Protein C and protein S levels in uremic patients before and after dialysis

The inhibitory capacity of the natural protein C (PC)/protein S (PS) system was evaluated measuring both the functional activity and the antigen level of both these inhibitors in 30 uremic patients before and after a dialytic treatment and in 30 healthy normal volunteers. PC functional activity was determined by two methods, one clotting and one chromogenic. PS antigen level was measured both as free protein and as total content. Unlike previous authors, we found that PC functional activity and the antigen level were normal in patients before dialysis, with a significant increase after. PS functional activity and free and total antigen levels were all normal before dialysis, and all except free antigen showed a significant post-treatment rise.     

Determination of functional and antigenic protein C inhibitor and its complexes with activated protein C in plasma by ELISA''s

Enzyme-linked immunosorbent assays (ELISA's) were developed for the measurement of protein C inhibitor (PCI) antigen and activity and for its complexes with activated protein C (APC) in plasma. For PCI activity and antigen, APC or anti-PCI, respectively, was immobilized to microtiter plates and PCI bound was detected with labelled anti-PCI antibodíes. For APC:PCI complexes, two different antibodies directed against protein C and PCI were used. The assays for PCI were calibrated with pooled normal human plasma (NHP) and with purified PCI, and for APC:PCI complexes with known concentrations of purified pre-formed complexes added to buffer or to plasma. The lower limit of sensitivity of the PCI activity and antigen assays was 10 ng/ml and 0.5 ng/ml, respectively and for plasma APC:PCI complexes 12 ng/ml. Mean coefficients of variation of 1.5 % to 5.8 % (intro-assay) and 2.1 % to 9.8 % (inter-assay) were found for the assays. For PCI antigen, a range of 56 % to 162 % of the NHP value was obtained in samples from 70 healthy donors (mean ±SD = 98.6 % ±23.1%). For PCI activity, the range was 59 % to 148 % (94.3 % ± 20.2). A good correlation (0.92) was obtained when both assays were compared. Plasma levels of APC:PCI complexes in 30 normals were under the detection limit (< 12 ng/ml). In plasma samples from 10 patients with disseminated intravascular coagulation (DIC) PCI antigen concentrations were decreased (55.6% ±20%) and 8 of the patients had APC-PCI complex levels between 32 and 240 ng/ml (median, 35 ng/ml). After addition of 20 ωg/ml APC to NHP or to protein C depleted plasma, 6.1 μg/ml complexes were recovered after 90 min incubation. Incubation of 10 μg/ml APC with NHP in the presence of 10 U/ml heparin yielded 11 μg/ml complexes after 90 min, which represent more than 90 % of the maximum possible value. Thus, the method should be adequate to study complexes of APC in vivo in clinical conditions in which activation of protein C pathway may occur.     

Enzyme immunoassay of human protein C by using monoclonal antibodies

An enzyme-linked immunosorbent assay (ELISA) for measuring human protein C by using two monoclonal antibodies directed toward the heavy chain of protein C is reported. This assay enabled the determination of protein C in concentrations of 10 to 400 ng/ml in less than 3 hours with a single antigen-antibody reaction. Within-run and between-run coefficients of variation were less than 8%. The mean concentrations of protein C in plasma of 42 normal subjects, 24 patients with liver disease, 27 with DIC, 48 with warfarin therapy and 15 with congenital protein C deficiency, were 4.2, 3.0, 2.3, 2.1 and 1.9 μg/ml, respectively. The results obtained with the present ELISA correlated well with those of radioimmunoassay (r=0.935, N=81) as well as those of Laurell's Rocket method (r=0.910, N=81) by using rabbit anti-human protein C serum. The present method was sensitive and specific for measurement of protein C and also PIVKA-protein C in plasma.     

Protein C inhibitor: Purification and proteinase reactivity

Protein C inhibitor was purified from human plasma by a modification of a published procedure (Suzuki, K., Nishioka, J., and Hashimoto, S. J. Biol. Chem. 258, 163–168, 1983). Approximately 1 mg of pure protein was obtained from 1 L plasma, a yield of about 17%. The protein C inhibitor preparation did not lose activity over 4 weeks at 4°C. Second order rate constants were measured for the inhibition of activated protein C, thrombin, and urokinase, and bimolecular complexes of protein C inhibitor with activated protein C and thrombin were visualized by denaturing polyacrylamide gel electrophoresis. Heparin accelerated the inhibition of the three proteinases in a manner consistent with a template mechanism. Plasma or pure protein C inhibitor (at the same concentration) showed the same effect of heparin on activated protein C inhibition, indicating that protein C inhibitor accounts for all the heparin-dependent inhibition of activated protein C in vivo.     

Effect of ketanserin on platelet function and bleeding time in uremic patients treated with erythropoietin

Hemostatic abnormalities are frequently observed in uremic patients and anemia is one of the clinical features of chronic renal failure and also a major factor in the pathogenesis of the uremic bleeding (1). There is a risk of bleeding and at the same time arterio-venous shunt thromboses occur (2). This may be due to a defective both platelet aggregation and platelet/vessel wall interactions (3). It is well established that treatment with recombinant human erythropoietin (rHuEPO) is efficacious in correcting anemia and improving hemostasis in uremic patients due to increased erythrocyte and thrombocyte counts, direct effect on hemostasis and/or improved platelet functions (4,5). Serotonin (5-hydroxytryptamine, 5-HT) released from platelets during their aggregation can play a role in hemostasis and platelet/vessel wall interactions (6). Although being a weak agonist, it markedly amplifies platelet response to variety of aggregating agents (7). Serotonin is also thought to contribute to the pathogenesis of vasospasm, thrombus formation,

(8). These effects may be attenuated by a blockade of vascular and platelet 5HT₂ receptors with ketanserin. On the other hand, erythropoietin therapy may increase a risk of thromboembolic complications in uremic patients. As reported previously, human recombinant erythropoietin (rHuEPO) led to an increase in blood and platelet serotonin (9). Therefore ketanserin might provide a protection against thrombosis (10). The purpose of the work was to study the effects of ketanserin on platelet function and bleeding time in uremic patients treated with rHuEPO.     

Assay of protein c in human plasma: Comparison of amidolytic, coagulation, and immunochemical assays

We studied functional protein C activity, both anticoagulant and amidolytic, as well as protein C antigen in 30 normal subjects, several members of a family with congenital protein C deficiency, 18 patients with severe preeclampsia, 27 patients with coronary heart disease, including 15 patients with myocardial infarction and 12 with angor pectoris, 20 patients on stable oral anticoagulant therapy (thrombotest values: 3–12%) and three patients with disseminated intravascular coagulation. Protein C values measured by the coagulant assay were compared to those obtained with amidolytic and immunochemical assays. In all the groups studied, the activity assays (amidolytic and coagulant) correlated significantly with each other as well as with the immunochemical assay. In patients on oral anticoagulant therapy the coagulant assay gave lower protein C values than amidolytic and immunochemical assays. A good correlation was found between immunological and amidolytic protein C assays (r=0.90, p < 0.001), immunological and coagulant protein C assays (r=0.93, p < 0.001), and amidolytic and coagulant protein C assays (r=0.95, p < 0.001) in all the samples studied without including the protein C values of patients on oral anticoagulant therapy. These results allow us to recommend the functional protein C coagulant assay in patients on stable oral anticoagulant therapy because only this assay evaluates the “in vivo” protein C function in these patients.     

Follow-up of nerve conduction in chronic uremic patients during hemodialysis

We studied to evolution of nerve conduction during hemodialysis following 21 patients with chronic renal failure. Mean motor nerve conduction velocity (MCV) was significantly different at hemodialysis onset and 3 years later for both common peroneal nerve (44.5 and 41.0 m./sec.) and ulnar nerve (52.5 and 47.1 m./ sec.). MCV decreased more in patients with low Kt/V (a depuration index) than in those with high Kt/V.

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Sommario Abbiamo studiato l'evoluzione a lungo termine della conduzione dei nervi periferici durante l'emodialisi valutando 21 pazienti affetti da insufficienza renale cronica in trattamento emodialitico da almeno 3 anni. I valori medi della velocità di conduzione motoria (VCM) all'inizio della dialisi e dopo 3 anni differivano significativamente, sia per il nervo sciatico popliteo esterno (media=44.5 e 41.0) che per il nervo ulnare (media=52.5 e 47.1). I pazienti con un basso Kt/V (un indice di depurazione) avevano una maggiore diminuzione della VCM rispetto ai pazienti con un elevato Kt/v.

     

Monoclonal antibodies against human plasma protein C and their uses for immunoaffinity chromatography

Human protein C, isolated by conventional multistep methods, was used for immunization of mice. Monoclonal antibodies were prepared and screening of antibodies to human protein C was achieved using an immunoblotting technique. Five monoclonal anti-protein C antibodies were compared as affinity ligands. Different parameters were studied (adsorption capacity, specificity of adsorption, possibility of desorption under mild conditions) and two antibodies were selected. One antibody allows preparation of highly purified protein C in a single-step procedure from a fraction of plasma containing high levels of coagulation factors whereas the other can be used for preparation of protein C deficient plasma.     

Isolation and characterization of a protein C activator from tropical moccasin venom

A protease from the venom of the tropical moccasin (Agkistrodon bilineatus) that activates protein C was purified to homogeneity by ion-exchange and gel permeation chromatography. The purified protease is a glycoprotein, and exhibited a molecular weight of 35,000 and 38,000 in SDS-PAGE under non-reducing and reducing conditions, respectively. The purified protease readily activated human protein C and steady-state kinetic parameters indicated an apparent Km for human protein C of 1.7 μM and an apparent kcat of 0.02 sec⁻¹. Calcium inhibited the activation of human protein C by the venom protease (K₁=93 μM). Amino-terminal sequence analysis revealed that the tropical moccasin protein C activator was highly homologous to the protein C activator isolated from Southern copperhead venom.     

Inhibitory effect of activated protein C on platelet aggregation induced by the prothrombin-converting reaction

The present study was undertaken to elucidate the effect on platelet aggregation of the prothrombin-converting reaction on platelets with or without activated protein C (APC). A reaction mixture of washed platelets from human individuals, Factor Xa and prothrombin markedly induced platelet aggregation; maximum aggregation rates, 31.3–92.5%, and times to reach to maximum aggregation, 11.6 to 20.1 min. This aggregation was inhibited by the addition of APC with 50% inhibition concentration (IC₅₀) value of 14.4 U/ml. APC also inhibited thrombin generation in the reaction mixture in a dose-dependent manner with IC50 value of 0.96 U/ml. However, APC did not inhibit the thrombin (0.1 CU/ml)-induced platelet aggregation at concentrations of up to 30 U/ml. These findings suggest that APC has no direct inhibitory effect on platelet aggregation and that APC inhibits platelet aggregation through inhibition of thrombin generation.     

Inhibition of activated protein C by benzamidine derivatives

In studies on the inhibition of activated protein C (APC) by benzamidine derivatives potent inhibitors of APC were found among anilides of 4-amidinophenyl--aminobutyric acid (K_i = 0.58 μmol/1). Several bis-benzamidine derivatives containing a cycloalkanone linking bridge inhibit APC with K_i values near the micromolar range.

Potent and selective inhibitors of thrombin derived from 3- and 4-amidinophenylalanine do not inhibit APC. This is of great importance for further development of these inhibitors as potential anticoagulant drugs.     

Recurrent venous thrombosis during warfarin treatment related to acquired protein S deficiency

Failure of warfarin to prevent new thrombotic processes was observed in three patients with very low free protein S concentrations and high C4b-binding protein (C4bBP) concentrations, and in one patient with hereditary protein S deficiency. We suggest that an increase in C4bBP reduces the free Protein S level, and warfarin treatment causes an additional decrease of free protein S. The four patients presented indicate that such reductions are of clinical importance. Heparin seems preferable as an anticoagulant in this situation, as warfarin given alone is ineffective, or may even be harmful. In a group of pancreatic cancer patients with advanced disease, subnormal mean free protein S was found, whereas mean total protein S concentration, and mean C4bBP concentrations were significantly higher (p<0.01) than in healthy controls. These findings indicate that an increase in C4bBP may induce free protein S deficiency contributing to the increased thrombotic tendency in this group of patients. The correlation between free protein S and C4bBP was 0.11, (n.s.), between total protein S and C4bBP 0.73 (p<0.0001).     

Mesenteric vein thrombosis in protein S congenital deficiency

Mesenteric vein thrombosis is considered an uncommon clinical presentation of protein S congenital deficiency. In the two patients with mesenteric vein thrombosis here reported an isolated deficiency of protein S was diagnosed; family investigation recognized protein S deficiency also in five relatives of one of them.     

L-asparaginase treatment reduces the anticoagulant potential of the protein C system without affecting vitamin K-dependent carboxylation

The changes in plasma levels of the vitamin K-dependent natural anticoagulants protein C (PC) and protein S (PS) and procoagulant factors II, IX and X were evaluated in 8 adult patients during treatment with L-asparaginase (L-ase i.v. 120,000 U/m² over 10 days). PC anticoagulant activity and factor IX, X and II coagulant activity decreased proportionally to their half-lives to a nadir of 50–60% of pretreatment values after 2–5 L-ase infusions, suggesting that inhibition of protein synthesis rather than consumption is the main mechanism responsible for the observed changes. Free PS antigen levels declined at a rate similar to total PS antigen, reaching a nadir of 56% of pretreatment values after 3 L-ase infusions; however, due to C4b-binding protein levels higher than total PS levels (p <0.05), they were constantly lower than the corresponding total PS antigen levels (0.05 < p <0.001). This implicates that total PS antigen levels cannot be taken as an indicator of PS activity. No differences between the antigenic levels and the anticoagulant activities of PC and free PS could be observed suggesting that L-ase does not affect the mechanisms of vitamin K-dependent carboxylation of Gla-residues. The faster rate of decline of PC and PS activities relative to that of factor II may be responsible for the onset of an hypercoagulable state during the early phase of L-ase treatment.     

Tissue factor pathway inhibitor (TFPI) activity in uremic patients during hemodialysis

We studied tissue factor pathway inhibitor (TFPI) activity during hemodialysis in 10 uremic patients who were not receiving anticoagulant for at least 120 minutes. TFPI activity before dialysis was normal (patients 107 ± 5.8%, controls 104 ± 4.5%). During extracorporeal circuit it rose progressively with a statistically significant difference, reaching a plateau between 60 and 120 minutes. Since thrombin induces a marked redistribution and release of TFPI from stimulated endothelial cells and platelets contain about 10% of TFPI activity that is secreted following activation it is possible that thrombin-induced release of TFPI by endothelium and platelets could account for the increased TFPI we found during hemodialysis. To investigate this possibility we measured during dialysis β-thromboglobulin (β-TG), thrombinantithrombin complex (TAT) and prothrombin fragment 1.2 (F 1.2). The increased levels of β-TG, TAT and F1.2 we noted during extracorporeal circuit are in keeping with this concept.

One hundred eighty minutes after initiation of dialysis, by which time all patients were receiving heparin there was a further increase in TFPI (to more than 200% of baseline), due to the presence of the glycosaminoglycan. This was due the previously reported displacement by heparin of the major intravascular pool of TFPI, from endothelial cell surfaces.     

Subacute uremic and diabetic polyneuropathy

We present 4 patients who had a subacute, predominantly motor polyneuropathy associated with diabetes mellitus and end-stage renal disease. Electrophysiological studies and muscle biopsy indicated a primary axonal degeneration of nerve with secondary segmental demyelination, and mild to moderate, acute and chronic denervation of muscle. A relative absence of denervation potentials on needle electromyography was an unusual feature. Three of our patients improved with a switch from conventional to high-flux hemodialysis. We speculate on possible mechanisms. © 1997 John Wiley & Sons, Inc.