Publications by authors named "Jeffrey D Keesee"

6 Publications

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Intravenous administration of mesenchymal stromal cells leads to a dose dependent coagulopathy and is unable to attenuate acute traumatic coagulopathy in rats.

J Trauma Acute Care Surg 2021 Nov 17. Epub 2021 Nov 17.

Blood and Shock Resuscitation, United States Army Institute of Surgical Research, Fort Sam Houston, TX 78234.

Background: Mesenchymal stromal cells (MSC) express surface tissue factor (TF), which may affect hemostasis and detract from therapeutic outcomes of MSCs if administered intravenously (IV). In this study, we determine a safe dose of MSCs for IV administration, and further demonstrate the impact of IV-MSC on acute traumatic coagulopathy (ATC) in rats.

Methods: TF expression of rat bone marrow derived MSC (BMSC) or adipose derived MSC (AMSC) was detected by immunohistochemistry and ELISA. The coagulation properties were measured in MSC-treated rat whole blood, and blood samples collected from rats after IV administration of MSCs. ATC-rats underwent polytrauma and 40% hemorrhage, followed by IV administration of 5 or 10million/kg BMSCs (BMSC-5, BMSC-10), or vehicle at 1 hr after trauma.

Results: Rat MSCs expressed TF, and incubation of rat BMSCs or AMSCs with whole blood in vitro led to a significantly shortened clotting time (CT). However, a dose-dependent prolongation of prothrombin time (PT) with reduction in platelet counts and fibrinogen was found in healthy rat treated with IV-MSCs. BMSCs at 5 million/kg or less led to minimal effect on hemostasis. MSCs were not found in circulation, but in the lungs after IV administration regardless of the dosage. ATC with prolonged PT was not significantly affected by 5 or 10million/kg BMSCs. BMSC-10 led to significantly lower fibrinogen and platelet counts; while significantly higher levels of lactate, wet/dry weight ratio and leukocyte infiltration in the lung were present compared to BMSC-5 or vehicle. No differences were seen in immune or inflammatory profiles with BMSC treatment in ATC-rats, at least in the acute timeframe.

Conclusions: Intravenous administration of MSCs leads to a risk of coagulopathy associated with a dose-dependent reduction in platelet counts and fibrinogen, and is incapable of restoring hemostasis of rats with ATC after polytrauma and hemorrhagic shock.

Level Of Evidence: Animal and laboratory studies; Study type: therapeutic.
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November 2021

Extracellular vesicles derived from cardiosphere-derived cells as a potential antishock therapeutic.

J Trauma Acute Care Surg 2021 08;91(2S Suppl 2):S81-S88

From the Coagulation and Blood Research (Blood) (T.C.C., X.W., J.D.K., J.G.-M., C.L.S., B.L., A.P.C., J.A.B.), United States Army Institute of Surgical Research, San Antonio, Texas; Capricor Therapeutics Institute (J.J.M., K.A.P., L.R.-B., N.A.A., L.S.M.), Beverly Hills, California; Department of Biological Chemistry (S.J.G.), Johns Hopkins, Baltimore, Maryland; and Department of Biomedical Engineering (C.R.R.), The University of Texas at San Antonio, San Antonio, Texas.

Background: Extracellular vesicles (EVs) isolated from cardiosphere-derived cells (CDC-EVs) are coming to light as a unique cell-free therapeutic. Because of their novelty, however, there still exist prominent gaps in knowledge regarding their therapeutic potential. Herein the therapeutic potential of CDC-EVs in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock is outlined.

Methods: Extracellular vesicle surface expression of procoagulant molecules (tissue factor and phosphatidylserine) was evaluated by flow cytometry. Extracellular vesicle thrombogenicity was tested using calibrated thrombogram, and clotting parameters were assessed using a flow-based adhesion model simulating blood flow over a collagen-expressing surface. The therapeutic efficacy of EVs was then determined in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock.

Results: Extracellular vesicles isolated from cardiosphere-derived cells are not functionally procoagulant and do not interfere with platelet function. In a rat model of multiple injuries and hemorrhagic shock, early administration of EVs significantly reduced the elevation of lactate and creatinine and did not significantly enhance coagulopathy in rats with acute traumatic coagulopathy.

Conclusion: The results of this study are of great relevance to the development of EV products for use in combat casualty care, as our studies show that CDC-EVs have the potential to be an antishock therapeutic if administered early. These results demonstrate that research using CDC-EVs in trauma care needs to be considered and expanded beyond their reported cardioprotective benefits.
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August 2021

Hemostatic capacity of canine chilled whole blood over time.

J Vet Emerg Crit Care (San Antonio) 2021 Mar 11;31(2):239-246. Epub 2021 Mar 11.

US Army Institute of Surgical Research, Joint Base San Antonio, Fort Sam Houston, Texas, USA.

Objective: To determine the hemostatic potential of canine chilled whole blood maintained at clinically relevant storage conditions.

Design: In vitro experimental study.

Setting: Government blood and coagulation research laboratory and government referral veterinary hospital.

Animals: Ten healthy Department of Defense military working dogs.

Interventions: One unit of fresh whole blood was collected from each of 10 military working dogs using aseptic technique. Blood was maintained in a medical-grade refrigerator for 28 days at 4°C (39°F) and analyzed before refrigeration (day 0) and after (days 2, 4, 7, 9, 11, 14, 21, and 28).

Measurements And Main Results: Ten units of canine blood were analyzed with whole blood platelet aggregation, thromboelastography, CBC, biochemical analysis, blood gas, and prothrombin/activated partial thromboplastin/fibrinogen assay. Clotting strength of chilled blood was maintained up to 21 days despite significant decreases in platelet aggregation to ADP, collagen, or γ-thrombin, significant prolongation of prothrombin and activated partial thromboplastin times, and reduced speed of clot formation (K time, alpha angle). Fibrinogen concentration, WBC, RBC, and platelet counts did not change over time.

Conclusions: Chilled canine whole blood loses a small percentage of clot strength through 21 days of refrigerated storage. Further research is needed to determine if this hemostatic potential is clinically relevant in hemorrhaging dogs who require surgical intervention or are exposed to traumatic events.
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March 2021

Effect of tranexamic acid administration on acute traumatic coagulopathy in rats with polytrauma and hemorrhage.

PLoS One 2019 3;14(10):e0223406. Epub 2019 Oct 3.

Coagulation and Blood Research Program, United States Army Institute of Surgical Research, Fort Sam Houston, Texas, United States of America.

Trauma and hemorrhagic shock can lead to acute traumatic coagulopathy (ATC) that is not fully reversed by prehospital resuscitation as simulated with a limited volume of fresh whole blood (FWB) in a rat model. Tranexamic Acid (TXA) is used as an anti-fibrinolytic agent to reduce surgical bleeding if administered prior to or during surgery, and to improve survival in trauma if given early after trauma. It is not clear from the existing clinical literature whether TXA has the same mechanism of action in both settings. This study sought to explore the molecular mechanisms of TXA activity in trauma and determine whether administration of TXA as a supplement to FWB resuscitation could attenuate the established ATC in a rat model simulating prehospital resuscitation of polytrauma and hemorrhagic shock. In a parallel in-vitro study, the effects on clotting assays of adding plasmin at varying doses along with either simultaneous addition of TXA or pre-incubation with TXA were measured, and the results suggested that maximum anti-fibrinolytic effect of TXA on plasmin-induced fibrinolysis required pre-incubation of TXA and plasmin prior to clot initiation. In the rat model, ATC was induced by polytrauma followed by 40% hemorrhage. One hour after trauma, the rats were resuscitated with FWB collected from donor rats. Vehicle or TXA (10mg/kg) was given as bolus either before trauma (TXA-BT), or 45min after trauma prior to resuscitation (TXA-AT). The TXA-BT group was included to contrast the coagulation effects of TXA when used as it is in elective surgery vs. what is actually feasible in real trauma patients (TXA-AT group). A single dose of TXA prior to trauma significantly delayed the onset of ATC from 30min to 120min after trauma as measured by a rise in prothrombin time (PT). The plasma d-dimer as well as plasminogen/fibrinogen ratio in traumatized liver of TXA-BT were significantly lower as compared to vehicle and TXA-AT. Wet/dry weight ratio and leukocytes infiltration of lungs were significantly decreased only if TXA was administrated later, prior to resuscitation (TXA-AT). In conclusion: Limited prehospital trauma resuscitation that includes FWB and TXA may not correct established systemic ATC, but rather may improve overall outcomes of resuscitation by attenuation of acute lung injury. By contrast, TXA given prior to trauma reduced levels of fibrinolysis at the site of tissue injury and circulatory d-dimer, and delayed development of coagulopathy independent of reduction of fibrinogen levels following trauma. These findings highlight the importance of early administration of TXA in trauma, and suggest that further optimization of dosing protocols in trauma to exploit TXA's various sites and modes of action may further improve patient outcomes.
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March 2020

Severe Trauma and Hemorrhage Leads to Platelet Dysfunction and Changes in Cyclic Nucleotides in The Rat.

Shock 2020 04;53(4):468-475

US Army Institute of Surgical Research, Fort Sam Houston, Texas.

Introduction: Rats subjected to polytrauma and hemorrhage develop a coagulopathy that is similar to acute coagulopathy of trauma in humans, and is associated with a rise in prothrombin time and a fall in clot strength. Because platelet aggregation accounts for a major proportion of clot strength, we set out to characterize the effects of polytrauma on platelet function.

Methods: Sprague-Dawley rats were anesthetized with isoflurane. Polytrauma included laparotomy and damage to 10 cm of the small intestines, right and medial liver lobes, right leg skeletal muscle, femur fracture, and hemorrhage (40% of blood volume). No resuscitation was given. Blood samples were taken before and after trauma for the measurement of impedance electrode aggregometry, and intracellular levels of cyclic adenosine and guanosine monophosphate (cAMP, cGMP), inositol trisphosphate (IP3), and adenosine and guanosine triphosphates (ATP, GTP).

Results: Polytrauma significantly increased the response of collagen (24%) and thrombin (12%) to stimulate platelet aggregation. However, aggregation to adenosine diphosphate (ADP) or arachidonic acid (AA) was significantly decreased at 2 (52% and 46%, respectively) and 4 h (45% and 39%). Polytrauma and hemorrhage also led to a significant early rise in cAMP (101 ± 11 to 202 ± 29 pg/mL per 1,000 platelets), mirrored by a decrease in cGMP (7.8 ± 0.9 to 0.6 ± 0.5). In addition, there was a late fall in ATP (8.1 ± 0.7 to 2.2 ± 0.6 ng/mL per 1,000 platelets) and GTP (1.5 ± 0.2 to 0.3 ± 0.1). IP3 rose initially, and then fell back to baseline.

Conclusions: Polytrauma and hemorrhage led to a deficit in the platelet aggregation response to ADP and AA after trauma, likely due to the early rise in cAMP, and a later fall in energy substrates, and may explain the decrease in clot strength and impaired hemostasis observed after severe trauma.
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April 2020

Platelets derived from fresh and cold-stored whole blood participate in clot formation in rats with acute traumatic coagulopathy.

Br J Haematol 2017 12 22;179(5):802-810. Epub 2017 Nov 22.

Coagulation and Blood Research Program, United States Army Institute of Surgical Research, Fort Sam Houston, TX, USA.

The in vitro haemostatic functions of fresh whole blood (FWB) are well preserved after cold storage. This study aimed to determine whether platelets derived from FWB and stored whole blood (SWB) contribute to clot formation in tissue injury after transfusion into coagulopathic rats with polytrauma/haemorrhage (T/H). The rats were resuscitated 1 h after trauma with FWB or SWB collected from green fluorescence protein (GFP) transgenic rats. After transfusion, a liver incision was made and the tissue was collected 10 min after injury to identify GFP platelets by immunohistochemistry. In comparison to FWB, platelet aggregation to adenosine diphosphate and protease-activated receptor-4 was reduced by 35% and 20%, and clotting time was shortened by 25% in SWB. After transfusion, SWB led to a significant increase in platelet activation as measured by an elevation of CD62P and phosphatidylserine expression. The platelets from SWB were in a higher activation state, and showed higher clearance rate and formation of platelet-leucocyte aggregates than those from FWB after transfusion. Platelets from both FWB and SWB were equivalently incorporated into the clot at the incisional site, as determined by co-localization of CD61 and GFP. This study suggests that SWB contributes to haemostatic function and is an effective alternative resource to treat trauma patients.
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December 2017