Use of human protein C for prevention and treatment of depositions of thrombocytes

The use of human Protein C for the prevention and treatment of deposition or aggregation of thrombocytes, microparticles of thrombocytes, and leucocytes is described. In addition, an improved method for the extra-corporeal treatment of body fluids is disclosed.

The invention relates to a new range of application of human Protein C and 
a pharmaceutical preparation for the treatment of thrombo-embolic 
conditions. 
BACKGROUND OF THE INVENTION 
Protein C is a vitamin K-dependent protein that is synthesized in the liver 
and circulates as an inactive zymogen in a concentration of 4 mg/l. It is 
transformed by the thrombin-thrombomodulin complex into the active serine 
protease (activated Protein C) on the vessel wall surface (endothelium). 
It is known that activated Protein C has profibrinolytic properties. It 
also has anticoagulatory effects because it inactivates Factor Va, the 
co-factor for the Factor Xa-induced prothrombin activation (thrombin 
formation), and Factor VIIIa, the co-factor for Factor IXa-induced Factor 
X activation, by proteolysis. 
The activation of Protein C in vivo constitutes a negative feedback 
reaction of thrombin generation. In order to develop optimal biological 
activity, a co-factor (Protein S) is necessary. 
In the European patent application EP 0 406 216 a pharmaceutical 
preparation is described which contains Protein S, optionally, in 
combination with activated Protein C, and can be employed for the 
treatment or prevention of thrombosis and thrombo-embolic complications. 
According to EP 0 519 900 the use of a Protein C-containing pharmaceutical 
preparation together with a thrombolytically effective substance for the 
treatment of thrombosis and for the prevention of re-occlusion is 
possible. It was found that during the thrombolysis therapy a deficiency 
of Protein C results wherefore the substitution with Protein C is 
recommended. 
The effect of the inactive zymogen of Protein C differs fundamentally from 
the active enzyme, activated Protein C. 
Activated Protein C enables the prevention of arterial thrombosis or 
stenosis, preferably in combination with a thrombolytically effective 
agent (tissue plasminogen activator, tPA); for this, see EP 0 318 201. 
It is also known that in blood platelet-enriched plasma (PRP) activated 
Protein C suppresses platelet aggregation which is induced by thrombin 
activation. However, a higher concentration of activated Protein C leads 
to an opposite effect, namely to the aggregation of the blood platelets 
(E. N. Santander et al., Acta Physiologica Latino-Americana 3.3. (2), 
1983). 
Activated or stimulated blood platelets possess the glycoprotein IIb-IIIa 
complex which functions as a receptor for various adhesion molecules. 
Among the adhesion proteins that bind to GP IIb-IIIa of stimulated blood 
platelets are fibrinogen, von Willebrand Factor, and fibronectin. It is 
supposed that a tri-peptide sequence, namely Arg-Gly-Asp (RGD), of the 
adhesion proteins binds to the receptor. Among the proteins with a RGD 
sequence are also human Protein C and activated Protein C. However, the 
interaction of Protein C or activated Protein C with blood platelets is 
uncertain. It was found, for example, that activated Protein C binds to 
non-stimulated blood platelets in the presence of Protein S, and by this, 
the inactivation of Factor Va is potentiated. Protein C does not, however, 
bind to the blood platelets (J. Biol. Chem., 260 (4), 2007-10, 1985). 
With the help of flow cytometry for thrombocytes, surfaces of thrombocytes 
can be examined. Flow Cytometry allows the fast and sensitive analysis of 
receptor proteins on a single cell. During the examinations, a flow rate 
from 400-1000 thrombocytes per second is employed. The size of the 
thrombocytes is expressed by the light scattering. By the use of 
antibodies coupled with fluorescein-isothiocyanate, the binding of ligands 
(adhesion proteins) on a thrombocyte can be measured (description of the 
method in Blood, 86 (1), 173-179, 1986). 
The binding of fibrinogen to stimulated thrombocytes leads finally to the 
aggregation and/or deposition of these on injured endothelium and 
therewith to occlusion of the wound. Thrombo-embolic complications or 
stenosis are also attributable to the binding of fibrinogen to stimulated 
thrombocytes. For example, balloon angioplasty leads to an injury of the 
endothelium and therewith to a predisposition for arterial restenosis. 
SUMMARY OF THE INVENTION 
The object of the invention is to extend the range of application of human 
Protein C and to make a preparation available that can be administered for 
the prevention and treatment of the deposition and/or aggregation of 
thrombocytes. 
The object is solved according to the invention by a new use of human 
Protein C for the production of a pharmaceutical preparation which is 
suitable for the prevention and treatment of deposition and/or aggregation 
of thrombocytes, microparticles of thrombocytes (dust), and leucocytes 
with pro-coagulatory activity. Protein C prevents the deposition on vessel 
surfaces or vessel stenoses, in particular on injured, virus-infected or 
damaged endothelium and/or on exposed subendothelium or artificial vessel 
surfaces or vessel prostheses with or without endothelium. It was found 
that the binding of fibrinogen on stimulated blood platelets can be 
prevented by the addition of human Protein C in a dose-dependent manner. 
PRP was stimulated by addition of adenosine diphosphate (ADP) and the 
fibrinogen binding measured. The addition of a 
fluorescein-isothiocyanate-coupled antibody against fibrinogen allowed the 
detection of the fibrinogen found in PRP bound on thrombocytes by 
measurement on the flow cytometer. The prevention of the fibrinogen 
binding on stimulated thrombocytes was all the more surprising because 
activated Protein C had no influence on the fibrinogen binding. Therefore, 
it is probable that the RGD sequence in a protein can not alone be 
responsible for the prevention of the fibrinogen binding.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
Experiments with monoclonal antibodies (for example, -1, an IgM antibody 
that only recognizes the activated form of the IIb-IIIa complex; S. J. 
Shattil, J. Biol. Chem. 260, 11107, 1985) make it clear that the GP 
IIb-IIIa complex exposed by the activation of thrombocytes is not bound by 
the antibodies in the presence of Protein C. A competitive binding of 
protein C to GP IIb-IIIa is therefore supposed. Instead of -1, however, 
the use of other monoclonal antibodies against thrombocyte 
activation-dependent epitopes is also possible, for example, PADGEM (J. 
Clin. Invest., 78, 130, 1986), GMP (J. Biol. Chem., 264, 1816, 1989), and 
2.28 (Blood 70, 838, 1987). Hence, the binding site of fibrinogen is 
blocked. In contrast to the other adhesion proteins, the aggregation of 
the thrombocytes is not promoted, but instead prevented by this. 
Experiments with a perfusion chamber demonstrated that Protein C prevents 
the adhesion of thrombocytes in a rabbit aorta that is flushed with blood. 
The endothelium is stabilized then in the presence of Protein C, whereby 
the adhesion of stimulated thrombocytes is prevented. 
Protein C, as a native protein, as a derivative thereof, or mutant with an 
RGD sequence can be used according to the invention. Protein C in its 
inactive form as a zymogen is usable. This has the advantage that the 
protein does not have to be activated in order to prevent the fibrinogen 
binding or von Willebrand Factor binding on stimulated blood platelets. 
Therefore, Protein C can also be used according to the invention in 
certain conditions, like homocyteinurea, diabetes, or uremia, in which the 
endogenous activation of Protein C is inhibited. The zymogen Protein C 
also does not have the disadvantage of the activated Protein C, namely 
that it leads in a high concentration to an aggregation of the 
thrombocytes. 
The experiments with the help of flow cytometry confirm that human Protein 
C is suitable above all for the prevention of arterial restenosis. 
According to the invention, Protein C can therefore be used with all 
interventions of angioplasty, but also with use of a catheter, in order to 
prevent negative interactions with stimulated thrombocytes among 
themselves, with leucocytes, and with the endothelium. 
On the basis of the found mechanism of action of Protein C, a process 
according to the invention is possible for the extra-corporeal treatment 
of body fluids, such as blood or ascites, with which the deposition and/or 
aggregation of thrombocytes in a circulation apparatus should be 
prevented. This method finds application, for example, in a dialysis 
apparatus or an artificial kidney. 
By the following examples, the invention is further described. 
Fibrinogen binding to activated PRP 
10 ml of blood was drawn in citrate. PRP was obtained by conventional 
centrifugation. The addition of ADP resulted in the stimulation of 
thrombocytes. Subsequently, a fluorescein-isothiocyanate-coupled antibody 
against fibrinogen was added, and the fibrinogen bound on thrombocytes was 
determined by measurement on the flow cytometer (FACScan.RTM., Becton 
Dickinson). The fibrinogen binding was determined by the Green 
Fluorescence Intensity (one parameter statistics, x-axis "Log Green 
Fluorescence" (bound anti-fibrinogen), y-axis "Number of Cells") as well 
as by the light scattering (two parameter analysis, x-axis "Log-Green 
Fluorescence", y-axis "Log-Light Scatter" (size of the thrombocytes)). 
FIGS. 1-3 show the intensity of the fluorescence, i. e. the fibrinogen 
binding on stimulated thrombocytes as well as the size of the 
thrombocytes. FIG. 1 shows the fibrinogen binding in the PRP after ADP 
stimulation. The application of the agonist leads to a fibrinogen binding 
on the cell surface, but also to a change of the size of the thrombocytes. 
In comparison to this, FIG. 2 shows the fibrinogen binding after ADP 
stimulation in the presence of Protein C in a concentration that 
corresponds to the 4-fold plasma concentration. A significant reduction of 
the fibrinogen binding is recognizable. As opposed to this, activated 
Protein C in this test mixture leads to no decrease of the fibrinogen 
binding on thrombocytes. 
Binding of -1 on activated thrombocytes 
The monoclonal antibody -1 reacts with the glycoprotein IIb-IIIa complex 
on the surface of thrombocytes after activation of the thrombocytes with 
ADP. 
The drawing of blood occurred in EDTA/Trasylol. Thereafter, 
platelet-enriched blood plasma (PRP) and plasma in which the blood 
platelets were depleted (PPP) was produced by conventional centrifugation. 
PRP was diluted 1:40 in PPP. Thereafter, -1 was added with ADP and, 
optionally, Protein C or activated Protein C and incubated at room 
temperature for 10 minutes. After addition of an anti-mouse antibody 
(fluorescence conjugated, FITC from Sigma Chemical Co.) and a further 
incubation time of 20 minutes, the binding of the -1 on thrombocytes 
was detected by flow cytometric examination. 
FIG. 4 
Binding of the -1 to PRP/ADP 
FIG. 5 
The -1 Binding to Thrombocytes is Prevented in the Presence of Protein 
C. 
FIG. 6 
Activated Protein C Leads to no Influencing of the -1 Binding on 
ADP-stimulated Thrombocytes. 
Blood Platelet Deposition on the Subendothelium in the Perfusion Experiment 
Venous blood was anticoagulated with citrate-phosphate-dextrose (19 mM). 
Protein C (Immuno AG) was reconstituted and added to 20 ml of the 
anticoagulated blood before the perfusion. Perfusions were performed at 
37.degree. C. in a perfusion chamber (experimental description in Thromb. 
Haemost., 37, 1-16, 1977). Enzymatically-treated aorta samples from rabbit 
were mounted in a perfusion chamber. With the aid of a haemodialysis blood 
pump (Renal Systems, Minneapolis, Minn., USA) an appropriate flow rate was 
set. After 5 minutes of perfusion, the aorta samples were washed with 
buffer and fixed (glutaraldehyde: formaldehyde=2%:3% (v/v). Thereafter, 
these were embedded in JB4 (Polyscience, Warrington, USA), stained with 
Toluidine Blue, and morphometrically examined. With the help of a computer 
program (described in Haemostasis, 16, 8-14, 1986), the blood platelets 
were classified as follows: adhesion (blood platelet layer&lt;5 .mu.m), 
thrombi (aggregation.gtoreq.5 .mu.m), both expressed in percent of the 
total length of the blood vessel. The results are given in Table 1. At 
concentrations of less than 16 .mu.g/ml, Protein C demonstrates no effect 
on the adhesion of blood platelets in comparison to controls. At 
concentrations of 16 and 32 .mu.g/ml, the covered surface was 
significantly reduced. 
TABLE 1 
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Blood Platelet Deposition on Subendothelium in the Perfusion 
Experiment with Citrate-anticoagulated Blood in Dependence on 
the Protein C Concentration (n = 3, x .+-. SD) (flow rate = 800 
s.sup.-1) 
coated surface 
Adhesion Clots 
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Control 18.1 .+-. 5.3 9.3 .+-. 4.5 
8.8 .+-. 1.9 
Protein C 13.1 .+-. 3.5 4.8 .+-. 0.9 
8.3 .+-. 2.9 
16 .mu.g/ml 
Protein C 9.6 .+-. 5.7 4 .+-. 2.5 
5.6 .+-. 3.4 
32 .mu.g/ml 
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