Jeffrey N. Harr

Early platelet dysfunction: an unrecognized role in the acute coagulopathy of trauma.

BACKGROUND Our aim was to determine the prevalence of platelet dysfunction using an end point of assembly into a stable thrombus after severe injury. Although the current debate on acute traumatic coagulopathy has focused on the consumption or inhibition of coagulation factors, the question of early platelet dysfunction in this setting remains unclear. STUDY DESIGN Prospective platelet function in assembly and stability of the thrombus was determined within 30 minutes of injury using whole blood samples from trauma patients at the point of care using thrombelastography-based platelet functional analysis. RESULTS There were 51 patients in the study. There were significant differences in the platelet response between trauma patients and healthy volunteers, such that there was impaired aggregation to these agonists. In trauma patients, the median ADP inhibition of platelet function was 86.1% (interquartile range [IQR] 38.6% to 97.7%) compared with 4.2 % (IQR 0 to 18.2%) in healthy volunteers. After trauma, the impairment of platelet function in response to arachidonic acid was 44.9% (IQR 26.6% to 59.3%) compared with 0.5% (IQR 0 to 3.02%) in volunteers (Wilcoxon nonparametric test, p < 0.0001 for both tests). CONCLUSIONS In this study, we show that platelet dysfunction is manifest after major trauma and before substantial fluid or blood administration. These data suggest a potential role for early platelet transfusion in severely injured patients at risk for postinjury coagulopathy.

Fibrinolysis greater than 3% is the critical value for initiation of antifibrinolytic therapy

BACKGROUND The acute coagulopathy of trauma is present in up to one third of patients by the time of admission, and the recent CRASH-2 and MATTERs trials have focused worldwide attention on hyperfibrinolysis as a component of acute coagulopathy of trauma. Thromboelastography (TEG) is a powerful tool for analyzing fibrinolyis, but a clinically relevant threshold for defining hyperfibrinolysis has yet to be determined. Recent data suggest that the accepted normal upper bound of 7.5% for 30-minute fibrinolysis (LY30) by TEG is inappropriate in severe trauma, as the risk of death rises at much lower levels of clot lysis. We wished to determine the validity of this hypothesis and establish a threshold value to treat fibrinolysis, based on prediction of massive transfusion requirement and risk of mortality. METHODS Patients with uncontrolled hemorrhage, meeting the massive transfusion protocol (MTP) criteria at admission (n = 73), represent the most severely injured trauma population at our center (median Injury Severity Score [ISS], 30; interquartile range, 20–38). Citrated kaolin TEG was performed at admission blood samples from this population, stratified by LY30, and evaluated for transfusion requirement and 28-day mortality. The same analysis was conducted on available field blood samples from all non-MTP trauma patients (n = 216) in the same period. These represent the general trauma population. RESULTS Within the MTP-activating population, the cohort of patients with LY30 of 3% or greater was shown to be at much higher risk for requiring a massive transfusion (90.9% vs. 30.5%, p = 0.0008) and dying of hemorrhage (45.5% vs. 4.8%, p = 0.0014) than those with LY30 less than 3%. Similar trends were seen in the general trauma population. CONCLUSION LY30 of 3% or greater defines clinically relevant hyperfibrinolysis and strongly predicts the requirement for massive transfusion and an increased risk of mortality in trauma patients presenting with uncontrolled hemorrhage. This threshold value for LY30 represents a critical indication for the treatment of fibrinolysis. LEVEL OF EVIDENCE Prognostic study, level III.

Functional Fibrinogen Assay Indicates That Fibrinogen is Critical in Correcting Abnormal Clot Strength Following Trauma

ABSTRACT Thromboelastography (TEG) is emerging as the standard in the management of acute coagulopathies in injured patients. Although TEG is sensitive in detecting abnormalities in clot strength, one shortcoming is differentiating between fibrinogen and platelet contributions to clot integrity. Current American algorithms suggest platelet transfusion, whereas European guidelines suggest fibrinogen concentrates for correcting low clot strength. Therefore, we hypothesized that a TEG-based functional fibrinogen (FF) assay would assess the contribution of fibrinogen and platelets to clot strength and provide insight to transfusion priorities. Blood samples were obtained from trauma patients on arrival to the emergency department or who were admitted to the surgical intensive care unit (n = 68). Citrated kaolin TEG, FF, and von Clauss fibrinogen levels (plasma-based clinical standard) were measured. Correlations were assessed using linear regression models. In vitro studies were also performed with adding fibrinogen concentrates to blood collected from healthy volunteers (n = 10). Functional fibrinogen and citrated kaolin TEG parameters were measured. Functional fibrinogen strongly correlated with von Clauss fibrinogen levels (R2 = 0.87) and clot strength (R2 = 0.80). The mean fibrinogen contribution to clot strength was 30%; however, there was a direct linear relationship with fibrinogen level and percent fibrinogen contribution to clot strength (R2 = 0.83). Traditional TEG parameters associated with fibrinogen activity (&agr; angle and kinetic time) had significantly lower correlations with FF (R2 = 0.70 and 0.35). Furthermore, platelet count had only a moderate correlation to clot strength (R2 = 0.51). The addition of fibrinogen concentrate in in vitro studies increased clot strength (MA) (60.44 ± 1.48 to 68.12 ± 1.39) and percent fibrinogen contribution to clot strength (23.8% ± 1.8% to 37.7% ± 2.5%). Functional fibrinogen can be performed rapidly with TEG and correlates well with the standard von Clauss fibrinogen assay. Both fibrinogen and platelet contribution of clot strength can be derived from FF. Moreover, FF had a stronger correlation to clot strength, and increased levels were directly associated with increased percent contribution to clot strength. In vitro studies also demonstrated an increase in FF, clot strength, and percent fibrinogen contribution to clot strength with the addition of fibrinogen concentrate. These data suggest that fibrinogen should be addressed early in trauma patients manifesting acute coagulopathy of trauma.

Platelets are dominant contributors to hypercoagulability after injury.

BACKGROUND Venous thromboembolic (VTE) disease has a high incidence following trauma, but debate remains regarding optimal prophylaxis. Thrombelastography (TEG) has been suggested to be optimal in guiding prophylaxis. Thus, we designed a phase II randomized controlled trial to test the hypothesis that TEG-guided prophylaxis with escalating low–molecular weight heparin (LMWH), followed by antiplatelet therapy would reduce VTE incidence. METHODS Surgical intensive care unit trauma patients (n = 50) were randomized to receive 5,000 IU of LMWH daily (control) or to TEG-guided prophylaxis, up to 5,000 IU twice daily with the addition of aspirin, and were followed up for 5 days. In vitro studies were also conducted in which apheresis platelets were added to blood from healthy volunteers (n = 10). RESULTS Control (n = 25) and TEG-guided prophylaxis (n = 25) groups were similar in age, body mass index, Injury Severity Score, and male sex. Fibrinogen levels and platelet counts did not differ, and increased LMWH did not affect clot strength between the control and study groups. The correlation of clot strength (G value) with fibrinogen was stronger on Days 1 and 2 but was superseded by platelet count on Days 3, 4, and 5. There was also a trend in increased platelet contribution to clot strength in patients receiving increased LMWH. In vitro studies demonstrated apheresis platelets significantly increased clot strength (7.19 ± 0.35 to 10.34 ± 0.29), as well as thrombus generation (713.86 ± 12.19 to 814.42 ± 7.97) and fibrin production (274.03 ± 15.82 to 427.95 ± 16.58). CONCLUSION Increased LMWH seemed to increase platelet contribution to clot strength early in the study but failed to affect the overall rise clot strength. Over time, platelet count had the strongest correlation with clot strength, and in vitro studies demonstrated that increased platelet counts increase fibrin production and thrombus generation. In sum, these data suggest an important role for antiplatelet therapy in VTE prophylaxis following trauma, particularly after 48 hours. LEVEL OF EVIDENCE Therapeutic study, level III.

Antiplatelet therapy is associated with decreased transfusion-associated risk of lung dysfunction, multiple organ failure, and mortality in trauma patients.

Objective:To determine whether prehospital antiplatelet therapy was associated with reduced incidence of acute lung dysfunction, multiple organ failure, and mortality in blunt trauma patients. Design:Secondary analysis of a cohort enrolled in the National Institute of General Medical Sciences Trauma Glue Grant database. Setting:Multicenter study including nine U.S. level-1 trauma centers. Patients:A total of 839 severely injured blunt trauma patients at risk for multiple organ failure (age > 45 yr, base deficit > 6 mEq/L or systolic blood pressure < 90 mm Hg, who received a blood transfusion). Severe/isolated head injuries were excluded. Measurements and Main Results:Primary outcomes were lung dysfunction (defined as grades 2–3 by the Denver multiple organ failure score), multiple organ failure (Denver multiple organ failure score >3), and mortality. Patients were documented as on antiplatelet therapy if taking acetylsalicylic acid, clopidogrel, and/or ticlopidine. Fifteen percent were taking antiplatelet therapy prior to injury. Median injury severity score was 30 (interquartile range 22–51), mean age 61 + 0.4 yr and median RBCs volume transfused was 1700 mL (interquartile range 800–3150 mL). Overall, 63% developed lung dysfunction, 19% had multiple organ failure, and 21% died. After adjustment for age, gender, comorbidities, blood products, crystalloid/12 hrs, presence of any head injury, injury severity score, and 12 hrs base deficit > 8 mEq/L, 12 hrs RBC transfusion was associated with a significantly smaller risk of lung dysfunction and multiple organ failure among the group receiving antiplatelet therapy compared with those not receiving it (lung dysfunction p = 0.0116, multiple organ failure p = 0.0291). In addition, antiplatelet therapy had a smaller risk (albeit not significant, p = 0.06) of death for patients receiving RBC compared to those not on antiplatelet therapy after adjustment for confounders, Conclusions:Pre-injury antiplatelet therapy is associated with a decreased risk of lung dysfunction, multiple organ failure, and possibly mortality in high-risk blunt trauma patients who received blood transfusions. These findings suggest platelets have a role in organ dysfunction development and have potential therapeutic implications.

A standardized technique for performing thromboelastography in rodents.

Thromboelastography (TEG), used in liver transplant and cardiac surgery for nearly 50 years, has recently been applied to the trauma setting. Rodents are used widely for shock research, but are known to have differences in their coagulation system compared with humans. Consequently, the appropriate technique for performing TEG requires modification of the standard clinical protocol. Thromboelastography was performed with blood collected from the femoral artery of rodents, and technical modifications were tested to optimize results. Analysis of citrated whole blood using TEG revealed a more rapid onset of coagulation in rats compared with humans. The reference ranges of TEG parameters for Sprague-Dawley rats are detailed. Citrated native whole blood is the optimal TEG method in the assessment of coagulation in rodents. Investigators using TEG for research purposes should establish their own reference ranges to determine normal values for their target population.

Hemodilution is not critical in the pathogenesis of the acute coagulopathy of trauma.

BACKGROUND The acute coagulopathy of trauma is multifactorial, but generally believed to be aggravated by coexisting acidosis, hypothermia, and hemodilution. While acidosis and hypothermia have been extensively evaluated, there is a paucity of data on the independent role of hemodilution in this scenario. We therefore hypothesized that hemodilution will impair coagulation following experimental trauma and hemorrhagic shock. METHODS Adult male Spraque-Dawley rats underwent trauma and hemorrhagic shock, followed by resuscitation with 2 × SBV using normal saline (NS). Thrombelastography (TEG) was performed before and after shock. RESULTS In this trauma model, resuscitation resulted in a hemodilution of 50% (43% ± 4.05% versus 19.8% ± 3.96% Hct pre-shock versus post-shock , P < 0.0001). Despite the substantial hemodilution, there was no significant change in clot strength (12.96 ± 2.84 versus 11.79 ± 1.28 dynes/cm(2) G pre-shock versus post-shock, P = 0.13). Similarly, the onset of coagulation (R time) was not impaired (1.68 ± 1.74 s versus 1.75 ± 0.63 s R time pre-shock versus post-shock, P = 0.45). CONCLUSION In the absence of hypothermia and acidosis, hemodilution (≤ 50%) has a trivial effect on coagulation following trauma and hemorrhagic shock. These data call to question the commonly held belief that hemodilution per se is critical in the development of post-injury coagulopathy.

Nebulized hypertonic saline attenuates acute lung injury following trauma and hemorrhagic shock via inhibition of matrix metalloproteinase-13.

Objective:We hypothesized that aerosolized inhaled hypertonic saline given at the onset of resuscitation will decrease acute lung injury following hemorrhagic shock, by inhibiting the release of epithelial derived proinflammatory mediators. Design:Animal study. Setting:Animal-care facility procedure room in a medical center. Subjects:Adult male Sprague-Dawley rats. Interventions:Rats underwent hemorrhagic shock followed by 2 hrs of resuscitation and 1 hr of observation. In the study group, nebulized hypertonic saline was delivered at the end of the shock period and after 1 hr and 2 hrs of resuscitation. Measurements and Main Results:Shock provoked acute lung injury, which was attenuated with inhaled hypertonic saline (1.56 ± 0.2 mg protein/mL vs. 0.95 ± 0.3 mg protein/mL bronchoalveolar lavage fluid, shock vs. shock + hypertonic saline, p < .01). Nebulized hypertonic saline reduced inflammation (cytokine-induced neutrophil chemoattractant-1 accumulation in bronchoalveolar lavage fluid 5999 ± 1267 pg/mL vs. 3342 ± 859 pg/mL, shock vs. shock + hypertonic saline, p = .006). Additionally, nebulized hypertonic saline inhibited matrix metalloproteinase-13 accumulation in the bronchoalveolar lavage fluid (1513 ± 337 pg/mL bronchoalveolar lavage fluid vs. 230 ± 19 pg/mL, shock vs. shock + hypertonic saline, p = .009) and pretreatment with a matrix metalloproteinase-13 inhibitor was sufficient to attenuate postshock acute lung injury (1.42 ± 0.09 mg/mL vs. 0.77 ± 0.23 mg/mL bronchoalveolar lavage protein, shock vs. shock + matrix metalloproteinase-13 inhibitor CL-82198, p = .002). Conclusion:Inhaled hypertonic saline attenuates postshock acute lung injury by exerting an anti-inflammatory effect on the pulmonary epithelium, suggesting a new clinical strategy to treat acute lung injury/acute respiratory distress syndrome.

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.

Isoflurane prevents acute lung injury through ADP-mediated platelet inhibition.

BACKGROUND Growing evidence suggests platelets are essential in posttraumatic, acute lung injury (ALI). Halogenated ethers interfere with the formation of platelet-granulocyte aggregates. The potential benefit of halogenated ethers has not been investigated in models of trauma/hemorrhagic shock (T/HS). Therefore, we hypothesized that isoflurane decreases T/HS-mediated ALI through platelet inhibition. METHODS Sprague-Dawley rats (n = 47) were anesthetized by either pentobarbital or inhaled isoflurane and placed into (1) control, (2) trauma (laparotomy) sham shock, (3) T/HS (mean arterial pressure, 30 mmHg × 45 min), (4) pretreatment with an ADP receptor antagonist, or (5) T/HS with isoflurane initiated during resuscitation groups. ALI was determined by protein and pulmonary immunofluorescence bronchoalveolar lavage (BAL) fluid. Platelet Mapping specifically evaluated thrombin-independent inhibition of the ADP and AA pathways of platelet activation. RESULTS Pretreatment with isoflurane abrogated ALI as measured by both BAL fluid protein and pulmonary immunofluorescence (P < .001). Platelet Mapping revealed specific inhibition of the platelet ADP-pathway with isoflurane (P < .001). Pretreatment with an ADP receptor antagonist decreased ALI to sham levels, confirming that specific platelet ADP inhibition decreases ALI. Isoflurane initiated during resuscitation also decreased ALI (P < .001). CONCLUSION Isoflurane attenuates ALI through an antiplatelet mechanism, in part, through inhibition of the platelet ADP pathway. Isoflurane given postinjury also protects against ALI, and highlights the potential applications of this therapy in various clinical scenarios of ischemia/reperfusion.