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With the use of the protease inhibitor from bovine organs--Trasylol-- as a model, we studied the pinocytic transport of peptides in the kidney. Rats were injected with the 125I-labeled peptide and killed at different times thereafter. Kidney homogenates were subfractionated by differential and sucrose gradient centrifugation. Radioactivity was measured in the fractions in order to study the time-dependent fixation of the peptide to different cell organelles. With short survival periods, the protease inhibitor is recovered in the brush-border fraction, with longer periods, a shift towards the lysosome fraction takes place. Thus, the renal transport of the protease inhibitor consists of three steps: binding to the brush border, incorporation in micropinocytic vesicles and transport in phagolysosomes.

In this study, we report on the characterization of a patient with Glanzmann thrombasthenia (GT). Immunochemical analysis on platelets from the patient showed that the expression of alpha IIb beta 3 was only 25% of that in normal healthy controls, suggesting a case of GT. Functional analysis revealed a total lack of fibrinogen binding capacity. Molecular genetic analysis of the full-length cDNA sequences of alpha IIb and beta 3 subunits showed a novel point mutation C621T in alpha IIb cDNA, leading to a missense substitution of threonine for isoleucine at position 176. Coexpression of normal beta 3 and mutant alpha IIb(1176) isoform in mammalian cells showed a marked reduction in the expression of alpha IIb beta 3 heterodimer when compared to the wild-type and a decreased intracellular level of alpha IIb. The T176 I mutation is located in the N-terminal region in the W3:1-2 connecting strand of the beta-propeller. These data suggest that the N-terminal alpha IIb domain plays an important structural role in the formation of heterodimer and that it is also involved in fibrinogen binding.

Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a new promising class of antithrombotic agents. The binding of fibrinogen to the fibrinogen receptor depends on an Arg-Gly-Asp-Ser (RGDS) tetrapeptide recognition motif. Structural modifications of the RGDS lead have led to the discovery of a non-peptide RGD mimetic GP IIb/IIIa antagonist20 (S 1197). Compound20 inhibits dose-dependently and reversibly human platelet aggregation. Modeling studies based on structure-activity data revealed the following structural features of the drug as important for receptor binding: the amidino group, the carboxylate group, hydrophobic substitutions at the carboxyl-terminus and at the side chain carrying the positive charge, the carboxyl-terminal NH group of the β-amino acid as a hydrogen bond donor and one oxygen atom of the hydantoin as a hydrogen bond acceptor. The ethyl ester prodrug of20 (S 5740) is an orally active antithrombotic agent which has the potential to be used to treat and prevent thrombotic diseases in humans.

A series of novel, highly potent P2Y₁₂ antagonists as inhibitors of platelet aggregation based on a phenylpyrazole glutamic acid piperazine backbone is described. Exploration of the structural requirements of the substituents by probing the structure-activity relationship along this backbone led to the discovery of the N-acetyl-(S)-proline cyclobutyl amide moiety as a highly privileged motif. Combining the most favorable substituents led to remarkably potent P2Y₁₂ antagonists displaying not only low nanomolar binding affinity to the P2Y₁₂ receptor but also a low nanomolar inhibition of platelet aggregation in the human platelet rich plasma assay with IC₅₀ values below 50 nM. Using a homology and a three-dimensional quantitative structure-activity relationship model, a binding hypothesis elucidating the impact of several structural features was developed.

Platelets are a main target for endogenous and exogenous nitric oxide (NO). The sydnonimine derivative C 89-4095 slowly consumed oxygen inaqueous solution as a measure of oxidative NO release. The antiplatelet properties of the compound were evaluated in vitro and in vivo. In human platelet-rich plasma, C 89-4095 inhibited aggregation induced by various agonists (IC 50 of 2 to 7 μM); the effect was abolished by oxyhemoglobin. The concentration-response curve of prostacyclin was shifted to the left by a threshold concentration of C 89-4095.

Antagonists of the platelet fibrinogen receptor (GP IIb/IIIa receptor) are expected to be a promising new class of antithrombotic agents. The binding of fibrinogen to the fibrinogen receptor depends on an Arg-Gly-Asp-Ser (RGDS) tetrapeptide recognition motif. Structural modifications of the RGDS lead have led to the discovery of a non-peptide RGD mimetic GP IIb/IIIa antagonist 44 (S 1197). Compound 44 inhibited, in a dose dependent and reversible manner, human and dog platelet aggregation as well as 125I-fibrinogen binding to ADP-activated human gel filtered platelets and isolated GP IIb/IIIa with K(i) values of 9 nM and 0.17 nM, respectively. A pharmacophore mapping procedure with QXP and a 3D-QSAR analysis applying the GRID/GOLPE methodology yielded a stable, rather predictive model and revealed structural features which are important for binding. Hydrophobic substitutions both at the hydantoin nucleus and at the C-terminus increase the affinity toward the fibrinogen receptor. The crystalline ethyl ester prodrug 48 (HMR 1794) is an orally active antithrombotic agent which is a promising drug candidate for the treatment of thrombotic diseases in humans.

The effect of recombinant hirudin (r-hirudin; HBW 023) was studied in four different models of thrombosis as well as on bleeding time. Arteriolar thrombus formation was induced in rats either by argon laser injury or by photochemical reaction. r-Hirudin, given by intravenous infusion, significantly inhibited arteriolar thrombus formation and arterial bleeding time at a dose of 40 μg/kg·min. In rabbit jugular veins and carotid arteries, occluding thrombi were produced by stenosis and endothelial damage. r-Hirudin reduced the incidence of thrombosis dose dependently (ED50 0.7 and 1.0 mg/kg i.v. in arteries and veins, respectively). Caval vein thrombosis was initiated in rats by insertion of a stainless steel coil. This thrombosis was dose dependently reduced by the injection of r-hirudin (ED50 0.16 mg/kg i.v.). The duration of the antithrombotic effect of 0.25 mg/kg of r-hirudin in caval veins was about 60 min, whereas the arterial bleeding time was significantly prolonged in these rats for only 20 min. The plasma level of r-hirudin had dropped to 0.12 μg/ml 60 min after injection. These results suggest that the duration of the effect on bleeding time may be shorter than the antithrombotic action of r-hirudin.

Rats were injected with labeled Kunitz protease inhibitor and killed at various times thereafter. Radioactivity was measured in various fractions of kidney homogenates in order to study the time-dependent fixation to different cell organelles, expecially the transition from the brush border to lysosome fraction. With short survival periods (up to 5 min), the renal protease inhibitor is recovered nearly completely with the brush border fraction. With longer periods, a shift towards particles with higher densities and higher beta-glucuronidase activities takes place. Similar results have been achieved with insulin. Lysosomes were prepared and subfractionated following i. v. administration of the protease inhibitor or insulin. The radioactivity of the peptides was found in the lysosomal range of density. According to our present and previous results, the renal pathway of the protease inhibitor consists of 3 steps: binding to the brush border, reabsorption into micropinocytotic vesicles and phagosomes, and final enrichment in phagolysosomes with subsequent degradation. We suggest this type of transport to be representative for peptides in general.

The purpose of the present study was to examine the effect of molsidomine in stenosed coronary arteries of anesthetized dogs and pigs. Stenosis and vascular damage resulted in cyclical reductions of coronary blood flow due to periodic acute platelet thrombus formation in the narrowed lumen. Cyclical flow reductions were quantitated by their frequency and severity. In adult dogs with critical stenosis, molsidomine reduced significantly both the frequency and severity of the cyclical reductions in the first hour following injection, whereas both parameters remained constant in control dogs. Aspirin also significantly reduced the frequency and severity of flow reductions. In young dogs, cyclical flow reductions developed only when epinephrine was added to the combination of critical stenosis plus endothelial damage. In this model, molsidomine reduced the severity of flow reductions but not their frequency. Aspirin reduced both parameters. In young pigs, cyclical flow reductions were induced by subcritical stenosis. Molsidomine reduced the severity but not significantly the frequency of the cyclical flow reductions. A higher dose of molsidomine abolished them in three additional pigs; this effect was reversed by a subsequent injection of hemoglobin. It is concluded that molsidomine, like aspirin, possesses antithrombotic activity. This effect may entirely or in part, be due to the release of nitric oxide.

A brush border membrane fraction from rat renal cortex binds the polyvalent protease inhibitor (Trasylol®) from bovine organs. Saturation is complete in the presence of about 40 nanomoles/ml of protease inhibitor. The maximal binding capacity is about 18 nanomoles (or 117 μg) of protease inhibitor per mg of brush border membrane protein. The affinity constant, as calculated from the double reciprocal plot of binding, is 7 μM. Removal of the microvillar knobs by incubation with papain does not change the binding characteristics, whereas incubation of the brush border with sialidase markedly decreases the fixation of the peptide. Guanidination fails to alter the binding properties, whereas the tetramaleoyl derivative is not fixed at all. Therefore, the positive net charge of the peptide seems to play an important role. Insulin, glucagon and bradykinin interact with the brush border fraction in a manner entirely different from that of the protease inhibitor. Over a wide concentration range, about 6 to 10% of these peptides are bound independent of the concentrations added. Our present and previous results suggest that binding to the brush border membrane is the initial step in the pinocytotic reabsorption of the protease inhibitor and possibly of other peptides too. The tremendous concentration of the protease inhibitor by the kidney cortex is assumed to be due to its high affinity to the brush border membrane in vivo.

1. 1. The reaction of venom hemorrhagic principles (HR1, HR2a and HR2b) with isolated glomerular basement membrane was studied in vitro. For comparison, experiments were also done with a bacterial collagenase which is known to induce the hemorrhage. Upon incubation of the basement membrane preparation with either of the four agents, its supernatant fluid gave positive reactions for proteins (or peptides) and carbohydrates. The optimal pH of the protein-liberating activities of HR1, HR2a and HR2b was around 8.0. 2. 2. The approximate ratio of the proteins or peptides liberated by HR2a, HR2b, HR1 and collagenase, under standard conditions, was 1:1:2:4; and for the carbohydrates liberated 0.5:0.5:2:4, thus suggesting that HR2a or HR2b acted on the basement membrane in a different manner from HR1. This interpretation is supported by comparison between the split-products of the membrane by these principles in gel filtration experiments. 3. 3. Liberation of both proteins and carbohydrates from basement membrane by HR1, HR2a and HR2b was inhibited by EDTA, cysteine, antivenin and antihemorrhagic factor from snake serum, all of which inhibit the hemorrhagic activity of these principles. The results suggest that one and the same entity is responsible for both the hemorrhagic and liberating activities in each venom principle. 4. 4. Purified HR1 was resolved partially into four components by polyacrylamide gel electrophoresis. Both liberating and hemorrhagic potencies, as expressed by their specific activities, however, were constant throughout, which further supports the idea that the same factor is responsible for both activities. 5. 5. The hemorrhagic effect of HR1, HR2a, HR2b and collagenase is attributed to enzymatic destruction of the basement membrane with consequent lowering of the stability of the vessel wall.

The bovine protease inhibitor aprotinin (Trasylol®) has a high affinity to the kidney and is preferentially pinocytized in the proximal tubule. After i.v. injection of 1 μg 125I aprotinin the blood content decreases to 2.8% of the primary injected amount within 3 hrs, while simultaneously each kidney contains 29%. This substance was used to test whether or not a peptide which is pinocytized, is released in the intact form into the peritubular blood. By a cross circulation technique with two unilaterally nephrectomized rats we were unable to detect any transport of pinocytized, intact peptide through the proximal tubule cell over the observed cross circulation period of 1–8 hrs even when using 5000 times the above dosage. Since the total amount of aprotinin in the kidney is immunologically reactive (ca. 97%), and 65% of the radioactivity in the blood is not reactive after 6 hrs, we believe that the last step in the absorption process consists in digestion inside the lysosomes and instantaneous release of the split products into the blood.

Upon intravenous injection of 3H-gentamicin in rats, radioactivity in serum rapidly declined to 3% of total within 1 h. Kidneys accumulated a constant amount (14%) of the injected radioactivity between 2 and 6 h after injection.