Dirk Hendriks

Proline motifs in peptides and their biological processing.

Many biologically important peptide sequences contain proline. It confers unique conformational constraints on the peptide chain in that the side‐chain is cyclized back onto the backbone amide position. Inside an a‐helix the possibility of making hydrogen bonds to the preceding turn is lost and a kink will be introduced. The conformational restrictions imposed by proline motifs in a peptide chain appear to imply important structural or biological functions as can be deduced from their often remarkably high degree of conservation as found in many proteins and peptides, especially cytokines, growth factors, G‐protein‐coupled receptors, V3 loops of the HIV envelope glycoprotein gpl20, and neuro‐ and vasoactive peptides. Only a limited number of peptidases are known to be able to hydrolyze proline adjacent bonds. Their activity is influenced by the isomeric state (cis‐trans) as well as the position of proline in the peptide chain. The three proline specific metallo‐peptidases (aminopeptidase P. car‐boxroeptidase P and prolidase) are activated by Mn2+, whereas the three serine type peptidases cleaving a post proline bond (prolyl oligopeptidase, dipep‐tidyl peptidase IV, and prolylcarboxypeptidase) share the sequential order of the catalytic Ser‐Asp‐His triade, which differentiates them from the chy‐motrypsin (His‐Asp‐Ser) and subtilisin (Asp‐His‐Ser) families. An endo or C terminal Pro‐Pro bond and an endo pre‐Pro peptide bond possess a high degree of resistance to any mammalian proteolytic enzyme.—Vanhoof, G., Goossens, F., De Meester, I., Hendrike, D., Schärpé, S. Proline motifs in peptides and their biological processing. FASEB J. 9, 736‐744 (1995)

Characterisation of a Carboxypeptidase in Human Serum Distinct from Carboxypeptidase N

Arginine carboxypeptidase activity in human serum, measured with the hippuryl-L-arginine substrate, is about three times higher than in human plasma. This difference is much smaller when hippuryl-L-lysine is used as the substrate. When fresh serum is incubated at 30 degrees C, the arginine and lysine carboxypeptidase activity decreases until a stable activity, close to the plasma activity, is reached. This stable carboxypeptidase activity is attributed to carboxypeptidase N. The unstable carboxypeptidase differs from carboxypeptidase N in pH-optimum, esterase activity, substrate specificity, Co2+-activation and dithiotreitol activation. Blood cells are not responsible for the release of this enzyme during coagulation. No activator of carboxypeptidase N was detectable in human serum. Ion-exchange chromatography on DEAE-cellulose confirms the presence of two different molecular forms of arginine carboxypeptidase activity.

PURIFICATION AND CHARACTERIZATION OF A NEW ARGININE CARBOXYPEPTIDASE IN HUMAN SERUM

A carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates is present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its unstability at room temperature and at 37 degrees C, we named it unstable carboxypeptidase (carboxypeptidase U). Carboxypeptidase U was partially purified from fresh human serum by chromatography on DEAE-cellulose and Mono-Q sepharose and was found to be a 435 kDa protein. We compared this enzyme with carboxypeptidase N, purified from human serum by a two-step affinity chromatography on arginine-Sepharose 4B, followed by ion-exchange chromatography on Mono-Q sepharose. Carboxypeptidase U cleaves hippuryl-L-arginine and hippuryl-L-lysine, but at a different relative rate than carboxypeptidase N, and has no esterase activity on hippuryl-L-argininic acid. Its activity was inhibited by o-phenanthroline, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, CoCl2, 2-mercaptoethanol, dithiothreitol and 4-chloromercuribenzoic acid. These characteristics differentiate carboxypeptidase U from carboxypeptidase N and other known carboxypeptidases.

Carboxypeptidase U (TAFIa) prevents lysis from proceeding into the propagation phase through a threshold-dependent mechanism

Summary.  In an in vitro clot lysis model in human plasma, carboxypeptidase U (CPU) is generated by thrombin following the coagulation and by plasmin at the later stage of clot lysis. CPU is able to slow down clot lysis by suppressing the cofactor activity of partially degraded fibrin in the plasminogen activation by tissue‐type plasminogen activator (t‐PA). Making use of thrombomodulin and a thrombin inhibitor, the generation of CPU during the in vitro clot lysis can be manipulated both in terms of magnitude and time course. The data obtained demonstrate that CPU affects the clot dissolution through a threshold‐dependent mechanism: as long as the CPU activity remains above the threshold value, lysis is prevented from proceeding into the propagation phase. From the moment the CPU activity drops below this threshold value, the rate of lysis accelerates. This threshold value for CPU activity is dictated by the t‐PA concentration: increasing the t‐PA concentration increases the CPU threshold and vice versa. This implies that the effect of the CPU pathway will become more apparent at a lower fibrinolytic capacity. Our threshold‐based hypothesis indicates that the time course of proCPU activation, the stability of CPU and the t‐PA concentration all play a crucial role in determining the result of the in vitro clot lysis experiment. Furthermore, this hypothesis provides us with new insights into previously published data on the effects of CPU on in vitro clot lysis by high and low t‐PA concentrations.

Development of a Genotype 325–Specific proCPU/TAFI ELISA

Objective—A Thr/Ile polymorphism at position 325 in the coding region of proCPU has been reported. Immunological assays, fully characterized (including genotype dependency), are required for the quantitation of proCPU levels. Methods and Results—We have generated a panel of monoclonal antibodies against human, plasma-derived proCPU. Two combinations exhibiting distinct reactivities were selected for measurement of proCPU in plasma. T12D11/T28G6-HRP yielded values of 10.1±3.1 &mgr;g/mL (mean±SD, n=86; normal donors), and T32F6/T9G12-HRP yielded values of 5.4±3.0 &mgr;g/mL. Grouping according to the 325 genotype demonstrated that T12D11/T28G6-HRP was independent to this polymorphism whereas T32F6/T9G12-HRP revealed a complete lack of reactivity with the Ile/Ile genotype (ie, 0.0±0.0, 4.2±1.7, and 7.3±2.9 &mgr;g/mL for the Ile/Ile, Ile/Thr, and Thr/Thr isoforms, respectively). Commercially available antigen assays appeared to be partially dependent on the 325 genotype (eg, 44±8.9% and 100±30% for the Ile/Ile and Thr/Thr isoforms, respectively). Conclusions—Our data demonstrate that great care should be taken when evaluating proCPU antigen values as a putative causative agent or as a diagnostic risk marker for cardiovascular events.

EFFECTS OF PSYCHOLOGICAL STRESS ON SERUM IMMUNOGLOBULIN, COMPLEMENT AND ACUTE PHASE PROTEIN CONCENTRATIONS IN NORMAL VOLUNTEERS.

The aim of this study was to examine the effects of academic examination stress on serum immunoglobulins (Igs), i.e. IgA, IgG, IgM, complement factors, i.e. C3c and C4, and acute phase proteins, i.e. alpha 1-acid glycoprotein (alpha 1-S), haptoglobin (Hp) and alpha 2-macroglobulin (alpha 2-M). Thirty-seven university students participated in this study. Serum was sampled a few weeks before and after as well as one day before a difficult academic examination. On the same occasions, students completed the Perceived Stress Scale (PSS). Students were divided into two groups, i.e. those with high- and low-stress perception as defined by changes in the PSS score. Academic examination stress induced significant increases in serum IgA, IgG, IgM, and alpha 2-M in students with high-stress perception, but not in these with low-stress perception. The stress-induced changes in serum IgA, C3c, and alpha 1-S concentrations were significantly higher in students with high-stress perception than in those with a low-stress perception. The stress-induced changes in serum IgA, IgM, C3c, C4, alpha 1-S, Hp and alpha 2-M were normalized a few weeks after the stress condition, whereas IgG showed a trend toward normalization. There were significant positive relationships between the stress-induced changes in the PSS and serum IgA, IgG, IgM and alpha 2-M. These findings suggest that psychological stress is accompanied by an altered secretion of serum Igs, complement factors and some acute phase proteins.

Carboxypeptidase U (TAFIa): a metallocarboxypeptidase with a distinct role in haemostasis and a possible risk factor for thrombotic disease.

Since the discovery of Carboxypeptidase U (CPU) in 1988, considerable information has been gathered about its biochemistry and function in physiological and pathophysiological circumstances. A variety of tools such as assays to measure proCPU and CPU, antibodies raised against (pro)CPU, selective CPU inhibitors and knock-out mice have been developed and are currently being used to explore the role of this metallocarboxypeptidase in different in vivo and in vitro settings. The knowledge that proCPU can be activated by thrombin and plasmin, enzymes with a key function in coagulation and fibrinolysis, and the ability of CPU to remove C-terminal lysine residues has led to the hypothesis that the proCPU/CPU pathway plays a role in the balance between coagulation and fibrinolysis. The maintenance of the equilibrium between coagulation and fibrinolysis is crucial for normal haemostasis and disturbance of this delicate balance can lead either to bleeding tendency or thrombosis. This review provides an update on several aspects of CPU known at the moment, including an extensive overview on the clinical studies performed up till now.

Pyrrolidides: synthesis and structure-activity relationship as inhibitors of dipeptidyl peptidase IV

Summary Dipeptidyl peptidase IV cleaves specifically the peptide bond at the carboxyl side of a proline at the penultimate N-terminal position of a peptide. It is thought to be important for the regulation of biologically active peptides. Moreover, it has been identified as an activation marker of T-lymphocytes (CD26). Pyrrolidides and thiazolidides are known as reversible inhibitors of DPP IV. Several homologues, unsaturated, open and 3-substituted analogues were synthesized in order to determine the structure-activity relationship of the P-1 site. l -Isoleucine was taken as P-2 amino acid. 1-( l -Isoleucyl)-3( S )-fluoropyrrolidine is about as active as the non-fluorinated compound and behaves as a competitive inhibitor. Other changes decrease or abolish the activity.

Proteolytic activation of purified human procarboxypeptidase U.

Carboxypeptidase U (CPU, EC 3.4.17.20) is a recently described basic carboxypeptidase which circulates in plasma as an enzymatically inactive precursor procarboxypeptidase U (proCPU), also known as plasma carboxypeptidase B precursor or thrombin activatable fibrinolysis inhibitor (TAFI). The activation of the zymogen proceeds through a proteolytic cleavage at Arg-92. The active form - CPU - is able to retard the initial phase of fibrinolysis by cleaving C-terminal lysine residues exposed on fibrin partially degraded by the action of plasmin. These C-terminal lysine residues are essential for the high affinity binding of plasminogen to fibrin and the subsequent activation to plasmin. In this report, the activation of purified human proCPU was studied using trypsin and some key proteases of the coagulation and fibrinolytic cascade, i.e., kallikrein, plasmin and thrombin. The most efficient activation is obtained in the presence of thrombin in complex with thrombomodulin. After in vitro activation, CPU is unstable at 37 degrees C (T(1/2)=15 min). Its stability can be improved dramatically using lower temperatures.

Influence of academic examination stress on hematological measurements in subjectively healthy volunteers

Some recent reports showed that a brief exposure to a mental stressor during 3-20 min may induce hematological changes in humans. The aim of the present study was to examine the effects of academic examination stress on erythron variables, such as the number of red blood cells (RBC), hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean cell Hb (MCH), mean cell Hb concentration (MCHC), RBC distribution width (RDW), and serum iron and transferrin (Tf). The above variables were determined in 41 students in three conditions, i.e. the stress condition (the day before a difficult oral exam) and two baseline conditions, i.e. a few weeks earlier and later. At the same occasions, subjects completed the Perceived Stress Scale (PSS), the state version of the State-Trait Anxiety Inventory (STAI) and the Profile of Mood States (POMS). Academic examination stress significantly increased Ht, Hb, MCV, MCH and MCHC and significantly decreased RDW. There were significant relationships between the stress-induced changes in the PSS, STAI and POMS scores and those in Ht, Hb, MCV and MCH (allpositive) and RDW (negative). It is concluded that academic examination stress induces significant hematological changes indicative of an increased number of large RBC and increased hemoglobinisation, which cannot be explained by shifts of fluid out of the intravascular space, concentrating non-diffusible blood constituents.