Rapid in vivo induction of leukocyte tissue factor mRNA and protein synthesis following low dose endotoxin administration to rabbits.

INTRODUCTION: Disseminated intravascular coagulation in humans is frequently associated with Gram-negative bacterial sepsis. Therefore, to examine the role and time frame of the in vivo induction of tissue factor (TF) by bacterial endotoxin, a reverse transcription polymerase chain reaction and a solid-phase ELISA assay were developed to monitor the in vivo production in rabbits, of TF mRNA and TF antigen by peripheral blood leukocytes (PBL). METHODS: : Healthy rabbits were injected intravenously with either 1, 10 or 50 microg/kg of Salmonella endotoxin. Blood samples were obtained both before endotoxin administration and at various time points thereafter, up to 24 h. Some experiments were also done to determine whether all-trans retinoic acid would ameliorate the signs of the endotoxin-induced disseminated intravascular coagulation. RESULTS: PBL counts dropped significantly within 2 h of rabbits receiving the endotoxin, recovering to baseline levels by 24 h. Platelet counts decreased gradually over this same time frame. Fibrin deposition was noted in renal glomerular capillaries at 24 h. An increase (P<0.001) in PBL-associated TF mRNA levels was observed 2 h post-endotoxin (10 microg/kg, n = 8), followed by a gradual decline over the subsequent 24 h. The average increase in TF mRNA at 2 h was approximately 4.6-fold (P<0.001) over that seen at time 0. The amount of mononuclear cell associated TF antigen demonstrated a peak at 2 h post-endotoxin (10 microg/kg, n = 13), with levels approximately 9.6-fold greater than (P<0.001) baseline. Pre-treatment of rabbits with all-trans retinoic acid significantly (P<0.001) ameliorated the PBL-associated increase in TF mRNA and TF antigen levels. CONCLUSION: These results suggest that low dose endotoxin (10 microg/kg) faithfully reproduces the non-overt activation of coagulation observed in primates and human volunteers, supporting the hypothesis that TF expression is involved in the in vivo initiation and propagation of disseminated intravascular coagulation. Moreover, all-trans retinoic acid may be effective in modulating in vivo the TF transcription induced by endotoxin.

Potent antithrombin activity and delayed clearance from the circulation characterize recombinant hirudin genetically fused to albumin.

In this study we sought to extend the plasma half-life while maintaining the potent antithrombin activity of hirudin. We hypothesized that gene fusion of hirudin to albumin would result in the expression of a slowly cleared hirudin molecule. A hirudin variant 3 (HV3) cDNA was obtained by gene synthesis, while a 1,996-bp full-length rabbit serum albumin (RSA) cDNA was selected from a rabbit liver cDNA library. Expression of the former in COS-1 cells conferred antithrombin activity on media conditioned by the cells, while expression of the latter resulted in the secretion of a 67-kD protein that reacted with mono-specific anti-RSA antibodies. Having shown independent expression of the two proteins, we next expressed two fusion proteins: HV3 linked via its C-terminus to albumin (HLA), and HV3 linked via its N-terminus to albumin (ALH). The former, but not the latter, inhibited both the amidolytic and fibrinogenolytic activities of thrombin. HLA also retained the dye-binding characteristics of RSA, as judged by Affi-Gel Blue chromatography. Highly similar concentrations of either commercial HV1 (40 nmol/L) or HLA (30 nmol/L) were required to halve the initial rate of thrombin reaction with chromogenic substrate S2238, suggesting the retention of high-affinity inhibition of thrombin by the fusion protein. An His-tagged form of HLA was purified by Ni2+-chelate affinity and heparin-Sepharose chromatography. The purified, radioiodinated protein was injected into rabbits, and demonstrated a catabolic half-life of 4.60 +/- 0.16 days. This represents an extension of hirudin half-life in vivo of greater than two orders of magnitude; gel analysis of HLA(H)6 recovered from rabbits showed that it circulated in intact form. Our results provide a rationale for future testing of the biological effects of HLA, and support our initial hypothesis.

Probable regulation of factor VIIa-tissue factor and prothrombinase by factor Xa-TFPI and TFPI in vivo.

Given that factor VIIa-tissue factor (TF) probably initiates coagulation in vivo, this study investigated the relationship between plasma concentrations of factor VIIa and prothrombin fragment 1 + 2 in plasma (the latter as an index of prothrombinase activity in vivo). The relationships between these two parameters and the concentrations of tissue factor pathway inhibitor (TFPI) and factor Xa-TFPI in plasma were also investigated. TFPI inactivates factor Xa in a reaction accelerated by heparin, whereas factor Xa-TFPI inactivates factor VIIa-TF and prothrombinase. Established enzyme-linked immunosorbent assays (ELISAs) were used to quantify TFPI and prothrombin fragment 1 + 2, whereas we developed an ELISA to quantify factor Xa-TFPI using affinity purified rabbit (anti-human TFPI)-IgG and chicken anti-(human factor Xa-TFPI)-IgY as the capture and detector antibodies, respectively. Plasma factor VIIa was quantified using truncated tissue factor. The concentrations of factor VIIa and prothrombin fragment 1 + 2 increased in parallel in the plasmas of up to 145 healthy adults assayed (P = 0.007), as did the concentrations of factor VIIa and TFPI (P = 0.0039), and prothrombin fragment 1 + 2 and TFPI (P = 0.013). In contrast, there was an inverse relationship between the concentrations of free factor Xa-TFPI and factor VIIa (P < 0.0001) and free factor Xa-TFPI and prothrombin fragment 1 + 2 (P = 0.0095). These results are consistent with factor Xa-TFPI regulating factor VIIa-tissue factor and prothrombinase in vivo.

Catabolism of rabbit prothrombin in rabbits: uptake of prothrombin by the aorta wall before and after a de-endothelializing injury in vivo.

After an injury to the vascular endothelium, certain blood proteins collect rapidly at the site of damage to prevent blood loss and maintain blood flow. The uptake of fibrinogen, plasminogen, and antithrombin--but not prothrombin--have been measured previously at the rabbit aorta wall after injury in vivo. This report describes the clearance of rabbit iodine 131-labeled prothrombin from the rabbit circulation to measure the distribution and fractional catabolic rate and compares the behavior of 131I-labeled prothrombin with either iodine 125-labeled fibrinogen or 125I-labeled antithrombin at the balloon catheter-injured aorta wall. When injected into young rabbits, 131I-labeled prothrombin was cleared from the intravascular space to yield a plasma curve that was best described by three exponentials. Fractional plasma and whole body catabolic rates were 2.0 day-1 and 0.41 day-1, respectively, equivalent to a catabolic half-life of 1.7 days. Fractional distribution of prothrombin amounted to 0.21, 0.24, and 0.55 within the intravascular, vascular endothelial, and extravascular compartments, respectively. Samples of 131I-labeled prothrombin and either 125I-labeled fibrinogen or 125I-labeled antithrombin were injected into anesthetized rabbits before balloon de-endothelialization of the thoracic aorta. The uptake of each radiolabeled protein by the aorta intima-media was measured at various times (5 to 60 minutes) after injury. Whereas uptake of plasma fibrinogen by the balloon-injured intima-media was maximal (20 pmol/cm2) in less than 5 minutes after injury, maximum uptake of prothrombin (5 to 6 pmol/cm2) took approximately 15 minutes. Uptake of prothrombin was initially faster than that of antithrombin, although approximately equimolar amounts of prothrombin and antithrombin were bound by the intimamedia by 60 minutes. The results are discussed in relation to thrombin production and the demand for antithrombin by the damaged aorta wall in vivo.

Measurement of factor Xa-antithrombin III in plasma: relationship to prothrombin activation in vivo.

The M(r) of the complexes formed when factor Xa reacts with antithrombin III (ATIII) in plasma were estimated by gel filtration and SDS-polyacrylamide electrophoresis. The predominant species of factor Xa-ATIII detected after plasma and plasma to which factor Xa had been added were gel filtered on Sephadex G-200 and Sepharose 4B had apparent M(r) > 200,000, in which factor Xa-ATIII was associated with vitronectin. Addition of factor Xa-ATIII to ATIII-depleted plasma also resulted in the formation of factor Xa-ATIII-vitronectin complexes with M(r) > 200,000. Using polyclonal antibodies to human factor Xa-ATIII and ATIII as the capture and detector antibodies, respectively, a sensitive and specific enzyme-linked immunosorbent assay was developed to quantify factor Xa-ATIII in plasma. The relationship between factor Xa-ATIII production and prothrombinase activity in vivo was investigated by quantifying factor Xa-ATIII and prothrombin fragment 1 + 2 endogenous to the plasmas of blood donors and patients with Hodgkin's and non-Hodgkin's lymphoma. Whereas the concentrations of prothrombin fragment 1 + 2 in the 84 normal plasmas increased with age, those of factor Xa-ATIII (mean +/- SD of 34.7 +/- 13.8 pM) did not, and no correlation existed between the concentrations of the two parameters in normal plasmas. In contrast, a highly significant correlation between the concentrations of these two parameters was found in the plasmas of the cancer patients which coincidentally also had higher concentrations of both factor Xa-ATIII and prothrombin fragment 1 + 2 than the normal plasmas. Thus, ATIII may differentially influence prothrombinase formation and activity in normal individuals and cancer patients.

Inhibition of thrombin by antithrombin III and heparin cofactor II in vivo.

The critical role of thrombin in the pathogenesis of venous and arterial thrombosis, and the effectiveness of glycosaminoglycans as antithrombotic drugs are well known. Antithrombin III is a major inhibitor of thrombin and augmentation of its inhibitory actions by heparin is the basis for the clinical uses of heparin. Recent clinical and experimental studies have demonstrated that another glycosaminoglycan, dermatan sulfate, is an effective antithrombotic drug. Dermatan sulfate catalyses the inhibition of thrombin by heparin cofactor II. The concentrations of heparin cofactor II are higher in the plasmas of individuals with congenital antithrombin III deficiency and pregnant women than controls. The role of heparin cofactor II as a physiologic thrombin inhibitor is unknown. Enzyme-linked immunosorbent assays were used to quantify thrombin-heparin cofactor II and thrombin-antithrombin III endogenous to the plasmas of adult antithrombin III-Hamilton deficient subjects, their siblings with normal antithrombin III levels, pregnant women at term and 3 to 5 days after delivery. Both thrombin-antithrombin III and thrombin-heparin cofactor II complexed with vitronectin were detected in all the plasmas. Significantly, the concentrations of thrombin-heparin cofactor II-vitronectin were higher in the plasmas of congenital antithrombin III deficient subjects and in pre- and post-delivery plasmas than those of normal subjects. In addition, the concentrations of thrombin-heparin cofactor II decreased 3 to 5 days after delivery, reflecting the disappearance of the catalytically active dermatan sulfate elaborated by the placenta. Thus, heparin cofactor II normally inactivates thrombin in vivo, with its role increasing in conditions associated with high levels of heparin cofactor II and/or dermatan sulfate.

Fibrin moderates the catalytic action of heparin but not that of dermatan sulfate on thrombin inhibition in human plasma.

Three recent studies have reported that fibrin in solution significantly inhibits the ability of heparin to catalyze the inhibition of thrombin by antithrombin III. In addition, heparin inhibits the release of fibrinopeptide A by clot-bound thrombin less effectively than it inhibits the release of fibrinopeptide A by thrombin in solution. We have also reported that dermatan sulfate, which catalyzes thrombin inhibition by heparin cofactor II, inhibits thrombus growth in rabbits more effectively than heparin. Because the results of these studies suggest that fibrin inhibits the reactivity of thrombin with antithrombin III-heparin but not with heparin cofactor II-dermatan sulfate, we compared the relative catalytic effects of heparin and dermatan sulfate on thrombin inhibition in plasma, both in the presence and absence of fibrin. We quantitated the rates of thrombin inhibition by antithrombin III and heparin cofactor II by specific enzyme-linked immunosorbent assays. When it was generated, fibrin was kept in solution by adding 2 mmol/L Gly-Pro-Arg-Pro to plasma. Fibrinogen-fibrin reduced the reactivity of thrombin with plasma antithrombin III, both in the presence of and in the absence of heparin. In contrast, the catalytic action of dermatan sulfate on thrombin inhibition by plasma heparin cofactor II was unimpaired by fibrinogen-fibrin. Based on the ability of dermatan sulfate to inhibit thrombus growth in rabbits, failure of fibrinogen-fibrin to moderate the catalytic action of dermatan sulfate may account for its greater antithrombotic effectiveness relative to that of heparin.