John E. Kaplan

Deficits in reticuloendothelial humoral control mechanisms in patients after trauma.

Plasma opsonic activity as expressed by an alpha-2-globulin which stimulates hepatic Kupffer cell phagocytosis, and thus modulates RES clearance, was determined in patients at varying intervals following whole-body trauma. Plasma opsonic activity decreased markedly following trauma in both nonsurviving (NS) and surviving (S) trauma patients as compared to an age- and sex-matched group of healthy volunteers. The initial post-traumatic hypoopsonemia (0-72 hr) was more severe (p less than 0.01) in nonsurviving patients than surviving patients. Survivors following trauma manifested restoration of opsonin levels with a definite transient rebound hyperopsonemia during the recovery phase (11-30 days); nonsurviving patients exhibited persistent systemic alpha-2-globulin opsonic deficiency. On the basis of previous animal and human studies, the presently observed humoral deficits following trauma in patients could contribute to impairment of reticuloendothelial Kupffer cell clearance of blood-borne particulate matter such as fibrin, damaged platelets, and other altered autologous tissue. The importance of post-trauma RES dysfunction to survival following severe injury warrants further investigation and clinical consideration.

Reversal of fibronectin and opsonic deficiency in patients. A controlled study.

Plasma fibronectin is an opsonic glycoprotein which augments reticuloendothelial phagocytic clearance of nonbacterial participates. We evaluated the influence of intravenous infusion of plasma cryoprecipitate on circulating immunoreactive fibronectin and associated opsonic activity at 0.5, 2.0, 4.0, 10, and 21 hr postinfusion in septic (n = 8) and nonseptic (n = 6) surgical and/or trauma patients with documented plasma fibronectin deficiency. The study was a randomized, double-blind, crossover clinical protocol in which fibronectin-poor (0.116 ± 0.025 mg/ ml) cryoprecipitate extracted plasma (placebo) was compared to fibronectin-rich (2.139 ± 0.161 mg/ml) plasma cryoprecipitate. Septic injured patients (149.37 ± 17.11 μg/ml) had lower (p < 0.05) plasma fibronectin levels than nonseptic injured patients (212.17 ± 7.14 μg/ml) and both were less (p < 0.05) than normal (330 ± 30 μg/ml). As tested in vitro with a peritoneal macrophage monolayer assay, cryoprecipitate manifested opsonic activity related to its fibronectin concentration. Intravenous infusion of fibronectin rich cryoprecipitate reversed both the immunoreactive fibronectin and opsonic deficiency, while infusion of the placebo at a comparable total protein load did not reverse either deficient parameter. Reversal of fibronectin deficiency was more sustained in nonseptic injured patients as compared to septic injured patients. Thus, reversal of opsonic deficiency in septic and nonseptic injured patients is observed after infusion of plasma cryoprecipitate and not with infusion of fibronectin deficient plasma at comparable protein loads. Also, cryoprecipitate extracted plasma may serve as an appropriate control solution for randomized studies evaluating the therapeutic value of fibronectin-rich plasma cryoprecipitate.

Disturbances in circulating opsonic activity in man after operative and blunt trauma.

A humoral factor deficit (α-2-opsonic glycoprotein) important for reticuloendothelial (RE) phagocytosis has been previously identified in animals as a major etiologic mechanism for RE host defense failure following trauma or operation. Assessment of this opsonic factor in man during the 72-hr period following extensive multiple trauma (n = 20) and major elective surgery (n = 13) revealed a transient depression of this opsonic protein at 24-hr postinjury in both surgical patients and surviving multiple trauma patients. Recovery of normal opsonic activity occurred by 48–72 hr postinjury in both groups. Nonsurviving multiple trauma patients had very low levels of circulating opsonic activity with no tendency toward recovery. The influence of an overnight fast in both preoperative patients (n = 8) and normal volunteers (n = 9) did not adversely affect the level of this humoral factor. Recovery of normal opsonic activity occurred during the 72-hr postoperative period, a time of diminished calorie intake, suggesting that a brief nutritional deficit is not solely responsible for postoperative RE depression. Altered RE function in patients following multiple trauma and major elective surgery may have important consequences in terms of the intravascular clearance of blood-borne particulate material such as platelet aggregates, fibrin, microthrombi, and other potentially noxious substances.

Plasma fibronectin synthesis in normal and injured humans as determined by stable isotope incorporation.

In humans, plasma fibronectin decreases early after operative injury, burn, or trauma, followed by a rapid restoration with a secondary decline typically observed if such patients become septic. We determined the rate of plasma fibronectin and plasma fibrinogen synthesis in normal subjects and injured patients using a stable isotope incorporation technique with [15N]glycine. During a constant 14-h infusion of [15N]glycine, the enrichment of [15N]glycine in both the free plasma glycine precursor pool as well as the urinary hippurate pool was determined; the latter used as an estimate of intracellular hepatic precursor enrichment. [15N]Glycine enrichment in both plasma fibronectin and fibrinogen was also quantified. The synthesis rate (Js/V) expressed in micrograms per milliliter of plasma per hour and the fractional synthesis rate (FSR) expressed as percentage of the plasma pool produced per day were determined. In normal subjects, the FSR for plasma fibronectin using 15N enrichment into urinary hippurate was 35.35 +/- 1.46%/d, whereas the Js/V was 4.45 +/- 0.19 micrograms/ml plasma per h. In normal subjects, the FSR for plasma fibronectin using 15N enrichment into free plasma glycine was 14.73 +/- 0.63%/d, whereas the Js/V was 1.98 +/- 0.09 micrograms/ml plasma per h. Early (2-3 d) after burn injury, fibronectin synthesis was increased (Js/V = 5.74 +/- 0.36; P less than 0.05), whereas later after injury, fibronectin synthesis began to decline (Js/V = 3.52 +/- 0.24; P less than 0.05) based on 15N enrichment of urinary hippurate. In contrast, the Js/V and FSR of plasma fibrinogen, a well-documented acute-phase plasma protein, revealed a sustained elevation (P less than 0.05) after injury in both the trauma and burn patients. Thus, plasma fibronectin synthesis is elevated early postinjury, which may contribute to the rapid restoration of its blood level. However, once fibronectin levels have normalized, the synthesis of plasma fibronectin appears to decline.

Thrombin induced platelet adhesion to endothelium is modified by endothelial derived relaxing factor (EDRF)

We investigated the hypothesis that thrombin-induced attachment of platelets to endothelial cells is modulated by EDRF. Thrombin significantly increased binding of radiolabelled platelets to cultured endothelium and to an intact pulmonary vasculature under flow conditions. These increases in binding were potentiated with hemoglobin (HB) and inhibited by superoxide dismutase (SOD) in both systems. We suggest that thrombin, in addition to enhancing platelet adhesion, elicits EDRF release from endothelial cells and that EDRF serves an antithrombotic function in the down regulation of platelet adhesion.

Effect of 13-hydroxyoctadeca-9,11-dienoic acid (13-hode) on thrombin induced platelet adherence to endothelial cells in vitro

The effect of exogenous 13-HODE on alpha-thrombin induced adherence of platelets to monolayers of cultured pulmonary artery endothelial cells was determined using homologous sheep cells. In a separate series of experiments, endogenous 13-HODE was demonstrated in sheep endothelial cells by reverse phase high pressure liquid chromatography. Levels of endogenous 13-HODE were decreased by alpha-thrombin preincubation. Exogenous 13-HODE (10 microM) reduced the augmented platelet adherence produced by coincubation of alpha-thrombin with platelets and endothelial monolayers, and eliminated the enhancement of platelet adherence produced by preincubation of alpha-thrombin with endothelial monolayers. 13-HODE also reduced the alpha-thrombin induced adherence of platelets to monolayers pretreated with aspirin and to fixed monolayers indicating a direct effect of 13-HODE as opposed to secondary effects mediated by the release of prostacyclin (PGI2) or endothelial derived relaxing factor (EDRF). Platelet adherence to subendothelial matrix was also reduced by 13-HODE. 13-HODE inhibited platelet aggregation initiated by 0.2 U/ml alpha-thrombin but did not affect aggregation initiated by 2.0 U/ml alpha-thrombin. These data provide evidence for the ability of exogenous 13-HODE to attenuate the interaction of thrombin activated platelets with endothelial cells as well as with other platelets.

Correlation of plasma lysosomal enzyme levels with hepatic reticuloendothelial function after trauma.

Summary Plasma lysosomal enzyme levels and hepatic phagocytosis were determined following Noble-Collip drum trauma in the rat. Circulating cathepsin and acid phosphatase activity increased after suble-thal trauma (300 rev), reaching maximal levels at 1-3 hr and returning to pretrauma levels at 24 hr after trauma. Hepatic phagocytosis was decreased maximally at 1 hr and recovered to control levels at 24 hr after sublethal trauma. Increasing trauma intensity (100-500 rev) resulted in a progressive failure in hepatic Kupffer cell phagocytosis and a progressive increase in plasma lysosomal enzyme levels when tested at 60-min post-trauma. A significant inverse correlation was found between the plasma lysosomal enzyme levels and Kupffer cell phagocytosis after trauma. The functional significance of the relationship between these two parameters and its importance in shock survival remain to be determined.

Fibrin degradation products increase lung transvascular fluid filtration after thrombin-induced pulmonary microembolism

The effect of fibrin degradation products (FDP) on pulmonary transvascular fluid and protein exchange was examined in the sheep lung lymph fistula preparation. The pulmonary lymph was used to assess changes in pulmonary lymph flow (Qlym) (a measure of net transvascular fluid filtration rate) and the lymph/plasma protein concentration ratio (L/P) (a measure of protein seiving across the microvascular barrier). Studies were made in 3 groups: Control-Thrombin (n = 7) received 96.9 +/- 9.4 U/kg of alpha-thrombin, Control-FDP (n = 6) received infusion of FDP prepared by plasmin digestion of fibrin, and Thrombin-FDP (n = 5) received thrombin (102.0 +/- 7.5 U/kg) and then an FDP infusion was begun at 60 min after the thrombin. In the Control-Thrombin animals, Qlym increased without a change in the L/P after thrombin, indicating an increase in vascular permeability to proteins. In the Control-FDP group, infusion of FDP had no effect on Qlym and L/P. In the Thrombin-FDP group, thrombin increased Qlym with no change in the L/P and the subsequent infusion of FDP further increased Qlym but slightly decreased the L/P, an effect not seen in the Control-FDR group. The results indicate that FDP infusion alone does not alter pulmonary transvascular fluid and protein exchange. However, in the presence of thrombin-induced pulmonary microembolization FDP infusion enhances the net transvascular fluid filtration rate, probably by increasing the capillary hydrostatic pressure.

PLATELET ACTIVATING FACTOR AND SHEEP PLATELETS: A SENSITIVE NEW BIOASSAY

The in vitro response of sheep platelets to platelet activating factor (PAF) was investigated. Sheep platelet-rich plasma aggregated in response to PAF with an EC50 of 10 nM. Platelets isolated via arabinogalactan density gradient centrifugation displayed an EC50 of 50 pM with a threshold response at 0.1 pM. PAF-induced release of 14C-serotonin from isolated sheep platelets was comparable with an EC50 of 50 pM and threshold release at 10 fM. PAF-induced aggregation was specific in that it could be blocked by the competitive receptor antagonists Alprazolam (Upjohn, IC50 = 40 microM), L-652,731 (MSD, IC50 = 6 microM), and WEB 2086 (Boehringer Ingelheim, IC50 = 0.8 microM). At micromolar concentrations, WEB 2086 did not inhibit ADP- or thrombin-induced aggregation nor thrombin-induced serotonin release. However, at higher concentrations of WEB 2086 some inhibition of thrombin-induced platelet aggregation and release was observed. Subsequent experiments demonstrated that high concentrations of WEB 2086 can inhibit thrombin-induced clotting (Ki = 866 microM) and cleavage of the chromogenic substrate Spectrozyme-TH (Ki = 842 microM). In summary, the response of isolated sheep platelets to PAF was specifically inhibitable and was 10 to 100 times more sensitive than washed rabbit platelets, the most popular bioassay currently in use.

Influence of prostaglandin I2 on fibronectin-mediated phagocytosis in vivo and in vitro.

This study evaluated the effect of prostaglandin I2 (PGI2)on fibronectin‐me‐ diated macrophage phagocytosis in vivo and in vitro. Phagocytosis measured in vivo in rats by the vascular clearance rate and hepatic localization of gelatinized sheep erythrocytes was inhibited in a dose‐dependent manner after intravenous administration of PGI2. Phagocytosis was assessed in vitro in terms of uptake of fibronectin‐dependent gelatinized sheep erythrocytes by monolayers of casein‐elicited rat peritoneal macrophages. Concentrations of 1 ng/ml PGI2 or greater resulted in inhibition of particle internalization but not attachment to macrophages. This inhibitory effect was enhanced by amino‐ phylline, a phosphodiesterase inhibitor. PGI2 increased cAMP levels and these were further increased in the presence of aminophylline. These data indicate that PGI2 inhibits macrophage uptake of gelatinized particles and support the idea that this is mediated by increased intracellular levels of cyclic AMP. PGI2 should thus be considered a potential etiologic factor in the phagocytic depression observed in association with thrombosis.