title: Activated platelets in heparinized shed blood:  the "second hit" of acute lung injury in trauma/hemorrhagic shock models.
abstract: The return of heparinized shed blood (SB) in trauma/hemorrhagic shock (T/HS) models remains controversial because of potential anti-inflammatory properties. Although ubiquitous as an anticoagulant, heparin is ineffective on cellular coagulation as an antithrombotic agent. Therefore, we hypothesized that returning heparinized SB would paradoxically enhance acute lung injury (ALI) after T/HS because of the infusion of activated platelets. Sprague-Dawley rats, anesthetized with pentobarbital, underwent laparotomy and hemorrhage-induced shock (MAP of 30 mmHg × 45 min). Animals were resuscitated with a combination of normal saline and returned SB. Shed blood was collected in either 80 U/kg of heparin, 800 U/kg of heparin, or citrate or diluted 1:8 with normal saline. An additional group of animals were pretreated with a platelet P2Y12 receptor antagonist (clopidogrel) before T/HS. Bronchoalveolar lavage, lung myeloperoxidase assays, pulmonary immunofluorescence, and blood smears were conducted. Bronchoalveolar lavage protein increased in animals resuscitated with heparinized SB (T/HS + 80 U/kg Hep 1.62 ± 0.29, T/HS + 800 U/kg Hep 1.30 ± 0.15 vs. T/SS 0.51 ± 0.16 and T/HS Citrate 0.7 ± 0.09) (P < 0.0001). Blood smears and platelet function assays revealed platelet aggregates and increased platelet activation. Animals pretreated with a platelet P2Y12 receptor antagonist were protected from postinjury ALI (P < 0.0001). Animals with return of SB had increased pulmonary polymorphonuclear leukocyte sequestration (P < 0.0001). Pulmonary immunofluorescence demonstrated microthrombi only in the T/HS group receiving heparinized SB (P < 0.0001). The return of heparinized SB functions as a "second hit" to enhance ALI, with activated platelets propagating microthrombi and pulmonary polymorphonuclear leukocyte recruitment.
authors.affiliation: Department of Surgery, University of Colorado-Denver, Denver, Colorado, USA.
authors.forename: Jeffrey N
authors.initials: JN
authors.lastname: Harr
authors.forename: Ernest E
authors.initials: EE
authors.lastname: Moore
authors.forename: Max V
authors.initials: MV
authors.lastname: Wohlauer
authors.forename: Miguel
authors.initials: M
authors.lastname: Fragoso
authors.forename: Fabia
authors.initials: F
authors.lastname: Gamboni
authors.forename: Xiayuan
authors.initials: X
authors.lastname: Liang
authors.forename: Anirban
authors.initials: A
authors.lastname: Banerjee
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Silliman
mesh_terms: D055371:Acute Lung Injury / Q000473:pathology* / Q000628:therapy*; D000818:Animals; D001792:Blood Platelets / Q000276:immunology* / Q000502:physiology; D001804:Blood Transfusion, Autologous / Q000009:adverse effects*; D008297:Male; D015539:Platelet Activation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012771:Shock, Hemorrhagic / Q000473:pathology / Q000628:therapy*; D014947:Wounds and Injuries / Q000473:pathology / Q000628:therapy*