Investigation of activated prothrombin complex concentrate as potential hirudin antidote in animal models.

The specific thrombin inhibitor r-hirudin (HBW 023) has been demonstrated to be effective in preventing thrombosis in preclinical models. Up to now, no bleeding complications have been observed using therapeutically effective doses in animals studies. However, in case of inadvertent overdosing the occurrence of undesired impairment of coagulation cannot be excluded. As a potential antidote an activated prothrombin complex concentrate (APC) was tested on its ability to normalize blood coagulation. APC given s bolus injections 5 min and 3.0 mg/kg neutralized the r-hirudin-induced prolongation and 3.0 mg/kg neutralized the r-hirudin-induced prolongation of whole blood coagulation time in rabbits completely within 5 min without any clot formation in the blood vessels or capillaries of the heart, kidneys, or lungs. Furthermore, bleeding time prolongation induced by bolus application of 3.0 and 30.0 mg/kg r-hirudin was significantly inhibited by APC within 5 min. These results suggest that administration of APC may be an effective way to reverse the effects of r-hirudin in the coagulation system in case of inadvertent overdosing of r-hirudin.

Inhibition of in vitro clot growth by r-hirudin is more effective and longer sustained than by an analogous peptide.

The specific thrombin inhibitors r-hirudin and a synthetic peptide (I) D-FPRP(G)4-NGDFEEIPEEYL were compared in in vitro tests. r-hirudin proved to be the superior compound with respect to inhibition of amidolytic small substrate turnover that is catalysed by soluble and immobilised thrombin as well as to inhibition of fibrinogen activation. In an in vitro clot model significantly higher molar concentrations of peptide I are needed to achieve fibrin bound thrombin inhibition equivalent to that of r-hirudin. Stable complexes consisting of thrombin and hirudin oppose labile complexes containing the synthetic peptide. The latter leads to a regaining of thrombin activity with subsequent additional fibrin accretion. Analyses of the mixtures of thrombin and peptide I display a time dependent release of amino-terminal D-FPR peptide (III) exhibiting, similar to the residual fragment (peptide II), only weak inhibitory activity. Peptide I and the carboxy-terminal fragment induce, within a certain concentration range, an increase in thrombin activity and clot growth.

Localization of annexins in normal and diseased human skin.

Annexins (AX) or lipocortins are a family of calcium and phospholipid binding proteins that have been implicated to play a role in the regulation of inflammation and cellular differentiation. To investigate a potential role of AX in skin disorders we studied the distribution of six different AX in normal human skin (NHS) and several inflammatory and hyperproliferative skin diseases. A distinct staining pattern could only be shown for AX-1 and AX-2. In NHS AX-1-antibody (Ab) displayed a very strong reactivity with eccrine sweat ducts. In the diseases investigated we found a highly increased expression of AX-1 in keratinocytes (KCs) in the vicinity of inflammatory processes such as psoriasis. Furthermore, the AX-1 expression was increased in differentiated squamous cell carcinoma (SCC) whereas undifferentiated SCC and basal cell carcinoma were negative. AX-3, -4, -5, and -6 showed no distinctive expression pattern. Our data demonstrate an abnormal distribution of AX-1 in association with proliferating KCs under inflammatory and neoplastic conditions. Its pattern of reactivity shows similarities to the known distribution of the EGF-receptor kinase, which has been demonstrated to phosphorylate AX-1 with high activity in various cellular systems. These results support the concept that the appearance of AX-1 is linked to a certain level of KC differentiation.

Comparison of in vitro and in vivo properties of rhirudin (HBW 023) and a synthetic analogous peptide.

Recombinant hirudin and a shortened synthetic analogue, with the amino acid sequence of D-Phe-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-Glu- Glu-Ile-Pro-Glu-Glu-Tyr-Leu, are specific thrombin inhibitors which in a concentration-dependent manner inhibit thrombus formation as well as clot propagation both in vitro and in vivo. In comparison to the analogue, lower molar concentrations of rhirudin affected doubling of aPTT and TT as well as inhibition of thrombin amidolytic activity or thrombin-induced platelet aggregation in vitro. In the rat wire coil-induced thrombosis model, a 50% thromboprotective effect may be brought about with doses of 0.043 mumol/kg of rhirudin and 1.43 mumol/kg of the synthetic peptide. However, doubling of bleeding times is caused, on average, by dosages of between 0.143 and 0.43 mumol/kg rhirudin or approximately 0.143 mumol/kg of the analogue. Treatment groups included animals revealing significant prolongation of bleeding times as well as nonresponders. Despite the 10-fold longer impact on aPTT after application of rhirudin, the extent of mean bleeding time prolongation is identical to that of the analogue.

Inhibition of human skin phospholipase A2 by "lipocortins" is an indirect effect of substrate/lipocortin interaction.

Proteins of the annexin/lipocortin family have been claimed to mediate the anti-inflammatory action of glucocorticosteroids by the inhibition of phospholipases A2. This hypothesis has been challenged by the finding that annexins do not directly interact with the enzyme in a classical enzyme/inhibitor behavior, but more likely block the access of the phospholipase A2 to its substrate by binding to phospholipids. Because former studies with skin phospholipase A2 suggested a specific regulation by annexin-1, we investigated the substrate dependence of this effect. For this purpose phospholipase A2 activities in human epidermis and dermis homogenates were measured in the presence of various amounts of annexins-1, -2, or -5. The respective annexin was preincubated in separate series either with the substrate or with the enzyme. We found a partial inhibition of both epidermal and dermal phospholipase A2 activities with all annexins tested (annexin-5 >> annexin-2 > annexin-1). The inhibitory effect was absolutely dependent on the annexin/phospholipid ratio and occurred only at very high annexin concentrations relative to the amount of substrate. Our data demonstrate that the inhibition of human skin phospholipase A2 by annexins depends on the substrate concentrations, as has been shown for phospholipases A2 of other origins as well. All observations can be explained by the current "substrate depletion model" characterizing the indirect effects of annexins on phospholipase A2 activities. It is therefore rather unlikely that annexins are directly involved in the regulation of phospholipase A2 activity of human skin under physiologic conditions.

Reduction of mortality with antithrombin III in septicemic rats: a study of Klebsiella pneumoniae induced sepsis.

Experimental gram-negative sepsis was induced in the rat by Klebsiella pneumoniae. Although bacteria are susceptible to the treatment with the antibiotic Tobramycin, DIC could not be prevented. DIC was manifested by a leuko- and thrombocytopenia, decreases in fibrinogen and AT III and an increase of the aPTT. In this model the therapeutic treatment with human AT III was evaluated. To determine the optimal concentration of AT III a prestudy in a LPS induced DIC in the rat was performed. It was shown that a bolus i.v. injection of 500 U/kg improved survival and DIC, and was thus chosen for the Klebsiella sepsis model. The infectious load was adjusted to yield a mortality rate of 90-100% in the untreated Klebsiella group and a reduction to about 40-50% of the mortality rate by Tobramycin. It was found that AT III reduced mortality in the Klebsiella induced sepsis not only when given prophylactically but was effective even when administrated in a late stage of the DIC, i.e. 3 or 5 h post infection.

Coagulation and fibrinolysis in cancer.

Haemostasis is a system of finely adjusted interactions between cells, enzymatic reaction cascades and inhibitors. Disturbances of this balance occur in many disorders, especially in inflammatory processes, septicaemia and cancer. In such cases malignant cells and infectious organisms activate the plasmatic enzyme cascades, especially of the coagulation and fibrinolysis cascades. The resulting consumption and proteolytic degradation of the regulatory proteins contribute to hypercoagulability and secondarily to reactive fibrinolysis, and these may then lead to local thromboses and haemorrhages. These pathogenic events culminate in disseminated intravascular coagulation (DIC), frequently with organ failure and death. Factors of both plasmatic systems are also "misused" by malignant cells for the purposes of growth and metastasis. Prominent examples of this misuse are the formation of a protective fibrin shield against the endogenous defence mechanisms and the local degradation of tissues for tumor proliferation as well as for cell permeation and invasion. In the search for a potential therapy a number of protease inhibitors, predominantly of enzymes of coagulation and fibrinolysis, have been tested in vivo with regard to their efficacy. So far, however, it has not been possible to find a new uniform treatment principle to inhibit the growth and/or metastasis of different types of tumor. The haemorrhagic diathesis and thromboses frequently associated with tumors are generally treated by substitution with plasma components, especially concentrates of coagulation factors and inhibitors.

Annexins I to VI: quantitative determination in different human cell types and in plasma after myocardial infarction.

Concentrations of annexins I to VI were quantitatively determined in extracts of placenta and different human cell types. They were detectable in all extracts studied, but lymphocytes/monocytes, endothelial cells and fibroblasts had very high annexin contents. The results indicate cell type specific annexin-repertoires. Annexins are intracellular proteins lacking signal sequences but which are detectable in trace amounts in plasma of healthy humans. The majority of plasma samples drawn from 14 patients suffering from myocardial infarction had elevated annexin III, IV and V concentrations. Shortly after infarction increased annexin levels were detected, reaching maximal values 24 to 48 h later. In the course of the following days annexin concentrations returned towards normal plasma levels.

Detection of human anti-annexin autoantibodies by enzyme immunoassays.

Annexins belong to a family of proteins characterized by calcium-dependent binding to the cytoskeleton and phospholipid surfaces. Basing on these properties annexins are discussed to be involved in the regulation of cytodynamic, anticoagulatory and antiinflammatory processes. Since autoantibodies against annexin I had been detected in patients suffering from inflammatory or autoimmune diseases, an impact on the pathophysiological outcome was assumed. Therefore we developed solid phase, enzyme-linked immunoassays for the quantitative determination of autoantibodies directed against six members of the annexin family. Some preliminary results obtained from sera of patients with malignant melanoma show a quite frequent presence of such autoantibodies. These data suggest that autoantibodies are generated against all annexins. Furthermore, in the individual patient autoantibodies of the IgG-type are monospecific, while about 1/4 of the IgM-type are directed against several annexins. These observations imply that for investigation of anti-annexin autoantibodies in inflammatory and autoimmune diseases as well as cancer all members of the annexin family have to be taken into consideration.

Comparative study on the in vitro effectiveness of antithrombotic agents.

The synthetic low molecular weight inhibitors MD 805, FUT-175 and FOY as well as heparin and r-Hirudin were compared for their in vitro antithrombotic potencies and protease specificities. The amidolytic activity of thrombin and the plasma coagulation were effectively inhibited by MD 805 and, in particular, by r-Hirudin. FUT-175 and FOY revealed only weak inhibition. None of the synthetic substances discriminated between alpha-, beta- and gamma-thrombin. Beside r-Hirudin only MD 805 revealed a relatively good specificity for thrombin. On the contrary, FUT-175 and FOY are unspecific and can not be classified as thrombin inhibitors.

Thrombin-hirudin complex stability: a comparison with the thrombin-antithrombin III complex.

In the present in vitro study the stabilities of the thrombin-hirudin and the thrombin-antithrombin III complexes were investigated. After incubation of the complexes with free inhibitors the thrombin-antithrombin III levels were determined by ELISA. The thrombin-hirudin complex proved to be stable in the presence of antithrombin III or heparin. However, in the presence of heparin and plasma equivalent concentrations of antithrombin III, the thrombin-hirudin complex dissociated and hirudin was displaced. In contrast, both thrombin-antithrombin III and thrombin-antithrombin III/heparin complexes are very stable even in the presence of a large excess of hirudin.

[Lipocortins and phospholipases: new aspects in the physiology of glucocorticosteroid effect]. / Lipokortine und Phospholipasen: neue Aspekte in der Physiologie der Glukokortikosteroidwirkung.

Glucocorticosteroids are among the most useful and most widely prescribed anti-inflammatory drugs. Despite their wide use little is yet known about their mode of action. In the last 10 years a group of proteins called lipocortins or annexins has been characterized. Those proteins exert an inhibitory effect on the synthesis of lipid mediators by way of an important proinflammatory enzyme, phospholipase A2. Phospholipases are known to be involved in cell-signal transduction and generation of inflammatory mediators like prostaglandins, leukotrienes and platelet-activating-factor. The cellular expression of lipocortins is induced by glucocorticosteroids. The inhibition of cellular phospholipases via lipocortins may account for some aspects of the action of glucocorticosteroids.

In-vivo antithrombotic potency of placenta protein 4 (annexin V).

The antithrombotic properties of Placenta Protein 4 (PP4) were investigated in laser or photochemically induced thrombus formation models in rats. In both in-vivo test-systems PP4 displayed a significant antithrombotic effect at dose levels as low as 0.3 and 1.0 mg/kg body weight. Bleeding times, surprisingly, were not prolonged significantly at these dose regimens. Maximal inhibition of thrombus formation in the laser-model was observed 15 min after intravenous administration of PP4, but was not recognizable in a clear-cut reaction in the second model. Determination of PP4 plasma levels in two monkeys revealed a half-life of 11.5 and 14.9 min, respectively. The maximal anticoagulant effect was observed between 15 and 30 min after administration of PP4 as determined functionally by means of thrombelastography.

The crystal and molecular structure of human annexin V, an anticoagulant protein that binds to calcium and membranes.

Human annexin V (PP4), a member of the family of calcium, membrane binding proteins, has been crystallized in the presence of calcium and analysed by crystallography by multiple isomorphic replacement at 3 A and preliminarily refined at 2.5 A resolution. The molecule has dimensions of 64 x 40 x 30 A3 and is folded into four domains of similar structure. Each domain consists of five alpha-helices wound into a right-handed superhelix yielding a globular structure of approximately 18 A diameter. The domains have hydrophobic cores whose amino acid sequences are conserved between the domains and within the annexin family of proteins. The four domains are folded into an almost planar array by tight (hydrophobic) pair-wise packing of domains II and III and I and IV to generate modules (II-III) and (I-IV), respectively. The assembly is symmetric with three parallel approximate diads relating II to III, I to IV and the module (II-III) to (I-IV), respectively. The latter diad marks a channel through the centre of the molecule coated with charged amino acid residues. The protein has structural features of channel forming membrane proteins and a polar surface characteristic of soluble proteins. It is a member of the third class of amphipathic proteins different from soluble and membrane proteins.

Anticoagulant properties of placenta protein 4 (annexin V).

A placenta protein, originally termed PP4, was found to inhibit the aPTT in a concentration-dependent manner. PP4 which turned out to be identical with a vascular anticoagulant of the annexin type, inhibits the blood clotting process by binding of the essential lipids in a reaction which is dependent on calcium ions. Also in the presence of calcium PP4 combines with platelet membranes neutralizing their procoagulant effect. By fluorescence-microscopy binding of PP4 to stimulated macrophages is shown. The antithrombotic effect of PP4 is demonstrated by means of thrombelastography of human blood. Coagulation triggered by the addition of thromboplastin/lipid-mixtures is extinguished by PP4.

The calcium binding sites in human annexin V by crystal structure analysis at 2.0 A resolution. Implications for membrane binding and calcium channel activity.

Crystal structure analysis and refinement at 2.0 A resolution of a rhombohedral crystal form of human annexin V at high calcium concentration revealed a domain motion compared to the previously analysed hexagonal crystal form. Five calcium ions were located on the convex face of the molecule. Three strongly bound calciums are liganded at protruding interhelical loops and Asp or Glu residues in homologous positions in repeats I, II and IV. Five proteinaceous oxygens and one solvent molecule form the coordination polyhedron in each case. The unoccupied seventh site is suggested as the phospholipid headgroup binding site. Two more weakly bound sites were identified by lanthanum labelling. The structural features suggest that annexin V attaches with its convex face to membranes by specific calcium mediated interactions with at least three phospholipids. The adjacent membrane bilayer may thus become locally disordered and permeable to allow calcium inflow through the central polar channel of the molecule.

Carbohydrate structures of a human tissue plasminogen activator variant expressed in recombinant Chinese hamster ovary cells.

The carbohydrate structures of a genetically engineered human tissue plasminogen activator variant bearing a single N-glycosylation site at Asn 448 are reported. After isolation of the tryptic glycopeptide and liberation of the N-linked carbohydrates by polypeptide:N-glycosidase F, 6 major oligosaccharide fractions were separated by HPLC on NH2-bonded phase. Their structures were determined by compositional and methylation analyses combined with fast atom bombardment mass spectrometry. Seventy percent of the carbohydrates were of the biantennary complex type with fucose at the proximal GlcNAc and zero, one or two alpha 2-3 linked NeuAc. The remainder were triantennary structures with one, two or three NeuAc.

[Hemostasis, fibrinolysis, proteolysis: interaction with inflammatory reactions]. / Hämostase, Fibrinolyse, Proteolyse: Wechselwirkungen mit Entzündungsreaktionen.

Many physiological processes are based on the finely regulated interaction between cells and enzymatic reaction cascades. Mainly proteinases are involved in these processes, which are regulated by inhibitors, principally proteins. If this sensitive balance is disturbed, uncontrolled pathophysiological events can be induced, which are often associated with inflammatory reactions. Characteristic for inflammation are events like contact activation of hemostasis, increasing permeability of blood vessels caused by activation of the Kallikrein-Kinin- and the Complement-system and Plasmin-release induced by activation of fibrinolysis. The following uncontrolled proteolysis, leading to tissue destruction, is mainly associated with the degree of illness. Inflammatory cells excrete besides proteinases also mediators maintaining and increasing these processes. Only when the balance between proteinases and inhibitors is restored, inflammation subsides. Afterwards the controlled course of physiological reactions is possible again.

Variants of human tissue-type plasminogen activator substituted at the protease cleavage site and glycosylation sites, and truncated at the N- and C-termini.

Mutations were directed to specific regions of the human tissue-type plasminogen activator (t-PA) gene in an effort to better define structure-function relationships of the enzyme. Three types of modifications were effected by in vitro mutagenesis: elimination of glycosylation sites; substitutions of amino acids at the cleavage site for conversion of single-chain t-PA to two-chain t-PA; and truncations of the N- and C-termini. Thirteen variants were purified from permanent CHO cell lines and analyzed for specific activity, fibrin stimulation, fibrin binding, inhibition by plasminogen activator inhibitor-2 (PAI-2) and half-life. The results of these analyses are: (i) variants with carbohydrate-depleted kringle domains possessed higher specific activities than wild-type t-PA; (ii) a cleavage site variant substituted at Arg275 with Gly had greatly reduced specific activity; (iii) two variants substituted at Lys277 exhibited altered interactions with PAI-2; (iv) the variant with a truncated C-terminus had reduced activity in the absence of fibrin; and (v) no variants had significantly altered half-lives. In order to test the effects of combining mutations, four additional variants were produced. Each combination variant retained at least one of the altered properties observed in the original variants, and in three of the variants the diverse properties were additive.