Altered glucose regulation of insulin biosynthesis in insulinoma cells: mouse beta TC3 cells secrete insulin-related peptides predominantly via a constitutive pathway.

The effects of glucose on insulin gene expression and proinsulin biosynthesis, processing, and secretion were studied in mouse beta TC3 cells, an established insulinoma cell line derived from transgenic mice carrying a hybrid insulin promotor-simian virus-40 tumor antigen. The level of insulin mRNA was maintained at high levels by culture in 11 mM glucose, but essentially disappeared after 48 h of culture without glucose. The rate of insulin biosynthesis in beta TC3 cells was also dependent on glucose concentration over periods of 24 or 48 h (but not during 3 h) of stimulation. Insulin biosynthesis decreased about 50% after 24 h and about 85% after 48 h of incubation without glucose. When beta TC3 cells were incubated without glucose for 48 h, the rate of conversion of proinsulin to insulin was decreased compared to that at 11 mM glucose. Insulin secretion was sustained by medium glucose and also exhibited a much lower threshold for maximal stimulation; 2-deoxyglucose uptake decreased about 50% after 48 h of incubation without glucose. Studies on the secretion of newly synthesized proinsulin/insulin revealed that up to 80% of the total cellular pool of labeled proinsulin was released during a 60-min chase compared to only 10% of labeled insulin. The release of immunoreactive insulin (IRI) during the chase period was stimulated by forskolin and phorbol-12-myristate-13-acetate 1.6- and 10-fold, respectively. However, the release of newly synthesized proinsulin was insensitive to these secretagogues. It is concluded that 1) as in normal islets, glucose influences the steady state levels of proinsulin mRNA in beta TC3 cells; 2) the rate of proinsulin biosynthesis reflects only the level of insulin mRNA; translational control is absent; 3) cellular conversion of proinsulin to insulin is up-regulated by glucose as in normal rat islets; 4) newly synthesized proinsulin is released predominantly via a constitutive, rather than a regulated pathway, in contrast to normal beta-cells.     

Differential regulation by cytokines of constitutive and stimulated secretion of von Willebrand factor from endothelial cells

We examined the effect of cytokines on basal and agonist-stimulated release of von Willebrand factor (vWf) by human endothelial cells. Treatment of endothelial cells for up to 48 hours with human recombinant or purified interleukin 1 (IL-1) or human recombinant tumor necrosis factor-alpha (TNF-alpha) did not significantly affect constitutive secretion of vWf or intracellular levels of vWf, although basal prostacyclin (PGI2) production was markedly enhanced. In contrast, both IL-1 and TNF-alpha modulated vWf release in response to thrombin or phorbol ester. Pretreatment of endothelial cells for 2 hours with either cytokine enhanced by up to threefold the stimulatory effect of a subsequent 60-minute exposure to thrombin. Addition of cycloheximide (5 micrograms/mL) during the preincubation abolished this enhancement. Moreover, if the cytokine pretreatment time was extended to 24 hours, agonist-stimulated vWf release was significantly suppressed. Cytokine treatment for 2 or 24 hours had no detectable effect on levels of vWf messenger RNA. The effects of cytokines were not the result of contamination with bacterial lipopolysaccharide and were not attributable to endothelial cell injury. These results show that cytokines have little or no direct effect on vWf release from endothelial cells but can significantly modulate its acute release in response to other stimuli in a complex time- and dose-dependent manner.     

In vivo regulation of von willebrand factor synthesis: von Willebrand factor production in endothelial cells after lung transplantation between normal pigs and von Willebrand factor-deficient pigs

To evaluate the regulation of plasma von Willebrand factor (vWF) and its in situ production by endothelial cells (ECs), 12 swine leukocyte antigen (SLA)-compatible left lung transplantations were performed. Normal lungs were transplanted into 10 pigs homozygous for von Willebrand disease and into 2 normal pigs. Additionally, 1 normal pig underwent pneumonectomy, and 1 SLA-incompatible lung transplantation between normal pigs was performed. None of the transplanted animals received immunosuppressive therapy. Plasma vWF level was evaluated by ELISA and multimeric pattern. EC vWF content was assessed by immunohistochemistry. Global hemostasis was assessed by standardized ear bleeding time. Six of 12 SLA-compatible lung transplantations and the incompatible transplantation were successful and were used for the study. The functions and the viability of ECs, reflected by their ability to produce vWF and normal multimeric plasma vWF pattern, were preserved in SLA-compatible and -incompatible lung transplantations. vWF production was preserved in ECs that initially synthesized it. EC constitutive and storage pathways are modulated differently according to transplantation compatibility and severity of rejection. In SLA-compatible lung transplantations without histological evidence of rejection, the production of vWF was preserved, whereas constitutive vWF secretion appeared to be altered in cases with minor histological signs of rejection. In pigs with von Willebrand disease that were transplanted with normal lungs without sign of rejection, plasma vWF was significantly increased in an amount expected from the estimated production of a normal lung. In the transplanted normal lung, there was no vWF overexpression by the ECs and no recruitment of ECs that initially did not express vWF. In SLA-incompatible transplantation, ECs were morphologically normal with increased and blurred vWF labeling, whereas plasma vWF levels remained normal, reflecting that EC activation is associated with an increased vWF production with probable diversion to storage pathway. This model depicts the changes of EC regulation of vWF secretion in pig lung transplants. However, this model cannot be directly extrapolated to human organ transplantation because animals did not receive any immunosuppressive therapy, which may be toxic to ECs.     

Unique expression of von Willebrand factor by type IIA von Willebrand''s disease endothelial cells

Endothelial cells (EC) were cultured from the umbilical cord of a male neonate whose mother was previously diagnosed with type IIA von Willebrand's disease (vWd). The diagnosis of type IIA vWd in the proband was confirmed by low ristocetin activity and the absence of the highest molecular weight (MW) forms of von Willebrand factor (vWf) in his platelet poor plasma. The vWf of EC cultured from the neonate's umbilical cord differed from that of control EC and the cell line EA.hy926 in two respects. Firstly, the full range of molecular weight forms was present in the patient EC lysate and, secondly, vWf:Ag expression was approximately seven-fold greater than that of control cells. Platelet lysates prepared from other affected members of the type IIA vWd family in the presence or absence of proteolytic inhibitors demonstrated a near normal vWf multimeric distribution. Resistance of these high MW forms to heat degradation was conferred by the presence of proteolytic inhibitors. Moreover, the full plasma vWf multimeric distribution could not be restored by the inclusion of EDTA. N-ethylmaleimide and leupeptin in the anticoagulant during the rapid preparation of platelet poor plasma. These findings lend support to the heterogeneous nature of type IIA vWd and has possible implications in the understanding of the intracellular processes involved in the biosynthesis and storage of the vWf macromolecular complex as well as the pathogenesis of type IIA vWd.     

von Willebrand factor released from Weibel-Palade bodies binds more avidly to extracellular matrix than that secreted constitutively

Large multimers of von Willebrand factor (vWf) are released from the Weibel-Palade bodies of cultured endothelial cells following treatment with a secretagogue (Sporn et al, Cell 46:185, 1986). These multimers were shown by immunofluorescent staining to bind more extensively to the extracellular matrix of human foreskin fibroblasts than constitutively secreted vWf, which is composed predominantly of dimeric molecules. Increased binding of A23187-released vWf was not due to another component present in the releasate, since releasate from which vWf was adsorbed, when added together with constitutively secreted vWf, did not promote binding. When iodinated plasma vWf was overlaid onto the fibroblasts, the large forms bound preferentially to the matrix. These results indicated that the enhanced binding of the vWf released from the Weibel-Palade bodies was likely due to its large multimeric size. It appears that multivalency is an important component of vWf interaction with the extracellular matrix, just as has been shown for vWf interaction with platelets. The pool of vWf contained within the Weibel-Palade bodies, therefore, is not only especially suited for platelet binding, but also for interaction with the extracellular matrix.     

In vitro correction of the abnormal multimeric structure of von Willebrand factor in type IIa von Willebrand''s disease.

Type IIa von Willebrand's disease (vWd) has been characterized by the absence of the largest and a reduction in the intermediate-sized multimers of the plasma and platelet von Willebrand factor (vWf) and by the diminished response of the platelet-rich plasma of these patients to ristocetin. Other recently demonstrated abnormalities include the presence of an abnormal triplet structure of vWf. We have studied the plasma and platelets from three patients with this form of vWd and have found that both their plasma and platelets manifest the previously described abnormalities. Because of the heterogeneity of the multimeric structure of the vWf in these patients, we considered the possibility that postsynthetic events may have modified the vWf. When blood was collected in 5 mM EDTA or 5 mM EDTA/leupeptin/N-ethylmaleimide, the abnormal multimeric structure of the plasma and platelet vWf was partially normalized in that the intermediate and the largest vWf multimers were increased, the abnormal multimer structure was no longer as apparent, and the fastest migrating band (an abnormality seen only in the type IIa vWd plasma and platelets) disappeared. The enzymatic activity responsible for this degradation can be classified as a calcium-dependent protease. Studies of normal radiolabeled vWf incubated with platelet lysates from normal subjects and these patients revealed that the patients' platelets did not contain increased amounts of calcium-dependent protease activity as assessed by degradation of normal vWf. These data suggest that patients with type IIa vWd synthesize an abnormal vWf protein that is susceptible to in vitro proteolytic degradation and that proteolytic degradation can play a significant role in the phenotypic expression of vWd by modifying the plasma and platelet vWf multimeric structure.     

Storage of tissue-type plasminogen activator in Weibel-Palade bodies of human endothelial cells.

Tissue-type plasminogen activator (t-PA) is acutely released by endothelial cells. Although its endothelial storage compartment is still not well defined, t-PA release is often accompanied by release of von Willebrand factor (vWf), a protein stored in Weibel-Palade bodies. We investigated, therefore, whether t-PA is stored in these secretory organelles. Under basal culture conditions, a minority of human umbilical vein endothelial cells (HUVEC) exhibited immunofluorescent staining for t-PA, which was observed only in Weibel-Palade bodies. To increase t-PA expression, HUVEC were infected with a t-PA recombinant adenovirus (AdCMVt-PA). Overexpressed t-PA was detected in Weibel-Palade bodies and acutely released together with endogenous vWf by thrombin or calcium ionophore stimulation. In contrast, plasminogen activator inhibitor type 1 and urokinase were not detected in Weibel-Palade bodies after adenovirus-mediated overexpression. Infection of HUVEC with proinsulin recombinant adenovirus resulted in the storage of insulin in Weibel-Palade bodies, indicating that these organelles can also store nonendothelial proteins that show regulated secretion. Infection of AtT-20 pituitary cells, a cell type with regulated secretion, with AdCMVt-PA resulted in the localization of t-PA in adrenocorticotropic hormone-containing granules, indicating that t-PA can be diverted to secretory granules independently of vWf. Coinfection of AtT-20 cells with AdCMVt-PA and proinsulin recombinant adenovirus resulted in the colocalization of t-PA and insulin in the same granules. Taken together, these results suggest that HUVEC have protein sorting mechanisms similar to those of other regulated secretory cells. Although the results did not exclude an alternative storage site for t-PA in HUVEC, they established that t-PA can be stored in Weibel-Palade bodies. This finding may explain the acute coordinate secretion of t-PA and vWf.     

A variant of type II von Willebrand disease with an abnormal triplet structure and discordant effects of protease inhibitors on plasma and platelet von Willebrand factor structure

We have characterized the plasma and platelet von Willebrand factor (vWf) multimeric structure of a patient with von Willebrand disease (vWd) as having a long bleeding time, no aggregation of her platelet-rich plasma (PRP) to ristocetin, and very low plasma and platelet von Willebrand antigen (vWf Ag) and vWf activity. The abnormalities of the plasma and platelet vWf have not been previously described. In particular, the patient's plasma and platelet vWf lacked the intermediate and largest vWf multimers and the slowest migrating minor band (band 1) of the triplet was markedly diminished compared to the major band (band 2) and the fastest migrating band of the triplet (band 3). A similar multimeric structure was seen in the platelet vWf. Collection of the patient's blood in protease inhibitors prior to the analysis of the platelet and plasma vWf structure revealed that the plasma vWf multimeric structure did not change, while the patient's platelet vWf showed a marked change with the appearance of the intermediate and large multimers and almost total disappearance of the abnormal multimeric structure. Direct comparison of this patient's plasma with previously reported cases of type IIA, IIC, and IID revealed marked differences in their multimeric organization compared to our patient. Employing the presently accepted convention, we have designated this variant type of vWd type IIG.     

Use of DDAVP in inherited and acquired platelet dysfunction

Twenty-one patients with prolonged bleeding times secondary to inherited disorders of platelet function and eight patients with prolonged bleeding times secondary to acquired platelet dysfunction were given 0.3 micrograms per kilogram of DDAVP, 1-deamino-8-D-arginine vasopressin, intravenously. Sixteen of twenty-two DDAVP trials in patients with inherited platelet dysfunction (73%) and seven of the nine DDAVP trials in patients with acquired platelet dysfunction (78%) resulted in normalization or shortening of the prolonged bleeding times by at least 4 min. The bleeding time response did not correlate with changes in the levels of von Willebrand factor (vWf) antigen or ristocetin cofactor activity, nor was it associated with changes in vWf multimeric analysis or in vitro platelet aggregations following the administration of DDAVP. Shortening of the bleeding time with DDAVP was seen in patients with a failure to release/storage pool type defect, thromboxane synthesis type defect, Bernard-Soulier syndrome, Glanzmann's thrombasthenia, the May-Hegglin anomaly, liver disease, nonuremic renal disease, myelofibrosis, and Tangier's disease.     

The high molecular weight form of endothelial cell von Willebrand factor is released by the regulated pathway

We have previously reported that two forms of von Willebrand factor (vWf) exist in cultured human umbilical vein endothelial cells: a high molecular weight (HMW) form that is released and can be proteolytically cleaved into a series of plasma-like multimers, and a non-secreted low molecular weight (LMW) form. In this study, the mode of vWf release and the relationship between the two forms were examined. As determined by two-dimensional analysis as well as by immunoreactivity with an antibody to the propolypeptide, the LMW form of endothelial cell vWf consisted of a 260 kD pro-vWf polypeptide, while the HMW form consisted of a 225 kD mature polypeptide. Only the 260 kD polypeptide was susceptible to digestion with endoglycosidase H. Release of the HMW form into the culture media was accompanied by a decrease in cellular vWf. Treatment of endothelial cells with cycloheximide or tunicamycin caused a decrease in the LMW form but did not affect the secretion of the HMW form. These results suggest that two pools of vWf exist in endothelial cells--a LMW form of pro-vWf in the endoplasmic reticulum and a HMW form of mature vWf in the storage compartment. Released vWf derives only from the storage pool.     

Proteoglycan metabolism in normal and inflammatory human macrophages

To study proteoglycan metabolism in inflammatory macrophages, primary cultures of human macrophages were cultured in the absence and presence of bacterial lipopolysaccharide (LPS). When exposed to [35S]sulfate, the cells incorporated the label almost exclusively into chondroitin sulfate proteoglycan (CSPG), which was recovered from the culture medium and the cell layer. Cells stimulated with LPS secreted approximately three times more [35]CSPG into the culture medium than control cells. Furthermore, cell adhesion was also found to promote proteoglycan secretion; when nonadherent monocytic cells were induced to adhere, the release of proteoglycan increased two times. The increased secretion seen in LPS-stimulated macrophages was partly due to increased biosynthesis, but was mostly due to increased sorting of CSPG to the secretory pathway. Only about 20% of the CSPG synthesized in unstimulated cells was secreted, whereas the corresponding figure in LPS-treated cells was 35%. In both cell types, the remaining [35S]CSPG was degraded, probably in the lysosomes. The degradation was a two-step process. First, the [35S]CSPG was rapidly cleaved to yield free glycosaminoglycan (GAG) chains (t1/2 = 15 to 30 minutes). Secondly, the GAG chains were completely depolymerized (t1/2 = 2 to 3 hours). Neither resting nor LPS-stimulated cells sorted CSPG to intracellular storage, as is evident in many hematopoietic cells. The LPS-treated cells synthesized [35S]CSPG of smaller molecular size than did control cells, with GAG chains of approximate molecular mass of 12 kD versus 16 kD in control cells. No difference was seen in the disaccharide composition of the GAG chains; both LPS-stimulated and unstimulated cells expressed a mixture of 80% to 90% chondroitin 4-sulfate and 10% to 20% chondroitin 4,6-disulfate. N-terminal sequence and Northern blot analysis indicate that the core protein of the CSPG secreted by human macrophages is serglycin.     

Vicinal cysteines in the prosequence play a role in von Willebrand factor multimer assembly.

von Willebrand factor (vWf) is synthesized as a large precursor that dimerizes in the endoplasmic reticulum and forms multimers in the trans- and post-Golgi compartments of megakaryocytes and endothelial cells. The disulfide-bonded multimers are stored in alpha granules of platelets and Weibel-Palade bodies of endothelial cells. The prosequence, composed of two homologous D domains, is required for vWf multimerization and storage. Each D domain contains vicinal cysteines (159Cys-Gly-Leu-162Cys and 521Cys-Gly-Leu-524Cys) that are similar to those at the active site of disulfide isomerases that catalyze thiol protein disulfide interchange. As in disulfide isomerases, a positively charged amino acid (lysine) is also found in close proximity to the vicinal cysteines. Although conserved, the lysine present in thioredoxin was shown not to be essential for its redox activity. We investigated the role of the vicinal cysteines and the lysine residue in the vWf propolypeptide by site-directed mutagenesis and expression of the resulting constructs in mammalian cells. Insertion of an extra glycine between the vicinal cysteines in either D domain inhibited multimerization of dimers, whereas alteration of lysine to glycine in both domains (residues 157 and 519) had no effect. This suggests the importance of the vicinal cysteines but not the lysines in vWf multimerization. Expression of the mutant with an additional glycine in the D1 domain in AtT-20 cells, a mouse pituitary cell line that can store vWf, led to the storage of the resulting dimers. This demonstrates that the mutation did not effect the capacity of the propolypeptide to direct vWf storage while its ability to promote interchain disulfide bonding was eliminated.     

Ribose accelerates the repletion of the ATP pool during recovery from reversible ischemia of the rat myocardium

It is a characteristic feature of the myocardium that the derangement in function [6] and the depletion of the ATP pool [1, 2, 9] that occur subsequent to oxygen deficiency persist when blood flow is restored. Of renewed interest is the inability of the heart to replenish rapidly its adenine nucleotide pool once it has been diminished during a brief period of regional ischemia [2, 9]. A hypothesis that could explain this metabolic insufficiency of the myocardium is that the biosynthesis of adenine nucleotides is very slow in the normal heart and is increased only moderately during postischemic recovery [15] so that the replenishment of adenine nucleotides is not affected appreciably. To substantiate such a hypothesis it is necessary to provide evidence that the restitution of the ATP pool can be accelerated by stimulation of this biosynthetic process. In previous studies ribose has been recognized as a substrate that enhances markedly adenine nucleotide biosynthesis in the rat heart [11, 12]. We now demonstrate that continuous i.v. infusion of ribose during recovery from a 15-min period of myocardial ischemia in rats leads to restoration of the cardiac ATP pool within 12 h, whereas 72 h are needed for ATP normalization without any intervention.     

Canine platelet von Willebrand factor: quantification and multimeric analysis

Washed canine platelets were shown to express a significant level of von Willebrand factor (vWf). Canine platelet vWf differed from canine plasma vWf by the absence of satellite bands associated with each multimer when resolved by SDS-agarose gel electrophoresis. Expression and multimeric composition of canine platelet vWf was quite similar to that of human platelet vWf. Quantification in both lysed, washed canine platelets and in releasate of washed canine platelets yielded estimates of platelet vWf at approximately 2% of circulating vWf in this species, with approximately 15% of this being released into the fluid phase on activation. This contrasts with findings in humans, in which approximately 10%-25% of circulating vWf is compartmentalized in platelets. The difference in relative levels of canine and human platelet vWf could not be accounted for by differences in platelet ultrastructure. The decreased relative level may account for reports that canine platelets contain no vWf.     

Enhanced ex vivo proteolysis of plasma von Willebrand factor in disseminated intravascular coagulation.

Plasma levels of von Willebrand factor (vWf) are frequently elevated in patients with disseminated intravascular coagulation (DIC). To investigate the qualitative abnormalities of vWf and the possibility of its ex vivo modification in DIC, we analysed the multimeric composition of vWf in citrated plasma from 15 patients with DIC in the presence or absence of serine protease inhibitors (aprotinin and soybean trypsin inhibitor) and/or cysteine protease inhibitors (leupeptin, N-ethylmaleimide and EDTA). The proportion of large vWf multimers in plasma prepared in the presence of cysteine protease inhibitors was higher than those without such inhibitors. The addition of serine protease inhibitors during the preparation of plasma had no effect on the relative amounts of large multimers. The relative proportion of large multimers in plasma prepared without inhibitors and the difference between plasmas prepared with and without cysteine protease inhibitors correlated with plasma plasmin-alpha 2-plasmin inhibitor complex values, but not with other plasma or serum markers of DIC (platelet count, fibrinogen, FDP, D-dimer or thrombin-antithrombin III complex). We conclude that ex vivo proteolysis of plasma vWf occurs frequently in patients with DIC and cysteine protease inhibitors can protect this degradation.     

Purification and characterization of human platelet von Willebrand factor

Summary. Platelet von Willebrand factor (vWf) was purified from human platelet concentrates. The multimeric structure of the purified platelet vWf was similar to that observed in the initial platelet lysate, and, like the platelet lysate, the purified platelet vWf contained higher molecular weight multimers than plasma vWf. The apparent molecular weight of the reduced platelet vWf subunit was similar to the plasma vWf subunit. The N-terminal amino acid of the purified platelet and plasma vWf was blocked. In concentration dependent binding to botrocetin- or ristocetin-stimulated platelets, ¹²⁵I-plasma vWf bound with a higher affinity than platelet. The ristocetin cofactor activity per mg of purified plasma vWf was 5-fold greater than the platelet vWf activity. Platelet and plasma vWf bound to collagen with similar affinities; however, platelet vWf bound to thrombin-stimulated platelets and to heparin with a higher affinity than plasma vWf. The differences in the binding affinity(s) of plasma and platelet vWf to platelet GPIb and GPIIb/IIIa and extracellular matrix proteins may reflect different roles for plasma and platelet vWf in the initial stages of haemostasis and thrombosis.     

Biosynthesis of human von Willebrand factor

Endothelium forms the inner lining of all blood vessels and, as a consequence, is in direct contact with the blood. Because of this and the synthesis and secretion of hemostatic components, the endothelium is able to modulate coagulation and fibrinolysis. An important hemostatic factor synthesized by endothelial cells is the von Willebrand factor (vWF). vWF is a large plasma glycoprotein which promotes the adhesion of platelets to the vessel wall after a vascular injury. vWF is initially synthesized as a pre-pro-polypeptide. During its transport to the outside of the cell, the single-chain polypeptides are assembled into multimers. The pro-polypeptide can be cleaved and also be secreted. Free pro-polypeptide is identified as von Willebrand antigen II, a plasma glycoprotein of unknown function. Plasma vWF consists of a heterogenous series of multimers, composed of an apparently single-type glycoprotein subunit, linked together by disulfide bonds. The hemostatic potency of vWF was shown to increase with increasing multimer size. Therefore, the multimeric assembly of vWF is a crucial aspect in vWF biosynthesis. Furthermore, vWF synthesized by endothelial cells can either be secreted constitutively or stored and released upon stimulation of the endothelial cell. In this review, data are presented which contribute to the understanding of the biosynthetic pathway and complex processing which vWF has to undergo before it is secreted by the endothelial cell. These data have allowed a prediction of the sequential events underlying vWF biosynthesis, processing, multimer assembly, and secretion.     

Basal secretion of von Willebrand factor from human endothelial cells

Endothelial cells store the adhesive glycoprotein von Willebrand factor (VWF) in Weibel-Palade bodies (WPBs), distinctively shaped regulated secretory organelles that undergo exocytosis in response to secretagogue. A significant proportion of newly synthesized VWF is also secreted spontaneously from nonstimulated cells, through what is thought to be the constitutive secretory pathway. To learn more about VWF trafficking, we performed kinetic analyses of the storage and nonstimulated secretion of VWF in cultured human endothelial cells. We found that most VWF was secreted through a route that was significantly delayed compared with constitutive secretion, although this pathway was responsible for secretion of a small amount of uncleaved VWF precursor. Disruption of pH-dependent sorting processes with ammonium chloride converted the secretion kinetics of mature VWF to that of its precursor. Conversely, preventing constitutive secretion of nascent protein with brefeldin A had only a modest effect on the spontaneous release of VWF, showing that most VWF secreted by nonstimulated cells was not constitutive secretion but basal release of a post-Golgi storage organelle, presumably the WPB. These data suggest that VWF is sorted to the regulated secretory pathway in endothelial cells much more efficiently than previously reported.     

Kallikrein activates prothrombin.

Kallikrein is a multitalented enzyme in hemostasis and inflammation. Normally, kallikrein is formed in intrinsic hemostasis and activates factor XII. A total of 10 microL of 0 to 100 microg/mL human plasma kallikrein in 6% human albumin-PBS were incubated with 90 microL 111.1 microg/mL prothrombin in 6% human albumin in absence and presence of 23 mM Ca(++). After 0 to 64 minutes (37 degrees C), 100 microL of 2.5 M arginine, pH 9, were added. Fifty microliters of 0.72 mM HD-CHG-Ala-Arg-pNA in 1.36 M arginine were added and increase in absorbance at 405 nm was determined. Within 8 minutes (37 degrees C), 1 microg/mL kallikrein, ie, 2.5% of the normal plasmatic prekallikrein concentration, generates approximately 3 mIU/mL thrombin in absence and 27 mIU/mL thrombin in presence of Ca(++). Kallikrein can directly activate prothrombin; there is a shortcut in the intrinsic hemostasis system that generates catalytic amounts of thrombin without following the known intrinsic clotting pathway.     

Spontaneous production of mediators by mononuclear human blood cells (author's transl)

Human mononuclear blood cells incubated in vitro without any intended stimulus produce lymphokine-like activities. Separated lymphocytes release a leukocyte migration stimulatory factor spontaneously. The spontaneous production of a leukocyte migration inhibitory factor in unstimulated cultures during the first 20 hours needs for a cooperation between lymphocytes and monocytes. As well lymphocytes as enriched monocytes elaborate leukocyte chemotactic activity. --The spontaneous activation is caused by the increasing acidity of the culture medium following metabolic processes. The dextran sedimentation and the Ficoll content of the separation solution were without importance for the stimulation.