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.

How inflammation modulates central nervous system vessel activation and provides targets for intervention--a personal perspective.

I here describe a line of research that grew out of studies of spinal cord-damaging decompression sickness, focused on the blood-endothelial interface, that was influenced by the local Shwartzman phenomenon, addressed innate immune and inflammatory mechanisms, and ultimately arrived at mucosal tolerance approaches to prevent stroke. Intranasal instillation of E-selectin is under development as a novel means of targeting immunomodulation to activating blood vessels within the vascular tree supplying the brain. The goal of this form of focused immunomodulation is to prevent recurrent strokes in patients that have previously suffered transient ischemic attacks or strokes.

Role of hypercoagulability in steroid-induced femoral head necrosis in rabbits.

BACKGROUND: The pathogenesis of femoral head necrosis is still uncertain. Both steroid treatment and hypercoagulopathy are considered risk factors. To investigate possible changes in coagulability and histology during steroid-induced osteonecrosis, we used a combination of the Shwartzman reaction and corticoid injections in rabbits to develop an animal model of femoral head necrosis. We studied blood coagulability and histopathological characteristics of the femoral head and liver. METHODS: A total of 30 rabbits were divided into three groups. In group A, rabbits were given two injections of lipopolysaccharide (40 microg/kg) at an interval of 24 h and were then immediately given one injection of prednisolone acetate (20 mg/kg). In group B, 10 rabbits were given one injection of prednisolone acetate (20 mg/kg). In group C, 10 rabbits were given no treatment and served as controls. At 1, 3, 7, 14, and 21 days after prednisolone injection, coagulability, blood lipid levels, and blood platelet levels were measured; and the femoral head and liver were removed for histopathological examination. RESULTS: At 24 h after the prednisolone injection in group A, coagulability and blood lipid levels were increased (P < 0.01), and blood platelet levels were decreased (P < 0.01). These abnormal levels were maintained throughout the entire observation period. Histologically, degeneration and necrosis of hepatocytes and osteocytes were found at day 21 after prednisolone injection in group A. In group B, coagulability and blood lipid levels were significantly increased by day 3 after treatment. CONCLUSIONS: Abnormal hypercoagulability might potentiate the thrombotic status and induce thrombus formation in the presence of steroid-induced necrosis of the femoral head.

Factors that control extravascular fibrinolysis.

The physiological and pathophysiological state of tissues determines the exudation of plasma proteins, hemostasis, and fibrinolysis, i.e., inflammation, injury, and malignancy. The physiological controls of extravascular fibrinolysis ultimately rest on a balance between generation of the fibrinolytic enzyme(s), i.e., plasmin, elastase, cathepsins, etc., and inhibitors of the fibrinolytic enzyme(s), i.e., plasminogen activator inhibitors, alpha-2 plasmin inhibitor, alpha 1-protease inhibitors, etc. Moreover, it is the structural modification of fibrin that determines its stability toward proteolytic enzymes and physical duress. The structural modification of fibrin involves factor XIIIa-mediated cross-linking of interfibrin chains and alpha 2-plasmin inhibitor to fibrin. In turn, there are a number of agents that influence factor XIIIa catalytic activity (e.g., sulfhydryl agents, albumin, erythrocytes). The two key proenzymes, factor XIII and plasminogen, are tightly bound with the circulating fibrinogen molecules. Such high selective affinity for fibrin(ogen) provides the reaction specificity in a complex tissue fluid milieu and governs the kinetics of fibrinolysis. Any agents that interfere with such binding reactions, e.g., autoantibodies, may also affect the fibrinolytic reactions. Understanding these unique biochemical controls of factors involved in fibrinolysis may provide an insight into the complex regulatory process of extravascular fibrinolysis.

Characterization of neutrophil activation by repeated injection of endotoxin in rabbits. Role of neutrophils in the generalized Shwartzman reaction.

The relationship between activated neutrophils and end-organ injury in endotoxemia was studied. The function of peripheral blood neutrophils (PMNs) in rabbits with the generalized Shwartzman reaction (GSR) was compared to that of PMNs rabbits receiving a single injection of endotoxin. The following results were obtained: (1) PMNs from rabbits with the GSR demonstrated enhanced adherence to endothelial cells and increased mitochondrial ATP production; (2) the GSR did not enhance chemotaxis and oxygen radical production of PMNs; (3) a single injection of endotoxin did not cause necrosis of visceral organs; (4) in vitro detachment of endothelial cells by PMNs was increased in rabbits with the GSR; (5) in vivo administration of monoclonal antibody (mAb) against CD11b/CD18 (Mac-1) suppressed the increase in PMN adherence; and (6) hemorrhagic necrosis did not occur when mAb to Mac-1 was injected. Thus, enhanced adherence of PMNs to endothelial cells appears to play a key role in endotoxin-induced end-organ injuries in this animal model.

Shwartzman reaction.

The local and generalized Shwartzman reactions are models of thrombohemorrhagic skin necrosis and DIC, respectively. An intravenous preparatory injection of endotoxin followed by an intradermal injection of endotoxin 24 hours later elicits a thrombohemorrhagic lesion only at the site of intradermal injection of endotoxin in the local Shwartzman reaction. Two intravenous injections of endotoxin spaced 24 hours apart induced a systemic generalized Shwartzman reaction characterized by coagulopathy, petechial hemorrhages, microthrombi, and decreased circulating platelets similar to DIC. Of particular interest is the observation that thrombohemorrhagic lesions of the Shwartzman reaction only develop at sites of intradermal injections of endotoxin. Microthrombi composed of platelets and leukocytes only adhere or accumulate in dermal vessels after an intradermal injection of endotoxin. Prior to the endotoxin injection, biopsies of skin show normal vessels without microthrombi or significant inflammation. Since endothelial cells line the small vessels in the dermis, where a Shwartzman reaction appears to be initiated, it is likely that endothelial cells are important for initiating a local Shwartzman reaction. IL-1 and TNF can substitute for the intradermal injection of endotoxin in the local Shwartzman reaction, induce endothelial cells to become thrombogenic, and can induce the expression of cell adhesion molecules on endothelial cells making endothelial cells more sticky for leukocytes. These observations suggest that endothelial cells play a central role in the local Shwartzman reaction and may be important in understanding diseases associated with thrombohemorrhagic skin necrosis.

[The similarity of the biological effects from the parenteral administration of ascarid antigenic complexes and Salmonella typhi endotoxins]. / Skhodstvo biologicheskikh éffektov pri parenteral'nom vvedenii antigennykh kompleksov askarid i éndotoksinov briushnotifoznykh bakterii.

The experiment with rabbits showed that it was basically possible to develop local and generalized Shwartzman's phenomenon under the combined effect of ascaris antigens and typhoid bacterial endotoxins. Intracutaneous (0.2 ml.) or intravenous (0.1 ml/kg) sensitization of animals by ascaris antigens or microbe endotoxins after repeated intravenous crossover, intravenous injection of the antigens used to result in the development of classical local and generalized Shwartzman's phenomenon.

Effect of cyclosporin on generalized Shwartzman reaction in diabetic rats.

The effect of cyclosporin (CsA) on the endotoxin-induced generalized Shwartzman reaction (GSR) was studied in diabetic and nondiabetic rats. After 4.5 wk of diabetes, CsA (20 mg/kg) or intralipid as a control substance was given intraperitoneally daily for 10 days. Next, diabetic rats were given either high-dose (2 mg/kg or low-dose (0.1 mg/kg) endotoxin (Escherichia coli 026:B6 lipopolysaccharide B) as a single injection. The rats were killed at intervals of 1, 4, 8, and 24 h. No significant glomerular thrombi were seen in the nondiabetic control animals, whereas the severity of glomerular thrombi in the diabetic animals was dependent on the presence or absence of CsA, endotoxin dose, and degree of glycemic control. In the diabetic rats, glomerular thrombi occurred maximally at 4 h but were no longer present at 24 h. The CsA/high-dose-endotoxin rats had fewer glomerular thrombi than rats receiving the intralipid/high-dose endotoxin, but this difference was not statistically significant. The CsA/low-dose-endotoxin rats had increased glomerular thrombi compared with the intralipid/low-dose-endotoxin rats (P less than 0.01). Insulin treatment reduced the glomerular capillary thrombi in the CsA/low-dose-endotoxin diabetic animals. Thus, CsA aggravates the GSR with low-dose endotoxin but has no significant effect when high-dose endotoxin is given. Improved glycemic control reduces the GSR in CsA-treated rats. Thus, the interrelationships of diabetes, endotoxin, and CsA on the GSR are complex, and the pathogenesis of these events is unclear.

Stroke risk factors prepare rat brainstem tissues for modified local Shwartzman reaction.

Stroke risk factors such as hypertension, diabetes, advanced age, and genetic predisposition to stroke were demonstrated to prepare rat brainstem tissues for a modified local Shwartzman reaction. A single intracisternal injection of endotoxin provoked the reaction, and affected rats manifested neurologic deficits accompanied by pathologic lesions. Brainstem infarcts developed in only a small proportion of rats without recognized risk factors after intracisternal injection of endotoxin. Thus, stroke risk factors, which are ordinarily regarded as operating through acceleration of atherosclerosis, may predispose to brain ischemia by local effects on brain microcirculation such as those thought to underlie preparation of a tissue for the local Shwartzman reaction.

Vasoactive agents and production of thrombosis during intravascular coagulation. 3. Comparative effects of catecholamines.

Epinephrine (E), isoproterenol (I), and dopamine (D) were compared with norepinephrine (N) for production of microthrombi during thrombin-induced disseminated intravascular coagulation (DIC) in rabbits. Only catecholamines acting on alpha-adrenoreceptors produced glomerular capillary thrombosis (GCT) typical of the generalized Shwartzman reaction (GSR). Epinephrine produced GCT three times (P less than 0.05) less severe than that produced by N, but beta-blockade with propranolol (P) rendered E equal to N in potency. I and D reduced fibrinogen consumption produced by thrombin. I (0.5-0.66 microgram/kg/min), as opposed to D, prevented the GSR produced by endotoxin in the pregnant rat and the cortisone-sensitized rabbit, and P increased the severity of the GSR in the pregnant rat. Alpha-adrenergic blockade with dibenzyline prevented the GSR produced by endotoxin in rats, whether pregnant, diabetic, or having a unilateral ureteral occlusion, and the classic reaction in rabbits, but not that produced in renal-hypertensive rats. Simultaneous alpha + beta stimulations by E triggered coronary and hepatic microthrombi, which were prevented by P. It is concluded that beta-adrenergic stimulation, as opposed to D-adrenergic stimulation, counterbalances alpha-adrenergic effects occurring in endotoxemia, which are required for production of the GSR in most models. These studies stress the risks and benefits of beta-blockade and provide additional evidence for the role of vasoactive agents and microcirculatory changes on selection of target organs for production of microthrombi during DIC.

Enhanced sensitivity to endotoxin induced by 6-sulfanilamidoindazole.

6-Sulfanilamidoindazole (6-SAI) is a sulfonamide that induces inflammation in the ankles and hind paws of older rats and sensitizes the animals to the lethal actions of endotoxin. Sensitization is associated with reductions in plasma glucose concentration but not changes in phagocytic function of the RES or increases in lysosomal or hepatic enzymes. Death in 6-SAI-pretreated but not control rats was associated with fibrin deposition in the glomerular capillaries (generalized Shwartzman reaction) and inflamed paws. Endotoxin shock in both 6-SAI-pretreated and control rats was associated with increases in activities of lysosomal and hepatic enzymes and hypoglycemia. Methylprednisolone but not desoxycorticosterone pretreatment protected 6-SAI-fed rats against endotoxin lethality and the generalized Shwartzman reaction and maintained the plasma glucose concentration but did not prevent loss of lysosomal integrity. Simultaneous administrations of small doses of phenylbutazone with 6-SAI suppressed inflammation but did not protect against endotoxin lethality or the generalized Shwartzman reaction.

Procoagulant activity of lymphocyte-macrophage populations in rabbits: selective increases in marrow, blood, and spleen cells during Shwartzman reactions.

The present experiments examine leukocyte procoagulant activity using mononuclear cell populations purified or enriched from rabbit bone marrow, blood, spleen, lymph node, thymus, and pulmonary alveoli. Cells from these six sites, obtained from control and endotoxemic animals and assayed without an intermediate culture step, were found to have procoagulant activity identified as tissue factor. Under control conditions, tissue factor activity was found to be at low levels in marrow and blood populations compared to median activities 3- and 11-fold higher in populations from spleen and lymph node, and 33- and 45-fold higher in thymus and alveolar populations. By contrast to respective controls, significantly increased amounts of tissue factor (35-, 15-, and 12-fold at median levels) were found in marrow, blood, and spleen populations from endotoxemic animals. The types of leukocytes in these latter three populations were morphologically and histochemically indistinguishable from respective controls, indicating that endotoxin induced increases of activity in cells with relatively low amounts under control conditions. Activity did not change significantly in lymph node, thymus, or alveolar populations after endotoxemia. These studies show that tissue factor is present in a range of leukocyte populations not previously reported to have procoagulant activity. In addition, the finding of widespread gains of tissue factor in the marrow-blood-spleen pool due to endotoxemia provides new evidence supporting the importance of leukocyte procoagulants in Shwartzman-like reactions.

Glomerular dynamics and morphologic changes in the generalized Shwartzman reaction in postpartum rats.

The roles of glomerular functional and morphologic changes were examined in the acute renal failure associated with generalized Shwartzman reaction in postpartum Munich Wistar rats. The susceptibility of postpartum rats to acute deterioration in renal function after a 2-h endotoxin infusion was found to be greater than in virgin litter mates: glomerular filtration rate fell by 93% in the former vs. 24% in the latter group (P less than 0.001). In postpartum rats there were marked changes in platelet count and fibrinogen level (P less than 0.025) compatible with consumption coagulopathy. Renal blood flow and glomerular filtration rate fell from 5.5 +/- 0.9 and 0.74 +/- 0.12 to 2.0 +/- 0.7 and 0.12 +/- 0.01 ml/min, respectively (both P less than 0.001). Blood pressure did not change. Results of glomerular dynamics studies showed decreases in single nephron filtration rate from 28 +/- 7 to 6 +/- 4 nl/min and in glomerular plasma flow rate from 77 +/- 26 to 23 +/- 12 nl/min (both P less than 0.001). Afferent net ultrafiltration pressure fell from 20 +/- 3 to 5 +/- 4 mm Hg due to a fall in glomerular capillary hydraulic pressure from 47 +/- 1 to 29 +/- 5 mm Hg (P less than 0.001). There were four- and twofold increases in afferent and efferent arteriolar resistances, respectively. Less than 20% of glomeruli had evidence of fibrin deposition after 2 h of endotoxin infusion, a time when glomerular filtration rate was reduced by greater than 90%. [1-Sar, 5-Ile, 8-Gly] angiotensin II infusion before endotoxin significantly protected glomerular filtration rate, 62 vs. 7% of control in rats with no preinfusion (P less than 0.01) despite consumption coagulopathy and glomerular fibrin deposition similar to rats without pretreatment. These data suggest that the early deterioration in renal function in the generalized Shwartzman reaction in the postpartum rat is due to major changes in glomerular dynamics induced by neurohumoral agents and that glomerular fibrin deposition plays a lesser pathogenetic role at this time in this disorder. The study does not address the pathogenesis of renal failure in pregnancy nor peripartum renal failure in another species.