Initiation of contact system activation in plasma is dependent on factor XII autoactivation and not on enhanced susceptibility of factor XII for kallikrein cleavage

Various mechanisms have been hypothesized to explain the initiation of contact system activation in plasma. We investigated the capability of dextran sulphate (DS) of different molecular weights to initiate contact system activation in normal human plasma, and compared this with their capability to support factor XII autoactivation and to enhance factor XII susceptibility for cleavage by kallikrein.
Dextran sulphate of M_r 500 000 (DS500) and 50 000 (DS50) was able to initiate contact system activation in plasma (determined by measuring the amount of factor XIIa–C1-inhibitor, kallikrein–C1-inhibitor and factor XIa–C1-inhibitor complexes generated) as well as to support factor XII autoactivation and to enhance factor XII susceptibility for cleavage by kallikrein (as measured with amidolytic assays using purified proteins). In contrast, dextran sulphate of M_r 15 000 (DS15) and 5000 (DS5) neither induced contact system activation in plasma, nor supported autoactivation of factor XII, although both of these DS species enhanced the rate of activation of factor XII by kallikrein in the purified system. Based on these properties (i.e. binding of factor XII without inducing autoactivation), DS15 and DS5 were predicted to be inhibitors of contact system activation induced in plasma by DS500, which indeed was observed.
We conclude that enhanced factor XII susceptibility for kallikrein activation and factor XII autoactivation are distinct phenomena, the latter being necessary to support activation of the contact system in plasma.     

Activation of factor XII by dextran sulfate: the basis for an assay of factor XII

A system was developed for studying the activation of factor XII (Hageman factor) in the presence of dextran sulfate (DS). Salient features of the system included low ionic strength (0.08), low concentration of factor XII (approximately 1/10,000 that in normal plasma), and an excess of exogenous prekallikrein (PK). In this system, factor XII was rapidly converted to the 80,000 molecular weight (mol wt) form of factor XIIa (alpha-factor-XIIa). Once formed, the factor XIIa converted PK to kallikrein at a rate that was proportional to the amount of factor XII originally present in the incubation mixture. This system was used to construct a simple sensitive assay for factor XII in plasma and other biologic samples. The kallikrein produced was measured spectrophotometrically with the chromogenic substrate (H-D-Pro-Phe-Arg- p-nitroanilide (S-2302). This assay was shown to be independent of the high molecular weight kininogen and the PK content of the sample being analyzed. The measurements obtained were consistent with fundamental enzymologic principles and, if desired, could be processed with a simple calculator program to achieve linear standard curves. When applied to the quantitation of factor XII in plasma, the assay yielded values in close agreement with those determined by coagulant assay or by radial immunodiffusion.     

Gene induction by coagulation factor Xa is mediated by activation of protease-activated receptor 1

Cell signaling by coagulation factor Xa (Xa) contributes to pro-inflammatory responses in vivo. This study characterizes the signaling mechanism of Xa in a HeLa cell line that expresses protease-activated receptor 1 (PAR-1) but not PAR-2, -3, or -4. Xa induced NF-kappaB in HeLa cells efficiently but with delayed kinetics compared to thrombin. This delay caused no difference in gene expression patterns, as determined by high-density microarray analysis. Both proteases prominently induced the angiogenesis-promoting gene Cyr61 and connective tissue growth factor. Inhibition of PAR-1 cleavage abolished MAP kinase phosphorylation and gene induction by Xa, demonstrating that Xa signals through PAR-1 and not through a novel member of the PAR family. Activation of cell surface prothrombin with the snake venom enzyme Ecarin also produced PAR-1-dependent signaling. However, though the response to Ecarin was completely blocked by the thrombin inhibitor hirudin, the response to Xa was not. This suggests that the Xa response is not mediated by locally generated thrombin. The concentration dependence of Xa for PAR-1 activation is consistent with previously characterized Xa-mediated PAR-2 signaling, suggesting that local concentration of Xa on the cell surface, rather than sequence-specific recognition of the PAR scissile bond, determines receptor cleavage. This study demonstrates that PAR-1 cleavage by Xa can elicit the same cellular response as thrombin, but mechanistic differences in receptor recognition may be crucial for specific roles for Xa in signaling during spatial or temporal separation from thrombin generation.     

Activation of factor XI in plasma is dependent on factor XII [see comments]

The activation of factor XI initiates the intrinsic coagulation pathway. Until recently it was believed that the main activator of factor XI is factor XIIa in conjunction with the cofactor high molecular weight kininogen on a negatively charged surface. Two recent reports have presented evidence that in a purified system factor XI is activatable by thrombin together with the soluble polyanion dextran sulfate. To assess the physiological relevance of these findings we studied the activation of factor XI in normal and factor XII-deficient plasma. We used either kaolin/cephalin or dextran sulfate as a surface for the intrinsic coagulation pathway, tissue factor to generate thrombin via the extrinsic pathway, or the addition of alpha-thrombin directly. 125I-factor XI, added to factor XI-deficient plasma at physiologic concentrations (35 nmol/L), is rapidly cleaved on incubation with kaolin. The kinetics appear to be exponential with half the maximum cleavage at 5 minutes. Similar kinetics of factor XI cleavage are seen when 40 nmol/L factor XIIa (equal to 10% of factor XII activation) is added to factor XII-deficient plasma if an activating surface is provided. Tissue factor (1:500) added to plasma did not induce cleavage of factor XI during a 90-minute incubation, although fibrin formation within 30 seconds indicated that thrombin was generated via the extrinsic pathway. Adding 1 mumol/L alpha-thrombin (equivalent to 50% prothrombin activation) directly to factor XII deficient or normal plasma (with or without kaolin/cephalin/Ca2+ or dextran sulfate) led to instantaneous fibrinogen cleavage, but again no cleavage of factor XI was observable. We conclude that in plasma surroundings factor XI is not activated by thrombin, and that proposals of thrombin initiation of the intrinsic coagulation cascade are not supportable.     

Activated factor V is a cofactor for the activation of factor XI by thrombin in plasma

The mechanism by which the intrinsic pathway of coagulation contributes to physiological hemostasis is enigmatic. Thrombin activates factor XI, a key zymogen in this pathway, which leads to increased thrombin generation. As thrombin-dependent activation of factor XI in vitro is relatively inefficient, we hypothesized that a physiological cofactor supports this reaction in a plasma environment. We therefore investigated whether the cofactors of coagulation, activated factor V, activated factor VIII, high-molecular weight kininogen, or protein S, influenced activation of factor XI by thrombin. Only activated factor V stimulated activation of factor XI by thrombin in a purified system. Binding studies demonstrated that factor XI specifically interacts with both factor V and factor Va through multiple binding sites. We further investigated this cofactor function of activated factor V in plasma. Depletion of factor V, or the addition of activated protein C, decreased the activation of the intrinsic pathway by thrombin in plasma. However, activated protein C did not exert this effect in the plasma of a homozygous carrier of the prothrombotic factor V Leiden mutation. In conclusion, we propose a role for (activated) factor V as a cofactor in the activation of factor XI by thrombin. These findings offer insights into the coagulation system in both health and disease.     

Inhibition of the activation of hageman factor (factor XII) by eosinophils and eosinophilic constituents

Several syndromes characterized by striking eosinophilia may be complicated by thrombosis. The experiments described indicate that, paradoxically, eosinophils and certain of their constituents inhibit the activation of Hageman factor (HF, factor XII). In earlier studies, suspensions of mixed types of granulocytes, other nucleated peripheral blood cells, and platelets inhibited activation of Hageman factor by ellagic acid, glass, and sulfatides. After these cells were sedimented by centrifugation, the supernatant fluids were also inhibitory. No attempt had been made earlier to distinguish among different granulocytic species. In the present study, suspensions of eosinophils and the supernatant fluid after eosinophils had been separated by centrifugation inhibited activation of Hageman factor by ellagic acid. The protein concentration of that amount of supernatant fluid that inhibited activation by about half was 16 μg/ml, approximately the same as had been described for suspensions of peripheral blood mononuclear cells. Activation of Hageman factor by ellagic acid was also inhibited by certain constituents of eosinophils, including eosinophil peroxidase, eosinophil major basic protein and eosinophil cationic protein. Inhibition was not specific for ellagic acid-induced activation of Hageman factor, as inhibition was also observed with sulfatide-induced activation. Inhibition was presumably related to neutralization of the negative charge of activators of Hageman factor. Thus, bismuth subgallate, a particulate activator of Hageman factor, was no longer effective after it had been exposed to eosinophil cationic protein. The observations reported here raise the question of whether in vivo eosinophils modulate certain of the defense reactions ascribed to Hageman factor. © 1993 Wiley-Liss, Inc.     

Activation of the kallikrein-kinin system by clonidine

Our results show that after the application of clonidine an activation of the kallikrein kinin system develops. The activation is characterized by the proof of active kallikrein and the decrease of kallikreinogen and the inhibitor capacity against kallikrein and trypsin. The extent of the activation seems to depend on the dosage of clonidine. The more pronounced reaction of the kallikrein kinin system might be connected with the side effects observed in the clonidine test.     

Amino-acid sequence of fragment A, an enzymically active fragment from diphtheria toxin.

The amino-acid sequence of Fragment A from diphtheria toxin is reported. Fragment A (molecular weight, Mr, 21,145) is the major enzymically active fragment produced upon activation of the intact toxin (Mr about 60,000) by limited tryptic digestion and reduction. It, or a similar fragment, is believed responsible for the inhibition of protein synthesis in animal cells exposed to the toxin. Fragment A, which corresponds to the amino terminus of the toxin, is shown here to consist of three major forms (190, 192, and 193 residues) resulting from cleavage by trypsin adjacent to any of three closely spaced arginine residues. All three forms are enzymically active.     

Oxytocin-induced activation of eukaryotic elongation factor 2 in myometrial cells is mediated by protein kinase C

The nonapeptide oxytocin (OT) mediates a wide spectrum of biological action, many of them related to reproduction. Recently, we have shown that OT exerts a trophic effect on uterine smooth muscle cells and induces dephosphorylation, and thus activation, of the translation elongation factor eukaryotic elongation factor 2 (eEF2). The present study was designed to elucidate the mechanisms underlying this novel action of OT in the well-characterized human myometrial cell line hTERT-C3. Pathways known to induce eEF2 dephosphorylation are mammalian target of rapamycin (mTOR), and the MAPKs ERK1/2 and p38. Using a panel of chemical inhibitors of specific signaling pathways, we determined that none of these pathways played a role in OT-mediated eEF2 dephosphorylation. Because the OT receptor is a G protein-coupled receptor linked to Galphaq, we tested the possibility that this OT action was mediated via protein kinase C (PKC). PKC activity was blocked by application of the general PKC chemical inhibitor Go6983 or by incubation with the cell-permeable PKC inhibitor peptide myr-psi PKC. With either approach, the effect of OT on eEF2 dephosphorylation was suppressed, indicating that the PKC pathway is essential for this OT action. Consistent with this idea, we also found that direct stimulation of PKC with the phorbol ester phorbol 12-myristate 13-acetate induced eEF2 dephosphorylation. Moreover, we observed that the stimulatory effect of OT on [(35)S]methionine incorporation into nascent proteins was blocked by PKC inhibition. Overall, these results define a novel hormonal signaling pathway that leads to eEF2 dephosphorylation and activation of protein synthesis.     

Inhibition of the activation of Hageman factor (factor XII) by human vascular endothelial cell culture supernates.

The supernatant fluid (conditioned medium) of cultured human vascular endothelial cells inhibits activation of Hageman factor (factor XII), whether by ellagic acid, bovine brain sulfatides, or bismuth subgallate; inhibition appears to be a property of one or more proteins in the culture supernates. This phenomenon may contribute to maintaining the fluidity of circulating blood by inhibiting surface activation of the intrinsic pathway of coagulation.     

Macrovascular thrombosis is driven by tissue factor derived primarily from the blood vessel wall

Leukocytes and leukocyte-derived microparticles contain low levels of tissue factor (TF) and incorporate into forming thrombi. Although this circulating pool of TF has been proposed to play a key role in thrombosis, its functional significance relative to that of vascular wall TF is poorly defined. We tested the hypothesis that leukocyte-derived TF contributes to thrombus formation in vivo. Compared to wild-type mice, mice with severe TF deficiency (ie, TF(-/-), hTF-Tg+, or "low-TF") demonstrated markedly impaired thrombus formation after carotid artery injury or inferior vena cava ligation. A bone marrow transplantation strategy was used to modulate levels of leukocyte-derived TF. Transplantation of low-TF marrow into wild-type mice did not suppress arterial or venous thrombus formation. Similarly, transplantation of wild-type marrow into low-TF mice did not accelerate thrombosis. In vitro analyses revealed that TF activity in the blood was very low and was markedly exceeded by that present in the vessel wall. Therefore, our results suggest that thrombus formation in the arterial and venous macrovasculature is driven primarily by TF derived from the blood vessel wall as opposed to leukocytes.     

Transfer of tissue factor from leukocytes to platelets is mediated by CD15 and tissue factor

We describe thrombogenic tissue factor (TF) on leukocyte-derived microparticles and their incorporation into spontaneous human thrombi. Polymorphonuclear leukocytes and monocytes transfer TF(+) particles to platelets, thereby making them capable of triggering and propagating thrombosis. This phenomenon calls into question the original dogma that vessel wall injury and exposure of TF within the vasculature to blood is sufficient for the occurrence of arterial thrombosis. The transfer of TF(+) leukocyte-derived particles is dependent on the interaction of CD15 and TF with platelets. Both the inhibition of TF transfer to platelets by antagonizing the interaction CD15 with P-selectin and the direct interaction of TF itself suggest a novel therapeutic approach to prevent thrombosis.     

Cerebrospinal fluid angiotensin II immunoreactivity is not derived from the plasma

To elucidate whether the presence of angiotensin II immunoreactivity (ANG II-ir) in the cerebrospinal fluid (CSF) of the dog is in part due to passage of the peptide across the CSF-blood-brain barrier, [Ile5] angiotensin II (ANG II) was infused intravenously for 7 days in conscious, trained dogs at a rate of 10 micrograms/kg/day. Mean arterial pressure (MAP) and heart rate were monitored each day, and samples of arterial blood and CSF (with a catheter secured into the cisterna magna) were drawn at regular intervals for determination of catecholamine levels, ANG II-ir, and electrolyte levels. Within 2 days after ANG II infusion, MAP stabilized at 35 +/- 1 mm Hg (mean +/- SE, p less than 0.001) above control values. The hypertension was associated with bradycardia, suppressed plasma renin activity, and a fall in both plasma and CSF Na+ concentrations. These changes coincided with a considerable and sustained decrease in the levels of plasma and CSF norepinephrine. On the other hand, levels of epinephrine and K+ in the two compartments remained unchanged. Although concentration of ANG II-ir in plasma was augmented markedly (368% above control values, p less than 0.001), ANG II-ir in the CSF remained within the low values measured in the control period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coronary no-reflow is caused by shedding of active tissue factor from dissected atherosclerotic plaque

Defined angiographically, no-reflow (NR) manifests as an acute reduction in coronary flow in the absence of epicardial vessel obstruction. One candidate protein to cause coronary NR is tissue factor (TF), which is abundant in atherosclerotic plaque and a cofactor for activated plasma coagulation factor VII. Scrapings from atherosclerotic carotid arteries contained TF activity (corresponding to 33.03 +/- 13.00 pg/cm(2) luminal plaque surface). Active TF was sedimented, indicating that TF was associated with membranes. Coronary blood was drawn from 6 patients undergoing coronary interventions with the distal protection device PercuSurge GuardWire (Traatek, Miami, FL). Fine particulate material that was recovered from coronary blood showed TF activity (corresponding to 91.1 +/- 62.16 pg/mL authentic TF). To examine the role of TF in acute coronary NR, blood was drawn via a catheter from coronary vessels in 13 patients during NR and after restoration of flow. Mean TF antigen levels were elevated during NR (194.3 +/- 142.8 pg/mL) as compared with levels after flow restoration (73.27 +/- 31.90 pg/mL; P =.02). To dissect the effects of particulate material and purified TF on flow, selective intracoronary injection of atherosclerotic material or purified relipidated TF was performed in a porcine model. TF induced NR in the model, thus strengthening the concept that TF is causal, not just a bystander to atherosclerotic plaque material. The data suggest that active TF is released from dissected coronary atherosclerotic plaque and is one of the factors causing the NR phenomenon. Thus, blood-borne TF in the coronary circulation is a major determinant of flow.     

Attenuation of insulin secretion by insulin-like growth factor 1 is mediated through activation of phosphodiesterase 3B

Both insulin and insulin-like growth factor 1 (IGF-1) are known to reduce glucose-dependent insulin secretion from the β cells of pancreatic islets. In this paper we show that the mechanism by which IGF-1 mediates this effect is in large part through activation of a specific cyclic nucleotide phosphodiesterase, phosphodiesterase 3B (PDE3B). More specifically, in both isolated pancreatic islets and insulin-secreting HIT-T15 cells, IGF-1 inhibits insulin secretion that has been increased by glucose and glucagonlike peptide 1 (GLP-1). Moreover, IGF-1 decreases cAMP levels in parallel to the reduction of insulin secretion. Insulin secretion stimulated by cAMP analogs that activate protein kinase A and also are substrates for PDE3B is also inhibited by IGF-1. However, IGF-1 does not inhibit insulin secretion stimulated by nonhydrolyzable cAMP analogs. In addition, selective inhibitors of PDE3B completely block the ability of IGF-1 to inhibit insulin secretion. Finally, PDE3B activity measured in vitro after immunoprecipitation from cells treated with IGF-1 is higher than the activity from control cells. Taken together with the fact that pancreatic β cells express little or no insulin receptor but large amounts of IGF-1 receptor, these data strongly suggest a new regulatory feedback loop model for the control of insulin secretion. In this model, increased insulin secretion in vivo will stimulate IGF-1 synthesis by the liver, and the secreted IGF-1 in turn feedback inhibits insulin secretion from the β cells through an IGF-1 receptor-mediated pathway. This pathway is likely to be particularly important when levels of both glucose and secretagogues such as GLP-1 are elevated.