Platelet Activation Triggers Rapid Progression of Cardiopulmonary Collapse and Consumptive Coagulopathy: A Severe Amniotic Fluid Embolism Rabbit Model Study

Platelet Activation Triggers Rapid Progression of Cardiopulmonary Collapse and Consumptive Coagulopathy: A Severe Amniotic Fluid Embolism Rabbit Model Study | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 4 December 2025 V1 Latest version Share on Platelet Activation Triggers Rapid Progression of Cardiopulmonary Collapse and Consumptive Coagulopathy: A Severe Amniotic Fluid Embolism Rabbit Model Study Authors : Yuka Otsuka 0009-0007-1094-2316 , Kohsuke Hagisawa [email protected] , Manabu Kinoshita , Kouki Kaneko , Ruka Sasa , Kimiya Sato , Katsuo Terui , Soko Nishimura , Morikazu Miyamoto , Hidenori Sasa , and Masashi Takano Authors Info & Affiliations https://doi.org/10.22541/au.176486790.02715791/v1 215 views 142 downloads Contents Abstract Abstract Introduction 2. Materials and Methods Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Objective : To establish an animal model of severe amniotic fluid embolism (AFE) that reproduces both hemodynamic changes and coagulopathy to elucidate the underlying pathophysiology and time course of AFE progression. Design: Prospective, controlled study. Setting: Laboratory. Population and sample: New Zealand white rabbits (n=34, 28 th day of pregnancy) Methods : Cesarean sections were performed, followed by intravenous administration of amniotic fluid (AF). Vascular resistivity index was assessed using transthoracic ultrasonography, and arterial blood pressure was monitored. Blood samples were collected over time until critical organs were harvested at the time of cardiac arrest or euthanasia after 60 min of observation. Main Outcome Measures: Acute prognosis, hemodynamics, and blood coagulation status. Results : The acute mortality rate of AF-injected animals was 55.9% (19 out of 34). All AF-injected rabbits exhibited fibrin thrombi in the capillaries of the lungs, liver, and uterus. They also showed thrombocytopenia and hypofibrinogenemia, with the dead group demonstrating a significant increase in plasma D-dimer levels, suggestive of obstetrical disseminated intravascular coagulation. Notably, this group also exhibited a significant increase in the plasma levels of platelet factor 4, syndecan 1, neutrophil elastase, and histamine levels at 5 min after AF injection. Conclusions : We established a novel animal model of severe AFE and demonstrated that anaphylactoid reactions and severe coagulopathy occur simultaneously with platelet and neutrophil activation, which are essential factors in the acute phase of severe AFE during the first few minutes. Platelet Activation Triggers Rapid Progression of Cardiopulmonary Collapse and Consumptive Coagulopathy: A Severe Amniotic Fluid Embolism Rabbit Model Study Yuka Otsuka, MD 1 , Kohsuke Hagisawa, MD, PhD 2* , Manabu Kinoshita, MD, PhD 3 , Kouki Kaneko, MD 4 , Ruka Sasa, MD 5 , Kimiya Sato MD, PhD 6 , Katsuo Terui, MD, PhD 4 , Soko Nishimura, MD 1 , Morikazu Miyamoto, MD, PhD 1 , Hidenori Sasa, MD, PhD 1 , Masashi Takano, MD, PhD 1 1 Department of Obstetrics and Gynecology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan 2 Department of Physiology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan 3 Department of Immunology and Microbiology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan 4 Division of Anesthesiology, Saitama Medical Center, Saitama Medical University, 1981 Kamoda, Kawagoe, 350-8550, Japan 5 Division of Traumatology, National Defense Medical College Research Institute, 3-2 Namiki, Tokorozawa 359-8513, Japan 6 Department of Basic Pathology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan *Corresponding author : Kohsuke Hagisawa, M.D., Ph.D., Department of Physiology, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan Tel.: +81-4-2995-1483; Fax: +81-4-2996-5188; E-mail address: [email protected] Short Title: Hemodynamics and Coagulation in Amniotic Fluid Embolism Abstract Objective : To establish an animal model of severe amniotic fluid embolism (AFE) that reproduces both hemodynamic changes and coagulopathy to elucidate the underlying pathophysiology and time course of AFE progression. Design: Prospective, controlled study . Setting: Laboratory. Population and sample: New Zealand white rabbits (n=34, 28 th day of pregnancy) Methods : Cesarean sections were performed, followed by intravenous administration of amniotic fluid (AF). Vascular resistivity index was assessed using transthoracic ultrasonography, and arterial blood pressure was monitored. Blood samples were collected over time until critical organs were harvested at the time of cardiac arrest or euthanasia after 60 min of observation. Main Outcome Measures: Acute prognosis, hemodynamics, and blood coagulation status. Results : The acute mortality rate of AF-injected animals was 55.9% (19 out of 34) . All AF-injected rabbits exhibited fibrin thrombi in the capillaries of the lungs, liver, and uterus. They also showed thrombocytopenia and hypofibrinogenemia, with the dead group demonstrating a significant increase in plasma D-dimer levels, suggestive of obstetrical disseminated intravascular coagulation. Notably, this group also exhibited a significant increase in the plasma levels of platelet factor 4, syndecan 1, neutrophil elastase, and histamine levels at 5 min after AF injection. Conclusions : We established a novel animal model of severe AFE and demonstrated that anaphylactoid reactions and severe coagulopathy occur simultaneously with platelet and neutrophil activation, which are essential factors in the acute phase of severe AFE during the first few minutes. Funding: JSPS KAKENHI Grant Number 22H03179 (to K.H., M.K., K.K., K.T., and M.M.). Key words: amniotic fluid embolism, consumptive coagulopathy, cardiopulmonary collapse. Introduction Amniotic fluid embolism (AFE) is an unpredictable and occasionally fatal complication of childbirth. It occurs in approximately 6 cases per 100,000 deliveries and increases coagulopathy risk (adjusted odds ratio, 24.7). The overall failure-to-rescue rate after AFE is approximately 17%. 1 AFE exhibits a sudden onset in pregnant women and is frequently associated with a poor prognosis owing to cardiopulmonary collapse with severe coagulopathy. Notably, its etiology remains unknown. Severe clinical AFE may involve anaphylactoid reactions, including IgE-mediated or non-IgE-mediated reactions, in which C3a or C5a directly stimulates mast cells. 2,3 Degranulation of activated mast cells induces the secretion of histamine and other substances, increasing vascular permeability and causing cardiopulmonary collapse. Nevertheless, the cause of coagulopathy in severe AFE remains unclear. 4 An in vitro study revealed that AF can activate neutrophils and induce platelet-neutrophil aggregation, 5 Such platelet-neutrophil aggregation may cause vascular endothelial damage, leading to coagulation and thrombus formation. Moreover, activated platelets release the platelet factor 4 (PF4), which can induce histamine release from mast cells in vitr o. 6 Therefore, AF-activated platelets and neutrophils may be involved in AFE progression. Animal models that accurately mimic severe clinical AFE are required to investigate its precise pathogenesis and facilitate therapy development. Such models should exhibit cardiopulmonary collapse and coagulopathy, as both are critical for patients with AFE. Several studies have attempted to establish animal models of AFE. 7 For example, a model of pregnant goats exhibited hemodynamic changes, in which the maximum effects of AF were observed immediately after administration; 8 however, this study did not account for coagulopathy. Pregnant minipig and rabbit models exhibited coagulopathy after AF administration, 9,10 reproducing coagulopathy induced by administering AF derived from the same species, respectively. However, these models did not address hemodynamic disturbance. Therefore, reproducing both cardiopulmonary collapse and severe coagulopathy resulting from AF administration to pregnant animals remains challenging. We have previously described that administering small amounts of AF in pregnant rabbits induces moderate coagulopathy without hemodynamic disturbance. 11 However, in our preliminary experiment, a single bolus with 5 mL of whole AF, including the meconium, led to severe hypotension and fatality in half of the animals within a few minutes. In the present study, we established an AFE rabbit model via a bolus administration of a larger volume of whole AF, which successfully induced both hemodynamic changes and coagulopathy. Using this model, we aimed to elucidate the underlying anaphylactoid reactions and simultaneous activation of platelets and neutrophils with hemodynamic changes. 2. Materials and Methods This study was conducted in accordance with the guidelines of the Institutional Review Board for the Care of Animal Subjects at the National Defense Medical College. The Institutional Review Board approved this study (approval number: 24014-2). This study used female New Zealand white rabbits in late gestation (3.8 ± 0.2 kg, 28 th day of pregnancy/normal gestation period of 29–36 days; Japan SLC, Hamamatsu, Japan). All rabbits were anesthetized with ketamine and xylazine, followed by intravenous injections of pentobarbital (15 mg/kg) at 30-min intervals during the experiment. Local anesthetic (1% lidocaine) was subcutaneously injected into the bilateral inguinal and the middle of the lower abdomen. The adequacy of general anesthesia was monitored based on the loss of the ear pinch reflex. Subsequently, each rabbit was placed on a warming plate to maintain body temperature at 37 °C. Aseptic techniques were applied for all surgical procedures. Surgical catheters (polyethylene indwelling needle 18G; Terumo Co., Tokyo, Japan) were bilaterally inserted into the femoral arteries and left femoral vein of each rabbit. After a midline lower abdominal incision, a cesarean section was performed, and all fetuses were delivered enclosed in the fetal sacs from the uterus. Subsequently, the uterus was sutured, followed by closure of the abdomen. AF collected from the fetal sac was administered via the femoral vein as a bolus at 1.8–2.3 mL/kg. Rabbits in the sham group received 2.3 mL/kg of normal saline instead of an AF injection (Figure 1A). Surgical catheters were inserted into the bilateral femoral arteries for continuous measurement of the mean arterial pressure (MAP) and collection of blood samples. Electrocardiograms (ECG) were obtained during the experiment (BSM-3592; Nihon Kohden, Tokyo, Japan). Transthoracic echocardiography (Voluson SWIFT; General Electric Healthcare Japan, Tokyo, Japan) was performed to assess the vascular resistivity index (RI), which was used as an indicator of downstream systemic vascular resistance 12 and was calculated as follows (Figure 1B): 13 RI = (Systolic Velocity − Diastolic Velocity) / Systolic Velocity Following AF injection, animals were initially divided into the stable and unstable hemodynamics groups, where unstable hemodynamics was defined as a decrease in MAP to below 40 mmHg or a more than 25% decrease from baseline. Animals whose MAP decreased to 30 mmHg received noradrenaline injections (0.1–1.0 mg, Alfresa Pharma, Osaka, Japan) (Figure 1A). Rabbits with unstable hemodynamics were further divided into the dead and hypotensive survival groups. Overall, the AF-injected rabbits were divided into the stable, hypotensive, and dead groups. (Figure 1A) Arterial blood samples (6 mL) were collected from the dead group (survival time: 8.3 ± 0.6 min) before the experiment, 2 and 5 min after AF injection, and upon cardiopulmonary collapse just before cardiac arrest. Cardiac arrest was defined as sinus arrest or pulseless electrical activity on ECG. Blood samples from the other groups were collected before the experiment and at 2, 5, 15, 30, and 60 min after AF injection. (Figure 1B) Blood cell counts and hemoglobin concentrations were measured using an Erma PCE 170 hematology analyzer (Erma, Tokyo, Japan). Plasma creatinine levels were assessed using the Fuji Dry-Chem System (Fujifilm Medical, Saitama, Japan). 2.4. Analyses of Whole Blood Coagulation Activity and Platelet Function Coagulation activity was evaluated using a Sonoclot coagulation analyzer (model SCP2; Sienco, Morrison, CO, USA). First, a small amount of whole blood (400 µL) without anticoagulant was placed in a cuvette containing a glass-bead activator and a stir bar, in which a vertically vibrating probe was suspended. As the sample clotted, increasing impedance due to probe vibration was detected by the sensor and converted to an output signal. The time until onset of fibrin formation was recorded as the activated clotting time (ACT). 14 For platelet function testing, multiple electrode impedance platelet aggregometry was applied using a multiplate analyzer (Roche Diagnostics, Mannheim, Germany), followed by assessment of collagen-induced platelet aggregation. 15 Whole blood samples and reagents were pipetted into the test cells using an electric pipette. Platelet adhesion and aggregation were measured based on changes in the between-sensor electrical resistance over 6 min. The impedance change was plotted against time, and the area under the curve was calculated. To evaluate coagulation, the activity of plasma antithrombin III was measured at Sanritsu Zel-kova Laboratory (Tokyo, Japan). 2.5. Enzyme-Linked Immunosorbent Assay (ELISA) Plasma samples were collected at the indicated time points and stored at −80 °C until ELISA. Measurements were performed using commercially available ELISA kits following the manufacturer’s instructions (Table S1). 2.6. Histopathological Examination The lungs, heart, liver, spleen, kidney, and uterus were collected at the time of death or euthanasia after 60 min of observation (n=3). Subsequently, they were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 5-um serial sections for hematoxylin-eosin and Alcian blue staining. 2.7. Transmission Electron Microscopic Study Lung specimens were prefixed with fixative containing 4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 mol/L phosphate buffer (pH 7.4) for 3 h at 4 ℃, followed by postfixing in 1% osmium tetroxide dissolved in 0.1 mol/L phosphate buffer (pH 7.4) for 2 h at 4 ℃, dehydration, and embedding in epoxy resin. Ultrathin tissue sections were stained with uranyl acetate and lead citrate, followed by examination using an electron microscope (JEM 1030; JEOL, Tokyo, Japan) at an accelerating voltage of 80 kV. 2.8. Statistical Analysis Statistical analyses were performed using JMP software Pro 14.0.0 (SAS Institute Inc., Tokyo, Japan, https://www.jmp.com/ja_jp/software/data-analysis-software.html). Between-group comparisons were performed using Student’s t -test, whereas among-group comparisons were performed using one-way analysis of variance, followed by the Bonferroni post-hoc test. Data are presented as mean standard error. Statistical significance was set at p < 0.05. 3. Results Within 2 min after AF injection, 26 of the 34 animals exhibited unstable hemodynamics. Among them, 19 animals showed a marked decrease in the MAP (23.0 ± 1.9 mmHg) at 3 min after AF injection due to decreased vascular resistance despite administration of noradrenaline; all these animals died and were designated as the dead group. Consequently, the acute mortality rate of AF-injected animals was 55.9% (19 out of 34), with an average survival duration of 8.3 ± 0.6 min. The remaining seven animals (hypotensive group) exhibited a significant decrease in MAP (45.6 ± 6.2 mmHg) at 3 min after AF injection ( p = 0.028); however, their hemodynamics recovered either spontaneously or after noradrenaline administration, and they survived for 60 min (Figure 2A, B). All groups demonstrated a significant decrease in the minimum RI, with this decrease being especially prominent in the hypotensive and dead groups (Figure 2C). The heart rate increased at 2 min after AF injection in the dead group and 3 min after AF injection in the stable and hypotensive groups (Figure 2D). ECG monitoring revealed no evident ST changes in any group. Lung tissues demonstrated thrombi with AF-derived acid mucin, which were positive for Alcian blue staining and surrounding inflammatory cells (Figure 3A-1, 2). 16 Specifically, the dead group showed extensive thrombus formation in the lung capillaries and alveolar hemorrhage. Furthermore, thrombi with inflammatory cell infiltration were observed in the spleen (Figure 3B), uterus (Figure 3C), and liver (Figure 3D). Platelet aggregation adjacent to inflammatory cells was observed in the splenic blood vessels in the dead group, whereas this aggregation was not noted in the sham group (Figure 3E-1, 2). Transmission electron microscopy confirmed that activated neutrophils migrated close to damaged endothelial cells with platelet aggregation in the lungs of rabbits in the dead group (Figure 3F-1). Furthermore, activated platelets were observed adjacent to the basophils (Figure 3F-2). Significant thrombocytopenia was observed in the dead group at 2 min after AF injection. Moreover, the stable and hypotensive groups exhibited a significant decrease in platelet count at 15 min after AF injection (Figure 3G). Injection of AF or normal saline led to anemia. Furthermore, the dead group demonstrated a hematocrit decrease due to hemodilution caused by resuscitation infusions. Notably, injection of AF, but not saline, caused leukocytopenia (Table S2). All groups exhibited decreased platelet function of aggregation (Figure S1A). ACT was significantly prolonged in the stable and hypotensive groups (Figure 3H), whereas it could not be measured in the dead group owing to severe coagulopathy (Figure S2D). Notably, the dead group demonstrated a marked increase in plasma D-dimer levels at 5 min after AF injection, and the plasma fibrinogen levels and antithrombin III activity were significantly decreased 2 min after AF injection (Figure 3I-K). These findings indicate that the dead group met the criteria for obstetric disseminated intravascular coagulation (DIC). 17 As the dead group received more infusions for resuscitation than the other groups, resulting in greater hemodilution, plasma measurements were normalized to plasma creatinine levels (Figure 3L). The dead group exhibited a significant increase in plasma histamine levels at 5 min after AF injection, with a concomitant significant increase in IgE levels; however, this was not observed in the other groups (Figure 4A, B). None of the groups demonstrated significant changes in C3 levels (Figure S1B). 3.6. Activation of Platelets and Neutrophils, Vascular Endothelial Damage in the Acute Phase of AFE The dead group showed an increase in plasma PF4 levels at 2 min after AF injection ( p = 0.045 vs. baseline), which continued to increase at 5 min after AF injection ( p < 0.001 vs. baseline). Notably, these changes were not observed in the other groups (Figure 4C). Furthermore, the dead group exhibited an increase in plasma syndecan-1 levels at 2 min after AF injection ( p = 0.025 vs. baseline), whereas no significant changes were observed in the other groups (Figure 4D). It also demonstrated an increase in plasma neutrophil elastase complex levels at 10 min after AF injection ( p = 0.009 vs. baseline), whereas the other groups did not exhibit significant changes (Figure 4E). Plasma platelet-activating factor (PAF) levels did not exhibit changes in any of the groups (Figure S1C). 4. Discussion 4.1. Main Findings We established a novel AFE model that successfully replicated coagulopathy. In the dead group, a severe anaphylactoid reaction was triggered by platelet activation, which caused irreversible cardiopulmonary collapse. Furthermore, our findings demonstrated that activated platelets are crucially involved in the rapid progression of consumptive coagulopathy during the acute phase of AFE. We initially developed a protocol for AF administration, which caused both hemodynamic changes and severe coagulopathy. We previously demonstrated that an injection of 3 mL (0.8 mL/kg) of AF, divided into four doses for 9 min, caused moderate coagulopathy without hemodynamic changes. 11 This volume of AF injection was relatively lower than those reported in several previous studies (1.8−8.2mL/kg). 7-10 In the present study, we performed bolus administration of non-centrifuged AF, revealing that a threshold dose of 1.8–2.3 mL/kg should be used to induce severe coagulopathy and cardiopulmonary collapse. Our histopathological examination of AF-injected animals revealed positivity for Alcian blue staining, which was consistent with a diagnosis of AFE. 16 The capillaries of the lungs, liver, spleen, and uterus exhibited thrombus formations with inflammatory cell infiltration, notably that of neutrophils, suggesting the occurrence of DIC in multiple organs. Specifically, in the lung capillaries of the dead group, activated neutrophils migrated to the site of vascular endothelial damage along with activated platelets. We also used RI to simultaneously evaluate vascular resistance with continuous MAP monitoring, revealing an MAP decrease linked to a decrease in systemic vascular resistance. In humans, a cutoff RI value of 0.7–0.8 in renal arteries is used as a predictor of renal function by evaluating vascular resistance and blood flow. 13,18,19 In our model, the dead group exhibited a decrease in the RI (<0.7) of the ascending aorta at 2 min after AF injection, and the mean minimum value during the experiment was 0.6. This decrease in RI in the dead group was associated with an increase in plasma histamine levels. 20 This phenomenon may be explained by the fact that activated platelets and mast cells rapidly release stored histamine in dense granules. 21 In our study, all AF-injected animals exhibited thrombocytopenia, prolonged ACT, decreased platelet aggregation, and decreased plasma fibrinogen levels. Notably, ACT was not measurable in the dead group owing to severe coagulopathy, and even the other groups exhibited a 3–4-fold prolongation of ACT. Furthermore, only the dead group exhibited a significant increase in plasma D-dimer levels and a significant decrease in AT- III activity at 2 and 5 min after AF injection, respectively, enabling the detection of changes in coagulopathy during the acute phase within 15 min after AF injection. In the dead group, plasma PF4 and syndecan-1 levels significantly increased at 2 min after AF injection, followed by a significant increase in plasma neutrophil elastase complex levels at 10 min. These changes were correlated with an increase and decrease in plasma D-dimer and fibrinogen levels, respectively. Moreover, plasma histamine and IgE levels were significantly increased at 5 min after AF injection (Figure 4A, B). Taken together, our findings indicate that AF injection causes rapid platelet activation and vascular endothelial damage, followed by anaphylactoid reactions and neutrophil activation (Figure S3). AFE is difficult to predict and exhibits rapid progression. Therefore, it is necessary to promptly assess changes in the vital signs of patients. Ultrasonography provides the advantages of minimal invasiveness and real-time evaluation of hemodynamics. Notably, our results suggest that measuring RI may allow early detection of hypotension in patients with severe AFE. Moreover, AFE is characterized by rapid progression of coagulopathy with cardiopulmonary collapse; therefore, early detection of coagulopathy and intervention are crucial. 22 In our model, plasma fibrinogen levels and AT- III activity decreased within a few minutes of onset, highlighting the importance of promptly considering their replacement in patients exhibiting symptoms of AFE. In the dead group , plasma histamine and IgE levels increased during the acute phase of AFE, whereas changes in plasma C3 levels were unclear. Therefore, it is necessary to elucidate the role of anaphylatoxins, such as C3a and C5a, which are part of the non-IgE-mediated pathway, 7 in AFE. Furthermore, plasma histamine levels were not increased in the stable and hypotensive groups, suggesting that assessing pathways that inhibit histamine release may help improve survival outcomes in AFE. Our model, involving the injection of AF derived from the species into pregnant animals, successfully reproduced severe AFE. This model can detect acute changes, which lead to hemodynamic changes and coagulopathy, within a few minutes. However, severe atonic bleeding could not be reproduced, as none of our rabbits had uterine atony. 5. Conclusions We established a novel animal model of severe AFE. Using this model, we demonstrated that anaphylactoid reactions and severe coagulopathy, as well as the activation of platelets and neutrophils, are essential factors in the acute phase of severe AFE. The authors thank Ayako Suzuki, Mieko Katsuura, Mio Konno, and Yayoi Ichiki for their excellent technical assistance. Disclosure of Interests None Contribution to Authorship Y.O.: Writing – original draft, Conceptualization, Methodology, Investigation, Data curation, Formal analysis. K.H.: Writing – original draft, Writing – review and editing, Conceptualization, Methodology, Investigation, Supervision, Funding acquisition, Formal analysis, Project administration. M.K.: Writing – review and editing, Conceptualization, Supervision, Funding acquisition, Resource, Methodology. K.K.: Writing – review and editing, Funding acquisition, Investigation, Data curation. R.S.: Investigation, Data curation. K.S.: Writing – review and editing, Supervision, Resource, Data curation. K.T.: Writing – review and editing, Conceptualization, Supervision, Methodology. S.N.: Investigation, Data curation. M.M.: Investigation, Data curation. H.S.: Investigation, Data curation. M.T.: Writing – review and editing, Conceptualization, Methodology, Supervision, Formal analysis, Project administration. Details of Ethics Approval This study was conducted in accordance with the guidelines of the Institutional Review Board for the Care of Animal Subjects at the National Defense Medical College. The Institutional Review Board of National Defense Medical College approved this study (approval number: 24014-2). Funding This work was supported in part by JSPS KAKENHI Grant Number 22H03179 (to K.H., M.K., K.K., K.T., and M.M.). Data Availability The data used in this study are available upon request from the corresponding author. References 1 Mazza GR, Youssefzadeh AC, Klar M, et al. Association of Pregnancy Characteristics and Maternal Mortality with Amniotic Fluid Embolism. JAMA Netw Open 2022;5(11):e2242842. 2 Clark SL. Amniotic Fluid Embolism. Obstetrics & Gynecology 2014;123(2 PART 1):337-348.3 Hankins GD, Snyder R, Dinh T, Van Hook J, Clark S, Vandelan A. Documentation of amniotic fluid embolism via lung histopathology. Fact or fiction? J Reprod Med 2002;47(12):1021-4.4 Petroianu GA, Altmannsberger SH, Maleck WH, et al. Meconium and amniotic fluid embolism: effects on coagulation in pregnant mini-pigs. Crit Care Med 1999;27:348–355.5 Rannou B, Rivard GE, Gains MJ, Bédard C. Intravenous injection of autologous amniotic fluid induces transient thrombocytopenia in a gravid rabbit model of amniotic fluid embolism. Vet Clin Pathol 2011;40(4):524-529.6 R-L Yang, M-Z Lang, X-M Qiao, et al. Immune storm and coagulation storm in the pathogenesis of amniotic fluid embolism. Eur Rev Med Pharmacol Sci 2021;25(4):1796-1803.7 Busardò FP, Frati P, Zaami S, Fineschi V. Amniotic fluid embolism pathophysiology suggests the new diagnostic armamentarium: β-tryptase and complement fractions C3-C4 are the indispensable working tools. Int J Mol Sci 2015;16: 6557-6570.8 Benson MD. Current concepts of immunology and diagnosis in amniotic fluid embolism. Clin Dev Immunol 2012;2012:946576.9 Chen K-B, Chang S-S, Tseng Y-L, et al. Amniotic fluid induces platelet-neutrophil aggregation and neutrophil activation. Am J Obstet Gynecol 2013;208:318.e1-7.10 Suzuki R, Kimura T, Kitaichi K, et al. Platelet factor 4 fragment induces histamine release from rat peritoneal mast cells. Peptides 2002;23(10):1713-7.11 Kaneko K, Hagisawa K, Kinoshita M, et al. Early treatment with Fibrinogen γ-chain peptide-coated, ADP-encapsulated Liposomes (H12-(ADP)-liposomes) ameliorates post-partum hemorrhage with coagulopathy caused by amniotic fluid embolism in rabbits. AJOG Global Reports 2023;3(4):100280.12 Marzok M, Almubarak A.I., Al Mohamad Z, et al. Reference Values and Repeatability of Pulsed Wave Doppler Echocardiography Parameters in Normal Donkeys. Animals 2022;12(17):2296.13 Radermacher J, Mengel M, Ellis S et al. The renal arterial resistance index and renal allograft survival. N Engl J Med 2003;349(2):115-24.14 Hett DA, Walker D, Pilkington SN, Smith DC. Sonoclot analysis. Br J Anaesth 1995;75(6):771-6.15 Pluta J, Nicińska B, Trzebicki J. Multiple electrode aggregometry as a method for platelet function assessment according to the European guidelines. Anaesthesiol Intensive Ther 2018;50(3):230-233.16 Tamura N, Farhana M, Oda T, Itoh H, Kanayama N. Amniotic fluid embolism: Pathophysiology from the perspective of pathology. J Obstet Gynaecol Res 2017;43(4):627-632.17 Rabinovich A, Abdul-Kadir R, Thachil J, et al. DIC in obstetrics: diagnostic score, highlights in management, and international registry-communication from the DIC and Women’s Health SSCs of the International Society on Thrombosis and Haemostasis. J Thromb Haemost 2019;17:1562–6.18 van de Kuit A, Benjamens S, Sotomayor CG, et al. Postoperative Ultrasound in Kidney Transplant Recipients: Association Between Intrarenal Resistance Index and Cardiovascular Events. Transplant Direct 2020;6(8):e581.19 Naesens M, Heylen L, Lerut E, et al. Intrarenal Resistive Index after Renal Transplantation. N Engl J Med 2013;369:1797-806.20 Kaliner M, Shelhamer JH, Ottesen EA. Effects of infused histamine: correlation of plasma histamine levels and symptoms. J Allergy Clin Immunol 1982;69(3):283-9. 21 Michelson AD. Platelets Fourth edition 2019;Academic Press:355, 511-513.22 Oda T, Tamura N, Ide R, et al. Consumptive coagulopathy involving amniotic fluid embolism: the importance of earlier assessments for interventions in critical care. Crit Care Med 2020;48:e1251–9. Figure Caption List Figure 1. Experimental design and classification of groups by hemodynamic changes. A All fetuses were delivered without rupturing the fetal sacs. AF was aspirated from the fetal sac. Based on hemodynamic changes, animals were divided into three groups: stable, hypotensive, and dead groups. Unstable hemodynamics was defined as a decrease in MAP to below 40 mmHg or a more than 25% decrease from baseline. Noradrenaline injections were administered to animals whose MAP decreased to 30 mmHg. Cardiac arrest was defined as sinus arrest or pulseless electrical activity on ECG. The sham group received the same volume of normal saline (2.3 ml/kg) instead of AF. B Surgical catheters were inserted into bilateral femoral arteries for continuous MAP measurement and collection of blood samples. ECG was monitored during the experiment. Simultaneously, transthoracic echocardiographic examination was performed to measure the RI of the ascending aorta. Arterial blood samples (6 mL) from the dead group were collected before the experiment, 2 and 5 min after AF injection, at the time of cardiopulmonary collapse, and immediately before cardiac arrest. Blood samples from the other groups were collected before the experiment and at 2, 5, 15, 30, and 60 min after AF injection. AF, amniotic fluid; Ao, ascending aorta; BP, blood pressure; CS, cesarean section; ECG, electrocardiogram; MAP, mean arterial pressure; RI, resistivity index (V syst - V diast ) / V syst. Figure 2. Changes in hemodynamics. A The dead group exhibited a significant decrease in MAP at 2 min after AF injection. The unstable group demonstrated a significant decrease in MAP at 3 min after AF injection, which recovered within a few minutes. B The dead group showed a significant decrease in RI at 2 min after AF injection, with a simultaneous decrease in MAP. C All groups showed a significant decrease in the minimum RI from baseline values during the observation period. The dead and unstable groups exhibited a significantly lower minimum RI than the sham and stable groups. D The heart rate in the dead group significantly increased 2 min after AF injection. Data are presented as mean ± SE. AF, amniotic fluid; MAP, mean arterial pressure; RI, resistivity index; SE, standard error. Figure 3. Platelet aggregations in the capillaries and changes in whole blood coagulation parameters and coagulation factors. A-1, 2 Pulmonary capillaries had AF components that were positive on AB staining (arrows) and comprised thrombi with surrounding inflammatory cells. B–D Thrombus formation with inflammatory cell infiltration was also observed (arrowheads) in the spleen (B), uterus (C), and liver (D). E-1,2 Platelet aggregations adjacent to inflammatory cells were observed in the blood vessels of the spleen in the dead group (A-2) but not in the sham group (A-1). F-1 Transmission electron microscopy confirms that activated neutrophils (arrow) migrate closer to damaged endothelial cells with platelet aggregation (arrowheads) in the lungs of the dead group. F-2 Activated platelets (arrowheads) adjacent to basophils (dotted circles). G AF injection caused thrombocytopenia, especially in the dead group. H ACT at 15 min after AF injection was three times as prolonged as that at baseline and gradually recovered. ACT at 2 and 5 min after AF injection in the dead group were outside the measurement range owing to severe coagulopathy. All rabbits in the dead group had cardiac arrest within 15 min after AF injection. I,K The dead group exhibited a significant decrease in plasma antithrombin Ⅲ activity at 2 min after AF injection, followed by an increase in plasma D-dimer levels. J All groups demonstrated a significant decrease in plasma fibrinogen levels compared to baseline values, especially the dead group. L Plasma creatinine levels decreased after AF injection in the dead group due to hemodilution by resuscitative injection. Data are presented as mean ± SE. AB, Alcian blue; ACT, activated clotting time; AF, amniotic fluid; Cr, creatinine; HE, hematoxylin-eosin; SE, standard error. Figure 4. Changes in plasma levels of pathogenic factors in the acute phase of severe AFE. A, B The dead group exhibited a significant increase in plasma histamine levels at 5 min after AF injection, with a concomitant significant increase in IgE levels. C, D The dead group demonstrated an increase in plasma PF4 and syndecan-1 levels at 2 min after AF injection, which continued to increase at 5 min after AF injection. E Plasma neutrophil elastase complex levels increased 10 min after AF injection in the dead group. Plasma measurements were performed by calculating the ratios of the plasma Cr levels. Data are presented as mean ± SE. AF, amniotic fluid; Cr, creatinine; PF4, platelet factor 4; SE, standard error. Table S1. ELISA kit and measurement timepoints AF, amniotic fluid; PF4, platelet factor 4. Table S2. Changes in blood cell counts * p < 0.05, ** p < 0.01 vs. before the experiment Data are presented as mean ± SE. AF, amniotic fluid; Hb, hemoglobin; Hct, hematocrit; SE, standard error; WBC, white blood cell; WL, large white blood cell. Figure S1. Changes in platelet aggregation and pathogenetic factors. A All AF-injected animals exhibited a decrease in platelet function of aggregation (collagen-induced). B, C In all groups, there were no significant changes in the plasma C3 or PAF levels. Data are presented as mean ± SE. C3, complement 3; COL, collagen; Cr, creatinine; PAF, platelet-activating factor; SE, standard error. Figure S2. Activated clotting time changes in severe AFE. ACT was not prolonged in the sham group (A). In the stable and hypotensive groups, ACT was three to four times as prolonged as that at baseline (B,C). ACT in the dead group was prolonged and outside the measurement range (D). Representative samples from each group are shown. ACT, activated clotting time; AF, amniotic fluid; NS, normal saline Figure S3. Potential concept of severe AFE pathophysiology. AF and fetal components invade maternal circulation, resulting in early platelet activation, degranulation, and endothelial damage. Subsequently, neutrophil elastase levels are elevated, suggesting accelerated thrombus formation at the site of endothelial damage. Simultaneously, an IgE-mediated anaphylactoid reaction to AF occurs, and histamine is released from mast cells. Activated platelets also contribute to histamine release. During the acute phase of AFE, activated platelets are crucially involved in the rapid progression of anaphylactoid reactions and coagulopathy. AF, amniotic fluid; AFE, amniotic fluid embolism; NETs, neutrophil extracellular traps; PF4, platelet factor 4. Supplementary Material File (afe model figure 12.04.2025.pptx) Download 58.49 MB Information & Authors Information Version history V1 Version 1 04 December 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords basic science haematology: coagulation immunology obstetric haemorrhage translational research Authors Affiliations Yuka Otsuka 0009-0007-1094-2316 National Defense Medical College View all articles by this author Kohsuke Hagisawa [email protected] National Defense Medical College View all articles by this author Manabu Kinoshita National Defense Medical College View all articles by this author Kouki Kaneko Saitama Medical Center View all articles by this author Ruka Sasa National Defense Medical College Research Institute View all articles by this author Kimiya Sato National Defense Medical College View all articles by this author Katsuo Terui Saitama Medical Center View all articles by this author Soko Nishimura National Defense Medical College View all articles by this author Morikazu Miyamoto National Defense Medical College View all articles by this author Hidenori Sasa National Defense Medical College View all articles by this author Masashi Takano National Defense Medical College View all articles by this author Metrics & Citations Metrics Article Usage 215 views 142 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Yuka Otsuka, Kohsuke Hagisawa, Manabu Kinoshita, et al. Platelet Activation Triggers Rapid Progression of Cardiopulmonary Collapse and Consumptive Coagulopathy: A Severe Amniotic Fluid Embolism Rabbit Model Study. Authorea . 04 December 2025. DOI: https://doi.org/10.22541/au.176486790.02715791/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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