Immunology, 1975, 29, 131.
Platelet Function in C6-deficient Rabbits AGGREGATION AND SECRETION INDUCED BY COLLAGEN AND ZYMOSAN FELICITY A. CHRISTIAN AND J. L. GORDON Department of Pathology, University of Cambridge
(Received 6th January 1975; acceptedfor publication 15th January 1975) Summary. Platelet aggregation and the selective release of 5-hydroxytryptamine (5HT) and adenine nucleotides were measured in platelet-rich plasma (PRP) and washed platelet suspensions from control and C6-deficient rabbits. Aggregation and release induced by collagen were similar in both groups of animals. Aggregation responses to zymosan in control PRP samples were biphasic, and only the second phase was associated with release. In C6-deficient PRP samples, zymosan-induced aggregation lacked the second phase and there was no release of 5HT or adenine nucleotides. Zymosan induced no aggregation in washed platelet suspensions unless cell-free plasma was also added. C6-deficient plasma supported only primary aggregation, but the addition of control plasma resulted in biphasic aggregation and release. Zymosan which had been pre-activated in control or C6-deficient cell-free plasma induced primary aggregation in washed platelet suspensions. To obtain secondary aggregation and release, the addition of control plasma to the platelet suspension was necessary. INTRODUCTION Rabbits genetically deficient in the sixth component of complement (C6D rabbits) exhibit a defect in blood coagulation in vitro, characterized by a prolonged whole-blood clotting time in plastic tubes and poor prothrombin consumption (Zimmerman, Arroyave and Muller-Eberhardt, 1971). Cell-free plasma from C6D rabbits will not support the release of histamine from rabbit platelets by zymosan (Siraganian, 1972), but it was not established whether this histamine release was associated with the formation of platelet aggregates, or whether it was a result of cell lysis or of a specific secretory process. Such a process-the so-called 'release reaction' (Grette, 1962)-is known to occur in platelets and involves the selective exocytosis of storage granules in response to agents such as thrombin or collagen. The main constituents of these granules are adenine nucleotides and 5hydroxytryptamine (5HT), and these are conventionally used as markers for the release reaction. Rabbit platelets are unique in having substantial amounts of histamine, and the localization and release of platelet histamine have not been determined in detail. In the present study, platelet aggregation and the release of 5HT and adenine nucleotides were measured in response to collagen and zymosan. These experiments were performed using platelet-rich plasma (PRP) and washed platelet suspensions from control and Correspondence: Dr J. L. Gordon, University of Cambridge, Department of Pathology, Tennis Court Road, Cambridge CB2 IQP.
131
Felicity A. Christian and J. L. Gordon 132 C6D rabbits. Platelet responses to zymosan which had been preactivated in cell-free plasma were also determined, and the degree of cell lysis was determined by measuring lactate dehydrogenase activity in the cells and in the suspending medium. MATERIALS AND METHODS Rabbits Control rabbits were adult New Zealand Whites (Bunns Bank, Norfolk). Rabbits homozygous for C6 deficiency (Cambridge Strain) were the source of C6D plasma and platelets. Blood samples Heparin (Evans Medical Ltd, Liverpool) was used in a final concentration of 5 u/ml to anticoagulate blood taken for the preparation of platelet-rich plasma (PRP) and platelet-poor plasma (PPP). Acid-citrate-dextrose (Aster and Jandl, 1964) was used in a concentration of 6 per cent v/v to anticoagulate blood taken for the preparation of washed platelet suspensions. Blood samples were taken from the marginal ear veins of rabbits sedated with 0 4 ml/kg body weight i.m. of Hypnorm (Janssen Pharmaceutica, Beerse, Belgium). Plastic syringes containing anticoagulant and fitted with 23-gauge needles were used. PRP was prepared by differential centrifugation at room temperature (4000 g for 20 seconds). Samples of PRP contained 2-4 x 108 cells/ml. PPP was prepared by centrifuging PRP at 14,700 g for 5 minutes. Suspensions of washed platelets in calcium-free Tyrode's solution were prepared from 2-7 ml volumes of citrated PRP using an albumin density gradient (Walsh, 1972). Final suspensions contained 5-8 x 108 cells/ml. Platelets were counted with a Coulter model B electronic particle counter.
Platelet aggregation Collagen 'extract' (CE) was prepared from pig aortas (Gordon and Gresham, 1972). Final concentrations used in PRP and washed platelet suspensions were 3A4 ug/ml and 6-8 ,ug/ml of collagen protein. These concentrations were used because they gave approximately half-maximal rates of platelet aggregation. Zymosan (Z) from Sigma Chemical Company was prepared by boiling for 20 minutes in distilled water, washing three times with isotonic saline, and suspending at a final concentration of 4 mg/ml (approximately 3 x 108 particles/ml). Final concentrations used were 0-2 mg/ml and 0 4 mg/ml; at these concentrations, the aggregation rates in control PRP samples were similar to those obtained with CE (i.e. approximately half-maximal). Activated zymosan (ZA) was prepared by incubating one volume of the 4 mg/ml suspension for 10 minutes at 370 with four volumes of either normal PPP (ZAN) or C6D PPP (ZAc6D). The suspension was then washed three times with ice-cold isotonic saline, made up to a final concentration of 4 mg/ml in isotonic saline and kept in melting ice until required. Platelet aggregation responses were measured photometrically (Born, 1962) using 0-1 ml volumes of PRP or platelet suspensions (Gordon and Drummond, 1974). Measurement of released platelet constituents Total adenine nucleotides in platelets and plasma were assayed fluorimetrically
Platelet Function in C6 Deficiency 133 (Gordon and Drummond, 1974). 5-Hydroxytryptamine (5HT) in platelets and extracellular fluid was also assayed fluorimetrically (Drummond and Gordon, 1974). Lactic dehydrogenase (LDH) activity in platelets and extracellular fluid from aggregated and control (non-aggregated) samples was assayed photometrically (Wroblewski and La Due, 1955) using 0-2 ml sample volumes. Release of adenine nucleotides, 5HT and LDH from platelets was determined by calculating the percentage of these substances present in the extracellular fluid. Results obtained were corrected by subtracting background values for control samples (i.e. without any collagen or Z added). RESULTS AGGREGATION IN PRP SAMPLES
Collagen-induced aggregation responses were essentially the same in PRP samples from control and C6D rabbits. With both concentrations of CE used, there was initially a lag phase of a few seconds, then a transient increase in optical density indicative of a shape change in the platelets. This was followed by a progressive decrease in optical density associated with platelet aggregation (Fig. 1). Mean aggregation rates for groups of control and C6D rabbits were similar (Table 1). Platelet aggregation responses to Z in control PRP samples were characterized by three distinct phases: there was an initial lag, rather longer than that observed with collagen, and this was followed by two phases of aggregation with the second being much more rapid
CE
(n en
c
ao
IcT C:
0
t Tw
cr1
3.4 pg/ml minute I6-8
ag /ml
FIG. 1. Platelet aggregation induced by collagen extract (CE) in stirred samples of heparinized rabbit PRP. Aggregation responses are measured as the rate of increase in light transmission and are directly related to the collagen concentration. Aggregation is preceded by a lag of several seconds, then a transient decrease in transmission which indicates a change in platelet shape.
Felicity A. Christian and J. L. Gordon
134
TABLE 1 PLATELET AGGREGATION RESPONSES TO COLLAGEN (CE) AND ZYMOSAN (Z) IN PRP SAMPLES FROM CONTROL AND C6D RABBITS
Aggregation rate
Aggregating agent Control PRP C6D PRP
3-4ug/ml CE 6-8pg/ml CE 0 2 mg/ml Z 04 mg/ml Z
61-1+8-3 67-1+7-2 74-4+ 10.3 85-2+11-1
53-3+8-3 74-8+9-3 16-3+2*2 29-9+ 3-5
Results are shown as values + s.e. of fourteen to twenty-five determinations. Responses are expressed as aggregation rate (light transmission units/minute).
than the first. No shape change (i.e. no transient increase in optical density) was observed
(Fig. 2a).
In PRP samples from C6D rabbits, only the initial lag and the first phase of aggregation could be distinguished (Fig. 2b). No second phase of aggregation was seen even in samples observed for up to 20 minutes. Because aggregation responses were calculated from the maximum rate obtained, and because the second phase of aggregation (which was characterized by a faster rate) was absent in C6D samples, the aggregation responses to Z in the C6D group were much lower than those in the control group (Table 1). z z (a)
b
0-2 mg/mi
v
a
&~ ~ ~ ~ ~ 04 mg/mi
0
0-4 mg/m l0-2 mg/ml
minute
FIG. 2. Platelet aggregation induced by zymosan (Z) in heparinized PRP samples from (a) control rabbits and (b) C6D rabbits.
Platelet Function in C6 Deficiency
135
TABLE 2 RELEASE OF PLATELET CONSTITUENTS INDUCED BY COLLAGEN (CE) AND ZYMOSAN (Z) IN PRP SAMPLES FROM CONTROL AND C6D RABBITS Percentage release Aggregating agent
3 4 pg/ml CE 68,pg/mlCE
02mg/mlZ 0 4 mg/ml Z
Control PRP
C6D PRP
Nucleotides
5HT
Nucleotides
5HT
16 7+2-4 215+13 82+ 34 16 3+5 9
1+7 9-1i 249+20 44+1 1 10 3+1 5
18 4+3 1 172+20 13+1-1 1-5+ 12
8 8+1 9 291+26 -13+05 -2 0+0 3
Results are given as mean values + s.e. of six to eight determinations. Responses are expressed as the percentage of platelet 5HT or adenine nucleotides released into the plasma. RELEASE IN PRP SAMPLES
Collagen-induced aggregation was associated with the release of adenine nucleotides and 5HT in PRP samples from both control and C6D animals (Table 2). Z caused release of both adenine nucleotides and 5HT from control PRP samples, but there was no significant release in C6D samples (Table 2). There was no significant difference in the content of either 5HT or total nucleotides in platelets from the two groups of animals. Values for 5HT content (micrograms per 109 cells) in control and C6D animals were 10-7+0-9 s.e. and 11l0+0-5 s.e. respectively. Values for total adenine nucleotides (nmol/108 cells) in control and C6D animals were 8-3+0O8 s.e. and 7 I+0-7 s.e. respectively. To determine whether the reduced responses to Z in C6D samples were due to an abnormality in the platelets or in the plasma, a further series of experiments was conducted using washed platelet suspensions. AGGREGATION AND RELEASE IN WASHED PLATELET SUSPENSIONS
Three different groups of samples were tested. Collagen or Z was added to: (1) washed platelets alone (in calcium-free Tyrode's solution); (2) washed platelets+ 10 P1 of C6D PPP; (3) washed platelets+ 10 !I of normal PPP. Collagen alone (6-8 Mg/ml) induced only a small aggregation response, but larger responses were obtained with higher collagen concentrations. Aggregation responses could be increased by adding 10 p1 of cell-free plasma to the platelet suspensions, and PPP from control and C6D animals was equipotent in this respect. Collagen-induced aggregation in platelet suspensions was accompanied by release of 5HT from the platelets. Z alone caused no aggregation of washed platelets in suspension even in concentrations up to 2 mg/ml. However, 0-4 mg/ml of Z caused first-phase aggregation if 10 P1 of C6D PPP was present, and caused biphasic aggregation if 10 M1 of normal PPP was present (Fig. 3). The second phase of aggregation was accompanied by release of 5HT. When washed platelet suspensions from C6D rabbits were tested, the results were indistinguishable from those obtained with platelet suspensions from control animals.
Felicity A. Christian and J. L. Gordon
136
,0 en
.E
+
10
LI
.21 _-
Cyl
minute N PPP
FIG. 3. Platelet aggregation induced by zymosan (Z) in suspensions of washed rabbit platelets. Tise when zymosan was added. Where indicated, 10 Jul of cell-free plasma was added to the seconds before the zymosan. The plasma was obtained from either a C6D rabbit (C6DPPP) or a normal rabbit (NPPP).
arrow indicates suspension 10
The above experiments were repeated using washed platelet suspensions and Z which had been pre-activated by incubation in normal PPP or C6D PPP. THE EFFECT OF ACTIVATED Z ON WASHED RABBIT PLATELETS
Z (0 4 mg/ml) which had been pre-activated in C6D PPP (ZAC6D) or in normal PPP (ZAN) was added to platelet suspensions as described in the previous section and the responses obtained are shown in Fig. 4. Both activated preparations of Z induced first phase aggregation in suspensions without any PPP added. Prior addition of 10 [l of C6D PPP did not alter the aggregation response, but when 10 pl of normal PPP was present the responses were biphasic. The second phase of aggregation was accompanied by release of 5HT. ZAN and ZAC6D gave essentially identical results. LYSIS OF PLATELETS DURING THE RELEASE REACTION
To determine the extent of platelet lysis accompanying the release of 5HT and adenine nucleotides, intracellular and extracellular LDH activity was measured in samples from control animals, before and after platelet aggregation induced by CE or Z. Because of the low LDH activity in rabbit platelets, 0 2-ml sample volumes were used for these experiments, and release of platelet 5HT was measured in aliquots of the same samples. Preliminary experiments established that, with the concentrations of CE and Z used previously, LDH release ranged from 0 to 5 per cent for both agents. To clarify the relationship between the release of 5HT and LDH, samples were stirred for 2 minutes and 30 minutes at 370 in the presence of large amounts of the aggregating agents (18 ,ug/ml of
Platelet Function in C6 Deficiency
137
zA
AN
_
zAC6D
E
~~~zA
o
.0
c' F C6DPPP minute
NPPP
FIG. 4. Platelet aggregation induced by activated zymosan (ZA) in suspensions of washed rabbit platelets. The arrow indicates when zymosan was added. Zymosan was activated by incubating in cell-free plasma for 10 minutes at 370, then washing with ice-cold isotonic saline.
CE and 2 mg/ml of Z). Stirred control samples (i.e. without any aggregating agents) were also included. After 2 minutes, both CE and Z released a small amount of platelet LDH, accompanied by about 45 per cent of platelet 5HT. In the 30-minute samples there was substantial LDH release even in the absence of any aggregating agent, although this release was increased when CE or Z was present. In control samples the 5HT initially released had been taken up again by 30 minutes, with the result that there was then no extracellular 5HT to parallel the LDH released. In contrast, samples containing CE or Z still showed more 5HT release than LDH release after 30 minutes. Details of these results are given in Table 3. TABLE 3 PLATELET LYSIS AND SELECTIVE RELEASE INDUCED BY CE AND Z IN SAMPLES FROM CONTROL RABBITS, STIRRED FOR 2 MINUTES OR 30 MINUTES
Percentage release Sample
2 minutes stirred
30 minutes stirred
LDH
LDH
5HT
5HT
Control 19 8+3-5 -9 0+3-1 2 mg/ml Z 12 0+3-7 460+6 0 51-4+89 68-3+ 15 18 ug/ml CE 7-2 + 1-6 40 4+ 4-4 55-3 + 9-2 79 0+ 1-5
Results are given as mean + s.e. of four to six determinations.
138
Felicity A. Christian and J. L. Gordon
DISCUSSION Both collagen and zymosan induce platelet aggregation and release constituents from platelet storage granules. Although dissimilar in composition, these agents are both particulate-soluble tropocollagen does not interact with platelets (Muggli & Baumgartner, 1973; Jaffe & Deykin, 1974; Gordon & Dingle, 1974). This might suggest that their stimulatory actions on platelets were non-specific, and a consequence of their particulate nature, but the results of recent work (including the experiments reported here) indicate that the platelet responses to collagen and zymosan are mediated in very different ways. Collagen-induced platelet aggregation in stirred PRP is preceded by an initial lag of 10-60 seconds duration, then a transient increase in optical density as the cells change in shape. The aggregation response which follows the shape change is monophasic and is accompanied by the release of platelet 5HT and nucleotides from storage granules. Little LDH activity is released from the platelets. Identical responses are seen in PRP samples from control and C6D rabbits. Aggregation can also be induced by collagen in washed platelet suspensions, although the responses are usually smaller than in PRP samples. These responses can be increased by adding small amounts of cell-free plasma to the suspensions, and in this respect plasmas from control and C6D rabbits are equipotent. It is evident from these results that collagen can induce platelet aggregation and selective release in the absence oflate complement components or plasma proteins, although some plasma factor(s) which are present equally in normal and C6D animals can augment the platelet response to collagen. Zymosan-induced platelet aggregation in stirred PRP also follows an initial lag (longer than that observed with collagen) but there is no discernible shape change. In samples from control animals the aggregation response is biphasic; the second phase is more rapid, and release of 5HT and nucleotides is observed only during the second phase. As in the case of collagen, little LDH activity is released from platelets by zymosan during the first 2 minutes, although both agents release substantial amounts of LDH after 30 minutes. In C6D animals, zymosan induces only the primary phase of aggregation and there is no release of 5HT or nucleotides. Zymosan cannot induce aggregation or release in washed platelet suspensions, but adding small amounts of cell-free plasma restores the aggregation response. Addition of C6D plasma results in primary aggregation only, with no release, but normal plasma restores both phases of aggregation and the release reaction. Washed platelets from C6D animals respond identically to those from control animals. When zymosan is preactivated by incubation in cell-free plasma before adding it to the platelet samples, aggregation in PRP proceeds faster, after a negligible lag, but the patterns of response are unchanged. Activation of zymosan in control or C6D plasma produces identical results. In washed platelet suspensions, activated zymosan induces primary aggregation without any cell-free plasma being added. Aggregation responses are not altered by adding C6D plasma to the samples but the addition of cell-free plasma from control animals results in biphasic aggregation and release. The results of these experiments indicate that zymosan can induce a primary aggregation response (without any release) in PRP samples from control or C6D rabbits, but that the second phase of aggregation and the release reaction which accompanies it require the presence of late complement components including C6. Primary aggregation in PRP is preceded by a prolonged lag, which is abolished if the zymosan has been preactivated in
139 Platelet Function in C6 Deficiency cell-free plasma, and zymosan induces no aggregation in washed platelet suspensions unless it has been preactivated or cell-free plasma is added. This suggests that primary aggregation requires prior activation of the zymosan-that is, the fixation of early complement components (notably C3) on the zymosan particles. Our experimental observations provide a fairly clear picture of the interaction between zymosan and rabbit platelets, but some points merit comment in the light of results obtained by other research workers. The main points are first, the separation of zymosaninduced aggregation into two phases, with no release seen during the primary phase; and the second point concerns the dependence of zymosan-induced release on C6, despite the fact that the ratio of released 5HT to LDH indicates a selective secretory process. During previous work on zymosan-induced release from rabbit platelets (Siraganian, 1972), aggregation responses were not measured. Recent experiments by Zucker and Grant (1974) showed apparently biphasic aggregation of human platelets by zymosan, but these authors did not attempt to discriminate between the two phases. It is clear from our experiments that primary aggregation results from a direct interaction between rabbit platelets and activated zymosan, and that this interaction per se does not cause any release. The method used to activate the zymosan may affect the results obtained. We incubated zymosan in cell-free plasma for 10 minutes at 370, then washed the particles at 40 with isotonic saline. These conditions were chosen to allow fixation of C3 on the zymosan while minimizing the development of complement inactivators and also reducing the chance of IgG accumulating on the zymosan. Previous workers (Zucker & Grant, 1974; Pfueller and Luscher, 1974) who incubated zymosan for 60 minutes at 370 found that these activated particles induced release from washed human platelets in the absence of added plasma. Although this could well be due to differences between rabbit and human platelets, it is also possible that IgG on the zymosan could be the release-inducing stimulus, since aggregated IgG itself can cause release under these conditions, and significant amounts of IgG could have accumulated on the zymosan after 60 minutes at 37°. Recent results from our laboratory show that zymosan's capacity to induce platelet aggregation in human PRP is dramatically increased if it has been preactivated for 60 minutes rather than 10 minutes (J. L. Gordon, unpublished observations) and experiments are now in progress to determine whether this is due to the accumulation of IgG on the zymosan
particles. The absence of release during primary zymosan-induced aggregation deserves comment. It should be emphasized that in these experiments we did not stir the samples longer than necessary to achieve maximum primary aggregation; prolonged stirring alone can induce release, and this release is greatly increased if either collagen or zymosan is present. Also, in the washed platelet studies we used an albumin density gradient to prepare the platelet suspensions, and this is presumably more efficient than simple centrifugation at removing residual plasma from the cells. Hence, contamination of the suspensions by complement components would be minimized. It is unlikely that the lack of release reflects insensitivity in the assay procedure-the method we use will measure less than 1 ng of 5HT, and rabbit platelets contain around 1 f2 pug 5HT per 108 cells. The secondary platelet aggregation induced by zymosan in the presence of control plasma is invariably associated with release, and it seems likely that released platelet constituents are responsible for this aggregation. Since far more 5HT than LDH is released from the platelets under these conditions it appears that cell lysis is not primarily responsible for this release. A small amount of platelet LDH appears extracellularly after only 2
140 Felicity A. Christian and J}. L. Gordon minutes stirring with zymosan, but a similar pattern was observed with collagen. Day and Holmsen (1971) found that 2-10 per cent of cytoplasmic markers were released from platelets stimulated with epinephrine, ADP, collagen or thrombin, and commented that 'some degree of lysis invariably occurs with any inducers of the specific release reaction'. These authors also noted that prolonged contact of platelets with any release inducer might eventually result in complete cell lysis, and they concluded that specific release from platelets can justifiably be investigated only in the first few minutes after stimulation. Our results are in full agreement with this concept, since they show a considerable specificity of release (i.e. a high extracellular 5HT/LDH ratio) 2 minutes after stimulation with collagen or zymosan, but much more LDH release after 30 minutes even if no aggregating agent was present. Brown and Lachmann (1974) have recently shown that exposure of normal rabbit platelets to complement-activating particles such as inulin or endotoxin induce activation of PF3 and release cytoplasmic constituents (measured with 51Cr). Of particular relevance to our work, however, was their observation that endotoxin induced 100 per cent PF3 activation at a time when only 1-2 per cent of 51Cr had been released, although about 60 per cent of the "Cr later appeared extracellularly. It seems, therefore, that complement-activating particles may initially induce a highly selective release of constituents from rabbit platelets and cytoplasmic leakage occurs much later. This phenomenon merits further investigation. In summary, then, the experiments reported here permit three conclusions to be made about the interaction of rabbit platelets with zymosan. First, zymosan which has not been activated does not stimulate platelets, but the addition of unactivated zymosan to PRP induces platelet aggregation after a delay sufficient for activation to have occurred in situ. Secondly, activated zymosan induces a primary platelet aggregation response, modest in rate and extent, which is not accompanied by any release. Third, in the presence of late complement components (including C6) this primary response leads to a second, more vigorous aggregation which is associated with the selective release of platelet constituents. The precise mechanisms responsible for this release are still not clear, but it is apparently not mediated simply by damage to the cell membrane. Experiments are in progress to establish how zymosan can cause selective degranulation of rabbit platelets, and to determine whether these results may be applicable to other cells.
ACKNOWLEDGMENTS This study was supported in part by a grant from Beecham Research Laboratories. We thank Dr D. L. Brown of the Department of Immunology, Royal Postgraduate Medical School, Hammersmith for his helpful advice. REFERENCES ASTIER, R. H. and JANDI., J. H. (1964). 'Platelet sequestration in man. I. Methods.' 7. c/in. Invest., 43, 843. BORN, G. V. R. (1962). 'Quantitative investigations into the aggregation of blood platelets.'J. Pkysiol. (Lond.), 162, 67P. BROWN. D. L. and LACHMANN. P. J. (1974). 'The relationship of complement-mediated platelet damage to intravascular (coagulation: comparison
between endotoxin and inulin in the rabbit.' Advances in the Biosciences (ed. by G. Raspe), p. 300. Pergamon, Vieweg. DAY, H. J. and HOLMSEN, H. (1 97 1). 'Concepts of the blood platelet release reaction.' Ser. Haematol., 4, 3. DRUMMOND, A. H. and GORDON, J. L. (1974). 'Rapid, sensitive microassay for platelet 5HT.' Thrombos. Diathes. haemorrh. (Stuttg.), 31, 366. GORDON, J. L. and DINGLE, J. T. (1974). 'Binding of
141 Platelet Function in C6 Deficiency reaction induced by immunologic stimuli. II. radiolabelled collagen to blood platelets.'_7. Cell Sci., The effects of zymosan.'_7. Immunol., 112, 121 1. 16, 157. GORDON, L. and A.
J. DRUMMOND, H. (1974). 'A simple fluorimetric microassay for adenine compounds in platelets and plasma, and its application to studies on the platelet release reaction.' Biochem. j., 138, 165. GORDON, J. L. and GREsHAm, G. A. (1972). 'The measurement of platelet aggregation in small blood samples.' Atherosclerosis, 15, 383. GRETTE, K. (1962). 'Studies on the mechanism of thrombin-catalyzed hemostatic reactions in blood platelets.' Acta. physiol. scand., 56, supplement 195, p. 1. JAFFE, R. and DEYKIN, D. (1974) 'Evidence for a structural requirement for the aggregation of platelets by collagen.'3. din Invest., 58, 875. LOSCHER, E. F., PFUELLER, S. L. and MASSINI, P. (1973). 'Platelet aggregation by large molecules.' Ser. Haematol., 6, 382. MUGGLI, R. and BAUMGARTNER, H. R. (1973). 'Collagen-induced platelet aggregation: requirement for tropocollagen multimers.' Thrombos. Res. 3, 715. PFUELLER, S. L. and LUSCHER, E. F. (1974). 'Studies of the mechanisms of the human platelet release
SIRAGANIAN, R. P. (1972). 'Platelet requirement in the interaction of the complement and clotting systems.' Nature: New Biology, 239, 208. WALSH, P. N. (1972). 'Albumin density gradient separation and washing of platelets and the study of platelet coagulant activities.' Brit. J. Haemat., 22, 205. WROBLEWSKI, F. and LA DUE, J. S. (1955). 'Lactic dehydrogenase activity in blood.' Proc. Soc. exp. Biol. (N..r.), 90, 210.
ZIMMERMAN, T. S., ARROYAVE, C. M. and MULLER-
EBERHARDT, H. J. (1971). 'A blood coagulation abnormality in rabbits deficient in the sixth component of complement (C6) and its correction by purified C6.' J. exp. Med., 134, 1591. ZIMMERMAN, T. S. and MULLER-EBERHARDT, H. J.
(1972). 'Interaction of complement, platelets and the blood coagulation system.' J. cdin. Invest., 51, 107A. ZUCKER, M. B. and GRANT, R. A. (1974). 'Aggregation and release reaction induced in human blood platelets by zymosan.'_7. Immunol., 112, 1219.
Platelet Function in C6-deficient Rabbits AGGREGATION AND SECRETION INDUCED BY COLLAGEN AND ZYMOSAN FELICITY A. CHRISTIAN AND J. L. GORDON Department of Pathology, University of Cambridge
(Received 6th January 1975; acceptedfor publication 15th January 1975) Summary. Platelet aggregation and the selective release of 5-hydroxytryptamine (5HT) and adenine nucleotides were measured in platelet-rich plasma (PRP) and washed platelet suspensions from control and C6-deficient rabbits. Aggregation and release induced by collagen were similar in both groups of animals. Aggregation responses to zymosan in control PRP samples were biphasic, and only the second phase was associated with release. In C6-deficient PRP samples, zymosan-induced aggregation lacked the second phase and there was no release of 5HT or adenine nucleotides. Zymosan induced no aggregation in washed platelet suspensions unless cell-free plasma was also added. C6-deficient plasma supported only primary aggregation, but the addition of control plasma resulted in biphasic aggregation and release. Zymosan which had been pre-activated in control or C6-deficient cell-free plasma induced primary aggregation in washed platelet suspensions. To obtain secondary aggregation and release, the addition of control plasma to the platelet suspension was necessary. INTRODUCTION Rabbits genetically deficient in the sixth component of complement (C6D rabbits) exhibit a defect in blood coagulation in vitro, characterized by a prolonged whole-blood clotting time in plastic tubes and poor prothrombin consumption (Zimmerman, Arroyave and Muller-Eberhardt, 1971). Cell-free plasma from C6D rabbits will not support the release of histamine from rabbit platelets by zymosan (Siraganian, 1972), but it was not established whether this histamine release was associated with the formation of platelet aggregates, or whether it was a result of cell lysis or of a specific secretory process. Such a process-the so-called 'release reaction' (Grette, 1962)-is known to occur in platelets and involves the selective exocytosis of storage granules in response to agents such as thrombin or collagen. The main constituents of these granules are adenine nucleotides and 5hydroxytryptamine (5HT), and these are conventionally used as markers for the release reaction. Rabbit platelets are unique in having substantial amounts of histamine, and the localization and release of platelet histamine have not been determined in detail. In the present study, platelet aggregation and the release of 5HT and adenine nucleotides were measured in response to collagen and zymosan. These experiments were performed using platelet-rich plasma (PRP) and washed platelet suspensions from control and Correspondence: Dr J. L. Gordon, University of Cambridge, Department of Pathology, Tennis Court Road, Cambridge CB2 IQP.
131
Felicity A. Christian and J. L. Gordon 132 C6D rabbits. Platelet responses to zymosan which had been preactivated in cell-free plasma were also determined, and the degree of cell lysis was determined by measuring lactate dehydrogenase activity in the cells and in the suspending medium. MATERIALS AND METHODS Rabbits Control rabbits were adult New Zealand Whites (Bunns Bank, Norfolk). Rabbits homozygous for C6 deficiency (Cambridge Strain) were the source of C6D plasma and platelets. Blood samples Heparin (Evans Medical Ltd, Liverpool) was used in a final concentration of 5 u/ml to anticoagulate blood taken for the preparation of platelet-rich plasma (PRP) and platelet-poor plasma (PPP). Acid-citrate-dextrose (Aster and Jandl, 1964) was used in a concentration of 6 per cent v/v to anticoagulate blood taken for the preparation of washed platelet suspensions. Blood samples were taken from the marginal ear veins of rabbits sedated with 0 4 ml/kg body weight i.m. of Hypnorm (Janssen Pharmaceutica, Beerse, Belgium). Plastic syringes containing anticoagulant and fitted with 23-gauge needles were used. PRP was prepared by differential centrifugation at room temperature (4000 g for 20 seconds). Samples of PRP contained 2-4 x 108 cells/ml. PPP was prepared by centrifuging PRP at 14,700 g for 5 minutes. Suspensions of washed platelets in calcium-free Tyrode's solution were prepared from 2-7 ml volumes of citrated PRP using an albumin density gradient (Walsh, 1972). Final suspensions contained 5-8 x 108 cells/ml. Platelets were counted with a Coulter model B electronic particle counter.
Platelet aggregation Collagen 'extract' (CE) was prepared from pig aortas (Gordon and Gresham, 1972). Final concentrations used in PRP and washed platelet suspensions were 3A4 ug/ml and 6-8 ,ug/ml of collagen protein. These concentrations were used because they gave approximately half-maximal rates of platelet aggregation. Zymosan (Z) from Sigma Chemical Company was prepared by boiling for 20 minutes in distilled water, washing three times with isotonic saline, and suspending at a final concentration of 4 mg/ml (approximately 3 x 108 particles/ml). Final concentrations used were 0-2 mg/ml and 0 4 mg/ml; at these concentrations, the aggregation rates in control PRP samples were similar to those obtained with CE (i.e. approximately half-maximal). Activated zymosan (ZA) was prepared by incubating one volume of the 4 mg/ml suspension for 10 minutes at 370 with four volumes of either normal PPP (ZAN) or C6D PPP (ZAc6D). The suspension was then washed three times with ice-cold isotonic saline, made up to a final concentration of 4 mg/ml in isotonic saline and kept in melting ice until required. Platelet aggregation responses were measured photometrically (Born, 1962) using 0-1 ml volumes of PRP or platelet suspensions (Gordon and Drummond, 1974). Measurement of released platelet constituents Total adenine nucleotides in platelets and plasma were assayed fluorimetrically
Platelet Function in C6 Deficiency 133 (Gordon and Drummond, 1974). 5-Hydroxytryptamine (5HT) in platelets and extracellular fluid was also assayed fluorimetrically (Drummond and Gordon, 1974). Lactic dehydrogenase (LDH) activity in platelets and extracellular fluid from aggregated and control (non-aggregated) samples was assayed photometrically (Wroblewski and La Due, 1955) using 0-2 ml sample volumes. Release of adenine nucleotides, 5HT and LDH from platelets was determined by calculating the percentage of these substances present in the extracellular fluid. Results obtained were corrected by subtracting background values for control samples (i.e. without any collagen or Z added). RESULTS AGGREGATION IN PRP SAMPLES
Collagen-induced aggregation responses were essentially the same in PRP samples from control and C6D rabbits. With both concentrations of CE used, there was initially a lag phase of a few seconds, then a transient increase in optical density indicative of a shape change in the platelets. This was followed by a progressive decrease in optical density associated with platelet aggregation (Fig. 1). Mean aggregation rates for groups of control and C6D rabbits were similar (Table 1). Platelet aggregation responses to Z in control PRP samples were characterized by three distinct phases: there was an initial lag, rather longer than that observed with collagen, and this was followed by two phases of aggregation with the second being much more rapid
CE
(n en
c
ao
IcT C:
0
t Tw
cr1
3.4 pg/ml minute I6-8
ag /ml
FIG. 1. Platelet aggregation induced by collagen extract (CE) in stirred samples of heparinized rabbit PRP. Aggregation responses are measured as the rate of increase in light transmission and are directly related to the collagen concentration. Aggregation is preceded by a lag of several seconds, then a transient decrease in transmission which indicates a change in platelet shape.
Felicity A. Christian and J. L. Gordon
134
TABLE 1 PLATELET AGGREGATION RESPONSES TO COLLAGEN (CE) AND ZYMOSAN (Z) IN PRP SAMPLES FROM CONTROL AND C6D RABBITS
Aggregation rate
Aggregating agent Control PRP C6D PRP
3-4ug/ml CE 6-8pg/ml CE 0 2 mg/ml Z 04 mg/ml Z
61-1+8-3 67-1+7-2 74-4+ 10.3 85-2+11-1
53-3+8-3 74-8+9-3 16-3+2*2 29-9+ 3-5
Results are shown as values + s.e. of fourteen to twenty-five determinations. Responses are expressed as aggregation rate (light transmission units/minute).
than the first. No shape change (i.e. no transient increase in optical density) was observed
(Fig. 2a).
In PRP samples from C6D rabbits, only the initial lag and the first phase of aggregation could be distinguished (Fig. 2b). No second phase of aggregation was seen even in samples observed for up to 20 minutes. Because aggregation responses were calculated from the maximum rate obtained, and because the second phase of aggregation (which was characterized by a faster rate) was absent in C6D samples, the aggregation responses to Z in the C6D group were much lower than those in the control group (Table 1). z z (a)
b
0-2 mg/mi
v
a
&~ ~ ~ ~ ~ 04 mg/mi
0
0-4 mg/m l0-2 mg/ml
minute
FIG. 2. Platelet aggregation induced by zymosan (Z) in heparinized PRP samples from (a) control rabbits and (b) C6D rabbits.
Platelet Function in C6 Deficiency
135
TABLE 2 RELEASE OF PLATELET CONSTITUENTS INDUCED BY COLLAGEN (CE) AND ZYMOSAN (Z) IN PRP SAMPLES FROM CONTROL AND C6D RABBITS Percentage release Aggregating agent
3 4 pg/ml CE 68,pg/mlCE
02mg/mlZ 0 4 mg/ml Z
Control PRP
C6D PRP
Nucleotides
5HT
Nucleotides
5HT
16 7+2-4 215+13 82+ 34 16 3+5 9
1+7 9-1i 249+20 44+1 1 10 3+1 5
18 4+3 1 172+20 13+1-1 1-5+ 12
8 8+1 9 291+26 -13+05 -2 0+0 3
Results are given as mean values + s.e. of six to eight determinations. Responses are expressed as the percentage of platelet 5HT or adenine nucleotides released into the plasma. RELEASE IN PRP SAMPLES
Collagen-induced aggregation was associated with the release of adenine nucleotides and 5HT in PRP samples from both control and C6D animals (Table 2). Z caused release of both adenine nucleotides and 5HT from control PRP samples, but there was no significant release in C6D samples (Table 2). There was no significant difference in the content of either 5HT or total nucleotides in platelets from the two groups of animals. Values for 5HT content (micrograms per 109 cells) in control and C6D animals were 10-7+0-9 s.e. and 11l0+0-5 s.e. respectively. Values for total adenine nucleotides (nmol/108 cells) in control and C6D animals were 8-3+0O8 s.e. and 7 I+0-7 s.e. respectively. To determine whether the reduced responses to Z in C6D samples were due to an abnormality in the platelets or in the plasma, a further series of experiments was conducted using washed platelet suspensions. AGGREGATION AND RELEASE IN WASHED PLATELET SUSPENSIONS
Three different groups of samples were tested. Collagen or Z was added to: (1) washed platelets alone (in calcium-free Tyrode's solution); (2) washed platelets+ 10 P1 of C6D PPP; (3) washed platelets+ 10 !I of normal PPP. Collagen alone (6-8 Mg/ml) induced only a small aggregation response, but larger responses were obtained with higher collagen concentrations. Aggregation responses could be increased by adding 10 p1 of cell-free plasma to the platelet suspensions, and PPP from control and C6D animals was equipotent in this respect. Collagen-induced aggregation in platelet suspensions was accompanied by release of 5HT from the platelets. Z alone caused no aggregation of washed platelets in suspension even in concentrations up to 2 mg/ml. However, 0-4 mg/ml of Z caused first-phase aggregation if 10 P1 of C6D PPP was present, and caused biphasic aggregation if 10 M1 of normal PPP was present (Fig. 3). The second phase of aggregation was accompanied by release of 5HT. When washed platelet suspensions from C6D rabbits were tested, the results were indistinguishable from those obtained with platelet suspensions from control animals.
Felicity A. Christian and J. L. Gordon
136
,0 en
.E
+
10
LI
.21 _-
Cyl
minute N PPP
FIG. 3. Platelet aggregation induced by zymosan (Z) in suspensions of washed rabbit platelets. Tise when zymosan was added. Where indicated, 10 Jul of cell-free plasma was added to the seconds before the zymosan. The plasma was obtained from either a C6D rabbit (C6DPPP) or a normal rabbit (NPPP).
arrow indicates suspension 10
The above experiments were repeated using washed platelet suspensions and Z which had been pre-activated by incubation in normal PPP or C6D PPP. THE EFFECT OF ACTIVATED Z ON WASHED RABBIT PLATELETS
Z (0 4 mg/ml) which had been pre-activated in C6D PPP (ZAC6D) or in normal PPP (ZAN) was added to platelet suspensions as described in the previous section and the responses obtained are shown in Fig. 4. Both activated preparations of Z induced first phase aggregation in suspensions without any PPP added. Prior addition of 10 [l of C6D PPP did not alter the aggregation response, but when 10 pl of normal PPP was present the responses were biphasic. The second phase of aggregation was accompanied by release of 5HT. ZAN and ZAC6D gave essentially identical results. LYSIS OF PLATELETS DURING THE RELEASE REACTION
To determine the extent of platelet lysis accompanying the release of 5HT and adenine nucleotides, intracellular and extracellular LDH activity was measured in samples from control animals, before and after platelet aggregation induced by CE or Z. Because of the low LDH activity in rabbit platelets, 0 2-ml sample volumes were used for these experiments, and release of platelet 5HT was measured in aliquots of the same samples. Preliminary experiments established that, with the concentrations of CE and Z used previously, LDH release ranged from 0 to 5 per cent for both agents. To clarify the relationship between the release of 5HT and LDH, samples were stirred for 2 minutes and 30 minutes at 370 in the presence of large amounts of the aggregating agents (18 ,ug/ml of
Platelet Function in C6 Deficiency
137
zA
AN
_
zAC6D
E
~~~zA
o
.0
c' F C6DPPP minute
NPPP
FIG. 4. Platelet aggregation induced by activated zymosan (ZA) in suspensions of washed rabbit platelets. The arrow indicates when zymosan was added. Zymosan was activated by incubating in cell-free plasma for 10 minutes at 370, then washing with ice-cold isotonic saline.
CE and 2 mg/ml of Z). Stirred control samples (i.e. without any aggregating agents) were also included. After 2 minutes, both CE and Z released a small amount of platelet LDH, accompanied by about 45 per cent of platelet 5HT. In the 30-minute samples there was substantial LDH release even in the absence of any aggregating agent, although this release was increased when CE or Z was present. In control samples the 5HT initially released had been taken up again by 30 minutes, with the result that there was then no extracellular 5HT to parallel the LDH released. In contrast, samples containing CE or Z still showed more 5HT release than LDH release after 30 minutes. Details of these results are given in Table 3. TABLE 3 PLATELET LYSIS AND SELECTIVE RELEASE INDUCED BY CE AND Z IN SAMPLES FROM CONTROL RABBITS, STIRRED FOR 2 MINUTES OR 30 MINUTES
Percentage release Sample
2 minutes stirred
30 minutes stirred
LDH
LDH
5HT
5HT
Control 19 8+3-5 -9 0+3-1 2 mg/ml Z 12 0+3-7 460+6 0 51-4+89 68-3+ 15 18 ug/ml CE 7-2 + 1-6 40 4+ 4-4 55-3 + 9-2 79 0+ 1-5
Results are given as mean + s.e. of four to six determinations.
138
Felicity A. Christian and J. L. Gordon
DISCUSSION Both collagen and zymosan induce platelet aggregation and release constituents from platelet storage granules. Although dissimilar in composition, these agents are both particulate-soluble tropocollagen does not interact with platelets (Muggli & Baumgartner, 1973; Jaffe & Deykin, 1974; Gordon & Dingle, 1974). This might suggest that their stimulatory actions on platelets were non-specific, and a consequence of their particulate nature, but the results of recent work (including the experiments reported here) indicate that the platelet responses to collagen and zymosan are mediated in very different ways. Collagen-induced platelet aggregation in stirred PRP is preceded by an initial lag of 10-60 seconds duration, then a transient increase in optical density as the cells change in shape. The aggregation response which follows the shape change is monophasic and is accompanied by the release of platelet 5HT and nucleotides from storage granules. Little LDH activity is released from the platelets. Identical responses are seen in PRP samples from control and C6D rabbits. Aggregation can also be induced by collagen in washed platelet suspensions, although the responses are usually smaller than in PRP samples. These responses can be increased by adding small amounts of cell-free plasma to the suspensions, and in this respect plasmas from control and C6D rabbits are equipotent. It is evident from these results that collagen can induce platelet aggregation and selective release in the absence oflate complement components or plasma proteins, although some plasma factor(s) which are present equally in normal and C6D animals can augment the platelet response to collagen. Zymosan-induced platelet aggregation in stirred PRP also follows an initial lag (longer than that observed with collagen) but there is no discernible shape change. In samples from control animals the aggregation response is biphasic; the second phase is more rapid, and release of 5HT and nucleotides is observed only during the second phase. As in the case of collagen, little LDH activity is released from platelets by zymosan during the first 2 minutes, although both agents release substantial amounts of LDH after 30 minutes. In C6D animals, zymosan induces only the primary phase of aggregation and there is no release of 5HT or nucleotides. Zymosan cannot induce aggregation or release in washed platelet suspensions, but adding small amounts of cell-free plasma restores the aggregation response. Addition of C6D plasma results in primary aggregation only, with no release, but normal plasma restores both phases of aggregation and the release reaction. Washed platelets from C6D animals respond identically to those from control animals. When zymosan is preactivated by incubation in cell-free plasma before adding it to the platelet samples, aggregation in PRP proceeds faster, after a negligible lag, but the patterns of response are unchanged. Activation of zymosan in control or C6D plasma produces identical results. In washed platelet suspensions, activated zymosan induces primary aggregation without any cell-free plasma being added. Aggregation responses are not altered by adding C6D plasma to the samples but the addition of cell-free plasma from control animals results in biphasic aggregation and release. The results of these experiments indicate that zymosan can induce a primary aggregation response (without any release) in PRP samples from control or C6D rabbits, but that the second phase of aggregation and the release reaction which accompanies it require the presence of late complement components including C6. Primary aggregation in PRP is preceded by a prolonged lag, which is abolished if the zymosan has been preactivated in
139 Platelet Function in C6 Deficiency cell-free plasma, and zymosan induces no aggregation in washed platelet suspensions unless it has been preactivated or cell-free plasma is added. This suggests that primary aggregation requires prior activation of the zymosan-that is, the fixation of early complement components (notably C3) on the zymosan particles. Our experimental observations provide a fairly clear picture of the interaction between zymosan and rabbit platelets, but some points merit comment in the light of results obtained by other research workers. The main points are first, the separation of zymosaninduced aggregation into two phases, with no release seen during the primary phase; and the second point concerns the dependence of zymosan-induced release on C6, despite the fact that the ratio of released 5HT to LDH indicates a selective secretory process. During previous work on zymosan-induced release from rabbit platelets (Siraganian, 1972), aggregation responses were not measured. Recent experiments by Zucker and Grant (1974) showed apparently biphasic aggregation of human platelets by zymosan, but these authors did not attempt to discriminate between the two phases. It is clear from our experiments that primary aggregation results from a direct interaction between rabbit platelets and activated zymosan, and that this interaction per se does not cause any release. The method used to activate the zymosan may affect the results obtained. We incubated zymosan in cell-free plasma for 10 minutes at 370, then washed the particles at 40 with isotonic saline. These conditions were chosen to allow fixation of C3 on the zymosan while minimizing the development of complement inactivators and also reducing the chance of IgG accumulating on the zymosan. Previous workers (Zucker & Grant, 1974; Pfueller and Luscher, 1974) who incubated zymosan for 60 minutes at 370 found that these activated particles induced release from washed human platelets in the absence of added plasma. Although this could well be due to differences between rabbit and human platelets, it is also possible that IgG on the zymosan could be the release-inducing stimulus, since aggregated IgG itself can cause release under these conditions, and significant amounts of IgG could have accumulated on the zymosan after 60 minutes at 37°. Recent results from our laboratory show that zymosan's capacity to induce platelet aggregation in human PRP is dramatically increased if it has been preactivated for 60 minutes rather than 10 minutes (J. L. Gordon, unpublished observations) and experiments are now in progress to determine whether this is due to the accumulation of IgG on the zymosan
particles. The absence of release during primary zymosan-induced aggregation deserves comment. It should be emphasized that in these experiments we did not stir the samples longer than necessary to achieve maximum primary aggregation; prolonged stirring alone can induce release, and this release is greatly increased if either collagen or zymosan is present. Also, in the washed platelet studies we used an albumin density gradient to prepare the platelet suspensions, and this is presumably more efficient than simple centrifugation at removing residual plasma from the cells. Hence, contamination of the suspensions by complement components would be minimized. It is unlikely that the lack of release reflects insensitivity in the assay procedure-the method we use will measure less than 1 ng of 5HT, and rabbit platelets contain around 1 f2 pug 5HT per 108 cells. The secondary platelet aggregation induced by zymosan in the presence of control plasma is invariably associated with release, and it seems likely that released platelet constituents are responsible for this aggregation. Since far more 5HT than LDH is released from the platelets under these conditions it appears that cell lysis is not primarily responsible for this release. A small amount of platelet LDH appears extracellularly after only 2
140 Felicity A. Christian and J}. L. Gordon minutes stirring with zymosan, but a similar pattern was observed with collagen. Day and Holmsen (1971) found that 2-10 per cent of cytoplasmic markers were released from platelets stimulated with epinephrine, ADP, collagen or thrombin, and commented that 'some degree of lysis invariably occurs with any inducers of the specific release reaction'. These authors also noted that prolonged contact of platelets with any release inducer might eventually result in complete cell lysis, and they concluded that specific release from platelets can justifiably be investigated only in the first few minutes after stimulation. Our results are in full agreement with this concept, since they show a considerable specificity of release (i.e. a high extracellular 5HT/LDH ratio) 2 minutes after stimulation with collagen or zymosan, but much more LDH release after 30 minutes even if no aggregating agent was present. Brown and Lachmann (1974) have recently shown that exposure of normal rabbit platelets to complement-activating particles such as inulin or endotoxin induce activation of PF3 and release cytoplasmic constituents (measured with 51Cr). Of particular relevance to our work, however, was their observation that endotoxin induced 100 per cent PF3 activation at a time when only 1-2 per cent of 51Cr had been released, although about 60 per cent of the "Cr later appeared extracellularly. It seems, therefore, that complement-activating particles may initially induce a highly selective release of constituents from rabbit platelets and cytoplasmic leakage occurs much later. This phenomenon merits further investigation. In summary, then, the experiments reported here permit three conclusions to be made about the interaction of rabbit platelets with zymosan. First, zymosan which has not been activated does not stimulate platelets, but the addition of unactivated zymosan to PRP induces platelet aggregation after a delay sufficient for activation to have occurred in situ. Secondly, activated zymosan induces a primary platelet aggregation response, modest in rate and extent, which is not accompanied by any release. Third, in the presence of late complement components (including C6) this primary response leads to a second, more vigorous aggregation which is associated with the selective release of platelet constituents. The precise mechanisms responsible for this release are still not clear, but it is apparently not mediated simply by damage to the cell membrane. Experiments are in progress to establish how zymosan can cause selective degranulation of rabbit platelets, and to determine whether these results may be applicable to other cells.
ACKNOWLEDGMENTS This study was supported in part by a grant from Beecham Research Laboratories. We thank Dr D. L. Brown of the Department of Immunology, Royal Postgraduate Medical School, Hammersmith for his helpful advice. REFERENCES ASTIER, R. H. and JANDI., J. H. (1964). 'Platelet sequestration in man. I. Methods.' 7. c/in. Invest., 43, 843. BORN, G. V. R. (1962). 'Quantitative investigations into the aggregation of blood platelets.'J. Pkysiol. (Lond.), 162, 67P. BROWN. D. L. and LACHMANN. P. J. (1974). 'The relationship of complement-mediated platelet damage to intravascular (coagulation: comparison
between endotoxin and inulin in the rabbit.' Advances in the Biosciences (ed. by G. Raspe), p. 300. Pergamon, Vieweg. DAY, H. J. and HOLMSEN, H. (1 97 1). 'Concepts of the blood platelet release reaction.' Ser. Haematol., 4, 3. DRUMMOND, A. H. and GORDON, J. L. (1974). 'Rapid, sensitive microassay for platelet 5HT.' Thrombos. Diathes. haemorrh. (Stuttg.), 31, 366. GORDON, J. L. and DINGLE, J. T. (1974). 'Binding of
141 Platelet Function in C6 Deficiency reaction induced by immunologic stimuli. II. radiolabelled collagen to blood platelets.'_7. Cell Sci., The effects of zymosan.'_7. Immunol., 112, 121 1. 16, 157. GORDON, L. and A.
J. DRUMMOND, H. (1974). 'A simple fluorimetric microassay for adenine compounds in platelets and plasma, and its application to studies on the platelet release reaction.' Biochem. j., 138, 165. GORDON, J. L. and GREsHAm, G. A. (1972). 'The measurement of platelet aggregation in small blood samples.' Atherosclerosis, 15, 383. GRETTE, K. (1962). 'Studies on the mechanism of thrombin-catalyzed hemostatic reactions in blood platelets.' Acta. physiol. scand., 56, supplement 195, p. 1. JAFFE, R. and DEYKIN, D. (1974) 'Evidence for a structural requirement for the aggregation of platelets by collagen.'3. din Invest., 58, 875. LOSCHER, E. F., PFUELLER, S. L. and MASSINI, P. (1973). 'Platelet aggregation by large molecules.' Ser. Haematol., 6, 382. MUGGLI, R. and BAUMGARTNER, H. R. (1973). 'Collagen-induced platelet aggregation: requirement for tropocollagen multimers.' Thrombos. Res. 3, 715. PFUELLER, S. L. and LUSCHER, E. F. (1974). 'Studies of the mechanisms of the human platelet release
SIRAGANIAN, R. P. (1972). 'Platelet requirement in the interaction of the complement and clotting systems.' Nature: New Biology, 239, 208. WALSH, P. N. (1972). 'Albumin density gradient separation and washing of platelets and the study of platelet coagulant activities.' Brit. J. Haemat., 22, 205. WROBLEWSKI, F. and LA DUE, J. S. (1955). 'Lactic dehydrogenase activity in blood.' Proc. Soc. exp. Biol. (N..r.), 90, 210.
ZIMMERMAN, T. S., ARROYAVE, C. M. and MULLER-
EBERHARDT, H. J. (1971). 'A blood coagulation abnormality in rabbits deficient in the sixth component of complement (C6) and its correction by purified C6.' J. exp. Med., 134, 1591. ZIMMERMAN, T. S. and MULLER-EBERHARDT, H. J.
(1972). 'Interaction of complement, platelets and the blood coagulation system.' J. cdin. Invest., 51, 107A. ZUCKER, M. B. and GRANT, R. A. (1974). 'Aggregation and release reaction induced in human blood platelets by zymosan.'_7. Immunol., 112, 1219.
