Platelet aggregating material from equine arterial tissue

Novel hemostatic agent comprises equine arterial fibrillar collagen in a carrier. The agent is useful for the aggregation of platelets for clinical diagnostic tests and for the clotting of blood, such as for controlling bleeding in warm blooded species. The fibrillar collagen is obtained by extracting homogenized equine arterial tissue with aqueous solutions followed by extensive dialysis.

BACKGROUND OF THE INVENTION 
Platelet aggregation is of primary importance in the clotting mechanism of 
blood. Materials that stimulate platelet action also promote blood 
clotting and are called hemostatic agents. In the prior art, it has been 
suspected that certain components of the arterial wall are related to 
platelet action. The function of the particular components, either singly 
or in combination, has not been well understood. The study of platelet 
aggregation is useful for determining the role of platelets in a variety 
of hemostatic diseases, and in diagnostic tests for detecting the presence 
of diseases characterized by abnormal platelet function or activity. A 
potent, highly sensitive platelet aggregating agent has long been needed. 
It is an object of this invention to provide a hemostatic agent which is 
highly effective for promoting platelet aggregation and blood clotting. 
It is a further object to provide an agent useful for studying platelet 
function and detecting functional disorders in the platelet aggregating 
mechanism. 
It is a further object to provide a method for producing a highly potent 
hemostatic agent from arterial tissue of equine species. 
It is a further object to provide a method for clotting blood and for 
controlling bleeding from a wound or incision. 
It is a further object of this invention to provide a vasoconstrictive 
agent. 
SUMMARY OF THE INVENTION 
These and other objects have been achieved by providing a platelet 
aggregating hemostatic composition from equine arterial tissue. It has 
been discovered that the compositions of this invention will stimulate 
platelet aggregation when an effective amount contacts a platelet 
containing suspension such as platelet rich plasma or whole blood either 
in vitro or in vivo. A composition therefore is useful in a method for 
controlling external bleeding from a wound or incision by contacting the 
wound or incision with an effective amount of the composition either alone 
or with a suitable carrier. The invention also comprises a method of 
detecting abnormal platelet function comprising contacting a solution 
containing platelets suspected of abnormal function, such as plasma or 
whole blood, with a composition of the invention and comparing the 
aggregation response of the plasma or whole blood under study to the 
response of normal platelets. 
The objects of this invention are achieved therefore according to a method 
for preparing hemostatic compositions comprising the steps of (a) 
contacting homogenized equine arterial tissue with an aqueous solution to 
provide an aqueous mixture containing extracted collagen species and 
unextracted arterial tissue; and (b) separating unextracted arterial 
tissue from said aqueous mixture to provide a supernatant extract 
containing arterial fibrillar collagen, said extract being capable of 
stimulating the aggregation of platelets. In its compositional aspects 
this invention comprises hemostatic compositions comprising a suspension 
of equine arterial fibrillar collagen in a carrier. In its method of use 
aspects, this invention comprises a method of stimulating platelet 
aggregation comprising contacting a platelet containing suspension such as 
platelet rich plasma or whole blood with an equine arterial fibrillar 
collagen composition. The aggregation of platelets according to this 
method is useful in a method for controlling bleeding from a wound or 
incision comprising contacting the wound or the incision with a novel 
composition of the invention. In its diagnostic aspects, this invention 
comprises a method of detecting abnormal platelet function comprising 
contacting a solution containing platelets such as plasma or whole blood 
with a composition of the invention and comparing the aggregation response 
of said platelets to the response of normal platelets. 
DETAILED DESCRIPTION 
An aspect of this invention is the discovery that equine arterial tissue 
contains material which can be extracted with a balanced salt solution and 
is highly potent for stimulating the aggregarion of platelets of warm 
blooded animals, including humans. Equine arteries contain lining 
endothelium, elastin (non-collagenous), basement membrane (amorphous 
collagen), and smooth muscle cells, believed to be the site of synthesis 
of fibrillar arterial collagen. The hemostatic compositions of this 
invention are extractable from homogenized arterial tissue by aqueous 
solutions. The aqueous extract obtained from equine arterial tissue is 
several times as potent as similarly obtained extracts from swine or 
bovine species. Additionally, it has been found that the extracted equine 
collagenous material is even more effective for stimulating the 
aggregation of human platelets than the platelets of other animals, 
including a homologous species. 
In the first step of this invention, segments of equine arterial tissue 
(horse, donkey, mule, etc.) are cleaned of all loose connective tissue, 
adventitial layer, blood cellular fragments, platelets, plasma, etc. by 
extensive rinsing, is contacted with a balanced salt solution. The 
preferred arterial tissue is tissue from the upper thoracic aorta of the 
animal, extending to the diaphragm and including the common 
bronciocephalic trunk. The platelet aggregating material isolated in 
accordance with this invention is most concentrated in this portion of the 
arterial system. The active material appears to be present in higher 
amounts in older animals. Animals with overt disease symptoms or with 
arthero- or arterosclerosis lesions should not be used as sources of the 
hemostatic composition. 
In the presently preferred procedure for preparing the arterial tissue to 
undergo the isolation procedure of this invention, the tissue is cut into 
small segments (approximately 5.times.5 mm) and thoroughly cleansed by 
washing or rinsing with an appropriate wash liquid. Any of a variety of 
physiological safe liquids including water, but preferably balanced salt 
solutions can be used for cleansing. Physiological saline is useful. 
Tyrode's solution which is a balanced salt solution simulating the salt 
content of mammalian body fluids and including glucose, sodium bicarbonate 
and magnesium salts stabilized at a pH of about 7.4 is preferred. Other 
balanced salt solutions such as Hank's or Earle's can also be employed. 
The cleansed segments may be utilized immediately, but will normally be 
prespared in relatively large amounts and stored at -20.degree. C. to 
-85.degree. C. until ready for use. 
The first step of the isolation procedure is to homogenize and extract the 
segments at low temperature, i.e. about 0.degree. C. to 5.degree. C. in 
the selected aqueous solution. The suspension is collected, and may be 
again homogenized and extracted. The procedure may be repeated up to eight 
or more times to insure complete extraction. The insoluble segments are 
separated to leave the extract as a useful composition of this invention. 
The unextracted arterial tissue is readily separable by filtration. 
However, the preferred separation method is moderate centrifugation, i.e. 
650 g for ten minutes. Additional centrifugation of the hemogenate tends 
to increase loss of suspended material to the pellet. Upon centrifugation, 
the unextracted material concentrates in the pellet. The supernatant 
extract, containing extracted material contains the hemostatic composition 
of this invention, and is capable of stimulating platelet aggregation. The 
composition can, and preferably is further concentrated and purified by a 
series of alternate dialyses and centrifugations. 
The hemostatic compositions of this invention prepared as described in 
detail hereinafter contains a hemostatic agent capable of enhancing the 
rate of platelet aggregation in mammalian blood which is stable for 
extended periods of time at temperatures as low as -85.degree. C. It loses 
its platelet aggregating activity when exposed to collagenase or when 
heated at 100.degree. C. for fifteen minutes. It is stable at 56.degree. 
C. for up to sixty minutes or when exposed to .alpha.-chymotrypsin. With 
repeated spearation, extraction and dialysis, it is possible to isolate 
from different starting materials, products which all manifest the 
foregoing properties and which, while not always absolutely identical, can 
be recognized by these properties and by the fact that they contain the 
following average number of amino acid residues per 1000 total amino acid 
residues: 
______________________________________ 
Lysine 29.8 Glycine 206.5 
Histidine 31.2 Alanine 133.9 
Hydroxylysine 
4.8 1/2 Cysteine 
13.2 
Arginine 43.2 Valine 57.6 
Hydroxyproline 
20.1 Methionine 14.2 
Aspartic Acid 
65.4 Isoleucine 26.7 
Threonine 38.1 Leucine 52.5 
Serine 40.3 Tyrosine 15.3 
Glutamic Acid 
81.9 Phenylalanine 
27.1 
Proline 98.2 
______________________________________ 
The foregoing is consistent with the fact that as with many products 
isolated from natural sources, particularly animal sources, the exact 
analysis and properties of each isolated product will vary. The 
composition of this invention, is however, readily identified by its 
platelet aggregating activity, its hypertensive activity and of course, 
its source and method of isolation. The actual analysis of product will 
vary depending on the member of the equine species which serves as the 
source, for example horse versus donkey. It may even vary when different 
individuals of the same type are used. For example, the amounts of glycine 
in the products isolated from several different burros may vary. 
A series of tests were performed to more fully characterize the hemostatic 
fibrillar collagen composition of this invention. A homogenate of burro 
aortic tissue was incubated with .alpha.-chymotrypsin, dialyzed and the 
retentate pelleted by ultracentrifugation and resuspended in Tyrode's 
solution to form a stock solution. Aliquots of this stock solution were 
treated with Tyrode's solution containing purified collagenase. Other 
aliquots were treated with collagenase solution which had been boiled for 
fifteen minutes. After incubation with .alpha.-chymotrypsin, the platelet 
aggregating ability was retained. Incubation of the stock solution with 
collagenase produced rapid and complete destruction of the aggregator 
activity characterized by a prolongation of time to the onset of platelet 
aggregation, a conspicuous decrease in the velocity of aggregation, and a 
marked deterioration in the aggregator potency. The collagenase that had 
been boiled prior to incubation with the stock solution had no deleterious 
effects on platelet aggregating activity or potency. 
Histochemical staining was performed by first precipitating fibrillar 
collagen from a burro aorta extract (dialyzed). To 7.5 ml (15 mg dry 
solids) of the dialyzed extract was added an equal volume of Millonig's 
buffered 0.1 glutaraldehyde solution, pH 7.4. After overnight incubation, 
the precipitated fibrils were pelleted by centrifugation (650.times.g for 
30 minutes). The precipitate was washed with distilled water and directly 
stained with Verhoeff's elastica stain and Von Gieson's collagen 
counterstain. The stained fibrillar material was dehydrated and cleared 
and mounted on a glass slide for microscopic examination. Another aliquot 
of the dialyzed extract was precipitated and preserved with 10 ml of 
Millonig's phosphate buffered 0.1% glutaraldehyde. The microfibrillar 
material was washed and stained with aqueous 1% phosphotungstic acid (pH 
2.3). In high magnification scanning electron microscopy, the burro 
fibrillar aortic collagen of this invention appears ultra thin (about 500 
A diameter) fibers with multiple well stained subbands between the major 
bands. This ultrastructural banding is similar to that reported for other 
fibrillar collagen. Amino acid analysis has indicated that fibrillar 
collagen of this invention resembles typ I collagen species reported in 
rat skin and tendon and human aortas. These collagen species are described 
in the following references: Gallop et al, "Posttranslational protein 
modifications, with special attention to collagen and elastin", Physical 
Rev., 55:418-487, 1975; Barnes et al, "Platelet aggregating activity of 
type I and type III collagens from human aorta and chicken skin", Biochem 
J., 160:647-651, 1976; McCullagh et al, "Collagen characterization and 
cell transformation in human arteriosclerosis", Nature, 268:73-75, 1975; 
Trelstad, "Human aorta collagens: Evidence for three distinct species", 
Biochem Biophys. Res Comm, 57:717-725, 1974. 
While it is not understood just how the fibrillar collagen of this 
invention functions in its natural environment, it is believed that the 
collagen functions differently in its extracted form. The platelet 
aggregating action of the extracted collagen, even for autologous 
platelets, is accelerated over natural equine blood clotting. Internal 
injection in a guinea pig of a very dilute solution of extracted equine 
arterial fibrillar collagen (the highest dilution still domonstrating 
platelet aggregating proerties) produced a dramatic rise in blood 
pressure, accompanied by extensive thrombus formation and acute heart 
attack. The rapid rise in blood pressure illustrates the vasoconstrictor 
action of the extracted fibrillar arterial collagen, which enhances its 
utility for controlling bleeding. 
The extracted arterial fibrillar collagen composition of this invention is 
the fibrillar collagen separated from its native arterial structural 
surroundings, regardless of the particular separation method or extractant 
used. In nature, the fibrillar collagen is not known to exist separate 
from surrounding arterial tissue. Nevertheless, this invention in its 
composition and method of use aspects is not intended to include arterial 
fibrillar collagen which becomes separated from its natural structural 
surroundings without human assistance. 
The hemostatic agent of this invention comprises a suspension of platelet 
aggregating material in a carrier. Tyrode's solution is preferred as the 
carrier because it has been shown to contain nothing which deactivates the 
platelet aggregating function of the collagen. It will be apparent to 
workers in the art that many other aqueous solutions would be suitable 
carriers, for example, conventional saline solutions, balanced salt 
solutions such as Hank's solution, or Earle's solution. The preferred 
carrier for frozen storage is pure water, however, it is preferred for 
clinical testing purposes that the fibrillar collagen be resuspended in a 
balanced salt solution. For use to control bleeding, the carrier can be 
any externally administrable pharmaceutical carrier in which the fibrillar 
collagen can be dispersed sufficiently for use. The preferred carrier is 
an aqueous salt free solution that is provided as described above by 
dialysis against water. Antibiotics, such as penicillin and streptomycin, 
can be added to the salt free solution if desired for use in treating 
wounds, incisions, etc. It has been found that the hemostatic material of 
this invention is highly stable in the presence of antibiotics and when 
frozen at -85.degree. C. for long term storage. 
One use of the extracted equine arterial hemostatic compositions is as a 
diagnostic tool to provide information about platelet function in human 
and other warm blooded species, e.g. mammals. It appears that platelets 
are more sensitive to this material than to any known commercial product 
or arterial collagen of other species. By contacting a solution containing 
platelets (e.g. plasma, or whole blood) with a composition of this 
invention and comparing the aggregation response to that of normal 
patients, platelet dysfunctions or hemostatic disorders can be detected. 
Platelet dysfunction is characteristic of several diseases. Some drugs 
such as aspirin have shown to affect platelet activity. Minor differences 
in platelet activity can be detected by the response to the composition of 
this invention, thus indicating the course of treatment, e.g. avoidance of 
the drug. The products of this invention are highly sensitive for 
detecting hyperactive platelets such as are associated with coronary heart 
disease, myocardial ischemia, and myocardial infarction. In addition, the 
extractable product of this invention is a valuable research tool for 
studying the role of platelet activity in such diseases as 
arteriosclerosis, heart attack, stroke, pulmonary embolism, drug toxicity, 
and ingestion of toxic metal pollutants such as cadmium. 
Another utility for the hemostatic compositions of this invention is to 
stimulate the clotting of blood in wounds or surgical incisions, including 
skin grafts. The fibrillar collagen, like other animal collagen species, 
is expected to be compatible for internal applications, such as for 
controlling hemorrhaging from ruptured organs during veterinary surgery. 
For this use, the material should be in a pharmaceutically compatible 
carrier, e.g. Hank's, Earle's or Tyrode's solution. Alternatively, the 
isolated material can be separated from solution, i.e. by freeze drying 
and applied as a sponge or powder. 
The various methods of preparation and use of extracted products of this 
invention are illustrated by the following examples. It will be apparent 
to those skilled in the art that substantial variations can be made in the 
illustrated methods without destroying or interferring with platelet 
aggregating functions of the material and such variations are contemplated 
as equivalents of the invention herein described. 
To establish the platelet aggregating activity of the products of this 
invention as referred to in some of the following examples, it was 
necessary to prepare protein rich plasma (PRP) and protein poor plasma 
(PPP) from selected subjects. Human subjects, when selected, were not 
permitted to ingest aspirin or any other medication for ten days prior to 
testing. Whole blood was drawn from a juglar (animal) or forearm (human) 
vein through a disposable sterile silicone-treated needle, using a sterile 
plastic (35 ml) syringe previously wetted with a filter sterilized 
anticoagulant of 3.8% trisodium citrate dihydrate and 0.5% deterose in 
triple distilled water (pH 7.0). Collections of blood were immediately 
admixed with 0.1 volume of the anticoagulant in capped polystyrene tubes 
to avoid glass activation of platelets and plasma. Differential slow 
centrifugation at 22.degree. C. was used to prepare citrated PRP. Equine 
blood was centrifuged once at 95 G for fifteen minutes; other animal or 
human blood was centrifuged twice (when necessary) sequentially at 95 G 
for thirty minutes and the two fractions cooled. The PRP was collected 
with a disposable polyethylene bulb pipet. To prevent pH change and 
optimize the platelet function during tests, the PRP was stored at 
22.degree. C. in capped plastic tubes. PPP was obtained by centrifugation 
of the remaining blood at 650 G for twenty minutes. Platelet counts in the 
PPP were determined by phase contrast microscopy using 1% ammonium 
oxalate. 
Assays of platelet aggregation activity and potency were carried out in 
flat bottom silicone coated test tubes 8.75.times.50 mm size using a 
self-calibrating photoelectric apparatus (Platelet Aggregation Profiler, 
Model PAP-2 Bio/Data Corporation, Willow Grove, Pa.) with an integrator 
for turbidimetric curve recordings.

EXAMPLE I 
The platelet aggregating property of a group of aged burros (Equus asinus) 
were studied using three platelet stimulation materials; thrombin, 
adenosine diphosphate (ADP) and an aqueous extract of burro aortic tissue. 
Some of the group of burros had been irradiated by exposure to a nuclear 
device, some had been irradiated by exposute to .sup.182 Ta.gamma. and 
others were unirradiated controls. Platelet dysfunction has been 
postulated as a symptom of radiation sickness. Whole blood was collected 
from the animals by jugular venipuncture using plastic (35 ml) syringes 
previously wetted with a filter sterilized citrate anticoagulant composed 
of 3.8% trisodium cirtrate (0.013 M) and 0.5% dextrose in glass distilled 
water (pH 7.0). Platelet rich plasma (PRP), 45 ml, was obtained from 60 ml 
whole blood centrifuged at 150.times.g for ten minutes at 22.degree. C. 
Platelet poor plasma (PPP) was obtained by centrifugation of the remaining 
blood at 650.times.g for twenty minutes. Platelet aggregation stimulators 
were prepared as follows: stock ADP, grade I sodium salt from equine 
muscle (obtained from Sigma Chemical Company, St. Louis, Mo.) was 
dissolved in barbital buffer (pH 7.0) to produce a concentration of 
2.times.10.sup.-4 M. Aliquots of 0.5 ml were frozen (-20.degree. C.) and 
stored in capped plastic tubes. The contents of a tube was thawed and 0.05 
ml of stock ADP was added to 0.45 ml of burro citrated PRP to produce a 
final concentration of 20 micro moles of ADP. Stock thrombin, (grade II 
thrombin 1000 NIH units/ml in 0.05 M phosphate buffer at pH 7.0) from beef 
plasma (Sigma Chemical Company) was diluted in 0.15 M NaCl to produce a 
concentration of 25 units of thrombin/ml. The equine aortic extracts were 
prepared as follows: the aortic arch and upper thoracic aortas of each of 
two burros (one irradiated and one unirradiated) were excized and rinsed 
in 0.15 M NaCl solution to provide working material; the loose connective 
tissue was dissected from the exterior surface of the arterial wall. After 
more than five repetitive rinsings in large volumes of saline solution and 
dissection the remaining loose surface connective tissue was dissected 
away. The cleansed vessel walls, predominantly tunica media and tunica 
intima, were cut in 5 mm square pieces. The cut issues were exhaustively 
washed free of all visible loosely adhered blood elements with Tyrode's 
solution. Finally, the pieces of aorta were rinsed several times in 
twenty-five volumes of filter sterilized Tyrode's solution. The cut, 
washed and drained tissues were packed in plastic petri plates, frozen 
(-20.degree. C.) and stored. A day before extractable material was desired 
for testing, pieces of the frozen tissue were weighed, immersed in cold 
filter sterilized Tyorde's solution 1 g/20 ml) and blended in a 
macrohomogenizer (Vertis, Model 3, Scientific Products, Stone Mountain, 
Ga.). Moderate to high speed mincing (23,000 rpm) was performed for five 
minutes. Blending was in a Lucite cooling cup packed in wet ice to avoid 
heat denaturation. The suspension of blended tissue was held overnight at 
4.degree. C. and centrifuged at 650.times.g for ten minutes. The desired 
extractable platelet stimulator from the aorta was contained in the 
supernatant phase. The extract was packed in wet ice and held at 4.degree. 
C. and showed no deterioration in the platelet aggregating ability after 
thirty-six hours. 
The response of the platelets to ADP, thrombin and to the aortic extract 
solution was evaluated by means of a self-calibrating aggregometer 
(Platelet Aggregation Profiler, Bio/Data Corporation, Willo Grove, Pa.) 
with an integrated recorder. Such devices are typically used in clinical 
laboratories to study platelet action. The platelet aggregation response 
to ADP was calculated as the maximum percentage decrease in optical 
density (OD) of the PRP reaction mixture stirred at 37.degree. C. for 
periods up to ten minutes. The maximal velocity of platelet aggregation 
(OD decrease in percent) was calculated from the slope of the tangent line 
to the downward inclination of the optical density tracing during themost 
rapid phase of platelet flocculation. The response to thrombin was 
measured as (1) the delay time in seconds to the formation and contraction 
of a reactive platelet means followed by plasma clotting, and (2) the rate 
of platelet aggregation response expressed as maximum slope/sec.times.100. 
The platelet response to the extracted aortic material was measured as (1) 
the lag phase time in seconds to the induction of platelet aggregation, 
(2) the maximal % decrease in OD of the reaction and (3) the maximal 
velocity of the platelet flocculation (maximum slope/second.times.100). To 
calibrate the aggregation profiler, a series of measurements were taken at 
various platelet concentrations. The log of the platelet count varied 
essentially linearly with the optical density. 
The platelet aggregation results are shown in Tables I, II and III. For 
both the ADP additions and the aortic extract, between 200,000 and 240,000 
platelets/microliter of platelet rich plasma produced optimal reaction. 
As shown, it appears that the extract from the irradiated burro was 
measurably more effective than the extract from the unirradiated burro. 
This effect was believed to be caused by a radiation induced late somatic 
effect. It has since been shown that some unirradiated burros also have 
large amounts of fibrillar arterial collagen. It can be seen from Tables 
I, II and III that the aortic extract was substantially superior to the 
ADP and the throbmin solutions, particularly with respect to the velocity 
of aggregation. In humans, the platelet response to ADP is generally lower 
than response to the collagen. The interaction of the aortic extract with 
the platelets was characterized by (1) a distinctive delay time to the 
induction of platelet flocculation, (2) a high velocity of platelet 
clumping and (3) irreversible platelet aggregation. The response are 
typical of collagen-platelet interactions. 
The extracted platelet aggregating activity in suspension in Tyrode's 
solution was insensitive to heating in a water bath at 56.degree. C. for 
sixty minutes. The platelet aggregating activity was abolished by 
immersing the extract into a boiling water bath for fifteen minutes. Upon 
freezing at -20.degree. C. and thawing, platelet extract retains all of 
its platelet aggregating potency. Sonication of the material subsequent to 
freeze preservation and thawing is not required to unmask the activity and 
might even be harmful. The platelet aggregating material was nondialyzable 
from the Tyrode's solution against three changes in forty volumes of 
Tyrode's solution for seven days at 4.degree. C. with continuous magnetic 
stirring. The protein concentration of extractable aortic material in two 
preparations from the aorta from unirradiated burro was 2030 and 2080 
micrograms of protein/ml against bovine serum albumin as standard. Four 
preparations of extract from the aorta from the irradiated burro had 
concentrations of 1900, 1200, 2100 and 1380 micrograms of protein/ml. 
Ultracentrifugation of the aqueous supernatant at 105,000.times.g for one 
hour at 4.degree. C. produced a soluble phase and a pellet of gelatinous 
material. The soluble phase had 75% of the original protein. The platelet 
aggregating agent was entirely contained in the gelatinous pellet. The 
irradiated burro from which aortic extract was obtained was about 
twenty-six years old and had been exposed to 545 R of .sup.182 Ta total 
body .gamma.-radiation at 27.7 R/hr. about twenty-four years prior to 
death. The unirradiated burro was about twenty-one years old at death. 
TABLE I 
______________________________________ 
Burro Platelet-Aggregation Responsiveness to Strong ADP 
Average 
Maximal 
velocity 
of aggre- 
gation 
(ADP 
ADP Average OD decrease (in %) after 
slope/ 
(in .mu.mole/ 
1 3 9 Maxi- second) 
0.45 ml of PRP) 
minute minutes minutes 
mum .times. 100 
______________________________________ 
Unirradiated 
controls 
(3 burros) 
40 (n = 8) 
29 47 41 48 59 
20 (n = 8) 
33 52 48 55 64 
Irradiated 
Burros 
(.sup.182 Ta 
.gamma.-radiation) 
(3 burros) 
40 (n = 7) 
26 46 44 51 50 
20 (n = 7) 
27 47 49 55 58 
Irradiated 
Burros 
(nuclear 
device) 
(4 burros) 
40 (n = 10) 
29 52 50 56 59 
20 (n = 11) 
29 51 47 54 62 
______________________________________ 
n = No. of repetitive tests. 
TABLE II 
______________________________________ 
Burro Platelet Reactions to Strong and Weak Thrombin 
Average 
Average Maximal velocity 
Delay time to PRP- 
of aggregation 
Thrombin (in units/ 
plasma clotting 
((thrombin slope/ 
0.90 ml of PRP) 
(in seconds) second) .times. 100) 
______________________________________ 
Unirradiated Controls 
(3 burros) 
0.50 (n = 12) 
75 92 
0.25 (n = 9) &gt;300 17 
Irradiated Burros 
(.sup.182 Ta .gamma.-radiation) 
(3 burros) 
0.50 (n = 8) 60 73 
0.25 (n = 7) &gt;300 23 
Irradiated Burros 
(nuclear device) 
(4 burros) 
0.50 (n = 15) 
69 76 
0.25 (n = 14) 
199 57 
______________________________________ 
n = No. of repetitive tests. 
TABLE III 
______________________________________ 
Unirradiated Burro PRP Reaction to Extracts 
from Aortas of Unirradiated and Irradiated Burros 
Platelet reaction 
Maximal 
Aorta velocity of 
collagen aggregation 
stimulator(s) OD decrease 
(collagen 
(dose in ml/ 
Lag phase maximum ((slope/second) 
0.45 ml of PRP 
(in seconds) 
(in %) .times. 100) 
______________________________________ 
Burro A86 
(unirradiated) 
control) 
0.05 (n = 3) 
29 87 165 
0.10 (n = 2) 
29 89 170 
Burro R52 
(irradiated) 
0.05 (n = 3) 
17 88 211 
0.10 (n = 2) 
17 89 227 
______________________________________ 
n = No. of repetitive tests. 
EXAMPLE II 
This example illustrates the platelet response of various animals, 
including humans, to arterial extracts from different irradiated and 
unirradiated burros. The aortic arch with the upper thoracic aorta of each 
of these burros was dissected and rinsed in three or more changes of 1 L 
volumes of 0.15 M NaCl solution. All visible loosely organized connective 
tissue was dissected from the outer surface of the arterial walls. Square 
cut pieces (about 5 mm) of aorta were rinsed in saline solution and 
exhaustively washed in Tyrode's solution to remove all loosely adsorbed 
plasma proteins and ahered blood elements. The cleansed tissue was 
drained, packed in plastic petri plates and frozen at -20.degree. C. The 
aortic tissue (4 grms wt.) was blended in 80 ml of cold filter sterilized 
Tyrode's solution in the macrohomogenizer at 23,000 rpm for five minutes 
in a Lucite cooling cup packed in wet ice. The homogenate was stored 
overnight in 4.degree. C. and then centrifuged at 650.times.g for ten 
minutes. The supernatant was separated and stored at 4.degree. C. The 
remaining unextracted pelleted tissue was suspended in 40 ml of Tyrode's 
solution, again homogenized as above and stored overnight at 4.degree. C. 
The second homogenate was centrifuged and the supernatant was removed. The 
two supernatants were pooled and blended in the macrohomogenizer. The 
resulting suspension was placed in a telescoped cellophane bag. It was 
dialyzed against thirty to forty volumes of Tyrode's solution with 
continuous magnetic stirring for seven to ten days at 4.degree. C. 90 to 
100 ml of of the dialyzed burro aorta extracts were obtained. After 
dialysis, the crude extracts (retentate) were pelleted by 
ultracentrifugation (105,000.times.g for one hour at 4.degree. C.). The 
pellets were suspended in 1/2 volume of fresh Tyrode's solution and again 
blended in a macrohomogenizer. The preparation was designated as a 
2.times. concentrate. For some experiments, 2.times. concentrates were 
intermixed with 3.90 M ammonium sulphate solution to a saturation of 46.2% 
(1.80 M). The precipitate which formed after storing in wet ice overnight 
at 4.degree. C. was collected by centrifugation (650.times.g for thirty 
minutes). The pellet was resuspended in Tyrode's solution to the starting 
volume and again blended and dialyzed as described previously. All 
dilutions of the extracts were made in Tyrode's solutions. The diluted 
suspension can be stored in wet ice for seventy-two hours without 
undergoing any deterioration in platelet aggregation activity quality or 
potency. Table IV demonstrates the response of human PRP compared to horse 
and burro PRP. The aggregating agent was the retentate prior to 
ultracentrifugation. Table V demonstrates the response of burro platelets 
and human platelets for the more highly purified extracts. As seen, the 
morehighly purified reagents are consistently better at higher dilution 
than are the extracts shown in Table IV. Platelet response of humans and 
burros to like concentrations of extract contained in salt-free solution 
obtained by dialysis of the Tyrode's solution extract against water, were 
virtually the same as to the extract of Table V. In general for clinical 
testing and for controlling bleeding, the agent of this invention should 
be at the highest dilution which will provide the desired results, e.g. 
quick onset, 80+% aggregation, etc. 
TABLE IV 
______________________________________ 
Aggregability Effects on Equine and Human platelets 
Concentration of 
extract (in 0.05 
PRP 
ml/0.45 ml of a PRP of 8 burros 
of PRP) horse (avg. response) 
Hu-1 Hu-2 Hu-3 
______________________________________ 
Delay time from stimulator addition to onset 
of platelet aggregation (in seconds) 
Undiluted 29 28 12 12 12 
1:5 dilution 
36 37 17 14 14 
1:10 43 43 17 22 18 
1:20 55 52 19 24 19 
1:40 77 63 24 28 24 
1:80 -- 72 34 29 29 
1:160 -- -- 48 43 31 
1:320 -- -- -- -- 38 
Maximal optical density decrease of PRP (% aggregation) 
Undiluted 83 95 96 89 82 
1:5 87 92 98 96 93 
1:10 83 93 87 96 89 
1:20 86 86 94 96 92 
1:40 70 59 90 89 88 
1:80 -- 27 68 85 85 
1:160 -- -- 18 11 84 
1:320 -- -- -- -- 42 
Maximal velocity of the platelet aggregation 
(collagen slope/second .times. 100) 
Undiluted 139 166 220 209 154 
1:5 135 141 219 199 172 
1:10 119 122 198 208 154 
1:20 97 84 198 208 154 
1:40 60 56 155 174 146 
1:80 -- 32 89 116 116 
1:160 -- -- 30 25 83 
1:320 -- -- -- -- 32 
______________________________________ 
TABLE V 
______________________________________ 
Relative Affinity of Equine and Human Platelets for 
Purified Stimulator 
Reprecipitated 
Concentration 
Pelleted 2 .times. 
(at 1.80 M 
of aggregator 
fraction saturation 
in 0.05 ml/ 
(105,000 .times. g) 
(NH.sub.4).sub.2 SO.sub.4) 
0.45 ml PRP 
PRP of 3 PRP of same 
(estimated .mu.g 
burros burros 
dry weight) 
Avg. response 
Avg. response 
PRP of Hu-2 
______________________________________ 
Delay time from aggregator addition to onset of platelet 
aggregation (in seconds) 
Undiluted (52) 
9 8 6 
1:5 dilution (10.4) 
11 13 7 
1:10 (5.2) 17 22 7 
1:20 (2.6) 22 26 10 
1:40 (1.3) 30 32 12 
1:80 (0.65) 
33 42 14 
1:160 (0.325) 
42 52 16 
1:320 (0.163) 
59 71 (two*) 
19 
1:640 (0.082) 
59 (two*) -- 24 
Maximal optical density decrease of PRP (% aggregation) 
Undiluted 95 97 88 
1:5 97 98 89 
1:10 95 95 98 
1:20 92 95 92 
1:40 90 87 96 
1:80 83 79 94 
1:160 70 42 86 
1:320 34 12 87 
1:640 5 -- 54, 85** 
Maximum velocity of aggregation (collagen slope/second) 
.times. 100) 
Undiluted 195 241 262 
1:5 205 248 262 
1:10 181 171 262 
1:20 149 165 218 
1:40 125 123 218 
1:80 110 99 186 
1:160 84 43 155 
1:320 48 24 137 
1:640 14 -- 60, **25 
______________________________________ 
*No. of PRP specimens of 3 responding to added stimulus. 
**Values of secondary wave of platelet aggregation and primary aggregatio 
velocity response to the lowest concentration of the aggregator addition, 
respectively. 
Example III describes the use of aqueous extractable equine arterial 
collagen for the treatment of wounds and for controlling bleeding and 
promoting healing, particularly of skin grafts. 
EXAMPLE III 
Two patches of skin, about 5.times.5 cm were removed from opposite sides of 
the back of an adult sow, and each was grafted to the open wound on the 
opposite side. One graft was performed without medication. The other graft 
was performed by first bathing the open wound and the underside of the 
graft with about 1 ml of aqueous aortic extract containing equine arterial 
fibrillar collagen, about 2 mg dry weight/cc. Bleeding was minimal for the 
graft using the aortic extract. Leakage of fluids accompanied the 
untreated graft. Healing of the treated graft appeared to be accelerated 
compared to the untreated graft. 
EXAMPLE IV 
The Example compares the platelet aggregating potency of a composition of 
this invention with collagen obtained from other species of mammals. 
The procedure employed for studying the collagen/platelet interaction was 
the in vitro turbidimetric (optical density) technique as described in 
Born GVR:Nature, 194:927-929, (1962). In this example, platelet rich 
plasma (PRP) from anticoagulant treated, i.e. heparizined (Hep) or 
citrated (Citr) whole blood from a healthy burro was prepared by low speed 
centrifugation (150.times.g for ten minutes at 22.degree. C.). Changes in 
light transmission through samples of Hep PRP and Citr PRP were 
continuously recorded on a strip-chart recorder by a Platelet Aggregation 
Profiler (Bio/Data Corporation, Willow Grove, Pa.). On the basis of 
comparable dilutions, quantative measurements were made of the aqueous 
extractable microfibrillar collagenous agents from the aortas of a burro, 
a pig, and a calf. The three microfibrillar collagenous agents were 
prepared in the same manner by blending aortic tissue in a 
macrohomogenizer in Tyrode's solution and dialyzing the homogenate against 
Tyrode's solution. The retentate, which contained the fibrillar collagen, 
was ultracentrifuged and the pellet resuspended in Tyrode's solution. The 
suspension was rehomogenized and dialyzed against triple distilled water 
(burro and calf) or Tyrode's solution (pig). Successive dilutions of the 
retentates in Tyrode's solution were made and 0.05 ml aliquots of the 
dilutions were contacted with 0.45 ml of the PRP and the optical 
transmission continuously recorded. 
Table V displays the results of the three parameters measured. 
1. The delay time (sec) to the initiation of platelet aggregation. 
2. The intensity of platelet aggregation (% aggregation corresponds to % 
light transmitted). 
3. The velocity of platelet clumping as indicated by the steepest slope of 
the optical transmission trace on the chart paper. 
TABLE V 
__________________________________________________________________________ 
Amount Of 
Aggr. Agents 
Contained In Maximal Decrease 
0.05 ml Added 
Delay Time To In Optical Density 
Maximal Velocity 
To 0.45 ml Of 
Onset Of Aggr. Of PRP Of Aggregation 
Burro PRP 
(In Secs) (% Aggregation) 
[(Max Slope/Sec) .times. 100] 
Aortic Conc. 
Burro Pig 
Calf Burro Pig 
Calf Burro Pig 
Calf 
Burro PRP 
Hep 
Citr 
Hep 
Hep 
Citr 
Hep 
Citr 
Hep 
Hep 
Citr 
Hep 
Citr 
Hep 
Hep 
Citr 
__________________________________________________________________________ 
Undiluted 
22 36 46 26 36 94 100 
94 88 88 232 
149 
141 
126 
102 
1:2 dilution 
23 41 48 34 43 98 100 
92 94 70 189 
141 
104 
124 
77 
1:5 dilution 
23 43 67 43 43 98 100 
80 83 208 
181 
85 104 
95 
1:10 dilution 
24 53 96 48 48 96 100 
tr 70 72 189 
154 
tr 95 99 
1:20 dilution 
29 67 0 50 65 94 100 46 30 174 
144 61 58 
1:40 dilution 
41 89 60 108 
90 100 12 tr 139 
141 35 tr 
1:80 dilution 
34 96 0 0 94 100 144 
134 
1:160 dilution 
54 125 96 100 134 
110 
1:320 dilution 
43 221 94 66 149 
62 
1:640 dilution 
72 0 54 0 71 0 
1:1280 dilution 
108 
0 tr 0 tr 0 
__________________________________________________________________________ 
From an analysis of the table, it is apparent that the extracted equine 
arterial fibrillar collagen material of this invention is approximately 
100 times more reactive than extracted pig arterial fibrillar collagen and 
approximately thirty times more reactive than extracted calf arterial 
fibrillar collagen. 
EXAMPLE V 
Thoracic aortas were obtained from twenty-five to thirty year old burros 
(Equus asinus) that had died from natural causes. 
Isolation of Collagenous Fibrils--In experiment 1, eight 10 g samples of 
stock aortic segments were each placed in 50 ml of Tyrode buffer pH 7.2 
containing 100 U and 100 .mu.g of buffered pencillin G potassium and 
streptomycin sulfate per milliliter, respectively. The tissue, placed in a 
cooling cup packed in wet ice, was blended with a macrohomogenizer at 
23,000 rpm for five minutes. After overnight extraction (4.degree. C.), 
the homogenate was centrifuged (650.times.g for ten minutes at 0.degree. 
to 4.degree. C.). The viscous supernatant and the centrifugation 
"cushion", or "buffy", layer (which contained the platelet proaggregatory 
property) were separated with a plastic 5 ml pipet. The supernatant was 
filtered through a double layer, 2.times.2 inch, 12-ply sterile gauze and 
temporarily stored (4.degree. C.), and the remaining pelleted tissue was 
reblended with Tyrode buffer. The centrifuged supernatant and "cushion" 
layer were again separated and filtered as described. The extraction 
process was repeated eight times in five days. The combined supernatants 
from the 1st and 2nd extractions were designated fraction a; extract from 
3, 4, and 5 and 6, 7 and 8 sequential homogenization and steps of the 
remaining pelleted tissue were pooled and designated fractions b and c. 
The extracts (200 to 250 ml) were placed in telescoped Visking tubes and 
exhaustively dialyzed against two changes of Tyrode buffer (5 L) 
containing the added antibiotics, with constant stirring on a magnetic 
plate (4.degree. C., five days). 
After five days of dialysis, retentates in the tubes were fractionated by 
ultracentrifugation (105,000.times.g for thirty minutes at 0.degree. to 
2.degree. C.). The pelleted material (which contained the total aggregant 
activity for platelets) was resuspended in approximately one-fifth volume 
of Tyrode buffer. The tissue was reblended in the macrohomogenizer for 
eight minutes, described above and the finely blended tissue concentrates 
were again exhaustively dialyzed in the cold (4.degree. C.) against Tyrode 
buffer for five days. Finally, the extracts were dialyzed against triple 
distilled water with daily changes (5 L) for five days. The dialyzed, 
essentially salt-free tissue retentates were collected and distributed in 
small aliquots (0.5 to 3.5 ml) in plastic tubes with caps, frozen and 
stored (-85.degree. C.). 
In experiment 2, the same extraction steps were applied to seven 20 g 
samples of stock vascular tissue, and ten sequential blending and 
extraction steps were carried out in five days. The total filtered viscous 
aqueous extract plus the centrifugation "cushion" layer (4 L) were placed 
in telescoped tubing and exhaustively dialyzed an described above. 
Dialyzed retentates were fractionated twice by ultracentrifugation, and 
the pelleted vascular tissue was reblended with the macrohomogenizer 
(eight minutes) in one-fifth volume of Tyrode buffer. After exhaustive 
dialysis against buffer and distilled water, the essentially salt-free 
retentates were pooled and aliquots placed in plastic tubes with caps, 
frozen and stored (-85.degree. C.). 
In experiment 3, a 20 g sample of aortic segments was extracted with 200, 
100 and 100 ml of Tyrode buffer with antiboitics on three consecutive 
days. From the extracts, a single salt-free concentrate was obtained. This 
preparation, after having been stored (-85.degree. C.) for more than three 
years and repeatedly assayed for platelet aggregation effectiveness, still 
retained this biological property, both qualitatively and quantitatively. 
Physiochemical Characterization--Determination of dry weight of the 
salt-free aortic extracts, protein assays by the micro-Kjeldahl method 
(bovine serum albumin and crystalline ammonium sulfate served as protein 
standards), and amino acid composition determination by the JEOL-JLC-6AH 
analyzer and a one-column system were carried out. 
Total reducing carbohydrates were determined by a ferricyanide procedure 
with glucose as a standard; Part et al: Handbook of Micromethods For 
Biological Sciences, VanNostrand-Rheinhold Co., New York, 1974, pp 49-50. 
The results are shown in Table IV and VII. 
TABLE IV 
______________________________________ 
Analytic Data of Salt-Free Fibrous Collagen 
Containing Extracts From Burro Aortas 
dry mg dry Assays 
weight weight Protein Carbohydrate 
Preparation 
aorta isolated (in mg per 100 mg dry wt.) 
______________________________________ 
Expt. 1 80 
Fraction 
a 426 .sup. 47* 
14.9 
b 402 56 18.2 
c 760 85 5.5 
Expt. 2 140 2440 .sup. 96** 
1.6 
Expt. 3 20 247 84 ND 
______________________________________ 
*Precipitated at 40% saturation with ammonium sulfate. Precipitate 
reblended with Tyrode buffer and dialyzed until saltfree. 
**Twice fractionated by ultracentrifugation. 
ND = Not determined. 
TABLE VII 
______________________________________ 
Amino Acids in Fibrous-Collagen-Containing 
Protein Extracts From Burro Aortas 
Amino Acids As Residues 
Per 1000 Total Amino Acid Residues 
Expt. 1 
a b c Expt. 2 Expt. 3 
______________________________________ 
Lysine 45.9 38.1 29.8 38.8 49.2 
Histidine 27.1 25.4 31.2 19.8 11.2 
Hydroxylysine 
3.4 7.4 4.8 10.7 5.5 
Arginine 44.6 46.5 43.2 40.4 57.6 
Hydroxyproline 
9.7 24.6 20.1 14.5 40.6 
Aspartic Acid 
83.2 69.3 65.4 65.3 76.5 
Threonine 46.3 39.4 38.1 38.0 34.8 
Serine 45.1 49.7 40.3 37.3 35.5 
Glutamic Acid 
84.5 91.3 81.9 71.1 90.8 
Proline 75.5 79.4 98.2 81.2 79.1 
Glycine 139.3 200.6 206.5 182.0 178.2 
Alanine 106.5 110.1 133.9 125.8 95.0 
1/2 Cysteine 
39.4 16.3 13.2 35.2 5.8 
Valine 66.1 58.4 57.6 67.9 56.2 
Methionine 
16.2 13.6 14.2 16.4 12.8 
Isoleucine 
40.0 31.7 26.7 38.5 35.3 
Leucine 71.0 57.8 52.5 62.0 81.2 
Tyrosine 21.7 12.7 15.3 24.1 19.3 
Phenylalanine 
34.6 27.6 27.1 30.9 35.2 
______________________________________ 
EXAMPLE VI 
To compare sensitivities of platelets from two normal human subjects toward 
the fibrous collagen on a dry weight basis, the activity of three of the 
vascular collagen preparations were measured by aggregometers, using ten 
comparable doses of the fibrous collagens, toward the human platelets. By 
analysis of variance of the multiple data collections (Table VII), 
differences in the platelet sensitivities for the two human subjects were 
found to be significant (P&lt;0.01) for nine levels of the three collagen 
preparations. These differences were reflected in all three parameters of 
the platelet responses measured (i.e., lag time to onset of flocculation, 
platelet aggregation intensity and aggregation velocity). The data 
indicated that platelets from the older of the two subjects were 
substantially more reactive. The data also clearly establishes the utility 
of the compositions of this invention for humans. 
TABLE VII 
__________________________________________________________________________ 
Fibrous Collagen Activity Toward Platelets from Two Healthy Human 
Subjects* 
Platelet reactivity (mean .+-. SE) 
Delay time Intensity Velocity 
Collagen 
to onset (%) (%/min) 
dose** 
Hu (30)*** 
Hu (48) 
Hu (30) 
Hu (48) 
Hu (30) 
Hu (48) 
__________________________________________________________________________ 
ng 
15.6 31.2 .+-. 2.1 
0 64 .+-. 14 
0 66 .+-. 11 
31.25 
40.8 .+-. 2.8 
25.6 .+-. 2.1 
4 .+-. 3 
84 .+-. 3 
15 .+-. 4 
97 .+-. 6 
62.5 27.2 .+-. 2.9 
20 .+-. 1.7 
63 .+-. 4 
89 .+-. 1 
70 .+-. 3 
106 .+-. 3 
125 21.2 .+-. 0.8 
16.8 .+-. 0 
77 .+-. 2 
89 .+-. 1 
111 .+-. 4 
110 .+-. 4 
250 16.4 .+-. 0.4 
15.2 .+-. 0.4 
81 .+-. 1 
90 .+-. 1 
102 .+-. 6 
114 .+-. 14 
500 14.4 .+-. 1.2 
10.8 .+-. 0 
81 .+-. 1 
90 .+-. 0 
96 .+-. 5 
123 .+-. 14 
.mu.g 
1 11.2 .+-. 1.4 
8.4 .+-. 0.7 
76 .+-. 2 
92 .+-. 1 
113 .+-. 4 
134 .+-. 9 
2 9.2 .+-. 1.1 
8.4 .+-. 0 
81 .+-. 2 
88 .+-. 1 
102 .+-. 3 
126 .+-. 5 
5 7.6 .+-. 0.4 
6.4 .+-. 1.1 
77 .+-. 1 
87 .+-. 1 
90 .+-. 7 
127 .+-. 10 
10 7.2 .+-. 1.2 
6 .+-. 0.7 
75 .+-. 1 
80 .+-. 8 
93 .+-. 3 
89 .+-. 10 
__________________________________________________________________________ 
*PRP concentrates were prepared from blood sample collections from the tw 
donors on successive days. 
**Three different preparations of burro aortic fibrous collagen 
preparations were evaluated (see Tables VI and VII, experiments 1c, 2, an 
3). An appropriate amount of the fibrous collagens in fine suspension in 
50 .mu.l of Tyrode buffer when added to 450 .mu.l of the PRP concentrate 
and constantly stirred in the aggregometer at 37 C. for 7 min produced th 
observed platelet reactivity. 
***Number in parentheses indicates age of male subject.