Contribution of protein Z and protein Z-dependent protease inhibitor in generalized Shwartzman reaction.

OBJECTIVE: Sepsis, a leading cause of mortality in critically ill patients, is closely linked to the excessive activation of coagulation and inflammation. Protein Z, a cofactor for the protein Z-dependent protease inhibitor, enhances the inhibition of coagulation factor Xa, and protein Z-dependent protease inhibitor inhibits factor XIa in a protein Z-independent fashion. The functions of protein Z and protein Z-dependent protease inhibitor in the inflammatory and coagulant responses to septic illness have not been evaluated. DESIGN: For induction of generalized Shwartzman reaction, dorsal skinfold chamber-equipped mice were challenged twice with lipopolysaccharide (0.05 mg/kg on day -1 and 5 mg/kg body weight 24 hr later). Time-matched control animals received equal volumes of saline. SETTING: University research laboratory. SUBJECTS, INTERVENTIONS, AND MEASUREMENTS: Using intravital fluorescence microscopy in protein Z-dependent protease inhibitor deficient (ZPI) and protein Z deficient (PZ) mice, as well as their wild-type littermates (ZPI, PZ), kinetics of light/dye-induced thrombus formation and microhemodynamics were assessed in randomly chosen venules. Plasma concentrations of chemokine (C-X-C motif) ligand 1, interleukin-6, and interleukin-10 were measured. Liver and lung were harvested for quantitative analysis of leukocytic tissue infiltration and thrombus formation. MAIN RESULTS: After induction of generalized Shwartzman reaction, all mice showed significant impairment of microhemodynamics, including blood flow velocity, volumetric blood flow, and functional capillary density, as well as leukocytopenia and thrombocytopenia. Thrombus formation time was markedly prolonged after induction of generalized Shwartzman reaction in all mice, except of ZPI mice, which also had a significantly higher fraction of occluded vessels in liver sections. PZ mice developed the highest concentrations of interleukin-6 and interleukin-10 in response to generalized Shwartzman reaction and showed greater leukocytic tissue infiltration than their wild-type littermates. CONCLUSIONS: In this murine model of generalized Shwartzman reaction, protein Z-dependent protease inhibitor deficiency enhanced the thrombotic response to vascular injury, whereas protein Z deficiency increased inflammatory response.

Interferon gamma, a mediator of lethal lipopolysaccharide-induced Shwartzman-like shock reactions in mice.

The involvement of cytokines in the pathogenesis of a generalized, Shwartzman-like lethal inflammatory response to bacterial lipopolysaccharides (LPS) was studied by testing the ability of cytokines or neutralizing anticytokine antibodies to modify the course of the syndrome. The reaction was elicitable in non-SPF NMRI mice by two consecutive injections of S. marcescens LPS: a first injection in the footpad, followed after 24 h by an intravenous dose; the size and route of the preparatory LPS dose were found to be critical. Treatment with mAbs against IFN-gamma was found to completely prevent the reaction. Treatment with IFN-gamma on the other hand, rendered the mice more sensitive to elicitation of the reaction. In contrast, systemic administration of IFN-alpha/beta exerted a desensitizing effect. The role of endogenous cytokines in the pathogenesis of this generalized Shwartzman reaction was also documented by a study of the cytokine levels in the serum of the mice. In comparisons between mice given lethal and nonlethal induction schedules, a good correlation was found between mortality rates and height of IFN or TNF levels, but no correlation was seen with IL-6 levels. Also, in mice that were protected by anti-IFN-gamma antibody, serum IFN and TNF were undetectable, whereas IL-6 levels were as high as in unprotected mice. These data provide evidence that among the cytokines that govern the inflammatory response to LPS, endogenous IFN-gamma occupies a key position. These findings therefore also open perspectives for clinical application of IFN-gamma antagonists.

Prevention of the localized and generalized Shwartzman reactions by an anticomplementary agent, cobra venom factor.

Both localized and generalized Shwartzman reactions were induced in the same rabbits by simultaneous administration of preparatory intravenous and intradermal doses of endotoxin followed in 24 hr by the provocative dose. Control rabbits with more than 80% positive responses showed corresponding changes of platelet, white blood cell, fibrinogen, and hemolytic complement levels. Circulating fibrinogen and fibrin degradation products were detected shortly after the preparatory dose and persisted for at least 3 days. Rabbits given cobra venom anticomplementary factor showed hypocomplementemia (less than 10% of normal), leukocytosis, and elevated fibrinogen levels. After the administration of endotoxin, only one of 15 CVF-treated animals developed a Shwartzman reaction and that was mild. These rabbits showed only minor changes of platelet and fibrinogen levels throughout the experiment although their white blood cell responses were similar to those of the control group. No detectable fibrinogen and fibrin degradation products appeared in circulation, and the hemolytic complement activity increased gradually beginning with the preparatory dose of endotoxin. Thus depletion of terminal complement components (mainly C3) in rabbits is protective against the development of both localized and generalized Shwartzman reactions; its mechanism of action is probably through the sparing of platelets by inhibiting platelet-endotoxin interaction. The essential role of the complement system in Shwartzman reaction indicates that this coagulopathy probably represents a manifestation of immunologic injury.

Coagulant activity of leukocytes. Tissue factor activity.

Peritoneal leukocytes harvested from rabbits which have received two spaced doses of endotoxin have significantly greater (10-fold) coagulant activity than leukocytes from control rabbits. The coagulant activity accelerates the clotting of normal plasma and activates factor X in the presence of factor VII and calcium and is therefore regarded as tissue factor. A total of 40-80 mg tissue factor activity was obtained from the peritoneal cavity of single endotoxin-treated rabbits. In leukocyte subcellular fractions, separated by centrifugation, the specific tissue factor activity sedimented mainly at 14,500 g and above. The procoagulant activity was destroyed after heating for 10 min at 65 degrees C but was preserved at lower temperatures. Polymyxin B, when given with the first dose of endotoxin, reduced both the number of peritoneal leukocytes and their tissue factor activity by two-thirds. When given immediately before the second dose of endotoxin, polymyxin B had no inhibitory effect.

Effect of platelet antiserum on the precipitation of soluble fibrin by endotoxin.

Rabbits were injected with a platelet antiserum to examine the role of thrombocytopenia of the precipitation of soluble fibrin by endotoxin.dotoxin. Platelet antiserum removed more than 98% of the circulating platelets, and the resulting thrombocytopenia with platelet counts below 10,000 per mul persisted during the entire 10 hr-period of the experiment. Leukocyte counts were not significantly influenced by the platelet antiserum. Since the rabbits were treated with high doses of heparin, activation of intravascular coagulation by the antiserum did not occur. Precipitation of soluble fibrin was achieved by injection of endotoxin into rabbits with ancrod-induced circulating soluble fibrin. Thrombocytopenia did not prevent the occurrence of glomerular microclots after ancrod and endotoxin administration. On the contrary, if endotoxin was injected into antiserum- and heparin-treated rabbits with circulating soluble fibrin, glomerular microclot formation occurred even in a higher percentage than in control rabbits treated with absorbed platelet antiserum. This investigation indicates that platelet antiserum-induced thrombocytopenia does not protect against precipitation of soluble fibrin by en

Depletion of plasma factor XIII prevents disseminated intravascular coagulation-induced organ damage.

The impact of clot stability affecting the vasculopathy and tissue necrosis in Shwartzman reaction was investigated using plasma Factor XIII A2-depleted rabbit (FXIII-DR). Plasma Factor XIIIA2 (FXIIIA2) was depleted by infusion of the mono-specific goat anti-rabbit FXIIIA2 IgG. Generalized Shwartzman reaction (GSR) was induced by priming and challenged by i.v. injection of LPS and local Shwartzman reaction (LSR) was primed by intradermal injection of LPS and challenged by i.v. injection of LPS. Histological examination of the GSR animals showed, extensive thrombi accumulation in renal tubules and bilateral cortical necrosis of kidney in 8 out of 10 rabbits but none in the FXIII-DR. Fibrinogen levels were elevated to 3 approximately 4 fold at 24 h and lowered at 48 h whereas a steady rise was seen in the FXIII-DR. FDP levels in GSR animals were significantly elevated at 24 h and further increased at 48 h but only slightly elevated in the FXIII-DR. Examination of the LSR tissues after 48 h showed an acute onset of progressive cutaneous vascular thrombosis, purpura, and secondary hemorrhagic necrosis whereas neither fibrin deposit nor necrosis of tissue were detected in FXIII-DR despite of an early edema formation. Fibrinogen levels were also increased two fold at 24 h but returned to basal levels at 48 h in control LSR animals but not affected at all in FXIII-DR. These results suggest that during the severe inflammatory conditions such as sepsis, the fibrinolytic system is functionally sufficient to dissipate the pathogenic accumulation of disseminated intravascular clots and exudated fibrin clots if those clots were prevented from getting crosslinked in plasma.

Vasoactive agents and production of thrombosis during intravascular coagulation 1--comparative effects of norepinephrine in thrombin and adenosine diphosphate (ADP) treated rabbits.

Pathogenesis of the microthrombi produced during intravascular coagulation was investigated in rabbits given intravenous infusions of thrombin or adenosine diphosphate (ADP). Thrombin, at a dosage producing a fibrinogen consumption of 70% within 4 h (1 unit/kg/min), failed to produce extrapulmonary microthrombi unless fibrinolysis inhibition (epsilon-aminocaproic acid-EACA) or alpha-adrenergic stimulation (norepinephrine) were provided simultaneously. The mechanism whereby norepinephrine initiated glomerular capillary thrombosis was not related to interference with fibrinolysis nor to potentiation of platelet aggregation and blood coagulation, as indicated by similar consumption of plasminogen and platelets in animals given thrombin alone or combined with norepinephrine, and by the lack of correlation between fibrinogen consumption and the incidence and severity of glomerular thrombosis produced with various dosages (1 to 3 mu/kg/min) of norepinephrine. With norepinephrine, microthrombi were also observed in the adrenals, the spleen and the gastric mucosa. Aspirin prevented the phenomena in the latter two organs, but was inactive or aggravating on thrombogenesis elsewhere. ADP associated with thrombin failed to trigger formation of microthrombi but initiated platelet-rich thrombi in the pulmonary vasculature when associated with norepinephrine. We conclude that unlike thrombin, ADP cannot be held responsible for the microthrombi elicited during experimental intravascular coagulation. Furthermore, the ability of norepinephrine to elicit glomerular thrombosis in thrombin injected rabbits may provide an explanation for requirement of alpha-adrenergic stimulation in the endotoxin-induced generalized Shwartzman reaction.

[The disseminated intravascular coagulation syndrome in endotoxin shock]. / Sindrom disseminirovannogo vnutrisosudistogo svertyvaniia krovi pri éndotoksinovom shoke.

Dynamics of blood coagulation and morphological changes after the intravenous administration of Salmonella typhimurium endotoxin is studied experimentally in Chinchilla rabbits. The stages of blood coagulation are established characteristic for the above syndrome. The signs of the developing blood stasiopathy with the domination of vascular wall damage and thrombocyte aggregation are observed. No considerable consumption of the blood coagulation factors is found. It is concluded that a leading role in the pathogenesis of the endotoxin shock microcirculatory disturbances belongs to the vascular damage and aggregation thrombocytopenia and not to the coagulopathy consumption.

Inhibition of the local Shwartzman reaction by turpentine, prostaglandin E2 and carbon tetrachloride.

The conventional local Shwartzman reaction (LSR) did not occur in rabbits whose plasma haptoglobin (Hp) was lowered by treatment with carbon tetrachloride, nor in animals in whom Hp was elevated after treatment with turpentine, or PGE2 or endotoxin. The possible mechanisms of such inhibition of the LSR are discussed.

Local Shwartzman-like phenomenon prepared with haptoglobin-hemoglobin complex and provoked with prostaglandin.

Intradermal injection of haptoglobin-hemoglobin (Hp-Hb) complex followed by intravenous injection of prostaglandin (PG) F2a or E2 produced a local Shwartzman-like phenomenon (LSLP) in rabbits, whereas PGE1 has no provoking action. Intradermal injection of hemoglobin or erythrocytes also produced LSLP when provoked with endotoxin or PGF2alpha. The provoking action of endotoxin in the local shwartzman reaction may not be entirely mediated through PGs.

[The role of prostaglandins in the pathogenesis of the Shwartzman phenomenon]. / O roli prostaglandinov v patogeneze fenomena Shvartsmana.

Schwartzman's phenomena, namely local and generalized reactions (SLR and SGR) were reproduced in two groups of rabbits weighing 1.5-2.5 kg. Use was made of endotoxin S. typhimurium obtained by the method of Bowen. Before endotoxin injection half of the animals of each group were given indomethacin (10 mg/kg). The coagulograms and thromboelastograms were examined after each endotoxin injection. Light and electron microscopy were used to study the animals' kidneys. Animals with the SLR and SGR demonstrated marked changes in the coagulogram and thromboelastogram characteristic of the development of the disseminated intravascular blood coagulation syndrome (DIC-syndrome). Animals' kidneys, particularly those in SGR, manifested gross structural and ultrastructural disorders, pronounced changes in microcirculation. Administration of indomethacin prevented the development of the DIC-syndrome. Besides, microscopic examination of the kidneys did not show fibrin release to the microcirculatory bloodstream. The data obtained indicate that the changes in hemostasis occurring in the SLR and SGR and microcirculatory disorders in the kidneys are linked with a dramatic intensification of prostaglandin synthesis, that develops in response to endotoxin injection.

The effects of infusion of thrombin or endotoxin in rabbits treated with cortisone.

Rabbits treated for 4 days with cortisone to prepare for the generalized Shwartzman reaction (GSR) were infused with thrombin or endotoxin. Whereas endotoxin induced the GSR, infusion of from 120--400 U/kg of thrombin over 1 to 2 1/2 hr failed to induce the GSR. Mean values for fibrinogen consumption after thrombin or endotoxin, calculated from changes in plasma fibrinogen concentration and plasma 125I-fibrinogen radioactivity, were as follows: for rabbits infused with thrombin, from 43 to 61 mg/kg over a 3 hr period; for rabbits infused with endotoxin, 58.5 mg/kg over a 6 hr period. A small peak of non-clottable protein radioactivity, indicative of secondary fibrinolysis, was found in animals infused with thrombin but not in animals infused with endotoxin. A striking late rise in plasma fibrinogen levels was noted in animals infused with endotoxin. It was not noted in animals infused with thrombin. This observation provides further evidence that endotoxin stimulates fibrinogen synthesis by mechanisms independent of intravascular clotting or fibrinolysis. The failure to produce the GSR with thrombin in cortisone-treated rabbits leads us to conclude that depression of reticuloendothelial cell clearance of fibrin can not account for the preparatory effect of cortisone for the GSR after endotoxin.