Coagulation Status in Dogs Naturally Infected with Angiostrongylus vasorum

Angiostrongylus vasorum infection has been associated with coagulopathies including hyperfibrinolysis. We compared coagulation status including thromboelastometry (ROTEM) parameters in dogs naturally infected with A. vasorum versus healthy dogs to determine clinicopathological parameters associated with bleeding, hypocoagulopathy, and hyperfibrinolysis. Clinical signs, white blood cell count, platelet count, hematocrit, plasmatic coagulation tests (PT, aPTT, fibrinogen concentration), D-dimer, and ROTEM S parameters (Ex-tem, In-tem, Fib-tem, Ap-tem) were analysed and compared between bleeding, nonbleeding, and control dogs and between hypo- and normocoagulable animals. Clinical signs of bleeding were present in 6/9 (67%) hypocoagulable and 1/9 (11%) normocoagulable dogs. PT, fibrinogen concentration, and several ROTEM parameters were significantly different between hypocoagulable and normocoagulabe A. vasorum infected dogs. Hyperfibrinolysis was identified in 44% of infected dogs and was significantly more common in bleeding and hypocoagulable dogs. Hyperfibrinolysis was significantly associated with low MCFFib-tem but not with low fibrinogen concentration or increased D-dimers. CFTEx-tem > 248 swas 100% sensitive and 89% specific to predict hyperfibrinolysis. Hyperfibrinolysis, hypocoagulability and bleeding are common in A. vasorum infected dogs. Only Ex-tem and Fib-tem parameters and potentially PT were associated with bleeding or hypocoagulability. Ex-tem analysis enables detection of bleeding, hypocoagulability and hyperfibrinolysis within minutes.

Mild Therapeutic Hypothermia Alters Hemostasis in ST Elevation Myocardial Infarction Patients

Background and Rationale: Mild therapeutic hypothermia (MTH) is a concept to reduce infarct size and improve outcome after ST-segment elevation myocardial infarction (STEMI). In the STATIM trial, we investigated MTH as an additional therapy for STEMI patients. In the intention-to-treat set, 101 patients were included. No difference in primary and secondary endpoints measured by cardiac magnetic resonance imaging was found. Platelet activation and plasmatic coagulation are key in the pathophysiology of STEMI. In the present study, we investigated the effect of MTH on primary and secondary hemostasis in STEMI patients.Methods and Results: Platelet function and morphology were assessed by routine blood count, aggregometry testing, and flow cytometry. Soluble platelet markers were determined by enzyme-linked immunosorbent assay (ELISA) testing. Plasmatic coagulation was measured throughout the study. Platelet count remained unchanged, irrespective of treatment, whereas platelet size decreased in both patient groups. Platelet aggregometry indicated increased platelet reactivity in the MTH group. Furthermore, higher adenosine diphosphate (ADP) plasma levels were found in MTH patients. Expression of glycoprotein IIb/IIIa was increased on platelets of STEMI patients treated with MTH. Lower patient temperatures correlated with longer clotting times and resulted in reduced pH. Lower pH values were positively correlated with longer clotting times.Conclusion: Present data indicate longer clotting times and higher platelet reactivity in STEMI patients treated with MTH. These changes did not correspond to clinical bleeding events or larger infarct size.

Plasmatic and cell-based enhancement by microparticles originated from platelets and endothelial cells under simulated in vitro conditions of a dilutional coagulopathy

Background Aggressive fluid management and other external factors may lead to hypothermia, acidosis and hemodilution (defined as Lethal Triad, LT) contributing to a trauma-induced coagulopathy (TIC) that worsens patients’ outcomes. Procoagulant microparticles (MP) are crucial players at the interface of cellular and plasmatic coagulation. However, their functions remain largely unexplored. This study aimed to characterize effects of MP subtypes and concentrations on functional coagulation under in vitro simulated conditions. Methods Blood from eleven volunteers were collected to simulate in vitro conditions of hemodilution (HD) and LT, respectively. HD was induced by replacing a blood volume of 33% by crystalloids and for LT, samples were further processed by reducing the temperature to 32 °C and lowering the pH to 6.8. MP were obtained either from platelet concentrates (platelet-derived MP, PDMP) or from cell culture (ECV304 cells for endothelial-derived MP, EDMP) by targeted stimulation. After introducing MP to in vitro conditions, we measured their concentration-dependent effects (1.000, 10.000 and 15.000 MP/μl blood) on coagulation compared to whole blood (WB). For each condition, coagulation was characterized by flow cytometric platelet activation and by quantification of fibrin clot propagation using Thrombodynamics® technology. Results MP originated from platelets and endothelial cells affected blood coagulation in a concentration-dependent manner. Particularly, high PDMP quantities (10.000 and 15.000 PDMP/μl blood) significantly induced platelet activation and fibrin clot growth and size in HD conditions. In LT conditions as well, only high PDMP concentration induced platelet activation, clot growth and size. In contrast, EDMP did not induce platelet activation, but resulted in enhanced formation of spontaneous clots, irrespective of simulated condition. With increasing EDMP concentration, the time until the onset of spontaneous clotting decreased in both HD and LT conditions. Discussion The study demonstrates an essential role of MP within the coagulation process under simulated coagulopathic conditions. PDMP affected platelets promoting clot formation likely by providing a surface enlargement. EDMP presumably affected clotting factors of the plasmatic coagulation resulting in an increased formation of spontaneous clots. Conclusion Under simulated conditions of a dilutional coagulopathy, MP from different cellular origin indicate a divergent but both procoagulant mechanism within the coagulation process.

Plasmatic and cell-based enhancement by microparticles originated from platelets and endothelial cells under simulated in vitro conditions of a dilutional coagulopathy

Background: Aggressive fluid management and other external factors may lead to hypothermia, acidosis and hemodilution (defined as Lethal Triad, LT) contributing to a trauma-induced coagulopathy (TIC) that worsens patients’ outcomes. Procoagulant microparticles (MP) are crucial players at the interface of cellular and plasmatic coagulation. However, their functions remain largely unexplored. This study aimed to characterize effects of MP subtypes and concentrations on functional coagulation under in vitro simulated conditions. Methods: Blood from eleven volunteers were collected to simulate in vitro conditions of hemodilution (HD) and LT, respectively. HD was induced by replacing a blood volume of 33% by crystalloids and for LT, samples were further processed by reducing the temperature to 32 °C and lowering the pH to 6.8. MP were obtained either from platelet concentrates (platelet-derived MP, PDMP) or from cell culture (ECV304 cells for endothelial-derived MP, EDMP) by targeted stimulation. After introducing MP to in vitro conditions, we measured their concentration-dependent effects (1.000, 10.000 and 15.000 MP/µl blood) on coagulation compared to whole blood (WB). For each condition, coagulation was characterized by flow cytometric platelet activation and by quantification of fibrin clot propagation using Thrombodynamics® technology.Results: MP originated from platelets and endothelial cells affected blood coagulation in a concentration-dependent manner. Particularly, high PDMP quantities (10.000 and 15.000 PDMP/µl blood) significantly induced platelet activation and fibrin clot growth and size in HD conditions. In LT conditions as well, only high PDMP concentration induced platelet activation, clot growth and size. In contrast, EDMP did not induce platelet activation, but resulted in enhanced formation of spontaneous clots, irrespective of simulated condition. With increasing EDMP concentration, the time until the onset of spontaneous clotting decreased in both HD and LT conditions.Discussion: The study demonstrates an essential role of MP within the coagulation process under simulated coagulopathic conditions. PDMP affected platelets promoting clot formation likely by providing a surface enlargement. EDMP presumably affected clotting factors of the plasmatic coagulation resulting in an increased formation of spontaneous clots.Conclusion: Under simulated conditions of a dilutional coagulopathy, MP from different cellular origin indicate a divergent but both procoagulant mechanism within the coagulation process.

Natural IgM antibodies inhibit microvesicle-driven coagulation and thrombosis

Thrombosis and the complications associated with it are a major cause of morbidity and mortality worldwide. Microvesicles (MVs), a class of extracellular vesicles, are increasingly recognized as mediators of coagulation and biomarkers of thrombotic risk. Thus, identifying factors targeting MV-driven coagulation may help in the development of novel antithrombotic treatments. We have previously identified a subset of circulating MVs that is characterized by the presence of oxidation-specific epitopes and bound by natural IgM antibodies targeting these structures. Here, we investigated whether natural IgM antibodies, which are known to have important anti-inflammatory house-keeping functions, inhibit the procoagulatory properties of MVs. We found that the extent of plasma coagulation is inversely associated with the levels of both free and MV-bound endogenous IgM. Moreover, the oxidation epitope-specific natural IgM antibody LR04, which recognizes malondialdehyde adducts, reduced MV-dependent plasmatic coagulation and whole blood clotting without affecting thrombocyte aggregation. Intravenous injection of LR04 protected mice from MV-induced pulmonary thrombosis. Of note, LR04 competed the binding of coagulation factor X/Xa to MVs, providing a mechanistic explanation for its anticoagulatory effect. Thus, our data identify natural IgM antibodies as hitherto unknown modulators of MV-induced coagulation in vitro and in vivo and their prognostic and therapeutic potential in the management of thrombosis.

Local blood coagulation drives cancer cell arrest and brain metastasis in a mouse model

Clinically relevant brain metastases (BM) frequently form in cancer patients, with limited options for effective treatment. Circulating cancer cells must first permanently arrest in brain microvessels to colonize the brain, but the critical factors are not well understood. Here, in vivo multiphoton laser-scanning microscopy (MPLSM) of the entire brain metastatic cascade allowed unprecedented insights into how blood clot formation and von Willebrand factor (VWF) deposition determine the arrest of circulating cancer cells and subsequent brain colonization in mice. Clot formation in brain microvessels occurred frequently (>95%) and specifically at intravascularly arrested cancer cells, allowing their long-time arrest. An extensive clot embedded approximately 20% of brain-arrested cancer cells, and those were more likely to successfully extravasate and form a macrometastasis. Mechanistically, tissue factor-mediated thrombin generation by cancer cells accounted for local activation of plasmatic coagulation in the brain. Thrombin inhibition by treatment with low-molecular weight heparin or dabigatran and an anti-VWF antibody prevented clot formation, cancer cell arrest, extravasation, and brain macrometastasis formation. In contrast, tumor cells were not able to directly activate platelets, and antiplatelet treatments did reduce platelet dispositions at intravascular cancer cells but did not reduce overall BM formation. In conclusion, our data shows that plasmatic coagulation is activated early by intravascular tumor cells in the brain, with subsequent clot formation, discovering a novel and specific mechanism that is crucial for brain colonization. Direct or indirect thrombin and VWF inhibitors emerge as promising drug candidates for BM prevention trials.

Plasmatic and cell-based enhancement of in vitro induced coagulopathy by microparticles originated from platelets and endothelial cells

Background: Aggressive trauma management and other external factors lead to hypothermia, acidosis and hemodilution (defined as Lethal Triad, LT) contributing to coagulopathy after trauma (Trauma-induced coagulopathy, TIC) that worsens patients’ outcomes. Procoagulative microparticles (MP) are crucial players at the interface of cellular and plasmatic coagulation. However, their functions remain largely unexplored. This study aimed to characterize effects of MP subtypes and concentrations on functional coagulation under in vitro simulated conditions. Methods: Blood from eleven volunteers were collected to simulate in vitro conditions of haemodilution (HD) and LT, respectively. HD was induced by replacing a blood volume of 33% by crystalloids and for LT, samples were further processed by reducing the temperature to 32 °C and lowering the pH to 6.8. MP were obtained either from platelet concentrates (platelet-derived MP, PDMP) or from cell culture (ECV304 cells for endothelial-derived MP, EDMP) by targeted stimulation. After introducing MP to in vitro conditions, their concentration-dependent effects (1.000, 10.000 and 15.000 MP/µl blood) on coagulation compared to whole blood (WB) were characterized by flow cytometric platelet activation and by quantification of fibrin clot propagation and spontaneous clotting using Thrombodynamics® technology.Results: MP originated from platelets and endothelial cells affected blood coagulation in a concentration-dependent manner. Particularly, high PDMP quantities significantly induced platelet activation and fibrin clot growth and size in HD conditions. In LT conditions, the highest PDMP concentration enhanced platelet activation, clot growth and size. In contrast, EDMP supplementation did not affect platelet activation, but resulted in enhanced formation of spontaneous clots, irrespective of simulated condition. With increasing EDMP concentration, the time until the onset of spontaneous clotting decreased in both HD and LT conditions.Discussion: The study demonstrates an essential role of MP within the coagulation process under simulated coagulopathic conditions. While PDMP affected platelets promoting clot formation likely by providing a surface enlargement, EDMP presumably affected clotting factors of the plasmatic coagulation resulting in an increased formation of spontaneous fibrin clots.Conclusion: The diverse effects of in vitro generated MP from different cellular origin indicate a divergent mechanism of action exhibiting distinct functions within the coagulation process.