Plasma High Molecular Weight Research Articles

Role of kinins in the cardioprotective effect of preconditioning: study of myocardial ischemia/reperfusion injury in B2 kinin receptor knockout mice and kininogen-deficient rats.

Kinins acting on the B2 receptor appear to be involved in the cardioprotective effect of preconditioning on myocardial ischemia/reperfusion injury. We tested the hypothesis that in mice lacking the gene encoding for the B2 kinin receptor (B2 knockout mice; B2-KO) as well as in rats deficient in high-molecular-weight (HMW) kininogen (Brown Norway Katholiek rats; BNK), the cardioprotective effect of preconditioning is diminished or abolished. 129SvEvTac (SV129) mice and Brown Norway rats (BN) served as controls. We confirmed that plasma HMW kininogen in BNK rats was 100-fold lower than in BN and 140-fold lower than in Sprague-Dawley rats (33+/-4 versus 1814+/-253 and 2397+/-302 ng/mL, P<.01). Each strain of mice was divided into (1) controls (without preconditioning); (2) one cycle of preconditioning (3 minutes ligation and 5 minutes reperfusion); and (3) three cycles of preconditioning. Each strain of rats was divided into (1) controls; and (2) three cycles of preconditioning. All animals were subjected to 30 minutes of ischemia and 120 minutes of reperfusion. In SV129 controls, the ratio of infarct size to risk area (IS/AR) was 55.6+/-4.6%. One and three cycles of preconditioning reduced IS/AR to 38.6+/-3.2% and 31.1+/-2.3%, respectively (P<.05 and P<.01 versus control). This protective effect was absent in B2-KO mice: IS/AR was 54.8+/-2.9% in controls, 58.5+/-3.6% with one cycle of preconditioning, and 58.5+/-3.4% with three cycles. In BN rats without preconditioning, IS/AR was 84.7+/-3.9%; preconditioning reduced it to 61.6+/-3.4% (P<.01). In BNK rats, IS/AR was 87.1+/-4.8% in controls and 84.3+/-4.1% with preconditioning. Preconditioning also prevented reperfusion arrhythmias in BN but not BNK rats. Within species, risk area, mean blood pressure, and heart rate were similar between strains. We concluded that (1) preconditioning protects the heart against ischemia/reperfusion injury in mice and rats; (2) activation of prekallikrein, which in turn generates kinins from HMW kininogen, may contribute to the effect of preconditioning; and (3) an intact kallikrein-kinin system is necessary for the cardioprotective effect of preconditioning.

Cleavage of plasma high molecular weight kininogen in surgical ICU patients

Cleavage of plasma high molecular weight kininogen in surgical ICU patients

Cleavage of plasma high molecular weight kininogen in surgical ICU patients.

To characterize kininogens in plasma from surgical patients in the intensive care unit (ICU). Prospective study. Surgical ICU. 35 patients aged 19-79 years, divided into two groups: sepsis (defined by standard criteria) and nonsepsis. Studies of proteolytic degradation of H-kininogen showed degradation in both patient groups compared to healthy controls. Functional quantification of prekallikrein showed a reduction of prekallikrein in plasma from both patients groups. Functional quantification of kininogens by a cysteine proteinase inhibitor assay showed no significant differences between the patients and the controls. Immunological levels of H-kininogen and total kininogen were not significantly different from normal plasma. No differences could be detected between the two patient groups in any of the parameters studied. This study showed that contact activation took place in surgical ICU patients: partial kinin release and consumption of prekallikrein took place in vivo.

Direct evidence for multifacial contacts between high molecular weight kininogen and plasma prekallikrein.

HK31 (S565-K595) has previously been shown to encompass the binding domain for plasma prekallikrein (PK) within domain 6 of high molecular weight kininogen (HK). The complementary binding domain for HK within PK is mapped to PK56 (F56-G86), in the apple 1 domain, and to PK266 (K266-C295), in the apple 4 domain. Isothermal titration calorimetry was used to directly monitor binding among HK31, PK56, and PK266. Either PK peptide binds to HK31 in 1:1 stoichiometry, regardless of whether a binary complex is first formed between PK266 and HK31 or between PK56 and HK31. Binding of the alternate PK peptide into a ternary complex is facilitated nearly 2-fold. The ternary complex consists of 1:1:1 HK31:PK56:PK266. Furthermore, binary and ternary complex formation is entropically driven and thermodynamically favored, suggesting that the conformational changes accompany binding. Fluorescence emission spectroscopy revealed that binding of PK56 caused a limited decrease in intrinsic tryptophan fluorescence emission intensity of HK31 while binding of PK266 to HK31 or the complex of HK31/PK56 had no such effect. We conclude that the two PK peptides bind to the HK peptide at different sites. The binding between HK and PK is likely due to conformational changes which serve to juxtapose the PK binding domain within HK with the HK binding site involving two spatial proximity segments.

Pharmacological manipulation of the complement system in human diseases.

Complement is one of the powerful effector systems involved in the body's defense. When present in a dormant state it can, in concert with other components of immune system, protect the individual from foreign pathogens. However, inappropriately activated complement can cause disease. Several disease states such as immune complex and autoimmune diseases and deficiencies of some complement regulators are associated with inappropriate activation of complement. In some diseases complement is activated for a long or indefinite period while in others for a comparatively short time; in some it is activated systemically, in others locally; in some whole cascade is activated, in others only a few components are activated; in some classical pathway is activated, in others alternative pathway. In some diseases activation of complement takes place on cell and tissue surfaces. In many complement activating diseases biological activities of complement fragments become detrimental resulting in tissue injury and disease. Inhibition of complement by specific inhibitors is likely to arrest complement mediated disease processes. From this point of view, some laboratories are developing low molecular weight synthetic inhibitors whereas others are focusing on the development of high molecular weight plasma or cell surface complement inhibitors in their natural or recombinant forms for therapeutic purposes. A review concerning development of low molecular weight inhibitors with the eventual aim of manipulating complement system in human diseases was recently published (1,2). This review is concerned with high molecular weight natural or recombinant complement inhibitory molecules in human plasma or cell membranes, some of which are already in clinical use.

Activation of the plasma contact system and hemodynamic changes after graft revascularization in liver transplantation.

In this study, the relation between activation of the plasma contact system and hemodynamic changes during orthotopic liver transplantation was evaluated. Nineteen consecutive courses of OLT in 17 adult patients were investigated. Veno-venous bypass was used in all patients. Blood samples were drawn through all phases of the procedure, and analyzed for the following parameters using functional techniques (chromogenic peptide substrate assays): plasma kallikrein (KK), prekallikrein, functional plasma kallikrein inhibition, C1 inhibitor, and alpha 2-macroglobulin. Plasma high molecular weight kininogen (HK) degradation was evaluated using the immunoblotting technique. An abrupt rise in KK activities occurred within 1 min after portal reperfusion of the liver graft (7-16 U/L, P < 0.05). Simultaneously, proteolytic breakdown of HK was seen. The elevated KK activities were maintained the next 1 1/2 hr. Ten min after graft reperfusion, a significant increase in cardiac output compared with the anhepatic phase (7.2-12.4 L/min, P < 0.05) was found. At the same time, systemic vascular resistance fell significantly (817-408 dynes x sec/cm-5, P < 0.05). The increase in plasma KK activities accompanied by simultaneous degradation of HK seen immediately after reperfusion of the liver graft may be due to contact activation as recipient blood contacts with the underlying basement membrane of injured sinusoidal endothelium in the transplanted liver. We suggest that hemodynamic changes associated with the postreperfusion syndrome seen after revascularization of the liver in OLT could at least be caused in part by bradykinin release due to contact activation.

Conformational Changes in Low Molecular Weight Kininogen Alters Its Ability to Bind to Endothelial Cells

The plasma kininogens, high (HK) and low (LK) molecular weight kininogens, are the parent proteins for bradykinin, a potent vasoactive peptide that locally influences vascular biology. Binding of both HK and LK to the endovascular wall contributes to bradykinin delivery. Recently, we found one preparation of LK (LKd) which had reduced inhibition of biotin-HK binding to endothelium. The functional defect in LKd was not merely due to bradykinin loss because two preparations of bradykinin-free LK blocked biotin-HK binding. However, using two different particular monoclonal antibodies to bradykinin, LKd, but no other preparation of LK, had its epitope to bradykinin exposed on non-reduced samples on immunoblot. These data suggested that LKd had an altered conformation which exposed the amino terminal arginine of bradykinin to antigenic detection. The altered conformation of LKd allowed it to be more susceptible to trypsin proteolysis. On circular dichroism, the percentage of alpha-helix was significantly increased, indicating an alteration in the protein. This alteration in LKd was not due to a loss of molecular mass of the protein. On laser desorption mass spectroscopy, the molecular mass of LKd was similar to the other preparations of LK. Investigations were performed to ascertain the mechanism by which LKd had altered ability to bind to cells. LKd was found to be proteolyzed by an unknown protease at the beginning of domain 2 between threonine119 and alanine120. Reduction of functional LK with dithiothreitol to expose its bradykinin epitope did not produce the LKd defect. Proteolysis of functional LK with plasma kallikrein, elastase followed by plasma kallikrein, chymotrypsin, or bromelain also did not produce the defect seen in LKd. These combined data indicated that LK maintains a particular conformation that allows the protein to orient itself such that it can bind to endothelial cells. Proteolysis in the surface exposed region between domains 1 and 2 probably allows for the protein to unfold and contributes to its lost ability to bind to endothelial cells.

Characterization of protein involvement in rabies virus binding to BHK-21 cells.

Prior studies established the specificity of rabies virus receptors on BHK-21 cells based on the saturability of the receptors and on competitive binding. In the present study, we used protease-treated cells to identify the involvement of protein in the specific binding of rabies virus to these cells. In addition, biochemical characterization of n-octylglucoside solubilized BHK-21 plasma membranes demonstrated the involvement of a protease sensitive, heat insensitive, integral membrane protein or protein complex in rabies virus binding to these cells. The membrane component that binds rabies virus is associated with a high molecular weight fraction of the n-octylglucoside-plasma membrane extract isolated by gel filtration. This high molecular weight fraction (approximately 450 KDa) is enriched with a cell surface integral membrane component that comigrates with denatured bovine serum fibronectin (220 KDa). This cellular component did not bind polyclonal antisera to fibronectin in Western blot (native or denatured) or immunoprecipitation experiments. Direct and specific virus binding to high molecular weight plasma membrane protein(s) separated on Western blots further confirmed the role of a protein receptor in rabies virus binding to these cells.

Renal function and hyperfiltration capacity in lead smelter workers with high bone lead.

The study was undertaken to assess whether the changes in urinary excretion of eicosanoids (a decrease of 6-keto-PGF1 alpha and PGF2 and an increase of thromboxane) previously found in lead (Pb) exposed workers may decrease the renal haemodynamic response to an acute oral protein load. The renal haemodynamic response was estimated by determining the capacity of the kidney to increase the glomerular filtration rate (in terms of creatinine clearance) after an acute consumption of cooked red meat (400 g). A cross sectional study was carried out in 76 male Pb workers (age range 30 to 60 years) and 68 controls matched for age, sex, socioeconomic state, general environment (residence), and workshift characteristics. The Pb workers had been exposed to lead on average for 18 (range 6-36) years and showed a threefold higher body burden of Pb than the controls as estimated by in vivo measurements of tibial Pb concentration (Pb-T) (geometric mean 66 v 21 micrograms Pb/g bone mineral). The geometric mean concentrations of Pb in blood (Pb-B) and Pb in urine (Pb-U) were also significantly higher in the Pb group (Pb-B: 430 v 141 micrograms Pb/l; Pb-U: 40 v 7.5 micrograms Pb/g creatinine). These conditions of chronic exposure to Pb did not entail any significant changes in the concentration of blood borne and urinary markers of nephrotoxicity, such as urinary low and high molecular weight plasma derived proteins (beta 2-microglobulin, retinol binding protein, albumin, transferrin), urinary activities of N-acetyl-beta-D-glucosaminidase and kallikrein, and serum concentrations of creatinine, beta 2-microglobulin, urea, and uric acid. All participants also had normal baseline creatinine clearances (> 80 ml/min/1.73 m2) amounting on average to 115.5 in the controls v 121.3 ml/min/1.73 m2 in the Pb group. Both control and Pb exposed workers showed a significant increment in creatinine clearance (on average 15%) after oral protein load suggesting that the previously found changes in secretion of urinary eicosanoids apparently has no deleterious effect on renal haemodynamics in the examined Pb workers. The finding that both baseline and stimulated creatinine clearance rates were not only significantly higher in the Pb workers but also positively correlated with Pb-T, suggests that moderate exposure to Pb may be associated with a slight hyperfiltration state, which has been found to attenuate the age related decline in baseline creatinine clearance by a factor of two. Although the relevance of this effect for the worker's health is unknown, it can be concluded that adverse renal changes are unlikely to occur in most adult male Pb workers when their blood Pb concentration is regularly kept below 700 micrograms Pb/l. One should, however, be cautious in extra-polating this conclusion to the general population because of pre-employment screening of the Pb workers for the absence of renal risk factors.

Abnormal Multimeric and Oligomeric Composition Is Associated With Enhanced Endothelial Expression of von Willebrand Factor in Pulmonary Hypertension

Abnormalities in endothelial von Willebrand factor (vWF) structure have been reported in pulmonary hypertension. These include loss of high molecular weight plasma multimers, resulting in decreased biologic activity. If endothelial processing of vWF is altered in this disorder, abnormalities in oligomeric composition may also be expected. We examined this possibility in ten adult patients with primary pulmonary hypertension. Enhanced endothelial vWF expression in these patients was indicated by increased plasma levels of vWF antigen (vWF:Ag) (214 +/- 91 vs 99 +/- 51 percent activity in controls, p < 0.001) and intense immunoperoxidase stain of pulmonary arterial endothelium for vWF (autopsy, 1 patient). Plasma from these patients also had a decreased capacity of inducing platelet aggregation in the presence of ristocetin, relative to vWF:Ag levels (57 +/- 20 percent activity). In addition to mild loss of the largest multimers, changes in oligomeric composition of plasma vWF were observed in most patients using both agarose and polyacrylamide gel electrophoresis. These included decreased concentration of dimeric (470 kDa) vWF in most patients, variable concentration of the 860-kDa fraction, and a relative decrease in subunit (223 kDa) density in subjects with elevated vWF:Ag. These findings provide additional information on the mechanisms responsible for endothelial production of dysfunctional vWF in patients with pulmonary hypertension.

Predominance of High Molecular Weight Plasma Na+-K+-ATPase Inhibitor in Essential Hypertension

Circulating inhibitors of the transport enzyme, sodium-potassium-activated adenosine triphosphatase (Na(+)-K(+)-ATPase), have been shown to be of possible pathogenetic importance in the mechanism of essential hypertension. Although previous studies have demonstrated the presence of both high molecular weight (HMW) and low molecular weight (LMW) natriuretic plasma Na(+)-K(+)-ATPase inhibitors, no previous attempts have been made to ascertain whether HMW or LMW forms predominate in hypertension. In this study, plasma samples obtained from 26 patients with essential hypertension, 12 normotensive controls, and six normotensives with a family history of hypertension, were separated into HMW and LMW moieties by passage through a 1 kDa Amicon membrane. The LMW moiety was separated on C18 Sep-Pak cartridges, applying a 10% step-wise acetonitrile trifluoroacetic acid gradient. The HMW moiety was further separated on Sephadex G-75. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the fraction with inhibitory activity contained a distinct 12 kDa protein band, with staining intensity depending on the presence or absence of hypertension. Na(+)-K(+)-ATPase inhibitory activity was found in several LMW fractions, but differences between hypertensives and normotensives were observed in only one fraction (0.29 +/- 0.12 SD v 0.11 +/- .12 mumol/L ouabain equivalents, P < .01). Na(+)-K(+)-ATPase inhibitory activity in the HMW fraction was 38 x the inhibitory activity in the LMW fraction and was significantly increased in hypertensives as compared to normotensive controls (10.9 +/- 8.9 v 1.3 +/- 0.8 mumol/L ouabain equivalents, P < .01). Inhibitory activity in both HMW and LMW fractions correlated positively with mean blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of microbial and mite proteases on low and high molecular weight kininogens. Generation of kinin and inactivation of thiol protease inhibitory activity.

Kinin release from guinea pig plasma high molecular weight kininogen (HMWK) induced by various microbial and mite proteases has been demonstrated previously (Molla, A., Yamamoto, T., Akaike, T., Miyoshi, S., and Maeda, H. (1989) J. Biol. Chem. 264, 10589-10594; Maruo, K., Akaike, T., Matsumura, Y., Kohmoto, S., Inada, Y., Ono, T., Arao, T., and Maeda, H. (1991) Biochim. Biophys. Acta 1074, 62-68). In this paper, we describe the effects of various microbial and mite proteases on low molecular weight kininogen (LMWK) and HMWK from human plasma. A protease from the house dust mite Dermatophagoides farinae (Df-protease) directly liberated kinin from both LMWK and HMWK to a significant degree. The Km, kcat, and kcat/Km values for kinin generation from LMWK were 3.24 microM, 0.61 s-1, and 1.9 x 10(5) M-1 x s-1, respectively, and those for kinin generation from HMWK were 0.56 microM, 0.12 s-1, and 2.1 x 10(5) M-1 x s-1, respectively; kcat/Km values for Df-protease were comparable with that for glandular kallikrein. In contrast, microbial proteases showed only weak kinin-releasing activity from both human plasma kininogens. Four of ten different microbial proteases liberated kinin from LMWK, and only serratial 56-kDa protease released kinin from HMWK. Furthermore, Df-protease markedly inactivated the thiol protease inhibitory activity of LMWK and HMWK, whereas all microbial proteases (as well as the endogenous protease trypsin) did not affect this inhibitory activity of both kininogens from human plasma.

Biological regulation by the kallikrein-kinin system: a study with a kininogen-deficient rat strain

It is well known that the kallikrein-kinin system expresses potent biological activities through its final product, bradykinin. However, bradykinin has an extremely short half life in biological fluids, so that it is difficult to quantitate the amount of bradykinin released in relevant pathological samples. Therefore investigators have attempted to prove its involvement or importance by measuring the precursor proteins, such as prekallikrein, kininogens, and glandular kallikreins. In this review, I would like to focus the discussion on a study of the kallikrein-kinin system in B/N Katholiek rats, a strain that has a congenital deficiency in plasma high molecular weight and low molecular weight kininogens. When experimental inflammation induced in the mutant deficient rats are compared to that induced in the normal rats (B/N-Kitasato), there was a significant difference; i.e., the deficient rats showed less swelling in the carrageenin-induced paw edema and less exudate accumulation in carrageenin-induced rat pleurisy. These results indicate that bradykinin may be released from kininogens and it may cause exudate formation in above inflammation. Furthermore, when experimental hypertension was induced by DOCA-salt loading, the blood pressure of the deficient rats rose faster than that of the normal rats. From the above findings, it is concluded that the plasma kallikrein-kinin system could be an important regulatory system in body defense mechanisms such as inflammation and blood pressure control.

Activation of the plasma kallikrein-kinin system in respiratory distress syndrome.

Components of the plasma kallikrein-kinin and fibrinolytic systems together with antithrombin III were measured the first days postpartum in 13 premature babies with severe respiratory distress syndrome (RDS). Seven of the patients received a single dose of porcine surfactant (Curosurf) as rescue treatment. Nine premature babies without lung disease or any other complicating disease served as controls. There were no differences in prekallikrein values between surfactant treated and non-treated RDS babies during the first 4 d postpartum. The controls had, however, significantly higher prekallikrein values than the RDS babies already at the first day of age (mean +/- SD 32 +/- 8% in controls versus 22 +/- 6 and 21.5 +/- 5% in the treated and nontreated RDS groups, respectively). Plasma kallikrein activities did not differ between RDS and control patients. Plasma kallikrein inhibition values, which increased steadily in all groups, were lower in the RDS babies treated with surfactant than in controls at d 2 and 4. The degree of degradation of plasma high molecular weight kininogen was measured in RDS patients treated with surfactant and was significantly higher when compared with controls at d 1, demonstrating an increased proteolysis of kininogen to kinin early in RDS. There were no differences in plasminogen and plasmin values between RDS and control babies. This study shows that the plasma kallikrein-kinin system is activated in RDS. This system as well as the fibrinolytic system does not seem to be influenced by rescue instillation of a single dose of porcine surfactant into the lungs of premature babies with RDS.

Determination of serum levels of complement component C4b-binding protein: influence of age and inflammation

C4b-binding protein (C4b-BP) is a high molecular weight plasma protein which inhibits the activity of the classical complement pathway C3 convertase. In addition to multiple binding sites for C4b, C4b-BP possesses a single binding site for vitamin K-dependent protein S, an inhibitor of blood coagulation. As protein S bound to C4b-BP has no anticoagulant activity, C4b-BP participates in the regulation of both the complement and the coagulation pathways. We have produced and immunochemically characterized a series of murine monoclonal antibodies to human C4b-BP. A mixture of four monoclonal antibodies precipitating C4b-BP both in agarose gel and in solution was used to develop a highly reproducible radial immunodiffusion method for the measurement of C4b-BP in human serum. C4b-BP levels were measured in sera from 284 patients referred to our central laboratory. Samples from subjects with an increased erythrocyte sedimentation rate (ESR), a1-acid glycoprotein (a1-AGP) or C-reactive protein (CRP) had significantly higher C4b-BP levels (307 mg/l, 292-322 mg/l, geometric mean and 95% confidence limits of the mean) than those from subjects without elevation of the aforementioned established acute phase reactants (231 mg/l, 226-237 mg/l, P less than 0.00001). C4b-BP was significantly (P less than 0.001) correlated with ESR (r = 0.715), a1-AGP (r = 0.692) and CRP (r = 0.567). There was no gender-related difference in C4b-BP levels. In subjects with no increased acute phase reactants there was a significant correlation between C4b-BP levels and age (r = 0.387, P less than 0.001). High C4b-BP might contribute to the increased thrombotic risk associated with inflammation and aging.

Impaired intracellular transport produced by a subset of type IIA von Willebrand disease mutations.

Type IIA von Willebrand disease (vWD) results from abnormalities in von Willebrand factor (vWF) characterized by absence of plasma high molecular weight (HMW) vWF multimers. In this report, 5 distinct point mutations were identified in 6 Type IIA vWD families. A total of 7 mutations, all clustered within a 124-amino acid segment of the vWF A2 domain, now account for 9 of a panel of 11 Type IIA families. In COS-7 cells, 3 single amino acid substitutions, Val844----Asp, Ser743----Leu, and Gly742----Arg, impaired the transport of vWF multimers between the endoplasmic reticulum and the Golgi complex, with more profound effects on the secretion of HMW multimers than lower molecular weight forms. In contrast, 2 substitutions, Arg834----Trp and Gly742----Glu, resulted in secretion of HMW multimers similar to wild-type vWF. The vWF structure observed within patient platelets correlated closely with the synthesis pattern seen for the corresponding mutants in COS-7 cells. These findings demonstrate that structural alterations within the A2 domain of vWF can produce the characteristic phenotype of Type IIA vWD via two distinct molecular mechanisms.

Characterization of the heredity of kininogen deficiency in Brown Norway katholiek strain rats

Brown Norway rat strain has been studied for mode of inheritance of its congenital deficiency in plasma high molecular weight (HMW)- kininogen and low molecular weight (LMW)- kininogen, and low plasma level of prekallikrein. We examined the genetics of the deficiency by performing a mixed breeding experiment between B/N- Katholiek (B/N-Ka, deficient) and B/N-Kitasato (B/N-Ki, normal) strains. Incidence of the deficiency was judged by the plasma level of HMW-kininogen. Plasma level of HMW-kininogen was around 50% of the normal level in all Fl generations of the hybrid between male B/N-Ka and female B/N-Ki (Exp. 1), and between female B/N-Ka and male B/N-Ki (Exp. 2). Incidence of deficiency (plasma HMW-kininogen level < 5% in Exp. 1 was 23.8 % in male F2 and 20.0% in female F2 generations. By Exp. 2 also the incidence was 25.0% in male and 30.0% in female F2 generations. There was no significant difference of the incidence between the two experiments or sexes. These results indicate the inheritance of the kininogen-deficiency to be Mendelian autosomal recessive, the same as for the reported cases of human kininogen deficiency. Gel filtration study suggests that prekallikrein in the B/N-Ka plasma may be free form, while that in the B/N-Ki plasma may form complex with HMW-kininogen.

Comparison of cysteine-proteinases inhibitory properties of human high-molecular-weight-kininogen (HMrK) purified from plasma and serum

We have purified both plasma and serum High Molecular Weight Kininogens (HMrK) and studied their inhibitory properties against members of the cysteine proteinases super-family. The clotting pathway liberates two different fragments by limited proteolysis of HMrK. The inhibitory properties of these fragments against the cysteine proteinases of the papain super-family are conserved after blood clotting, i.e when the procoagulant domain of HMrK was destroyed.

Synthesis and secretion of alpha 2-macroglobulin by human glioma established cell lines.

Human alpha 2-macroglobulin (alpha 2M) is a high molecular weight plasma proteinase inhibitor exhibiting a broad specificity; in fact it is capable of binding endopeptidases from all known classes of proteases (Barret 1981). Two human glioma cell lines, namely an astrocytoma and a glioblastoma, were found to synthesize and secrete in the culture medium a protein which resembles the serum alpha 2M for immunological, biochemical and biological features. Using polyclonal antibodies to serum alpha 2M, an alpha 2M-like factor could be detected in the cytoplasm and in the culture medium of the tumor cells. Furthermore this factor accumulated in cytoplasmic granules if cells were incubated with monensin and its production was dramatically reduced following a treatment with cycloheximide. This protein behaved like the serum alpha 2M in immunoblotting analysis and exhibited the same antiproteolytic activity. Its role in human brain is unknown at present. Since interactions of proteinases and proteinase-inhibitors appear to influence the host-tumor immune response and to play a crucial role during the migration of metastasizing tumor cells, alpha 2M expression observed in these glioma cells could be involved in tumor cell proliferation and invasion.

On the glomerular mechanism of the renal protein excretion. Studies in the isolated perfused rat kidney.

The glomerular permeability of the isolated perfused kidney is manifold higher than in vivo, although structural changes at the glomerular filtration barrier are not described. It has been proposed that in vivo high molecular weight plasma proteins are concentrated at the glomerular endothelium forming an additional filtration barrier ('concentration polarization'). To test this hypothesis, isolated rat kidneys were perfused with substrate-enriched Krebs-Henseleit solutions containing albumin (50 g/l) or albumin/globulin (40/10 g/l), or with plasma. The pO2 was 675 mm Hg. Furthermore, experiments were carried out with the addition of erythrocytes with and without a reduced O2 supply (pO2 35 mm Hg). Independent of the composition of the perfusate a continuously increasing glomerular permeability was observed immediately after perfusion was started. After about 10 min, a steady state value for the glomerular permeability was reached. The addition of globulin to the perfusate or perfusion with plasma did not prevent the initial increase of the permeability. However, after addition of 5% erythrocytes to the perfusate the increase of permeability was much less pronounced. Neither the reduction of the O2 supply nor the addition of erythrocytes to a higher concentration than 5% had any further effect on the glomerular permeability. These data show that concentration polarization of high molecular weight proteins does not take place at the endothelium of the glomerulus. Independent of their ability to carry O2 erythrocytes play an important role in the glomerular filtration process.