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MECHANISM OF BLOOD CLOTTING
18
MECHANISM OF BLOOD
vascular upsets may be of first-rate importance, the adrenal changes being merely contributory to the usually fatal- outcome ; it is well known that adrenalectomised animals withstand shock badly. On these grounds he urges the importance of energetic attempts to restore normal vascular conditions by the administration of plasma as well as adrenal substitution, which he relegates to second place. The use of plasma or blood in addition to cortical therapy was also recommended by GRACE, HARRisoN and DAVIE,4 who recorded a success with this treatment. Two lessons are to be learned from this work. First, meningococcal infection is often, or even usually, septiceemic, and meningitis is only one manifestation of infection. Secondly, impending adrenal and vascular failure must be looked out for in meningoeoccal (and other) infections, and combated actively. when it occurs ; the prognosis in cases with sudden adrenal failure is grave but not hopeless.
MECHANISM OF BLOOD CLOTTING propounded the theory
i ORTY years ago MORAWITZ that blood-clotting is a
two-stage mechanism : in the first stage the prothrombin of blood-plasma in the presence of calcium ions and a tissue or platelet factor (thrombokinase, thromboplastin) is transformed into thrombin ; in the second stage the thrombin acts on the plasma-protein fibrinogen, converting it to the relatively insoluble fibrin which forms the scaffold of the clot. The framework of this theory is now generally accepted, but there is still much controversy about the detail. Useful information has been obtained from studying the blood-clotting activity of certain proteolytic enzymes, and by following the kinetics of the reactions. EAGLE 5 showed that trypsin and certain proteolytic snake-venoms in proper concentration " activated prothrombin to thrombin, and in this reaction calcium was not necessary. The concentration of trypsiri had to b optimal ; if it was - too weak -there was no action at all; if it was too strong both prothrombin and thrombin were destroyed. He compared the rates at which thrombin was formed from prothrombin by calcium-platelet mixture, crystalline trypsin, and the snake-venoms ; very similar curves were obtained. He therefore suggested that the normal action of the calcium-platelet combination was to form an enzyme that converted prothrombin to thrombin ; much as the trypsinogen .Qf the pancreatic secretion is converted by the enterokinase of the intestinal secretion into the actively "
-proteolytic trypsin. In the early days of blood-coagulation studies, ScHMlDT suggested that thrombin was a proteolytic enzyme acting on fibrinogen ; though not accepted by many, this has remained a possibility, and here :again study of the action of known proteolytic enzymes
has brought support. EAGLE has shown that some nake-venoms clot blood by acting directly on fibrinogen and converting it to fibrin ; they are active in very high dilutions, their power varies as their
proteolytic activity (estimated by their action on gelatin), and their optimum pH is the same as that ,of thrombin ; the proteolytic enzyme papain’shows similar properties. EAGLE therefore looks on blood4.
Grace, W. H., Harrison, C. V. 102.
5.
and
Davie, T.
B.
Lancet, 1940, ii,
Eagle, H. Medicine, Baltimore, 1937, 16, 95 ; J. exp. Med. 1937, 65, 613; Eagle, H. and Harris, T. N. J. gen. Physiol. 1937, 20, 543.
CLOTTING
the outcome of the reaction of two enzymes : (1) Inactive prothrombin z- activating proteolytic enzyme—s-
clotting as proteolytic
active
(2) Fibrinogen z-
thrombin
proteolytic —
enzyme thrombin.
fibrin.
The system whereby the precursor of a proteolytic enzyme is activated by a second proteolytic enzyme has already been found by KUNITZ and NoRTHROP6 in the activation of chymotrypsinogen by trypsin, and the blood-clotting system appears to be
similar. All this left unanswered two interlocking problems : ,Why does the blood not clot in vivo, and how is reaction (1) set off ? HowELi/s theory was that cir-
culating heparin acted as an anti-prothrombin and prevented blood from clotting in vivo, but when blood was shed a neutralising thromboplastin formed by disintegrating platelets had to be postulated. EAGLE also allbws that it remains doubtful how the platelets and calcium ions are induced to form the activating enzyme only when blood is shed. FERGUSON7 now suggests an explanation for some of the difficulties. He thinks that the activating proteolytic enzyme is to be found in the non-specific proteases that can be thus
demonstrated in tissue, blood-cells and serum, those with a trypsin-like action-tryptases-being important. Under certain conditions this enzyme can also digest prothrombin and thrombin-thus explaining the variable anti-thrombic activity of -serum-and can digest fibrinogen and fibrin causing " fibrinolysis." the enzyme is inactive ; " damage," he says, Normally " i.e. colloidal disturbance, introduces new conditions favourable to activation of the enzyme." When it is free, the tryptase in conjunction with ionised calcium and free phospholipid acts as the agent for conversion of prothrombin to thrombin. FERGUSON points out that tryptase-inhibitors exist in normal plasma and serum, and there is evidence that they are chemically similar to heparin, but similar inhibition can be produced by several other substances. These tryptaseinhibitors are thought to be present in sufficient concentration to prevent in-vivo coagulation. The main differences between the two theories are as follows. EAGLE believes that the first link in the is formed when blood is shed by combination chain of disintegrating platelets and calcium ; he assigns
place to heparin or phospholipid (cephalin), accepting the evidence that these are unnecessary and their importance unproven. FERGUSON believes that the initial enzyme is the normal tryptase present in
no
the blood in an inactive form ; it is activated when blood is shed by cephalin and calcium, but he does not know precisely how ; since cephalin and calcium are present in the blood, some tryptase may be activated in vivo and is kept in check by natural anti-tryptases of which heparin may be a type. FERGUSON asserts that serum-tryptase variations account for difficulties in the preparation of plasma products ; but others might retort, and quote figures in support, that platelet disintegration is responsible. While the crux of blood-clotting-the means whereby the chain of events is set in train when the blood is shed -remains unsolved, FERGUSON’S claim to a comprehensive hypothesis seems premature. But the physician will gather that the names he has learnt to 6. Kunitz, M. and Northrop, J. H. J. gen. 7. Ferguson, J. H. Science, 1943, 97, 319.
Physiol. 1935, 18, 433.
TOPOGRAPHY
OF DIPHTHERIA
associate with the twofold reaction of blood-clotting conceal a system of enzyme and activator such as he has met with elsewhere in physiology.
TOPOGRAPHY OF DIPHTHERIA BACILLI A
medium has many advanthe Loeffler slope in the investigation of diphtheria. Besides improving bacteriological diagnosis (the Loeffler slope misses 10-30% of clinical infections), the newer medium eliminates false positive reports on cultures from nose, ear or wound, it facilitates the detection of carriers, and by typedetermination it obviates the need for virulence tests in a large proportion of cases since practically all gravis and intermedius strains are toxigenic. Type-determination also helps towards a better understanding of the epidemiology of diphtheria and explains why the fatality is low in one area and high in another, according to the prevalence of the milder mitis infections or the more severe gravis and intermedius diphtheria. The Emergency Public Health Laboratory Service in its April Bulletin (p. 31) gives an analysis of the distribution of mitis, intermedius and gravis types of diphtheria bacilli during 1941, based on swabs from 8457 diphtheria patients examined at twenty laboratories, including a number of independent but associated laboratories such as those at Liverpool and Bristol- Their material is drawn from varied, but not all, parts of the country, and uafortunately the numbers examined at individual laboratories are often too small to be significant. Liverpool was not only heavily infected with gravis but contributed about a third of the total cases and therefore loaded the gravis figure ; excluding Liverpool, the average percentages become : mitis 27, intermedius 39-6 and gra,vis 334. In individual centres distribution was very variable ; thus Leeds showed a high incidence of gravis and a low of intermediusi Cardiff a very low gravis incidence with high figures for the other two strains; Bristol and Carmarthen were unique in having a very high intermedius rate. In Cambridge, Conway and Oxford the gravis percentages were about 50 ; in Liverpool and Leicester gravis accounted for
tellurite-containing
tages
over
Leeds,
over
70%. Geographical
distribution was not clearsuggest that gravis is the type in north, east and central
cut ; altogether the data
prevalent infecting England, while intermedius common
in the
and
mitis
are
south-west, but figures for other
more areas
including London are needed to fill in the gaps. It was possible to compare the types found in three cities in 1940 and 1941, for
Bristol, Leeds and Liver-
pool preserved complete figures derived from much the The change in frequency was same area in each year. statistically significant. To quote one example, the percentages in Leeds in the two years were mitis, 52’1 and 33-4 ; intermedius, 8-8 and 15-3 ; and gravis, 39-2 and 51-3.
Practical difficulties prevent a fair comparison of fatality with incidence of types. Thus every laboratory examined swabs from districts outside the town in which it was situated and not all cases notified in a
particular borough were necessarily reported on by the laboratory in that borough. Again, notifications included cases diagnosed clinically before bacteriological examinations had been carried out, and the diagnosis of such cases, if reversed on bacteriological examination, would
not be corrected in the returns
for
BACILLI
19
time to come. Anything from a third to half of cases diagnosed clinically by practinearly tioners failed to yield diphtheria bacilli in the laboratory, but this does not mean that none of these bacteriologically negative children had diphtheria ; although they are always present not every clinically indubitable case yields up its diphtheria bacilli to the swab, and to wait for bacteriological confirmation before taking action may, as every clinician knows, spell tragedy for the patient. Centres with the highest fatality also experienced a high gravis rate, but a fatality of under 4% was recorded in Leeds, Cardiff and Bristol with gravis rates 51-3, 6-8 and 8-2%. The fatality from the three types varies considerably in different areas and at different times, and it is not usually possible to pick out the particular The factors that have to be concause operating. sidered are general, social, and economic conditions, virulence of the prevalent type, stage of the epidemic (waxing, waning or at its peak), standard ofadministrative and medical treatment, degree of immunisation of the community, and age-distribution of the cases. Careful analysis of the experience of Liverpool during 1937-401 showed that the percentage incidence of mitis_fell from 41-1to 17-1and of intermedius from 24-7 to 13-3, whereas that of gravis rose from 34’2 to 69-6 in these four years. The fatality from each of the types remained remarkably constant at about 2% for mitis, rather more than 10% for intermedius and 6% for gravis infections. The high fatality from intermedius infections in this Liverpool inquiry bears out the general experience that in clinical severity, other things being equal, there is little to choose between gravis and intermedius infections. Scotland has lately experienced an epidemic wave of gravis infection with an increased fatality, a ’greater attack-rate in older age-groups, and in certain areas a high incidence among inoculated children-all evidence of infection by an epidemic invasive strain. From a consideration of these and other data RUSSELL2 thinks it possible that the type change is related in some way to the number of susceptible children in the population-gravis type prevailing when the proportion of protected children is low, mitis type when it is high. Yet it is not proven that the gravis infection always connotes high mortality. The Bulletin hopes that further work will reveal what effect artificial immunisation, if it becomes universal, will have on the type-distribution of diphtheria bacilli. Some localities should already be approaching, the degree of immunisation when we may see a change in the prevailing types, although as D. T. ROBINSON3 foresaw atypical strains and difficulties of classification are likely to arise where the disease is mild and none of the types has an epidemic distribution. If so, news of such increased technical difficulties of typing will be welcome because it will imply that immunisation has reached a pitch when, extended to the greater part of the most susceptible population, it will reduce the incidence of clinical cases. That such a reduction in incidence occurring in conjunction with large-scale immunisation has in fact been a case of cause and effect has some a
1. 2.
Wright, H. D. J. Path. Bact. 1941, 52, 283. Epidemiology of diphtheria during the last forty years. By W. T. Russell, D SC, of the statistical staff, Medical Research Council. Spec. 3. J. Path. Bact. 1934, 39, 551. Rep. Ser., 247. HMSO, 1s.
/
MECHANISM OF BLOOD
vascular upsets may be of first-rate importance, the adrenal changes being merely contributory to the usually fatal- outcome ; it is well known that adrenalectomised animals withstand shock badly. On these grounds he urges the importance of energetic attempts to restore normal vascular conditions by the administration of plasma as well as adrenal substitution, which he relegates to second place. The use of plasma or blood in addition to cortical therapy was also recommended by GRACE, HARRisoN and DAVIE,4 who recorded a success with this treatment. Two lessons are to be learned from this work. First, meningococcal infection is often, or even usually, septiceemic, and meningitis is only one manifestation of infection. Secondly, impending adrenal and vascular failure must be looked out for in meningoeoccal (and other) infections, and combated actively. when it occurs ; the prognosis in cases with sudden adrenal failure is grave but not hopeless.
MECHANISM OF BLOOD CLOTTING propounded the theory
i ORTY years ago MORAWITZ that blood-clotting is a
two-stage mechanism : in the first stage the prothrombin of blood-plasma in the presence of calcium ions and a tissue or platelet factor (thrombokinase, thromboplastin) is transformed into thrombin ; in the second stage the thrombin acts on the plasma-protein fibrinogen, converting it to the relatively insoluble fibrin which forms the scaffold of the clot. The framework of this theory is now generally accepted, but there is still much controversy about the detail. Useful information has been obtained from studying the blood-clotting activity of certain proteolytic enzymes, and by following the kinetics of the reactions. EAGLE 5 showed that trypsin and certain proteolytic snake-venoms in proper concentration " activated prothrombin to thrombin, and in this reaction calcium was not necessary. The concentration of trypsiri had to b optimal ; if it was - too weak -there was no action at all; if it was too strong both prothrombin and thrombin were destroyed. He compared the rates at which thrombin was formed from prothrombin by calcium-platelet mixture, crystalline trypsin, and the snake-venoms ; very similar curves were obtained. He therefore suggested that the normal action of the calcium-platelet combination was to form an enzyme that converted prothrombin to thrombin ; much as the trypsinogen .Qf the pancreatic secretion is converted by the enterokinase of the intestinal secretion into the actively "
-proteolytic trypsin. In the early days of blood-coagulation studies, ScHMlDT suggested that thrombin was a proteolytic enzyme acting on fibrinogen ; though not accepted by many, this has remained a possibility, and here :again study of the action of known proteolytic enzymes
has brought support. EAGLE has shown that some nake-venoms clot blood by acting directly on fibrinogen and converting it to fibrin ; they are active in very high dilutions, their power varies as their
proteolytic activity (estimated by their action on gelatin), and their optimum pH is the same as that ,of thrombin ; the proteolytic enzyme papain’shows similar properties. EAGLE therefore looks on blood4.
Grace, W. H., Harrison, C. V. 102.
5.
and
Davie, T.
B.
Lancet, 1940, ii,
Eagle, H. Medicine, Baltimore, 1937, 16, 95 ; J. exp. Med. 1937, 65, 613; Eagle, H. and Harris, T. N. J. gen. Physiol. 1937, 20, 543.
CLOTTING
the outcome of the reaction of two enzymes : (1) Inactive prothrombin z- activating proteolytic enzyme—s-
clotting as proteolytic
active
(2) Fibrinogen z-
thrombin
proteolytic —
enzyme thrombin.
fibrin.
The system whereby the precursor of a proteolytic enzyme is activated by a second proteolytic enzyme has already been found by KUNITZ and NoRTHROP6 in the activation of chymotrypsinogen by trypsin, and the blood-clotting system appears to be
similar. All this left unanswered two interlocking problems : ,Why does the blood not clot in vivo, and how is reaction (1) set off ? HowELi/s theory was that cir-
culating heparin acted as an anti-prothrombin and prevented blood from clotting in vivo, but when blood was shed a neutralising thromboplastin formed by disintegrating platelets had to be postulated. EAGLE also allbws that it remains doubtful how the platelets and calcium ions are induced to form the activating enzyme only when blood is shed. FERGUSON7 now suggests an explanation for some of the difficulties. He thinks that the activating proteolytic enzyme is to be found in the non-specific proteases that can be thus
demonstrated in tissue, blood-cells and serum, those with a trypsin-like action-tryptases-being important. Under certain conditions this enzyme can also digest prothrombin and thrombin-thus explaining the variable anti-thrombic activity of -serum-and can digest fibrinogen and fibrin causing " fibrinolysis." the enzyme is inactive ; " damage," he says, Normally " i.e. colloidal disturbance, introduces new conditions favourable to activation of the enzyme." When it is free, the tryptase in conjunction with ionised calcium and free phospholipid acts as the agent for conversion of prothrombin to thrombin. FERGUSON points out that tryptase-inhibitors exist in normal plasma and serum, and there is evidence that they are chemically similar to heparin, but similar inhibition can be produced by several other substances. These tryptaseinhibitors are thought to be present in sufficient concentration to prevent in-vivo coagulation. The main differences between the two theories are as follows. EAGLE believes that the first link in the is formed when blood is shed by combination chain of disintegrating platelets and calcium ; he assigns
place to heparin or phospholipid (cephalin), accepting the evidence that these are unnecessary and their importance unproven. FERGUSON believes that the initial enzyme is the normal tryptase present in
no
the blood in an inactive form ; it is activated when blood is shed by cephalin and calcium, but he does not know precisely how ; since cephalin and calcium are present in the blood, some tryptase may be activated in vivo and is kept in check by natural anti-tryptases of which heparin may be a type. FERGUSON asserts that serum-tryptase variations account for difficulties in the preparation of plasma products ; but others might retort, and quote figures in support, that platelet disintegration is responsible. While the crux of blood-clotting-the means whereby the chain of events is set in train when the blood is shed -remains unsolved, FERGUSON’S claim to a comprehensive hypothesis seems premature. But the physician will gather that the names he has learnt to 6. Kunitz, M. and Northrop, J. H. J. gen. 7. Ferguson, J. H. Science, 1943, 97, 319.
Physiol. 1935, 18, 433.
TOPOGRAPHY
OF DIPHTHERIA
associate with the twofold reaction of blood-clotting conceal a system of enzyme and activator such as he has met with elsewhere in physiology.
TOPOGRAPHY OF DIPHTHERIA BACILLI A
medium has many advanthe Loeffler slope in the investigation of diphtheria. Besides improving bacteriological diagnosis (the Loeffler slope misses 10-30% of clinical infections), the newer medium eliminates false positive reports on cultures from nose, ear or wound, it facilitates the detection of carriers, and by typedetermination it obviates the need for virulence tests in a large proportion of cases since practically all gravis and intermedius strains are toxigenic. Type-determination also helps towards a better understanding of the epidemiology of diphtheria and explains why the fatality is low in one area and high in another, according to the prevalence of the milder mitis infections or the more severe gravis and intermedius diphtheria. The Emergency Public Health Laboratory Service in its April Bulletin (p. 31) gives an analysis of the distribution of mitis, intermedius and gravis types of diphtheria bacilli during 1941, based on swabs from 8457 diphtheria patients examined at twenty laboratories, including a number of independent but associated laboratories such as those at Liverpool and Bristol- Their material is drawn from varied, but not all, parts of the country, and uafortunately the numbers examined at individual laboratories are often too small to be significant. Liverpool was not only heavily infected with gravis but contributed about a third of the total cases and therefore loaded the gravis figure ; excluding Liverpool, the average percentages become : mitis 27, intermedius 39-6 and gra,vis 334. In individual centres distribution was very variable ; thus Leeds showed a high incidence of gravis and a low of intermediusi Cardiff a very low gravis incidence with high figures for the other two strains; Bristol and Carmarthen were unique in having a very high intermedius rate. In Cambridge, Conway and Oxford the gravis percentages were about 50 ; in Liverpool and Leicester gravis accounted for
tellurite-containing
tages
over
Leeds,
over
70%. Geographical
distribution was not clearsuggest that gravis is the type in north, east and central
cut ; altogether the data
prevalent infecting England, while intermedius common
in the
and
mitis
are
south-west, but figures for other
more areas
including London are needed to fill in the gaps. It was possible to compare the types found in three cities in 1940 and 1941, for
Bristol, Leeds and Liver-
pool preserved complete figures derived from much the The change in frequency was same area in each year. statistically significant. To quote one example, the percentages in Leeds in the two years were mitis, 52’1 and 33-4 ; intermedius, 8-8 and 15-3 ; and gravis, 39-2 and 51-3.
Practical difficulties prevent a fair comparison of fatality with incidence of types. Thus every laboratory examined swabs from districts outside the town in which it was situated and not all cases notified in a
particular borough were necessarily reported on by the laboratory in that borough. Again, notifications included cases diagnosed clinically before bacteriological examinations had been carried out, and the diagnosis of such cases, if reversed on bacteriological examination, would
not be corrected in the returns
for
BACILLI
19
time to come. Anything from a third to half of cases diagnosed clinically by practinearly tioners failed to yield diphtheria bacilli in the laboratory, but this does not mean that none of these bacteriologically negative children had diphtheria ; although they are always present not every clinically indubitable case yields up its diphtheria bacilli to the swab, and to wait for bacteriological confirmation before taking action may, as every clinician knows, spell tragedy for the patient. Centres with the highest fatality also experienced a high gravis rate, but a fatality of under 4% was recorded in Leeds, Cardiff and Bristol with gravis rates 51-3, 6-8 and 8-2%. The fatality from the three types varies considerably in different areas and at different times, and it is not usually possible to pick out the particular The factors that have to be concause operating. sidered are general, social, and economic conditions, virulence of the prevalent type, stage of the epidemic (waxing, waning or at its peak), standard ofadministrative and medical treatment, degree of immunisation of the community, and age-distribution of the cases. Careful analysis of the experience of Liverpool during 1937-401 showed that the percentage incidence of mitis_fell from 41-1to 17-1and of intermedius from 24-7 to 13-3, whereas that of gravis rose from 34’2 to 69-6 in these four years. The fatality from each of the types remained remarkably constant at about 2% for mitis, rather more than 10% for intermedius and 6% for gravis infections. The high fatality from intermedius infections in this Liverpool inquiry bears out the general experience that in clinical severity, other things being equal, there is little to choose between gravis and intermedius infections. Scotland has lately experienced an epidemic wave of gravis infection with an increased fatality, a ’greater attack-rate in older age-groups, and in certain areas a high incidence among inoculated children-all evidence of infection by an epidemic invasive strain. From a consideration of these and other data RUSSELL2 thinks it possible that the type change is related in some way to the number of susceptible children in the population-gravis type prevailing when the proportion of protected children is low, mitis type when it is high. Yet it is not proven that the gravis infection always connotes high mortality. The Bulletin hopes that further work will reveal what effect artificial immunisation, if it becomes universal, will have on the type-distribution of diphtheria bacilli. Some localities should already be approaching, the degree of immunisation when we may see a change in the prevailing types, although as D. T. ROBINSON3 foresaw atypical strains and difficulties of classification are likely to arise where the disease is mild and none of the types has an epidemic distribution. If so, news of such increased technical difficulties of typing will be welcome because it will imply that immunisation has reached a pitch when, extended to the greater part of the most susceptible population, it will reduce the incidence of clinical cases. That such a reduction in incidence occurring in conjunction with large-scale immunisation has in fact been a case of cause and effect has some a
1. 2.
Wright, H. D. J. Path. Bact. 1941, 52, 283. Epidemiology of diphtheria during the last forty years. By W. T. Russell, D SC, of the statistical staff, Medical Research Council. Spec. 3. J. Path. Bact. 1934, 39, 551. Rep. Ser., 247. HMSO, 1s.
/