of the outcomes in clinical cases and in vivo rat model showed the coagulopathy by wasp stung-induced endogenous heparinization.

		Stings	CK-standard			CK-heparinase
			R (min)	K (min)	Angle (deg)	R (min)	K (min)	Angle (deg)
In clinical	Patient-A	>80	13.6	5.5	32.8	6.7	2.5	58.8
—	Patient-B	>80	11.7	4.3	41.2	5.6	2.4	59.5
In vivo	Rat model	0 (pre-sting)	1.0 ± 0.2	0.8 ± 0.1	80.4 ± 6.1	0.9 ± 0.3	0.8 ± 0.2	81.0 ± 0.2
—	—	8 (0 h)	0.8 ± 0.3	0.8 ± 0.1	78.2 ± 5.8	0.9 ± 0.2	0.8 ± 0.1	79.4 ± 6.8
—	—	8 (1 h)	9.5 ± 3.3	2.7 ± 1.1	53.2 ± 7.7	3.6 ± 2.3	1.7 ± 0.7	73.3 ± 6.1
—	—	8 (3 h)	10.2 ± 3.3	3.6 ± 1.3	48.2 ± 7.9	4.4 ± 1.5	1.7 ± 0.6	67.4 ± 5.5
—	—	8 (6 h)	5.2 ± 1.4	2.3 ± 0.9	65.2 ± 6.8	2.3 ± 1.1	1.1 ± 0.3	75.4 ± 5.8

Human normal range is R (min): 3.8-10.2; K (min): 1.2-4.1; Angle (deg): 49.0-72.6, respectively. Rat normal range is R (min): 1.0 ± 0.2; K (min): 0.8 ± 0.1; Angle (deg): 80.4 ± 6.1, respectively.

Remarkably, the data showed that the R values of different times without wasp-stung of rats were not significantly different. For the wasp-stung rats, the data found that the coagulation time was prolonged with stung times regardless of the sting numbers. On a progressively prolonged maintenance interval (PPMI) up to 6 h, there continuously appeared the low coagulation in various stings; it reached the climax at 3 h and declined at 6 h after wasp-stung. To determine whether the procedures of anesthetic and cardiac puncture caused the alteration of the coagulation time, the data revealed that the procedure did not result in any change at 0, 1, 3, 6 h with 0 sting. Additionally, compared to the various stings at various times 1, 3, and 6 h, the coagulation time was prolonged with increasing sting numbers. Interestingly, there was found the maximum coagulopathy at eight stings given for 3 h (Table 1, Figure 4). Figure 5 depicts the typical TEG profiles of rats and human patients, the results illustrated that the TEG profile of the wasp-stung rats was resemble to that of the wasp-stung human patients, implying that the responses of wasp-stung rats are comparable to the human victims. Summary of the outcomes in clinical cases including human and rat normal ranges, the data showed that the incidence of endogenous heparinization was induced by wasp-stung caused the coagulopathy (Table 1). Taken all results together demonstrated the coagulation abnormality without DIC manifestations in these wasp-stung rats, suggesting this model is resembling to the coagulopathy of the wasp-stung human victims.

Figure 4.

The heparin reaction time of various stings treatment was along with various times in wasp stung rats. *p < 0.05, **p < 0.01 present a significant difference in the treatment group at 0 h. #p < 0.05, ##p < 0.01 present the significant difference at 3 h compared to 6 h.

Figure 5.

The typical TEG profiles of rat and human patient. The black curve represents CK-standard test. The purple curve represents CK-heparinase test. (a) RAT model and (b) Clinical case.

Discussion

Wasp sting envenomation is a public health concern, and its symptom depicts variable manifestations depending on the stung site, number of attacks, and individual sensitivities. Massive wasp envenomation would cause severe and systemic allergic reactions and anaphylaxis; it also results in severe delayed toxin-mediated systemic reactions, including coagulopathy, ¹⁸ hemolysis, ¹⁹ rhabdomyolysis, ⁷ acute renal failure, ²⁰ and hepatotoxicity. ⁸ This phenomenon is probably due to the activated mast cells or basophils that they release the mediators such as heparin and tryptase. ²¹ Subsequently, heparin can act as an anticoagulant by binding to anti-thrombin. Once mast cells and basophils are activated in allergic reactions, the production and release of heparin or heparin-like substances may be a major source for hyper-endogenous heparinization. This resulting hyper-endogenous heparinization is considered to be the vital reason for the coagulation dysfunction. ¹⁷ This “heparinization” would be explained by the anti-Xa activity that is attributed to the prolonged APTT detected in our patients. Secondly, we noted an extremely low fibrinogen level in the presence of normal platelet count and only a slight increase of D-dimers (absence of DIC). ¹¹ This could be ascribed to serum tryptase released during massive mast cell activation. This tryptase also directly acts on the fibrinolytic pathway by activating the single-chain urinary-type plasminogen activator, resulting in the conversion of plasminogen into plasmin and subsequent the degradation of fibrinogen and other coagulation factors. ¹¹ This hyper-fibrinogenolysis may elucidate both the prolonged clotting times and low fibrinogen level. Nevertheless, with the high occurrence of wasp-stung, the studies on the pathophysiological modalities of envenomation are still emerging.

For the treatment of wasp-stung victim, the main clinical approaches of coagulation disorders include blood transfusion, anti-fibrinolytic drugs, and other hemostatic drugs. In this study, the analysis of the clinical laboratory data from two patients with severe wasp-stung admitted to the emergency room in our hospital revealed that the inpatients manifested coagulation disorders in the acute phase after wasp-stung, and the key finding was the endogenous heparinization in blood by the measurement of TEG. After confirming the etiology of the coagulation disorder, intravenous protamine treatment was given to neutralize this endogenous heparinization of the patient. Consequently, the persistent hypo-coagulability of two patients was rapidly recovered. This protocol of clinical diagnosis and treated strategy for massive wasp stings patients are not only greatly shortened the requirements of clinical treatment but also avoided the waste of a large number of blood transfusion products requisite by the routine treatment. In the past, due to insufficient clinical laboratory tests and lack knowledge of the physician, the clinical treatment for the coagulation disorders of patients with the same or similar manifestations after hospitalization were seen in this study, the endogenous heparinization of patients may respond to the pathogenesis of persistent hypo-coagulation, and which is extremely easy to overlook in the clinic and subsequently lead to more serious complications.

Notably, Hymenoptera stings cause the toxic response and both local and systemic allergic reactions that Hymenoptera venom consists of a mixture of biologically active substances, e.g. enzymes (phospholipase A, hyaluronidase, lipase, esterase, protease, acid phosphatase, alkaline phosphatase, and phosphodiesterase), peptides (melittin, apamin, mastoparans, bombolitins), and low-molecular-weight compounds (biogenic amines, acetylcholine, carbohydrates, lipids, free amino acids). ⁹ Phospholipase A₂ (PLA₂) is a predominant component of bee venoms, while PLA₁ is highly abundant in wasps and ants. ²² The activity of PLA₁ in venoms of P. paulista affects directly hemolytic action against washed red blood cells is similar to that of Cobra cardiotoxin from Naja naja atra. ²² Furthermore, magnvesin of Polistes dominulus wasp shares 52% identity with allergen serine protease and exerts its anti-coagulant function by hydrolyzing coagulant factors TF, VII, VIII, IX, and X. ²³ The toxic substances from various species venoms play an important role in the different pathological response of the cause.

Mostly, Hymenoptera stings only led local inflammation in most victims. Nevertheless, most deaths related to Hymenoptera stings are the result of immediate hypersensitivity reactions, causing anaphylaxis. Massive envenomation can also cause death in non-allergic individuals. The estimate lethal dose of stings is about 500 stings in human adults, but 20 stings/kg body weight in most smaller mammals, ²⁴ and the lethal dose of estimated stinging in mammals differs by body size and correlates with the ratio of surface area over body weight. In this massive wasp-stung animal model, the lethal dose is more than 12 stings in adult rat and the stinging toxic response including coagulopathy is very comparable to human cases. In wasp-stung rats plus control rats, blood samples were directly collected from the left ventricle by cardiac punch that can provide a multiple blood sampling from the same individual not only truly simulates the coagulated dynamic of the post-stung but also evades other coagulating interference with less trauma. The coagulation results of TEG test were consistent with the dynamic change trend of coagulation in clinical patients, and combined with other parameters, a multiple blood sampling with different post-stung time from the same individual could be better imitation for the clinical change of wasp-stung patient and simultaneously decrease the experimental animal use.

Of note anaphylactic reactions to Hymenoptera stings are not dose-dependent or related to the number of stings. Accordingly, four possible reactions are seen after insect stings: local reactions, regional reactions, systemic anaphylactic responses, and less commonly delayed-type hypersensitivity. ²⁴ There are rare complications found in patients multiple wasp stings with DIC, acute renal failure, and thrombotic microangiopathy. ²⁵ Serious toxic reactions usually occur after numerous stings. In mild cases, patients may only have isolated and prolonged APTT and normal prothrombin time (PT), clinically without a tendency to hemorrhage. The clinical reports show the severity of bee stings has the effect on the prolong APTT, and the patient’s APTT was significantly prolonged as the increase of the numbers and severity degree of stings.^26,27 The post-sting phenomenon of isolated prolongation on APTT was observed in a case report of three wasp-stung patients. ²⁸ Those clinical symptom of coagulopathy on stings-induced prolong APTT is consistent with the results of our wasp stings animal model.

In this study, male rats were chosen for the modeling setup and female rats were also tested by the same protocol. Surprisingly, in comparison to the results of male rats, the blood coagulation of female rats exhibited inconsistent level during the experimental period. It is possible that female sex hormone could interfere the wasp envenomation. In the reviewed literature, while female patients showed more sensitivity (sensitive to Hymenoptera-derived allergens) than that of male patients in skin prick test, but the difference was not statistically significant in a study population at West Bengal, India. ²⁹ Contrarily, a clinical pattern study revealed that there is a gender difference in cardiovascular symptoms induced by wasp-stung with more frequency in males than that of females. ³⁰ The sex difference of animals in response of Hymenoptera-derived allergens needs to be further clarified.

Conclusions

This study refers to the clinical hyper-endogenous heparinization or coagulopathy induced by massive wasp stings to establish a rodent disease model, and these modeling animal data showed that the transient endogenous heparinization by massive wasp stings is moderated with most clinical laboratory data in a dose-dependent. This diseased model rules out the manifestations of DIC according to published diagnostic criteria. Most patients with multiple wasp stings present the coagulopathy caused by the venoms before toxic reactions and multiple organ dysfunction. It is evidenced that this massive wasp sting animal model can be applied to the studies of pathological mechanisms and provides clinical evaluation and strategy for clinical intervention.