Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase

In previous studies, we have shown that administration of monoclonal antibody (MoAb) C6B7 against human factor XII to baboons challenged with a lethal dose of Escherichia coli abrogates activation of the contact system and modulates secondary hypotension. To evaluate the contribution of activated contact proteases to the appearance of other inflammatory mediators in this experimental model of sepsis, we studied the effect of administration of MoAb C6B7 on activation of complement and fibrinolytic cascades, stimulation of neutrophil degranulation, and release of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Activation of the complement system, as reflected by circulating C3b/c and C4b/c levels, was significantly reduced in five animals that had received MoAb C6B7 before a lethal dose of E coli as compared with five control animals that had been given a lethal challenge only. Inhibition of contact activation also modulated the fibrinolytic response, since the release of tissue-type plasminogen activator (t-PA) and the appearance of plasmin-alpha2-antiplasmin (PAP) complexes into the circulation was significantly attenuated upon pretreatment with anti-factor XII MoAb. In contrast, plasma levels of plasminogen activator inhibitor (PAI) were modestly enhanced in the treatment group. Degranulation of neutrophils, as assessed by circulating elastase-alpha1-protease inhibitor complexes, and release of IL-6 but not of TNF-alpha was decreased in anti-factor XII-treated animals. Observed differences in the inflammatory response between treatment and control groups were not likely due to different challenges, since the number of E coli that had been infused, as well as circulating levels of endotoxin after the challenge, were similar for both groups. These data suggest that activation of the contact system modulates directly or indirectly various mediator systems involved in the inflammatory response during severe sepsis in nonhuman primates.     

High molecular weight kininogen: localization in the unstimulated and activated platelet and activation by a platelet calpain(s)

High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha-granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme- linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti-HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium-activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35-fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK.     

Minactivin expression in human monocyte and macrophage populations

Adherent monolayer cultures of human blood monocytes, peritoneal macrophages, bone marrow macrophages, and colonic mucosa macrophages were examined for their ability to produce and secrete minactivin, a specific inactivator of urokinase-type plasminogen activator. All except colonic mucosa macrophages produced and secreted appreciable amounts of minactivin, but only blood monocytes were stimulated by muramyl dipeptide (adjuvant peptide) to increase production. The minactivin from each of these populations could be shown to preferentially inhibit urokinase-type plasminogen activator and not trypsin, plasmin, or "tissue"-type plasminogen activator (HPA66). A plasminogen-activating enzyme present in monocyte cultures appeared unaffected by the presence of minactivin and could be shown to be regulated independently by dexamethasone.     

Development and characterization of monoclonal antiplatelet autoantibodies from autoimmune thrombocytopenic purpura-prone (NZW x BXSB)F1 mice

Male (NZW x BXSB)F1 (W/BF1) mice develop systemic autoimmunity involving autoantibodies, progressive thrombocytopenia, lupus nephritis, and degenerative coronary vascular disease with myocardial infarction. Platelet-associated IgG (PAIgG) on the platelet surface mediates platelet destruction by the reticuloendothelial system in the autoimmune thrombocytopenic purpura (ATP) of W/BF1 mice. Because the epitopes targeted in ATP by PAIgG have not been identifiable using serum from thrombocytopenic W/BF1 mice, we developed seven hybridomas secreting antiplatelet monoclonal antibodies (MoAbs) using splenocytes of thrombocytopenic W/BF1 mice. Epitopes recognized by three MoAbs were similar to those recognized by PAIgG, because eluted IgG from platelets of thrombocytopenic W/BF1 mice inhibited platelet binding by MoAbs in competitive micro-enzyme-linked immunosorbent assay. Hybridoma cells or purified Ig from the ascites of two clones (2A12 and 6A6), when injected into nude mice produced acute thrombocytopenia, elevated the levels of PAIgG, purpura, and megakaryocytosis. MoAbs of two clones also reacted with single-stranded DNA or double-stranded DNA, and one of these clones (4-13) bound to cardiolipin (CL) but was nonpathogenic in nude mice, suggesting that anti-CL and antiplatelet autoantibodies can be distinct. On immunoblotting analysis, antiplatelet MoAbs frequently bound a 100-Kd platelet protein. These MoAbs contribute to an understanding of the etiopathogenesis of ATP and the several antigens and autoantibodies involved.     

Reflex control of sympathetic nerve activity in dopamine beta-hydroxylase deficiency

Patients with autonomic failure secondary to dopamine beta-hydroxylase deficiency lack the enzyme activity necessary for the conversion of dopamine to norepinephrine in sympathetic nerve terminals and the adrenal medulla. These patients have virtually undetectable norepinephrine and epinephrine in plasma and cerebrospinal fluid. The presence of intact sympathetic nerve activity in these patients has been suggested by the enhanced release of dopamine (but not norepinephrine) in response to maneuvers that augment sympathetic outflow in normal subjects. In the present study, we recorded sympathetic nerve traffic by using microneurography in a patient with dopamine beta-hydroxylase deficiency and measured sympathetic neural responses to static exercise, the cold pressor test, and pharmacological alterations of blood pressure. At rest, sympathetic nerve activity was abundant and was modulated in a normal manner by handgrip (+278%), the cold pressor test (+169%), hypotension induced with isoproterenol (+102%), and hypertension induced with phenylephrine (-85%). These results provide the first electrophysiological evidence for intact regulation of sympathetic neural outflow in a patient with dopamine beta-hydroxylase deficiency and suggest that central norepinephrine and epinephrine pathways believed essential for the control of sympathetic neurotransmission in humans may be supplanted by alternative redundant mechanisms.     

Granulocyte colony-stimulating factor versus placebo in addition to penicillin G in a randomized blinded study of gram-negative pneumonia sepsis: analysis of survival and multisystem organ failure

Sepsis is a common cause of morbidity and mortality. Neutrophils are the major defense against bacterial invasion, and granulocyte colony- stimulating factor (G-CSF) augments both neutrophil number and function. In our study, 160 rabbits were inoculated transtracheally with 0.5 mL of a solution containing 10(4) colony forming units per milliliter of Pasteurella multocida. Twenty-four hours later, chest x- rays and quantitative blood cultures demonstrated pneumonia and bacteremia. Therapy was then begun with penicillin G and either recombinant human G-CSF (rG-CSF; 5 to 8 micrograms/kg subcutaneously) or placebo every day for 5 days. Arterial blood gases and 23 other parameters of organ function were performed before inoculation and serially thereafter. All rabbits underwent histologic examination of organs at the time of septic death or when sacrificed on day 6. A total of 149 rabbits survived long enough to initiate therapy. A significant increase in leukocytes by day 4 was found in the rG-CSF-treated group. There was a trend towards improved survival in the rG-CSF group (77% v 67%; P = .13, n = 149). Analysis of pretreatment variables revealed sepsis-induced leukopenia (< or = 2,800/microL) as the only predictor of significantly improved survival with rG-CSF treatment (57% v 39%; P = .04, n = 73). The majority of the survival benefit occurred within the first 24 hours of treatment. This was before the time that a significant difference in mean white blood cell (WBC) count was observed between the study groups, making intravascular leukocytosis an unlikely explanation for the survival advantage in the rG-CSF group. No significant difference in laboratory variables reflecting organ function was demonstrated between the groups. Histologic grading of inflammation (0, normal, to 6, necrosis) in seven organs revealed that the surviving rabbits had mild but statistically significant increased inflammation in the liver, spleen, and noninoculated lung in the rG-CSF versus placebo groups (liver: 2.6 v 1.5, P < or = .0001; spleen: 3.2 v 2.3, P < or = .0001; and noninoculated lung: 2.9 v 2.5, P = .04). Administration of rG-CSF, in addition to penicillin G, in immune competent rabbits with gram-negative sepsis complicated by leukopenia significantly improved survival over antibiotics alone. The administration of rG-CSF in early sepsis for a short therapeutic duration was not associated with any clinically evident toxicity. Clinical trials using rG-CSF in septic patients with leukopenia are indicated.     

Factors influencing the acceleration of human factor XIa inactivation by antithrombin III

Controversy exists in the literature concerning the potentiating effect of heparin on the inactivation rate of factor XIa by antithrombin III (AT III) in both purified systems and in plasma. We have analyzed the factors that could influence this reaction and found that ionic strength of the medium, as well as the type and concentration of the heparin preparations accounted for the major discrepancies in the literature. At I = 0.43 N, a preparation of bovine lung heparin at 1 U/mL did not augment the inactivation rate of factor XIa by inhibitors in plasma or by purified AT III. However, when ionic strength was decreased, a progressive increase in the potentiating effect was observed, reaching 6.5-fold at I = 0.15 N. At saturating concentrations of heparin, which results in the formation of 100% AT III-heparin complex, (greater than ten-fold molar excess over AT III) in purified systems, all heparin preparations (porcine, bovine, low molecular weight [LMW], and high affinity) yielded an approximately 30-fold augmentation of the factor XIa inactivation rate. However, when heparin was less than saturating, we observed that various heparin preparations affected the AT III-induced inactivation of factor XIa to different degrees even though they exhibited the same inhibitory activity (1 U/mL) against thrombin. This variation resulted from differences in the number of AT III binding sites in each heparin preparation, despite a similar Kd for each. Addition of high molecular weight kininogen (HK) to AT III-heparin complexes did not enhance their ability to inhibit factor XIa, and high concentrations of HK decreased the inactivation rate. A high therapeutic dose of heparin only permits the formation of 2.5% to 16.5% of the AT III-heparin complexes that can be achieved at saturation. We observed that 1 U/mL heparin (bovine lung heparin) (high therapeutic concentration) in virtually undiluted plasma only accelerated the inactivation rate of factor XIa (in the absence of other active enzymes) less than two-fold. These new observations further support our previous conclusion that therapeutic levels of heparin have little to no influence on the inactivation rate of factor XIa in plasma.     

Adherence to Antiretroviral Therapy and Clinical Outcomes Among Young Adults Reporting High-Risk Sexual Behavior, Including Men Who Have Sex with Men, in Coastal Kenya

African men who have sex with men (MSM) face significant stigma and barriers to care. We investigated antiretroviral therapy (ART) adherence among high-risk adults, including MSM, participating in a clinic-based cohort. Survival analysis was used to compare attrition across patient groups. Differences in adherence, weight gain, and CD4 counts after ART initiation were assessed. Among 250 HIV-1-seropositive adults, including 108 MSM, 15 heterosexual men, and 127 women, patient group was not associated with attrition. Among 58 participants who were followed on ART, 40 % of MSM had less than 95 % adherence, versus 28.6 % of heterosexual men and 11.5 % of women. Although MSM gained less weight after ART initiation than women (adjusted difference ?3.5 kg/year), CD4 counts did not differ. More data are needed on barriers to adherence and clinical outcomes among African MSM, to ensure that MSM can access care and derive treatment and prevention benefits from ART.