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Enhanced platelet aggregability in dogs following myocardial infarction
Life Sciences Vol. 20, pp . 843-848, 1977 . Printed in the U.S .A .
Pergamon Press
ENHANCED PLATELET AGGREGABILITY Ill DOGS POLLOWIIIG MYOCARDIAL INFARCTION J. Stuart Fleming and J. P. Buyniski Pharmacology Department Bristol Laboratories, Syracuse, N.Y . 13201 (Received in final form January 24, 1977) Summary Coronary artery ligation in beagle dogs led to an abnormal platelet response that maybe similar to that observed in patients with ischemic heart disease . Platelet rich plasma (AP) obtained from these animals was markedly more responsive to collagen induced platelet aggregation than was M obtained from control animals which had undergone similar surgery but without having had a coronary artery ligated. Platelet sensitivity was followed after ligation for periods up to four months . The peak response to collagen occurred three weeks following surgery, remained high for three additional weeks and then gradually re turned toward normal over the ensuing ten weeks . Crossover experiments employing platelets and platelet poor plasma (PPP) from ligated and control animals demonstrated tkat this observation was due to a plasma factor abnormality . Further studies established that BL-3459 and certain other inhibitors of platelet aggregation were capable of normalising this exaggerated response to collagen . There are a number of reports which indicate that platelet aggregation abnormalities are associated with coronary artery disease (1-4) . Salkey and Dugdale (3) reported an abnormal response to collagen in patients soon after suffering myocardial infarction in which platelet sensitivity to collagen increased significantly and remained above normal for varying periods of three months or more . We have observed a similar abnormality in beagle dogs following surgical ligation of the left anterior descending coronary artery . Materials and Methods Ligation of the anterior descending coronary artery was accomplished according to the method described by Harris (5) in beagle dogs in which anesthesia was induced with thiopental sodium (30 mg/kg iv) and maintained with meth osyfiurane by means of a Mark 8 respirator during the open chest procedure (6) . A control group of animals was also studiedin which the same vessel isolation procedure was performed except that the coronary artery was not actually ligated. Coronary artery ligation of this kind is sufficient to produce significant infarction with associated cardiac arrhythmias and electrocardiographic abnormalities as shown in our laboratories (6) . 843
84 4
Enhanced Plateleg Aggregability
Vol . 20, No . 5, 1977
A Payton aggregometer, model 300BD, and the optical density method of Born (7) as modified by Mustard et al . (8) were employed to test increasing collagen dilutions on platelet rich plasma (PRP) samples obtained from beagle dogs (7-9 kg) before and at various times after coronary artery ligation . The collagen suspension was prepared by homogenising bovine achilles tendon (Sigma Chemical Company, St . Louis, Mo .) in Tyrode's solution according to the method described by Evans et al . (9) . Aggregation was then induced by adding 0 .050 .1 ml of the stock collagen suspension or a dilution of this in Tyrode's solution to the 0 .9 ml PRP sample . PRP was prepared from blood drawn from a jugular vein into syringes containing 3 .8% sodium citrate (1 ml citrate/9 ml blood) and then centrifuged for 5 minutes at 1000 RPM (140 x g) . The undiluted collagen produced maximal aggregation while various dilutions ranging from 1 :4 to 1 :30 produced progressively less aggregation during the pre-surgery control period . After coronary artery ligation, platelet sensitivity increased with time so that higher dilutions produced a strong aggregation response (i .e . 50% of the maximal response) . The highest dilution capable of producing such a response was then recorded for each animal at each point in time . Crossover studies were also conducted in which platelets from coronary artery ligated dogs were resuspended in platelet poor plasm (PPP) from control dogs and platelets from control dogs were resuspended in PPP from ligated dogs . For these experiments, platelet buttons were separated from PRP by centrifugation and then resuspended by gently mixing in the appropriate PPP sample . Once again various dilutions of the collagen suspension were tested for their ability to induce aggregation and the highest dilution to produce aggregation equal to or greater than 50% of the maximal response was recorded . Single oral doses of various platelet aggregation inhibitors (aspirin, sulfinpyrazone, dipyridamole and 6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b} quinazolin-2-one hydrochloride monohydrate (BL-3459) were tested in dogs during the period when their response to collagen was elevated following coronary artery ligation. These compounds were evaluated for their ability to normalize the exaggerated collagen response . Results Platelet sensitivity to collagen was followed for 118 days after coronary artery ligation in a group of five beagle dogs . The peak response to collagen occurred approximately three weeks following surgery, remained high for three additional weeks and then gradually returned toward normal over the ensuing ten weeks . A control series was conducted in five similar dogs experiencing the same type of surgery but without having their coronary arteries actually ligated. This group demonstrated only minor fluctuations in platelet sensitivity to collagen and was followed through sixty days . These results are depicted in Figure 1. A second series of experiments employing the same five coronary artery ligated dogs and five other unoperated control dogs revealed that the abnormality was associated with the plasma of the coronary artery ligated dogs . Platelets from ligated animals were resuspended in PPP from control dogs and platelets from control dogs were resuspended in PPP from ligated dogs . These samples were then subjected to collagen induced aggregation using various dilutions of collagen as described above. The results depicted in Figure 2 indicate that a normal aggregation response was obtained with platelets from ligated animals when they were resuspended in PPP from control dogs while an abnormal aggregapattern was seen with platelets from control dogs resuspended in PPP from ligated dogs . Thus, the observed abnormality appears to be associated with the plasma of ligated animals rather than with the platelets themselves .
Vol . 20, No . 5,
1977
Enhanced Platelet Aggregability
L %I~1
~_I
I
845
~ COI RONARYAR7FRY `LIGATED GROUP
Z W
S
1a SURóGlC=L CONTROL GROUP
0
20
40
60
80
100
DAYS AFTER SURGERY
120
FIG. 1 Effect of coronary artery ligation on collagen induced platelet aggregation in PRP obtained from beagle dogs at various times after surgery. Each point represents the highest collagen dilution able to produce a strong aggregation response . 0---* coronary artery ligated animals, o--0 surgical control group, N - 5 dogs/group . TÀBLE 1 Effect of BL-3459, Aspirin, Sulfinpyrasone and Dipyridamole on increased Platelet Sensitivity to Collagen in Beagle Dogs Following Coronary Artery Ligation . Compound á
Doge
BL-3459
(mgh~ po) 0.025 0.10 0 .40
N
A Normalisation of Response
7 7 5
9 83 100
Aspirin
1 .25 5 20
5 5 5
0 83 100
Sulfinpyrasone
1 .23 5 20
5 5 5
0 84 92
Dipyridamole
1 .25 5 20
5 5 5
0 47 47
84 6
Enhanced Platelet Aggregability
Vol. 20, No . 5, 1977
It was of further interest to investigate the ability of several antiaggregation agents to inhibit the exaggerated collagen response . Aspirin, sulfinpyrasone, dipyridamole and the now antithrombotic agent, BL-3459 (10), were all tested following oral administration to conscious coronary artery ligated dogs, for their ability to normalise this response . The results of these studies are summarised in Table 1 . BL-3459 resulted in approximately 80% normalisation of the platelet response at a dose of 0.1 mg/kg and was about 50 times more potent than aspirin and sulfinpyrazone . In contrast, oral doses of dipyridamole as high as 20 mg/kg did not produce effects reaching the 80% normalization level .
1 :20
z
0 1 :15
H _J
2 w 1 :10
00
c7 Q U
00
1 :5
CONTROL DOGS
LIGATED DOGS
LI GATED PLATELETS CONTROL PLASMA
CONTROL PLATELETS LIGATED PLASMA
FIG. 2 In Vitro collagen induced aggregation crossover study of platelets and PPP obtained from coronary artery ligated and control beagle dogs . Each point represents the highest collagen dilution able to produce a strong aggregation response in each sample of the indicated test groups . N - 5 samples/group. Discussion The association between enhanced platelet aggregability or adhesiveness and various disease states has been the subject of numerous studies (1-4, 1115) . In view of the fact that platelet aggregation is now regarded as a key event in the initiation of arterial thrombosis, one would expect that abnormalities in platelet function might wall be observed in patients experiencing thrombotic episodes . While studies such as those reported by Salkey and Dugdale (3) have identified certain platelet function abnormalities, the cause and effect relationship between the disease state and the observed abnormality is not at all well understood .
Vol . 20, No . 5, 1977
Enhanced Platelet Aggregability
847
Results of various studies indicate that thromboembolic conditions can be induced in laboratory animal models by altering platelet aggregability through infusion of agents such as adenosine diphosphate or epinephrine (16-18) . On the other hand, the data reported here indicate that enhanced platelet aggregability can be brought about as a result of mechanically produced myocardial infarction . Thus, it is possible, at least in esperimantal situations, for enhanced platelet aggregability to be both a cause and an effect of various conditions or disease states . Alterations in normal plasm composition seems to be involved in determining platelet aggregability not only in post myocardial infarction, as reported here, but also in diabetes . While the watt nature of the defect in not know, the work of Kraan et al . (13) indicates that enhanced platelet aggregability associated with diabetes is related to a plasma abnormality . In the case of diabetes, it is also suspected that this situation is responsible for the appearance of retinopathy and other angiopathies often associated with the disease . Likewise, the increased risk of repeat attack in post myocardial infarction patients may be related to enhanced aggregability of blood platelets . Thus, it appears that there may be a number of clinical conditions which give rise to enhanced platelet aggregability which might, in turn, be responsible for the dsvolopment of secondary problems . Pharmacologic inhibition of platelet aggregation may be useful, not only for prevention of the primary event (i .e . myocardial infarction, stroke, etc .), but also, following diagnosis of the primary disease, for the prevention of secondary sequelae such as diabetic retinopathy and rspeat .attack in post myocardial infarction patients . BL-3459 is an agent showing an exceptionally broad range of antithrcmbotic activity in various laboratory models of throubosis (10, 18) and, overall, represents one of the most potent and effective oral antithrembotic agents yet discovered . The effect of BL-3459 in the prosent model of platelet dysfunction is further evidence that this class of compounds may find utility in the treatment of human thromboembolie disease . Acknoulsdaments The authors gratefully acknowledge the technical assistance of Beverly Cornish, John Buchanan, Charles Baca and Mark Smith . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
S . RE]iAUD, K . KUBA, C . GOULET, Y . LEKIRE and C. ALLARD, Circulation Ras . 26 333-564 (1970) . P. B . GOLDBIMFA®, M. B . CATBEY, 8 . ZUCKER, P . WILBBR mad J . J . CORRIGAU, Circulation 43 538-546 (1971) . N. SAIM andM . DOGOAIX, Am. J . Cardiol . 32 612-617 (1973) . W . FRISM», J . CHRISTODOULOU, B . VMIER, C . SMITBMB and S . HOMITZ . Am. J . Cardiol. 3 3 137 (1974) . A . S . BARRIS, Circulation 1 1318-1328 (1950) . J . P . BUYNISKI and R. W . GARDM, J . Pharmcol . exo . Thar . 190 289-299 (1974) . G . V . R . BORE, J . Physiol . (London) 162 67P-68P (1962) . J . F . NUSTARD, B . BEGA=, B. C . ROWSELL and R. L . MACQII.AN, J . Lb . Clin. Mad . 6 4 548-559 (1964) . G. EVANS, M. G . main, M . A. PACKWM, E . E . NISEIZA,WA, J . F . MUSTM and E. A . MURM , J . Esp . Med . 128 877-894 (1968) . J . S . FIEKING, J . P. BUYNISKI, R. L. CAVANAGB and M. E . B , ~. Pharmacol . mm . Thar . _194 435-449 (1975) . B . A . CASPARY, J . PRIIMS, B . MILIaR and E . J . FIELD, Lancet 1108-1109 (1965) .
848
12 . 13 . 14 . 15 . 16 . 17 . 18 .
Enhanced Platelet Aggregability
Vol . 20, No . 5, 1977
J . LEVI, H. I . BICHER and J . ROSENFELD, J . Mad . 1 132-141 (1970) . H . C . KWAAN, J . A . COLWELL, S . CRUZ, N . SUWANW1LA and J . G . BOBBIE, J . Lab . Clin . Mod . 8 0 236-246 (1972) . K . K . WU and J . C . HOAK, Stroke 6 521-524 (1975) . K . K . WU, R . W . BARNES and J . C . HOAK, Circulation 53 687-691 (1976) . L . JORGENSEN, H . C . ROWSELL, T . HOVIG, M . F . GLYNN and J . F . MUSTARD, Lab . Invest . 17 616-644 (1967) . L . JORGENSEN, T . HOVIG, H . C . ROWSELL and J . F . MUSTARD, Am . J . Path . 61 161-170 (1970) . J. S . FLElfNG, J . 0 . BUCHANAN and J . P . BUYNISKI, Eur . J . Pharoac . 40 57-62 (1976) .
Pergamon Press
ENHANCED PLATELET AGGREGABILITY Ill DOGS POLLOWIIIG MYOCARDIAL INFARCTION J. Stuart Fleming and J. P. Buyniski Pharmacology Department Bristol Laboratories, Syracuse, N.Y . 13201 (Received in final form January 24, 1977) Summary Coronary artery ligation in beagle dogs led to an abnormal platelet response that maybe similar to that observed in patients with ischemic heart disease . Platelet rich plasma (AP) obtained from these animals was markedly more responsive to collagen induced platelet aggregation than was M obtained from control animals which had undergone similar surgery but without having had a coronary artery ligated. Platelet sensitivity was followed after ligation for periods up to four months . The peak response to collagen occurred three weeks following surgery, remained high for three additional weeks and then gradually re turned toward normal over the ensuing ten weeks . Crossover experiments employing platelets and platelet poor plasma (PPP) from ligated and control animals demonstrated tkat this observation was due to a plasma factor abnormality . Further studies established that BL-3459 and certain other inhibitors of platelet aggregation were capable of normalising this exaggerated response to collagen . There are a number of reports which indicate that platelet aggregation abnormalities are associated with coronary artery disease (1-4) . Salkey and Dugdale (3) reported an abnormal response to collagen in patients soon after suffering myocardial infarction in which platelet sensitivity to collagen increased significantly and remained above normal for varying periods of three months or more . We have observed a similar abnormality in beagle dogs following surgical ligation of the left anterior descending coronary artery . Materials and Methods Ligation of the anterior descending coronary artery was accomplished according to the method described by Harris (5) in beagle dogs in which anesthesia was induced with thiopental sodium (30 mg/kg iv) and maintained with meth osyfiurane by means of a Mark 8 respirator during the open chest procedure (6) . A control group of animals was also studiedin which the same vessel isolation procedure was performed except that the coronary artery was not actually ligated. Coronary artery ligation of this kind is sufficient to produce significant infarction with associated cardiac arrhythmias and electrocardiographic abnormalities as shown in our laboratories (6) . 843
84 4
Enhanced Plateleg Aggregability
Vol . 20, No . 5, 1977
A Payton aggregometer, model 300BD, and the optical density method of Born (7) as modified by Mustard et al . (8) were employed to test increasing collagen dilutions on platelet rich plasma (PRP) samples obtained from beagle dogs (7-9 kg) before and at various times after coronary artery ligation . The collagen suspension was prepared by homogenising bovine achilles tendon (Sigma Chemical Company, St . Louis, Mo .) in Tyrode's solution according to the method described by Evans et al . (9) . Aggregation was then induced by adding 0 .050 .1 ml of the stock collagen suspension or a dilution of this in Tyrode's solution to the 0 .9 ml PRP sample . PRP was prepared from blood drawn from a jugular vein into syringes containing 3 .8% sodium citrate (1 ml citrate/9 ml blood) and then centrifuged for 5 minutes at 1000 RPM (140 x g) . The undiluted collagen produced maximal aggregation while various dilutions ranging from 1 :4 to 1 :30 produced progressively less aggregation during the pre-surgery control period . After coronary artery ligation, platelet sensitivity increased with time so that higher dilutions produced a strong aggregation response (i .e . 50% of the maximal response) . The highest dilution capable of producing such a response was then recorded for each animal at each point in time . Crossover studies were also conducted in which platelets from coronary artery ligated dogs were resuspended in platelet poor plasm (PPP) from control dogs and platelets from control dogs were resuspended in PPP from ligated dogs . For these experiments, platelet buttons were separated from PRP by centrifugation and then resuspended by gently mixing in the appropriate PPP sample . Once again various dilutions of the collagen suspension were tested for their ability to induce aggregation and the highest dilution to produce aggregation equal to or greater than 50% of the maximal response was recorded . Single oral doses of various platelet aggregation inhibitors (aspirin, sulfinpyrazone, dipyridamole and 6-methyl-1,2,3,5-tetrahydroimidazo[2,1-b} quinazolin-2-one hydrochloride monohydrate (BL-3459) were tested in dogs during the period when their response to collagen was elevated following coronary artery ligation. These compounds were evaluated for their ability to normalize the exaggerated collagen response . Results Platelet sensitivity to collagen was followed for 118 days after coronary artery ligation in a group of five beagle dogs . The peak response to collagen occurred approximately three weeks following surgery, remained high for three additional weeks and then gradually returned toward normal over the ensuing ten weeks . A control series was conducted in five similar dogs experiencing the same type of surgery but without having their coronary arteries actually ligated. This group demonstrated only minor fluctuations in platelet sensitivity to collagen and was followed through sixty days . These results are depicted in Figure 1. A second series of experiments employing the same five coronary artery ligated dogs and five other unoperated control dogs revealed that the abnormality was associated with the plasma of the coronary artery ligated dogs . Platelets from ligated animals were resuspended in PPP from control dogs and platelets from control dogs were resuspended in PPP from ligated dogs . These samples were then subjected to collagen induced aggregation using various dilutions of collagen as described above. The results depicted in Figure 2 indicate that a normal aggregation response was obtained with platelets from ligated animals when they were resuspended in PPP from control dogs while an abnormal aggregapattern was seen with platelets from control dogs resuspended in PPP from ligated dogs . Thus, the observed abnormality appears to be associated with the plasma of ligated animals rather than with the platelets themselves .
Vol . 20, No . 5,
1977
Enhanced Platelet Aggregability
L %I~1
~_I
I
845
~ COI RONARYAR7FRY `LIGATED GROUP
Z W
S
1a SURóGlC=L CONTROL GROUP
0
20
40
60
80
100
DAYS AFTER SURGERY
120
FIG. 1 Effect of coronary artery ligation on collagen induced platelet aggregation in PRP obtained from beagle dogs at various times after surgery. Each point represents the highest collagen dilution able to produce a strong aggregation response . 0---* coronary artery ligated animals, o--0 surgical control group, N - 5 dogs/group . TÀBLE 1 Effect of BL-3459, Aspirin, Sulfinpyrasone and Dipyridamole on increased Platelet Sensitivity to Collagen in Beagle Dogs Following Coronary Artery Ligation . Compound á
Doge
BL-3459
(mgh~ po) 0.025 0.10 0 .40
N
A Normalisation of Response
7 7 5
9 83 100
Aspirin
1 .25 5 20
5 5 5
0 83 100
Sulfinpyrasone
1 .23 5 20
5 5 5
0 84 92
Dipyridamole
1 .25 5 20
5 5 5
0 47 47
84 6
Enhanced Platelet Aggregability
Vol. 20, No . 5, 1977
It was of further interest to investigate the ability of several antiaggregation agents to inhibit the exaggerated collagen response . Aspirin, sulfinpyrasone, dipyridamole and the now antithrombotic agent, BL-3459 (10), were all tested following oral administration to conscious coronary artery ligated dogs, for their ability to normalise this response . The results of these studies are summarised in Table 1 . BL-3459 resulted in approximately 80% normalisation of the platelet response at a dose of 0.1 mg/kg and was about 50 times more potent than aspirin and sulfinpyrazone . In contrast, oral doses of dipyridamole as high as 20 mg/kg did not produce effects reaching the 80% normalization level .
1 :20
z
0 1 :15
H _J
2 w 1 :10
00
c7 Q U
00
1 :5
CONTROL DOGS
LIGATED DOGS
LI GATED PLATELETS CONTROL PLASMA
CONTROL PLATELETS LIGATED PLASMA
FIG. 2 In Vitro collagen induced aggregation crossover study of platelets and PPP obtained from coronary artery ligated and control beagle dogs . Each point represents the highest collagen dilution able to produce a strong aggregation response in each sample of the indicated test groups . N - 5 samples/group. Discussion The association between enhanced platelet aggregability or adhesiveness and various disease states has been the subject of numerous studies (1-4, 1115) . In view of the fact that platelet aggregation is now regarded as a key event in the initiation of arterial thrombosis, one would expect that abnormalities in platelet function might wall be observed in patients experiencing thrombotic episodes . While studies such as those reported by Salkey and Dugdale (3) have identified certain platelet function abnormalities, the cause and effect relationship between the disease state and the observed abnormality is not at all well understood .
Vol . 20, No . 5, 1977
Enhanced Platelet Aggregability
847
Results of various studies indicate that thromboembolic conditions can be induced in laboratory animal models by altering platelet aggregability through infusion of agents such as adenosine diphosphate or epinephrine (16-18) . On the other hand, the data reported here indicate that enhanced platelet aggregability can be brought about as a result of mechanically produced myocardial infarction . Thus, it is possible, at least in esperimantal situations, for enhanced platelet aggregability to be both a cause and an effect of various conditions or disease states . Alterations in normal plasm composition seems to be involved in determining platelet aggregability not only in post myocardial infarction, as reported here, but also in diabetes . While the watt nature of the defect in not know, the work of Kraan et al . (13) indicates that enhanced platelet aggregability associated with diabetes is related to a plasma abnormality . In the case of diabetes, it is also suspected that this situation is responsible for the appearance of retinopathy and other angiopathies often associated with the disease . Likewise, the increased risk of repeat attack in post myocardial infarction patients may be related to enhanced aggregability of blood platelets . Thus, it appears that there may be a number of clinical conditions which give rise to enhanced platelet aggregability which might, in turn, be responsible for the dsvolopment of secondary problems . Pharmacologic inhibition of platelet aggregation may be useful, not only for prevention of the primary event (i .e . myocardial infarction, stroke, etc .), but also, following diagnosis of the primary disease, for the prevention of secondary sequelae such as diabetic retinopathy and rspeat .attack in post myocardial infarction patients . BL-3459 is an agent showing an exceptionally broad range of antithrcmbotic activity in various laboratory models of throubosis (10, 18) and, overall, represents one of the most potent and effective oral antithrembotic agents yet discovered . The effect of BL-3459 in the prosent model of platelet dysfunction is further evidence that this class of compounds may find utility in the treatment of human thromboembolie disease . Acknoulsdaments The authors gratefully acknowledge the technical assistance of Beverly Cornish, John Buchanan, Charles Baca and Mark Smith . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 .
S . RE]iAUD, K . KUBA, C . GOULET, Y . LEKIRE and C. ALLARD, Circulation Ras . 26 333-564 (1970) . P. B . GOLDBIMFA®, M. B . CATBEY, 8 . ZUCKER, P . WILBBR mad J . J . CORRIGAU, Circulation 43 538-546 (1971) . N. SAIM andM . DOGOAIX, Am. J . Cardiol . 32 612-617 (1973) . W . FRISM», J . CHRISTODOULOU, B . VMIER, C . SMITBMB and S . HOMITZ . Am. J . Cardiol. 3 3 137 (1974) . A . S . BARRIS, Circulation 1 1318-1328 (1950) . J . P . BUYNISKI and R. W . GARDM, J . Pharmcol . exo . Thar . 190 289-299 (1974) . G . V . R . BORE, J . Physiol . (London) 162 67P-68P (1962) . J . F . NUSTARD, B . BEGA=, B. C . ROWSELL and R. L . MACQII.AN, J . Lb . Clin. Mad . 6 4 548-559 (1964) . G. EVANS, M. G . main, M . A. PACKWM, E . E . NISEIZA,WA, J . F . MUSTM and E. A . MURM , J . Esp . Med . 128 877-894 (1968) . J . S . FIEKING, J . P. BUYNISKI, R. L. CAVANAGB and M. E . B , ~. Pharmacol . mm . Thar . _194 435-449 (1975) . B . A . CASPARY, J . PRIIMS, B . MILIaR and E . J . FIELD, Lancet 1108-1109 (1965) .
848
12 . 13 . 14 . 15 . 16 . 17 . 18 .
Enhanced Platelet Aggregability
Vol . 20, No . 5, 1977
J . LEVI, H. I . BICHER and J . ROSENFELD, J . Mad . 1 132-141 (1970) . H . C . KWAAN, J . A . COLWELL, S . CRUZ, N . SUWANW1LA and J . G . BOBBIE, J . Lab . Clin . Mod . 8 0 236-246 (1972) . K . K . WU and J . C . HOAK, Stroke 6 521-524 (1975) . K . K . WU, R . W . BARNES and J . C . HOAK, Circulation 53 687-691 (1976) . L . JORGENSEN, H . C . ROWSELL, T . HOVIG, M . F . GLYNN and J . F . MUSTARD, Lab . Invest . 17 616-644 (1967) . L . JORGENSEN, T . HOVIG, H . C . ROWSELL and J . F . MUSTARD, Am . J . Path . 61 161-170 (1970) . J. S . FLElfNG, J . 0 . BUCHANAN and J . P . BUYNISKI, Eur . J . Pharoac . 40 57-62 (1976) .