Severe Trauma Leads to a Fall in Intracellular Triphosphates and a Rise in cAMP in Platelets

Background: Severe trauma and hemorrhage leads to an acute coagulopathy as exemplified by a rise in prothrombin time, and a fall in clotting firmness and platelet aggregation. Because platelets contribute 70-80% of the clot strength, evaluating the intracellular mechanisms that regulate aggregation may lead to strategies to mitigate the development of coagulopathy. Platelet aggregation requires energy, and energy stores may be depleted after hemorrhagic shock and tissue trauma. Also, aggregation is initiated by elevations in inositol trisphosphate and calcium, and inhibited by elevations in cyclic adenosine and guanosine monophosphates. Platelet dysfunction after trauma may involve one or more of these intracellular intermediates.

Objective: To measure intracellular levels of mono- and tri-phosphates in platelets from rats subjected to polytrauma and hemorrhagic shock.

Methods: Polytrauma was induced in isoflurane anesthetized Sprague-Dawley rats (n=10 per group) by damage to the small intestines, right and medial lobes of the liver, the right leg skeletal muscle, and by fracturing the right femur. 40% hemorrhage was then performed immediately after trauma. Blood samples were taken before, immediately after trauma, and at 2 and 4hrs. Platelet rich plasma PRP was generated by centrifugation of whole blood at 200g for 10min, no brakes. Cyclic AMP and cGMP were extracted from 100μl of PRP after adding to 1ml of EtOH, 10mM ammonium formate, with 10μg/ml cGMP-Br as an internal control. Triphosphates were extracted from another 100μl PRP by addition of 50% EtOH, 500mM Formic Acid with 10μg/ml ATP-C10 as an internal control. Samples were centrifuged at 20K g for 10min, and supernatant dried. All Samples were brought up in 200ul of 0.1% formic acid for analysis by LC-MS/MS.

Results: In platelets, the intracellular levels of Adenosine Triphosphate (ATP), Guanosine Triphosphate (GTP) and Inositol Trisphosphate (IP3) all fell significantly by 4hrs after polytrauma and hemorrhage (ATP: 5.8±0.9 vs. 0.9±0.7μg/ml, GTP: 1.23±0.22 vs 0.01±0.01 μg/ml and IP3: 625±310 vs 125±36 ng/ml). Cyclic AMP was significantly elevated over baseline (10.4±0.7 vs 15.1±1.63ng/ml), however cGMP was down (1.17±0.39 vs 0.10±0.12 ng/ml).

Conclusion: The platelet dysfunction that accompanies polytrauma and hemorrhagic shock may be a result of falling energy stores (ATP, GTP), a loss of IP3 mediation of Ca dependent aggregation, and a rise in the inhibitory mediator cAMP. These results suggest that the addition of metabolic substrates to standard resuscitation fluids may help restore platelet function after trauma and hemorrhage.